Synthesis and biological evaluation of 2′,5′-dimethoxychalcone derivatives as microtubule-targeted anticancer agents

Article abstract of DOI:10.1016/j.bmc.2010.02.012

A series of novel 2′,5′-dimethoxylchalcone derivatives including 18 new compounds were synthesized and evaluated for cytotoxicities against two human cancer cell lines, NTUB1 (human bladder cancer cell line) and PC3 (human prostate cancer cell line). All these derivatives except for 21 exhibited significant cytotoxic effect against NTUB1 and PC3 cell lines. Compounds 13 and 17 with 4-carbamoyl moiety showed potent inhibitory effect on growth of NTUB1 and PC3 cells. Flow cytometric analysis demonstrated that treatment of NTUB1 cells with 1 μM 13 and 17 induced G1 phase arrest accompanied by an increase in apoptotic cell death of NTUB1 cells after 24 h. Treatment of PC3 cells with 1 μM and 3 μM 13, and 1 μM and 3 μM 17 induced S and G1, and G1 and G2/M phase arrests, respectively, accompanied by an increase in apoptotic cell death. These data suggested that 13 and 17 with different 4-carbamoyl moiety displayed same cell cycle arrest in NTUB1 cells while different doses of 13 and 17 revealed different cell cycle arrest in PC3 cells. Cell morphological study of 17 indicated that more cells rounding up or dead associated with tubulin polymerization. Compound 17 showed an increased α-tubulin level in polymerized microtubule fraction in a dose-dependent manner while 500 nM paclitaxel also showed similar effect in NTUB1 cells by Western blot analysis. The result suggested that 17 may be used as microtubule-targeted agents.

Full text of DOI:10.1016/j.bmc.2010.02.012

Bioorganic & Medicinal Chemistry 18 (2010) 2089–2098
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 20,50-dimethoxychalcone derivatives
as microtubule-targeted anticancer agents
c
Huang-Yao Tu a, A-Mei Huang b, Tzyh-Chyuan Hour b, Shyh-Chyun Yang a, , Yeong-Shiau Pu ,
*
Chun-Nan Lin a,d,
*
a Faculty of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan
b Graduate Institute of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
c Department of Urology, College of Medicine, National Taiwan University, Taipei 100, Taiwan
d Department of Life Science, National Taitung University, Taitung 950, Taiwan
a r t i c l e i n f o
a b s t r a c t
A series of novel 20,50-dimethoxylchalcone derivatives including 18 new compounds were synthesized
and evaluated for cytotoxicities against two human cancer cell lines, NTUB1 (human bladder cancer cell
line) and PC3 (human prostate cancer cell line). All these derivatives except for 21 exhibited significant
cytotoxic effect against NTUB1 and PC3 cell lines. Compounds 13 and 17 with 4-carbamoyl moiety
showed potent inhibitory effect on growth of NTUB1 and PC3 cells. Flow cytometric analysis demon-
Article history:
Received 8 December 2009
Revised 4 February 2010
Accepted 5 February 2010
Available online 12 February 2010
strated that treatment of NTUB1 cells with 1
an increase in apoptotic cell death of NTUB1 cells after 24 h. Treatment of PC3 cells with 1
M 13, and 1 M and 3 M 17 induced S and G1, and G1 and G2/M phase arrests, respectively, accom-
panied by an increase in apoptotic cell death. These data suggested that 13 and 17 with different 4-car-
bamoyl moiety displayed same cell cycle arrest in NTUB1 cells while different doses of 13 and 17 revealed
different cell cycle arrest in PC3 cells. Cell morphological study of 17 indicated that more cells rounding
l
M 13 and 17 induced G1 phase arrest accompanied by
Keywords:
Synthesis
Claisen–Schmidt condensation
Chalcone
Anticancer
lM and
3
l
l
l
Microtubule
Tubulin polymerization
Paclitaxel
Anti-mitotic
up or dead associated with tubulin polymerization. Compound 17 showed an increased a-tubulin level in
polymerized microtubule fraction in a dose-dependent manner while 500 nM paclitaxel also showed
similar effect in NTUB1 cells by Western blot analysis. The result suggested that 17 may be used as micro-
tubule-targeted agents.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Chalcones have been characterized with multiple biological
activities including anti-inflammatory, anti-malaria, anti-proto-
Microtubules display an important role in a variety of cellular
process including mitosis and cell division. Various anti-mitotic
agents interfering with the natural dynamics of tubulin, the major
protein component of microtubules, polymerization, and depoly-
merization inhibit cancer cell growth.1
Anti-mitotic agents such as paclitaxel stabilize microtubules by
preventing the depolymerization of tubulin. The vinca alkaloids
and colchicine inhibit the polymerization of tubulin. Disruption
of tubulin dynamics leads to cell cycle arrest in the G2/M phase
and induction of apoptosis.1 Anti-mitotic compounds have been
used clinically in the treatment of different cancers, a major
problem of anti-mitotic agent, such as taxanes and vinca alkaloids
in clinical application is the development of drug resistance.1
Therefore, it needs to find and develop effective tubulin inhibitors
for treating multidrug-resistant (MDR) tumors.
zoal, anti-bacterial, nitric oxide inhibitory, tyrosinase inhibitory,
cytotoxic, anti-leishmanial, and anti-oxidant activities in the past
decade.2–8 In our previous study, various 20,50-dialkoxylchalcones
were synthesized and demonstrated their structures and cyto-
toxic relationships against A549, Hep 3B, HT-29, and MCF-7
cells.8,9 Several reports have delineated the SAR of colchicine–
tubulin binding. These studies have shown that the important
structural features of colchicine molecule (Fig. 1) for binding to
tubulin are the methoxy groups of the A ring and the carbonyl
of the C ring.10 In addition, a (E)-1-(2,5-dimethoxyphenyl)-3-[4-
(dimethylamino)phenyl]-2-methyl-2-propen-1-one was reported
to be an effective antimitotic agent at concentration of 4 nM in
an in vitro HeLa cell test system.10 Based on the above reason,
we further synthesized a series of 20,50-dialkoxylchalcones with
a carbonyl or carbamoyl group substituted at C-4 of the B ring,
evaluated their cytotoxicities against human NTUB1 (human
bladder cancer cell line) and PC3 (human prostate cancer cell
line) cells, and discussed the structures and cytotoxicity and
mechanism of action.
* Corresponding authors. Tel.: +886 7 3121101; fax: +886 7 3210638 (S.-C.Y.);
tel.: +886 7 3121101; fax: +886 7 5562365 (C.-N.L.).
E-mail addresses: scyang@kmu.edu.tw (S.-C. Yang), lincna@cc.kmu.edu.tw (C.-N.
Lin).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.012

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H.-Y. Tu et al. / Bioorg. Med. Chem. 18 (2010) 2089–2098
H3CO
H3CO
O
O
O
O
OH
B
A
NHCCH3
a
S
H
OH
O
S
O
O
O
O
O
C
OCH3
20
21
1
O
Scheme 2. Reagents: (a) 8% KOH in H2O, MeOH
Colchicine
OCH3
except for 0.3 lM 17 against NTUB1 and PC3 cells. It suggested that
17 was a good candidate for developing as cancer chemotherapeu-
Figure 1. Structure of colchicine.
tic agents for treating cancer related to urinary system.
The effect of positive control, cisplatin, 13, and 17 on cell cycle
progression was determined by using fluorescence-activated cell
sorting (FACS) analysis in propidium iodide-stained NTUB1 and
PC3 cells. As shown in Figures 3 and 4, NTUB1 cells treated with
2. Chemistry
As shown in Schemes 1 and 2, chalcones 3 and 21 were pre-
pared by using Claisen–Schmidt condensation and hydrolysis with
methanolic 8% KOH solution. The compound 3 in acetone was
added potassium carbonate and various alkyl halides in appropri-
ate conditions to esterify C-4-COOH of 3 and afford various esters
4–9. Treatment of 3 in CH2Cl2 with various amines in the presence
of N-hydroxybenzotriazole (HOBt) and 1-ethyl-3-(3-dimethylami-
nopropyl)carbodiimide (EDC) afford various amides 10–19. These
procedures gave products with good yields (Table 1).
20
and S phases with the concomitant increase of the population of
sub-G1 phase. NTUB1 cells were exposed to 1 M 13 and 17,
lM cisplatin for 24 h led to an accumulation of cells in G1
l
respectively, for 24 h induced G1 phase arrest compared to the
respective control value, accompanied by an increase in apoptotic
cell death, respectively. PC3 cells treated with 20 lM cisplatin for
24 h led to an accumulation of cells in S phase with the concomi-
tant increase of apoptotic cell death. Treatment of PC3 cells with
1 lM 13 and 17, respectively, induced S and G1 phase arrests,
respectively, compared to respective control value, accompanied
3. Results and discussion
by an increase of apoptotic cells while exposure of PC3 cells to
Cisplatin shows more cytotoxicity against human transitional
carcinoma but about 30–50% of advanced bladder cancer did not
respond to cisplatin in chemotherapy.11 Based on resistances of
chemotherapy with cisplatin, it would like to find other new agents
for cancer treatment. Cytotoxicities of 3 derivatives against NTUB1
and PC3 cell lines were studied and cisplatin was used as the posi-
tive control. As shown in Table 2, compound 3 and most of its
derivatives showed significantly cytotoxic activities against NTUB1
cells while 21 had no significant cytotoxicity. The esterification of
C-4-COOH of 3 enhanced the cytotoxicity against NTUB1 and PC3
cells except for 6, 8, and 9. It indicated that increase of alkyl chain,
unsaturated or branched chain, and aromatic functional group in
the ester moiety attenuate the cytotoxicity against the same cancer
cell lines. The amidation of C-4-COOH of 3 enhanced the cytotoxic
effect against the two human cancer cell lines used in Table 2 while
12, 13, and 17 significantly enhanced the cytotoxicity against
NTUB1 and PC3 cells. It clearly revealed that compounds possessed
of 4-propyl, 4-(2-methylethyl), and 4-tetrahydropyrrolyl carbam-
oyl groups significantly enhanced the cytotoxicity against cancer
cell lines used in Table 2.
3 lM 13 and 17, respectively, induced G1 and G2/M phase arrests,
respectively, compared to the respective control value, accompa-
nied by an increase of the apoptotic cells death (Figs. 5 and 6). It
suggested that the increase of cell number of G2/M phase, accom-
panied by an increase of apoptotic cell death. It also suggested that
13 and 17 at lower dose revealed same cell cycle arrest in NTUB1
cells while their at higher dose indicated different cell cycle arrest
in PC3 cells. In addition, cell morphological and immuno-fluores-
cence studies were also performed for demonstrating the vital role
in the cell differentiation.
As shown in Figure 7, NUTB1 cells treated with different con-
centrations of 17 for 24 h showed more cell death and rounding
up after treatment. Microtubules play an important role in the
cell differentiation. If the function of microtubules is blocked by
chemicals or compounds, such as paclitaxel, cell differentiation
will arrest and be not progressed into the normal pattern of the
cell cycle control. Paclitaxel was reported as a microtubule-tar-
geted tubulin-polymerizing agent (MTPA) blocks the function of
microtubules.12 To determine whether 17, like paclitaxel, induced
the formation of stable microtubules bundles in cultured cells, the
immuno-fluorescent localization of tubulin was assessed.13 As
shown in Figure 8, compound 17 induced microtubule bundle for-
mation in NTUB1 cells and mimicked the effect of paclitaxel used
As shown in Figure 2, compound 17 was selected to assay two
additional cell lines, A549 and SV-HUC1. Compound 17 showed
more potent effect against NTUB1 and PC3 cell lines than that of
A549 cell line. It indicated that 17 displayed the specific cytotoxic-
ity against cancer cells in the urinary system. In addition, com-
pound 17 exhibited less cytotoxicity against human normal
uroepithelial SV-HUC1 cells than those of NTUB1 and PC3 cell lines
as the positive control.14 NTUB1 cells treated with 1
showed more mitotic arrest cell and influence on the microtubule
formation than those of 0.3 M 17 while paclitaxel showed more
potent stabilization of the tubulin assembly and inhibition on the
lM 17
l
O
O
O
O
O
O
O
OH
O
O
R
a
b or c
O
H
O
O
O
R'
2
4-19
1
3
Scheme 1. Reagents: (a) 8% KOH in H2O, MeOH; (b) various alkyl halides, K2CO3, acetone; (c) various amines, anhydrous HOBt, EDCI.

H.-Y. Tu et al. / Bioorg. Med. Chem. 18 (2010) 2089–2098
2091
Table 1
Structures and yields of all synthesized compounds
O
O
O
O
O
R
OH
S
O
R'
O
3-19
21
Compound
R
R0
Mp (°C)
Yield (%)
Molecular formula
3
4
5
6
7
–OH
–OCH3
–OCH2CH3
O
O
O
O
O
O
O
O
O
O
O
O
214–215
118–119
118–119
Oil
Oil
Oil
63.0
92.8
45.9
40.8
85.6
87.6
85.5
50.6
47.8
30.0
60.7
48.8
C18H16O5
C19H18O5
C20H20O5ꢀ1/2H2O
–OCH2CH2CH3
–OCH(CH3)2
–OCH2C6H5
–OCH2CH@CH(CH3)2
–NHCH3
–NHCH2CH3
–NHCH2CH2CH3
–NHCH(CH3)2
–N(CH3)2
C21H22O5
C21H22O5ꢀ1/4H2O
C25H22O5
C23H24O5
C19H19NO4
C20H21NO4
8
9
Oil
10
11
12
13
14
217–218
213–214
Oil
227–228
Oil
C21H23NO4ꢀ1/2H2O
C20H23NO4
C20H21NO4
15
O
280–281
66.7
C24H27NO4
N
H
16
17
–N(CH2CH3)2
O
O
Oil
Oil
98.2
68.4
C22H25NO4ꢀ1/4H2O
C22H23NO4ꢀ1/4H2O
N
N
18
O
Oil
79.1
C21H23NO5
OH
O
19
21
282–283
237–238
76.2
59.1
C24H26N2O3ꢀ1/4H2O
N
H
N
C16H14O5S
Table 2
Cytotoxicities of chalcone derivatives against NTUB1 and PC3 cancer cell linesa
Compound
NTUB1
IC50 M) SD
PC3
IC50
(
l
(lM) SD
Cisplatin
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
21
3.27 0.10
5.70 0.68
3.51 0.45
4.49 0.98
6.01 0.30
4.42 0.50
13.26 2.86
13.41 0.78
2.20 0.37
3.76 0.40
2.26 0.31
1.97 0.39
3.89 0.21
2.25 0.57
2.69 0.47
1.26 0.25
4.42 0.15
3.57 0.47
4.56 0.76
7.24 0.58
4.85 0.11
5.70 0.43
26.93 0.00
6.74 1.34
17.95 0.50
>50 lM
2.13 0.21
4.18 0.23
1.50 0.22
1.58 0.08
4.58 1.05
6.30 0.59
3.79 0.56
0.53 0.00
5.75 1.27
4.26 0.81
n.d
Figure 2. Cytotoxicities of 17 against NTUB1, PC3, A549, and SV-HUC1 cells. Cell
viability was assessed by the MTT assay after treating with different concentrations
of 17 for 72 h. The data shown represent mean SD (n = 3) for one experiment
performed in triplicate. *P <0.05, **P <0.01, and ***P <0.001 compared to the
respective control values, respectively.
>50
lM
a
Positive control: cisplatin; n.d, not determined. n = 3–5.
cell division. Following 17 binding to
a
-tubulin, it inhibited
centrations of 17 for 6 h (short time treatment) and collected the
microtubule (MT) dynamic instability, cell cycle G2/M phase
transition, mitotic arrest, and NTUB1 cell death through apoptosis
(Fig. 4).
supernatant and pellet for assays. Paclitaxel, used as the positive
control, certainly stabilized the tubulin assembly.
a-Tubulin in
the supernatant showed a decreased level in a dose-dependent
manner after treatment with various concentrations of 17 while
in the pellet increased in a dose-dependent manner except for that
In the Western blot analysis of
a-tubulin (Fig. 9), NTUB1 cells
were treated with 500 nM paclitaxel and more high different con-

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H.-Y. Tu et al. / Bioorg. Med. Chem. 18 (2010) 2089–2098
Figure 3. Flow cytometry analysis of the cell cycle with the treatment of 13 and 17. NTUB1 cells (7 ꢂ 105 cells/10 cm dish) was treated with no compound (A), cisplatin
20 lM (B), 1 lM 13 (C), 3 lM 13 (D), 1 lM 17 (E), and 3 lM 17 (F) for 24 h. At the time indicated, cells were stained with propidium iodide (PI), and DNA contents were
analyzed by flow cytometry, apoptosis was measured by the accumulation of sub-G1 DNA contents in cells. The control cells were treated with medium. Results were
representative of three independent experiments.
substituted at C-4 of B ring was found to have strongest cytotoxic-
ity against NTUB1 and PC3 cancer cell lines and showed less
cytotoxicity against SV-HUC1 and A549 cells. It indicated 17
specifically inhibited the growth of cancer cells in urinary system
and less toxicity for normal cells. It also indicated that the carbam-
oyl group substituted at C-4 of the B ring of chalcone moiety may
also important structural feature for binding to tubulin. It sug-
gested that 17 may be used as MTPAs.
5. Experimental section
5.1. General experimental procedures
IR spectra were determined with a Perkin–Elmer system 2000
FTIR spectrophotometer. 1H (400 MHz) and 13C (100 MHz) NMR
were recorded on a Varian UNITY-400 spectrometer, and mass
were obtained on a JMX-HX 100 mass spectrometer. Elemental
analyses measured with Vario EL III were within 0.4% of the the-
oretical values for all elements listed. Chromatography was per-
formed using a flash-column technique on Silica Gel 60 supplied
by E. Merck.
Figure 4. Cell cycle distribution of NTUB1 cells treated for 24 h with 13 and 17 in
different concentrations. Cisplatin was used as reference drug. Percentages of sub-
G1 cells and cells in G0/G1, S, and G2/M phase are shown. Data refer to
a
representative experiment one of three. CT (control), *P <0.05, **P <0.01 compared
to the respective control values.
5.2. 4-Carboxyl-20,50-dimethoxychalcone (3)
of 10 lM. It suggested that 17 could influence tubulin assembly in
the molecular action similar to paclitaxel.
2,5-Dimethoxyacetophenone (1) (450.0 mg, 2.5 mmol) and
methyl 4-formylbenzoate (2) (410.4 mg, 2.5 mmol) were dissolved
in MeOH (50 mL), and added 8% KOH in H2O (50 mL). The reaction
mixture was stirred at room temperature for 24 h and neutralized
with 10% HCl solution (100 mL) to form yellow precipitate. The yel-
low precipitate was filtered and washed with appropriate amount
of water. The crude product was purified by chromatography using
EtOAc/n-hexane (2:1), and crystallized by EtOAc/n-hexane (1:4) to
afford 3 (491.6 mg, 63.0%) as a yellow solid, IR (KBr): 1681,
4. Conclusion
A series of 20,50-dimethoxylchalcones including 18 new com-
pounds were synthesized and examined their biological activities.
In the MTT assays, all derivatives except for 21 showed signifi-
cantly cytotoxic effect against NTUB1 and PC3 cancer cell lines.
Among all these derivatives, compound 17 with a carbamoyl group
1597 cmꢁ1 1H NMR (CDCl3): d 3.82 (3H, s, OCH3), 3.89 (3H, s,
.

H.-Y. Tu et al. / Bioorg. Med. Chem. 18 (2010) 2089–2098
2093
Figure 5. Flow cytometry analysis of the cell cycle with the treatment of 13 and 17. PC3 cells (7 ꢂ 105 cells/10 cm dish) was treated with no compound (A), cisplatin 20
lM
(B), 1 lM 13 (C), 3 lM 13 (D), 1 lM 17 (E), and 3 lM 17 (F) for 24 h. At the time indicated, cells were stained with propidium iodide (PI), and DNA contents were analyzed by
flow cytometry, apoptosis was measured by the accumulation of sub-G1 DNA contents in cells. The control cells were treated with medium. Results were representative of
three independent experiments.
5.3. Procedure A for synthesis of C-4 ester compounds 4–9
To a solution of 3 (60 mg, 0.19 mmol) in acetone was added
K2CO3 (52.5 mg, 0.38 mmol) and alkyl halide (0.38 mmol). The
reaction mixture was stirred at the room temperature for prepar-
ing 4–6 or refluxed for preparing 7–9 overnight. The mixture was
concentrated to dryness under reduced pressure, neutralized with
10% HCl solution, and extracted with CH2Cl2. The organic phase
was dried over Na2SO4, filtered, and concentrated in vacuo to give
the crude product. The crude product was purified by chromatog-
raphy with EtOAc/n-hexane, and crystallized with acetone/n-hex-
ane (1:4) to afford purified products.
5.3.1. 4-Methoxycarbonyl-20,50-dimethoxychalcone (4)
To a solution of 3 (60 mg, 0.19 mmol) in acetone was added
K2CO3 (52.5 mg, 0.38 mmol) and methyl iodide (0.38 mmol). The
mixture was treated as procedure A for synthesis of C-4 ester to af-
ford
4 (58.2 mg, 92.8%) as a yellow solid, IR (KBr): 1721,
Figure 6. Cell cycle distribution of PC3 cells treated for 24 h with 13 and 17 in
different concentrations. Cisplatin was used as reference drug. Percentages of sub-
1598 cmꢁ1
.
1H NMR (CDCl3): d 3.81 (3H, s, OCH3), 3.87 (3H, s,
OCH3), 3.92 (3H, s, COOCH3), 6.95 (1H, d, J = 8.8 Hz, H-30), 7.05
(1H, dd, J = 8.8, 3.2 Hz, H-40), 7.21 (1H, d, J = 3.2 Hz, H-60), 7.51
G1 cells and cells in G0/G1, S, and G2/M phase are shown. Data refer to
a
representative experiment one of three. CT (control), *P <0.05, **P <0.01 compared
to the respective control values.
(1H, d, J = 16.0 Hz, H-a), 7.64 (2H, d, J = 8.0 Hz, H-2 and 6), 7.65
(1H, d, J = 16.4 Hz, H-b), 8.05 (2H, d, J = 8.4 Hz, H-3 and 5). 13C
NMR (CDCl3): d 52.2 (COOCH3), 55.8 (OCH3), 56.4 (OCH3), 113.4
OCH3), 6.96 (1H, d, J = 8.8 Hz, H-30), 7.06 (1H, dd, J = 9.0, 3.2 Hz,
H-40), 7.23 (1H, d, J = 3.2 Hz, H-60), 7.55 (1H, d, J = 16.0 Hz, H-
(C-60), 114.4 (C-30), 119.6 (C-10 and 40), 119.6 (C-
a), 128.1 (C-2
and 6), 128.9 (C-3 and 5), 130.0 (C-4), 139.5 (C-1), 141.2 (C-b),
152.8 (C-50), 153.6 (C-20), 166.5 (COOCH3), 191.8 (C@O). ESIMS:
m/z 327 [M+H]+. Anal. Calcd for C19H18O5: C, 69.93; H, 5.56. Found:
C, 69.95; H, 5.62.
a
), 7.68 (1H, d, J = 15.6 Hz, H-b), 7.69 (2H, dd, J = 8.8, 1.6 Hz, H-2
and 6), 8.13 (2H, dd, J = 8.8, 1.6 Hz, H-3 and 5). 13C NMR
(CDCl3): d 55.9 (OCH3), 56.5 (OCH3), 113.4 (C-60), 114.4 (C-30),
119.8 (C-10 and 40), 119.8 (C-
a), 128.3 (C-2 and 6), 129.3 (C-3
and 5), 130.2 (C-4), 140.4 (C-1), 141.0 (C-b), 152.8 (C-50),
153.7 (C-20), 171.0 (–COOH), 191.8 (C@O). ESIMS: m/z 313
[M+H]+. Anal. Calcd for C18H16O5: C, 69.22; H, 5.16. Found: C,
69.10; H, 5.18.
5.3.2. 4-Ethoxycarbonyl-20,50-dimethoxychalcone (5)
To a solution of 3 (100 mg, 0.32 mmol) in acetone was added
K2CO3 (88.5 mg, 0.64 mmol) and ethyl iodide (0.64 mmol). The
mixture was treated as procedure A for synthesis of C-4 ester to

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H.-Y. Tu et al. / Bioorg. Med. Chem. 18 (2010) 2089–2098
Figure 7. Cell morphologies of NTUB1 cells after treatment without compound (control), 10 nM paclitaxel, and different concentrations of 17 for 24 h. NTUB1 cells (7 ꢂ 105
cells/10 cm dish) were treated with 0.3, 1, and 3
cells rounded up and died.
lM of 17 for 24 h while treatment without compound was used as the control. As the concentration increased, most NTUB1
Figure 8. (A) Immuno-fluorescent microscopy of a-tubulin after treatment with only medium, 10 nM of paclitaxel, and different concentrations of compound 17. NTUB1 cells
were coated on cover slide with 100% serum in six-well plate (105 cells/well) before incubating overnight and treated with compounds for 24 h. Cells were fixed by 2%
formaldehyde/PBS, washed with PBS, fixed with cold methanol, washed with PBS, added a-tubulin monoclonal antibody, washed with PBS, added several conjugated mouse
antihuman antibody, washed with PBS, mounted on the slide with 80% glycerol/PBS, and microscoped. (B) Immuno-fluorescent microscopy of DNA after treatment with only
medium, 10 nM of paclitaxel, and different concentrations of 17. The protocol was performed as mentioned above while the antibody was replaced by DAPI stain.
J = 8.4, 1.6 Hz, H-3 and 5). 13C NMR (CDCl3): d 14.3 (CH3), 55.8
(OCH3), 56.4 (OCH3), 61.1 (COOCH2–), 113.4 (C-60), 114.4 (C-30),
119.6 (C-10 and 40), 119.6 (C-
a), 128.1 (C-2 and 6), 128.8 (C-3 and
5), 130.0 (C-4), 139.4 (C-1), 141.3 (C-b), 152.8 (C-50), 153.6 (C-20),
166.0 (COOCH2–), 191.9 (C@O). ESIMS: m/z 341 [M+H]+. Anal. Calcd
for C20H20O5ꢀ1/2H2O: C, 68.75; H, 6.06. Found: C, 68.71; H, 6.49.
5.3.3. 4-Propoxycarbonyl-20,50-dimethoxychalcone (6)
To a solution of 3 (60 mg, 0.19 mmol) in acetone was added
K2CO3 (52.5 mg, 0.38 mmol) and propyl iodide (0.38 mmol). The
mixture was treated as procedure A for synthesis of C-4 ester to af-
ford 6 (27.8 mg, 40.8%) as a yellow oil, IR (KBr): 1715, 1602 cmꢁ1
.
Figure 9. Western blotting of
with 500 nM paclitaxel or different concentrations of 17 for 6 h, collected,
suspended with PBS, and assayed for -tubulin assembly in the supernatant and
a-tubulin in NTUB1 cells. NTUB1 cells were treated
1H NMR (CDCl3): d 1.04 (3H, t, J = 7.2 Hz, CH3), 1.80 (2H, m,
CH2CH3), 3.81 (3H, s, OCH3), 3.88 (3H, s, OCH3), 4.29 (2H, t,
J = 6.4 Hz, COOCH2–), 6.95 (1H, d, J = 9.2 Hz, H-30), 7.05 (1H, dd,
J = 9.2, 3.2 Hz, H-40), 7.22 (1H, d, J = 3.2 Hz, H-60), 7.51 (1H, d,
a
pellet by western blotting. In this assay, b-actin was used as the internal control.
afford 5 (50.0 mg, 45.9%) as a yellow solid, IR (KBr): 1715, 1599 cmꢁ1
.
J = 16.0 Hz, H-a), 7.65 (2H, d, J = 8.4 Hz, H-2 and 6), 7.66 (1H, d,
1H NMR (CDCl3): d 1.34 (3H, t, J = 7.2 Hz, CH3), 3.81 (3H, s, OCH3),
3.87 (3H, s, OCH3), 4.38 (2H, q, J = 7.2 Hz, COOCH2–), 6.95 (1H, d,
J = 8.8 Hz, H-30), 7.05 (1H, dd, J = 8.8, 3.2 Hz, H-40), 7.21 (1H, d,
J = 16.4 Hz, H-b), 8.06 (2H, d, J = 8.4 Hz, H-3 and 5). 13C NMR
(CDCl3): d 10.5 (CH3), 22.1 (CH2CH3), 55.8 (OCH3), 56.4 (OCH3),
66.7 (COOCH2–), 113.4 (C-60), 114.4 (C-30), 119.7 (C-10 and 40),
J = 3.2 Hz, H-60), 7.51 (1H, d, J = 16.0 Hz, H-
a
), 7.64 (2H, d,
119.7 (C-
a), 128.1 (C-2 and 6), 128.8 (C-3 and 5), 130.0 (C-4),
139.4 (C-1), 141.3 (C-b), 152.8 (C-50), 153.7 (C-20), 166.1
J = 8.4 Hz, H-2 and 6), 7.65 (1H, d, J = 16.4 Hz, H-b), 8.06 (2H, dd,

H.-Y. Tu et al. / Bioorg. Med. Chem. 18 (2010) 2089–2098
2095
(COOCH2–), 191.9 (C@O). ESIMS: m/z 355 [M+H]+. Anal. Calcd for
product was purified by chromatography with EtOAc/n-hexane,
C21H22O5: C, 71.17; H, 6.26. Found: C, 71.15; H, 6.39.
and crystallized with EtOAc to afford purified products.
5.3.4. 4-Isopropoxycarbonyl-20,50-dimethoxychalcone (7)
To a solution of 3 (60 mg, 0.19 mmol) in acetone was added
K2CO3 (52.5 mg, 0.38 mmol) and isopropyl iodide (0.38 mmol).
The mixture was treated as procedure A for synthesis of C-4 ester
to afford 7 (58.2 mg, 85.6%) as a yellow oil, IR (KBr): 1711,
5.4.1. 4-Methylcarbamoyl-20,50-dimethoxychalcone (10)
A mixture of 3 (156 mg, 0.5 mmol), HOBt (135.1 mg, 1.0 mmol),
EDC (191.7 mg, 1.0 mmol) was dissolved with CH2Cl2, and stirred
for 10 min. The mixture was added methylamine (1.0 mmol) and
treated as procedure B to afford 10 (82.3 mg, 50.6%) as a yellow so-
lid, IR (KBr): 3326, 1652, 1546 cmꢁ1. 1H NMR (CDCl3): d 3.10 (3H, d,
J = 4.8 Hz, NHCH3), 3.80 (3H, s, OCH3), 3.86 (3H, s, OCH3), 6.39 (1H,
br s, CONH–), 6.94 (1H, d, J = 9.2 Hz, H-30), 7.04 (1H, dd, J = 9.2,
3.2 Hz, H-40), 7.19 (1H, d, J = 3.2 Hz, H-60), 7.47 (1H, d, J = 16.0 Hz,
1603 cmꢁ1
.
1H NMR (CDCl3): d 1.37 (6H, d, J = 6.0 Hz, CH3 ꢂ 2),
3.81 (3H, s, OCH3), 3.87 (3H, s, OCH3), 5.26 (1H, m, COOCH(CH3)2),
6.95 (1H, d, J = 9.2 Hz, H-30), 7.05 (1H, dd, J = 8.8, 3.2 Hz, H-40), 7.21
(1H, d, J = 3.2 Hz, H-60), 7.51 (1H, d, J = 16.0 Hz, H-
a), 7.64 (2H, d,
J = 8.8 Hz, H-2 and 6), 7.66 (1H, d, J = 16.0 Hz, H-b), 8.05 (2H, d,
J = 8.4 Hz, H-3 and 5). 13C NMR (CDCl3): d 21.9 (CH3 ꢂ 2), 55.9
(OCH3), 56.5 (OCH3), 68.6 (COOCH(CH3)2), 113.4 (C-60), 114.4 (C-
H-a), 7.61 (2H, d, J = 8.4 Hz, H-2 and 6), 7.62 (1H, d, J = 16.0 Hz,
H-b), 7.78 (2H, d, J = 8.4 Hz, H-3 and 5). 13C NMR (CDCl3): d 26.9
(NHCH3), 55.8 (OCH3), 56.5 (OCH3), 113.4 (C-60), 114.4 (C-30),
30), 119.7 (C-10 and 40), 119.7 (C-
a
), 128.1 (C-2 and 6), 128.8 (C-3
119.5 (C-10 and 40), 119.5 (C-
a), 127.4 (C-2 and 6), 128.4 (C-3 and
and 5), 130.0 (C-4), 139.3 (C-1), 141.4 (C-b), 152.8 (C-50), 153.7
(C-20), 166.1 (COOCH(CH3)2), 191.9 (C@O). ESIMS: m/z 355
[M+H]+. Anal. Calcd for C21H22O5ꢀ1/4H2O: C, 70.28; H, 6.32. Found:
C, 70.64; H, 6.26.
5), 135.7 (C-4), 138.0 (C-1), 147.5 (C-b), 152.7 (C-50), 153.6 (C-20),
167.5 (CONH–), 192.0 (C@O). ESIMS: m/z 326 [M+H]+. Anal. Calcd
for C19H19NO4: C, 70.14; H, 5.89; N, 4.31. Found: C, 69.23; H,
5.95; N, 4.21.
5.3.5. 4-Benzyloxycarbonyl-20,50-dimethoxychalcone (8)
To a solution of 3 (60 mg, 0.19 mmol) in acetone was added
K2CO3 (52.5 mg, 0.38 mmol) and benzyl bromide (0.38 mmol).
The mixture was treated as procedure A for synthesis of C-4 ester
to afford 8 (67.7 mg, 87.6%) as a yellow oil, IR (KBr): 1717,
5.4.2. 4-Ethylcarbamoyl-20,50-dimethoxychalcone (11)
A mixture of 3 (156 mg, 0.5 mmol), HOBt (135.1 mg, 1.0 mmol),
EDC (191.7 mg, 1.0 mmol) was dissolved with CH2Cl2, and stirred
for 10 min. The mixture was added ethylamine (1.0 mmol) and
treated as procedure B to afford 11 (81.0 mg, 47.8%) as a yellow so-
lid, IR (KBr): 3326, 1641, 1544 cmꢁ1. 1H NMR (CDCl3): d 1.25 (3H, t,
J = 7.2 Hz, CH3), 3.50 (2H, m, NHCH2–), 3.80 (3H, s, OCH3), 3.86 (3H,
s, OCH3), 6.26 (1H, br s, CONH–), 6.94 (1H, d, J = 8.8 Hz, H-30), 7.04
(1H, dd, J = 8.8, 3.2 Hz, H-40), 7.20 (1H, d, J = 3.2 Hz, H-60), 7.47 (1H,
1601 cmꢁ1 1H NMR (CDCl3): d 3.81 (3H, s, OCH3), 3.87 (3H, s,
.
OCH3), 5.38 (2H, s, COOCH2–), 6.95 (1H, d, J = 8.8 Hz, H-30), 7.05
(1H, dd, J = 8.8, 3.2 Hz, H-40), 7.22 (1H, d, J = 3.2 Hz, H-60), 7.35
(5H, m, –C6H5), 7.51 (1H, d, J = 16.0 Hz, H-a), 7.64 (2H, d,
J = 8.4 Hz, H-2 and 6), 7.66 (1H, d, J = 16.0 Hz, H-b), 8.09 (2H, d,
J = 8.4 Hz, H-3 and 5). 13C NMR (CDCl3): d 55.9 (OCH3), 56.5
(OCH3), 66.9 (COOCH2–), 113.4 (C-60), 114.4 (C-30), 119.7 (C-10
d, J = 16.0 Hz, H-a), 7.62 (2H, d, J = 8.4 Hz, H-2 and 6), 7.63 (1H, d,
J = 16.0 Hz, H-b), 7.78 (2H, d, J = 8.0 Hz, H-3 and 5). 13C NMR
(CDCl3): d 14.8 (CH3), 35.0 (NHCH2–), 55.8 (OCH3), 56.5 (OCH3),
and 40), 119.7 (C-
a
), 128.2–128.3 (tertiary aromatic carbon ꢂ 5),
113.4 (C-60), 114.4 (C-30), 119.5 (C-10 and 40), 119.5 (C-
a), 127.4
128.6 (C-2 and 6), 129.0 (C-3 and 5), 130.2 (C-4), 135.9 (quaternary
aromatic carbon ꢂ 1), 139.6 (C-1), 141.2 (C-b), 152.8 (C-50), 153.7
(C-20), 165.8 (COOCH2–), 191.8 (C@O). ESIMS: m/z 403 [M+H]+.
Anal. Calcd for C25H22O5: C, 74.61; H, 5.51. Found: C, 74.63; H, 5.63.
(C-2 and 6), 128.4 (C-3 and 5), 135.9 (C-4), 138.0 (C-1), 141.5 (C-
b), 152.7 (C-50), 153.6 (C-20), 166.7 (CONH–), 192.0 (C@O). EIMS
(70 eV) m/z (% rel. int.): 339 (100). Anal. Calcd for C20H21NO4: C,
70.48; H, 6.24; N, 4.13. Found: C, 69.87; H, 6.28; N, 4.07.
5.3.6. 4-(3-Methyl-but-2-enyloxycarbonyl)-20,50-dimethoxy-
chalcone (9)
5.4.3. 4-Propylcarbamoyl-20,50-dimethoxychalcone (12)
A mixture of 3 (156 mg, 0.5 mmol), HOBt (135.1 mg, 1.0 mmol),
EDC (191.7 mg, 1.0 mmol) was dissolved with CH2Cl2, and stirred
for 10 min. The mixture was added propylamine (1.0 mmol) and
treated as procedure B to afford 12 (52.9 mg, 30.0%) as a yellow
To a solution of 3 (60 mg, 0.19 mmol) in acetone was added
K2CO3 (52.5 mg, 0.38 mmol) and 1-bromo-3-methyl-2-butene
(0.38 mmol). The mixture was treated as procedure A for synthesis
of C-4 ester to afford 9 (62.5 mg, 85.5%) as a yellow oil, IR (KBr):
oil, IR (KBr): 3329, 1641, 1544 cmꢁ1 1H NMR (CDCl3): d 0.99 (3H,
.
1714, 1600 cmꢁ1
.
1H NMR (CDCl3): d 1.78 (3H, s, CH3), 1.79 (3H,
t, J = 7.6 Hz, CH3), 1.65 (2H, m, CH2CH3), 3.43 (2H, m, NHCH2–),
3.81 (3H, s, OCH3), 3.87 (3H, s, OCH3), 6.22 (1H, br s, CONH–),
6.95 (1H, d, J = 8.8 Hz, H-30), 7.02 (1H, dd, J = 8.8, 3.2 Hz, H-40),
s, CH3), 3.81 (3H, s, OCH3), 3.88 (3H, s, OCH3), 4.83 (2H, d,
J = 7.2 Hz, COOCH2–), 5.47 (1H, m, CH@C(CH3)2), 6.95 (1H, d,
J = 8.8 Hz, H-30), 7.05 (1H, dd, J = 8.8, 3.2 Hz, H-40), 7.21 (1H, d,
7.20 (1H, d, J = 3.2 Hz, H-60), 7.48 (1H, d, J = 16.0 Hz, H-
a), 7.63
J = 3.2 Hz, H-60), 7.51 (1H, d, J = 15.6 Hz, H-
a), 7.63 (2H, d,
(2H, d, J = 8.8 Hz, H-2 and 6), 7.64 (1H, d, J = 16.0 Hz, H-b), 7.78
(2H, d, J = 8.8 Hz, H-3 and 5). 13C NMR (CDCl3): d 11.4 (CH3), 22.9
(CH2CH3), 41.8 (NCH2–), 55.9 (OCH3), 56.5 (OCH3), 113.4 (C-60),
J = 8.0 Hz, H-2 and 6), 7.65 (1H, d, J = 16.0 Hz, H-b), 8.06 (2H, d,
J = 8.4 Hz, H-3 and 5). 13C NMR (CDCl3): d 18.1 (CH3), 25.8 (CH3),
55.9 (OCH3), 56.5 (OCH3), 62.1 (COOCH2–), 113.4 (C-60), 114.4 (C-
114.4 (C-30), 119.6 (C-10 and 40), 119.6 (C-
a), 127.4 (C-2 and 6),
30), 118.5 (CH@C(CH3)2), 119.7 (C-10 and 40), 119.7 (C-
a
), 128.1
128.4 (C-3 and C-5), 136.0 (C-4), 138.0 (C-1), 141.5 (C-b), 152.7
(C-50), 153.7 (C-20), 166.8 (CONH–), 192.0 (C@O). EIMS (70 eV)
m/z (% rel. int.): 353 (100). Anal. Calcd for C21H23NO4ꢀ1/2H2O: C,
69.58; H, 6.68; N, 3.87. Found: C, 70.60; H, 6.68; N, 3.85.
(C-2 and 6), 128.8 (C-3 and 5), 130.1 (C-4), 137.1 (CH@C(CH3)2),
139.4 (C-1), 141.4 (C-b), 152.8 (C-50), 153.7 (C-20), 166.1
(COOCH2–), 191.9 (C@O). ESIMS: m/z 381 [M+H]+. Anal. Calcd for
C23H24O5: C, 72.61; H, 6.36. Found: C, 72.60; H, 6.48.
5.4.4. 4-(2-Methylethyl)carbamoyl-20,50-dimethoxychalcone
(13)
5.4. Procedure B for synthesis of C-4 amide compounds 10–19
A
mixture of
3
(100 mg, 0.32 mmol), HOBt (86.5 mg,
A mixture of 3 (156 mg, 0.5 mmol), HOBt (135.1 mg, 1.0 mmol),
EDCI (191.7 mg, 1.0 mmol) was dissolved with CH2Cl2, and stirred
for 10 min. The mixture was added methylamine (1.0 mmol), and
stirred at the room temperature for 2 h. The reaction mixture
was concentrated in vacuo to give the crude product. The crude
0.64 mmol), EDC (122.7 mg, 0.64 mmol) was dissolved with
CH2Cl2, and stirred for 10 min. The mixture was added isopropyl-
amine (1.0 mmol) and treated as procedure
(68.7 mg, 60.7%) as yellow solid, IR (KBr): 3320, 1638,
1538 cmꢁ1 1H NMR (CDCl3): d 1.26 (6H, d, J = 6.4 Hz, –CH(CH3)2),
B to afford 13
a
.

2096
H.-Y. Tu et al. / Bioorg. Med. Chem. 18 (2010) 2089–2098
4.29 (1H, m, NHCH(CH3)2), 6.02 (1H, d, J = 7.6 Hz, CONH–), 6.94
(1H, d, J = 8.8 Hz, H-30), 7.04 (1H, dd, J = 9.0, 3.2 Hz, H-40), 7.20
126.9 (C-2 and 6), 128.4 (C-3 and 5), 135.9 (C-4), 138.7 (C-1),
141.9 (C-b), 152.6 (C-50), 153.6 (C-20), 170.5 (–CONH–), 192.1
(C@O). EIMS (70 eV) m/z (% rel. int.): 367 (100). Anal. Calcd for
C22H25NO4ꢀ1/4H2O: C, 71.04; H, 6.91; N, 3.77. Found: C, 71.23; H,
6.93; N, 3.65.
(1H, d, J = 3.2 Hz, H-60), 7.47 (1H, d, J = 16.0 Hz, H-
a), 7.62 (2H, d,
J = 8.0 Hz, H-2 and 6), 7.63 (1H, d, J = 16.8 Hz, H-b), 7.77 (2H, d,
J = 8.4 Hz, H-3 and 5). 13C NMR (CDCl3): d 22.8 (CH3), 29.7 (CH3),
42.0 (NHCH(CH3)2), 55.8 (OCH3), 56.5 (OCH3), 113.4 (C-60), 114.4
(C-30), 119.5 (C-10 and 40), 119.5 (C-
a), 127.4 (C-2 and 6), 128.4
5.4.8. 4-Tetrahydropyrrolylcarbamoyl-20,50-dimethoxychalcone
(17)
(C-3 and 5), 136.1 (C-4), 138.0 (C-1), 141.5 (C-b), 152.7 (C-50),
153.6 (C-20), 165.9 (CONH–), 192.0 (C@O). EIMS (70 eV) m/z (%
rel. int.): 353 (100). Anal. Calcd for C20H23NO4: C, 71.37; H, 6.56;
N, 3.96. Found: C, 71.57; H, 6.68; N, 3.86.
A
mixture of
3
(100 mg, 0.32 mmol), HOBt (86.5 mg,
0.64 mmol), EDC (122.7 mg, 0.64 mmol) was dissolved with
CH2Cl2, and stirred for 10 min. The mixture was added pyrrolidine
(1.0 mmol) and treated as procedure B to afford 17 (80.0 mg, 68.4%)
5.4.5. 4-Dimethylcarbamoyl-20,50-dimethoxychalcone (14)
as a yellow oil, IR (KBr): 3466, 1619 cmꢁ1 1H NMR (CDCl3): d 1.89
.
A
mixture of
3
(100 mg, 0.32 mmol), HOBt (86.5 mg,
(4H, m, –CH2CH2–), 3.42 (2H, m, NCH2–), 3.60 (2H, m, NCH2–), 3.79
(3H, s, OCH3), 3.85 (3H, s, OCH3), 6.94 (1H, d, J = 8.8 Hz, H-30), 7.02
(1H, dd, J = 8.0, 3.2 Hz, H-40), 7.18 (1H, d, J = 3.2 Hz, H-60), 7.44 (1H,
0.64 mmol), EDC (122.7 mg, 0.64 mmol) was dissolved with
CH2Cl2, and stirred for 10 min. The mixture was added dimethyl-
amine (1.0 mmol) and treated as procedure
B
to afford 14
d, J = 16.0 Hz, H-a), 7.52 (2H, d, J = 8.0 Hz, H-2 and 6), 7.59 (2H, d,
(53.0 mg, 48.8%) as a yellow oil, IR (KBr): 3485, 1633 cmꢁ1
.
1H
J = 8.4 Hz, H-3 and 5), 7.62 (1H, d, J = 15.6 Hz, H-b). 13C NMR
(CDCl3): d 24.3 (–CH2CH2–), 26.3 (–CH2CH2–), 46.2 (NCH2–), 49.5
(NCH2–), 55.8 (OCH3), 56.4 (OCH3), 113.3 (C-60), 114.3 (C-30),
NMR (CDCl3): d 3.09 (6H, m, –N(CH3)2), 3.81 (3H, s, –OCH3), 3.86
(3H, s, –OCH3), 6.94 (1H, d, J = 9.2 Hz, H-30), 7.03 (1H, dd, J = 9.2,
3.2 Hz, H-40), 7.19 (1H, d, J = 3.2 Hz, H-60), 7.44 (2H, d, J = 8.4 Hz,
119.4 (C-10 and 40), 119.4 (C-
a), 127.6 (C-2 and 6), 128.1 (C-3 and
5), 136.5 (C-4), 138.5 (C-1), 141.8 (C-b), 152.6 (C-50), 153.5 (C-20),
168.9 (CONH–), 192.0 (C@O). EIMS (70 eV) m/z (% rel. int.): 365
(97), 295 (100). Anal. Calcd for C22H23NO4ꢀ1/4H2O: C, 71.43; H,
6.40; N, 3.79. Found: C, 71.67; H, 6.71; N, 3.63.
H-2 and 6), 7.45 (1H, d, J = 15.6 Hz, H-a), 7.51 (2H, d, J = 8.0 Hz,
H-3 and 5), 7.63 (1H, d, J = 16.0 Hz, H-b). 13C NMR (CDCl3): d 35.3
and 39.5 (N(CH3)2), 55.8 (OCH3), 56.5 (OCH3), 113.4 (C-60), 114.4
(C-30), 119.5 (C-10 and 40), 119.5 (C-
a), 127.6 (C-2 and 6), 128.3
(C-3 and 5), 136.3 (C-4), 137.7 (C-1), 141.8 (C-b), 152.7 (C-50),
153.6 (C-20), 170.9 (CONH–), 192.0 (C@O). EIMS (70 eV) m/z (%
rel. int.): 339 (100). Anal. Calcd for C20H21NO4: C, 70.48; H, 6.24;
N, 4.13. Found: C, 70.10; H, 6.45; N, 4.16.
5.4.9. 4-(2-Hydroxyethyl)methylcarbamoyl-20,50-dimethoxy-
chalcone (18)
A
mixture of
3
(100 mg, 0.32 mmol), HOBt (86.5 mg,
0.64 mmol), EDC (122.7 mg, 0.64 mmol) was dissolved with
CH2Cl2, and stirred for 10 min. The mixture was added N-methy-
lethanolamine (1.0 mmol) and treated as procedure B to afford
5.4.6. 4-Cyclohexylcarbamoyl-20,50-dimethoxychalcone (15)
A
mixture of
3
(100 mg, 0.32 mmol), HOBt (86.5 mg,
18 (93.6 mg, 79.1%) as a yellow oil, IR (KBr): 3403, 1610 cmꢁ1 1H
.
0.64 mmol), EDC (122.7 mg, 0.64 mmol) was dissolved with
CH2Cl2, and stirred for 10 min. The mixture was added cyclohexyl-
amine (1.0 mmol) and treated as procedure B to afford 15 (84.0 mg,
NMR (CDCl3): d 2.45 (OH), 3.05 (3H, s, NCH3), 3.10 (1H, m,
NCHH–), 3.42 (1H, m, NCHH–), 3.71 (2H, m, CH2OH), 3.80 (3H, s,
OCH3), 3.85 (3H, s, OCH3), 6.93 (1H, d, J = 9.2 Hz, H-30), 7.03 (1H,
dd, J = 8.8, 3.2 Hz, H-40), 7.19 (1H, d, J = 3.2 Hz, H-60), 7.45 (1H, d,
66.7%) as a yellow solid, IR (KBr): 3312, 1637, 1540 cmꢁ1 1H NMR
.
(CDCl3): d 1.24 (2H, m, –CH2–), 1.44 (2H, m, –CH2–), 1.66 (2H, m,
–CH2–), 1.76 (2H, m, –CH2–), 2.04 (2H, m, –CH2–), 3.81 (3H, s, OCH3),
J = 16.0 Hz, H-a), 7.46 (2H, d, J = 8.0 Hz, H-2 and 6), 7.61 (2H, d,
J = 9.2 Hz, H-3 and 5), 7.62 (1H, d, J = 15.6 Hz, H-b). 13C NMR
(CDCl3): d 38.6 (NCH3), 51.0 (NCH2–), 55.8 (OCH3), 56.4 (OCH3),
60.6 (–CH2OH), 113.3 (C-60), 114.4 (C-30), 119.5 (C-10 and 40),
NHCH
3.94 (3H, s, OCH3), 3.98 (1H, m,
), 6.00 (1H, d, J = 7.6 Hz,
CONH–), 6.95 (1H, d, J = 8.8 Hz, H-30), 7.05 (1H, dd, J = 8.8, 3.2 Hz,
H-40), 7.21 (1H, d, J = 3.2 Hz, H-60), 7.48 (1H, d, J = 16.0 Hz, H-
a
),
7.63 (2H, d, J = 7.6 Hz, H-2 and 6), 7.64 (1H, d, J = 16.4 Hz, H-b),
119.5 (C-a), 127.8 (C-2 and 6), 128.3 (C-3 and 5), 136.7 (C-4),
7.77 (2H, d, J = 8.4 Hz, H-3 and 5). 13C NMR (CDCl3): d 24.9
137.0 (C-1), 141.6 (C-b), 152.7 (C-50), 153.6 (C-20), 172.5 (–CON–),
192.0 (C@O). EIMS (70 eV) m/z (% rel. int.): 369 (31), 295 (100).
Anal. Calcd for C21H23NO5: C, 68.28; H, 6.28; N, 3.79. Found: C,
66.78; H, 6.52; N, 3.45.
NHCH
(–CH2– ꢂ 2), 25.5 (–CH2– ꢂ 1), 33.2 (–CH2– ꢂ 2), 48.8 (
),
55.9 (OCH3), 56.5 (OCH3), 113.4 (C-60), 114.4 (C-30), 119.6 (C-10
and 40), 119.6 (C-
a
), 127.4 (C-2 and 6), 128.4 (C-3 and 5), 136.2
(C-4), 138.0 (C-1), 141.5 (C-b), 152.7 (C-50), 153.7 (C-20), 165.8
(CONH–), 192.0 (C@O). EIMS (70 eV) m/z (% rel. int.): 393 (100).
Anal. Calcd for C24H27NO4: C, 73.26; H, 6.92; N, 3.56. Found: C,
73.31; H, 7.03; N, 3.55.
5.4.10. N-Cyclopropyl-4-[3-cyclopropylimino-3-(20,50-dimeth-
oxy-phenyl)-propenyl]-benzamide (19)
A
mixture of
3
(100 mg, 0.32 mmol), HOBt (86.5 mg,
0.64 mmol), EDC (122.7 mg, 0.64 mmol) was dissolved with
CH2Cl2, and stirred for 10 min. The mixture was added cyclopro-
pylamine (1.0 mmol) and treated as procedure B to afford 19
5.4.7. 4-Diethylcarbamoyl-20,50-dimethoxychalcone (16)
A
mixture of
3
(100 mg, 0.32 mmol), HOBt (86.5 mg,
0.64 mmol), EDC (122.7 mg, 0.64 mmol) was dissolved with
CH2Cl2, and stirred for 10 min. The mixture was added diethyl-
(95.3 mg, 76.2%) as
a yellow solid, IR (KBr): 3286, 1640,
1536 cmꢁ1 1H NMR (CDCl3): d 0.60 (2H, m, –CH2CH2–), 0.84 (4H,
.
amine (1.0 mmol) and treated as procedure
B
to afford 16
m, –CH2CH2–), 0.96 (2H, m, –CH2CH2–), 2.67 (1H, m, NHCH–),
2.87 (1H, m, @NHCH–), 6.28 (1H, br s, CONH–), 6.42 (1H, d,
(115.5 mg, 98.2%) as a yellow oil, IR (KBr): 3568, 1627 cmꢁ1
.
1H
J = 16.0 Hz, H-a
), 6.70 (1H, d, J = 2.4 Hz, H-60), 6.95 (2H, m, H-30
NMR (CDCl3): d 1.12 (3H, m, CH3), 1.24 (3H, m, CH3), 3.25 (2H,
m, NCH2–), 3.54 (2H, m, NCH2–), 3.80 (3H, s, OCH3), 3.86 (3H, s,
OCH3), 6.94 (1H, d, J = 8.8 Hz, H-30), 7.03 (1H, dd, J = 9.2, 3.2 Hz,
H-40), 7.19 (1H, d, J = 2.8 Hz, H-60), 7.39 (2H, d, J = 8.4 Hz, H-2 and
and 40), 7.12 (1H, d, J = 16.4 Hz, H-b), 7.42 (2H, d, J = 8.4 Hz, H-2
and 6), 7.66 (2H, d, J = 8.4 Hz, H-3 and H-5).13C NMR (CDCl3): d
6.8 (–(–CH2– ꢂ 2), 9.9 (–CH2–), 10.0 (–CH2–), 23.1 (–NHCH–),
36.2 (@NCH–), 55.8 (OCH3), 56.4 (OCH3), 112.6 (C-60), 114.5 (C-
6), 7.44 (1H, d, J = 15.6 Hz, H-a), 7.60 (2H, d, J = 8.0 Hz, H-3 and
H-5), 7.63 (1H, d, J = 15.6 Hz, H-b). 13C NMR (CDCl3): d 12.9
30), 115.0 (C-10 and 40), 125.7 (C-
a), 127.1 (C-2), 133.5 (C-5 and
C-6), 133.7 (C-3), 135.6 (C-1), 139.6 (C-b), 150.5 (C-50), 153.6 (C-
20), 164.6 (C=N–), 168.3 (CONH–). EIMS (70 eV) m/z (% rel. int.):
(CH3), 14.2 (CH3), 39.3 (NCH2–), 43.2 (NCH2–), 55.8 (OCH3), 56.4
(OCH3), 113.3 (C-60), 114.4 (C-30), 119.4 (C-10 and 40), 119.4 (C-
a),

H.-Y. Tu et al. / Bioorg. Med. Chem. 18 (2010) 2089–2098
2097
390 (31). Anal. Calcd for C24H26N2O3ꢀ1/4H2O: C, 72.98; H, 6.76; N,
FACScan flow cytometer and Cell Quest software (Becton
Dickinson).
7.09. Found: C, 73.01; H, 6.69; N, 7.10.
5.5. 3-(3-Thiophene)carboxyl-1-(2,5-dimethoxyphenyl)prop-2-
en-1-one (21)
5.8. Immuno-fluorescence staining
NTUB1 cells were seeded onto serum-coated coverslides. The
cells were treated as indicated conditions and processed as
described previously.17 Briefly, The cells were fixed with 2%
formaldehyde/PBS for 20 min followed by cold methanol
permeabilization for 3 min. The cells were washed with 1ꢂ PBS
five minutes for two times. The cells were then incubated with
2,5-Dimethoxyacetophenone (450.0 mg, 2.5 mmol) and 5-for-
myl-2-thiophenecarboxylic acid (20) (390.4 mg, 2.5 mmol) were
dissolved in MeOH (50 mL), and added 8% KOH in H2O (50 mL).
The reaction mixture was stirred at room temperature for 72 h
and neutralized with 10% HCl solution (100 mL) to form yellow
precipitate. The yellow precipitate was filtered and washed with
appropriate amount of water. The crude product was purified by
chromatography using EtOAc/n-hexane (2:1), and crystallized with
EtOAc to afford 21 (469.6 mg, 59.1%) as a yellow solid. 1H NMR
(CD3OD): d 3.80 (3H, s, OCH3), 3.90 (3H, s, OCH3), 7.12 (1H, m, H-
30), 7.14 (1H, m, H-40), 7.15 (1H, m, H-60), 7.40 (1H, d, J = 15.6 Hz,
a
-tubulin (Oncogene, CP06) primary antibody overnight. The cells
were washed with 1ꢂ PBS five minutes for three times. Goat-
anti-rabbit-rhodamine secondary antibody and
g/mL 40-6-
1
l
diamidino-2-phenylindole (DAPI) were incubated for 1 h at room
temperature. The slides were washed with 1ꢂ PBS, mounted with
mounting solution (80% glycerol/PBS) and photographed by
microscopy.
H-
a), 7.42 (1H, d, J = 3.2 Hz, H-5), 7.70 (1H, d, J = 15.6 Hz, H-b),
7.71 (1H, d, J = 3.2 Hz, H-4). 13C NMR (CD3OD): d 56.2 (OCH3),
56.9 (OCH3), 114.8 (C-60), 115.5 (C-30), 120.7 (C-40), 129.1 (C-
a),
5.9. Western blot analysis for a-tubulin
132.8 (C-5), 135.1 (C-4), 135.8 (C-b), 147.5 (C-1), 154.3 (C-50),
155.2 (C-20), 193.2 (C@O). ESIMS: m/z 319 [M+H]+. Anal. Calcd for
C16H14O5S: C, 60.37; H, 4.43; S, 10.07. Found: C, 60.09; H, 4.42; S,
10.27.
NTUB1 cells were seeded at a density of 1 ꢂ 106/100-mm2
dishes and treated with indicated concentrations of test agents
for 6 h. The cells were then washed with cold PBS and lysed by
adding lysis buffer (20 mM Tris–HCl (pH 6.8), 1 mM MgCl2, 2 mM
EGTA, 20 g/mL aprotinin, 20 g/mL leupeptin, 1 mM phenylmethyl-
sulfonyl fluoride, 1 mM orthovanadate, and 0.5% Nonidet P-40).
The cell lysates were centrifuged at 15,000g for 10 min at 4 °C. Sol-
uble and assembled tubulin could be separated in supernatant and
pellet fraction, respectively.19 The supernatants and pellets were
dissolved in an SDS–PAGE sampling loading buffer and heated at
95 °C for 10 min; the resulting material was subjected to electro-
phoresis on 10% SDS–polyacrylamide gels. After electrophoresis,
the proteins were transferred to a nitrocellulose membrane, and
the membrane was blocked with 5% skim milk/TBST overnight at
4 °C. The relative amounts of tubulins were detected by anti-tubu-
lin monoclonal antibody (Oncogene) and horseradish peroxidase-
conjugated secondary antibody (Jackson). b-Actin (AC-15, Novus
Biologicals) was used for control. Detection of immunoreactive
signal was accomplished with Western blot chemiluminescent
reagent (Millipore).
5.6. Cell culture and MTT assay for cell viability/proliferation
NTUB1, an immortalized human urothelial carcinoma cell line,
was established from a high-grade bladder cancer.15 PC3 (a human
prostate cancer cell line), A549 (a human lung adenocarcinoma
epithelial cell line), and SV-HUC1 (a SV-40 immortalized human
uroepithelial cell line) were obtained from ATCC. The cells were
maintained in RPMI 1640 (for NTUB1, PC3, and A549 cells) or
F12 medium (for SV-HUC1) supplemented with 10% fetal bovine
serum (FBS), 100 unit/mL penicillin-G, 100
lg/mL streptomycin,
and 2 mM -glutamine. The cells were cultured at 37 °C in a humid-
L
ified atmosphere containing 5% CO2.
For evaluating the cytotoxic effect of tested compounds and cis-
platin, a modified 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltet-
razolium bromide (MTT, Sigma Chemical Co.) assay was
performed.16,17 Briefly, the cells were plated at a density of 1800
cells/well in 96-well plates and incubated at 37 °C overnight before
drug exposure. Cells were then cultured in the presence of graded
concentrations of tested compounds at 37 °C for 72 h. At the end of
5.10. Statistical analysis
the culture period, 50
each well and allowed to react for 3 h. Following centrifugation of
plates at 1000g for 10 min, media were removed and 150 L DMSO
were added to each well. The proportions of surviving cells were
determined by absorbance spectrometry at 540 nm using MRX
(DYNEXCO) microplate reader. The cell viability was expressed as
a percentage to the viable cells of control culture condition. The
IC50 value of each group were calculated by the median-effect anal-
ysis and presented as mean standard deviation (SD).
lL of MTT (2 lg mg/mL in PBS) was added to
Data were expressed as mean SD. Statistical analysis were
performed using the Bonferroni t-test method after ANOVA for
multigroup comparison and student’s t-test method for two group
comparison, with P <0.05 was considered to be statistically
significant.
l
Acknowledgment
This work was partially supported by a research grant from the
National Science Council of the Republic of China (NSC97-2320-B-
037-014 and NSC97-2320-B-037-018-MY3).
5.7. Cell cycle analysis
DNA content was determined following propidium iodide (PI)
staining of cells as previously described.17,18 Briefly, 6 ꢂ 105 cells
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