Bis-chalcone analogues as potent NO production inhibitors and as cytotoxic agents

Article abstract of DOI:10.1016/j.ejmech.2011.10.026

Chalcones have a distinctive 1,3-diarylpropenone skeleton and exert numerous biological effects. Using a one-step Claisen-Schmidt condensation, we synthesized eleven bis-chalcones (3-13) and three acetyl chalcones (14-16) from substituted aldehydes and diacetylresorcinol. The compounds were tested for in vitro cytotoxic activity against four human cancer cell lines (A549, DU145, KB, and KB-VIN) and inhibition of NO production in lipopolysaccharide (LPS)-activated microglial cells. Among them, four compounds (3, 5, 6, and 13) showed significant cytotoxic activity with EC₅₀ values ranging from 1.57 to 5.14 μM, and seven compounds (3, 5-8, 10, and 13) displayed potent anti-inflammatory activity by inhibiting NO production with IC₅₀ values ranging from 0.95 to 8.65 μM. A mechanism of action study of active compounds 6 and 7 discovered that these compounds down-regulated iNOS expression by inhibiting p65 NF-κB activation/nuclear translocation due to prevention of IκBα degradation. Structure-activity relationship (SAR) findings are also discussed.

Full text of DOI:10.1016/j.ejmech.2011.10.026

European Journal of Medicinal Chemistry 47 (2012) 97e103
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Bis-chalcone analogues as potent NO production inhibitors and as cytotoxic
agents
,
,
M. Vijaya Bhaskar Reddy a 1, Yuh-Chiang Shen b 1, Emika Ohkoshi c, Kenneth F. Bastow d, Keduo Qian c,
Kuo-Hsiung Lee c e, Tian-Shung Wu a
,
,e,f,
*
a Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan, ROC
b National Research Institute of Chinese Medicine, Taipei 112, Taiwan, ROC
c Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
d Division of Medicinal Chemistry and Natural Products, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
e Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan, ROC
f College of Pharmacy, China Medical University, Taichung, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
Chalcones have a distinctive 1,3-diarylpropenone skeleton and exert numerous biological effects. Using
a one-step ClaiseneSchmidt condensation, we synthesized eleven bis-chalcones (3e13) and three acetyl
chalcones (14e16) from substituted aldehydes and diacetylresorcinol. The compounds were tested for
in vitro cytotoxic activity against four human cancer cell lines (A549, DU145, KB, and KB-VIN) and
inhibition of NO production in lipopolysaccharide (LPS)-activated microglial cells. Among them, four
compounds (3, 5, 6, and 13) showed significant cytotoxic activity with EC50 values ranging from 1.57 to
Received 2 June 2011
Received in revised form
11 October 2011
Accepted 12 October 2011
Available online 20 October 2011
5.14
inhibiting NO production with IC50 values ranging from 0.95 to 8.65
active compounds 6 and 7 discovered that these compounds down-regulated iNOS expression by
inhibiting p65 NF- B activation/nuclear translocation due to prevention of I degradation. Structure-
activity relationship (SAR) findings are also discussed.
m
M, and seven compounds (3, 5e8, 10, and 13) displayed potent anti-inflammatory activity by
Keywords:
Bis-chalcones
Cytotoxicity
NO production inhibition
Structure activity relationships
m
M. A mechanism of action study of
k
kBa
Crown Copyright Ó 2011 Published by Elsevier Masson SAS. All rights reserved.
1. Introduction
angiogenesis, and metastasis are regulated by these two pathways
[5]. Thus, the molecular and cellular pathways that connect
inflammation and cancer have emerged as attractive targets for
cancer prevention and therapy.
Despite recent advances in our understanding of the biological
processes leading to cancer development and the consequent
improvement in drug development, new and more effective agents
are still needed to help bring these diseases under control. Chronic
inflammation and inflammatory mediators play critical roles in the
carcinogenesis of cancers [1,2]. The interactions of tumor cells with
their microenvironment and inflammation within the tumor
environment are correlated with tumor growth and metastasis and
poor prognosis in many cancer types [3,4]. Two major pathways for
inflammation, the transcription factors nuclear factor kappa B (NF-
Chalcones, with a 1,3-diarylpropenone skeleton, constitute an
important group of natural products as they possess a wide range of
biological activities [6], including anti-inflammatory [7,8], antimi-
crobial (antibacterial, antifungal) [8-10], antiparasitic (antimalarial,
antileishmanial) [8,11,12], antituberculosis [13], antioxidant [14,15],
antimitotic [16], anti-invasive [17], anticancer [18-21], as well as
modulation of P-glycoprotein-mediated multidrug resistance [22].
In a continued study of this important biologically active compound
class, we report herein the synthesis and in vitro biological evalu-
ation of nine new (5, 6, 8, 9, and 11e15) and five known (3, 4, 7, 10,
[23] and 16 [24]) bis-chalcones as NO production inhibitors and as
cytotoxic agents. All 14 synthesized analogues were tested for
cytotoxic activity against A549 (lung cancer), DU-145 (prostate
cancer), KB (nasopharyngeal carcinoma), and KB-VIN (vincristine-
resistant KB subline) cancer cell lines. In addition, representative
biomarkers involved in the inflammatory or metastasis pathways
k
B) and signal transducer and activator of transcription 3 (STAT3),
are constitutively active in most cancers; and most gene products
linked to inflammation, survival, proliferation, invasion,
* Corresponding author. Department of Chemistry, National Cheng Kung
University, Tainan, Taiwan, ROC. Tel.: þ886 6 2757575x65333; fax: þ886 2 2740552.
E-mail address: tswu@mail.ncku.edu.tw (T.-S. Wu).
1
These authors made equal contributions.
0223-5234/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.026

98
M. Vijaya Bhaskar Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 97e103
2. Chemistry
O
HO
O
OH
O
HO
OH
a
HO
OH
In a previously reported synthetic method, 2,4-diacetylphenol
was prepared by acetylation of 4-hydroxyacetophenone followed
by Fries rearrangement of the resulting 4-acetylphenyl acetate with
aluminum chloride [25]. A similar synthetic approach [26] was
applied to the reaction of resorcinol (1) with acetic anhydride in the
presence of zinc chloride under acidic (HCl) conditions (140 ꢀC,
24 h) to afford a mixture of diacetylresorcinols 2a and 2b in 72% and
9% yields, respectively (Scheme-1). Bis-chalcones 3e13 were
prepared by reaction of 2a with two equivalents of different
substituted aldehydes in a 50% KOH solution. Reaction with one
equivalent of aldehyde gave the mono-addition products 14e16
(Scheme 2).
+
O
1
2a
2b
Regents and conditions: (a) Ac2O, ZnCl2, reflux at 140 0C, 24 h
Scheme 1. Synthesis of precursor 2a.
[e.g., NF-
k
B, nitric oxide (NO), etc.] were characterized after treat-
The structures of all compounds were confirmed by IR, 1H NMR,
ment with the newly synthesized bis-chalcone analogues. Specifi-
cally, we evaluated the compounds as inhibitors of nitric oxide (NO)
production in lipopolysaccharide (LPS)-activated microglial cells.
13C NMR, EIMS, HREIMS and elemental analysis. A sharp two proton
downfield signal was observed at
d
13.62 in the 1H NMR spectrum
R4
B
R4
B
R3
R2
HO
O
OH
O
R3
R2
A
R1
R1
3. R1 = R2 = R3 = R4 = H
4. R1 = R2 = R4 = H, R3 = OMe
5. R1 = R3 = R4 = H, R2 = OMe
6. R1 = R2 = OMe, R3 = R4 = H
7. R1 = R4 = H, R2 = R3 = OMe
8. R1 = R2 = R3 = OMe, R4 = H
9. R1 = R4 = OMe, R2 = R3 = H
two moles of aldehyde
50% KOH, r.t, MeOH
2a (for 3-13)
R1
R2
HO
OH
X
A
B
B
R1
X
R2
O
O
10. X = O, R1 = R2 = H
11. X = O, R1 = Me, R2 = H
12. X = S, R1 = H, R2 = Me
HO
OH
A
B
B
N
N
O
O
13
HO
OH
O
O
O
O
14
OMe
OMe
HO
OH
OMe
one mole of aldehyde
50% KOH, r.t, MeOH
2a (for 14-16)
O
O
15
HO
OH
O
O
16
Scheme 2. Synthesis of target compounds 3e9, 10e12 and 14e16.

M. Vijaya Bhaskar Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 97e103
99
Table 1
Compound 3, which has two un-substituted phenyl B-rings,
showed the highest potency against DU145, KB, and KB-VIN tumor
cell lines. It was slightly less potent against A549 cells (Table 1). Bis-
chalcone 5, which has 3-methoxyphenyl B-rings, also showed
significant cytotoxic activity. In contrast, bis-chalcone 4, which has
4-methoxyphenyl B-rings, was inactive against all cancer cell lines.
Thus, the position of the methoxy group on the B-ring had great
importance; position 3 was favourable, while position 4 was
unfavourable for cytotoxic activity.
Three of the new bis-chalcone analogues have dimethoxy
substituted phenyl B-rings, 6 (2,3-dimethoxy), 7 (3,4-dimethoxy),
and 9 (2,5-dimethoxy). Compound 6 was significantly active
against DU145 and KB cell lines, while 7 showed only weak activity,
and 9 was inactive. The 2,3,4-trimethoxy bis-chalcone 8 was also
inactive. These findings demonstrate that the positions and
numbers of the methoxy substituents affect cytotoxic activity.
When the B-rings were changed from phenyl to heteroaromatic
rings, only analogue 13, with pyridine B-rings, showed significant
potency against DU145 and KB cancer cell lines, although it was less
potent than 3. Compounds 10e12, which have five-membered
pyrrole or thiophene B-rings, did not show appreciable cytotoxic
activity. Finally, compounds 14e16, which have only one arylpro-
penone group, were essentially inactive in the cytotoxicity assays.
Regarding inhibition of NO production, bis-chalcone 3 was more
potent than the positive control L-NAME. The 3-methoxyphenyl
analogue 5 also showed significant inhibition of NO production,
while the 4-methoxy analogue 4 exhibited only weak inhibition.
These results paralleled those found in the cytotoxicity assay.
The most potent inhibitors of NO production were the 2,3-
dimethoxyphenyl analogue 6 and 3,4-dimethoxyphenyl analogue 7.
These two compounds were more potent than the corresponding
Cytotoxic Activity Data for Compounds 3e16.
Compound
EC50 (mM)
A549
DU145
KB
KB-VIN
3
4
5
6
7
9
13
14
4.08
NA
8.76
4.38
9.14
NA
1.70
NA
1.86
2.45
8.63
NA
1.62
NA
1.57
33.97
2.07
5.14
7.02
NA
2.88
2.06
8.18
40.08
2.93
17.18
13.66
19.38
2.85
16.32
11.04
17.21
NA: no activity (compounds did not reach 50% inhibition at 20
Compounds not listed in the table were not active against any cell line.
mg/mL).
of 3, suggesting the presence of two chelated hydroxy groups. This
assignment was further supported by carbon resonances at 191.9
in the 13C NMR spectrum of 3. In addition, two sharp aromatic
proton signals were observed with 3 at 8.53 and 6.53, and two
sets of protons with a trans coupling constant by (J ¼ 15.3 Hz) were
present at 7.97 and 7.57.
d
d
d
d
d
3. Results and discussion
All synthesized compounds were tested for in vitro cytotoxic
activity against four human cancer cell lines, (A549, DU145, KB, and
KB-VIN), and the results are shown in Table 1. In addition, we also
evaluated the compounds for inhibition of NOX-dependent ROS
production and NOS dependent inhibition of NO production in
microglial cells, as well as for 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical scavenging capacity. None of the compounds showed
significant inhibition of NOX-dependent ROS production or direct
mono-methoxy substituted analogues
4 and 5, and control
compound L-NAME. Although bis-chalcone 7 was less potent
than 6 in the cytotoxicity assay, it was more potent in the NO
production inhibition assay. Three additional compounds (8, 10, 13)
showed greater potency than L-NAME, while 14 showed
slightly lower potency. In mechanistic studies, compounds 6 and 7
radical scavenging in
compounds 3e14 did inhibit NO production in microglial cells with
IC50 values ranging from 0.95 to 43.27 M, compared with an IC50
value of 13.35 M for -nitro-arginine methyl ester (L-NAME),
a cell-free DPPH solution. However,
m
m
L
a non-specific NOS inhibitor (Table 2).
Table 2
Summary of the effects of compounds 3e16 on iNOSa and NOXb activity in murine
microglial cells.
Compound
IC50
(
m
M) iNOSa
IC50 (m
M) NOXa
3
4
5
6
7
8
9
5.44 ꢁ 0.17
25.69 ꢁ 5.18
8.09 ꢁ 0.72
1.82 ꢁ 0.01
0.95 ꢁ 0.01
7.04 ꢁ 0.16
23.29 ꢁ 3.4
4.00 ꢁ 0.43
36.31 ꢁ 3.36
43.27 ꢁ 2.18
8.65 ꢁ 0.40
17.74 ꢁ 2.54
NA
121.84 ꢁ 11.36
149.59 ꢁ 11.76
127.05 ꢁ 13.19
89.02 ꢁ 7.92
49.08 ꢁ 0.97
70.92 ꢁ 3.18
94.97 ꢁ 12.86
49.61 ꢁ 1.74
60.42 ꢁ 2.60
81.41 ꢁ 7.70
76.80 ꢁ 0.36
72.05 ꢁ 3.91
72.97 ꢁ 15.56
120.06 ꢁ 1.14
NT
10
11
12
13
14
15
16
L-NAME
DPI
NA
13.35 ꢁ 0.81
NT
0.65 ꢁ 0.01
NT: Not tested; NA: Not active.
a
iNOS activity was measured by NO production in the presence of 1e50
m
M of
Fig. 1. Effects of compounds 6 and 7 on the lipopolysaccharide (LPS)-induced iNOS
test drug. L-NAME (N-nitro-l-arginine methyl ester, a non-specific NOS inhibitor)
expression, degradation of cytosolic I
murine microglial BV2 cells. Upper panel, representative immunoblots of LPS-induced
iNOS up-expression; lower panel, degradation of cytosolic I [IkB (c)] and the time-
dependent translocation of p65NF- B from the cytosol [p65(c)] to the nucleus [p65(n)]
in microglial cells pre-treated with solvent only (LPS) or compound 6 (cpd 6) (2, 4 M)
and cpd7 (1, 2 M) followed by stimulation with LPS (0.5 g/mL) for different times
(from 15 to 120 min). Cells in control group (control) received solvent only (drug free).
The -actin signal was used as a reference for normalization. At least three indepen-
dent experiments per compound were examined.
kBa, and nuclear translocation of p65NF-kB in
was included as a positive control.
b
NOX activity was measured by ROS production (chemiluminescence), and DPI (a
k
B
a
a
NOX inhibitor) was included as a positive control. Data were calculated as 50%
inhibitory concentration (IC50) and expressed as mean ꢁ SEM from three experi-
ments performed on different days using microglial cells from different passages.
Compounds were also tested in DPPH assay, but none of the compounds showed
significant activity. For the DPPH assay, Trolox (an antioxidant) was the positive
k
m
m
m
b
control (IC50 30.24 ꢁ 3.2
mM).

100
M. Vijaya Bhaskar Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 97e103
down-regulated iNOS expression by inhibiting p65NF-
k
B activa-
chromatography on silica gel, eluting with 50-50% EtOAc in
hexanes, to afford bis-chalcones 3e13. Reaction of 2a with one
equivalent of aldehyde under the above conditions gave
compounds 14e16.
tion/nuclear translocation due to prevention of I degradation
kBa
(Fig. 1). These results indicate that dimethoxy substitution on the
phenyl B-rings leads to the highest potency for the inhibition of NO
production of these compounds, most likely due to inhibition of
Ik
Ba
-dependent NF-
k
B activation.
5.3.1. (2E, 20E)-1,10-[4,6-Dihydroxy-1,3-phenylene]bis(3-
phenylprop-2-en-1-one) (3)
4. Conclusions
Pale yellow solid (1.08 g), 58% yield, mp 190e192 ꢀC. IR (neat)
nmax 2916, 1630, 1553, 1485, 1345, 1300, 1279, 1245, 1193, 1031, 973,
In this paper we described the synthesis and screening of bis-
chalcones against four human cancer cell lines (A549, DU-145,
KB, and KB-VIN) and for inhibition of NO production in LPS-
activated microglial cells. The results showed that compounds
with un-substituted (3) and 3-methoxy substituted (5) phenyl rings
generally exhibited the most potent cytotoxicity, and compounds 3,
5-8, 10, and 13 were more potent inhibitors of cellular NO
production in LPS-activated microglial cells than the control drug L-
NAME, a non-specific NOS inhibitor. Active compounds 6 and 7
exerted their potent anti-inflammatory effects by inhibiting
841, 760, 732, 711 .1H NMR (300 MHz, CDCl3)
d 13.62 (2H, s, OH),
8.53 (1H, s), 7.97 (2H, d, J ¼ 15.3 Hz), 7.69 (4H, m), 7.57 (2H, d,
J ¼ 15.3 Hz), 7.48 (6H, m), 6.53 (1H, s).13C NMR (75 MHz, CDCl3):
d
191.9, 169.9, 146.0, 134.3, 134.0, 131.1, 129.1, 128.7, 119.3, 113.8,
105.4.EIMS m/z 370 [M]þ (5), 282 (100), 281 (78), 205 (65), 196 (16),
163 (63), 129 (17), 103 (20), 77 (23).HREIMS m/z 370.1205 (calcd for
C24H18O4, 370.1205). Elemental analysis: Found C 76.68%, H 4.80%;
Calcd for C 77.82%, H 4.90%.
5.3.2. (2E, 20E)-1,10-[4,6-Dihydroxy-1,3-phenylene]bis(3-(4-
methoxyphenyl)prop-2-en-1-one) (4)
p65NFkB-dependent iNOS expression, and thus, reducing NO
production. Based on the above mentioned results, these active bis-
chalcones may be useful for further development as anticancer and
anti-inflammatory agents.
Bright yellow solid (1.1 g), 53% yield, mp 187e189 ꢀC. IR (neat)
nmax 2934, 2838, 1627, 1569, 1509, 1422, 1356, 1308, 1286, 1198,
1166, 1031, 974, 844, 704 .1H NMR (300 MHz, CDCl3)
d 13.73 (2H, s),
8.48 (1H, s), 7.93 (2H, d, J ¼ 15.3 Hz), 7.63 (4H, d, J ¼ 9.0 Hz), 7.43
5. Experimental section
(2H, d, J ¼ 15.3 Hz), 6.96 (4H, d, J ¼ 9.0 Hz), 6.49 (1H, s), 3.88 (6H,
s).13C NMR (75 MHz, CDCl3)
d 191.9, 169.8, 162.2, 145.8, 133.6, 130.6,
5.1. General procedure
127.1, 116.8, 114.6, 113.8, 105.2, 55.4.EIMS m/z: 430 [M]þ (81), 429
(29), 325 (16), 163 (20), 134 (100), 121 (65), 119 (17), 91 (14).HREIMS
m/z 430.1413 (calcd for C26H22O6, 430.1416). Elemental analysis:
Found C 71.68%, H 5.10%; calcd for C 72.55%, H 5.15%.
Melting points were determined using a Yanagimoto MP-S3
micro-melting point apparatus and are uncorrected. IR spectra
were determined on a Shimadzu FT-IR Prestige 21 spectropho-
tometer. 1H and 13C NMR spectra were recorded on a Bruker Avance
300 spectrometer, using tetramethylsilane (TMS) as internal stan-
5.3.3. (2E, 20E)-1,10-[4,6-Dihydroxy-1,3-phenylene]bis(3-(3-
methoxyphenyl)prop-2-en-1-one) (5)
dard; all chemical shifts are reported in parts per million (ppm,
d
).
Bright yellow solid (1.34 g), 62% yield, mp 144e146 ꢀC. IR (neat)
nmax 2917, 2835,1632,1565,1483,1432,1345,1316,1266, 1192,1049,
EIMS and HREIMS spectra were obtained on a VG-70-250S mass
spectrometer. Elemental analyses were determined by Elementer
Vario EL III and reported as combustion values for C, H, N, and S.
Column chromatography was performed on silica gel (70e230
mesh, 230e400 mesh). TLC was conducted on precoated Kieselgel
60 F254 plates (Merck), and the spots were detected by examina-
tion under a UV lamp.
1031, 841, 775, 729 .1H NMR (300 MHz, CDCl3)
d 13.56 (2H, s, OH),
8.48 (1H, s), 7.88 (2H, d, J ¼ 15.3 Hz), 7.52 (2H, d, J ¼ 15.3 Hz), 7.35
(2H, dd, J ¼ 9.0, 9.0 Hz), 7.24 (2H, d, J ¼ 8.4 Hz), 7.16 (2H, d,
J ¼ 1.8 Hz), 7.00 (2H, dd, J ¼ 1.8, 7.8 Hz), 6.49 (1H, s), 3.85 (6H, s).13C
NMR (75 MHz, CDCl3)
d 191.8, 169.9, 160.0, 145.7, 135.7, 134.1, 130.1,
121.3, 119.5, 116.8, 113.7, 113.5, 105.3, 55.2.EIMS m/z 430 [M]þ (100),
429 (47), 399 (11), 323 (51), 312 (43), 295 (25), 281 (15), 205 (30),
163 (40).HREIMS m/z 430.1415 (calcd for C26H22O6, 430.1416).
Elemental analysis: Found C 72.12%, H 5.12%; calcd for C 72.55%, H
5.15%.
5.2. Synthesis of diacetylresorcinol (2a)
A mixture containing resorcinol 1 (22 g, 200 mmol), acetic
anhydride (20.4 mL, 200 mmol), zinc chloride (13.6 g, 100 mmol)
and a drop of concentrated hydrochloric acid was heated to reflux
at 140 ꢀC for 24 h. Additional excess acetic anhydride (5.1 mL,
50 mmol) was added during the reaction. The progress of the
reaction was monitored by TLC. After the reaction was complete,
the residue was purified by column chromatography (CHCl3:hex-
ane, 1:1) to yield 2a (28.0 g, 72%) and 2b (3.5 g, 9%). 2a: 1H NMR
5.3.4. (2E, 20E)-1,10-[4,6-Dihydroxy-1,3-phenylene]bis(3-(2,3-
dimethoxyphenyl)prop-2-en-1-one) (6)
Yellow solid (1.72 g), 70% yield, mp 168e170 ꢀC. IR (neat) nmax
2938, 2836, 1634, 1566, 1480, 1428, 1342, 1299, 1267, 1229, 1200,
1073, 999, 849, 783, 737, 694 cmꢂ1.1H NMR (300 MHz, CDCl3)
d
13.62 (2H, s), 8.55 (1H, s), 8.18 (2H, d, J ¼ 15.3 Hz), 7.68 (2H, d,
(300 MHz, CDCl3)
d
12.88 (2H, s), 8.17 (1H, s), 6.36 (1H, s), 2.59 (6H,
12.79 (2H, brs), 7.71 (1H, d,
J ¼ 15.3 Hz), 7.26 (2H, dd, J ¼ 9.0 Hz, 2.1 Hz), 7.11 (2H, t, J ¼ 9.0 Hz),
s). 2b: 1H NMR (300 MHz, CDCl3)
d
7.01 (2H, dd, J ¼ 9.0, 2.1 Hz), 6.51 (1H, s), 3.90 (12H, s).13C NMR
J ¼ 8.4 Hz), 6.50 (1H, d, J ¼ 8.4 Hz), 2.57 (6H, s).
(75 MHz, CDCl3) d 192.5, 169.9, 153.2, 149.2, 141.0, 134.3, 128.5,
124.2, 121.0, 120.2, 114.8, 113.9, 105.3, 61.2, 55.9.EIMS m/z 490 [M]þ
(32), 459 (100), 295 (35), 214 (11), 163 (14).HREIMS m/z 490.1631
(calcd for C28H26O8, 490.1628). Elemental analysis: Found C 68.22%,
H 5.30%; calcd for C 68.56%, H 5.34%.
5.3. General procedure for synthesis of Bis-chalcones (3e13)
A solution of 50% KOH (40 mL) was added drop-wise to a well-
stirred mixture of 2a (5.0 mmol) and different substituted alde-
hydes (10.0 mmol) in MeOH at room temperature. After 24e36 h,
the solvent was removed under reduced pressure, and 5% HCl
(50e70 mL) was added to the residue. After extraction with EtOAc,
the organic layer was washed with brine, dried (Na2CO3), and then
concentrated in vacuo. The residue was purified by column
5.3.5. (2E, 20E)-1,10-[4,6-Dihydroxy-1,3-phenylene]bis(3-(3,4-
dimethoxyphenyl)prop-2-en- 1-one) (7)
Bright yellow solid (1.58 g), 65% yield, mp 188e190 ꢀC. IR (neat)
nmax 2937, 2839, 1632, 1557, 1510, 1463, 1422, 1338, 1311, 1267, 1235,
1196, 1160, 1139, 1022, 973, 796, 701 .1H NMR (300 MHz, CDCl3)

M. Vijaya Bhaskar Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 97e103
101
d
13.66 (2H, s, OH), 8.51 (1H, s), 7.88 (2H, d, J ¼ 15.3 Hz), 7.42 (2H, d,
1036, 968, 871, 844, 712, 682 .1H NMR (300 MHz, CDCl3)
d
13.71
J ¼ 15.3 Hz), 7.28 (2H, dd, J ¼ 9.0 1.2 Hz), 7.14 (2H, d, J ¼ 1.2 Hz), 6.91
(2H, s), 8.45 (1H, s), 8.14 (2H, d, J ¼ 15.0 Hz), 7.39 (2H, d, J ¼ 5.1 Hz),
(2H, d, J ¼ 9.0 Hz), 6.56 (1H, s), 3.95 (12H, s).13C NMR (75 MHz,
7.28 (2H, d, J ¼ 15.0 Hz), 6.95 (2H, d, J ¼ 5.1 Hz), 6.48 (1H, s), 2.44
CDCl3)
d
191.8, 169.8, 152.0, 149.3, 145.8, 133.8, 127.4, 123.0, 117.3,
(6H, s).13C NMR (75 MHz, CDCl3)
d 191.2, 169.9, 143.8, 136.5, 134.2,
113.7, 111.2, 110.9, 105.2, 56.06, 56.01.EIMS m/z 490 [M]þ (100), 489
(16), 326 (10), 191 (13), 164 (67), 163 (19), 151 (73), 149 (15).HREIMS
m/z 490.1625 (calcd for C28H26O8, 490.1628). Elemental analysis:
Found C 67.94%, H 5.31%; calcd for C 68.56% H 5.34%.
133.6, 131.7, 128.4, 116.9, 113.6, 105.2, 14.4.EIMS m/z 410 [M]þ(100),
395 (15), 374 (16), 286 (17), 271 (25), 268 (54), 260 (21), 163 (48),
151 (32), 124 (72), 111 (53), 95 (30).HREIMS m/z 410.0645 (calcd for
C22H18O4S2, 410.0647). Elemental analysis: Found C 64.10%, H
4.43%, S 15.53%; calcd for C 64.37%, H 4.42%, S 15.62%.
5.3.6. (2E, 20E)-1,10-[4,6-Dihydroxy-1,3-phenylene]bis(3-(2,3,4-
trimethoxyphenyl)prop-2-en-1-one) (8)
5.3.11. (2E, 20E)-1,10-[4,6-Dihydroxy-1,3-phenylene]bis(3-(pyridin-
2-yl)prop-2-en-1-one) (13)
Pale yellow solid (1.4 g), 51% yield, mp 161e163 ꢀC. IR (neat) nmax
2941, 2840,1634,1556,1493,1464,1414,1365,1287,1201,1098,1060,
Pale yellow solid (1.2 g), 64% yield, mp 145e147 ꢀC. IR (neat) nmax
3057, 2952,1649,1574,1468, 1423,1350,1312,1260,1224,1212, 976,
990, 945, 842, 800, 696 .1H NMR (300 MHz, CDCl3)
d 13.77 (2H, s),
8.58(1H, s), 8.06(2H, d, J¼ 15.3 Hz), 7.76 (2H, d, J ¼15.3 Hz), 7.38 (2H,
869, 843, 777, 740, 591 .1H NMR (300 MHz, DMSO-d6)
d 12.96 (2H,
d, J ¼ 9.0 Hz), 6.76 (2H, d, J ¼ 9.0 Hz), 6.51 (1H, s), 3.93e3.97 (18H,
s, OH), 8.71 (2H, d, J ¼ 4.2 Hz), 8.63 (1H, s), 8.27 (2H, d, J ¼ 15.3 Hz),
m).13C NMR (75 MHz, CDCl3)
d 192.5, 169.8, 156.3, 154.1, 142.5, 141.6,
7.92 (4H, m), 7.77 (2H, d, J ¼ 15.3 Hz), 7.45 (2H, m), 6.62 (1H, s).13C
134.0, 125.5, 121.5, 118.8, 113.9, 107.6, 105.1, 61.2, 60.9, 56.1.EIMS m/z
550 [M]þ (11), 519 (100), 325 (32), 179 (8), 163 (13).HREIMS m/z
550.1837 (calcd for C30H30O10, 550.1839). Elemental analysis: Found
C 64.58%, H 5.45%; calcd for, C 65.45%, H 5.49%.
NMR (75 MHz, DMSO-d6) d 191.0, 166.9, 152.5, 150.0, 142.4, 137.3,
135.8, 126.2, 125.4, 124.9, 115.9, 103.8.EIMS m/z 372 [M]þ (100), 354
(24), 327 (15), 294 (27), 239 (30), 154 (11), 132 (31), 106 (22), 104
(30), 78 (22).HREIMS m/z 372.1113 (calcd for C22H16 N2O4, 372.1110).
Elemental analysis: Found C 70.61%, H 4.28%, N 7.45%; calcd for C
70.96%, H 4.33%, N 7.52%.
5.3.7. (2E, 20E)-1,10-[4,6-Dihydroxy-1,3-phenylene]bis(3-(2,5-
dimethoxyphenyl)prop-2-en-1-one) (9)
Yellow solid (1.32 g), 54% yield, mp 205e207 ꢀC. IR (neat) nmax
2936, 2834, 1625, 1543, 1492, 1314, 1244, 1220, 1188, 1047, 1020,
5.3.12. (E)-1-[5-Acetyl-2,4-dihydroxyphenyl]-3-(benzo[d] [1,3]
dioxol-5-yl)prop-2-en-1-one (14)
982, 951, 877, 848, 798 .1H NMR (300 MHz, DMSO-d6)
d
13.81 (2H,
Yellow solid (980 mg), 63% yield,166e168 ꢀC. IR (neat) nmax 3012,
2911,1635,1553,1489,1447,1356,1313,1243,1206,1104,1063,1035,
s, OH), 8.99 (1H, s), 8.21 (2H, d, J ¼ 15.9 Hz), 8.15 (2H, d, J ¼ 15.9 Hz),
7.69 (2H, d, J ¼ 1.8 Hz), 7.09 (4H, m), 6.44 (1H, s), 3.83e3.80 (12H,
929, 753, 662, 588 .1H NMR (300 MHz, CDCl3)
d 13.68 (1H, s, OH),
m).13C NMR (75 MHz, DMSO-d6)
d
192.2, 168.8, 153.2, 152.9, 138.8,
12.95 (1H, s, OH), 8.34 (1H, s), 7.86 (1H, d, J ¼ 15.0 Hz), 7.34 (1H, d,
136.0, 123.2, 121.0, 118.6, 114.1, 113.0, 112.9, 104.0, 56.1, 55.6.EIMS m/
z 490 [M]þ (50), 459 (100), 295 (39), 163 (20).HREIMS m/z 490.1631
(calcd for C28H26O8, 490.1628). Elemental analysis: Found C 68.19%,
H 5.33%; calcd for C 68.56%, H 5.34%.
J ¼ 15.0 Hz), 7.14 (2H, dd, J ¼ 9.0, 3.0 Hz), 6.88 (1H, d, J ¼ 9.0 Hz), 6.46
(1H, s), 6.06 (2H, s), 2.68 (3H, s).13C NMR (75 MHz, CDCl3):
d 202.4,
191.6, 170.0, 168.7, 148.5, 145.8, 134.8, 128.7, 125.9, 116.8, 113.7, 113.5,
108.6, 106.6, 105.1, 101.8, 26.1.EIMS m/z: 326 [M]þ (100), 325 (37),
205 (11), 179 (10), 163 (15), 148 (76), 135 (59), 89 (15).HREIMS m/z
326.0792 (calcd for C18H14O6, 326.0790). Elemental analysis: Found
C 66.65%, H 4.21%; calcd for, C 66.26%, H 4.32%.
5.3.8. (2E, 20E)-1,10-[4,6-Dihydroxy-1,3-phenylene]bis(3-(furan-2-
yl)prop-2-en-1-one) (10)
Orange solid (1.42 g), 81% yield, mp 203e205 ꢀC. IR (neat) nmax
3031,1631,1583,1550,1499,1473,1360,1301,1283,1259,1236,1204,
5.3.13. (E)-1-[5-Acetyl-2,4-dihydroxyphenyl]-3-(2,4,5-
trimethoxyphenyl)prop-2-en-1-one (15)
1190,1015, 978, 924, 882, 758 .1H NMR (300 MHz, DMSO-d6)
d 13.34
(2H, s, OH), 8.67 (1H, s), 7.95 (2H, s), 7.76 (2H, d, J ¼ 15.0 Hz), 7.63
Yellow solid (1.2 g), 65% yield, mp 206e208 ꢀC. IR (neat) nmax
2934, 2836, 1632, 1584, 1548, 1438, 1384, 1353, 1307, 1275, 1213,
(2H, d, J ¼ 15.0 Hz), 7.16 (2H, d, J ¼ 3.3 Hz), 6.71 (2H, t, J ¼ 1.5 Hz),
6.44 (1H, s).13C NMR (75 MHz, DMSO-d6)
d
190.9, 167.6, 151.2, 146.6,
1046, 1029, 998, 833, 760 .1H NMR (300 MHz, CDCl3)
d 13.85 (1H, s,
135.4,130.6,119.0,117.9,115.0,113.3,103.8.EIMS m/z 350 [M]þ (100),
256 (43), 228 (11), 163 (20), 121 (15), 95 (10), 65 (17).HREIMS m/z
350.0790 (calcd for C20H14O6, 350.0790). Elemental analysis: Found
C 68.31%, H 4.00%; calcd for C 68.57%, H 4.03%.
OH),12.89 (1H, s, OH), 8.35 (1H, s), 8.11 (1H, d, J ¼ 15.3 Hz), 7.53 (1H,
d, J ¼ 15.3 Hz), 7.07 (1H, s), 6.52 (1H, s), 6.43 (1H, s), 3.96 (3H, s),
3.94 (3H, s), 3.90 (3H, s), 2.65 (3H, s).13C NMR (75 MHz, CDCl3)
d
202.3, 192.4, 170.1, 168.5, 155.3, 153.2, 143.3, 141.7, 135.0, 117.3,
114.9, 114.0, 113.4, 113.1, 104.9, 96.6, 56.8, 56.2, 56.1, 25.9.EIMS m/z
372 [M]þ (43), 341 (100), 181 (16), 179 (13).HREIMS m/z 372.1212
(calcd for C20H20O7, 372.1209). Elemental analysis: Found C 64.16%,
H 5.39%; calcd for C 64.51%, H 5.41%.
5.3.9. (2E, 20E)-1,10-[4,6-Dihydroxy-1,3-phenylene]bis(3-(5-
methylfuran-2-yl)prop-2-en-1-one) (11)
Yellow solid (1.28 g), 68% yield, mp 185e187 ꢀC. IR (neat) nmax
2919, 2849, 1633, 1557, 1494, 1366, 1259, 1193, 1025, 960, 785, 771,
696, 636, 596 .1H NMR (300 MHz, CDCl3)
d
13.87 (2H, s), 8.57 (1H,
5.3.14. (2E, 4E)-1-[5-Acetyl-2,4-dihydroxyphenyl]-5-phenylpenta-
2,4-dien-1-one (16)
s), 7.67 (2H, d, J ¼ 15.0 Hz), 7.47 (2H, d, J ¼ 15.0 Hz), 6.73 (2H, d,
J ¼ 3.0 Hz), 6.49 (1H, s), 6.21 (2H, d, J ¼ 3.0 Hz), 2.45 (6H, s).13C NMR
Yellow solid (750 mg), 49% yield, mp 178e180 ꢀC. IR (neat) nmax
2918, 2842, 1630, 1554, 1487, 1451, 1353, 1323, 1241, 1230, 1199,
1150,1063,1000, 953, 844, 758, 745 .1H NMR (300 MHz, Me2CO-d6)
(75 MHz, CDCl3) d 191.5, 169.9, 156.6, 150.1, 133.7, 131.3, 119.7, 114.9,
113.8, 109.9, 105.1, 14.0.EIMS m/z 378 [M]þ (100), 270 (58), 255 (11),
163 (19),108 (42), 95 (29).HREIMS m/z 378.1106 (calcd for C22H18O6,
378.1103). Elemental analysis: Found C 69.49%, H 4.72%; calcd for C
69.84%, H 4.79%.
d
13.69 (1H, s, OH), 12.95 (1H, s, OH), 8.32 (1H, s), 7.76 (1H, dd,
J ¼ 15.0,15.0 Hz), 7.64 (3H, m), 7.43 (3H, d, J ¼ 15.0 Hz), 7.25 (2H, m),
6.35 (1H, s), 2.78 (3H, s).13C NMR (75 MHz, Me2CO-d6)
204.7,
d
193.2, 170.7, 146.4, 143.8, 137.2, 137.0, 130.2, 129.7, 128.2, 127.6,
124.2, 114.3, 104.7, 26.4.EIMS m/z 308 [M]þ (100), 307 (20), 265 (12),
231 (12), 179 (24), 163 (13), 152 (7).HREIMS m/z 308.1046 (calcd for
C19H16O4, 308.1049). Elemental analysis: Found C 73.21%, H 5.20%;
calcd for C 74.01%, H 5.23%.
5.3.10. (2E, 20E)-1,10-[4,6-Dihydroxy-1,3-phenylene]bis(3-(3-
methylthiophen-2-yl)prop-2-en-1-one) (12)
Yellow solid (1.52 g), 74% yield, mp 229e231 ꢀC. IR (neat) nmax
2917, 2849, 1626, 1554, 1486, 1384, 1341, 1298, 1273, 1243, 1193,

102
M. Vijaya Bhaskar Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 97e103
5.4. Cytotoxic activity assay
Acknowledgments
All stock cultures were grown in T-25 flasks. Freshly trypsinized
cell suspensions were seeded in 96-well microtiter plates at
densities of 1500e7500 cells per well with compounds added from
DMSO-diluted stock. After 3 days in culture, attached cells were
fixed with cold 50% trichloroacetic acid and then stained with 0.4%
sulforhodamine B (SRB). The absorbance at 562 nm was measured
using a microplate reader after solubilizing the bound dye. The
mean IC50 is the concentration of agent that reduces cell growth by
50% under the experimental conditions; and it is the average from
at least three independent determinations that were reproducible
and statistically significant. The following human tumor cell lines
were used in the assay: DU-145 (prostate cancer), A549 (lung
cancer), KB (nasopharyngeal carcinoma) and KB-VIN (vincristine-
resistant KB subline). All cell lines were obtained from the Line-
berger Comprehensive Cancer Center (UNC-CH) or from ATCC
(Rockville, MD) and were cultured in RPMI-1640 medium supple-
This work was supported by grants from the National Science
Council, Taiwan (NSC 96-2628-M-006-002) and National Cheng
Kung University, Tainan, Taiwan (OUA 95-3-2-021) awarded to T. S.
Wu and Y.C. Shen (NSC 97-2320-B-077-004 - MY3). This study was
also supported in part by a grant from the National Cancer Institute,
NIH (CA17625-32) awarded to K. H. Lee and by a grant from Taiwan
Department of Health Clinical Trial and Research Center of Excel-
lence (DOH100-TD-B-111-004).
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