New bichalcone analogs as NF-κB inhibitors and as cytotoxic agents inducing Fas/CD95-dependent apoptosis

Article abstract of DOI:10.1016/j.bmc.2011.02.004

A series of novel bichalcone analogs were synthesized and evaluated in lipopolysaccharide (LPS)-activated microglial cells as inhibitors of nitric oxide (NO) and for in vitro anticancer activity using a limited panel of four human cancer cell lines. All a

Full text of DOI:10.1016/j.bmc.2011.02.004

Bioorganic & Medicinal Chemistry 19 (2011) 1895–1906
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New bichalcone analogs as NF-
Fas/CD95-dependent apoptosis
jB inhibitors and as cytotoxic agents inducing
M. Vijaya Bhaskar Reddy a, , Yuh-Chiang Shen b, , Jai-Sing Yang c, , Tsong-Long Hwang d,
,
a,g,
Kenneth F. Bastow e, Keduo Qian f, Kuo-Hsiung Lee f,g, , Tian-Shung Wu
⇑
⇑
a Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan
b National Research Institute of Chinese Medicine, Taipei, Taiwan 112
c Department of Pharmacology, China Medical University, Taichung, Taiwan
d Graduate Institute of Natural Products, College of Medicine, Chang Gung University, 259, Taoyan, Taiwan
e Division of Medicinal Chemistry and Natural Products, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, USA
f Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, USA
g Chinese Medicine Research and Development Center and Department of Pharmacy, China Medical University and Hospital, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel bichalcone analogs were synthesized and evaluated in lipopolysaccharide (LPS)-
activated microglial cells as inhibitors of nitric oxide (NO) and for in vitro anticancer activity using a
limited panel of four human cancer cell lines. All analogs inhibited NO production. Compounds 4 and
Received 22 November 2010
Revised 28 January 2011
Accepted 1 February 2011
Available online 4 March 2011
11 exhibited optimal activity with IC50 values of 0.3 and 0.5
better than the positive control. A mechanism of action study showed that both compounds significantly
blocked the nuclear translocation of NF- B p65 and up-regulation of iNOS at 1.0 M. Compound 4 and
three other analogs (3, 20, and 23) exerted significant in vitro anticancer activity GI50 values ranging from
0.70 to 13.10 M. A mode of action study using HT-29 colon cancer cells showed that 23 acts by inducing
apoptosis signaling.
lM, respectively, and were at least 38-fold
j
l
Keywords:
Bichalcone analogs
Nitric oxide production inhibition
Cytotoxicity
l
Ó 2011 Elsevier Ltd. All rights reserved.
Apoptosis
1. Introduction
The Mannich base reaction is an important carbon–carbon
bond-forming reaction in organic synthesis, and it has been widely
utilized in the synthesis of nitrogen-containing drugs, natural
products and biologically active compounds.8,9 Considering the
pharmacological importance of bichalcones, we synthesized a ser-
ies of bichalcone analogs through the piperazine Mannich base
linkage with different substitutions in the B-ring of the chalcone
moiety. Target compounds were examined as inhibitors of nitric
oxide (NO) production in lipopolysaccharide (LPS)-activated
microglial cells, which are important free radical-producing cells
in the central nervous system. Rapid production of reactive oxygen
species (ROS) by NADPH oxidase (NOX) and nitric oxide (NO) by
NO synthase (NOS) can be generated experimentally.10 Thus, we
Dietary flavonoids, commonly present in edible plants, are
known to have beneficial effects, such as antioxidative effects,
tumor cell growth inhibitory activity, and apoptosis induction in
cancer cell lines. Therefore, dietary flavonoids have attracted atten-
tion as chemopreventive agents.1 Chalcones are the immediate
precursors in the biosynthesis of flavonoids, and their structure dif-
fers considerably from the other members of the flavonoid family.
Chalcones are reported with diverse biological activities including
anti-inflammatory, anti-malarial, anti-protozoal, anti-bacterial, ni-
tric oxide inhibition, tyrosinase inhibition, cytotoxic, anticancer,
and anti-leishmanial activities.2–6 The bichalcones are well repre-
sented in the Anacardiacea family. The Rhus genus is also a rich
source for biflavonoids and bichalcones. In general, naturally
occurring bichalcones carry either C–O–C or C–C linkage between
the two chalcone units. Although natural bichalcones from Rhus
pyroides demonstrated varying degrees of cytotoxic activity against
different cancer cell lines, they showed more selectivity toward co-
lon cancer cell lines, especially the HT29 and HCT-116 cell lines.7
investigated the expression of iNOS protein and NF-jB p65 (C)
and p65 (N) in presence of bichalcone analogs. In addition, we fur-
ther evaluated the in vitro anticancer activity of the newly synthe-
sized compounds against four human cancer cell lines, and
explored the mechanism of action of a selected compound (23)
in the HT-29 human colon adenocarcinoma cell line.
2. Chemistry
⇑
Corresponding authors. Tel.: +1 919 9620066; fax: +1 919 9663893 (K.-H.L.);
tel.: +886 6 2747538; fax: +886 2 2740552 (T.-S.W.).
4-Hydroxyacetophenone (1) and 4-hydroxy-3-methoxyaceto-
phenone (2) were reacted with 4-piperazinoacetophenone and
E-mail addresses: khlee@unc.edu (K.-H. Lee), tswu@mail.ncku.edu.tw (T.-S. Wu).
These authors contribute equally to this article.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.004

1896
M. Vijaya Bhaskar Reddy et al. / Bioorg. Med. Chem. 19 (2011) 1895–1906
R
ditions using 30% KOH in MeOH at rt (Scheme 2). Compounds 1b
O
HO
and 1c were obtained by the reaction of 1 with pyrrole-2-carbox-
aldehyde and N-methylpyrrole-2-carboxaldehyde, and 2b was ob-
tained by the reaction of 2 with 3-pyridinecarboxaldehyde under
Claisen–Schmidt conditions. The target chalcones 21, 22, and 23
were prepared by the further reaction of 1b, 1c, and 2b with 4-
piperazinoacetophenone and paraformaldehyde in EtOH at 120 °C
for 18–22 h (Scheme 3).
N
N
O
OH
a
R
O
1. R = H
1a. R = H
2a. R = OMe
2. R = OMe
Scheme 1. Reagents and conditions: (a) 4-piperizino acetophenone, paraformal-
dehyde, EtOH reflux at 120 °C in 18–22 h.
3. Results and discussion
paraformaldehyde in EtOH at 120 °C for 18–22 h to obtain C-5
substituted Mannich base derivatives 1a and 2a, respectively
(Scheme 1). Target compounds 3–20 were obtained by the reaction
of 1a or 2a with substituted aldehydes under Claisen–Schmidt con-
Other laboratories have found that chalcone compounds can
function as potent NF-
j
B inhibitors11 and inhibit NO production.12
O
B-ring
R
A
aldehyde
1a (for 3-14)
R1
2a (for 15-20)
HO
N
30% KOH, rt
R1
N
B-ring
O
3
. R = H, R1 = 2-pyridyl
4. R = H, R1 = 3-pyridyl
5. R = H, R1 = 2-furan
6
. R = H, R1 = 2-thiophene
7. R= H, R1 = 3-methyl-2-thiophene
8
. R = H, R1 = 5-methyl-2-furan
9. R = H, R1 = N-methyl pyrrole
10. R = H, R1 = phenyl
11
. R = H, R1 = 4-methoxybenzene
12. R = H, R1 = 3,4-methylenedioxybenzene
13
. R = H, R1 = 2-chlorobenzene
14. R = H, R1 = 2,4-dichlorobenzene
15. R = OMe, R1 = 2-thiophene
16.
R = OMe, R1 = 3-methyl-2-thiophene
17. R = OMe, R1 = phenyl
18
. R = OMe, R1 = 4-methoxybenzene
19. R = OMe, R1 = 2-chlorobenzene
20. R = OMe, R1 = 2-pyridyl
Scheme 2.
Scheme 3. Reagents and conditions: (a) for 1b from 1, pyrrole-2-carboxaldehyde, MeOH, 30% KOH, rt, 24 h, for 1c from 1, N-methylpyrrole-2-carboxaldehyde, MeOH, 30%
KOH, rt, 24 h; (b) 4-piperizino acetophenone, paraformaldehyde, EtOH reflux at 120 °C in 18–22 h; (c) for 2b from 2, 3-pyridinecarboxaldehyde, MeOH, 30% KOH, rt, 24 h.

M. Vijaya Bhaskar Reddy et al. / Bioorg. Med. Chem. 19 (2011) 1895–1906
1897
Based on these precedents, our new compounds 1a–23 were
evaluated for inhibition of NOX-dependent ROS production and
NOS-dependent NO production in microglial cells as well as for
1,1-diphenyl-2-picrylhydrozyl (DPPH) radical scavenging capacity.
None of the compounds showed inhibition of NOX-dependent ROS
production or ability for direct radical-scavenging in a cell-free
DPPH solution. In contrast, 1a–23 were potent inhibitors of NO
production in microglial cells with IC50 values ranging from 0.3
C-3 or C-5 position of the thiophene or furan rings decreased the
compounds’ activity, as seen with 7 and 8. The introduction of a
methoxy group at the C-4 position of the phenyl B-ring of 11 re-
sulted in increased inhibition of NO production (IC50: 0.5 lM),
while addition of chloro substituents on the B-ring as in 13 and
14 led to decreased inhibition of NO production (IC50: 30.5 and
25.7
The influence of the most potent NO production inhibitors 4 and
11 on the iNOS protein expression and NF- B signaling pathway
lM, respectively).
to 30.5
lM, compared with L-nitro-arginine methyl ester
j
(L-NAME) (IC50: 18.9
lM), a specific NOS inhibitor (Table 1).
was further explored in LPS-activated BV2 cells ( Fig. 1). Both
To summarize the initial structure–activity observations on the
bichalcone analogs, we found that a methoxy group at the C-3 po-
sition in the A-ring resulted in weaker NO production inhibition
activity compared with no substitution at this position, as seen
compounds significantly blocked the nuclear translocation of NF-
j
B p65 at 1.0
expression. These results suggested that 4 and 11 may target the
NF- B signaling pathway to block iNOS up-regulation, which in
lM concentration and decreased the iNOS protein
j
with 1b (IC50: 15.9
B-ring, 3 and 4 with 2-pyridyl and 3-pyridyl B-rings, respectively,
showed improved activity with IC50 values of 1.4 and 0.3 M,
respectively. Compounds 5 (IC50: 1.7 M) and 6 (IC50: 1.9 M) with
l
M) versus 1a (IC50: 6.6
l
M). Concerning the
turn suppresses the NO production.
l
Table 2
l
l
Cytotoxic activity data for 1a–23a
2-furanyl and 2-thiophenyl B-rings, respectively, showed similar
Compound
GI50
(
lM)
NO inhibition activity to that of 3. Adding methyl groups at the
KB
A549
HCT-8
DU145
Table 1
3
4
1.23
1.66
1.08
1.47
1.26
1.40
1.25
2.19
Summary of the effects of 1–23 on NOS activity in murine microglial cellsa,b
6
17
20
21
22
23
Paclitaxel
>37.00
>35.80
3.98
19.00
39.10
13.10
5.16 nM
>37.00
>35.80
1.00
20.10
31.20
9.77
>37.00
25.80
0.70
24.70
>45.10
11.25
>100 nM
29.20
>35.80
1.30
17.90
>45.10
9.98
Compound
IC50
(
l
M)
Compound
IC50 (lM)
1a
2a
3
4
5
6
7
8
9
6.6 1.3*
15.9 1.5
1.4 0.2
0.3 0.0
1.7 0.2
1.9 0.0
7.5 1.6
2.4 0.2
1.2 0.4
2.8 0.5
0.5 0.0
2.1 0.2
13
14
15
16
17
18
19
20
21
22
23
L-NAME
30.5 11.4
25.7 4.6
2.7 0.3
3.1 0.1
2.1 0.4
3.5 0.8
10.9 2.8
1.6 0.3
1.7 0.3
1.9 0.3
3.5 0.4
18.9 3.2
7.6 nM
5.23 nM
a
Compounds that are not shown did not reach 50% inhibition at 20
are considered inactive.
lg/mL and
10
11
12
a
NOS activity was measured by NO production in the presence of 1–50 lM of
drug. DPI (diphenyleneiodonium, a NOX inhibitor) was included as a positive
control. Data were calculated as 50% inhibitory concentration (IC50) and expressed
as means SEM from
3 to 6 experiments performed on different days using
microglial cells from different passages.
b
Compounds were also tested in NOX and DPPH assays, but none of the com-
pounds were active. NOX activity was measured by ROS production, and DPI (a NOX
inhibitor) was included as positive control (IC50 0.4 0.2
l
M). For the DPPH assay,
M).
Trolox (an antioxidant) was the positive control (IC50 36.0 3.5
l
*
p <0.05 as compared with relative positive controls, respectively.
Figure 1. Effects of compounds 4 and 11 on the LPS-induced iNOS expression and
nuclear translocation of NF-
immunoblots of iNOS expression and nuclear (N) translocation of NF-
[p65(N)] from cytosol [p65(C)] in microglial cells receiving water only (control),
0.5 g/mL LPS (L) for 2 h, or 0.5 g/mL LPS plus 0.5–1.0 M of compound 4 or 11
(L+cpd4 or L+cpd11). The b-actin was included as reference for protein
j
B
p65 in murine microglial cells. Representative
jB
p65
Figure 2. Effects of compound 23 on cell viability of human colon adenocarcinoma
cell line HT-29. Cells were treated with 0, 10, 20, 30, 40 and 50 M of compound 23
for 24 h by MTT assay (A). Cells were treated with 30 M of compound 23 for 12, 24,
l
l
l
l
a
l
normalization. Similar results were observed at least in three independent
experiments.
48 and 72 h by MTT assay (B) Results are presented as mean SD. The experiments
were done in triplicate. ⁄⁄⁄p <0.001.

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M. Vijaya Bhaskar Reddy et al. / Bioorg. Med. Chem. 19 (2011) 1895–1906
Figure 3. Effects of compound 23 on cell morphological changes (A), and DNA condensation by DAPI staining (B) in human colon adenocarcinoma cell line HT-29. After
incubation with compound 23 (30 and 50 M) for 24 and 48 h, cells exhibited nuclear shrinkage and chromatin condensation (A). DNA condensation was detected by DAPI
l
staining (B). Cells were examined and photographed under fluoresce microscopy (Â200) as described in Section 4.1.
at a fixed concentration (30 lM), and cell viability was determined
over time up to three days. As shown in Figure 2B, 23 inhibited HT-
29 cell viability in a time-dependent manner. Figure 3A and B show
the results from phase-contrast microscopy of 23-treated HT-29
cells coupled with DAPI staining for the occurrence of morpholog-
ical changes and DNA condensation. Following treatment with 23
(30 and 50 lM), HT-29 cells exhibited nuclear shrinkage and chro-
matin condensation, compared to the untreated control cells. To
further investigate 23’s effect on the DNA of HT-29 cells, we as-
sessed the DNA fragmentation by terminal deoxynucleotidyl trans-
ferase-mediated dUTP nick end labeling staining (TUNEL) coupled
with flow cytometry. As shown in Figure 4, 23 (30 lM) induced
DNA fragmentation in HT-29 cells in a time-dependent manner.
This response is associated with apoptosis, so the effects of 23 on
the signaling pathway were investigated.
The activities of caspase-3, -8, and -9 in 23-treated HT-29 cells
detected by colorimetric enzymatic assay are shown in Figure 5A.
All of the active caspases were detected after 24 h treatment with
23. To verify the involvement of caspase-3, -8, and -9 in 23-in-
duced apoptosis of HT-29 cells, inhibitors of each caspase
(caspase-3, Z-DEVE-FMK; caspase-8, Z-IETD-FMK; caspase-9, Z-
LEHD-FMK) were used. The results shown in Figure 5B are
consistent with 23 inducing apoptosis through the activation of
caspase-3, -8, and -9. The relative levels of 12 key apoptosis-asso-
ciated protein levels in 23-treated HT-29 cells were then analyzed
using Western Blot Analysis. Compound 23 increased the protein
expression of Fas/CD95, FADD (Fig. 6A), cytosolic cytochrome c,
Apaf-1, AIF, Endo G (Fig. 6B), caspase-3 (Fig. 6C), Bax (Bcl2-associ-
ated X protein), and t-Bid (Fig. 6D), and decreased the protein levels
of pro-caspase-8, pro-caspase-9 (Fig. 6C) and Bcl-2 (Fig. 6D). The
analysis results suggested that 23 induces apoptosis in HT-29 cells
via both death receptor (Fas/CD95) and mitochondrial-dependent
pathways.13–15 The effects of 23 on the proteins analyzed and the
Figure 4. Effects of compound 23 on DNA fragmentation in human colon
adenocarcinoma cell line HT-29. HT-29 cells were treated with compound 23
(30 lM) for 24 and 48 h, then cells were stained with TUNEL and analyzed by
FACScan flow cytometry. The experiments were done in triplicate. ⁄⁄⁄p <0.001.
Compounds 1a–23 were also evaluated for cytotoxicity against
four human cancer cell lines, DU145 (prostate cancer), A549 (non
small cell lung cancer), KB (nasopharyngeal carcinoma) and HCT-
8 (ileocecal) (Table 2). It was found that the B-ring pyridyl moiety
is important for significant activity of the bichalcone analogs 3
(GI50: 1.08–1.26
3.98 M). Compound 23, bearing a single B-ring pyridyl moiety,
also showed activity, but with higher GI50 values (9.77–13.1 M).
lM), 4 (GI50: 1.40–2.19 lM), and 20 (GI50: 0.70–
l
l
Compounds 21 and 22 with one pyrrole B-ring showed marginal
cytotoxicity, and all the other analogs tested were essentially
inactive.
As shown in Figure 2A, 23 inhibited HT-29 cell viability in a
dose-dependent manner. HT-29 cells were then treated with 23

M. Vijaya Bhaskar Reddy et al. / Bioorg. Med. Chem. 19 (2011) 1895–1906
1899
300 spectrometer, using tetramethylsilane (TMS) as internal stan-
dard; all chemical shifts are reported in parts per million (ppm, d).
FABMS and HRFABMS spectra were obtained on a JEOL JMS-700
mass spectrometer. Column chromatography was performed on
silica gel (70–230 mesh, 230–400 mesh). TLC was conducted on
precoated Kieselgel 60 F254 plates (Merck), and the spots were de-
tected by UV. Elemental analyses were determined by Elementer
Vario EL III and gave combustion values for C, H, N and S. Concen-
tration of the reaction solutions involved the use of rotary evapo-
rator under reduced pressure. All other chemicals were obtained
from Aldrich, Inc. Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4’,6-diamidi-
no-2-phenylindole (DAPI), Tris–HCl and Triton X-100 were
obtained from Sigma Chemical Co. (St. Louis, MO, USA). Caspase-
8 inhibitor, z-Ile-Glu-Thr-Asp-fluoromethyl ketone (Z-IETD-FMK),
Caspase-9 inhibitor, z-Leu-Glu-His-Asp-fluoromethyl ketone (Z-
LEHD-FMK), Caspase-3 inhibitor z-Asp-Met-Gln-Asp-fluoromethyl
ketone (Z-DEVD-FMK) (R&D, USA) were dissolved in DMSO and
diluted in cell culture medium before use. RPMI 1640, penicillin–
streptomycin, trypsin-EDTA, fetal bovine serum (FBS), and gluta-
mine were obtained from Gibco BRL (Invitrogen, Grand Island, NY).
4.2. General procedure for the synthesis of Mannich bases of
acetophenones
To a solution of hydroxy-substituted acetophenone and parafor-
maldehyde in EtOH (75 mL) was added 4-piperazinoacetophenone
at rt as per reported earlier literature.16–19 Then the resulting mix-
ture was heated to reflux for 18–22 h at 120 °C. On completion, the
reaction mixture was concentrated under reduced pressure and
the crude product was purified by column chromatography to yield
the ortho substituted Mannich base in good yield (Scheme 1).
Figure 5. Effects of compound 23 on caspase-3, caspase-8 and caspase-9 activity in
human colon adenocarcinoma cell line HT-29 (A) and effects of caspase-3, caspase-
8, and caspase-9 inhibitors on cell viability in compound 23-treated HT-29 cells (B).
Cells were pretreated with the caspase-3 inhibitor (Z-DEVE-FMK), caspase-8
inhibitor (Z-IETD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) for 1 h and then
4.2.1. 1-(4-(4-(5-Acetyl-2-hydroxybenzyl)piperazin-1-yl)-
phenyl)ethanone (1a)
Compound
1 (13.6 g, 100 mmol), paraformaldehyde (3.0 g,
treated with 30
l
M of compound 23 for 24 h. The total cell extracts were incubated
100 mmol) and 4-piperizinoacetophenone (20.4 g, 100 mmol)
were treated as described above. The crude product was purified
by CC eluting with (i-Pr)2O/MeOH (9:1) to yield 1a (colorless crys-
talline solid, 24.5 g, 69.6%), mp 146–148 °C. IR (neat) 3005, 2711,
1666, 1597, 1519, 1442, 1357, 1284, 1238, 1195, 1118, 1002,
with caspase-3, caspase-8, and caspase-9 specific substrates, respectively (Ac-
DEVD-pNA, Ac-LEHD-pNA, and Ac-IETD-pNA). The release of pNA was measured at
405 nm by a spectrophotometer. For cell viability assay, cells were pretreated with
the caspase-3, caspase-8 and caspase-9 inhibitor for 1 h and then treated with
30 lM of compound 23 for 24 h by MTT assay. The experiments were done in
triplicate. ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001.
925, 825, 756, 690, 563, 513 cmÀ1 1H NMR (CDCl3, 300 MHz): d
.
7.87 (3H, d, J = 8.7 Hz), 7.74 (1H, s), 6.86 (3H, d, J = 8.7 Hz), 3.86
(2H, s), 3.47 (4H, s), 2.77 (4H, s), 2.54 (3H, s), 2.52 (3H, s).13
C
interactions of these proteins within the Fas/CD95 and mitochon-
drial-dependent pathways are shown graphically on the left and
right sides, respectively, of Figure 7.
In summary, a series of bichalcones with a piperazine Mannich
base linkage was prepared and evaluated for inhibition of NO pro-
duction in microglial cells and for in vitro anticancer activity. Com-
pounds 4 and 11 were potent inhibitors of cellular NO production
in LPS-activated microglial cells, likely indirectly via blockade of
NMR (CDCl3, 75 MHz): d 196.6, 196.4, 162.3, 153.6, 130.4, 130.3
(Â2), 129.4, 129.1, 128.2, 120.5, 116.0, 113.8 (Â2), 61.0, 52.0
(Â2), 47.2 (Â2), 26.2, 26.1.FABMS, m/z (% rel. intensity): 353
(100) [M+H]+, 352 (76), 351 (39), 205 (27), 204 (15), 203 (26),
177 (14), 174 (11), 162 (23), 154 (13), 149 (37), 148 (12), 136
(11).HRFABMS m/z calcd for
C21H25O3N2, 353.1865; found,
353.1866. Elemental Anal. Calcd C, 71.57; H, 6.86; N, 7.95. Found:
C, 71.08; H, 6.88; N, 7.80.
NF-jB p65 nuclear translocation. Several bichalcone analogs,
including 4 as well as 3, 20, and 23, with a signature pyridyl moiety
also showed significant activity against human tumor cell replica-
tion. Exploration of the mechanism of action showed that 23 likely
acted via the Fas/CD95 apoptosis signaling pathway.
4.2.2. 1-(4-(4-(5-Acetyl-2-hydroxy-3-methoxybenzyl)piperazin-
1-yl)phenyl)ethanone (2a)
Compound
2 (8.3 g, 50 mmol), paraformaldehyde (1.5 g,
50 mmol) and 4-piperizinoacetophenone (10.2 g, 50 mmol) were
treated as described above. The crude product was purified by CC
eluting with (i-Pr)2O/MeOH (7:3) to yield 2a (colorless crystalline
solid, 14.5 g, 76%), mp 130–132 °C. IR (neat) 2831, 1666, 1597,
1516, 1489, 1450, 1411, 1537, 1296, 1234, 1141, 1083, 1002,
956, 921, 821, 752, 594, 559 cmÀ1.1H NMR (CDCl3, 300 MHz): d
7.83 (2H, d, J = 9.0 Hz), 7.44 (1H, s), 7.28 (1H, s), 6.83 (2H, d,
J = 9.0 Hz), 3.89 (3H, s), 3.81 (2H, s), 3.36 (4H, s), 2.72 (4H, s),
2.51 (3H, s), 2.48 (3H, s). 13C NMR (CDCl3, 75 MHz): d 196.4,
196.3, 153.5, 151.8, 147.8, 130.2 (Â2), 128.7, 128.1, 122.5, 119.8,
4. Experimental section
4.1. Materials and methods
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 spectrophotom-
eter. 1H and 13C NMR spectra were recorded on a Bruker Avance

1900
M. Vijaya Bhaskar Reddy et al. / Bioorg. Med. Chem. 19 (2011) 1895–1906
Figure 6. Effects of compound 23-induced apoptotic relative protein levels on HT-29 cells. Western blotting analysis for (A) Fas/CD95 and FADD; (B) cytosolic cytochrome c,
Apaf-1, AIF and Endo G; (C) pro-caspase-8, pro-caspase-9 and caspase-3; (D) Bax, Bcl-2 and t-Bid protein levels in compound 23 treated HT-29 cells. For Western blotting
analysis, total or cytosolic protein extracts were analyzed by immunoblotting.
Figure 7. A proposed model for the apoptosis signaling pathways in 23-treated HT-29 cells.
113.7 (Â2), 110.4, 60.7, 55.8, 51.9 (Â2), 47.2 (Â2), 25.9 (Â2). FAB-
MS, m/z (% rel. intensity): 383 (100) [M+H]+, 382 (75), 381 (28), 307
(12), 217 (12), 207 (13), 205 (25), 204 (11), 203 (22), 179 (40), 162
(20), 155 (17), 154 (67), 138 (20), 137 (41), 136 (51), 120 (11), 107
(16), 91 (11), 89 (13), 77 (11).HRFABMS m/z calcd for C22H27O4N2,
383.1971; found, 383.1968. Elemental Anal. Calcd C, 69.09; H,
6.85; N, 7.32. Found: C, 68.42; H, 7.07; N, 7.37.
4.3. General procedure for the synthesis of bichalcones through
the piperazine Mannich base linkage
The general synthetic strategy employed to prepare the bichal-
cones through the piperazine Mannich base linkage analogs were
based on the Claisen–Schmidt condensation. As shown in Scheme
2, a series of 23 bichalcones were prepared by base-catalyzed con-

M. Vijaya Bhaskar Reddy et al. / Bioorg. Med. Chem. 19 (2011) 1895–1906
1901
densation of substituted Mannich bases of acetophenones with
appropriate aldehydes in MeOH. To a stirred reaction mixture at
0 °C was added a 30% solution of KOH (40 mL) dropwise over
30 min. The reaction mixture was kept at rt for 24 h, then diluted
with water and extracted with EtOAc. Pure target compounds were
obtained by silica gel column chromatography (cc) of the residue
eluting with various solvent mixtures as indicated below. The
structures of all the 23 bichalcones through the piperazine Man-
nich base linkage analogs were established on the basis of IR, 1H,
13CNMR, FABMS and HRFABMS.
was purified by CC eluting with hexanes/EtOAc (7:3) to yield 5
(pale yellow solid, 780 mg, 77%), mp 169–171 °C. IR (neat) 3417,
2920, 1651, 1600, 1550, 1527, 1473, 1388, 1350, 1280, 1230,
1192, 1111, 1018, 925, 817, 744, 644 cmÀ1 1H NMR (CDCl3,
.
300 MHz): d 8.00 (2H, d, J = 9.0 Hz), 7.94 (1H, s), 7.81 (1H, s),
7.58 (2H, dd, J = 15.0, 15.0 Hz), 7.51 (2H, m), 7.46 (2H, d,
J = 15.6 Hz), 6.91 (3H, d, J = 9.0 Hz), 6.68 (2H, d, J = 4.5 Hz), 6.50
(2H, s), 3.86 (2H, s), 3.44 (4H, s), 2.76 (4H, s). 13C NMR (CDCl3,
75 MHz): d 187.8, 187.5, 162.4, 153.6, 151.9, 151.8, 144.6, 144.5,
130.5 (Â2), 130.4, 130.0, 129.9 (Â2), 129.5, 129.0, 120.8, 119.2,
119.0, 116.2, 115.8, 115.5, 114.0 (Â2), 112.6, 112.5, 61.1, 52.1
(Â2), 47.3 (Â2). FABMS, m/z (% rel. intensity): 509 (24) [M+H]+,
508 (11), 307 (27), 289 (14), 283 (9), 240 (7), 227 (7), 155 (36),
154 (100), 153 (9), 152 (13), 147 (8), 139 (17), 138 (42), 137
(82), 136 (100), 135 (11), 124 (11), 121 (23), 120 (18), 115 (9),
108 (10), 107 (33), 106 (11), 105 (12), 91 (24), 90 (26), 89 (30),
78 (12), 77 (23), 73 (20).HRFABMS m/z calcd for C31H29O5N2,
509.2076; found, 509.2073. Elemental Anal. Calcd C, 73.21; H,
5.55; N, 5.51. Found: C, 72.97; H, 5.44; N, 5.32.
4.3.1. (E)-1-(4-Hydroxy-3-((4-(4-((E)-3-(pyridin-2-yl)acryloyl)-
phenyl)piperazin-1-yl)methyl)phenyl)-3-(pyridin-2-yl)prop-2-
en-1-one (3)
Compound 1a (704 mg, 2.0 mmol) and 2-pyridinecarboxalde-
hyde (428 mg, 4.0 mmol) were treated as described above. The
crude product was purified by CC eluting with hexanes/EtOAc
(1:1) to yield 3 (pale yellow solid, 680 mg, 64%), mp 59–61 °C. IR
(neat) 2874, 1657, 1608, 1589, 1522, 1468, 1432, 1387, 1329,
1225, 1193, 1111, 1027, 993, 927, 830, 782, 661, 569 cmÀ1 1H
.
NMR (DMSO-d6, 300 MHz): d 8.67 (2H, s), 8.14 (2H, dd, J = 15.3,
15.3 Hz), 7.99 (4H, t, J = 5.7 Hz), 7.87 (4H, s), 7.66 (2H, dd,
J = 15.3, 15.3 Hz), 7.41 (2H, d, J = 3.9 Hz), 7.03 (3H, dd, J = 8.4 Hz),
3.75 (2H, s), 3.43 (4H, s), 2.63 (4H, s). 13C NMR (DMSO-d6,
75 MHz): d 187.3, 186.5, 161.7, 153.8, 153.1, 152.9, 149.9 (Â2),
141.8, 141.3, 137.1 (Â2), 130.9, 130.6 (Â2), 129.9, 128.7, 126.9,
125.3, 125.1, 124.8, 124.6 (Â2), 124.4, 123.0, 115.5, 113.3 (Â2),
57.2, 51.9 (Â2), 46.4 (Â2).FABMS, m/z (% rel. intensity): 531 (11)
[M+H]+, 530 (8), 481 (1), 460 (1), 437 (1), 391 (4), 307 (10), 294
(14), 292 (10), 291 (8), 251 (9), 239 (13), 238 (19), 210 (4), 207
(5), 195 (5), 194 (5), 180 (6), 167 (9), 155 (25), 154 (100), 152
(9), 149 (17), 139 (14), 138 (35), 137 (65), 136 (87), 132 (45),
124 (11), 120 (15), 107 (40), 91 (26), 90 (32), 89 (51), 77 (44).
HRFABMS m/z calcd for C33H31O3N4, 531.2396; found, 531.2397.
Elemental Anal. Calcd C, 74.70; H, 5.75; N, 10.56. Found: C,
74.52; H, 5.92; N, 10.34.
4.3.4. (E)-1-(4-Hydroxy-3-((4-(4-((E)-3-(thiophen-2-yl)acryloyl)-
phenyl)piperazin-1-yl)methyl)phenyl)-3-(thiophen-2-yl)prop-2-
en-1-one (6)
Compound 1a (704 mg, 2.0 mmol) and 2-thiophenecarboxalde-
hyde (448 mg, 4.0 mmol) were treated as described above. The
crude product was purified by CC eluting with hexanes/EtOAc
(1:1) to yield 6 (pale yellow solid, 750 mg, 69%), mp 126–128 °C.
IR (neat) 2874, 1648, 1600, 1511, 1450, 1426, 1365, 1285, 1232,
1190, 1125, 1105, 1028, 998, 925, 817, 708, 657, 566 cmÀ1.1H
NMR (CDCl3, 300 MHz): d 7.97 (2H, d, J = 9.0 Hz), 7.86 (2H, d,
J = 15.0 Hz), 7.75 (2H, s), 7.35 (2H, d, J = 15.0 Hz), 7.32 (3H, m),
7.07 (2H, s), 6.91 (4H, d, J = 8.1 Hz), 3.87 (2H, s), 3.45 (4H, s), 2.78
(4H, s). 13C NMR (CDCl3, 75 MHz): d 187.4 (Â2), 153.5, 140.6,
140.5, 136.3, 135.8, 131.7, 131.5, 130.5 (Â3), 130.3, 129.8, 129.4,
128.8, 128.4, 128.2 (Â2), 128.1 (Â2), 120.6, 120.4, 113.9 (Â3),
61.0, 52.0 (Â2), 47.2 (Â2).FABMS, m/z (% rel. intensity): 541 (12)
[M+H]+, 540 (9), 523 (2), 505 (3), 447 (21), 446 (12), 431 (8), 391
(6), 300 (6), 299 (20), 297 (16), 281 (11), 256 (17), 255 (5), 243
(11), 242 (12), 221 (12), 217 (11), 207 (15), 189 (13), 167 (15),
165 (16), 155 (20), 154 (67), 152 (19), 150 (14), 149 (48), 137
(66), 136 (84), 115 (23), 111 (18), 107 (46), 97 (34), 95 (50), 91
(67), 90 (42), 89 (51), 81 (59), 73 (100), 71 (40), 67 (45), 55
4.3.2. (E)-1-(4-Hydroxy-3-((4-(4-((E)-3-(pyridin-3-yl)acryloyl)-
phenyl)piperazin-1-yl)methyl)phenyl)-3-(pyridin-3-yl)prop-2-
en-1-one (4)
Compound 1a (704 mg, 2.0 mmol) and 3-pyridinecarboxalde-
hyde (428 mg, 4.0 mmol) were treated as described above. The
crude product was purified by CC eluting with CHCl3/MeOH (9:1)
to yield 4 (pale yellow solid, 720 mg, 68%), mp 69–71 °C. IR (neat)
3420, 2835, 1654, 1600, 1527, 1419, 1346, 1307, 1284, 1226, 1192,
1122, 1026, 995, 925, 806, 736, 702, 628 cmÀ1. 1H NMR (DMSO-d6,
300 MHz): d 9.00 (2H, d, J = 4.5 Hz), 8.60 (2H, s), 8.33 (2H, d,
J = 9.0 Hz), 8.06 (2H, d, J = 15.9 Hz), 8.05 (4H, m), 7.70 (2H, dd,
J = 15.6, 15.6 Hz), 7.48 (2H, t, J = 3.9 Hz), 7.04 (2H, d, J = 8.4 Hz),
6.93 (1H, d, J = 9.0 Hz), 3.77 (2H, s), 3.44 (4H, s), 2.64 (4H, s). 13C
NMR (DMSO-d6, 75 MHz): d 186.7, 186.3, 161.8, 158.5, 152.8,
149.3, 148.6, 148.2, 139.1, 138.2, 137.1, 136.6, 135.0, 132.7,
131.7, 131.3, 130.8 (Â2), 128.8, 127.8, 124.9, 124.6, 124.5, 124.4,
116.4, 115.7, 113.9 (Â2), 53.6, 50.5 (Â2), 43.8 (Â2).FABMS, m/z (%
rel. intensity): 531 (5) [M+H]+, 307 (35), 289 (18), 280 (8), 165
(8), 156 (9), 155 (48), 154 (100), 153 (11), 152 (15), 150 (6), 139
(21), 138 (59), 137 (98), 136 (96), 124 (15), 121 (13), 120 (23),
108 (15), 107 (45), 106 (13), 105 (14), 91 (25), 90 (31), 89 (43),
77 (34), 65 (12).HRFABMS m/z calcd for C33H31O3N4, 531.2396;
found, 531.2393. Elemental Anal. Calcd C, 74.70; H, 5.75; N,
10.56. Found: C, 74.08; H, 5.92; N, 10.34.
(87).HRFABMS m/z calcd for
C31H29O3N2S2, 541.1620; found,
541.1617. Elemental Anal. Calcd C, 68.86; H, 5.22; N, 5.18; S,
11.86. Found, C, 68.36; H, 5.52; N, 5.45; S, 11.15.
4.3.5. (E)-1-(4-Hydroxy-3-((4-(4-((E)-3-(3-methylthiophen-2-yl)
acryloyl)phenyl)-piperazin-1-yl)methyl)phenyl)-3-(3-methyl-
thiophen-2-yl)prop-2-en-1-one (7)
Compound 1a (704 mg, 2.0 mmol) and 3-methyl-2-thiophene-
carboxaldehyde (504 mg, 4.0 mmol) were treated as described
above. The crude product was purified by CC eluting with hex-
anes/EtOAc (7:3) to yield 7 (pale yellow solid, 860 mg, 76%), mp
179–181 °C. IR (neat) 2875, 1648, 1600, 1516, 1493, 1452, 1384,
1288, 1220, 1189, 1110, 1034, 926, 820, 752, 629 cmÀ1.1H NMR
(CDCl3, 300 MHz):
d 8.04 (1H, d, J = 3.9 Hz), 7.97 (2H, d,
J = 15.0 Hz), 7.96 (2H, d, J = 9.0 Hz), 7.81 (1H, s), 7.29 (2H, d,
J = 9.0 Hz), 7.28 (2H, d, J = 15.0 Hz), 6.90 (5H, m), 3.90 (2H, s),
3.46 (4H, s), 2.80 (4H, s), 2.38 (6H, s). 13C NMR (CDCl3, 75 MHz):
d 187.8, 187.5, 162.2, 153.4, 142.3, 142.0, 134.7 (Â2), 134.5,
134.3, 131.3 (Â2), 130.4 (Â2), 130.3, 130.0, 129.9, 129.0, 126.6,
120.5, 119.6, 119.4, 116.2, 113.9 (Â2), 60.8, 52.0 (Â2), 47.1 (Â2),
14.2 (Â2). FABMS, m/z (% rel. intensity): 569 (29) [M+H]+, 568
(18), 553 (2), 461 (2), 445 (10), 313 (23), 312 (9), 311 (20), 309
(8), 307 (10), 281 (11), 270 (17), 258 (11), 257 (17), 207 (15),
189 (12), 165 (10), 155 (23), 154 (89), 152 (19), 151 (54), 149
4.3.3. (E)-3-(Furan-2-yl)-1-(4-(4-(5-((E)-3-(furan-2-yl)acryloyl)-
2-hydroxybenzyl)piperazin-1-yl)phenyl)prop-2-en-1-one (5)
Compound 1a (704 mg, 2.0 mmol) and 2-furaldehyde (384 mg,
4.0 mmol) were treated as described above. The crude product

1902
M. Vijaya Bhaskar Reddy et al. / Bioorg. Med. Chem. 19 (2011) 1895–1906
(18), 147 (30), 138 (30), 137 (56), 136 (100), 107 (41), 105 (24), 91
(36), 90 (36), 89 (48), 77 (48), 73 (78), 71 (19), 69 (31), 57 (38), 55
7.80 (2H, dd, J = 15.6, 15.6 Hz), 7.82 (1H, s), 7.65 (4H, d,
J = 6.0 Hz), 7.55 (2H, d, J = 15.6 Hz), 7.40 (6H, m), 6.93 (3H, d,
J = 9.0 Hz), 3.88 (2H, s), 3.45 (4H, s), 2.77 (4H, s). 13C NMR (CDCl3,
75 MHz): d 188.4, 188.1, 162.4, 153.6, 143.8, 143.4, 135.2, 135.0,
130.6 (Â2), 130.4, 130.3, 130.1, 129.9, 129.0 (Â3), 128.8 (Â2),
128.4 (Â2), 128.3 (Â3), 121.8, 121.7, 120.8, 116.2, 114.0 (Â2),
61.1, 52.1 (Â2), 47.3 (Â2). FABMS, m/z (% rel. intensity): 529 (11)
[M+H]+, 308 (8), 307 (33), 293 (7), 289 (19), 189 (5), 178 (5), 166
(7), 165 (11), 156 (9), 155 (47), 154 (100), 153 (11), 152 (16),
139 (22), 138 (58), 137 (98), 136 (92), 131 (14), 124 (16), 121
(22), 120 (24), 115 (10), 108 (15), 107 (46), 105 (16), 91 (27), 90
(32), 89 (43), 79 (12), 77 (37), 65 (12), 63 (11).HRFABMS m/z calcd
for C35H33O3N2, 529.2491; found, 529.2494. Elemental Anal. Calcd
C, 9.52; H, 6.10; N, 5.30. Found: C, 78.47; H, 6.16; N, 5.04.
(38).HRFABMS m/z calcd for
C33H33O3N2S2, 569.1933; found,
569.1931. Elemental Anal. Calcd C, 69.69; H, 5.67; N, 4.93; S,
11.58. Found, C, 69.48; H, 5.71; N, 4.90; S, 11.46.
4.3.6. (E)-1-(4-Hydroxy-3-((4-(4-((E)-3-(5-methylfuran-2-yl)
acryloyl)phenyl)piperazin-1-yl)methyl)phenyl)-3-(5-methyl-
furan-2-yl)prop-2-en-1-one (8)
Compound 1a (704 mg, 2.0 mmol) and 5-methyl-2-furaldehyde
(440 mg, 4.0 mmol) were treated as described above. The crude
product was purified by CC eluting with hexanes/EtOAc (7:3) to
yield 8 (pale yellow solid, 750 mg, 70%), mp 94–96 °C. IR (neat)
3390, 2873, 1651, 1600, 1527, 1446, 1350, 1222, 1192, 1118,
1022, 802, 732, 671, 617 cmÀ1.1H NMR (CDCl3, 300 MHz): d 8.01
(2H, d, J = 9.0 Hz), 7.97 (1H, dd, J = 8.4, 2.1 Hz), 7.82 (1H, d,
J = 2.1 Hz), 7.53 (2H, dd, J = 15.3, 15.3 Hz), 7.38 (2H, dd, J = 15.3,
15.3 Hz), 6.92 (2H, d, J = 9.0 Hz), 6.90 (1H, d, J = 3.3 Hz), 6.59 (2H,
dd, J = 3.3, 6.0 Hz), 6.12 (2H, t, J = 3.3 Hz), 3.88 (2H, s), 3.57 (4H,
s), 2.77 (4H, s), 2.40 (6H, s). 13C NMR (CDCl3, 75 MHz): d 187.8,
187.5, 162.1, 155.5, 155.3, 153.4, 150.5, 150.4, 130.4 (Â2), 130.2,
130.1, 130.0, 129.7, 129.6, 129.2, 120.7, 117.7, 117.5, 117.4,
117.2, 116.0, 113.9 (Â2), 109.2, 109.1, 61.1, 52.1 (Â2), 47.3 (Â2),
13.9 (Â2). FABMS, m/z (% rel. intensity): 537 (61) [M+H]+, 536
(39), 535 (13), 429 (13), 307 (22), 297 (28), 296 (13), 295 (21),
254 (19), 240 (14), 155 (26), 154 (100), 139 (11), 138 (28), 137
(52), 136 (73), 135 (50), 120 (12), 107 (26), 91 (12), 90 (14), 89
(20), 77 (19).HRFABMS m/z calcd for C33H33O5N2, 537.2389; found,
537.2387. Elemental Anal. Calcd C, 73.86; H, 6.01; N, 5.22. Found:
C, 73.25; H, 6.08; N, 5.07.
4.3.9. (E)-1-(4-Hydroxy-3-((4-(4-((E)-3-(4-methoxyphenyl)
acryloyl)phenyl)piperazin-1-yl)methyl)phenyl)-3-(4-methoxy-
phenyl)prop-2-en-1-one (11)
Compound 1a (704 mg, 2.0 mmol) and 4-methoxybenzalde-
hyde (544 mg, 4.0 mmol) were treated as described above. The
crude product was purified by CC eluting with hexanes/EtOAc
(7:3) to yield 11 (pale yellow solid, 720 mg, 61%), mp 127–
129 °C. IR (neat) 2831, 1647, 1597, 1508, 1450, 1423, 1342, 1292,
1253, 1222, 1172, 1126, 1029, 983, 925, 813, 767, 651, 551,
520 cmÀ1.1H NMR (CDCl3, 300 MHz): d 7.99 (2H, d, J = 9.0 Hz),
7.95 (1H, dd, J = 1.8, 9.0 Hz), 7.82 (1H, s), 7.78 (2H, dd, J = 15.0,
15.0 Hz), 7.60 (4H, dd, J = 8.7, 8.7 Hz), 7.44 (2H, dd, J = 15.0,
15.0 Hz), 6.92 (7H, m), 3.87 (2H, s), 3.84 (6H, s), 3.48 (4H, s), 2.76
(4H, s). 13C NMR (CDCl3, 75 MHz): d 188.3, 188.1, 162.1, 161.4,
161.3, 153.4, 143.6, 143.1, 130.4 (Â3), 130.3, 130.1, 130.0 (Â2),
129.9 (Â3), 129.1, 127.8, 127.6, 120.6, 119.4, 119.2, 116.0, 114.2
(Â3), 113.9 (Â2), 60.9, 55.3 (Â2), 52.0 (Â2), 47.1 (Â2).FABMS, m/z
(% rel. intensity): 589 (37) [M+H]+, 588 (17), 472 (34), 471 (100),
470 (47), 455 (21), 337 (16), 324 (14), 323 (53), 322 (17), 321
(42), 320 (14), 319 (12), 307 (15), 280 (41), 267 (20), 266 (26),
189 (19), 161 (61), 155 (15), 154 (67), 151 (13), 149 (34), 137
(32), 136 (53) 133 (19), 107 (19), 105 (10), 91 (13), 90 (14), 89
(18), 77 (20).HRFABMS m/z calcd for C37H37O5N2, 589.2702; found,
589.2705. Elemental Anal. Calcd C, 75.49; H, 6.16; N, 4.76. Found:
C, 74.61; H, 6.19; N, 4.80.
4.3.7. (E)-1-(4-Hydroxy-3-((4-(4-((E)-3-(1-methyl-1H-pyrrol-2-
yl)acryloyl)phenyl)piperazin-1-yl)methyl)phenyl)-3-(1-methyl-
1H-pyrrol-2-yl)prop-2-en-1-one (9)
Compound 1a (704 mg, 2.0 mmol) and N-methylpyrrole-2-
carboxaldehyde (436 mg, 4.0 mmol) were treated as described
above. The crude product was purified by CC eluting with hex-
anes/EtOAc (7:3) to yield 9 (pale yellow solid, 780 mg, 73%), mp
90–92 °C. IR (neat) 2877, 1642, 1599, 1525, 1480, 1416, 1383,
1336, 1279, 1223, 1192, 1111, 1059, 1033, 998, 927, 825, 744,
627, 605, 573 cmÀ1.1H NMR (DMS0-d6, 300 MHz): d 7.99 (2H, d,
J = 8.4 Hz), 7.98 (2H, s), 7.65 (2H, dd, J = 15.0, 15.0 Hz), 7.51 (2H, d,
J = 15.0 Hz), 7.02 (6H, m), 6.90 (1H, d, J = 9.0 Hz), 6.16 (2H, d,
J = 2.7 Hz), 3.76 (6H, s), 3.75 (2H, s), 3.40 (4H, s), 2.63 (4H, s). 13C
NMR (DMSO-d6, 75 MHz): d 186.6, 186.0, 161.1, 153.4, 131.1,
130.5 (Â2), 130.1 (Â2), 129.9, 129.8, 129.6, 129.5, 128.2, 128.0,
127.9, 122.6, 116.2, 116.0, 115.3, 113.4 (Â2), 112.7, 112.5, 109.2,
109.1, 57.7, 51.9 (Â2), 46.7 (Â2), 33.9 (Â2). FABMS, m/z (% rel. inten-
sity): 535 (9) [M+H]+, 428 (7), 307 (5), 296 (22), 295 (9), 294 (21),
253 (19), 241 (13), 240 (25), 239 (14), 189 (18), 155 (16), 154
(68), 153 (13), 149 (17), 147 (14), 137 (47), 136 (68), 134 (100),
133 (18), 131 (10), 121 (13), 120 (20), 107 (42), 106 (37), 105
(19), 91 (34), 90 (34), 89 (54), 81 (24), 78 (32), 77 (62), 73 (54),
71 (16), 69 (19), 65 (23), 63 (23), 57 (34), 55 (35).HRFABMS m/z
calcd for C33H35O3N4, 535.2709; found, 535.2709. Elemental Anal.
Calcd C, 74.13; H, 6.41; N, 10.48. Found: C, 73.97; H, 6.55; N, 10.14.
4.3.10. (E)-3-(Benzo[d][1,3]dioxol-5-yl)-1-(4-(4-(5-((E)-3-
(benzo[d][1,3]dioxol-5-yl)acryloyl)-2-hydroxybenzyl)piperazin-
1-yl)phenyl)prop-2-en-1-one (12)
Compound 1a (704 mg, 2.0 mmol) and 3,4-methylenedioxy-
benzaldehyde (600 mg, 4.0 mmol) were treated as described
above. The crude product was purified by CC eluting with (i-
Pr)2O/MeOH (9:1) to yield 12 (pale yellow solid, 765 mg, 62%),
mp 83–85 °C. IR (neat) 3005, 2893, 2831, 1654, 1597, 1492,
1446, 1361, 1307, 1249, 1192, 1111, 1037, 991, 929, 810, 756,
659, 601, 528 cmÀ1.1H NMR (CDCl3, 300 MHz): d 7.98 (2H, d,
J = 8.7 Hz), 7.93 (1H, dd, J = 2.1, 8.7 Hz), 7.71 (2H, dd, J = 15.6,
15.6 Hz), 7.38 (2H, d, J = 15.6 Hz), 7.14 (4H, m), 6.90 (2H, d,
J = 8.7 Hz), 6.82 (4H, m), 6.00 (4H, s), 3.85 (2H, s), 3.47 (4H, s),
2.74 (4H, s). 13C NMR (CDCl3, 75 MHz): d 188.1, 187.8, 162.2,
153.4, 149.6, 149.5, 148.2, 143.6, 143.1, 130.4 (Â2), 130.2, 130.0,
129.7, 129.5, 129.4, 129.0, 124.9, 124.8, 120.7, 119.8, 119.6,
116.0, 113.9 (Â2), 108.5 (Â2), 106.5 (Â2), 101.4 (Â2), 61.0, 52.0
(Â2), 47.2 (Â2). FABMS, m/z (% rel. intensity): 617 (18) [M+H]+,
616 (11), 486 (17), 485 (52), 484 (32), 483 (15), 337 (33), 335
(22), 307 (22), 294 (21), 281 (15), 280 (14), 189 (12), 175 (30),
165 (16), 155 (24), 154 (100), 152 (11), 149 (28), 138 (27), 137
(49), 136 (75), 135 (12), 120 (14), 107 (27), 91 (15), 90 (17), 89
(27), 77 (22).HRFABMS m/z calcd for C37H33O7N2, 616.2288; found,
616.2286. Elemental Anal. Calcd C, 72.07; H, 5.23; N, 4.54. Found:
C, 71.85; H, 5.20; N, 4.52.
4.3.8. (E)-1-(4-(4-(5-Cinnamoyl-2-hydroxybenzyl)piperazin-1-
yl)phenyl)-3-phenylprop-2-en-1-one (10)
Compound 1a (704 mg, 2.0 mmol) and benzaldehyde (424 mg,
4.0 mmol) were treated as described above. The crude product
was purified by CC eluting with hexanes/EtOAc (8:2) to yield 10
(pale yellow solid, 625 mg, 59%), mp 179–181 °C. IR (neat) 3414,
2831, 1651, 1600, 1527, 1492, 1446, 1346, 1280, 1226, 1188,
1033, 995, 925, 825, 767, 736, 694 cmÀ1.1H NMR (CDCl3,
300 MHz): d 8.01 (2H, d, J = 9.0 Hz), 7.96 (1H, dd, J = 9.0, 1.8 Hz),

M. Vijaya Bhaskar Reddy et al. / Bioorg. Med. Chem. 19 (2011) 1895–1906
1903
4.3.11. (E)-3-(2-Chlorophenyl)-1-(4-(4-(5-((E)-3-(2-chloro-
phenyl)acryloyl)-2-hydroxybenzyl)piperazin-1-yl)phenyl)prop-
2-en-1-one (13)
(11), 138 (29), 137 (61), 136 (70), 120 (11), 107 (20), 91 (11), 90
(13), 89 (18), 77 (16).HRFABMS m/z calcd for C32H31O4N2S2,
571.1725; found, 571.1726. Elemental Anal. Calcd C, 67.34; H,
5.30; N, 4.91; S, 11.24. Found: C, 66.75; H, 5.38; N, 4.90; S, 10.76.
Compound 1a (704 mg, 2.0 mmol) and 2-chlorobenzaldehyde
(560 mg, 4.0 mmol) were treated as described above. The crude
product was purified by CC eluting with benzene/acetone (9:1) to
yield 13 (pale yellow solid, 680 mg, 57%), mp 34–36 °C. IR (neat)
3012, 2831, 1654, 1597, 1442, 1388, 1319, 1276, 1222, 1192,
1122, 1029, 995, 925, 864, 825, 756, 667, 570 cmÀ1.1H NMR (CDCl3,
300 MHz): d 8.31 (2H, dd, J = 15.6, 15.6 Hz), 7.99 (2H, d, J = 9.0 Hz),
7.94 (1H, dd, J = 2.1, 9.0 Hz), 7.80 (2H, d, J = 15.0 Hz), 7.73 (2H, m),
7.54 (4H, m), 7.31 (4H, m), 6.91 (2H, d, J = 9.0 Hz), 3.87 (2H, s),
3.44 (4H, s), 2.76 (4H, s). 13C NMR (CDCl3, 75 MHz): d 188.3,
187.8, 162.5, 153.6, 139.6, 139.1, 135.3, 135.2, 133.5, 133.3, 131.0,
130.9 (Â3), 130.5, 130.2, 130.0, 129.6, 128.6, 127.6 (Â3), 127.0,
126.9, 124.6, 124.5, 120.8, 116.1, 113.9 (Â2), 61.0, 52.0 (Â2), 47.1
(Â2).FABMS, m/z (% rel. intensity): 597 (39) [M+H]+, 596 (22), 459
(8), 327 (17), 325 (12), 307 (21), 289 (12), 271 (13), 165 (20), 155
(27), 154 (100), 139 (11), 138 (26), 137 (54), 136 (64), 120 (11),
107 (19), 106 (7), 91 (11), 89 (17), 77 (15).HRFABMS m/z calcd for
4.3.14. (E)-1-(4-Hydroxy-3-methoxy-5-((4-(4-((E)-3-(3-methyl-
thiophen-2-yl)acryloyl)phenyl)piperazin-1-yl)methyl)phenyl)-
3-(3-methylthiophen-2-yl)prop-2-en-1-one (16)
Compound 2a (955 mg, 2.5 mmol) and 3-methyl-2-thiophene-
carboxaldehyde (630 mg, 5.0 mmol) were treated as described
above. The crude product was purified by CC eluting with ben-
zene/acetone (9:1) to yield 16 (pale yellow solid, 975 g, 65%), mp
76–78 °C. IR (neat) 2831, 1647, 1597, 1523, 1450, 1388, 1346,
.
1288, 1222, 1188, 1037, 825, 752, 524 cmÀ1 1H NMR (DMS0-d6,
300 MHz):
d
7.96 (2H, d, J = 9.0 Hz), 7.86 (2H, dd, J = 15.3,
15.3 Hz), 7.65 (3H, m), 7.53 (1H, s), 7.40 (2H, dd, J = 15.0, 15.0 Hz),
7.02 (4H, m), 3.88 (3H, s), 3.79 (2H, s), 3.42 (4H, s), 2.64 (4H, s),
2.36 (6H, s). 13C NMR (DMSO-d6, 75 MHz): d 186.3, 185.7, 153.4,
151.1, 147.4, 141.8, 141.6, 133.9, 133.8, 133.6, 133.1, 131.2 (Â2),
130.0 (Â2), 128.5, 127.6, 127.4, 127.3, 123.1, 121.4, 119.3, 119.2,
113.3 (Â2), 110.1, 58.1, 55.6, 51.6 (Â2), 46.4 (Â2), 13.8 (Â2). FABMS,
m/z (% rel. intensity): 599 (87) [M+H]+, 598 (55), 597 (22), 475 (22),
394 (29), 393 (49), 391 (35), 323 (20), 322 (68), 313 (38), 311 (33),
307 (16), 289 (15), 288 (13), 287 (34), 270 (29), 189 (20), 154 (100),
151 (66), 149 (93), 137 (58), 123 (18), 113 (16), 109 (24), 108 (11),
107 (34), 95 (39), 91 (41), 83 (33), 81 (38), 77 (33), 69
C35H31O3N2Cl2, 597.1712; found, 597.1714. Elemental Anal. Calcd
C, 70.35; H, 5.06; N, 4.69. Found: C, 70.03; H, 5.65; N, 4.40.
4.3.12. (E)-3-(2,4-Dichlorophenyl)-1-(4-(4-(5-((E)-3-(2,4-
dichlorophenyl)acryloyl)-2-hydroxybenzyl)piperazin-1-yl)-
phenyl)prop-2-en-1-one (14)
Compound 1a (704 mg, 2.0 mmol) and 2,4-chlorobenzaldehyde
(692 mg, 4.0 mmol) were treated as described above. The crude
product was purified by CC eluting with benzene/acetone (9:1) to
yield 14 (pale yellow solid, 710 mg, 53%), mp 41–43 °C. IR (neat)
3417, 2843, 2349, 1651, 1597, 1527, 1469, 1384, 1346, 1315,
1222, 1192, 1103, 1049, 995, 925, 813, 732, 671 cmÀ1.1H NMR
(CDCl3, 300 MHz): d 8.04 (2H, dd, J 15.6, 15.6 Hz), 7.99 (2H, d,
J = 9.0 Hz), 7.93 (1H, dd, J = 2.1, 9.0 Hz), 7.81 (1H, d, J = 1.8 Hz),
7.68 (2H, d, J = 9.0 Hz), 7.47 (2H, dd, J = 15.0, 15.0 Hz), 7.45 (1H,
s), 7.36 (1H, s), 7.29 (2H, s), 6.92 (3H, d, J = 9.0 Hz), 3.89 (2H, s),
3.47 (4H, s), 2.78 (4H, s). 13C NMR (CDCl3, 75 MHz): d 188.0,
187.6, 162.6, 153.7, 138.4, 137.9, 136.2, 136.0, 135.9, 135.8,
132.1, 132.0, 130.7 (Â2), 130.6, 130.1, 130.0, 129.5, 128.5, 128.4
(Â2), 128.3 (Â2), 127.4 (Â2), 124.9, 124.8, 120.9, 116.2, 113.9,
61.1, 52.1 (Â2), 47 (Â2). FABMS, m/z (% rel. intensity): 667 (13),
666 (10), 665 (11), [M+H]+, 307 (27), 289 (12), 199 (5), 155 (25),
154 (100), 153 (5), 139 (10), 138 (27), 137 (55), 136 (66), 120
(10), 107 (17), 90 (10), 9 (14), 77 (12).HRFABMS m/z calcd for
(44).HRFABMS m/z calcd for C34H35O4N2S2, 599.2038; found,
599.2038. Elemental Anal. Calcd C, 68.20; H, 5.72; N, 4.68; S, 10.7.
Found, C, 67.86; H, 6.03; N, 4.34; S, 10.65.
4.3.15. (E)-1-(4-(4-(5-Cinnamoyl-2-hydroxy-3-methoxy-
benzyl)piperazin-1-yl)phenyl)-3-phenylprop-2-en-1-one (17)
Compound 2a (955 mg, 2.5 mmol) and benzaldehyde (530 mg,
5.0 mmol) were treated as described above. The crude product
was purified by CC eluting with (i-Pr)2O/MeOH (9:1) to yield 15
(pale yellow solid, 820 mg, 59%), mp 43-45 °C. IR (neat) 3437,
2873, 1651, 1600, 1527, 1492, 1450, 1350, 1222, 1192, 1103,
999, 925, 806, 767, 690, 675, 563 cmÀ1.1H NMR (CDCl3,
300 MHz):
d 8.01 (2H, d, J = 9.0 Hz), 7.81 (2H, dd, J = 15.0,
15.0 Hz), 7.65 (5H, m), 7.56 (2H, dd, J = 15.0, 15.0 Hz), 7.42 (7H,
m), 6.92 (2H, d, J = 9.0 Hz), 3.98 (3H, s), 3.90 (2H, s), 3.45 (4H, s),
2.77 (4H, s). 13C NMR (DMSO-d6, 75 MHz): d 188.2, 188.0, 153.5,
152.0, 148.2, 143.8, 143.3, 135.1, 135.0, 130.6 (Â2), 130.3, 130.1,
129.6, 128.8 (Â4), 128.3 (Â2), 128.2 (Â3), 122.6, 121.8, 121.5,
120.0, 113.9 (Â2), 111.0, 60.8, 56.0, 52.0 (Â2), 47.2 (Â2). FABMS,
m/z (% rel. intensity): 559 (72) [M+H]+, 558 (46), 557 (17), 455
(13), 396 (23), 307 (18), 295 (12), 293 (36), 292 (13), 291 (29),
290 (11), 289 (19), 267 (40), 189 (13), 174 (19), 155 (24), 154
(100), 152 (13), 138 (28), 136 (78), 131 (73), 121 (20), 107 (29),
106 (10), 105 (15), 91 (24), 77 (32).HRFABMS m/z calcd for
C35H29O3N2Cl4, 665.0932; found, 665.0929. Elemental Anal. Calcd
C, 63.08; H, 4.23; N, 4.20. Found: C, 62.79; H, 4.44; N, 4.00.
4.3.13. (E)-1-(4-Hydroxy-3-methoxy-5-((4-(4-((E)-3-(thiophen-
2-yl)acryloyl)phenyl)piperazin-1-yl)methyl)phenyl)-3-
(thiophen-2-yl)prop-2-en-1-one (15)
Compound 2a (955 mg, 2.5 mmol) and 2-thiophenecarboxalde-
hyde (560 mg, 5.0 mmol) were treated as described above. The
crude product was purified by CC eluting with (i-Pr)2O/MeOH
(9:1) to yield 15 (pale yellow solid, 1.02 g, 72%), mp 109–111 °C.
IR (neat) 3082, 2831, 1647, 1597, 1523, 1454, 1419, 1350, 1288,
C36H35O4N2, 559.2597; found, 559.2600. Elemental Anal. Calcd C,
77.40; H, 6.13; N, 5.01. Found: C, 76.81; H, 6.36; N, 4.67.
4.3.16. (E)-1-(4-Hydroxy-3-methoxy-5-((4-(4-((E)-3-(4-meth-
oxyphenyl)acryloyl)phenyl)piperazin-1-yl)methyl)phenyl)-3-
(4-methoxyphenyl)prop-2-en-1-one (18)
1226, 1192, 1165, 1033, 999, 968, 921, 817, 709, 567 cmÀ1 1H
.
NMR (CDCl3, 300 MHz): d 7.99 (2H, d, J = 9.0 Hz), 7.91 (2H, dd,
J = 15.3, 15.3 Hz), 7.57 (1H, s), 7.39 (3H, m), 7.33 (2H, dd, J = 15.3,
15.3 Hz), 7.32 (2H, d, J = 3.6 Hz), 7.06 (2H, d, J = 4.2 Hz), 6.91 (2H,
d, J = 9.0 Hz), 3.97 (3H, s), 3.94 (2H, s), 3.44 (4H, s), 2.78 (4H, s).
13C NMR (CDCl3, 75 MHz): d 187.5, 187.3, 153.4, 151.9, 148.1,
140.5, 140.4, 136.2, 135.7, 131.7, 131.4, 130.4 (Â2), 129.5, 128.7,
128.3, 128.2, 128.1 (Â2), 122.4, 120.5, 120.1, 119.9, 113.8 (Â2),
110.8, 60.8, 55.9, 51.9 (Â2), 47.1 (Â2). FABMS, m/z (% rel. intensity):
571 (24) [M+H]+, 570 (15), 477 (20), 476 (13), 307 (25), 299 (13),
297 (11), 289 (15), 273 (11), 256 (11), 155 (25), 154 (100), 139
Compound 2a (955 mg, 2.5 mmol) and 4-methoxybenzalde-
hyde (680 mg, 5.0 mmol) were treated as described above. The
crude product was purified by CC eluting with benzene/acetone
(9:1) to yield 18 (pale yellow solid, 1.08 gm, 70%), mp 89–91 °C.
IR (neat) 2931, 2835, 1651, 1597, 1512, 1454, 1419, 1388, 1346,
1300, 1257, 1168, 1111, 1029, 995, 925, 821, 756, 671, 528 cm
À1.1H NMR (DMS0-d6, 300 MHz): d 7.97 (2H, d, J = 9.0 Hz), 7.72
(2H, dd, J = 15.6, 15.6 Hz), 7.59 (2H, d, J = 15.6 Hz), 7.56 (3H, m),
7.42 (3H, m), 6.90 (6H, m), 3.95 (3H, s), 3.82 (8H, s), 3.41 (4H, s),
2.75 (4H, s). 13C NMR (DMSO-d6, 75 MHz): d 188.2, 188.0, 161.4,

1904
M. Vijaya Bhaskar Reddy et al. / Bioorg. Med. Chem. 19 (2011) 1895–1906
161.2, 153.3, 151.7, 148.0, 143.6, 143.1, 130.4 (Â2), 130.0 (Â2),
129.9 (Â2), 129.1, 127.8, 127.6, 122.3, 119.9, 119.4, 119.0, 114.2
(Â5), 113.9 (Â2), 110.9, 60.9, 56.0, 55.2 (Â2), 52.0 (Â2), 47.2
(Â2). FABMS, m/z (% rel. intensity): 619 (44) [M+H]+, 618 (29),
617 (11), 323 (17), 322 (6), 321 (15), 307 (24), 298 (5), 297 (14),
293 (4), 289 (12), 280 (13), 161 (32), 155 (24), 154 (100), 153
(6), 152 (8), 139 (9), 138 (25), 137 (52), 136 (67), 121 (9), 120
(10), 107 (18), 91 (10), 90 (12), 89 (17), 77 (15).HRFABMS m/z calcd
for C38H39O6N2, 619.2808; found, 619.2808. Elemental Anal. Calcd
C, 73.77; H, 6.19; N, 4.53. Found: C, 73.21; H, 6.37; N, 4.44.
uct was used directly in the Mannich base reaction. Compound 1b
(1.065 g, 5 mmol), 4-piperizinoacetophenone (1.02 g, 5 mmol) and
paraformaldehyde (150 mg, 5 mmol) were treated as described
above. The crude product was purified by CC eluting with hex-
anes/EtOAc (7:3) to yield 21 (pale yellow solid, 1.5 g, 70%), mp
66-68 °C. IR (neat) 1670, 1643, 1597, 1570, 1531, 1450, 1411,
1357, 1284, 1242, 1126, 1029, 975, 821, 752, 686, 605 cmÀ1.1H
NMR (DMS0-d6, 300 MHz): d 11.64 (1H, s, NH), 7.94 (2H, d,
J = 9.0 Hz), 7.79 (2H, dd, J = 8.1, 1.5 Hz), 7.57 (1H, d, J = 15.3 Hz),
7.50 (1H, d, J = 15.3 Hz), 7.09 (1H, s), 7.03 (2H, d, J = 9.0 Hz), 6.87
(1H, d, J = 8.1 Hz), 6.65 (1H, s), 6.20 (1H, s), 3.70 (2H, s), 3.40
(4H,s), 2.61 (4H, s), 2.50 (3H, s). 13C NMR (DMSO-d6, 75 MHz): d
196.3, 186.2, 161.3, 153.5, 132.7, 130.5, 130.0 (Â2), 129.6, 129.3,
128.5, 128.0, 123.6, 122.5, 115.5, 115.3, 114.9, 113.5 (Â2), 110.4,
57.4, 51.9 (Â2), 46.6 (Â2), 26.3. FABMS, m/z (% rel. intensity):
430 (11) [M+H]+, 307 (35), 289 (18), 155 (25), 154 (100), 139
(10), 138 (26), 137 (48), 136 (63), 107 (16), 89 (13).HRFABMS m/
z calcd for C26H28O3N3, 430.2131; found, 430.2130. Elemental Anal.
Calcd C, 72.71; H, 6.34; N, 9.78. Found: C, 71.94; H, 6.45; N, 9.52.
4.3.17. (E)-3-(2-Chlorophenyl)-1-(4-(4-(5-((E)-3-(2-chloro-
phenyl)acryloyl)-2-hydroxy-3-methoxybenzyl)piperazin-1-
yl)phenyl)prop-2-en-1-one (19)
Compound 2a (955 mg, 2.5 mmol) and 2-chlorobenzaldehyde
(700 mg, 5.0 mmol) were treated as described above. The crude
product was purified by CC eluting with benzene/acetone (9:1) to
yield 20 (pale yellow solid, 925 gm, 59%), mp 42–44 °C. IR (neat)
2873, 1654, 1593, 1450, 1350, 1292, 1222, 1192, 1168, 1107,
1053, 999, 921, 759, 671, 570 cmÀ1.1H NMR (CDCl3, 300 MHz): d
8.13 (2H, d, J = 15.6 Hz), 7.97 (2H, d, J = 9.0 Hz), 7.73 (2H, m),
7.57 (1H, d, J = 1.8 Hz), 7.50 (2H, dd, J = 15.3, 15.3 Hz), 7.47 (3H,
m), 7.32 (4H, m), 6.90 (2H, d, J = 9.0 Hz), 3.96 (3H, s), 3.85 (2H, s),
3.44 (4H, s), 2.78 (4H, s). 13C NMR (CDCl3, 75 MHz): d 188.3,
187.8, 153.5, 152.1, 148.2, 139.6, 139.0, 135.2, 133.5, 133.3,
130.9, 130.7 (Â3), 130.1, 129.3 (Â2), 127.6 (Â3), 127.0, 126.9,
124.6, 124.5, 122.8, 120.0, 113.9 (Â3), 111.1, 60.9, 56.0, 52.0
(Â2), 47.2 (Â2). FABMS, m/z (% rel. intensity): 627 (37) [M+H]+,
626 (23), 329 (10), 327 (20), 325 (16), 307 (18), 301 (20), 289
(12), 284 (12), 167 (11), 165 (29), 155 (27), 154 (100), 139 (13),
138 (30), 137 (61), 136 (74), 120 (14), 107 (23), 105 (10), 91
(15), 90 (14), 89 (20), 77 (17).HRFABMS m/z calcd for
4.3.20. (E)-1-(3-((4-(4-Acetylphenyl)piperazin-1-yl)methyl)-4-
hydroxyphenyl)-3-(1-methyl-1H-pyrrol-2-yl)prop-2-en-1-one
(22)
Compound 1 (1.36 gm, 10 mmols) and N-methyl-pyrrole-2-
carboxaldehyde (1.09 g, 10 mmol) were treated as described above
in the synthesis of chalcones, to obtain 1c in 79% yield. The crude
product was used directly in the Mannich base reaction. Com-
pound 1c (1.14 g, 5 mmol), 4-piperizinoacetophenone (1.02 g,
5 mmol), and paraformaldehyde (150 mg, 5 mmol) were treated
as described above. The crude product was purified by CC eluting
with (i-Pr)2O/MeOH (9:1) to yield 22 (pale yellow solid, 1.45 gm,
65%), mp 166-168 °C. IR (neat) 3390, 2873, 1597, 1527, 1350,
1284, 1192, 1103, 1037, 925, 806, 675 cmÀ1.1H NMR (DMS0-d6,
300 MHz): d 7.99 (2H, d, J = 9.0 Hz), 7.83 (1H, s), 7.79 (1H, s),
7.64 (1H, d, J = 15.0 Hz), 7.51 (1H, d, J = 15.0 Hz), 7.02 (3H, d,
J = 9.0 Hz), 6.99 (1H, d, J = 3.3 Hz), 6.88 (1H, d, J = 9.0 Hz), 6.16
(1H, t, J = 3.3 Hz), 3.76 (3H, s), 3.72 (2H, s), 3.40 (4H, s), 2.62 (4H,
s), 2.49 (3H, s). 13C NMR (DMSO-d6, 75 MHz): d 196.1, 186.0,
161.2, 153.4, 130.5, 130.3, 130.1 (Â2), 129.8, 129.5, 128.4, 128.0,
127.8, 122.5, 116.2, 115.1, 113.3 (Â2), 112.4, 109.0, 57.3, 51.9
(Â2), 46.6 (Â2), 33.9, 26.2. FABMS, m/z (% rel. intensity): 444 (66)
[M+H]+, 443 (35), 442 (15), 337 (12), 307 (21), 296 (12), 294
(13), 289 (11), 253 (12), 239 (13), 155 (25), 149 (24), 139 (11),
138 (28), 137 (53), 136 (71), 134 (40), 120 (14), 107 (25), 106
(14), 90 (15), 89 (21), 77 (19).HRFABMS m/z calcd for
C36H33O4N2Cl2, 627.1817; found, 627.1815. Elemental Anal. Calcd
C, 68.09; H, 5.14; N, 4.46. Found: C, 67.87; H, 5.27; N, 4.34.
4.3.18. (E)-1-(4-Hydroxy-3-methoxy-5-((4-(4-((E)-3-(pyridin-2-
yl)acryloyl)phenyl)piperazin-1-yl)methyl)phenyl)-3-(pyridin-2-
yl)prop-2-en-1-one (20)
Compound 2a (955 mg, 2.5 mmol) and 2-pyridinecarboxalde-
hyde (535 mg, 5.0 mmol) were treated as described above. The
crude product was purified by CC eluting with benzene/acetone
(9:1) to yield 19 (pale yellow solid, 1.02 gm, 73%), mp 84–86 °C.
IR (neat) 2831, 1658, 1589, 1527, 1492, 1465, 1431, 1330, 1222,
1192, 1165, 995, 921, 783, 748, 667 cmÀ1.1H NMR (CDCl3,
300 MHz): d 8.66 (2H, t, J = 4.2 Hz), 8.14 (1H, d, J = 3.0 Hz), 8.06
(2H, d, J = 16.2 Hz), 8.05 (1H, d, J = 4.2 Hz), 7.75 (1H, d,
J = 15.6 Hz), 7.73 (1H, d, J = 15.6 Hz), 7.68 (2H, d, J = 1.2 Hz), 7.62
(1H, s), 7.52 (1H, s), 7.45 (2H, m), 7.28 (2H, m), 6.89 (2H, d,
J = 9.0 Hz), 3.96 (3H, s), 3.87 (2H, s), 3.44 (4H, s), 2.76 (4H, s). 13C
NMR (CDCl3, 75 MHz): d 187.9, 187.8, 153.5, 153.3, 153.1, 152.2,
149.9 (Â2), 148.1, 141.6, 141.2, 136.8, 136.7, 130.8 (Â2), 129.3,
128.4, 125.4 (Â2), 125.1, 124.9, 124.1, 124.0, 123.0, 119.9, 113.7
(Â2), 110.8, 60.7, 55.9, 51.9 (Â2), 47.0 (Â2). FABMS, m/z (% rel.
intensity): 561 (77) [M+H]+, 560 (47), 559 (20), 456 (11), 380
(17), 307 (13), 295 (12), 294 (40), 293 (13), 292 (35), 291 (20),
290 (14), 268 (50), 251 (26), 155 (24), 154 (81), 138 (29), 136
(68), 132 (100), 109 (21), 107 (33), 106 (40), 105 (26), 91 (30),
81 (35), 77 (26), 69 (48), 57 (47), 55 (46).HRFABMS m/z calcd for
C27H30O3N3, 444.2287; found, 444.2287. Elemental Anal. Calcd C,
73.11; H, 6.59; N, 9.47. Found: C, 72.63; H, 6.69; N, 9.47.
4.3.21. (E)-1-(3-((4-(4-Acetylphenyl)piperazin-1-yl)methyl)-4-
hydroxy-5-methoxyphenyl)-3-(pyridin-3-yl)prop-2-en-1-one
(23)
Compound 2 (1.66 g, 10 mmol) and 3-pyridinecarboxaldehyde
(1.07 g, 10 mmol) were treated as described above in the general
procedure for chalcones to obtain 2b in 75% yield. The crude prod-
uct was used directly in the Mannich base reaction. Compound 2b
(1.28 g, 5.0 mmol), 4-piperizinoacetophenone (1.02 g, 5.0 mmol),
and paraformaldehyde (150 mg, 5.0 mmol) were treated as de-
scribed above. The crude product was purified by CC eluting with
(i-Pr)2O/MeOH (9:1) to yield 23 (pale yellow solid, 1.45 gm, 62%),
mp 146–148 °C. IR (neat) 2831, 1658, 1589, 1489, 1450, 1411,
1357, 1300, 1222, 1192, 1145, 1107, 1083, 999, 921, 806, 752,
678, 628, 563 cmÀ1.1H NMR (CDCl3, 300 MHz): d 8.85 (1H, s),
8.60 (1H, s), 7.99 (2H, d, J = 9.0 Hz), 7.93 (1H, d, J = 7.5 Hz), 7.75
(1H, d, J = 15.6 Hz), 7.60 (1H, d, J = 15.6 Hz), 7.47 (1H, d,
J = 1.2 Hz), 7.35 (1H, m), 7.32 (1H, s), 6.91 (2H, d, J = 9.0 Hz), 3.93
(3H, s), 3.85 (2H, s), 3.45 (4H, s), 2.76 (4H, s), 2.54 (3H, s). 13C
C34H33O4N4, 561.2502; found, 561.2501. Elemental Anal. Calcd C,
72.84; H, 5.75; N, 9.99. Found: C, 72.40; H, 6.07; N, 9.55.
4.3.19. (E)-1-(3-((4-(4-Acetylphenyl)piperazin-1-yl)methyl)-4-
hydroxyphenyl)-3-(1H-pyrrol-2-yl)prop-2-en-1-one (21)
Compound 1 (1.36 g, 10 mmol) and pyrrole2-carboxaldehyde
(950 mg, 10 mmol) were treated as described above for the general
procedure for chalcones to obtain 1b in 82% yield. The crude prod-

M. Vijaya Bhaskar Reddy et al. / Bioorg. Med. Chem. 19 (2011) 1895–1906
1905
NMR (CDCl3, 75 MHz): d 196.5, 187.3, 153.7, 151.9, 150.7, 149.7,
147.9, 139.4, 134.4, 130.7 (Â3), 128.8, 128.4, 123.6, 122.6, 119.8,
113.9 (Â3), 110.5, 60.9, 56.0, 52.0 (Â2), 47.2 (Â2), 26.1.FABMS,
m/z (% rel. intensity): 472 (18) [M+H]+, 392 (13), 391 (46), 307
(11), 167 (25), 155 (25), 154 (87), 152 (12), 150 (17), 149 (100),
139 (17), 138 (35), 137 (64), 136 (66), 124 (10), 121 (15), 120
(14), 113 (23), 107 (28), 97 (22), 95 (29), 91 (23), 89 (18), 77
(18), 69 (40), 57 (51), 55 (38).HRFABMS m/z calcd for
Wellesley, MA, USA) and was quantitated using an image
program21 (Multi Gauge v2.2 software, Fujifilm, Tokyo, Japan).
4.8. Cytotoxic activity assay
All stock cultures were grown in T-25 flasks. Freshly trypsinized
cell suspensions were seeded in 96-well microtiter plates at densi-
ties of 1500–7500 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. The absorbance at 562 nm was measured
using a microplate reader after solubilizing the bound dye. The
mean EC50 is the concentration of agent that reduces cell growth
by 50% under the experimental conditions and 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: A549 (non small cell lung cancer), HCT-8
(ileocecal), KB (nasopharyngeal carcinoma) and DU145 (prostate
cancer). All cell lines were obtained from the Lineberger Compre-
hensive Cancer Center (UNC-CH) or from ATCC (Rockville, MD)
and were cultured in RPMI-1640 medium supplemented with
C28H30O4N3, 472.2236; found, 472.2239. Elemental Anal. Calcd C,
71.32; H, 6.20; N, 8.91. Found, C, 70.43; H, 6.16; N, 8.74.
4.4. Measurement of NADPH oxidase activity
NADPH oxidase activity was measured as described previ-
ously.10 Test compounds were added to the wells of a biolumines-
cence plate and incubated with 50
20 min at 37 °C in the dark. O2 production was stimulated with
200 M NADPH, and the chemiluminescence was monitored for
lg of cell homogenate for
l
30 min, after which the AUC (area under the curve) was calculated
to represent reactive oxygen species production (NADPH
activity).
25 lM HEPES, 0.25% sodium bicarbonate, and 10% fetal bovine
4.5. Measurement of 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical-scavenging capacity
serum.22
4.9. Cell culture
The DPPH radical-scavenging capacity assay was performed in
our previous report.20 The DPPH solution (200
tration of 200 lM in MeOH) was added to 10 lL of diluted drugs in
lL, at a final concen-
Human colon adenocarcinoma cell line HT-29 was obtained
from the American Type Culture Collection. Cells were maintained
in RPMI-1640 containing 100 M)mL/L FBS with 100,000 U/L
penicillin and 100 mg/L streptomycin.
each well of a 96-well microplate, and the resulting solution was
allowed to react for 30 min in the dark at rt. The absorbance
(A517 OD units) is defined as the optical density (OD) measured
at 517 nm caused by the DPPH radical as determined using a Power
Wave™ XS (BioTek) microplate-spectrophotometer. The radical-
scavenging capacity is expressed as the change in the OD517 over
4.10. MTT assay
HT-29 cells were plated onto 96-well plates and exposure to
compound 23 as detailed concentration in respective experiments
for 24 and 48 h. Then, MTT was added to each well then incubated
for an additional 4 h in the dark at 37 °C. The medium was then
aspirated from the wells and the blue formazon product was dis-
30 min (D A517 OD units/30 min). The antioxidant, Trolox (OXIS,
USA), was included as a reference compound.
4.6. Microglial cell culture and measurement of nitric oxide
(NO)
solved in 100 lL of DMSO. The plates were analyzed at O.D.
570 nm using a spectrophotometric plate reader (Bio-Rad, Tokyo,
Japan). Each data point was replicated in triplicate. Percentage of
cell viability was calculated as (O.D. of drug-treated sample/O.D.
of none treated sample) Â 100%.
A murine microglial cell line (BV2) was cultured in Dulbecco’s
modified Eagle medium (DMEM; Gibco, USA) supplemented with
5% fetal bovine serum (Hyclone, USA). The production of NO was
determined by measuring the accumulation of nitrite in the culture
medium 24 h after stimulation with LPS (0.5
lg/mL) by the Griess
4.11. DAPI staining
reagent as in our previous report.10
After compound 23 (30
paraformaldehyde for 30 min, and incubated with 1 l
l
M) treatment, cells were fixed in 4%
g/mL of DAPI
4.7. Western immunoblot analysis of iNOS and NF-
jB p65
staining solution for 30 min in the dark. The apoptotic cells were
observed through fluorescence microscopy (Zeiss, Oberköchen,
Germany).
Equal amounts of protein (50
g/mL LPS-treated samples in the absence or presence of
compound 4 or 11 (0.5–1.0 M) were subjected to sodium dode-
lg) at different time points from
0.5
l
l
cylsulfate polyacrylamide gel electrophoresis (SDS–PAGE) and
electro-transferred to a hydrophobic polyvinylidene difluoride
(PVDF) membrane. After blocking with 5% nonfat milk in
phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBST)
at 4 °C for 1 h, the membrane was washed three times with PBST
and incubated overnight at 4 °C with an antibody against iNOS
4.12. Assessment for apoptosis
The induction of cell apoptosis was assayed by Tdt-mediated
deoxyuridine triphosphate nick end labeling (TUNEL) staining.
Cells were seeded into each well of 6-well plate. After the com-
pound 23 time-dependent 24 and 48 h treatments, cells were har-
vested and were then immediately incubated with working
strength terminal deoxynucleotidyl transferase (Tdt) enzyme in a
humidified chamber at 37 °C for 1 h. Cells were immersed in
stop/wash buffer and gently rinsed with PBS. FITC-labeled antidi-
goxigenin conjugate was then applied to cells and incubated at
37 °C for 30 min in the dark. Cells were washed with PBS then
TUNEL positive cells were determined by flow cytometry (FACSCal-
ibur, Becton Dickinson).
(BD Pharmingen, BD Biosciences, San Diego, CA, USA) and NF-jB
p65 (BD Transduction Laboratories, BD Biosciences, San Diego,
CA, USA) at a properly titrated dilution (1:1000–2500). After
additional washes with PBST, the membrane was incubated with
a second antibody IgG conjugated with horseradish peroxidase
(Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at rt. The
immunoblot on the membrane was visible after development with
an enhanced chemiluminescence (ECL) system (Perkin–Elmer,

1906
M. Vijaya Bhaskar Reddy et al. / Bioorg. Med. Chem. 19 (2011) 1895–1906
4.13. Western blotting
the National Cheng Kung University, Tainan, Taiwan ROC awarded
to T. S. Wu. Partial support was received from NIH grant CA-17625
awarded to K. H. Lee.
Proteins (30 lg) were resolved on SDS–PAGE and transferred
onto a polyvinylidene fluoride membrane (PVDF; Millipore). After
blocking, the blots were incubated with an appropriate dilution
of specific monoclonal antibodies for Fas/CD95, FADD, cytochrome
c, Apaf-1, pro-caspase-9, pro-caspase-8, caspase-3, AIF, Endo G,
Bax, Bcl-2, t-Bid (Santa Cruz Biotechnology, USA) for 12 h. Blots
were washed three times and then incubated with horseradish
peroxidase-conjugated secondary antibody (Santa Cruz Biotech-
nology, USA). The specific protein was detected by using enhanced
chemiluminescence kits (Amersham, ECL Kits) (30).
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4.15. Statistical analysis
Student’s t-test was used to analyze differences between com-
pound 23 treated and control groups. ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p
<0.001.
Acknowledgments
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This work was supported by a grant of National Science Council,
Taiwan, Republic of China, and Grant No. (OUA 95-3-2-021) from