Discovery of potent cytotoxic ortho-aryl chalcones as new scaffold targeting tubulin and mitosis with affinity-based fluorescence

Article abstract of DOI:10.1021/jm500024v

A series of new ortho-aryl chalcones have been designed and synthesized. Many of these compounds were found to exhibit significant antiproliferation activity toward a panel of cancer cell lines. Selected compounds show potent cytotoxicity against several drug resistant cell lines including paclitaxel (Taxol) resistant human ovarian carcinoma cells, vincristine resistant human ileocecum carcinoma cells, and doxorubicin resistant human breast carcinoma cells. Further investigation revealed that active analogues could inhibit the microtubule polymerization by binding to colchicine site and thus induce multipolar mitosis, G₂/M phase arrest, and apoptosis of cancer cells. Furthermore, affinity-based fluorescence enhancement was observed during the binding of active compounds with tubulin, which greatly facilitated the determination of tubulin binding site of the compounds. Finally, selected compound 26 was found to exhibit obvious in vivo antitumor activity in A549 tumor xenografts model. Our systematic studies implied a new scaffold targeting tubulin and mitosis for novel antitumor drug discovery.

Full text of DOI:10.1021/jm500024v

Article
pubs.acs.org/jmc
Discovery of Potent Cytotoxic Ortho-Aryl Chalcones as New Scaffold
Targeting Tubulin and Mitosis with Affinity-Based Fluorescence
Cuige Zhu,† Yinglin Zuo,† Ruimin Wang, Baoxia Liang, Xin Yue, Gesi Wen, Nana Shang, Lei Huang,
Yu Chen, Jun Du, and Xianzhang Bu*
School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China
S
* Supporting Information
ABSTRACT: A series of new ortho-aryl chalcones have been
designed and synthesized. Many of these compounds were
found to exhibit significant antiproliferation activity toward a
panel of cancer cell lines. Selected compounds show potent
cytotoxicity against several drug resistant cell lines including
paclitaxel (Taxol) resistant human ovarian carcinoma cells,
vincristine resistant human ileocecum carcinoma cells, and
doxorubicin resistant human breast carcinoma cells. Further investigation revealed that active analogues could inhibit the
microtubule polymerization by binding to colchicine site and thus induce multipolar mitosis, G2/M phase arrest, and apoptosis of
cancer cells. Furthermore, affinity-based fluorescence enhancement was observed during the binding of active compounds with
tubulin, which greatly facilitated the determination of tubulin binding site of the compounds. Finally, selected compound 26 was
found to exhibit obvious in vivo antitumor activity in A549 tumor xenografts model. Our systematic studies implied a new
scaffold targeting tubulin and mitosis for novel antitumor drug discovery.
resistance and normally exhibit systematic toxicity, etc., which
limited their clinical applications11 and thus urged the
prompted development of novel microtubule targeting agents
for cancer treatment.
INTRODUCTION
■
Microtubules are ubiquitous slender filamentous dynamic
polymers of α-tubulin and β-tubulin heterodimers in cell and
serve as cell cytoskeleton for intracellular transports including
movement of organelles, vesicles, proteins, or signaling
molecules by motor proteins throughout the cell structure.1
In the cell mitosis, highly dynamic microtubules also play a key
role in completing the cell division by forming mitotic spindle,
driving the intricate movement of chromosomes and separating
sister chromatids.1a,2 Microtubule dynamics are tightly
regulated both spatially and temporally.3 Tubulin binding
molecules could induce abnormal mitotic spindles formation
through interference with the dynamic instability of micro-
tubules, thereby disrupting microtubules with the result of cell
cycle arrest and apoptotic cell death.4
To date, microtubule dynamics has become one of the most
successful anticancer targets.5 Many natural occurring and
synthetic compounds interfering with microtubule dynamics
have been developed. Paclitaxel, vinblastine, and colchicine
represent the well-known three types of microtubule targeting
agents which are distinct from their different binding domains
named taxane, vinca, and colchicine binding site of β-tubulin,
respectively.6 The vinca alkaloids7 and taxanes,8 identified
respectively over 50 and 40 years ago, are currently
administered in broad clinical indications.9 Recently, colchicine
domain binding combretastatin A-4P (CA4P) as vascular
disrupting agents has been approved for clinical trials.10 The
mentioned representative drugs are shown in Figure 1.
However, besides the rare resource and complex and expensive
syntheses of some natural drugs like paclitaxel, many clinically
applied microtubule targeting drugs easily acquire tumor
The natural occurring curcumin (1,7-bis(4-hydroxy-3-me-
thoxyphenyl)-1,6-heptadiene-3,5-dione), a dietary yellow spice
and pigment isolated from the rhizome of Curcuma longa, has
been found to exhibit a wide range of biological activities,
including anticancer, antimicrobial, anti-inflammatory, and
antioxidation activities.12 The promising biological profile and
easy synthetic accessibility have triggered design and develop-
ment of new curcumin derivatives as potential chemo-
therapeutical agents.12,13 In our previous studies, we reported
a series of 4-arylidene curcumin analogues that could target
nuclear factor κB (NF-κB) activation pathway with much
enhanced cytotoxic activity than curcumin.14 During the
structure optimization, meta-phenyl substituted chalcones
(Figure 1) were proposed by structure moiety deletion and
conformation restriction based on the preliminary analysis of
the interaction of 4-arylidene curcumin with IKKβ, in order to
obtain improved potency in cytotoxicity. Biological evaluation
revealed that most of these compounds showed moderate
cytotoxic and NF-κB inhibitory activity.15 Here we describe the
design and biological evaluation of a series of ortho-aryl
substituted chalcones (Figure 1) as extended efforts. Though
demonstrated nonactive on NF-κB inhibition, many of these
new types of chalcones showed unexpectedly higher cytotox-
icity than our previous reported 4-arylidene curcumins and
Received: January 6, 2014
Published: July 25, 2014
© 2014 American Chemical Society
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meta-phenyl chalcones, with IC50 values ranged from moderate
to low nanomolar concentrations against a panel of parental
and drug resistant cancer cell lines. The selected potent
compounds are significantly superior to colchicine, paclitaxel
(Taxol), doxorubicin, and vincristine in the growth inhibition of
several drug resistant cancer cell lines, and furthermore, tubulin
was identified as their potential molecular target by biological
evaluations coupled with fluorescent assay based on the
interesting affinity-induced fluorescent property of selected
compounds. Finally, selected compound with high potency was
found to exhibit obvious antitumor activity in vivo.
RESULTS AND DISCUSSION
■
Chemistry. The ortho-aryl substituted chalcone analogues
2−6, 8−17 were synthesized as illustrated in Scheme 1. In brief,
microwave assistant Suzuki coupling of 1-(2-bromophenyl)-
ethanone with (3,4-dimethoxylphenyl)boronic acid or (4-
fluorophenyl)boronic acid yields biphenyl intermediate 1 or
7, respectively, and then KOH catalyzed aldol condensation of
1 or 7 with various arylaldehydes affords 2−6 or 8−17,
respectively.
For the synthesis of 21−28, an aldol condensation followed
by Suzuki coupling procedure was adopted. As illustrated in
Scheme 2, chalcone intermediates 18−20 were prepared first
by NaOH catalyzed aldol condensation and then coupled with
various arylboronic acids by microwave assisted Suzuki reaction
to give final products 21−28, respectively. Similar procedures
were also used in the synthesis of analogues bearing reversed
α,β-unsaturated ketone moiety (35−45) (Scheme 3).
Figure 1. Structures of well-known microtubule-targeting agents and
the design of aryl chalcones.
a
Scheme 1. Synthesis of Compounds 2−6 and 8−17
a
Reagents and conditions: (a) PdCl2(dppf), 2 M K2CO3, dioxane, microwave heating, 150 °C, 20 min; (b) KOH, EtOH, rt.
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a
Scheme 2. Synthesis of Compounds 21−28
a
Reagents and conditions: (a) 6 M NaOH (aq), EtOH, rt; (b) PdCl2(dppf), 2 M K2CO3, dioxane, microwave heating, 150 °C, 20 min.
a
Scheme 3. Synthesis of Compounds 35−45
a
Reagents and conditions: (a) 6 M NaOH (aq), EtOH, rt; (b) PdCl2(dppf), 2 M K2CO3, dioxane, microwave heating, 150 °C, 20 min.
a
Scheme 4. Synthesis of Compounds 49−51
a
Reagents and conditions: (a) SOCl2, reflux; (b) toluene, TEA, reflux; (c) PdCl2(dppf), 2 M K2CO3, dioxane, microwave heating, 150 °C, 20 min.
Preparation of 49−51 was achieved respectively by acylation
of the different substituted benzylamine with 2-bromobenzoyl
chloride to afford 46−48 first, followed by Suzuki coupling with
4-fluorophenylboronic acid, as shown in Scheme 4.
Antiproliferative Activity Evaluation of Obtained
Compounds against a Panel of Cancer Cell Lines. To
evaluate the antiproliferative activity of obtained compounds,
sulforhodamine B (SRB) assay was performed. Human lung
carcinoma cell line A549, nasopharyngeal carcinoma cell line
CNE2, colon carcinoma cell line SW480, breast carcinoma cell
line MCF7, and hepatoma carcinoma cell line HepG2 were
chosen. In actual work, the design of these compounds was
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Table 1. Antiproliferative Activities of 2−6 against Different Cancer Cells
a
IC50, mean SE (μM)
compd
A549
CNE2
SW480
MCF-7
HepG2
2
3
4
5
6
8.6 1.4
1.6 0.1
8.5 1.4
4.1 0.7
6.8 0.9
5.4 0.1
0.32 0.02
2.5 0.25
2.4 0.5
4.8 0.8
6.8 0.6
0.89 0.21
7.6 0.6
5.2 0.2
6.5 0.2
7.1 0.1
1.1 0.0
4.9 0.9
3.1 0.4
8.3 0.0
4.3 0.3
0.60 0.16
3.2 0.5
2.1 0.3
3.3 1.6
a
Data are presented as the mean SE from the dose−response curves of at least two independent experiments.
Table 2. Antiproliferative Activities of 8 and 21−23 against Different Cancer Cells
a
IC50, mean SE (μM)
compd
A549
CNE2
SW480
MCF-7
HepG2
8
0.14 0.04
0.28 0.00
0.93 0.03
0.58 0.17
0.053 0.008
0.073 0.001
0.16 0.01
0.061 0.008
0.14 0.02
0.33 0.04
0.20 0.04
0.67 0.36
0.64 0.35
2.7 0.0
0.093 0.023
0.11 0.02
21
22
23
0.29 0.02
0.064 0.007
0.83 0.28
0.097 0.027
a
Data are presented as the mean SE from the dose−response curves of at least two independent experiments.
Table 3. Antiproliferative Activities of 9−17, 24, and 26 against Different Cancer Cells
a
IC50, mean SE (μM)
compd
A549
CNE2
SW480
MCF-7
HepG2
9
0.44 0.11
1.1 0.2
0.081 0.008
0.75 0.24
5.2 0.5
0.57 0.20
2.4 0.1
0.24 0.05
1.7 0.5
0.095 0.028
0.52 0.18
5.0 1.4
10
11
7.1 1.3
5.7 0.1
5.3 0.9
12
7.6 2.1
2.5 0.1
6.6 0.6
4.0 1.0
2.8 0.2
13
0.065 0.035
0.082 0.013
0.073 0.010
0.087 0.010
0.021 0.000
0.030 0.002
0.057 0.010
0.041 0.009
0.041 0.010
0.19 0.08
0.22 0.03
0.23 0.06
0.023 0.007
0.030 0.003
0.047 0.011
14
15
16
31
12
1
2
14
2
23
16
0
1
23
6
12
2
17
5.9 0.8
9.5 1.7
6.2 1.5
24
0.33 0.04
0.65 0.58
0.16 0.02
1.2 0.1
0.15 0.01
26
0.011 0.002
0.050 0.032
0.0050 0.0010
0.017 0.004
0.034 0.005
0.0030 0.0003
0.010 0.004
0.033 0.005
0.0070 0.0004
0.065 0.020
0.16 0.09
0.023 0.004
0.0080 0.0010
0.035 0.008
0.028 0.007
colchicine
paclitaxel
a
Data are presented as the mean SE from the dose−response curves of at least two independent experiments.
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Table 4. Antiproliferative Activities of 25, 27, and 28 against Different Cancer Cells
a
IC50, mean SE (μM)
compd
A549
CNE2
SW480
MCF-7
HepG2
25
27
28
1.2 0.1
1.6 0.9
1.3 0.2
2.8 0.5
1.0 0.3
0.29 0.00
0.19 0.06
0.79 0.16
0.16 0.06
0.30 0.05
0.17 0.03
0.68 0.16
0.48 0.06
0.19 0.02
0.23 0.03
a
Data are presented as the mean SE from the dose−response curves of at least two independent experiments.
Table 5. Antiproliferative Activities of 35−45 and 49−51 against Different Cancer Cells
a
IC50, mean SE (μM)
compd
A549
CNE2
SW480
MCF-7
HepG2
35
36
37
38
39
40
41
42
43
44
45
49
50
51
8.2 2.0
>50
9.0 3.0
8.4 1.5
8.8 2.2
6.3 1.1
8.2 0.9
6.6 0.7
13
7.6 1.9
17
7
10
8.3 0.5
15
1
13
7.1 1.2
41
9.3 0.0
12
5.4 0.2
27
3
7.5 0.6
17
5
5
1
2
17
1
6.9 1.3
6.9 2.6
4.6 1.3
7.3 2.1
8.7 3.5
4.8 1.1
8.7 0.1
7.4 0.9
4.2 0.4
8.5 3.4
0.34 0.15
1.6 0.3
2.2 0.0
>50
4.2 0.1
6.4 1.3
3.1 0.1
9.9 0.3
0.19 0.01
1.8 0.6
2
13
7
19
9
1
0.94 0.64
3.2 1.4
2.2 0.3
>50
0.75 0.53
2.1 1.3
3.8 0.5
>50
0.56 0.06
2.0 0.7
9.1 0.2
>50
12
2
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
a
Data are presented as the mean SE from the dose−response curves of at least two independent experiments.
based on an activity-guided approach. First, analogues 2−6 with
the 3,4-dimethoxyl substituted on ring C (rings A, B, and C are
defined in Figure 1 for convenience) were designed and
evaluated, and the results in Table 1 indicate that compound 3
which bears a 4-methoxyl substitution on ring B increases the
activity compared to the 3,4-dimethoxyl (4), 2,5-dimethoxyl
(5), or 4-halogen (F or Br, 2 and 6) substitutions, showing low
micromolar concentration of IC50 value against the five tested
cancer cell lines (IC50 of 0.32−1.6 μM).
This result leads to the design and evaluation of more
analogues with different substituted phenyl or pyridinyl of ring
C and the same 4-methoxyl substitution on ring B (8, 21−23).
The data summarized in Table 2 show that all of the four
compounds generally exhibit higher cytotoxicity than 3, with
submicromolar concentration of IC50 (0.053−0.93 μM) except
for the individual case of 22 against the MCF-7 cell line (IC50
of 2.7 μM), and the fluoro-substituted analogue 8 is
demonstrated to be the most potent of these derivatives.
From Table 2, we also learned that the replacement of
phenyl with pyridinyl at position of ring C decreased slightly
the cytotoxicity in general. Therefore, 3- or 4-fluoro substituted
phenyl of ring C was reserved and the moiety of ring B was
redesigned for evaluation. The inhibitory effects of the resulting
24, 26, and 9−17 were summarized in Table 3. Among the
analogues with 4-F substitution, compounds with 3,4-
dimethoxyl and 2,5-dimethoxyl on ring B (9 and 10,
respectively) exhibit generally lower activity (IC50 of 0.081−
2.4 μM) than 8 (IC50 of 0.053−0.67 μM), which is in
consistence with the rule revealed by comparing 3 with 4 and 5.
Meanwhile, 2,3-dimethoxyl (11) and 4-hydroxyl-3-methoxyl
(12) substitutions on ring B further decrease the activities (IC50
of 2.5−7.6 μM) than 9 and 10; all of the above information
indicated that the methoxyl substitutions in the meta or ortho
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Table 6. Drug Tolerances of Selected Compounds against Different Drug Resistant Cancer Cells
a
IC50, mean SE (μM)
b
compd
A2780
A2780/TAX
DRI
HCT-8
HCT-8/VCT
DRI
A549/CDDP
DRI
MCF-7/DOX
DRI
8
0.17 0.01
0.69 0.00
0.50 0.01
0.23 0.06
0.049 0.012
0.86 0.12
0.23 0.01
1.6 0.2
4.1
18
0.22 0.05
0.23 0.02
0.42 0.03
0.084 0.015
0.096 0.009
0.22 0.00
0.057 0.015
2.0 0.1
1.0
11
0.21 0.06
0.43 0.22
0.074 0.004
0.11 0.04
0.48 0.16
0.18 0.11
0.59 0.19
0.081 0.011
0.0090 0.0020
0.29 0.02
0.056 0.017
1.1
10
0.12 0.17
0.29
0.040
0.60
0.68
0.64
0.030
0.24
169
13
0.027 0.009
0.063 0.015
0.094 0.020
0.16 0.01
0.038 0.020
0.063 0.009
0.13 0.02
0.0070 0.0003
0.024 0.001
0.045 0.007
0.084 0.012
0.0020 0.0003
0.17 0.03
14
3.6
0.52
5.2
18
1.3
0.72
1.1
3.6
4.0
19
1.1
1.6
2.1
16
15
21
0.20 0.02
26
0.013 0.005
0.38 0.17
0.016 0.008
0.52 0.26
27
4.3
10
2.0
2.3
1.8
1.0
4.7
COL
TAX
DOX
VCT
0.057 0.003
0.015 0.009
0.59 0.08
2.9 0.5
0.059 0.007
0.021 0.013
1.1 0.4
3.2 0.2
196
2.6 0.9
124
7.6 1.0
2525
106
c
c
ND
1.9 0.3
ND
3.4 0.3
15
4
c
c
ND
0.93 0.05
ND
1.9 0.5
4.3 0.8
1063
a
Data are presented as the mean SE from the dose−response curves of at least two independent experiments. A2780 and HCT-8 were also
b
c
evaluated as the parental control cells in this assay. Drug resistant index: (IC50 of drug resistant cancer cell)/(IC50 of parental cancer cell). ND =
not determined.
position of the ethylenic linkage of ring B contritute negatively
to the activities. On the other hand, replacements of ring B by
heterocyclic furan (16) or thiophen (17) moiety largely
decreased their activity (IC50: of 5.9−31 μM).
41, and 22 vs 36, we concluded that the α,β-unsaturated ketone
moiety itself and its direction in the molecular scaffold were
crucial for their cytotoxicity.
A main drawback of clinically used anticancer drugs is the
drug resistance of tumor cells. Therefore, we also evaluate the
antiproliferation activities of selected compounds against a
panel of parental and drug resistant cancer cell lines to
demonstrate their potential in anticancer. We chose the
representative and commercially available human ovarian
carcinoma cell A2780 with the paclitaxel resistant cell A2780/
TAX and human ileocecum carcinoma cell HCT-8 with the
vincristine resistant cell HCT-8/VCT as the paclitaxel and
vincristine resistant models, respectively. Furthermore, the cis-
platinum resistant cell A549/CDDP and doxorubicin resistant
cell MCF-7/DOX were chosen as representative models for
well-known multidrug resistant cancer cells. Highly active
compounds 13−15 and 26 with relative less active compounds
8, 21, and 27 were selected for the examination. Colchicine,
paclitaxel, vincristine, and doxorubicin were employed as
positive controls. The results were summarized in Table 6.
The results show that the drug resistance of MCF-7/DOX
was overcome by all selected compounds 8, 13−15, 21, 26, and
27 with significantly lower IC50 value than that against MCF-7.
The drug resistant index (DRI), which were calculated from the
ratio of IC50 from drug resistant cancer cell over the IC50 from
parental cancer cell (Table 6), ranged from 0.03 to 0.68, and
Interestingly, compared to 8, a further substitution of 3-
hydroxyl (13), 3-boric acid (14), or replacement of 4-methoxyl
with 4-N,N-dimethyl (15) on ring B dramatically increased the
activities, and IC50 values closer or comparable to those of
colchicine (IC50 of 0.033−0.16 μM) and paclitaxel (IC50 of
0.0030−0.028 μM) were observed. Except for the inhibition of
MCF-7 (IC50 of 0.19−0.23 μM), all three compounds show
IC50 values in nanomolar concentration range (0.021−0.087
μM) on the other four cancer cell lines. At the same time,
compound 26, which was structurally different from 13 by the
position of fluoro substitution on ring C, generally demon-
strated the highest inhibitory activity (IC50 of 0.0080−0.065
μM), shows superior potency to the colchicine, and is less
potent in 4/5 cell lines but in one case (HepG2) has higher
potency than paclitaxel. Methylation of the 3-hydroxyl on ring
B of 26 yields much less active compound 24 (IC50 of 0.15−1.2
μM), which is consistent with the above conclusion that
methoxyl substitutions on ring B in the meta position of the
ethylenic linkage greatly decrease the activity.
More structure−activity relationship information was there-
fore investigated. 25, the pyridinyl containing compound,
exhibits obvious lower activity than 9, suggesting again that the
pyridine moiety on ring C does not favor the activity (Table 4).
Noticeably, trimethoxyl substituted phenyl moiety frequently
appeared in highly active tubulin binders such as colchicine,
CA4P, and many of their derivatives and mimics. We found
that the incorporated compounds 27 and 28 (IC50 of 0.16−
0.79 μM) display much lower activity than 13 and 26,
respectively, though with submicromolar concentration of IC50
(Table 4).
most active compounds 13 (IC50 = 0.0070 μM) and 26 (IC50
=
0.0020 μM) show dramatically high potency in the MCF-7/
DOX growth inhibition; similar results were observed in the
cases of 15 against A2780/TAX (IC50 = 0.049 μM, DRI = 0.52)
and HCT-8/VCT (IC50 = 0.096 μM, DRI = 0.72). No obvious
drug tolerance was found (DRI < 1.5) in the cases of 8 and 14
against HCT-8/VCT and A549/CDDP and of 21 against
HCT-8/VCT. Slight to moderate drug resistance was observed
in the cases of 8, 13, 14, 21, 26, 27 against A2780/TAX (DRI =
3.6−18), 13, 26, 27 against HCT-8/VCT (DRI = 3.6−11), and
13, 15, 21, 26, 27 against A549/CDDP (DRI = 1.6−16).
Nevertheless, all these compounds exhibit inhibitory effects
against the tested cancer cell lines with submicromolar
concentration of IC50 except for the individual case of 27.
Noticeably, though the positive controls colchicine, paclitax-
el, vincristine, and doxorubicin show high activity in all tested
parental cancer cell lines, all of them were greatly tolerated by
the tested drug resistant cancer cell lines A2780/TAX (DRI =
To probe the contribution of the α,β-unsaturated ketone
moiety to the cytotoxicity, analogues with directional reversed
α,β-unsaturated ketone moiety 35−45 and replacements of
amide moiety (49−51) were examined. The results were
summarized in Table 5. We found that 35−45 only show low
to moderate inhibitory activity against the tested five cancer cell
lines except the 3-hydroxyl-4-methoxyl substitution bearing
compound 43. Meanwhile, no obvious cytotoxicity was
observed on 49−51 at concentrations up to 50 μM. By further
comparing the activity of 8 vs 35 and 49, 9 vs 37 and 50, 12 vs
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Figure 2. Cell cycle distribution of A549 with or without treatment of different compounds. A549 cells were plated in 96-well plates for 24 h and
treated without and with different concentrations of tested compounds 8, 13, 14, 15, and 26 (0, 7.81, 15.63, 31.25, 62.5, 125, and 250 μM), and
colchicine treated with the same concentration as the control. After 24 h, cells were fixed by 4% paraformaldehyde and stained by Hoechst 33342
and then analyzed by ArrayScan VTI high content analysis system.
Figure 3. 15 induced apoptosis of A549 cells. Cells were seed in a six-well plate for 24 h. Then different concentrations of 15 (0.25, 0.50, 0.75, and
1.0 μM) and positive control colchicine (0.25 μM) were added for 48 h. Then 1 × 105 cells were collected by centrifugation and resuspended in 500
μL of 1× binding buffer and were analyzed by flow cytometer after being stained by 5 μL of annexin V-FITC and 10 μL of propidium iodide for 5
min in the dark.
10−196), HCT-8/VCT (DRI = 19−124), and MCF-7/DOX
(DRI = 106−2525) and slightly resisted by A549/CDDP (DRI
= 1.8−4.7) except for doxorubicin. Furthermore, the IC50
values of positive controls are significantly higher than those
of 13−15 and 26 by several to thousands times during the
growth inhibition of A2780/TAX, HCT-8/VCT, and MCF-7/
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Figure 4. Effects of selected compounds on the microtubule polymerization. Tubulin was mixed with selected compounds in general tubulin buffer
containing 1 mM GTP and 20% glycerol. Microtubule polymerization was monitored in kinetic model of FlexStation 3 multimode reader (excitation,
360 nm; emission, 450 nm).
DOX. To explore the potential mechanism under the
observation, we examined the intracellular P-gp function by
rho123 accumulation and efflux assay in A2780/TAX, HCT-8/
VCT, and MCF-7/DOX and their parental cell lines. We found
that rho123 accumulated very well in all three parental cell
lines, and no obvious efflux was observed during the examined
time (Supporting Information, Figures S1 and S2d). On the
contrary, much lower rho123 accumulation was observed in
three drug resistant cancer cells and the accumulated rho123
obviously eliminated along with the time consumption
(Supporting Information, Figures S1 and S2a−c). These results
indicated a much elevated intracellular P-glycoprotein function
in the investigated three drug resistant cancer cell lines.
Reasonably, our compounds may evade a drug efflux pump
mechanism which may affect the positive control drugs such as
paclitaxel, doxorubicin, and vincristine.
Taking the above results together, we discovered that 3′- or
4′-fluoro substituted 1-([1,1′-biphenyl]-2-yl)-3-phenylprop-2-
en-1-one may serve as potent cytotoxic scaffold, especially with
3-hydroxyl-4-methoxyl, 3-boric acid 4-methoxyl, or 4-N,N-
dimethyl substituted on ring B.
Active Analogues Induce G2/M Arrest and Apoptosis
of A549 Cells. As we mentioned before, the aryl-substituted
chalcones were originally designed to improve the potency to
inhibit the NF-κB activation pathway.15 Unfortunately, we
found that the o-aryl chalcones totally lost their ability of NF-κB
blockage (data not shown). However, the finding of highly
active compounds encouraged us to search for their potential
molecular target. Therefore, we first investigated the effects of
the found active compounds on the cell cycle. 8, 13−15, and 26
were chosen and evaluated on the A549 cancer cell line. A549
was treated with different concentrations of employed
compounds for 24 h. The nuclei were stained with Hoechst
33342, and the fluorescence was recorded by ArrayScanVTI
high content analysis system. The percentage of each cell cycle
phase was calculated by the ratio of the population of cells with
certain gated fluorescence intensity over the total counted cell
number. The results are shown in Figure 2. An obvious G2/M
phase arrest is observed in all 8, 13−15, and 26 treated A549
cells, which are similar to the positive control colchicine.
Colchicine induces significant G2/M arrest at 31.25 nM
concentration, the same as 26 achieved. 13−15 induced
obvious G2/M arrest at 62.5 nM, and the less active function
of 8 was observed at 125 nM.
To assess whether the active compound would induce cancer
cell apoptosis, 15 treated A549 cells were stained with annexin
V-FITC and propidium iodide (PI) and analyzed. The flow
cytometric data (Figure 3) show concentration-dependent high
activity on stimulating cell apoptosis, and the total numbers of
early and late apoptotic cells were 69.10%, 75.71%, 82.06%, and
83.88% in the cases of 0.25, 0.50, 0.75, and 1.0 μM 15 treated
A549 cells, respectively. Positive control colchicine (0.25 μM)
induced 36.19% and 37.72% early and late apoptosis cells,
respectively. In contrast, only 0.47% and 0.10% early and late
apoptotic cells were found in untreated control. This result
confirmed that 15 could induce efficient apoptosis of A549.
Microtubule Polymerization Inhibition of Selected
Compounds. The above data suggest that these molecules
may interfere with the mitotic phase and induce apoptosis of
cancer cells, which are typical properties of microtubule binding
agents.4 16 Therefore, we evaluated the effects of different
c,
compounds on the polymerization of tubulin by using the
colchicine as positive control. First, the employed compounds
were evaluated at 5 or 20 μM concentration (Figure 4). We
found that compounds with at least three of IC50 values below
1.0 μM (3, 8, 9, 13−15, 21−23, and 26−28) against the first
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Table 7. IC50 of Selected Compounds on Inhibition of Tubulin Polymerization
8
13
14
15
21
26
27
colchicine
2.6 0.6
a
IC50 SE (μM)
1.8 0.0
1.3 0.0
3.4 0.2
3.6 0.0
4.0 0.3
0.49 0.27
2.8 0.2
a
IC50 presented as mean SE from at least two independent experiments.
Figure 5. Effects of 26 on the cellular microtubule network and microtubule reassemble by immunofluorescence. Cells were incubated with 0.5%
DMSO (a, c−f) or 31 nM 26 (b, g−j) at 37 °C for 24 h, and then direct microscopy detection (a, b) of the fixed and stained cell was performed. In
the microtubule regrowth assay, cells were chilled on ice for 1 h first, then warm at 37 °C. Cells with 0 (c, g), 5 (d, h), 10 (e, (i), and 15 min (f, j)
warm-up time were fixed and stained and then recorded by confocal microscopy. Insets show the mitosis cell. The cellular microtubules were stained
with primary β-tubulin mouse antibody and Dylight 549 goat anti-mouse IgG (red). DNA was stained by 4′,6-diamidino-2-phenylindole (DAPI,
blue). Scale bars: 10 μm.
Figure 6. Affinity-induced fluorescence by the binding of 15 or 26 to tubulin. Various concentrations of 15 (a−c) or 26 (d−f) were mixed with or
without 4 μM (15) and 8 μM (26) tubulin and incubated in general tubulin buffer for 30 min. Fluorescent emission spectrum of each compound was
recorded under the optimal excitation wavelength of 362 nm (a, d) and at 510 nm (15, b) and 460 nm (26, e) emission wavelengths, respectively.
The concentrations of both the tubulin- or compound-only control are 4 μM tubulin for 10 μM 15 binding and 8 μM tubulin for 8 μM 26 binding,
respectively. (c) and (f) show that the enhanced fluorescence (ΔF), induced by various concentrations of 15 with 4 μM tubulin (c) or of 26 with 8
μM tubulin (f) at the optimal excitation and emission wavelengths, respectively, and calculated from the increments of stable fluorescence emission
over the fluorescence intensity with tubulin only in each case, varied with the concentrations of compounds.
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Figure 7. Competitive binding assay of colchicine, 26, and vincristine with 15 in the presence of tubulin. 8 μM 15 and 4 μM tubulin were incubated
until the fluorescence of tubulin−15 complex reach a stable level. Time-course fluorescence was detected at 430 and 510 nm as excision and
emission wavelengths, respectively, right after the addition of various concentrations of colchicine (a), 26 (b), and vincristine (c). The decreased
stable fluorescence ratio of colchicine, 26, and vincristine treated well (F) at each concentration to the original fluorescence intensity of 15−tubulin
(F0) was plotted and fitted (d).
screened five cancer cell lines obviously inhibited the
microtubule polymerization, and 8, 13, 26, and 27 show the
most potent activity. With the decrease of the cytotoxicity (10
and 25), the inhibitory activity toward microtubule polymer-
ization reduced accordingly. As expected, no significant
microtubule polymerization inhibition was observed after
treatment with low cytotoxic compounds (4, 12 at 5 μM or
2, 6 at 20 μM) except for the moderate inhibition of 5, 11, and
17 at 20 μM. This result suggested a general correlation
between the cytotoxicity with the microtubule polymerization
inhibitory activity.
Various concentrations of compounds with high cytotoxicity
or microtubule polymerization inhibitory activity (8, 13−15,
21, 26, 27) were therefore examined, and the IC50 was
calculated. The results (Table 7) indicate that 8 (IC50 = 1.8
μM), 13 (IC50 = 1.3 μM), and 26 (IC50 = 0.49 μM) exhibit
stronger activity than colchicine (IC50 = 2.6 μM). This work
revealed that these active cytotoxic molecules may target
microtubule to exhibit cytotoxicity.
26 Inhibits Microtubule Reassemble and Leads to
Multipolar Mitosis in Nanomolar Concentration. To
further confirm the molecular target of the found cytotoxic
compounds, we investigated the effects of 26 on the cellular
microtubule network, reassembly of microtubule,17 and the
mitosis in A549 cells by confocal microscopy (Figure 5).
Normal filiform and meshy microtubule network and bipolar
mitotic division were present in the DMSO treated cells
(Figure 5a). In contrast, low concentration of 26 (31 nM)
efficiently induced the disruption of microtubule network,
resulting in massive and dotted microtubule disorder formation
(Figure 5b, region pointed by yellow arrow), and the
multipolarization of spindle and multinucleation phenomena
were observed. In the microtubule regrowth assay, cold
treatment of the cells almost completely depolymerized the
microtubule, and the obvious microtubule regrowth occurred
along with the warm-up process. The microtubule network and
normal cell mitosis recovered after a 15 min warmup (Figure
5c−f). Meanwhile, 31 nM 26 almost completely inhibited the
reassembly of microtubule in both interphase and mitosis cells,
accompanied by disorganized mitosis as indicated by the
observed multipolarization of spindle and multinucleation
phenomena (Figure 5g−j).
This result validated that 26 can inhibit microtubule
organization and interfere the mitosis of A549 in much lower
effective concentration than that to microtubule polymerization
inhibition. These results implied that the microtubule dynamics
is easier to be interfered by the microtubule targeting agents
than the microtubule polymerization, which is consistent with
the finding of many previous reports.18
15 and 26 Show Affinity-Based Fluorescence En-
hancement during Binding to Tubulin. Interestingly, a
fluorescent emission phenomenon was observed during the
interaction of 15 or 26 with tubulin, respectively. At 362 nm
excition wavelength, 4 μM 15 showed slight fluorescent
emission at 530 nm; however, in the presence of 4 μM tubulin,
fluorescent emission appeared at 510 nm, with intensity
increasing with the increasing concentration of 15 (Figure
6a). A similar phenomenon was also obversed in the 26−
tubulin interaction. At 362 nm excition wavelength, 8 μM 26
showed no fluorescent emission and in the presence of 8 μM
tubulin, fluorescent emission appeared at 465 nm, which is
concetnration dependent (Figure 6d). Excitation wavelength
scanning revealed that the fluorescence intensity increased with
increasing concentration of 15 (Figure 6b) or 26 (Figure 6e) in
the presence of tubulin, while tubulin or compound alone
showed no obvious fluorescence upon the excitation of 15 and
26 at 430 and 365 nm, respectively, suggesting affinity-induced
fluorescent properties observed in tubulin−15 (or 26) complex.
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Figure 8. Antitumor activity of 26 toward A549 tumor xenografts.
Kinetic analysis was therefore carried out. Fluorescence
intensity was recorded right after the mixing of various
concentrations of 15 with 4 μM tubulin (excitation, 430 nm;
emission, 510 nm) or 26 with 8 μM tubulin (excitation, 365
nm; emission, 465 nm), respectively. Fluorescence intensity of
15−tubulin complex increased in a time dependent manner and
finally reached a stable plateau in less than 30 min, while 26
induced fast fluorescence saturation after quickly mixing with
tubulin (Supporting Information, Figure S3), indicating a rapid
binding of 26 with tubulin. The stable fluorescence showed a
dose dependent manner in both cases of 15 and 26, and the
time course of the fluorescence enhancements from 15−tubulin
(Figure 6c) or 26−tubulin binding (Figure 6f) suggested that
the fluorescence enhancements reached maximum when the
concentration ratio of compound with tubulin is close to 1:1.
15 and 26 Binding to Colchicine Domain of Tubulin. It
is well-known that colchicine also showed affinity-induced
fluorescence enhancement (excitation, 362 nm; emission, 435
nm) with tubulin,19 which was widely used to determine
whether other investigated compounds are binding to the
colchicine site.20 For the case of vincristine site binding,
however, 3H labeled vincristine is usually needed.20,21
Fortunately, the interesting fluorescent properties of our
compounds allow us to examine their binding site by
fluorescent competitive assay. Owing to the overlay of the
excision and emission wavelength of 26 with colchicine, 8 μM
15 was chosen, which is incubated with tubulin (4 μM) until
the fluorescence intensity reached the stable state after 30 min.
Then various concentrations of colchicine, 26, and vincristine
were added, respectively, and the kinetic curves were recorded
accordingly (Figure 7). Colchicine decreased the fluorescence
of 15−tubulin complex in a concentration dependent manner,
indicating a competitive binding of 15 with cochicine in the
same binding domain, and 20 μM cochicine efficiently replaced
15 from tubulin (Figure 7a and Figure 7d). Noticeably, 26 was
more potent than colchicine. Addition low concentration to 2
μM 26 induced dramatic decrease of the fluorescence of 15−
tubulin complex, which reflected the higher affinity of 26 than
that of colchicine (Figure 7b and Figure 7d) toward tubulin.
This result is consistent with the observed higher potency of 26
in cytotoxicity and tubulin polymerization than that of
colchicine. In contrast, addition of vincristine would not affect
the fluorescence of the 15−tubulin complex, which excluded
the possible binding of 15 in the vincristine domain (Figure
7c,d).
Interestingly, the employed aryl chalcones may also serve as
Michael acceptors to biological nucleophiles, mostly attributed
to the presence of an α,β-unsaturated ketone moiety, which is
readily modified by the free thiol group in the protein
structure.22 Accordingly, we found that preincubation of
tubulin with the 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)
disabled the affinity-induced fluorescence of 26−tubulin
complex dose-dependently (Supporting Information, Figure
S4),20,23 which indicated that the thiol groups in tubulin may
participate compound−tubulin binding. In antiproliferation
assay, the α,β-unsaturated ketone moiety indeed was proved to
be crucial to the cytotoxicity. IC50 values of compounds 49−51
were greater than 50 μM. We therefore cannot exclude the
possibility of a covalent modification of tubulin through these
active compounds. In fluorescent competitive assay, however,
the observation of 15 in tubulin replaced by colchicine and 26
suggested that these active aryl-substituted chalcones could
reversibly bind to the colchicine site. From this point of view,
the semirigid α,β-unsaturated ketone moiety may promote the
binding by maintaining a favorable conformation of active aryl
chalcones during the interaction, and in the cases of
compounds 49−51, the amide bonds are more elastic which
may thus disrupt the favorable conformation. We unfortunately
failed to obtain a proper binding pose of acitve compound in
tubulin by molecular docking analysis upon the reversible
binding model (data not shown). However, all of the above
information may imply a more complicated situation of the
investigated chalcone−tubulin interaction. Does a reversible
covalent interaction exist during the colchicine site binding?
The raised question may need systematic investigation in future
work.
26 Inhibits A549 Tumor Xenografts Growth in Vivo.
To evaluate the role of 26 in tumor proliferation in vivo, we
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conducted by flow cytometer (Beckman Coulter, USA). DMEM high
glucose medium, fetal bovine serum, and Hoechst 33342 were
purchased from Invitrogen (Life Technologies Corporation, China).
HepG2 (ATCCHB-8065), SW480 (ATCCCCL-228), and CNE2
(TCHu13) were purchased from National Center for Medical Culture
Collection (CMCC). A549/CDDP (BG034), A2780/TAX (BG028),
HCT8/VCT (BG208), and MCF7/DOX (BG337) along with their
parental cells A549 (BG033), A2780 (BG072), HCT8 (BG207), and
MCF7 (BG336) were purchased from Bogoo Biotechnology Co., Ltd.
(Shanghai, China). Annexin V/PI apoptosis kit (catalog no. AP101)
was purchased from Liankebio Corporation (Guangzhou, China).
Tubulin polymerization assay kit (Cytoskeleton, catalog no. BK011P)
and Cellomics cytoskeletal rearrangement kit were purchased from
Univ-Bio Corporation (Shanghai, China).
examined the ability of 26 to suppress the growth of A549
tumor xenografts in nude mice. Mice were treated with 26
every other day for 2 weeks. 26 significantly inhibits the growth
of tumor xenografts (Figure 8A). The excised tumors from the
control group weighed between 1.11 and 1.96 g (mean, 1.446
g), whereas those from the 2 mg/kg body weight 26-treated
animals ranged from 0.38 to 0.90 g (mean, 0.641 g) (Figure
8B). In addition, treatment with compound 26 at 2 mg/kg
body weight resulted in a significant decrease in final tumor
volume (mean, 965.6 mm3) when compared to the control
group (mean, 1806.9 mm3; Figure 8C), while keeping a stable
increase of mice body weights (Figure 8D). These data show
that 26 has the ability to inhibit lung cancer growth in nude
mice bearing A549 xenografts via proliferation suppression.
2. General Procedure for the Synthesis of 1 and 7. A mixture
of 1-(2-bromophenyl)ethanone (1 equiv), corresponding boronic acid
(1.3 equiv), dichloro(1,10-bis(diphenylphosphino)ferrocene)-
palladium(II)−dichloromethane adduct (0.06 equiv), 2 M K2CO3
(1.5 mL), and 1,4-dioxane (1.5 mL) was heated in a sealed vial in a
microwave reactor at 150 °C for 20 min. After that, 10 mL of water
was added and the mixture was extracted with EtOAc (10 mL) three
times. The combined organic layer was washed to neutral and dried
over anhydrous sodium sulfate. After removal of the solvent in
vacuum, the crude product was purified by column chromatography.
1-(3′,4′-Dimethoxy[1,1′-biphenyl]-2-yl)ethanone (1). Yellow
CONCLUSIONS
■
In summary, we designed and synthesized a series of
substituted ortho-aryl-substituted chalcones, and potent
cytotoxic agents were found with most IC50 values at
nanomolar concentration range against a panel of cancer cell
lines. The active compounds such as 13−15 and 26 show
superior potency by several to thousands times than those of
paclitaxel, doxorubicin, colchicine, and vincristine in the growth
inhibition of paclitaxel resistant A2780/TAX, vincristine
resistant HCT-8/VCT, cis-platinum resistant A549/CDDP,
and doxorubicin resistant MCF-7/DOX. The most potent
compounds 13 and 26 show dramatic low IC50 values of 7 and
2 nM, respectively, toward MCF-7/DOX. Furthermore, the in
vivo antitumor potency of 26 were confirmed in A549 tumor
xenografts model.
1
liquid, yield 88.02%; H NMR (500 MHz, CDCl3) δ 7.52−7.47 (m,
2H), 7.41−7.37 (m, 2H), 6.93 (d, J = 8.1 Hz, 1H), 6.89 (dd, J = 8.1,
2.0 Hz, 1H), 6.87 (d, J = 2.0 Hz, 1H), 3.93 (s, 3H), 3.89 (s, 3H), 2.02
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 205.00, 148.86, 148.80,
140.83, 139.94, 133.09, 130.40, 129.80, 127.48, 126.97, 121.14, 111.84,
111.16, 55.72, 55.70, 30.17. HRMS calcd for C16H16O3 [M + Na]+,
279.0997; found, 279.0990.
1-(4′-Fluoro[1,1′-biphenyl]-2-yl)ethanone (7). Yellow liquid, yield
82.15%; 1H NMR (500 MHz, CDCl3) δ 7.55 (dd, J = 7.6, 1.1 Hz, 1H),
7.51 (td, J = 7.5, 1.4 Hz, 1H), 7.42 (td, J = 7.5, 1.3 Hz, 1H), 7.36 (dd, J
= 7.6, 0.9 Hz, 1H), 7.33−7.28 (m, 2H), 7.15−7.09 (m, 2H), 2.05 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 204.40, 163.91, 161.45, 140.79,
139.43, 136.84, 136.80, 130.84, 130.52, 130.44, 130.35, 127.98, 127.63,
115.79, 115.57, 30.41. HRMS calcd for C14H11OF [M + Na]+,
237.0692; found, 237.0685.
3. General Procedure for the Synthesis of 2−6 and 8−17. A
mixture of the corresponding acetophenone (1 equiv) and aldehyde (1
equiv) in anhydrous EtOH was stirred at room temperature for 10
min. Then solid KOH (6 equiv) was added. The reaction mixture was
stirred at room temperature until aldehyde disappeared as monitored
by TLC. After that, HCl (10%) was added until pH 4 was obtained.
The mixture was concentrated in vacuum to remove EtOH and further
extracted with EtOAc. The EtOAc layer was dried over anhydrous
sodium sulfate. After removal of the solvent in vacuum, the residue was
purified by column chromatography to give corresponding chalcones.
(E)-1-(3′,4′-Dimethoxy[1,1′-biphenyl]-2-yl)-3-(4-fluorophenyl)-
prop-2-en-1-one (2). Yellow oil, yield 30.38%; HPLC tR = 12.23 min;
1H NMR (400 MHz, CDCl3) δ 7.63 (ddd, J = 7.6, 1.4, 0.5 Hz, 1H),
7.59−7.54 (m, 1H), 7.51−7.48 (m, 1H), 7.48−7.43 (m, 1H), 7.32 (d, J
= 15.9 Hz, 1H), 7.26−7.21 (m, 2H), 7.01−6.96 (m, 2H), 6.95 (dd, J =
8.2, 2.1 Hz, 1H), 6.90 (d, J = 2.0 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H),
6.48 (dd, J = 15.9, 0.5 Hz, 1H), 3.85 (s, 3H), 3.85 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 196.46, 165.18, 162.68, 149.04, 148.98, 141.94,
140.73, 139.81, 133.26, 131.04, 131.01, 130.81, 130.09, 130.00, 128.83,
127.29, 126.59, 126.57, 121.73, 116.10, 115.89, 112.74, 111.42, 56.05,
56.00. HRMS calcd for C23H19O3F [M + Na]+, 385.1216; found,
385.1209.
The cell cycle analysis, immunofluorescence visualization,
and tubulin polymerization inhibition assay further confirmed
that the selected active compounds could greatly inhibit in vitro
and cellular tubulin polymerization, interfere with the mitosis,
and thus lead to G2/M cell cycle arrest and apoptosis of the
treated cancer cells. Interestingly, compounds 15 and 26 were
found to exhibit affinity-induced fluorescence during the
binding to tubulin. On the basis of this special optical
characterization, we further found the active compounds may
interact with β-tubulin in the colchicine binding site. Our
finding revealed a new scaffold and opportunities for mitosis-
targeting drug discovery.
EXPERIMENTAL SECTION
■
1. General. All commercial reagents and solvents were purchased
from vendors and were used without further purification or distillation.
Microwave reactions were performed in a Biotage Initiator 2.5
microwave reactor. The purities of compounds used for biological
evaluation (>95%) were determined on a DIONEX Ultimate 3000
HPLC system (Chromeleon SR9 Build 2673): column, Acclaim 120
C18, 5 mm, 4.6 mm × 250 mm; flow rate, 1 mL/min. Samples were
eluted with a gradient up to 95% solvent A over 30 min and detected
at 254 nm, whereas the A is CH3CN in double-deionized H2O with
0.1% trifluoroacetic acid (TFA). For compounds 8, 16, and 17, start
from 80% A. For compounds 9−12, start from 70% A. For compounds
2−6 and 35−44, start from 60% A. For compounds 13−15, 21−28,
1
45, and 49−51, start from 40% A. H NMR and 13C NMR spectra
(E)-1-(3′,4′-Dimethoxy[1,1′-biphenyl]-2-yl)-3-(4-methoxyphenyl)-
prop-2-en-1-one (3). Yellow oil, yield 31.55%; HPLC tR = 11.44 min;
1H NMR (400 MHz, CDCl3) δ 7.59 (dd, J = 7.6, 1.0 Hz, 1H), 7.56−
7.51 (m, 1H), 7.47 (dd, J = 7.7, 0.9 Hz, 1H), 7.45−7.40 (m, 1H), 7.31
(d, J = 15.9 Hz, 1H), 7.25−7.19 (m, 2H), 6.94 (dd, J = 8.2, 2.1 Hz,
1H), 6.90 (d, J = 2.0 Hz, 1H), 6.85 (d, J = 8.2 Hz, 1H), 6.83−6.78 (m,
2H), 6.48 (d, J = 15.9 Hz, 1H), 3.84 (s, 3H), 3.83 (s, 3H), 3.80 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 196.70, 161.48, 148.83, 148.74,
were recorded using TMS as an internal standard with Burker BioSpin
Ultrashield 400 NMR system or Varian INOVA 500NB. High
resolution mass spectra were recorded on a Shimadzu LCMS-IT-TOF
instrument. Cell images were captured on LSM710 confocal
microscope from Carl Zeiss and ArrayScanVTI high content screening
(HCS) reader from Thermo Fisher. Fluorescence was detected on
TECAN Infinite M200 Pro multimode reader and FlexStation 3
multimode reader from Molecular Devices. Apoptosis assay was
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143.52, 140.47, 139.99, 133.24, 130.38, 129.87, 129.85, 128.57, 127.29,
127.06, 124.69, 121.55, 114.23, 112.60, 111.29, 55.90, 55.85, 55.27.
HRMS calcd for C24H22O4 [M + Na]+, 397.1416; found, 397.1412.
(E)-1-(3′,4′-Dimethoxy[1,1′-biphenyl]-2-yl)-3-(3,4-
dimethoxyphenyl)prop-2-en-1-one (4). Yellow oil, yield 31.19%;
HPLC tR = 9.25 min; 1H NMR (400 MHz, CDCl3) δ 7.63 (dd, J = 7.6,
1.1 Hz, 1H), 7.57−7.51 (m, 1H), 7.47 (dd, J = 7.7, 1.0 Hz, 1H), 7.46−
7.42 (m, 1H), 7.32 (d, J = 15.9 Hz, 1H), 6.96−6.92 (m, 2H), 6.89 (dd,
J = 8.3, 1.9 Hz, 1H), 6.87−6.84 (m, 1H), 6.77 (d, J = 8.3 Hz, 1H), 6.71
(d, J = 2.0 Hz, 1H), 6.44 (d, J = 15.9 Hz, 1H), 3.88 (s, 3H), 3.84 (s,
3H), 3.84 (s, 3H), 3.81 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
196.25, 151.27, 149.13, 148.92, 148.82, 143.36, 140.65, 139.96, 133.37,
130.58, 129.98, 128.80, 127.73, 127.18, 124.93, 123.16, 121.78, 112.55,
111.27, 110.96, 109.27, 55.94, 55.93, 55.90, 55.69. HRMS calcd for
C25H24O5 [M + Na]+, 427.1521; found, 427.1516.
153.48, 153.11, 140.07, 139.80, 139.45, 136.75, 136.72, 130.94, 130.86,
130.67, 130.19, 128.92, 127.55, 127.29, 124.09, 117.86, 115.57, 115.35,
112.71, 112.46, 55.97, 55.71. HRMS calcd for C23H19O3F [M + H]+,
363.1396; found, 363.1393.
(E)-3-(2,3-Dimethoxyphenyl)-1-(4′-fluoro[1,1′-biphenyl]-2-yl)-
prop-2-en-1-one (11). Yellow powder, yield 75.00%; HPLC tR = 8.61
1
min; H NMR (500 MHz, CDCl3) δ 7.66 (d, J = 16.2 Hz, 1H), 7.61
(dd, J = 7.6, 0.8 Hz, 1H), 7.55 (td, J = 7.5, 1.3 Hz, 1H), 7.46 (td, J =
7.6, 1.1 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.36−7.33 (m, 2H), 7.08−
7.02 (m, 2H), 6.96 (t, J = 8.0 Hz, 1H), 6.89 (d, J = 8.1 Hz, 1H), 6.79
(d, J = 7.9 Hz, 1H), 6.62 (d, J = 16.2 Hz, 1H), 3.84 (s, 3H), 3.72 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 196.86, 163.85, 161.39, 153.13,
148.78, 140.05, 139.76, 139.20, 136.67, 136.64, 130.93, 130.85, 130.72,
130.20, 128.84, 128.78, 128.05, 127.59, 124.18, 119.30, 115.69, 115.47,
114.33, 61.29, 55.93. HRMS calcd for C23H19O3F [M + H]+,
363.1396; found, 363.1391.
(E)-1-(4′-Fluoro[1,1′-biphenyl]-2-yl)-3-(4-hydroxy-3-
methoxyphenyl)prop-2-en-1-one (12). Yellow powder, yield 79.02%;
HPLC tR = 5.23 min; 1H NMR (500 MHz, CDCl3) δ 7.63 (dd, J = 7.6,
1.4 Hz, 1H), 7.55 (td, J = 7.5, 1.4 Hz, 1H), 7.46 (td, J = 7.5, 1.3 Hz,
1H), 7.43 (dd, J = 7.6, 1.2 Hz, 1H), 7.36−7.33 (m, 2H), 7.30 (d, J =
15.9 Hz, 1H), 7.07−7.03 (m, 2H), 6.87 (dd, J = 8.2, 1.7 Hz, 1H), 6.84
(d, J = 8.2 Hz, 1H), 6.74 (d, J = 1.7 Hz, 1H), 6.44 (d, J = 15.9 Hz,
1H), 5.86 (s, 1H), 3.86 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
196.29, 163.83, 161.37, 148.50, 146.91, 144.83, 139.99, 139.85, 136.82,
136.79, 130.92, 130.84, 130.70, 130.28, 128.91, 127.61, 127.20, 124.66,
123.79, 115.64, 115.43, 114.86, 109.30, 55.94. HRMS calcd for
C22H17O3F [M + H]+, 349.1240; found, 349.1229.
(E)-1-(3′,4′-Dimethoxy[1,1′-biphenyl]-2-yl)-3-(2,5-
dimethoxyphenyl)prop-2-en-1-one (5). Yellow powder, yield 16.09%;
1
HPLC tR = 12.15 min; H NMR (400 MHz, CDCl3) δ 7.71 (d, J =
16.1 Hz, 1H), 7.63 (dd, J = 7.6, 1.2 Hz, 1H), 7.56−7.51 (m, 1H),
7.49−7.45 (m, 1H), 7.43 (td, J = 7.4, 1.3 Hz, 1H), 6.95 (dd, J = 8.2,
2.0 Hz, 1H), 6.91 (d, J = 1.9 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.84
(dd, J = 9.0, 3.0 Hz, 1H), 6.76 (d, J = 9.0 Hz, 1H), 6.64 (d, J = 3.0 Hz,
1H), 6.59 (d, J = 16.1 Hz, 1H), 3.85 (s, 3H), 3.83 (s, 3H), 3.74 (s,
3H), 3.70 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 196.79, 153.47,
153.05, 148.97, 148.89, 140.73, 140.17, 138.36, 133.42, 130.63, 129.98,
128.91, 127.39, 127.22, 124.37, 121.86, 117.60, 112.78, 112.62, 112.43,
111.35, 56.02, 55.98, 55.94, 55.66. HRMS calcd for C25H24O5 [M +
H]+, 405.1702; found, 405.1711.
(E)-3-(4-Bromophenyl)-1-(3′,4′-dimethoxy[1,1′-biphenyl]-2-yl)-
prop-2-en-1-one (6). Yellow oil, yield 29.08%; HPLC tR = 16.54 min;
1H NMR (400 MHz, CDCl3) δ 7.62 (ddd, J = 7.6, 1.4, 0.4 Hz, 1H),
(E)-1-(4′-Fluoro[1,1′-biphenyl]-2-yl)-3-(3-hydroxy-4-
methoxyphenyl)prop-2-en-1-one (13). Yellow crystals, yield 67.38%;
1
HPLC tR = 16.79 min; H NMR (400 MHz, CDCl3) δ 7.60 (dd, J =
7.58−7.53 (m, 1H), 7.48 (ddd, J = 7.8, 1.3, 0.5 Hz, 1H), 7.46−7.42
(m, 1H), 7.42−7.38 (m, 2H), 7.25 (d, J = 15.9 Hz, 1H) 7.11−7.06 (m,
2H), 6.93 (dd, J = 8.2, 2.1 Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.86 (d, J
= 8.2 Hz, 1H), 6.51 (d, J = 15.9 Hz, 1H), 3.84 (s, 3H), 3.83 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 196.24, 149.04, 149.00, 141.57,
140.75, 139.68, 133.71, 133.17, 132.04, 130.90, 129.98, 129.47, 128.86,
127.30, 127.25, 124.53, 121.75, 112.71, 111.41, 56.04, 55.99. HRMS
calcd for C23H19O3Br [M + Na]+, 445.0415; found, 445.0408.
(E)-1-(4′-Fluoro[1,1′-biphenyl]-2-yl)-3-(4-methoxyphenyl)prop-2-
7.6, 1.3 Hz, 1H), 7.54 (td, J = 7.5, 1.4 Hz, 1H), 7.46 (dd, J = 7.5, 1.2
Hz, 1H), 7.42 (d, J = 7.5 Hz, 1H), 7.36−7.30 (m, 2H), 7.26 (d, J =
15.9 Hz, 1H), 7.08−7.01 (m, 2H), 6.88 (d, J = 2.0 Hz, 1H), 6.84 (dd, J
= 8.3, 2.0 Hz, 1H), 6.77 (d, J = 8.3 Hz, 1H), 6.46 (d, J = 15.9 Hz, 1H),
5.64 (s, 1H), 3.89 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 196.58,
163.68, 161.22, 149.08, 145.92, 144.78, 139.91, 139.69, 136.58, 136.54,
130.72, 130.64, 130.54, 130.15, 128.65, 127.99, 127.44, 125.00, 122.15,
115.54, 115.33, 113.29, 110.64, 55.90. HRMS calcd for C22H17O3F [M
+ H]+, 349.1234; found, 349.1219.
1
en-1-one (8). Yellow powder, yield 58.60%; HPLC tR = 6.91 min; H
(E)-(5-(3-(4′-Fluoro[1,1′-biphenyl]-2-yl)-3-oxoprop-1-en-1-yl)-2-
methoxyphenyl)boronic Acid (14). Light yellow powder, yield
70.45%; HPLC tR = 15.67 min; 1H NMR (400 MHz, CDCl3) δ
7.79 (d, J = 2.3 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.57−7.52 (m, 1H),
7.48−7.39 (m, 3H), 7.36−7.28 (m, 3H), 7.03 (t, J = 8.7 Hz, 2H), 6.86
(d, J = 8.7 Hz, 1H), 6.56 (d, J = 16.0 Hz, 1H), 5.81 (s, 2H), 3.93 (s,
3H); 13C NMR (100 MHz, DMSO) δ 195.47, 165.31, 162.92, 160.49,
144.67, 139.64, 139.02, 136.63, 135.64, 132.04, 130.75, 130.67, 130.47,
130.17, 128.20, 127.50, 126.18, 124.31, 115.39, 115.18, 110.84, 55.56.
HRMS calcd for C22H18O4BF [M + H]+, 377.1359; found, 377.1354.
(E)-3-(4-(Dimethylamino)phenyl)-1-(4′-fluoro[1,1′-biphenyl]-2-
NMR (500 MHz, CDCl3) δ 7.61 (dd, J = 7.6, 1.4 Hz, 1H), 7.54 (td, J
= 7.5, 1.4 Hz, 1H), 7.46 (td, J = 7.5, 1.2 Hz, 1H), 7.43 (dd, J = 7.6, 1.2
Hz, 1H), 7.36−7.32 (m, 2H), 7.31 (d, J = 15.9 Hz, 1H), 7.26−7.23
(m, 2H), 7.07−7.02 (m, 2H), 6.85−6.81 (m, 2H), 6.49 (d, J = 15.9
Hz, 1H), 3.81 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 196.30,
163.69, 161.68, 161.23, 144.35, 140.00, 139.68, 136.64, 136.61, 130.74,
130.66, 130.50, 130.15, 129.96, 128.68, 127.45, 127.17, 124.62, 115.53,
115.32, 114.34, 55.28. HRMS calcd for C22H17O2F [M + H]+,
333.1291; found, 333.1280.
(E)-3-(3,4-Dimethoxyphenyl)-1-(4′-fluoro[1,1′-biphenyl]-2-yl)-
prop-2-en-1-one (9). Yellow oil, yield 73.90%; HPLC tR = 6.82 min;
1H NMR (500 MHz, CDCl3) δ 7.64 (dd, J = 7.6, 1.4 Hz, 1H), 7.55
yl)prop-2-en-1-one (15). Yellow powder, yield 67.26%; HPLC tR
=
20.87 min; 1H NMR (400 MHz, DMSO) δ 7.61−7.56 (m, 1H), 7.54−
7.49 (m, 2H), 7.46 (d, J = 7.6 Hz, 1H), 7.39−7.30 (m, 4H), 7.23−7.14
(m, 3H), 6.64 (d, J = 8.8 Hz, 2H), 6.53 (d, J = 15.9 Hz, 1H), 2.96 (s,
6H); 13C NMR (100 MHz, CDCl3) δ 196.73, 163.58, 161.13, 152.05,
146.18, 140.38, 139.49, 136.75, 136.71, 130.65, 130.57, 130.20, 130.10,
128.52, 127.31, 122.02, 121.91, 115.39, 115.18, 111.70, 39.93. HRMS
calcd for C23H20NOF [M + H]+, 346.1602; found, 346.1590.
(td, J = 7.5, 1.4 Hz, 1H), 7.46 (td, J = 7.5, 1.2 Hz, 1H), 7.43 (dd, J =
7.6, 1.2 Hz, 1H), 7.38−7.34 (m, 2H), 7.32 (d, J = 15.9 Hz, 1H), 7.08−
7.03 (m, 2H), 6.91 (dd, J = 8.3, 2.0 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H),
6.74 (d, J = 2.0 Hz, 1H), 6.45 (d, J = 15.9 Hz, 1H), 3.89 (s, 3H), 3.83
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 196.07, 163.82, 161.36,
151.53, 149.27, 144.39, 140.00, 139.84, 136.80, 136.77, 130.90, 130.82,
130.70, 130.27, 128.91, 127.63, 127.60, 124.92, 123.28, 115.63, 115.42,
111.10, 109.54, 56.03, 55.86. HRMS calcd for C23H19O3F [M + H]+,
363.1396; found, 363.1395.
(E)-3-(2,5-Dimethoxyphenyl)-1-(4′-fluoro[1,1′-biphenyl]-2-yl)-
prop-2-en-1-one (10). Yellow oil, yield 71.55%; HPLC tR = 8.88 min;
1H NMR (500 MHz, CDCl3) δ 7.69 (d, J = 16.1 Hz, 1H), 7.63 (dd, J =
7.6, 1.1 Hz, 1H), 7.55 (td, J = 7.5, 1.4 Hz, 1H), 7.46 (td, J = 7.5, 1.3
Hz, 1H), 7.43 (dd, J = 7.6, 0.8 Hz, 1H), 7.37−7.33 (m, 2H), 7.07−
7.03 (m, 2H), 6.87 (dd, J = 9.0, 3.0 Hz, 1H), 6.77 (d, J = 9.0 Hz, 1H),
6.68 (d, J = 3.0 Hz, 1H), 6.61 (d, J = 16.1 Hz, 1H), 3.75 (s, 3H), 3.72
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 196.61, 163.79, 161.33,
(E)-1-(4′-Fluoro[1,1′-biphenyl]-2-yl)-3-(5-methylfuran-2-yl)prop-
1
2-en-1-one (16). Yellow oil, yield 40.84%; HPLC tR = 7.11 min; H
NMR (500 MHz, CDCl3) δ 7.58 (dd, J = 7.6, 1.0 Hz, 1H), 7.52 (td, J
= 7.5, 1.4 Hz, 1H), 7.43 (td, J = 7.6, 1.3 Hz, 1H), 7.41 (dd, J = 7.7, 0.7
Hz, 1H), 7.33−7.30 (m, 2H), 7.03 (d, J = 15.7 Hz, 1H), 7.06−7.01
(m, 2H), 6.45 (d, J = 3.3 Hz, 1H), 6.45 (d, J = 15.7 Hz, 1H), 6.04 (dd,
J = 3.3, 0.8 Hz, 1H), 2.29 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
196.18, 163.82, 161.37, 156.22, 149.81, 140.06, 139.80, 136.68, 136.65,
130.87, 130.81, 130.73, 130.47, 130.30, 128.60, 127.48, 122.75, 117.98,
115.59, 115.37, 109.38, 13.96. HRMS calcd for C20H15O2F [M + H]+,
307.1134; found, 307.1123.
6376
dx.doi.org/10.1021/jm500024v | J. Med. Chem. 2014, 57, 6364−6382

Journal of Medicinal Chemistry
Article
(E)-1-(4′-Fluoro[1,1′-biphenyl]-2-yl)-3-(thiophen-2-yl)prop-2-en-
7.8, 1.6 Hz, 1H), 7.67−7.61 (m, 3H), 7.42 (d, J = 15.6 Hz, 1H), 7.36
(t, J = 7.6 Hz, 1H), 7.24 (td, J = 7.5, 1.9 Hz, 1H), 6.97−6.92 (m, 1H),
5.70 (s, 1H), 3.99 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 188.79,
150.93, 145.77, 142.49, 135.33, 133.60, 131.63, 131.24, 127.97, 127.78,
125.88, 124.91, 122.38, 114.91, 110.16, 56.22. HRMS calcd for
C16H13O3Br [M + H]+, 333.0121; found, 333.0118.
(E)-3-(2-Bromophenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-
one (34). Light yellow crystal, yield 85.28%; HPLC tR = 21.35 min; 1H
NMR (400 MHz, acetone) δ 8.12 (d, J = 15.5 Hz, 1H), 8.03 (d, J = 7.8
Hz, 1H), 7.84 (d, J = 15.5 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.46 (d, J
= 9.6 Hz, 3H), 7.42−7.35 (m, 1H), 3.94 (s, 6H), 3.84 (s, 3H).
5. General Procedure for the Synthesis of 21−28 and 35−
45. These compounds were synthesized using Suzuki−Miyaura
coupling conditions as previously described in the synthesis of 1 and
7, in which a mixture of corresponding intermediate chalcones (1.0
mmol), corresponding boronic acid (1.3 mmol), dichloro(1,10-
bis(diphenylphosphino)ferrocene)palladium(II)−dichloromethane ad-
duct (0.06 mmol), 2 M K2CO3 (1.5 mL), and 1,4-dioxane (1.5 mL)
was used.
1
1-one (17). Yellow oil, yield 19.48%; HPLC tR = 6.98 min; H NMR
(500 MHz, CDCl3) δ 7.63 (dd, J = 7.6, 1.2 Hz, 1H), 7.55 (td, J = 7.5,
1.4 Hz, 1H), 7.49 (d, J = 15.6 Hz, 1H), 7.47 (td, J = 7.5, 1.3 Hz, 1H),
7.43 (dd, J = 7.6, 1.0 Hz, 1H), 7.35−7.31 (m, 3H), 7.13 (d, J = 3.6 Hz,
1H), 7.08−7.04 (m, 2H), 6.99 (dd, J = 5.1, 3.6 Hz, 1H), 6.37 (d, J =
15.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 195.41, 163.84, 161.38,
140.00, 139.91, 139.72, 136.52, 136.48, 136.24, 131.64, 130.83, 130.80,
130.74, 130.25, 129.06, 128.82, 128.27, 127.57, 125.58, 115.67, 115.45.
HRMS calcd for C19H13OFS [M + H]+, 309.0749; found, 309.0739.
4. General Procedure for the Synthesis of 18−20 and 29−
34. These intermediate chalcones were synthesized by the Claisen−
Schmidt condensation. In general, acetophenone (5 equiv) was added
to equimolar of aromatic aldehyde and dissolved in EtOH. To the
solution, 6 M NaOH (aq) (2 mL) was added, and the reaction mixture
was stirred at room temperature until the aldehyde disappeared, by
monitoring with TLC. Then HCl (10%) was added until pH 4 was
obtained. The mixture was concentrated in vacuum to remove EtOH
and further extracted with EtOAc. The EtOAc layer was dried over
anhydrous sodium sulfate. After removal of the solvent in vacuum, the
crude product was purified by recrystallization with EtOH to give
corresponding chalcones.
(E)-1-(3′-Fluoro[1,1′-biphenyl]-2-yl)-3-(4-methoxyphenyl)prop-2-
1
en-1-one (21). Orange oil, yield 69.04%; HPLC tR = 22.38 min; H
NMR (400 MHz, CDCl3) δ 7.62 (dd, J = 7.5, 1.4 Hz, 1H), 7.56 (td, J
= 7.5, 1.5 Hz, 1H), 7.48 (dd, J = 7.5, 1.3 Hz, 1H), 7.44 (d, J = 7.5 Hz,
1H), 7.34−7.28 (m, 1H), 7.31 (d, J = 15.9 Hz, 1H), 7.24−7.27 (m,
2H), 7.15−7.08 (m, 2H), 6.98 (ddd, J = 10.1, 7.7, 2.2 Hz, 1H), 6.86−
6.80 (m, 2H), 6.52 (d, J = 15.9 Hz, 1H), 3.81 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 196.00, 163.86, 161.67, 161.41, 144.47, 142.83,
142.76, 139.94, 139.46, 139.44, 130.51, 130.02, 130.00, 129.95, 129.92,
128.69, 127.77, 127.12, 125.00, 124.97, 124.54, 115.99, 115.78, 114.52,
114.31, 55.22. HRMS calcd for C22H17O2F [M + H]+, 333.1285;
found, 333.1281.
(E)-1-(2-Bromophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one
1
(18). Light yellow crystal, yield 85.68%; HPLC tR = 11.76 min; H
NMR (400 MHz, CDCl3) δ 7.64 (d, J = 7.9 Hz, 1H), 7.51 (d, J = 8.8
Hz, 2H), 7.43−7.29 (m, 3H), 7.38 (d, J = 16.0 Hz, 1H), 6.97 (d, J =
16.0 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 3.85 (s, 3H).
(E)-1-(2-Bromophenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one
(19). Light green crystal, yield 86.94%; HPLC tR = 9.12 min; 1H NMR
(400 MHz, CDCl3) δ 7.68−7.62 (m, 1H), 7.43−7.30 (m, 3H), 7.35
(d, J = 16.0 Hz, 1H), 7.13 (dd, J = 8.3, 2.0 Hz, 1H), 7.08 (d, J = 1.9
Hz, 1H), 6.96 (d, J = 16.0 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 3.92 (s,
3H), 3.91 (s, 3H). HRMS calcd for C17H15O3Br [M + H]+, 347.0277;
found, 347.0283.
(E)-1-(2-(6-Fluoropyridin-3-yl)phenyl)-3-(4-methoxyphenyl)prop-
2-en-1-one (22). Yellow powder, yield 80.65%; HPLC tR = 18.09 min;
1H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 2.5 Hz, 1H), 7.76 (ddd, J
(E)-1-(2-Bromophenyl)-3-(3-hydroxy-4-methoxyphenyl)prop-2-
= 8.4, 7.7, 2.6 Hz, 1H), 7.64 (dd, J = 7.5, 1.2 Hz, 1H), 7.58 (td, J = 7.5,
1.5 Hz, 1H), 7.52 (td, J = 7.5, 1.3 Hz, 1H), 7.42 (dd, J = 7.6, 0.9 Hz,
1H), 7.37−7.31 (m, 2H), 7.34 (d, J = 16.0 Hz, 1H), 6.90 (dd, J = 8.4,
3.0 Hz, 1H), 6.88−6.83 (m, 2H), 6.64 (d, J = 16.0 Hz, 1H), 3.83 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 195.09, 163.90, 161.71, 161.52,
146.86, 146.71, 145.48, 141.45, 141.37, 139.71, 135.63, 134.13, 134.09,
130.45, 130.22, 129.91, 128.49, 127.93, 126.60, 124.03, 114.21, 108.90,
108.53, 55.04. HRMS calcd for C21H16NO2F [M + H]+, 334.1238;
found, 334.1244.
en-1-one (20). Light yellow crystal, yield 65.70%; HPLC tR = 13.30
1
min; H NMR (400 MHz, CDCl3) δ 7.63 (d, J = 7.8 Hz, 1H), 7.43−
7.28 (m, 4H), 7.17 (d, J = 1.8 Hz, 1H), 7.05 (dd, J = 8.3, 1.6 Hz, 1H),
6.94 (d, J = 16.0 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 5.72 (s, 1H), 3.93
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 194.90, 149.43, 147.11,
146.05, 141.25, 133.37, 131.24, 129.09, 127.84, 127.32, 124.28, 122.79,
119.42, 113.54, 110.76, 56.04. HRMS calcd for C16H13O3Br [M + H]+,
333.0121; found, 333.0115.
(E)-3-(2-Bromophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one
(29). White solid, yield 41.04%; HPLC tR = 14.23 min; 1H NMR (400
MHz, CDCl3) δ 8.11 (d, J = 15.7 Hz, 1H), 8.04 (d, J = 8.8 Hz, 2H),
7.73 (d, J = 7.8 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 15.7 Hz,
1H), 7.36 (t, J = 7.4 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 6.99 (d, J = 8.8
Hz, 2H), 3.90 (s, 3H).
(E)-3-(4-Methoxyphenyl)-1-(2-(pyridin-3-yl)phenyl)prop-2-en-1-
1
one (23). Yellow oil, yield 68.23%; HPLC tR = 6.53 min; H NMR
(400 MHz, CDCl3) δ 8.65 (d, J = 0.9 Hz, 1H), 8.53 (d, J = 4.8 Hz,
1H), 7.69−7.63 (m, 2H), 7.59 (t, J = 7.5 Hz, 1H), 7.51 (t, J = 7.5 Hz,
1H), 7.45 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 15.9 Hz, 1H), 7.30−7.25
(m, 3H), 6.83 (d, J = 8.6 Hz, 2H), 6.56 (d, J = 15.9 Hz, 1H), 3.79 (d, J
= 9.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 195.10, 161.46,
149.00, 148.28, 144.86, 139.71, 136.74, 135.97, 135.93, 130.35, 130.04,
129.73, 128.46, 127.73, 126.58, 124.06, 122.76, 114.03, 54.93. HRMS
calcd for C21H17NO2 [M + H]+, 316.1332; found, 316.1320.
(E)-3-(2-Bromophenyl)-1-(3,4-dimethoxyphenyl)prop-2-en-1-one
1
(30). Yellow crystal, yield 77.93%; HPLC tR = 11.73 min; H NMR
(400 MHz, CDCl3) δ 8.11 (d, J = 15.7 Hz, 1H), 7.73 (dd, J = 7.7, 1.1
Hz, 1H), 7.68 (dd, J = 8.4, 2.0 Hz, 1H), 7.64 (dd, J = 8.0, 0.9 Hz, 1H),
7.62 (d, J = 1.9 Hz, 1H), 7.43 (d, J = 15.7 Hz, 1H), 7.36 (t, J = 7.3 Hz,
1H), 7.27−7.21 (m, 1H), 6.94 (d, J = 8.4 Hz, 1H), 3.97 (s, 6H).
HRMS calcd for C17H15O3Br [M + H]+, 347.0277; found, 347.0267.
(E)-3-(2-Bromophenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one
(31). Yellow solid, yield 62.65%; HPLC tR = 8.73 min; 1H NMR (400
MHz, CDCl3) δ 8.14 (d, J = 15.7 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H),
7.64 (d, J = 7.8 Hz, 1H), 7.58−7.56 (m, 2H), 7.40 (d, J = 15.6 Hz,
1H), 7.44−7.33 (m, 2H), 7.27−7.23 (m, 1H), 7.15−7.08 (m, 1H).
(E)-3-(2-Bromophenyl)-1-(4-hydroxy-3-methoxyphenyl)prop-2-
en-1-one (32). Yellow powder, yield 76.59%; HPLC tR = 8.67 min; 1H
NMR (400 MHz, CDCl3) δ 8.10 (d, J = 15.7 Hz, 1H), 7.73 (dd, J =
7.8, 1.6 Hz, 1H), 7.66−7.61 (m, 3H), 7.43 (d, J = 15.6 Hz, 1H), 7.38−
7.33 (m, 1H), 7.24 (td, J = 7.5, 1.6 Hz, 1H), 7.01−6.97 (m, 1H), 6.15
(s, 1H), 3.99 (s, 3H). HRMS calcd for C16H13O3Br [M + H]+,
333.0121; found, 333.0112.
(E)-3-(3,4-Dimethoxyphenyl)-1-(3′-fluoro[1,1′-biphenyl]-2-yl)-
prop-2-en-1-one (24). Orange oil, yield 72.36%; HPLC tR = 19.82
1
min; H NMR (400 MHz, CDCl3) δ 7.65 (dd, J = 7.6, 1.3 Hz, 1H),
7.56 (td, J = 7.5, 1.4 Hz, 1H), 7.49 (dd, J = 7.5, 1.2 Hz, 1H), 7.47−7.43
(m, 1H), 7.34−7.27 (m, 1H), 7.31 (d, J = 15.9 Hz, 1H), 7.16−7.11
(m, 2H), 6.99 (ddd, J = 8.1, 2.9, 1.5 Hz, 1H), 6.91 (dd, J = 8.3, 1.9 Hz,
1H), 6.79 (d, J = 8.3 Hz, 1H), 6.75 (d, J = 1.9 Hz, 1H), 6.48 (d, J =
15.9 Hz, 1H), 3.88 (s, 3H), 3.83 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 194.82, 163.43, 160.97, 151.09, 148.79, 143.80, 142.61,
142.53, 139.52, 139.08, 139.06, 130.18, 129.64, 129.61, 129.53, 128.39,
127.41, 127.05, 124.78, 124.75, 124.27, 122.81, 115.57, 115.35, 114.06,
113.86, 110.70, 109.14, 55.32, 55.22. HRMS calcd for C23H19O3F [M
+ H]+, 363.1391; found, 363.1393.
(E)-3-(3,4-Dimethoxyphenyl)-1-(2-(6-fluoropyridin-3-yl)phenyl)-
prop-2-en-1-one (25). Orange oil, yield 68.62%; HPLC tR = 15.29
min; 1H NMR (400 MHz, CDCl3) δ 8.23 (s, 1H), 7.80−7.73 (m, 1H),
7.65 (d, J = 7.5 Hz, 1H), 7.59 (t, J = 7.3 Hz, 1H), 7.52 (t, J = 7.4 Hz,
(E)-3-(2-Bromophenyl)-1-(3-hydroxy-4-methoxyphenyl)prop-2-
1
en-1-one (33). Yellow solid, yield 78.94%; HPLC tR = 8.53 min; H
NMR (400 MHz, CDCl3) δ 8.11 (d, J = 15.6 Hz, 1H), 7.72 (dd, J =
6377
dx.doi.org/10.1021/jm500024v | J. Med. Chem. 2014, 57, 6364−6382

Journal of Medicinal Chemistry
Article
1H), 7.42 (d, J = 7.5 Hz, 1H), 7.33 (d, J = 15.9 Hz, 1H), 6.98 (d, J =
8.2 Hz, 1H), 6.90 (dd, J = 8.4, 2.7 Hz, 1H), 6.86 (s, 1H), 6.82 (d, J =
8.3 Hz, 1H), 6.62 (d, J = 15.9 Hz, 1H), 3.90 (s, 3H), 3.86 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ 194.82, 163.69, 161.31, 151.39, 148.85,
146.60, 146.45, 145.58, 141.40, 141.32, 139.40, 135.45, 134.02, 133.98,
130.35, 130.05, 128.36, 127.77, 126.73, 124.01, 122.99, 110.78, 109.44,
108.75, 108.38, 55.39, 55.32. HRMS calcd for C22H18NO3F [M + H]+,
364.1343; found, 364.1350.
(s, 3H), 3.94 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 188.11, 163.56,
161.10, 153.24, 149.17, 142.65, 142.12, 135.99, 135.96, 133.25, 131.38,
131.30, 131.04, 130.48, 129.84, 127.71, 127.07, 122.98, 122.92, 115.31,
115.10, 110.70, 109.93, 55.93, 55.85. HRMS calcd for C23H19O3F [M
+ H]+, 363.1391; found, 363.1382.
(E)-1-(3,4-Dimethoxyphenyl)-3-(2-(6-fluoropyridin-3-yl)phenyl)-
prop-2-en-1-one (38). White powder, yield 76.38%; HPLC tR = 11.98
min; 1H NMR (400 MHz, CDCl3) δ 8.22 (s, 1H), 7.91−7.82 (m, 1H),
7.76 (td, J = 8.0, 2.4 Hz, 1H), 7.70 (d, J = 15.5 Hz, 1H), 7.62 (d, J =
8.4 Hz, 1H), 7.56 (d, J = 1.4 Hz, 1H), 7.52−7.42 (m, 3H), 7.40−7.32
(m, 1H), 7.02 (dd, J = 8.3, 2.8 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 3.96
(s, 3H), 3.95 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 188.00, 164.31,
161.92, 153.47, 149.33, 147.94, 147.80, 142.35, 142.27, 141.55, 138.10,
133.78, 133.75, 133.71, 130.96, 130.63, 130.18, 128.72, 127.37, 123.97,
123.13, 110.78, 110.05, 109.33, 108.96, 56.09, 56.00. HRMS calcd for
C22H18NO3F [M + H]+, 364.1343; found, 364.1334.
(E)-1-(3′-Fluoro[1,1′-biphenyl]-2-yl)-3-(3-hydroxy-4-
methoxyphenyl)prop-2-en-1-one (26). Yellow oil, yield 72.38%;
1
HPLC tR = 16.75 min; H NMR (400 MHz, CDCl3) δ 7.61 (d, J =
7.5 Hz, 1H), 7.54 (dd, J = 7.5, 1.2 Hz, 1H), 7.48 (dd, J = 7.4, 1.0 Hz,
1H), 7.44 (d, J = 7.7 Hz, 1H), 7.33−7.29 (m, 1H), 7.26 (d, J = 15.9
Hz, 1H), 7.15−7.11 (m, 1H), 7.09 (d, J = 9.7 Hz, 1H), 6.98 (td, J =
8.5, 1.5 Hz, 1H), 6.88 (s, 1H), 6.84 (d, J = 8.3 Hz, 1H), 6.77 (d, J = 8.3
Hz, 1H), 6.50 (d, J = 15.9 Hz, 1H), 5.65 (s, 1H), 3.89 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ 196.66, 163.65, 161.20, 149.32, 145.84,
145.39, 142.57, 142.49, 139.49, 139.31, 139.30, 130.50, 129.91, 129.84,
128.51, 127.62, 127.56, 124.77, 124.74, 124.56, 122.16, 115.80, 115.59,
114.37, 114.17, 113.35, 110.61, 55.59. HRMS calcd for C22H17O3F [M
+ H]+, 349.1234; found, 349.1230.
(E)-3-(4′-Fluoro[1,1′-biphenyl]-2-yl)-1-(3-hydroxyphenyl)prop-2-
en-1-one (39). White powder, yield 74.69%; HPLC tR = 11.55 min;
1H NMR (400 MHz, acetone) δ 8.67 (s, 1H), 8.09 (dd, J = 7.5, 1.6 Hz,
1H), 7.75 (d, J = 15.6 Hz, 1H), 7.70 (d, J = 15.6 Hz, 1H), 7.61−7.57
(m, 1H), 7.56−7.49 (m, 3H), 7.46−7.40 (m, 3H), 7.37 (t, J = 7.9 Hz,
1H), 7.31−7.24 (m, 2H), 7.10 (ddd, J = 8.1, 2.6, 0.9 Hz, 1H); 13C
NMR (100 MHz, acetone) δ 189.91, 164.50, 162.06, 158.62, 143.16,
143.13, 140.50, 137.28, 137.25, 133.92, 132.62, 132.54, 131.36, 131.02,
130.60, 128.86, 128.04, 124.29, 120.82, 120.74, 116.12, 115.91, 115.64.
HRMS calcd for C21H15O2F [M + H]+, 319.1129; found, 319.1131.
(E)-3-(2-(6-Fluoropyridin-3-yl)phenyl)-1-(3-hydroxyphenyl)prop-
2-en-1-one (40). White powder, yield 71.42%; HPLC tR = 7.13 min;
1H NMR (400 MHz, acetone) δ 8.67 (s, 1H), 8.24 (s, 1H), 8.16−8.11
(m, 1H), 8.00 (td, J = 8.1, 2.5 Hz, 1H), 7.76 (d, J = 15.6 Hz, 1H), 7.68
(d, J = 15.6 Hz, 1H), 7.63−7.52 (m, 3H), 7.52−7.46 (m, 2H), 7.37 (t,
J = 7.9 Hz, 1H), 7.24 (dd, J = 8.4, 2.9 Hz, 1H), 7.11 (dd, J = 8.1, 1.8
Hz, 1H); 13C NMR (100 MHz, acetone) δ 189.71, 165.24, 162.89,
158.67, 148.90, 148.75, 143.78, 143.70, 142.26, 140.41, 139.30, 134.95,
134.90, 134.43, 131.58, 131.23, 130.65, 129.66, 128.30, 125.06, 120.94,
120.79, 115.66, 110.11, 109.73. HRMS calcd for C20H14NO2F [M +
H]+, 320.1081; found, 320.1078.
(E)-3-(3-Hydroxy-4-methoxyphenyl)-1-(3′,4′,5′-trimethoxy[1,1′-
biphenyl]-2-yl)prop-2-en-1-one (27). Yellow oil, yield 79.14%; HPLC
1
tR = 14.01 min; H NMR (400 MHz, CDCl3) δ 7.61 (d, J = 7.6 Hz,
1H), 7.56−7.51 (m, 1H), 7.50−7.41 (m, 2H), 7.26 (d, J = 15.4 Hz,
1H), 6.84−6.77 (m, 2H), 6.74 (d, J = 8.3 Hz, 1H), 6.59 (s, 2H), 6.45
(d, J = 15.4 Hz, 1H), 5.66 (s, 1H), 3.87 (s, 3H), 3.80 (s, 6H), 3.77 (d, J
= 0.5 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 196.41, 152.74,
149.08, 145.75, 143.76, 140.34, 139.49, 137.36, 135.78, 130.15, 129.29,
128.15, 127.44, 127.05, 124.40, 121.57, 113.05, 110.43, 106.48, 60.27,
55.74, 55.35. HRMS calcd for C25H24O6 [M + H]+, 421.1646; found,
421.1653.
(E)-3-(3-Hydroxy-4-methoxyphenyl)-1-(2′,3′,4′-trimethoxy[1,1′-
biphenyl]-2-yl)prop-2-en-1-one (28). Yellow powder, yield 62.75%;
1
HPLC tR = 15.13 min; H NMR (400 MHz, DMSO) δ 9.11 (s, 1H),
7.63−7.55 (m, 2H), 7.47 (td, J = 7.5, 1.1 Hz, 1H), 7.38 (d, J = 7.6 Hz,
1H), 7.23 (d, J = 15.8 Hz, 1H), 6.94−6.81 (m, 5H), 6.51 (d, J = 15.8
Hz, 1H), 3.78 (s, 3H), 3.77 (s, 3H), 3.55 (s, 3H), 3.50 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ 194.80, 153.68, 151.10, 148.76, 145.82,
142.94, 142.02, 140.04, 136.99, 130.80, 130.46, 128.44, 128.27, 127.43,
127.08, 124.95, 124.09, 122.08, 112.81, 110.43, 107.37, 60.56, 60.50,
55.88, 55.73. HRMS calcd for C25H24O6 [M + H]+, 421.1646; found,
421.1655.
(E)-3-(4′-Fluoro[1,1′-biphenyl]-2-yl)-1-(4-methoxyphenyl)prop-2-
en-1-one (35). Light yellow powder, yield 80.23%; HPLC tR = 17.89
min; 1H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 8.8 Hz, 2H), 7.82 (d,
J = 7.1 Hz, 1H), 7.78 (d, J = 15.8 Hz, 1H), 7.49−7.39 (m, 3H), 7.36
(d, J = 7.4 Hz, 1H), 7.31 (dd, J = 8.4, 5.5 Hz, 2H), 7.13 (t, J = 8.6 Hz,
2H), 6.96 (d, J = 8.8 Hz, 2H), 3.88 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 188.65, 163.73, 163.53, 161.28, 142.89, 142.30, 136.09,
133.44, 131.52, 131.44, 130.89, 130.64, 129.99, 127.86, 127.14, 123.40,
115.48, 115.26, 113.90, 55.50. HRMS calcd for C22H17O2F [M + H]+,
333.1285; found, 333.1282.
(E)-3-(4′-Fluoro[1,1′-biphenyl]-2-yl)-1-(4-hydroxy-3-
methoxyphenyl)prop-2-en-1-one (41). Yellow powder, yield 76.98%;
1
HPLC tR = 11.31 min; H NMR (400 MHz, CDCl3) 7.82 (d, J = 7.1
Hz, 1H), δ 7.79 (d, J = 15.6 Hz, 1H), 7.62−7.56 (m, 2H), 7.45 (d, J =
15.5 Hz, 1H), 7.48−7.41 (m, 2H), 7.36 (dd, J = 7.1, 1.7 Hz, 1H),
7.34−7.28 (m, 2H), 7.13 (t, J = 8.6 Hz, 2H), 6.97 (d, J = 8.0 Hz, 1H),
6.09 (s, 1H), 3.96 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 188.55,
163.77, 161.32, 150.65, 147.05, 142.98, 142.35, 136.16, 136.12, 133.48,
131.55, 131.47, 130.89, 130.70, 130.04, 127.89, 127.25, 123.86, 123.18,
115.52, 115.30, 113.96, 110.66, 56.15. HRMS calcd for C22H17O3F [M
+ H]+, 349.1234; found, 349.1233.
(E)-3-(2-(6-Fluoropyridin-3-yl)phenyl)-1-(4-hydroxy-3-
methoxyphenyl)prop-2-en-1-one (42). Yellow powder, yield 70.38%;
HPLC tR = 7.12 min; 1H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 2.4
Hz, 1H), 7.85 (dd, J = 6.2, 3.0 Hz, 1H), 7.80−7.74 (m, 1H), 7.71 (d, J
= 15.5 Hz, 1H), 7.60 (dd, J = 8.2, 1.9 Hz, 1H), 7.58 (d, J = 1.8 Hz,
1H), 7.53−7.47 (m, 3H), 7.38−7.34 (m, 1H), 7.02 (dd, J = 8.4, 2.6
Hz, 1H), 6.98 (d, J = 8.1 Hz, 1H), 6.18 (s, 1H), 3.96 (s, 3H); 13C
NMR (100 MHz, DMSO) δ 186.88, 163.70, 161.34, 152.09, 147.80,
147.69, 147.54, 143.13, 143.05, 139.90, 137.80, 133.75, 133.70, 133.06,
130.60, 130.19, 129.28, 128.66, 127.55, 123.87, 123.78, 115.05, 111.74,
109.42, 109.05, 55.69. HRMS calcd for C21H16NO3F [M + H]+,
350.1187; found, 350.1181.
(E)-3-(2-(6-Fluoropyridin-3-yl)phenyl)-1-(4-methoxyphenyl)prop-
2-en-1-one (36). White powder, yield 75.68%; HPLC tR = 11.99 min;
1H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 2.4 Hz, 1H), 8.03−7.94
(m, 2H), 7.89−7.82 (m, 1H), 7.80−7.73 (m, 1H), 7.69 (d, J = 15.5
Hz, 1H), 7.53−7.44 (m, 2H), 7.48 (d, J = 15.6 Hz, 1H), 7.40−7.33
(m, 1H), 7.02 (dd, J = 8.4, 2.8 Hz, 1H), 7.00−6.92 (m, 2H), 3.89 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 188.25, 164.39, 163.65, 162.00,
148.01, 147.86, 142.43, 142.35, 141.60, 138.19, 133.89, 133.82, 133.78,
130.91, 130.81, 130.70, 130.24, 128.80, 127.39, 124.31, 113.98, 109.42,
109.04, 55.57. HRMS calcd for C21H16NO2F [M + H]+, 334.1238;
found, 334.1238.
(E)-3-(4′-Fluoro[1,1′-biphenyl]-2-yl)-1-(3-hydroxy-4-
methoxyphenyl)prop-2-en-1-one (43). Yellow crystals, yield 77.54%;
1
HPLC tR = 11.35 min; H NMR (400 MHz, CDCl3) δ 7.81 (dd, J =
7.4, 1.8 Hz, 1H), 7.76 (d, J = 15.7 Hz, 1H), 7.57 (d, J = 1.4 Hz, 1H),
7.56 (dd, J = 7.6, 1.8 Hz, 1H), 7.48−7.39 (m, 2H), 7.42 (d, J = 15.5
Hz, 1H), 7.38−7.34 (m, 1H), 7.33−7.27 (m, 2H), 7.17−7.10 (m, 2H),
6.93−6.88 (m, 1H), 5.70 (s, 1H), 3.97 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 188.92, 163.77, 161.32, 150.76, 145.70, 142.98, 142.37,
136.14, 136.11, 133.47, 131.78, 131.55, 131.47, 130.67, 130.02, 127.89,
(E)-1-(3,4-Dimethoxyphenyl)-3-(4′-fluoro[1,1′-biphenyl]-2-yl)-
prop-2-en-1-one (37). Light yellow powder, yield 83.56%; HPLC tR =
1
15.29 min; H NMR (400 MHz, CDCl3) δ 7.82 (d, J = 8.1 Hz, 1H),
7.79 (d, J = 15.2 Hz, 1H), 7.62 (dd, J = 8.5, 1.7 Hz, 1H), 7.57 (d, J =
1.6 Hz, 1H), 7.49−7.41 (m, 3H), 7.37 (dd, J = 6.8, 1.7 Hz, 1H), 7.34−
7.29 (m, 2H), 7.13 (t, J = 8.6 Hz, 2H), 6.90 (d, J = 8.5 Hz, 1H), 3.97
6378
dx.doi.org/10.1021/jm500024v | J. Med. Chem. 2014, 57, 6364−6382

Journal of Medicinal Chemistry
Article
127.15, 123.48, 122.14, 115.53, 115.31, 114.85, 110.12, 56.16. HRMS
calcd for C22H17O3F [M + H]+, 349.1234; found, 349.1231.
136.13, 135.98, 130.34, 130.26, 130.10, 129.97, 129.69, 129.12, 128.50,
127.59, 115.53, 115.31, 113.89, 55.21, 43.40. HRMS calcd for
C21H18NO2F [M + H]+, 336.1394; found, 336.1399.
(E)-3-(2-(6-Fluoropyridin-3-yl)phenyl)-1-(3-hydroxy-4-
methoxyphenyl)prop-2-en-1-one (44). Yellow powder, yield 70.05%;
HPLC tR = 7.12 min; 1H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 2.6
Hz, 1H), 7.88−7.82 (m, 1H), 7.76 (td, J = 7.9, 2.6 Hz, 1H), 7.67 (d, J
= 15.6 Hz, 1H), 7.59−7.55 (m, 2H), 7.53−7.48 (m, 2H), 7.44 (d, J =
15.5 Hz, 1H), 7.38−7.34 (m, 1H), 7.03 (dd, J = 8.3, 2.9 Hz, 1H), 6.92
(d, J = 9.1 Hz, 1H), 5.71 (s, 1H), 3.98 (s, 3H); 13C NMR (100 MHz,
DMSO) δ 187.24, 163.68, 161.32, 152.38, 147.68, 147.53, 146.64,
143.20, 143.12, 139.96, 137.87, 133.73, 133.68, 132.95, 130.65, 130.48,
130.27, 128.71, 127.47, 123.75, 121.87, 114.78, 111.16, 109.45, 109.08,
55.74. HRMS calcd for C21H16NO3F [M + H]+, 350.1187; found,
350.1185.
(E)-3-(4′-Fluoro[1,1′-biphenyl]-2-yl)-1-(3,4,5-trimethoxyphenyl)-
prop-2-en-1-one (45). Light yellow crystals, yield 81.42%; HPLC tR =
24.13 min; 1H NMR (400 MHz, CDCl3) δ 7.82 (d, J = 15.7 Hz, 1H),
7.81 (d, J = 7.2 Hz, 1H), 7.50−7.41 (m, 2H), 7.41−7.35 (m, 2H), 7.32
(d, J = 8.7 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.22 (s, 2H), 7.13 (t, J =
8.7 Hz, 2H), 3.93 (s, 3H), 3.92 (s, 6H); 13C NMR (100 MHz, CDCl3)
δ 188.58, 163.64, 161.18, 153.11, 143.45, 142.60, 142.24, 136.05,
136.02, 133.26, 133.13, 131.41, 131.33, 130.66, 130.07, 127.80, 127.41,
122.99, 115.42, 115.20, 106.11, 60.88, 56.31. HRMS calcd for
C24H21O4F [M + Na]+, 415.1316; found, 415.1308.
N-(3,4-Dimethoxybenzyl)-4′-fluoro[1,1′-biphenyl]-2-carboxa-
1
mide (50). White powder, yield 83.48%; HPLC tR = 12.15 min; H
NMR (400 MHz, CDCl3) δ 7.68 (d, J = 7.3 Hz, 1H), 7.50−7.44 (m,
1H), 7.41 (t, J = 7.4 Hz, 1H), 7.37−7.29 (m, 3H), 7.01 (t, J = 8.6 Hz,
2H), 6.73 (d, J = 8.2 Hz, 1H), 6.62 (s, 1H), 6.49 (d, J = 8.1 Hz, 1H),
5.43 (s, 1H), 4.28 (d, J = 5.5 Hz, 2H), 3.86 (s, 3H), 3.82 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ 169.12, 163.50, 161.04, 148.85, 148.36,
138.28, 135.99, 135.96, 135.78, 130.22, 130.14, 130.01, 129.91, 128.37,
127.46, 120.12, 115.34, 115.12, 111.38, 111.11, 55.76, 55.70, 43.74.
HRMS calcd for C22H20NO3F [M + Na]+, 388.1319; found, 388.1302.
4′-Fluoro-N-(3,4,5-trimethoxybenzyl)[1,1′-biphenyl]-2-carboxa-
1
mide (51). White powder, yield 85.61%; HPLC tR = 12.42 min; H
NMR (400 MHz, CDCl3) δ 7.68 (d, J = 7.7 Hz, 1H), 7.49−7.37 (m,
2H), 7.37−7.28 (m, 3H), 7.01 (t, J = 8.5 Hz, 2H), 6.29 (s, 2H), 5.51
(s, 1H), 4.25 (d, J = 5.6 Hz, 2H), 3.81 (s, 3H), 3.79 (s, 6H); 13C NMR
(100 MHz, CDCl3) δ 169.26, 163.48, 161.03, 153.14, 138.31, 137.28,
135.99, 135.96, 135.68, 133.24, 130.27, 130.19, 130.09, 130.03, 128.38,
127.51, 115.31, 115.10, 105.16, 60.60, 55.93, 44.36. HRMS calcd for
C23H22NO4F [M + Na]+, 418.1425; found, 418.1417.
7. Biology. Cell Culture. Cells (A549, CNE2, SW480, MCF-7,
HepG2, A2780, A2780/TAX, HCT-8, HCT-8/VCT, A549/CDDP,
and MCF-7/DOX) were grown in tissue culture flasks in complete
growth medium (DMEM high glucose medium, pH 7.4, supplemented
with 10% fetal bovine serum, 100 mg/mL streptomycin, and 100
units/mL penicillin) in a carbon dioxide incubator (37 °C, 5% CO2,
90% relative humidity (RH)).
8. SRB Cell Survival Assay. Cytotoxicity study was determined
using a 96-well microplate. SRB assay was performed to evaluate cell
viability.24 It measured cellular protein content to determine cell
density. Cells were plated at 5000 cells/well. The cells were allowed to
grow in carbon dioxide incubator (37 °C, 5% CO2, 90% RH) for 24 h.
Then one plate (plate day 0) was directly fixed by trichloroacetic acid
(50%, 50 μL) by means described in the following text. Meanwhile
different concentrations of test compounds in complete growth
medium (100 μL) were added to other plates in triplicate, and these
plates were further incubated for 48 h. The cell growth was stopped by
gently layering trichloroacetic acid (50%, 50 μL) on top of the
medium in all the wells. The plates were incubated at 4 °C for 1 h to
fix the cells attached to the bottom of the wells. The liquid of all the
wells was gently pipetted out and discarded. The plates were washed
five times with distilled water and air-dried. The plates were stained
with SRB dye (0.4% in 1% acetic acid, 100 μL) for 30 min. The plates
were washed five times with 1% acetic acid and then air-dried. The
adsorbed dye was dissolved in Tris base solution (150 μL,10 mM, pH
10.4), and plates were gently shaken for 1 h on an orbital shaker. The
optical density (OD) was recorded on a TECAN Infinite M200 Pro
multimode reader at 515 nm. The cell growth was determined by
subtracting mean OD value of plate day 0 from the mean OD value of
experimental set. From a plot of % control of cell viability against the
test compound concentration by plot concentrations on a log scale and
a fit of the data by using nonlinear regression in GraphPad Prism 5, the
compound concentration required to inhibit tumor cell proliferation
by 50% (IC50) was obtained from the dose−response curves. All data
are average values from triplicate samples, and the experiments were
repeated at least two times.
6. General Procedure for the Synthesis of 49−51. 2-
Bromobenzoic acid (5 mmol) was refluxed in 10 mL of SOCl2 for 1
h and then concentrated in vacuum to remove the solvent SOCl2. The
remainder in 20 mL of anhydrous toluene was added dropwise to a
mixture of phenylamine (5 mmol) and triethylamine (5 mmol) in 20
mL of toluene, and then the mixture was refluxed for 12 h. After
cooling, the mixture was evaporated to remove toluene and extracted
by CHCl3 from 10% HCl and 5% NaHCO3 three times. The
combined organic layer was dried over anhydrous sodium sulfate. After
removal of CHCl3 in vacuum, the crude product was purified by
column chromatography to yield the intermediates 46−48.
2-Bromo-N-(4-methoxybenzyl)benzamide (46). White solid, yield
1
71.21%; HPLC tR = 10.83 min; H NMR (400 MHz, acetone) δ 7.82
(s, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.46−7.30 (m, 5H), 6.89 (d, J = 8.4
Hz, 2H), 4.52 (d, J = 6.0 Hz, 2H), 3.78 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 167.48, 158.96, 137.79, 133.20, 131.04, 129.85, 129.32,
129.23, 127.37, 119.30, 113.99, 55.22, 43.45. HRMS calcd for
C15H14NO2Br [M + Na]+, 342.0100; found, 342.0092.
2-Bromo-N-(3,4-dimethoxybenzyl)benzamide (47). White solid,
1
yield 73.54%; HPLC tR = 8.58 min; H NMR (400 MHz, CDCl3) δ
7.58 (d, J = 8.0 Hz, 1H), 7.55 (dd, J = 7.6, 0.9 Hz, 1H), 7.35 (t, J = 7.4
Hz, 1H), 7.28−7.24 (m, 1H), 6.95 (s, 1H), 6.91 (d, J = 8.2 Hz, 1H),
6.83 (d, J = 8.1 Hz, 1H), 6.23 (s, 1H), 4.59 (d, J = 5.6 Hz, 2H), 3.88
(s, 3H), 3.87 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 167.55, 149.32,
148.69, 137.84, 133.44, 131.34, 130.41, 129.61, 127.63, 120.37, 119.36,
111.50, 111.37, 56.03, 44.06. HRMS calcd for C16H16NO3Br [M +
Na]+, 372.0206; found, 372.0202.
2-Bromo-N-(3,4,5-trimethoxybenzyl)benzamide (48). White solid,
1
yield 68.82%; HPLC tR = 8.85 min; H NMR (400 MHz, CDCl3) δ
7.59 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.36 (t, J = 7.4 Hz,
1H), 7.30−7.26 (m, 1H), 6.61 (s, 2H), 6.30 (s, 1H), 4.58 (d, J = 5.7
Hz, 2H), 3.85 (s, 6H), 3.82 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
167.60, 153.54, 137.88, 137.65, 133.53, 133.49, 131.39, 129.63, 127.67,
119.39, 105.23, 60.88, 56.30, 44.45. HRMS calcd for C17H18NO4Br [M
+ Na]+, 402.0311; found, 402.0296.
49−51 was synthesized using Suzuki−Miyaura coupling conditions
as previously described in the synthesis of 1 and 7, in which a mixture
of 46−48 (1.0 mmol), (4-fluorophenyl)boronic acid (1.3 mmol),
dichloro(1,10-bis(diphenylphosphino)ferrocene)palladium(II)−di-
chloromethane adduct (0.06 mmol), 2 M K2CO3(1.5 mL), and 1,4-
dioxane (1.5 mL) was used.
9. Cell Cycle Analysis. A549 cells were plated at 5000/well in a
96-well microplate and cultured overnight. Then various concen-
trations of tested compounds were added for 24 h. Cells were then
fixed with prewarmed (37 °C) paraformaldehyde for 10 min. Then
cells were permeabilized with permeabilization buffer (Triton X-100)
for 10 min and washed twice with PBS. Hoechst 33342 was added and
incubated at room temperature for 1 h, After washing, 50 μL of PBS
was added and the plates were evaluate on an ArrayScan HCS
reader.25
4′-Fluoro-N-(4-methoxybenzyl)[1,1′-biphenyl]-2-carboxamide
1
(49). White powder, yield 80.26%; HPLC tR = 14.56 min; H NMR
(400 MHz, acetone) δ 7.52−7.35 (m, 7H), 7.11−7.00 (m, 4H), 6.84−
6.76 (m, 2H), 4.30 (d, J = 6.0 Hz, 2H), 3.78 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 169.18, 163.69, 161.24, 158.96, 138.43, 136.17,
10. Apoptosis Assay. Annexin V/PI apoptosis kit was used. A549
cells (5 × 104/well) was incubated in asix-well plate for 24 h. Different
concentrations of 15 (0.25, 0.50, 0.75, 1.0 μM) and 0.25 μM
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Article
colchicine as control were added to cultured A549 cells and incubated
for 48 h. Approximately 1 × 105 cells were then stained for 5 min at
room temperature in the dark with fluorescein isothiocyanate (FITC)
conjugated annexin V (5 μL) and propidium iodide (PI) (10 μL) in a
1× annexin-binding buffer and analyzed by a two-color flow cytometric
assay. Annexin V and PI emissions were detected in the FL1 and FL2
channels of flow cytometer, respectively. Flow cytometer data show
three distinct populations of cells. The normal healthy cells, early
apoptosis, late apoptosis, and necrotic populations were represented
by annexin V-negative/PI-negative population, annexin V-positive/PI-
negative, annexin V-positive/PI-positive, and annexinV-negative/PI-
positive cells, respectively. The data were analyzed using Cell Quest
program from Beckman Coulter.26
11. In Vitro Tubulin Polymerization Assay. Tubulin polymer-
ization assay kit was used, and the enclosed experimental protocol was
followed. Tubulin (20 μM, 99% pure, 50 μL) was mixed with different
concentrations of tested compounds (10× in distilled water, 5 μL) in
general tubulin buffer (80 mM PIPES, pH 6.9, 2.0 mM MgCl2, and 0.5
mM EGTA), containing 1 mM GTP, 20% glycerol, and fluorescent
reporter DAPI.27 Microtubule polymerization was immediately
monitored in a kinetic model of FlexStation 3 multimode reader
followed by an increase in fluorescence emission at 450 nm at 37 °C
(excitation wavelength is 360 nm). Each compound was tested in
duplicate at each concentration in each experiment.28
measured everyday. The tumor volume was calculated using the
formula V = (larger diameter) × (smaller diameter)2/2, and growth
curves were plotted using average tumor volume within each
experimental group at the set time points. At the end of treatment,
the animals were sacrificed, and the tumors were removed and
weighed.
ASSOCIATED CONTENT
* Supporting Information
■
S
Rho123 accumulation and efflux assay; affinity-induced
fluorescence by 15 and 26; inhibition of affinity-induced
1
fluorescence by DTNB; H NMR, 13C NMR, HRMS, and
HPLC spectra of final compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone and fax: 020-39943054. E-mail: phsbxzh@mail.sysu.
edu.cn.
■
Author Contributions
†C.Z. and Y.Z. contribute equally.
Notes
12. Immunofluorescence Microscopy. Cellomics cytoskeletal
rearrangement kit was used, and the experiment protocol with slight
modification was followed. A549 cells were plated in a confocal culture
dish at 6 × 104 cells/dish and grown for 24 h. Tested compound 26
was added for 24 h, and then the dishes were placed in ice for 1 h to
make the microtubule depolymerized. After that, dishes were
reincubated in 37 °C incubator for 0, 5, 10, and 15 min to observe
whether the microtubule could reassemble, Cells were then fixed with
prewarmed (37 °C) paraformaldehyde for 15 min. Then cells were
permeabilized with permeabilization buffer (Triton X-100) for 15 min
and washed twice with PBS. Mouse anti-tubulin antibody was added
and incubated at room temperature for 1 h. Cells were washed three
times and incubated with DyLight 549-conjugated goat anti-mouse
IgG along with DAPI (stain nucleus) kept in darkness at room
temperature for 30 min. After washing, 1 mL PBS was added and
fluorescently stained cells were analyzed by confocal microscope.17
13. Fluorescence Measurement. Tubulin was incubated without
or with different concentrations of 15 and 26 in tubulin general buffer
and immediately evaluated in a FlexStation 3 multimode reader. The
fluorescence was found to increase when 15 and 26 were added to
tubulin, but no obvious fluorescence increase was observed in the
tubulin only control group. The optimal excitation wavelength and the
optimal emission wavelength were evaluated by a fluorescence
wavelength scanning model in Flexstation 3 multimode reader.29
14. Competitive Inhibition Assay. Tubulin−15 complex was
formed by incubating 4 μM tubulin in general tubulin buffer with 8
μM 15 at 37 °C in a kinetic model of FlexStation 3 multimode reader
(excitation at 430 nm, emission at 510 nm) until the fluorescence of
tubulin−15 came to a stable level. 26, colchicine, and vinctistine (0−
80 μM) were added to the preformed tubulin−15 complex, and
fluorescence was again determined at 37 °C using FlexStation 3
multimode reader (excitation at 430 nm, emission at 510 nm).20,29
15. Tumor Xenografts Growth Inhibition Assay. All animal
experiments complied with the Zhongshan School of Medicine Policy
on the Care and Use of Laboratory Animals. Female BALB/c nude
mice (5 weeks old) were purchased from the Experimental Animal
Center at Sun Yat-Sen University and maintained in pathogen-free
conditions. A549 cells were harvested during log-phase growth and
resuspended in RPMI-1640 medium at 6.67 × 107 cells/mL. Each
mouse was injected subcutaneously in the right flank with 1 × 107
cells. When the tumor volume reached approximately 100 mm3, the
mice were randomly divided into two groups of eight animals and
treated intraperitoneally (ip) with 26 at a dose of 2 mg/kg body
weight every other day for 2 weeks, whereas the control group was
treated with an equivalent volume of normal saline (including 0.5%
DMSO, 5% Kolliphor HS 15). Tumor size and body weight were
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are indebted to the National Nature Science Foundation of
China (Grants 30973619 and 81172931) and the Fundamental
Research Funds for the Central Universities (Grant 09ykpy66)
of China for financial support of this study.
ABBREVIATIONS USED
■
Rho123, rhodamine 123; NF-κB, nuclear factor κB; SRB,
sulforhodamine B; TAX, Taxol; VCT, vincristine; CDDP, cis-
platinum; DOX, doxorubicin; DRI, drug resistance index; RH,
relative humidity; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid)
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