Antiproliferative properties of piperidinylchalcones

Article abstract of DOI:10.1016/j.bmc.2005.08.006

Methoxylated chalcones bearing N-methylpiperidinyl substituents on ring A inhibited the growth of human tumour cell lines (MCF, HCT 116, and Jurkat) at IC₅₀ values of <5 μM. Investigations on a representative member (12) showed that antiproliferative activity was linked to the disruption of the cell cycle at G1 and G2/M phases. The effect was concentration dependent and was evident at the approximate IC₅₀ of 12. Down regulation of cell cycle regulatory components (CDK4, cyclin B, E2F, and phosphorylated Rb) were observed under similar conditions. Methoxylated chalcones without the piperidinyl substituent were found to exert equally potent and selective antiproliferative activity against HCT 116 tumour cells but did not interfere with cell cycle progression at their IC₅₀ concentrations. The presence of the piperidinyl substituent in the chalcone template is proposed to lend specificity to the mechanism of antiproliferative activity, in addition to promoting a more desirable physicochemical profile.

Full text of DOI:10.1016/j.bmc.2005.08.006

Bioorganic & Medicinal Chemistry 14 (2006) 153–163
Antiproliferative properties of piperidinylchalcones
Xiaoling Liu and Mei-Lin Go*
Department of Pharmacy, Medicinal Chemistry Program, Office of Life Sciences, National University of Singapore, Singapore
Received 1 July 2005; revised 30 July 2005; accepted 1 August 2005
Available online 26 September 2005
Abstract—Methoxylated chalcones bearing N-methylpiperidinyl substituents on ring A inhibited the growth of human tumour cell
lines (MCF, HCT 116, and Jurkat) at IC50 values of <5 lM. Investigations on a representative member (12) showed that antipro-
liferative activity was linked to the disruption of the cell cycle at G1 and G2/M phases. The effect was concentration dependent and
was evident at the approximate IC50 of 12. Down regulation of cell cycle regulatory components (CDK4, cyclin B, E2F, and phos-
phorylated Rb) were observed under similar conditions. Methoxylated chalcones without the piperidinyl substituent were found
to exert equally potent and selective antiproliferative activity against HCT 116 tumour cells but did not interfere with cell cycle
progression at their IC50 concentrations. The presence of the piperidinyl substituent in the chalcone template is proposed to lend
specificity to the mechanism of antiproliferative activity, in addition to promoting a more desirable physicochemical profile.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
tion of methoxy groups to activity, their presence may
serve to reduce aqueous solubility and drug-like charac-
ter. One approach to moderate the hydrophobic influ-
ence of the methoxy groups would be to introduce
hydrophilic substituents into the chalcone template.
Basic amino functions, which are protonated at physio-
logical pH, may be useful in this regard. In this investi-
gation, we have synthesized a series of methoxylated
chalcones bearing a basic N-methylpiperidinyl substitu-
ent on ring A (Scheme 1, Series A–C), with the aim of
understanding how the presence of an ionizable basic
function would influence the antiproliferative properties
of these compounds. From a physicochemical stand-
point, the N-methylpiperidine ring (pKa ꢀ 10), which is
protonated at physiological pH, enhances the aqueous
solubility of a generally hydrophobic chalcone template.
The basic character of the piperidine ring may also have
the added advantage of promoting hydrogen bonding
and other interactions at the putative target site. Inter-
estingly, others have reported enhanced selectivity and
potency in the biological properties of chalcones with
basic amino functions.17,18 In one report, 20-aminochal-
cones demonstrated a significantly increased anti-tu-
mour activity compared with chalcones that lack this
function.17 Another investigation showed how the intro-
duction of cationic aliphatic amino groups in the chal-
cone scaffold enhanced selectivity and potency of the
resulting compounds against Gram-positive and -nega-
tive pathogens.18 Thus, it is of interest to elucidate fur-
ther the contribution of the basic amino function to
the biological activity of chalcones.
Chalcones are a class of privileged structures that have a
wide range of biological properties.1,2 An area of partic-
ular interest is their potential as anticancer agents,3–6 for
which several modes of action have been proposed.
These include inhibition of angiogenesis,7 interfering
with p53–MDM2 interaction,8,9 induction of mitochon-
drial uncoupling and membrane collapse,10 and disrup-
tion of the cell cycle.11–16 The antimitotic activity of
chalcones has been attributed to the reactive enone moi-
ety in the molecule which interacts with a critical thiol
residue at the colchicine binding site on tubulin, leading
to the inhibition of tubulin polymerization and disrup-
tion of mitosis.14,15 The association of methoxylated
chalcones with antimitotic activity is also noted in the
literature, arising, in part, from the observation that
known inhibitors of tubulin polymerization, such as
combretastatin A and colchicine, are rich in methoxy
groups.11,14,15 The methoxylated chalcone, E-1-
(30,40,50-trimethoxy phenyl)-2-methyl-3-(3-hydroxyl-4-
methoxyphenyl)prop-2-en-1-one (1), is probably the
most potent cytotoxic chalcone reported to date.16 It
has an IC50 of 0.21 nM against K562 human leukaemia
cells and is a potent inhibitor of microtubule assembly
(IC50 0.5 lM). Notwithstanding the beneficial contribu-
Keywords: Piperidinylchalcones; Antiproliferative activity; Cell cycle;
Expression of cell cycle regulatory proteins.
*
Corresponding author. Tel.: +65 687 426 54; fax: +65 677 915 5;
e-mail: phagoml@nus.edu.sg
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.006

154
X. Liu, M.-L. Go / Bioorg. Med. Chem. 14 (2006) 153–163
OCH
3
O
OCH
3
O
OCH
3
O
OCH
3
O
CH
3
CH
3
(i)
(i)
R
R
H
CO
3
OH
H CO
3
H
CO
OH
H
CO
3
3
Series A
Series B
12: R = 2-Cl
N
5: R = 2-Cl
6: R = 4-Cl
7: R = H
N
N
CH
N
CH
13: R = 4-Cl
14: R = H
CH
CH
3
3
3
3
8: R = 2-F
4
11
OCH
O
3
OCH
O
3
OCH
3
O
OCH
3
O
CH
3
(i)
CH
3
R
(i)
R
R
1
R
1
Series C
Series D
Series E
N
CH
18: R = 2-Cl
19: R = 4-Cl
20: R = H
R1 = H
R1 = OCH3
21: R = 2-Cl
22: R = 4-Cl
23: R = H
N
24: R = 2-Cl
25: R = 4-Cl
26: R = H
3
CH
3
17
Scheme 1. Reagents and conditions: (i) Aldehyde, 3% w/v NaOH, methanol, 12 h, rt.
gas in glacial acetic acid to give the unsaturated product
2 in moderate yield (65%).19 Catalytic reduction of the
double bond in 2 afforded 3, which was treated with ace-
tic anhydride and boron triflouride for the introduction
of the acetyl function. In the course of reaction, it was
found that one of the methoxy groups was lost through
demethylation to give compound 4. This compound was
reacted with commercially available benzaldehydes to
give the Series A chalcones 5–8 (Scheme 1). In a similar
manner, 1,3-dimethoxybenzene and methoxybenzene
were reacted with N-methyl-4-piperidinone to give com-
pounds 9 and 15, respectively. Catalytic hydrogenation
gave compounds 10 and 16 in good yield and subsequent
acetylation reaction was achieved without the loss of a
methoxy group to give acetophenones 11 and 17, respec-
tively. Claisen–Schmidt condensation of compounds 11
and 17 with benzaldehydes gave the desired Series B
(12–14) and Series C (18–20) chalcones in acceptable
yields. Chalcones that lack the N-methylpiperidine ring
(Series D and E) were also synthesized by base-catalyzed
O
CH
3
H
H
CO
CO
3
OH
3
OCH
3
OCH
3
1
2. Results
2.1. Synthesis of chalcones
Five series of chalcones (A–E) were synthesized by a
base-catalyzed Claisen–Schmidt reaction between an
appropriately substituted benzaldehyde and a methoxy-
lated acetophenone (Scheme 1). In the case of the Series
A–C piperidinyl chalcones, the methoxylated 3-(N-
methylpiperidinyl)acetophenones (4, 11, and 17) were
synthesized according to procedures shown in Scheme
2. 1,3,5-Trimethoxybenzene was reacted with 1-methyl-
4-piperidinone in the presence of hydrogen chloride
OCH
OCH
O
3
3
OCH
3
OCH
3
CH
3
(iii)
(i)
(ii)
R
R'
R
R'
R
R'
R
R'
R = R' = OCH3
R = OCH3, R' = H
R = R' = H
N
N
N
CH
3
CH
3
CH
3
R = R' = OCH3
3:
4: R = OCH3, R' = OH
R = R' = OCH3
2:
9:
R = OCH3, R' = H
R = R' = H
10:
16:
R = OCH3, R' = H
R = R' = H
11:
17:
R = OCH3, R' = H
R = R' = H
15:
Scheme 2. Reagents and conditions: (i) N-methylpiperidinone, HCl, acetic acid, 95–100 ꢁC, 3 h; (ii) 10% Pd/C, 40 psi, rt; (iii) BF3, acetic anhydride,
DCM.

X. Liu, M.-L. Go / Bioorg. Med. Chem. 14 (2006) 153–163
155
Table 1. Antiproliferative IC50 values and selectivity indices of Series A–E chalcones
Antiproliferative activity IC50 lMa
Selectivity index
C50 CCL/IC50 average IC50 AG 1523/IC50 average
MCF7
HCT 116
Jurkat
Averageb
CCL 186
AG 1523
Series A
5
6
7
8
3.1
6.7
5.7
3.4
3.1
2.6
4.2
3.0
1.6
2.4
2.2
1.7
2.6
3.9
4.0
2.7
3.1
6.1
7.3
4.6
6.4
9.8
9.3
5.2
1.2
1.6
1.8
1.7
2.5
2.5
2.3
1.9
Series B
12
13
14
2.7
2.5
3.4
3.4
2.9
3.9
2.5
1.8
2.9
2.9
2.4
3.4
7.6
3.6
7.8
7.1
6.9
2.6
1.5
2.3
2.4
2.9
3.9
13.2
Series C
18
19
20
2.9
3.2
4.2
2.3
3.3
3.6
2.0
1.4
2.9
2.4
2.6
3.6
6.0
5.2
9.8
11.3
9.4
2.5
2.0
2.7
4.7
3.6
3.9
14.0
Series D
21
22
23
5.2
6.2
4.4
12.8
11.3
13.7
2.4
1.8
3.1
Series E
24
25
26
5.2
2.7
5.5
11.7
4.3
2.3
1.6
3.3
18.0
27c
25.0
30.0
1.2
a Determined by MTT assay, 72 h incubation with test compound. Mean of at least three separate determinations. Flavorpiridol was used as a
control: IC50 0.02 lM (HCT116); 0.17 lM (CCL 186).
b Average of IC50 values from MCF-7, HCT 116 and Jurkat cell lines.
c 20,30,40-Trimethoxy-2,4-dimethoxychalcone.20
condensation of 2,4-dimethoxyacetophenone or 2-meth-
oxyacetophenone with the relevant benzaldehydes
(Scheme 1).
stitution pattern on ring B) made little difference to
activity. However, this conclusion should be tempered
with the observation that substitution on ring B had
essentially been limited to halogens (chlorine, fluorine)
in this study. A different finding may emerge when other
groups are introduced to ring B.
2.2. Inhibition of cell proliferation
The IC50 values of the target chalcones were determined
against three human cancer cell lines (HCT 116, MCF-7
and Jurkat) and two human diploid fibroblast cell lines
(fetal lung CCL 186, foreskin AG 1523) by the microcul-
ture tetrazolium assay, which is based on the ability of
metabolically active cells to reduce the yellow tetrazoli-
um salt to a coloured formazan product.20 The results
are given in Table 1, together with the average IC50 val-
ues against the three cancer cell lines and the selectivity
indices of these compounds when evaluated against the
normal human cells.
The importance of the N-methylpiperidine substituent
on ring A was explored by comparing the antiprolifera-
tive activities of Series B and C compounds (with N-
methylpiperidine) with those in Series D and E (without
N-methylpiperidine). Based on IC50 values for HCT116,
it is seen that with one exception (compounds 19 and
25), the presence of the N-methylpiperidine substituent
served to enhance activity only to a modest degree. As
in the case of the Series A–C chalcones, Series D and
E compounds do not show marked differences in their
IC50 values. In this respect, the poor activity of com-
pound 27 (20,30,40-trimethoxy-2,4-dimethoxychalcone,
IC50 25 lM)21 is notable, as it suggests an important role
for the substitution pattern on ring B.
As seen from Table 1, the N-methylpiperidinyl chal-
cones (Series A–C) had moderately good antiprolifera-
tive activity, with average IC50 values less than 5 lM.
An interesting observation is the limited variation in
IC50 values across Series A–C, despite the different sub-
stitution patterns on their rings A and B. Thus, equally
potent members were found in all three series, as can be
seen from the IC50 average values of 5 (Series A, 2.6 lM),
13 (Series B, 2.4 lM) and 18 (Series 2.4 lM). Clearly,
the number of methoxy groups or the inclusion of an
additional hydroxyl group on ring A (for the same sub-
The selective activity of the target chalcones was also
evaluated and expressed as selectivity indices against
the relevant normal cell line (Table 1). The Series A–C
compounds were 1.9–4.7 times more selective against
cancer cell lines than AG 1523, but slightly less selective
(1.2–2.7) when compared against CCL 186. The absence
of the N-methylpiperidine ring in Series D and E

156
X. Liu, M.-L. Go / Bioorg. Med. Chem. 14 (2006) 153–163
chalcones did not alter selectivity to a great extent
(SICCL 186 = 1.6–3.3).
were exposed to different concentrations of the test com-
pound and progression through the cell cycle was mon-
itored for over 48 h. Twenty-four hours after release
from the G1 block, untreated cells get aligned at the
G2/M phase. These G2/M-synchronized cells were col-
lected and similarly exposed to the test compounds.
Of the five series evaluated, the compounds in Series B
and C were seen to combine the advantageous features
of potency and selectivity, as exemplified by compounds
12 and 18.
Figure 1 shows the cell cycle phase distribution in HCT
116 cells initially synchronized at G1 over a period of
48 h. Within 24 h of serum treatment, the control cells
showed significant progression into the G2/M phase
(Fig. 1, panel 3). This was not observed for cells exposed
to compounds 12 (10 lM), 22 (20 lM) and 27 (50 lM)
where the proportion of cells in the G1 phase remained
largely unchanged 24 h after the start of the experiment.
Lower concentrations of compound 12 (5 lM, but not
0.5 lM) also arrested cells at G1. Notably, compound
12 arrested the cell cycle at a concentration close to its
IC50 (3.4 lM). In contrast, compounds 22 and 27 caused
G1 arrest only at concentrations that were at least twice
their IC50 values (Table 2A).
2.3. Effect on cell cycle analysis
The antiproliferative activity of the chalcones may stem
from interference with different stages of cell cycle. Meth-
oxylated chalcones are known to block cells in the G2/M
phase of the cell cycle, which is consistent with their abil-
ity to disrupt the mitotic spindle by interaction with the
protein tubulin.11,16 It would be of interest to see if the
current methoxylated chalcones behave in a similar man-
ner. This aspect was investigated by fluorescence-activat-
ed cell sorter (FACS) analysis using flow cytometry. The
time-related changes in the distribution of cells at each
phase of the cell cycle were determined by monitoring
the DNA content of the cells. Three compounds were
investigated for their effects on the cell cycle: compound
12 (a representative N-methylpiperidinyl chalcone with
acceptable potency and selectivity), compound 22 (a rep-
resentative chalcone without the N-methylpiperidinyl
substituent) and compound 27 (a highly methoxylated
chalcone with low potency and selectivity).
When control cells were synchronized at G2/M, progres-
sion to the G1 phase was attained after 8 h (Fig. 2, panel
2). Compounds 12, 22 and 27 arrested the cells in the
G2/M phase and halted their progression to G1. This ef-
fect was observed at 10 and 20 lM of compound 12, but
not at lower concentrations (0.5, 5 lM) (Table 2B). One
possibility is that the G2/M ! G1 progression was less
sensitive to 12. As for compounds 22 and 27, the arrest
of cells at the G2/M phase was again observed at con-
centrations greater than their IC50 values (Table 2B).
The cell cycle progression experiments were designed to
examine synchronized cell populations at G1 or G2/M
phase through one transit of the cell cycle. G1-synchro-
nized cells were obtained by arresting the cells at the G1
phase by growing to confluence and then stimulating
their re-entry into the cell cycle by sub-culturing at lower
densities. Cells immediately released from a G1 block
The effect of compound 12 on the cell cycle was also
investigated on a normal cell line (CCL 186) to avoid
the confounding effects of loss of checkpoint control in
Control
10µM 12
20 µM 22
50 µM 27
Figure 1. FACS diagram showing the effect of compounds 12, 22 and 27 in G1-arrested HCT 116 cells.

X. Liu, M.-L. Go / Bioorg. Med. Chem. 14 (2006) 153–163
157
Table 2. Effect of compounds 12, 22 and 27 on distribution of HCT 116 and CCL 186 cells in various phases of the cell cycle
Concentration (lM)
% Cells in phase of cell cycle
24 ha
8 ha
48 ha
G1
S
G2/M
G1
S
G2/M
G1
S
G2/M
(A) HCT 116 cells synchronized at G1 phase
12b
0.5
5
62.0 (65.6) 6.4 (6.0)
61.4 (65.6) 5.8 (6.0)
74.3 (72.7) 3.8 (5.2)
69.2 (72.7) 5.8 (5.2)
27.8 (25.9) 32.9 (34.1) 14.8 (15.8) 48.4 (46.8) 40.0 (49.7) 22.7 (11.5) 31.6 (34.3)
30.7 (25.9) 50.2 (34.1) 13.0 (15.8) 33.7 (46.8) 49.1 (49.7) 11.1 (11.5) 36.4 (34.3)
21.7 (21.3) 75.8 (28.8) 4.7 (13.7) 18.0 (54.5) 57.2 (57.9) 7.6 (14.5) 33.1 (27.4)
18.6 (21.3) 78.4 (28.8) 3.6 (13.7) 17.0 (54.5) 41.4 (57.9) 6.6 (14.5) 11.8 (27.4)
10
20
22b
27b
10
20
— —
75.5 (72.7) 3.2 (5.2)
— 54.3 (35.5) 13.6 (27.9) 31.8 (36.8) 49.5 (54.3) 18.5 (11.4) 30.7 (33.7)
20.9 (21.3) 75.4 (28.8) 5.6 (13.7) 18.4 (54.5) 71.5 (57.9) 5.6 (14.5) 20.4 (27.4)
20
50
— —
74.9 (72.7) 4.1 (5.2)
— 45.8 (35.5) 19.5 (27.9) 34.5 (36.8) 51.1 (54.3) 17.4 (11.4) 31.2 (33.7)
19.9 (21.3) 73.5 (28.8) 3.0 (13.7) 23.0 (54.5) 76.7 (57.9) 3.4 (14.5) 19.6 (27.4)
Concentration (lM)
% Cells in phase of cell cycle
8 hc
24 hc
48 hc
G1
S
G2/M
G1
S
G2/M
G1
S
G2/M
(B) HCT 116 cells synchronized at the G2 phase
12b
0.5
5
39.4 (38.8) 12.5 (14.3) 44.0 (43.4) 42.2 (49.7) 14.8 (11.5) 38.4 (34.3) 54.6 (52.0) 7.1 (6.5) 33.4 (37.9)
38.7 (38.8) 12.5 (14.3) 45.1 (43.4) 41.8 (49.7) 13.4 (11.5) 41.3 (34.3) 46.9 (52.0) 22.7 (6.5) 22.9 (37.9)
24.0 (45.9) 13.4 (18.2) 62.4 (35.8) 22.2 (57.9) 8.4 (14.5) 68.8 (27.4) 28.6 (66.0) 9.1 (7.6) 60.4 (25.8)
24.2 (45.9) 11.2 (18.2) 64.4 (35.8) 20.0 (57.9) 7.7 (14.5) 72.1 (27.4) 21.7 (66.0) 8.6 (7.6) 68.0 (25.8)
10
20
22b
27b
10
20
36.6 (46.0) 16.0 (15.4) 47.4 (38.5) 53.0 (54.3) 17.1 (11.4) 29.6 (33.7) 54.8 (68.4) 13.3 (3.9) 31.7 (26.9)
13.8 (45.9) 8.2 (18.2) 77.2 (35.8) 20.9 (57.9) 12.3 (14.5) 64.8 (27.4) 27.7 (66.0) 16.4 (7.6) 42.7 (25.8)
20
50
46.5 (46.0) 15.7 (15.4) 37.6 (38.5) 53.6 (54.3) 13.5 (11.4) 32.7 (33.7) 60.0 (68.4) 4.2 (3.9) 34.6 (26.9)
32.7 (45.9) 12.1 (18.2) 54.8 (35.8) 46.1 (57.9) 4.0 (14.5) 48.9 (27.4) 48.3 (66.0) 3.7 (7.6) 46.2 (25.8)
Concentration lM
% Cells in phase of cell cycle
24 hd
48 hd
G1
S
G2/M
G1
S
G2/M
(C) CCL 186 cells
Cells synchronized at G1 phase
12e 0.5
5.0
56.9 (42.8) 10.3 (16.0) 32.7 (40.5) 79.0 (72.7) 3.4 (6.3) 16.2 (19.9)
69.3 (42.8) 8.5 (16.0) 21.2 (40.5) 76.3 (72.7) 3.0 (6.3) 17.2 (19.9)
Cells synchronized at G2 phase
12e 0.5
5.0
67.7 (72.7) 7.1 (6.3) 24.2 (19.9) 75.4 (75.1) 6.9 (5.7) 17.6 (19.0)
38.4 (72.7) 14.6 (6.3) 43.7 (19.9) 36.1 (75.1) 14.1 (5.7) 46.9 (19.0)
a Time (h) after cells were released from the G1 block. Values in parentheses represent control values in concurrent experiments.
b IC50 HCT 116: 12 3.4 lM; 22: 6.2 lM; 27: 25.0 lM.
c Time (h) after cells were synchronized at G2/M. Untreated cells progressed to G1 within 8–12 h. Values in parentheses represent control values in
concurrent experiments.
d Time (h) after cells were released from G1 block or time (h) after cells were synchronized at G2/M. Values in parentheses represent control values in
concurrent experiments.
e IC50 CCL 186: 7.6 lM.
tumour-derived cell lines. It was noted that compound
12 at 2 lM also interfered with cell cycle progression
in both G1-arrested and G2/M-synchronized cell
populations.
was examined by Western blot analysis. The proteins
investigated were cyclins D and B, CDK4, E2F and the
retinoblastoma protein Rb. Cyclin D and CDK4 are
important for initiating the phosphorylation of Rb (ret-
inoblastoma protein). In its unphosphorylated state,
Rb is bound to and inactivates the E2F family of tran-
scription factors, thereby repressing DNA synthesis.
Once Rb is phosphorylated, E2F dissociates from Rb
and activates the genes necessary for S phase entry and
progression. Progression from G2 to M phase is mainly
regulated by cyclin B and its CDK partner (CDK 1).
2.4. Effect of compound 12 on the expression of cell cycle
regulatory proteins
Since compound 12 affected cell cycle progression at con-
centrations that were close to its IC50, it was of interest to
see if its effect on the cell cycle was mediated through
interference with the expression of key cell cycle regula-
tory proteins. HCT116 cells synchronized at the G1
phase were incubated with compound 12 at 2 lM for
24 h and the expression of selected regulatory proteins
The results of the Western blot analysis are shown in
Figure 3. Serum-starved cells did not undergo the cell
cycle and showed light bands of CDK4, cyclin D, cyclin

158
X. Liu, M.-L. Go / Bioorg. Med. Chem. 14 (2006) 153–163
0 H
8 H
24 H
48 H
Control
10 µM 1
20 µM 22
50 µM 27
Figure 2. FACS diagram showing the effect of compounds 12, 22 and 27 in G2-synchronized HCT 116 cells.
CDK4
Cyclin D
Cyclin B
1
2
3
1
2
3
1
2
3
Rb
E2F
Actin
1
2
3
1
2
3
1
2
3
Figure 3. Expression of cell cycle regulatory proteins in HCT 116 cells synchronized at G1 phase. Lane 1, serum-starved cells; lane 2, 24 h after
addition of 10% serum to serum starved cells; lane 3, 24 h after addition of compound 12 (2 lM) and 10% serum to serum-starved cells. Protein
content of cellular extracts was standardized for uniformity of content.
B, E2F and Rb (hypophosphorylated). Upon addition
of serum, the intensity of these bands increased and both
states (phosphorylated and unphosphorylated) of Rb
were detectable. Incubation of the cells with compound
12 caused the CDK4, cyclin B and E2F bands to de-
crease noticeably in intensity. The band corresponding
to hyperphosphorylated Rb band diminished but the
hypophosphorylated Rb band remained detectable. On
the other hand, there was no discernible change in the
level of cyclin D. The decreases in CDK4 and E2F indi-
cated an interference with the early G1 phase while the
decrease in cyclin B suggested that the G2/M phase
was also affected. The unchanged levels of cyclin D
which is also involved in the G1 phase transition could
not be readily explained.
lines, with IC50 average values less than 5 lM. Omission
of the piperidinyl group to give Series D and E com-
pounds resulted in only a slight decrease in potency,
with little change in selective activity. Despite the limited
variations in activity, the Series B and C compounds
can be identified as useful leads for future structural
modifications because several members combine the
advantageous features of potency and selectivity.
While these results point to a limited contribution by the
piperidine ring to antiproliferative activity, their effects
on cell cycle progression suggested otherwise. Notably,
compound 12, a representative piperidinyl chalcone,
caused a buildup of cells in the G1 phase, blocking nor-
mal progression through G2/M, at 5 lM—a concentra-
tion that was close to its IC50 value (3.4 lM). On the
other hand, compounds 22 and 27, which lack the basic
functionality, had no effect on cell cycle progression at
their IC50 concentrations. The presence of the basic
piperidine ring in 12 may have contributed to this small
but significant difference.
3. Discussion
The piperidinylchalcones of Series A–C showed promis-
ing antiproliferative activity against three cancer cell

X. Liu, M.-L. Go / Bioorg. Med. Chem. 14 (2006) 153–163
159
At 5 lM, compound 12 slowed the progression of the
G1-synchronized HCT 116 cells to the G2/M stage but
had less effect on the transition of the G2-synchronized
cells to G1. Since higher concentrations of 12 blocked
the cell cycles of synchronized G1 and G2/M cells, it
may be that the G1 ! G2/M transition was more sensi-
tive to intervention by this compound. However, Wes-
tern blot analyses pointed to multiple effects of 12 on
the cell cycle, involving both the early G1 phase
(decrease in CDK4 and E2F) and the later G2/M phase
(decrease in cyclin B).
focus on the inclusion of other basic moieties besides
N-methylpiperidine in the chalcone template and inves-
tigate their effects on antiproliferative activity. Possible
candidates are other nitrogen-containing heterocycles
(N-methylpiperazine, N-methylpyrrolidine) and aliphat-
ic amines designed as open chain analogues of N-
methylpiperidine.
5. Experimental
5.1. Chemistry
The ability of methoxylated chalcones to arrest the cell
cycle at the G2/M phase had been reported by other
investigators.11,16 In one report, methoxylated chalcones
arrested G2/M progression when investigated at five
times their IC50 values on K562 human leukaemia
cells.11 These compounds were subsequently shown to
disrupt microtubule assembly. Licochalcone A, a natu-
rally occurring chalcone found in liquorice root, had
pronounced effects on cell cycle progression, arresting
a prostate cancer cell line (PC-3) in G2/M.12 The induc-
tion of G2/M arrest was associated with changes in the
levels of cell cycle regulatory proteins (a reduction of cy-
clin D1, CDK4 and CDK6) and the transcription factor
E2F. These observations were made at 25 lM licochal-
cone, a concentration that was approximately twice its
IC50 against PC-3 cells.
Melting points were determined in open glass capillary
tubes and are uncorrected. 1H NMR spectra were
recorded on a Bruker (DPX 300 MHz) spectrometer
and are reported in d (ppm), relative to TMS as an inter-
nal standard. Mass spectra were collected on a VG
Micromass 7035 E mass spectrometer by chemical ioni-
zation. Analytical thin-layer chromatography (TLC)
was carried out on precoated plates (silica gel F 254)
and column chromatography was performed with silica
gel 60 (70–230 mesh). Combustion analyses (C,H) were
carried out by the Department of Chemistry, National
University of Singapore.
5.1.1. 1-Methyl-4-(2,4,6-trimethoxyphenyl)-1,2,3,6-tetra-
hydropyridine (2). N-Methylpiperidone (0.15 mol) was
added with stirring to a solution of 1,3,5-trimethoxyben-
zene (0.15 mol) in glacial acetic acid (200 ml) with tem-
perature maintained below 25 ꢁC. At the end of
addition, HCl gas was bubbled through the reaction
mixture for 1 h. The reaction mixture was then stirred
for 24 h at 25 ꢁC, and at 95–100 ꢁC for 3 h. The solvent
was removed under reduced pressure and the residue
was diluted with water. The aqueous solution was
extracted with ether, the ethereal layer was separated
off and the aqueous layer was rendered alkaline with
1 M NaOH. A precipitate was obtained, filtered off,
washed with water and dried. Recrystallization from
ether/hexane (2/8) gave 2, mp 121–123 ꢁC (mp 118–
Like licochalcone, the antiproliferative activity by com-
pound 12 has been shown in this investigation to be
linked to disruption of the cell cycle and changes in
the expression of cell cycle regulatory proteins. Howev-
er, unlike licochalcone, these effects were observed at a
concentration close to its IC50 value and after a relative-
ly short incubation period (24 h). In addition, unusual
for a methoxylated chalcone, compound 12 interfered
with both G1 and G2/M phase transitions, with possibly
a greater effect on the former.
1
4. Conclusion
121 ꢁC19). H NMR (CDCl3): 6.15 (2H, s), 5.53–5.52
(1H, m), 3.83 (3H, s), 3.77 (6H, s), 3.11–3.08 (2H, m),
2.68–2.64 (2H, t), 2.41 (3H, s), 2.39–2.37 (2H, m).
This investigation has shown that presence of basic
piperidine ring on the methoxylated chalcone template
did not significantly affect antiproliferative potency but
may influence the mechanism of antiproliferative activ-
ity. This can be seen from the antiproliferative activity
of the piperidinylchalcone 12 which inhibited the cell
cycle at both G1 and G2/M phases at a concentration
that was close to its IC50. The downregulation of cell
cycle regulatory components (CDK4, cyclin B, E2F
and phosphorylated Rb) lends further support to this
conclusion. In contrast, chalcones 22 and 27 that lack
the basic functionality disrupted the cell cycle at higher
concentrations (P2 · IC50). It is possible that these
chalcones do not specifically disrupt cell cycle progres-
sion and interfere with cell replication by other means.
Thus, besides moderating the physicochemical charac-
teristics of solubility and lipophilicity, the basic piperi-
dine function may have an important role in
influencing the mechanism of antiproliferative activity
of methoxylated chalcones. Further investigations will
5.1.2. N-Methyl-4-(2,4-dimethoxyphenyl)-1,2,3,6-tetra-
hydropyridine (9). N-Methylpiperidone (0.15 mol) was
similarly reacted with 1,3-dimethoxybenzene (0.15 mol)
in glacial acetic acid (200 ml) under conditions described
for 2. On workup, 9 was obtained as a colourless oil
(65%) and purified by column chromatography with
0.1% triethylamine in ethyl acetate as mobile phase.
1H NMR (CDCl3): 7.10–7.07 (1H, m), 7.45–7.43 (2H,
m), 5.74–5.72 (1H, m), 3.80 (3H, s), 3.78 (3H, s), 3.10–
3.08 (2H, m), 2.65–2.62 (2H, m), 2.57–2.56 (2H, m),
2.40 (3H, s).
5.1.3. N-Methyl-4-(4-methoxyphenyl)-1,2,3,6-tetrahydro-
pyridine (15). As described for 2, N-methylpiperidone
(0.15 mol) was added with stirring to a solution of meth-
oxybenzene (0.15 mol) in glacial acetic acid (200 ml).
Crude 15 was recrystallized from hexane to give the pure
product in 72% yield (mp 102–104 ꢁC). 1H NMR

160
X. Liu, M.-L. Go / Bioorg. Med. Chem. 14 (2006) 153–163
(CDCl3) 7.33 (2H, d, J = 9.0 Hz), 6.87 (2H, d,
J = 9.0 Hz), 5.97–5.94 (1H, m), 3.81 (3H, s), 3.24–3.22
(2H, m), 2.81–2.77 (2H, m), 2.64 (2H, m), 2.50 (3H, s).
5.1.8. 4-(5-Acetyl-2,4-dimethoxy)phenyl-1-methyl-piperi-
dine (11). Compound 10 (3 g) was similarly reacted with
boron trifluoride dimethyl etherate (11.7 ml, 0.13 mol)
and acetic anhydride (9.6 ml) in CH2Cl2 (100 ml) as de-
scribed for 3. Compound 11 was obtained as a solid res-
idue and recrystallized with methanol–water (2:1) to give
5.1.4. N-Methyl-4-(2,4,6-trimethoxyphenyl)piperidine (3).
Compound 2 (5 g) was hydrogenated in acetic acid–
water (10:1, 150 ml) with 10% Pd/C (0.5 g) as catalyst
at normal pressure (30–40 psi) and room temperature
for 24 h. The reaction mixture was filtered over Celite
and the solvent was evaporated under reduced pressure
to give a residue that was diluted with water and ren-
dered alkaline with 10 M NaOH solution. The precipi-
tate thus obtained was filtered off, washed with water,
dried and used without further purification for the next
1
the desired product in 85% yield. Mp 181–182 ꢁC. H
NMR (CDCl3): 7.77 (1H, br s), 6.45 (1H, br s), 3.92
(3H, s), 3.89 (3H, s), 2.97–2.93 (2H, m), 2.82–2.72
(1H, m), 2.56 (3H, s), 2.30 (3H, s), 2.08–2.00 (2H, m),
1.87–1.76 (4H, m).
5.1.9. 4-(3-Acetyl-4-methoxy)phenyl-1-methyl-piperidine
(17). Compound 16 (1.8 g) was similarly reacted with
boron trifluoride dimethyl etherate (6 ml, 0.065 mol)
and acetic anhydride (5 ml) in CH2Cl2 (50 ml) as de-
scribed for 3. Compound 17 was obtained as a solid res-
idue and recrystallized with methanol–water (2:1) to give
the desired product in 81% yield. Mp 63–65 ꢁC. 1H
NMR (CDCl3): 7.62 (1H, d, J = 2.4 Hz), 7.36 (1H, dd,
J1 = 2.3 Hz, J2 = 8.4 Hz), 6.94 (1H, d, J = 8.4 Hz), 3.91
(3H, s), 3.00–2.96 (2H, m), 2.62 (3H, s), 2.56–2.44
(1H, m), 2.33 (3H, s), 2.10–2.01 (2H, m), 1.85–1.75
(4H, m).
1
step. Yield: 96%. Mp 109–111 ꢁC. H NMR (CDCl3)
6.15 (2H, s), 3.81 (3H, s), 3.79 (6H, s), 3.14–3.05 (1H,
m), 2.96–2.92 (2H, m), 2.48–2.34 (2H, m), 2.31 (3H, s),
2.05–1.97 (2H, m), 1.50–1.48 (2H, m).
5.1.5. N-Methyl-4-(2,4-dimethoxyphenyl)piperidine (10).
Compound 9 (10 g) was hydrogenated in acetic acid–
water (10:1, 200 ml) with 10% Pd/C (1 g) as catalyst under
similar conditions as described for 2. The aqueous solu-
tion was rendered alkaline with 10 M NaOH solution
and extracted with ether. The solvent ether was removed
and the residue was dissolved in 1 M HCl solution. The
product was purified by reverse-phase column chroma-
5.2. General procedure for the preparation of Series A–E
chalcones
˚
tography using a C18 stationary phase (125 A, 55–
105 lM) with water–methanol as mobile phase. Elution
was made progressively, starting with pure water and
moving to 5% methanol/water, 10% methanol/water
and finally 100% methanol. Compound 10 was obtained
as a colourless liquid (92% yield). Accurate mass
A solution of the aldehyde (1.2 mmol) in methanol
(5 ml) was added dropwise to a stirred solution of the
acetophenone (1 mmol) dissolved in 3% (w/v) NaOH
in methanol (20 ml). The solution was stirred at room
temperature (28 ꢁC) for 12 h, after which the solvent
was removed under reduced pressure. The resulting res-
idue was dissolved in 1 M HCl and extracted with ether.
The aqueous layer was rendered alkaline with saturated
aqueous Na2CO3 solution to give a precipitate that was
filtered, washed with water and dried. Recrystallization
from methanol–water gave the desired product.
1
[M+H]+ 236.1679 (236.1651). H NMR (CDCl3) 7.13–
7.10 (1H, m), 6.50–6.46 (2H, m), 3.82 (3H, s), 3.81 (3H,
s), 2.98–2.95 (2H, m), 2.86–2.83 (1H, mb), 2.33 (3H, s),
2.12–2.04 (2H, m), 1.80–1.76 (4H, m).
5.1.6. N-Methyl-4-(4-Methoxyphenyl)piperidine(16). Com-
pound 15 (5 g) was hydrogenated in acetic acid–water
(10:1, 150 ml) using 10% Pd/C (0.5 g) as catalyst as de-
scribed for 2 and worked up in a similar way. Compound
16 was obtained as a colourless liquid and used for the next
step of reaction without further purification. Yield: 93%.
1H NMR (CDCl3): 7.17 (2H, d, J = 8.6 Hz), 6.87 (2H, d,
J = 8.6 Hz), 3.80 (3H, s), 2.99–2.96 (2H, m), 2.46–2.42
(1H, m), 2.33(3H, s), 2.09–2.01(2H, m), 1.83–1.74 (4H, m).
5.2.1. 3-(2-Chlorophenyl)-1-[2-hydroxy-4,6-dimethoxy-3-
(N-methylpiperidin-4-yl)phenyl]-prop-2-en-1-one
(5).
Yield: 54%. Mp 194–195 ꢁC. Accurate mass: [M+H]+
416.1626 (416.1629). H NMR (CDCl3): 14.14 (1H, s),
1
8.15 (1H, d, J = 15.6 Hz), 7.86 (1H, d, J = 15.6 Hz),
7.73–7.64 (1H, m), 7.48–7.44 (1H, m), 7.35–7.31 (2H,
m), 6.01 (1H, s), 3.96 (3H, s), 3.92 (3H, s), 3.19–3.14
(1H, m), 2.99–2.90 (2H, m), 2.47–2.44 (2H, m), 2.33
(3H, s), 2.10–2.02 (2H, m), 1.59 (2H, m).
5.1.7.
4-(3-Acetyl-4,6-dimethoxy-2-hydroxy)phenyl-1-
methyl-piperidine (4). Boron trifluoride dimethyl ether-
ate (6 ml, 0.065 mol) was added dropwise to a stirred
solution of 3 (1.8 g, 0.007 mol) in CH2Cl2 (50 ml), which
had been cooled in an ice bath. Acetic anhydride (5 ml)
was then added dropwise to the solution and stirring
was continued for 24 h at room temperature. The reac-
tion mixture was diluted with water, rendered alkaline
with Na2CO3 and extracted with CH2Cl2. Removal of
solvent in vacuo gave a solid residue that was recrystal-
lized with methanol–water (2:1) to give 4 (83% yield, mp
5.2.2. 3-(4-Chlorophenyl)-1-[2-hydroxy-4,6-dimethoxy-3-
(N-methylpiperidin-4-yl)phenyl]-prop-2-en-1-one (6). 49%
yield. Mp 189–190 ꢁC. Accurate mass: [M+H]+ 416.1634
1
(416.1629). H NMR (CDCl3): 14.18 (1H, s), 7.85 (1H,
d, J = 15.8 Hz), 7.73 (1H, d, J = 15.8 Hz), 7.56–7.53
(2H, d, J = 8.6 Hz), 7.41–7.38 (2H, d, J = 8.4 Hz), 6.01
(1H, s), 3.97 (3H, s), 3.92 (3H, s), 3.23–3.18 (1H, m),
2.98–2.95 (2H, m), 2.48–2.41 (2H, m), 2.33 (3H, s),
2.10–2.02 (2H, m), 1.59 (2H, m).
1
208–210 ꢁC). H NMR (CDCl3): 14.17 (1H, OH), 5.94
(1H, s), 3.89 (3H, s), 3.87 (3H, s), 3.18–3.07 (1H, m),
2.94–2.90 (2H, m), 2.61 (3H, s), 2.48–2.34 (2H, m),
2.29 (3H, s), 2.05–1.96 (2H, m), 1.48–1.44 (2H, m).
5.2.3. 1-[2-Hydroxy-4,6-dimethoxy-3-(N-methylpiperidin-
4-yl)phenyl]-3-phenyl-prop-2-en-1-one (7). Yield: 56%.
Mp 199–200 ꢁC. Accurate mass: [M+H]+ 382.2017

X. Liu, M.-L. Go / Bioorg. Med. Chem. 14 (2006) 153–163
161
(382.2018). 1H NMR (CDCl3): 14.20 (1H, s), 7.86 (1H, d,
J = 15.6 Hz), 7.76 (1H, d, J = 15.6 Hz), 7.62–7.58 (2H,
m), 7.42–7.38 (3H, m), 5.99 (1H, s), 3.95 (3H, s), 3.89
(3H, s), 3.20–3.10 (1H, m), 2.96–2.92 (2H, m), 2.50–2.42
(2H, m), 2.30 (3H, s), 2.10–1.96 (2H, m), 1.53 (2H, m).
5.2.11. 3-(2-Chlorophenyl)-1-(2,4-dimethoxyphenyl)prop-
2-en-1-one (21). Yield: 36.9%. Mp 119–120 ꢁC. C (calcd
67.44, found 67.38) H (calcd 4.99, found 5.30). 1H NMR
(CDCl3): 8.04 (1H, d, J = 15.8 Hz), 7.78 (1H, d,
J = 8.7 Hz), 7.71–7.68 (1H, m), 7.49 (1H, d,
J = 15.8 Hz), 7.46–7.40 (1H, m), 7.30–7.26 (2H, m),
6.58 (1H, d, J = 8.7 Hz), 6.50 (1H, s), 3.90 (3H, s),
3.87 (3H, s).
5.2.4. 3-(2-Fluorophenyl)-1-[2-hydroxy-4,6-dimethoxy-3-
(N-methylpiperidin-4-yl)phenyl]-prop-2-en-1-one (8). Yield:
62%. Mp 192–194 ꢁC. Accurate mass: [M+H]+ 400.1930
(400.1924) 1H NMR (CDCl3,) 14.18 (1H, s), 7.99 (1H, d,
J = 15.8 Hz), 7.85 (1H, d, J = 15.8 Hz), 7.63–7.58 (1H,
m), 7.38–7.11 (3H, m), 6.01 (1H, s), 3.97 (3H, s), 3.92
(3H, s), 3.19–3.14 (1H, m), 2.95 (2H, m), 2.48–2.43 (2H,
m), 2.33 (3H, s), 2.09–2.02 (2H, m), 1.59 (2H, m).
5.2.12. 3-(4-Chlorophenyl)-1-(2,4-dimethoxyphenyl)prop-
2-en-1-one (22). Yield: 63.7%. Mp 118–120 ꢁC. C (calcd
67.44, found 67.41) H (calcd 4.99, found 5.34). 1H NMR
(CD3OD): 7.76 (1H, d, J = 8.7 Hz), 7.68 (1H, d,
J = 15.0 Hz), 7.65 (1H, d, J = 15.4 Hz), 7.58 (2H, d,
J = 8.6 Hz), 7.42 (2H, d, J = 8.6 Hz), 6.67–6.64 (2H,
m), 3.95 (3H, s), 3.91 (3H, s).
5.2.5. 3-(2-Chlorophenyl)-1-[2,4-dimethoxy-5-(N-methylpi-
peridin-4-yl)phenyl]-prop-2-en-1-one (12). Yield: 57%. Mp
99–100 ꢁC. Accurate mass: [M+H]+ 400.1687 (400.1679).
1H NMR (CDCl3): 8.06 (1H, d, J = 15.8 Hz), 7.51 (1H, d,
J = 15.8 Hz), 7.74–7.69 and 7.46–7.29 (5H, m), 6.48 (1H,
br), 3.95 (3H, s), 3.94 (3H, s), 3.01–2.97 (2H, m), 2.83
(1H, m), 2.34 (3H, s), 2.08–2.04 (2H, m), 1.86–1.79
(4H, m).
5.2.13. 1-(2,4-Dimethoxyphenyl)prop-2-en-1-one (23).
Yield: 43.2%. Mp 75–76 ꢁC. C (calcd 76.10, found
76.29) H (calcd 6.01, found 5.95). H NMR (CDCl3):
1
7.77 (1H, d, J = 8.7 Hz), 7.68 (1H, d, J = 15.8 Hz),
7.62–7.58 (2H, m), 7.51 (1H, d, J = 15.8 Hz), 7.41–7.38
(3 H, m), 6.59–6.55 (1H, dd, J = 8.7 Hz, 2.3 Hz), 6.51
(1H, d, J = 2.3 Hz), 3.91 (3H, s), 3.88 (3H, s).
5.2.6. 3-(4-Chlorophenyl)-1-[2,4-dimethoxy-5-(N-methylpi-
peridin-4-yl)phenyl]-prop-2-en-1-one (13). Yield: 52%. Mp
161–163 ꢁC. Accurate mass: [M+H]+ 400.1676
5.2.14. 3-(2-Chlorophenyl)-1-(2-methoxyphenyl)prop-2-
en-1-one (24). Yield: 27.8%. Mp 65–67 ꢁC. C (calcd
70.46, found 70.33) H (calcd 4.80, found 4.48). 1H
NMR (CD3OD): 8.01 (1H, d, J = 15.8 Hz), 7.72–7.69
(1H, m), 7.63 (1H, d, J = 7.5 Hz), 7.48 (1H, t,
J = 8.7 Hz), 7.44–7.41 (1H, m), 7.35 (1H, d,
J = 15.8 Hz), 7.34–7.29 (2H, m), 7.08–6.99 (2H, m),
3.91 (3H, s).
1
(400.1679). H NMR (CDCl3): 7.69–7.37 (7H, m), 6.48
(1H, br), 3.96 (3H, s), 3.94 (3H, s), 3.00–2.97 (2H, m),
2.83–2.82 (1H, m), 2.34 (3H, s), 2.08 (2H, m), 1.86–1.79
(4H, m).
5.2.7. 1-[2,4-Dimethoxy-5-(N-methylpiperidin-4-yl)phenyl]-
3-phenylprop-2-en-1-one (14). Yield: 60%. Mp 132–134 ꢁC.
1
Accurate mass: [M+H]+ 366.2072 (366.2069). H NMR
5.2.15. 3-(4-Chlorophenyl)-1-(2-methoxyphenyl)prop-2-
en-1-one (25). Yield: 41.7%. C (calcd 70.46, found 70.56)
(CDCl3) 7.70 (1H, d, J = 15.8 Hz), 7.54 (1H, d,
J = 15.8 Hz), 7.68, 7.62–7.59 (3H, m), 7.41–7.39 (3H,
m), 6.48 (1H, br), 3.95 (3H, s), 3.93 (3H, s), 3.00–2.96
(2H, m), 2.82–2.81 (1H, m), 2.32 (3H, s), 2.08–2.03 (2H,
m), 1.85–1.78 (4H, m).
1
H (calcd 4.80, found 4.90). H NMR (CDCl3): 7.64–
7.60 (1H, m), 7.53 (1H, d, J = 7.9 Hz), 7.52 (2H, d,
J = 8.3 Hz), 7.48 (1H, d, J = 15.8 Hz), 7.37 (2H, d,
J = 8.3 Hz), 7.35 (1H, d, J = 15.8 Hz), 7.06 (1H, dd,
J = 7.5 Hz, 1.1 Hz), 7.01 (1H, d, J = 7.9 Hz), 3.90 (3H, s).
5.2.8. 3-(2-Chlorophenyl)-1-[2-methoxy-5-(N-methylpiperi-
din-4-yl)phenyl]prop-2-en-1-one (18). Yield: 84%. Mp 90–
5.2.16. 1-(2-Methoxyphenyl)prop-2-en-1-one (26). Yield:
72.8%. C (calcd 80.65, found 80.47) H (calcd 5.92, found
1
91 ꢁC. Accurate mass: [M+H]+ 370.1577 (370.1574). H
1
5.68). H NMR (CD3OD): 7.63 (1H, d, J = 15.8 Hz),
NMR (CDCl3): 8.03 (1H, d, J = 16.1 Hz), 7.73–7.70 (1H,
m), 7.49 (1H, d, J = 16.2 Hz), 7.52–7.30 (5H, m), 6.99
(1H, d, J = 8.7 Hz), 3.91 (3H, s), 3.22 (2H, m), 2.54–2.44
(1H, m), 2.33 (3H, s, N-methyl), 2.40 (2H, m), 2.08–1.92
(4H, m).
7.65–7.56 (3H, m), 7.51–7.45 (1H, m), 7.38 (1H, d,
J = 15.8 Hz), 7.40–7.35 (3H, m), 7.06 (1H, d,
J = 7.5 Hz), 7.01 (1H, d, J = 8.3 Hz), 3.91 (3H, s).
5.3. Materials for cell culture
5.2.9. 3-(4-Chlorophenyl)-1-[2-methoxy-5-(N-methylpiperi-
din-4-yl)phenyl]prop-2-en-1-one (19). Yield: 42%. Mp 115–
HCT 116 (transformed human colon cancer cell line)
was a gift from Dr. Balram Chowbay, National Cancer
Centre, Singapore. MCF-7 (human breast cancer cell
line), Jurkat (human leukaemic cell line), CCL 186 (nor-
mal human diploid embryonic lung fibroblast) and AG
01523C (normal human foreskin diploid fibroblast) were
purchased from American Type Culture Collection
(Rockville, MD, USA). The following cell cycle regula-
tory proteins were obtained from BD Biosciences,
Pharmingen, Ont., Canada: cyclin D1, cyclin B, CDK
4, retinoblastoma pRb (a.a. 332–344) E2F-1 and b-
actin. pRb was also purchased from Santa Cruz Bio-
1
117 ꢁC Accurate mass: [M+H]+ 370.1582 (370.1574). H
NMR (CDCl3): 7.64–7.38 (8H, m), 7.01 (1H, d,
J = 8.3 Hz), 3.92 (3H, s), 3.57–3.53 (2H, m), 2.76 (3H,
s, N-methyl), 2.76–1.93 (7H, m).
5.2.10. 1-[2-Methoxy-5-(N-methylpiperidin-4-yl)phenyl]-
3-phenyl-prop-2-en-1-one (20). Yield: 48%. Accurate
1
mass: [M+H]+ 336.1970 (336.1964). H NMR (CDCl3):
7.69–7.40 (9H, m), 7.02–6.99 (1H, d, J = 8.3 Hz), 3.92
(3H, s), 3.59–3.55 (2H, m), 2.78 (3H, s, N-methyl),
2.78–2.71 (1H, m), 2.47–2.02 (6H, m).

162
X. Liu, M.-L. Go / Bioorg. Med. Chem. 14 (2006) 153–163
technology, Inc., CA, USA. Luminol was purchased
from Supersignal West Pico, Pierce, IL, US. RNase A
and propidium iodide were from Sigma Chemical Co,
USA.
adjusted by subtraction of blank values (wells without
cells) and expressed as a percentage of the average
absorbance obtained from control wells (in the absence
of test compound). IC50 values were determined from
logarithmic plots of the % absorbance versus concentra-
tion generated using Prism GraphPad (San Diego, CA,
USA).
5.4. MTT assay for determination of antiproliferative
activity
The growth inhibitory activity of Series A–C chalcones
was determined on the following cell lines: HCT 116
(human colon cancer cell line), MCF-7 (human breast
cancer cell line), Jurkat (human leukaemic cell line),
CCL 186 (normal human diploid embryonic lung fibro-
blast) and AG 01523C (normal human foreskin diploid
fibroblast) using a modified version of the microculture
tetrazolium assay.20 Series D and E compounds were
only tested on HCT116 and CCL 186 cells. HCT116
cells were cultured in McCoyÕs 5A modified medium
with 1.5 mM L-glutamine (ATCC) supplemented with
10% FBS and 1% antibiotics, and cells of passages 19–
22 were used. MCF-7 cells were cultured in DulbeccoÕs
modified EagleÕs medium (DMEM) supplemented with
10% FBS and 1% antibiotics, and cells of passages 3–8
were used and CCL 186 in minimum essential medium
a medium (MEM) with 2 mM L-glutamine (Gibco), sup-
plemented with 1% 100· non-essential amino acids,
1.5 g/L sodium bicarbonate, 10% FBS and 1% antibiot-
ics, and cells of passages 14–18 were used. AG 01523C
cells were cultured in minimum essential medium
(MEM) with non-essential amino acids, supplemented
with 10% FBS and 1% antibiotics but without L-gluta-
mine. Cells of passages 10–14 were used.
5.5. Cell cycle analysis
Human colon carcinoma cells (HCT 116) and normal
human lung fibroblast cells (CCL 186) were grown to
confluence and maintained in this state for a minimum
of 5 days. The cells were then trypsinized and subcul-
tured at 8000 cells/cm2 in growth medium containing
10% fetal calf serum and the test compound was added
either immediately (G1 release) or 24 h later (G2 syn-
chronized). The cells were incubated in the presence of
the test compound for up to 48 h. At indicated time
points after compound addition, the cells were harvest-
ed, trypsinized and fixed in 70% ice-cold ethanol for a
minimum of 24 h. After centrifugation, the supernatant
was discarded and the pellet was treated with RNase A
(200 lg/ml) for 30 min at room temperature, followed
by cell staining using propidium iodide at a final concen-
tration of 20 lg/ml. The stained cells were then analyzed
for cell cycle distribution in the Go/G1, S and G2/M
phases on a FACScan flow cytometer (Beckman Coulter
CA, USA) equipped with an argon-ion laser (488 nm)
using the WinMDI (version 2.8) software. Compound
12 was examined at 0.5, 2, 5, 10 and 20 lM on HCT
116 cells and at 0.5 and 2 lM on CCL 186 cells. Com-
pounds 22 and 27 were tested on HCT 116 cells at con-
centrations of 3, 6, 10 and 20 lM (22) and at 5, 10, 20
and 50 lM (27).
Once the cells reached 90% confluency, a cell suspension
was prepared by mechanical disaggregation of the float-
ing cells (Jurkat) or by trypsinization of monolayer cul-
tures (other cell lines). Cell counts were performed, and
the suspensions were diluted accordingly to give 1 · 105
cells/ml with the appropriate media. Aliquots (100 ll) of
the cell suspension were added to each well in a 96-well
microtitre plate. The cells were incubated for 24 h
(37 ꢁC, 5% CO2) for incubation. Stock solutions of the
test compound were prepared in DMSO and serial dilu-
tions were made with media to over a 100-fold concen-
tration range. Not more than 1% DMSO (final
concentration) was present in each well. The test sample
was incubated with the cells for 72 h. Wells without cells
and those with cells in culture media/DMSO were exam-
ined in parallel. 0.1% SDS and dextran were included as
positive and negative controls, respectively. At the end
of the incubation period, the medium was decanted
and replaced with 100 ll MTT solution (0.5 mg/ml in
1· phosphate-buffered saline solution (PBS)). The cells
were incubated for another 3 h after which the medium
was removed from each well by pipetting and the cells
were carefully washed with PBS (100 ll). DMSO
(150 ll) was added to each well to lyse the cells and to
dissolve the purple formazan crystals. The absorbance
of the formazan product was measured within 30 min
at 590 nm on a microtitre plate reader. The absorbance
values obtained at each concentration (triplicates for
each run and three independent runs were carried out
using cells of different passage numbers) were averaged,
5.6. Protein extraction and Western blot analysis
Human colon carcinoma cells (HCT 116) were grown to
confluence and maintained in this state for a minimum
of 5 days. The cells were then trypsinized and subcul-
tured at 8000 cells/cm2 in growth media (McCoy 5A)
containing 10% fetal calf serum. The test compound
was added at the specified concentration and incubated
with the cells for up to 24 h. Control or treated cells
were rinsed with ice-cold PBS and transferred to Eppen-
dorf tubes. The contents of each tube were suspended in
100 ll lysis buffer (PhosphataseTM extraction buffer,
Novagen) in the presence of protease inhibitors (15 ll,
protease inhibitor cocktail, Roche). The cell suspension
was incubated for 20 min (4 ꢁC) and centrifuged
(20 min, 10 K rpm) and the clear supernatants were
stored in aliquots at ꢁ80 ꢁC. Protein concentrations
were measured with protein assay reagent (Dye Reagent
Concentrate, Bio-Rad). For Western blot analysis, the
protein extract was diluted with Laemmli buffer (4·)
and analyzed by SDS gel electrophoresis on appropriate
polyacrylamide gels (6–12%) with 20 lg protein loaded
on each lane. The proteins on the gels were then trans-
ferred to polyvinylidene diflouride (PVDF) membranes
(Bio-Rad) by a semi-dry transfer method. After block-
ing with Tris-buffered saline containing 5% low-fat milk
and 0.1% Tween 20, the membranes were probed with

X. Liu, M.-L. Go / Bioorg. Med. Chem. 14 (2006) 153–163
163
6. Yamamoto, S.; Aizu, E.; Jiang, H.; Nakadate, T.; Kiyoto,
I.; Wang, J. C.; Kato, R. Carcinogenesis 1991, 12, 317.
7. Nguyen-Hai, N.; Kim, Y.; You, Y. J.; Hong, D. H.; Kim,
H. M.; Ahn, B. Z. Eur. J. Med. Chem. 2003, 38, 179.
8. Stoll, R.; Renner, C.; Hansen, S.; Palme, S.; Klein, C.;
Belling, A.; Zeslawski, W.; Kamionka, M.; Rehm, T.;
Muhlhahn, P.; Schumacher, R.; Hesse, F.; Kaluza, B.;
Voelter, W.; Engh, R. A.; Holak, T. A. Biochemistry 2001,
40, 336.
9. Kumar, S. K.; Hager, E.; Pettit, C.; Gurulingappa, H.;
Davidson, N. E.; Khan, S. R. J. Med. Chem. 2003, 46,
2813.
10. Sabzevari, O.; Galati, G.; Moridani, M. Y.; Siraki, A.;
OÕBrien, P. J. Chem. Biol. Interact. 2004, 148, 57.
11. Lawrence, N. J.; McGown, A. T.; Ducki, S.; Hadfield, J.
A. Anti-Cancer Drug Des. 2000, 15, 135–141.
12. Fu, Y.; Hsieh, T. C.; Guo, J.; Kunicki, J.; Lee, M. Y. W.
T.; Darzynkiewicz, Z.; Wu, J. M. Biochem. Biophys. Res.
Commun. 2004, 322, 263.
13. Won, S. J.; Cheng, C. T.; Tsao, L. T.; Weng, J. R.; Ko, H.
H.; Wang, J. P.; Lin, C. N. Eur. J. Med. Chem. 2005, 40, 103.
14. Edwards, M. L.; Stemerick, D. M.; Sunkara, P. S. J. Med.
Chem. 1990, 33, 1948.
antibodies against the following proteins: cyclin D1,
cyclin B, CDK 4, Rb, E2F-1 and b-actin using standard
techniques. The membrane blots previously probed with
protein specific primary antibodies were reacted (1 h)
with secondary antibodies (horseradish peroxidase
anti-mouse IgG or horseradish peroxidase anti-rabbit
IgG), washed extensively with Tris-buffered saline
(0.1% Tween 20) and submerged (5 min) in a mixture
of horseradish peroxidase substrate (luminol). Mem-
branes were lightly hand-blotted dry, exposed for vary-
ing periods (10 s to 5 min) to autoradiography film and
developed to visualize the antibody–antigen complexes.
Acknowledgments
X. L. Liu gratefully acknowledges research scholarship
support from the National University of Singapore.
This work was supported by Grant RP140000042112
from National University of Singapore.
15. Ducki, S.; Forrest, R.; Hadfield, J. A.; Kendall, A.;
Lawrence, N. J.; McGown, A. T.; Rennison, D. Bioorg.
Med. Chem. Lett. 1998, 8, 1051.
References and notes
16. Lawrence, N. J.; Rennison, D.; McGown, A. T.; Hadfield,
J. A. Bioorg. Med. Chem. Lett. 2003, 13, 3759.
17. Xia, Y.; Yang, Z. Y.; Xia, P.; Bastow, K. F.; Nakanishi,
Y.; Lee, K. H. Bioorg. Med. Chem. Lett. 2000, 10, 699.
18. Nielsen, S. F.; Larsens, M.; Boesen, T.; Schenning, K.;
Kromann, H. J. Med. Chem. 2005, 48, 2667.
19. Schoepfer, J.; Fretz, H.; Chaudhuri, B.; Muller, L.; Seeber,
E.; Meijer, L.; Lozach, O.; Vangrevelinghe, E.; Furet, P.
J. Med. Chem. 2002, 45, 1741.
1. Dimmock, J. R.; Elias, D. W.; Beazely, M. A.; Kandepu,
N,M. Curr. Med. Chem. 1999, 6, 1125.
2. Go, M. L.; Wu, X.; Liu, X. L. Curr. Med. Chem. 2005, 12,
483.
3. De Vincenzo, R.; Scambia, G.; Benedetti, P. P.; Ranelletti,
F. O.; Bonanno, G.; Ercoli, A.; Belle Monache, F.;
Ferrari, F.; Piantelli, M.; Mancuso, S. Anticancer Drug
Des. 1995, 10, 481.
20. Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M.
L.; Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo,
J. G.; Shoemaker, R. H.; Boyd, M. R. Cancer Res.
1988, 48, 589.
21. Liu, M. Ph.D. Thesis, National University of Singapore,
Singapore, 2003.
4. De Vincenzo, R.; Ferlini, C.; Distefano, M.; Gaggini, C.;
Riva, A.; Bombardelli, E.; Morazzoni, P.; Valenti, P.;
Belluti, F.; Ranelletti, F. O.; Mancuso, S.; Scambia, G.
Cancer Chemother. Pharmacol. 2000, 46, 305.
5. Park, E. J.; Park, H. R.; Lee, J. S.; Kim, J. Planta Med.
1998, 64, 464.