3-(3,4,5-Trimethoxyphenyl)-1-oxo-2-propene: A novel pharmacophore displaying potent multidrug resistance reversal and selective cytotoxicity

Article abstract of DOI:10.1016/j.bmc.2007.03.022

This study revealed that various alicyclic and acyclic compounds containing the 3-(3,4,5-trimethoxyphenyl)-2-propenoyl group displayed potent MDR reversal properties. In particular, a concentration of 4 μg/ml of 2,5-bis(3,4,5-trimethoxyphenylmethylene)cyc

Full text of DOI:10.1016/j.bmc.2007.03.022

Bioorganic & Medicinal Chemistry 15 (2007) 3373–3380
3-(3,4,5-Trimethoxyphenyl)-1-oxo-2-propene: A novel
pharmacophore displaying potent multidrug resistance
reversal and selective cytotoxicity
Umashankar Das,a Masami Kawase,b Hiroshi Sakagami,c Atsushi Ideo,c Jun Shimada,c
d
d
d
a,
*
´
´
´
Joseph Molnar, Zoltan Barath, Zsuzsanna Bata and Jonathan R. Dimmock
aCollege of Pharmacy and Nutrition, University of Saskatchewan, 110 Science Place, Saskatoon, Canada SK S7N 5C9
bFaculty of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan
cDepartment of Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry, Saitama 350-0283, Japan
dInstitute of Medical Microbiology and Immunobiology, Albert Szent-Gyo¨rgyi Medical Centre,
University of Szeged, Szeged H-6720, Hungary
Received 28 December 2006; revised 20 February 2007; accepted 8 March 2007
Available online 13 March 2007
Abstract—This study revealed that various alicyclic and acyclic compounds containing the 3-(3,4,5-trimethoxyphenyl)-2-propenoyl
group displayed potent MDR reversal properties. In particular, a concentration of 4 lg/ml of 2,5-bis(3,4,5-trimethoxyphenylmeth-
ylene)cyclopentanone was 31 times more potent than verapamil as a MDR revertant. In general, they were selectively toxic to malig-
nant rather than normal cells. Two representative compounds induced apoptosis in human HL-60 cells and markedly activated
caspase-3.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
reversing and cytotoxic properties.5 A subsequent study
confirmed the good potency of 1 (but not 2) towards
additional neoplastic cell lines.6 Clearly 1 is a novel lead
compound and the objective of the present study was to
prepare a limited number of prototypic molecules in
order to develop guidelines for a major expansion in this
area.
A major problem in cancer chemotherapy is multidrug
resistance (MDR) which is caused principally by the
increased efflux of drugs from malignant cells due to
increased expression of P-glycoprotein. A number of
structurally divergent drugs used in treating a variety
of pathological conditions possess MDR-reversing
properties such as verapamil,1 trifluoperazine,2 and qui-
nine3 as well as molecules designed as MDR-reversing
agents such as biricodar.4 A preliminary communication
from our laboratory disclosed that 6-(4-nitrophenylm-
ethylene)-2-(3,4,5-trimethoxyphenylmethylene)cyclohex-
anone 1 reversed MDR in murine L-5178 cells and
demonstrated greater toxicity toward various neoplasms
than non-malignant cells (Fig. 1 and Table 1).5 In this
series of compounds, the 3,4,5-trimethoxyphenyl group
was optimal, for example, under the test conditions uti-
lized, the 4-methoxy analog 2 was bereft of MDR-
The MDR-reversing properties of 1 are likely influ-
enced, inter alia, by hydrophobic bonding between the
aryl rings A and B and a complementary area on a bind-
ing site. In addition, bioactivity is likely mediated by
interactions between the olefinic carbon atoms of 1 with
cellular nucleophiles such as thiols. Hence the alicyclic
and acyclic analogs of 1, namely 3 and 4, were proposed
O
R
H3CO
A
B
NO2
R
Keywords: Unsaturated ketones; 3-(3,4,5-Trimethoxyphenyl)-2-prope-
noyl pharmacophore; Multidrug resistance; Cytotoxicity.
1
2
R = OCH3
R = H
*
Corresponding author. Tel.: +1 306 966 6331; fax: +1 306 966
6377; e-mail: jr.dimmock@usask.ca
Figure 1. Structures of compounds 1 and 2.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.022

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U. Das et al. / Bioorg. Med. Chem. 15 (2007) 3373–3380
Table 1. Evaluation of 1–10 for MDR reversal
colon cancer cells. The quest for identifying compounds
with preferential cytotoxicity for malignant cells used
HSC-2, HSC-3, and HSC-4 squamous cell carcinomas
and HL-60 promyelocytic leukemia cells as well as the
following non-malignant cells viz. HGF gingival fibro-
blasts, HPLF periodontal ligament fibroblasts, and
HPC pulp cells. The results for these assays are por-
trayed in Table 2. The unsaturated ketones 1 and 4 in-
duced bimodal changes in the caspase activation in
HL-60 and HSC-2 cells. They induced caspase activa-
tion more potently in HL-60 cells than in HSC-2 cells
(Fig. 4). They induced internucleosomal DNA fragmen-
tation in HL-60 cells, while in HSC-2 cells they induced
a large DNA fragment (indicated by an arrow in Fig. 5)
without the induction of internucleosomal DNA
fragmentation.
Compound
FAR valuesa
4 lg/mL
40 lg/mL
1b
2b
3
15.6
0.76
1.10
0.82
1.38
13.3
4.30
96.3
1.15
1.37
75.1
0.82
1.36
1.89
1.34
69.3
13.8
107
4
5
6
7
8
9
10
39.0
48.3
a FAR, fluorescence activity ratio. The FAR values of verapamil are
3.08 and 37.20 when 4 and 40 lg/mL of drug were employed.28
b The data were previously reported in Ref. 5 copyright (2005) with
permission of Elsevier.
3. Discussion
in which the relative positions of the aryl rings and ole-
finic carbon atoms of these three molecules should differ
which may be reflected by variations in MDR-reversing
potencies. For similar reasons, a comparison of the bio-
activities of 2 and the related acyclic analog 5 was
planned.
The 1H NMR spectra of the unsaturated alicyclic
ketones revealed that the compounds possess the E
stereochemistry based on the absorptions of the olefinic
protons10–13, while the acyclic molecules 4, 5, and 10
were the E isomers as revealed by the coupling constants
of the olefinic protons.14 These conclusions are in accord
with X-ray crystallographic determinations of related
compounds.15–17
The compounds related to 1 which had previously
shown MDR-reversing properties all contained
a
4-nitrophenylmethylene group.5 However, aryl nitro
groups can give rise to unwanted toxicity7–9 and the
ability of 1 to cause reversal of MDR may have been
due, either wholly or in part, to the presence of the
3,4,5-trimethoxyphenylmethylene group. Hence removal
of the nitro group from ring B of 1 and the introduction
of multiple methoxy substituents to form 6 were sug-
gested. In order to explore whether the mounting of
both 3,4,5-trimethoxyphenylmethylene groups on differ-
ent cyclic scaffolds would influence MDR-reversing
potencies, the preparation of two analogs of 6, namely
7 and 8, was planned. Finally the presence of one
3,4,5-trimethoxyphenylmethylene group could be suffi-
cient to reverse MDR and in order to explore this pos-
sibility, the synthesis and bioevaluation of 9 and 10
were proposed.
The evaluation of two concentrations of the enones 1–10
as MDR reversing agents is summarized in Table 1. In
this assay, rhodamine 123 is employed and its concen-
tration in treated and untreated cells is compared from
which data, fluorescence activity ratio (FAR) values
are computed. Compounds with a FAR value of 1 or
more indicate that reversal of MDR has taken place.
MDR modulators likely bind in the transmembrane do-
mains of Pgp thereby inducing a conformational change
in Pgp which inhibits the action of ABC transporters.18
The following conclusions may be drawn from the bio-
data. First, the 4-nitrophenylmethylene group is not
essential for inducing MDR-reversing properties. Thus,
replacement of this functionality in 1 by a 3,4,5-trimeth-
oxyphenylmethylene group led to 6 having a similar
MDR reversing potency. In fact, on occasions the
4-nitrophenylmethylene substituent exerted a dysthera-
peutic effect, for example, excision of this group from
the inactive compounds 3 and 4 led to 9 and 10, respec-
tively, which possess MDR reversing properties. Second,
an attempt was made to find reasons for the differences
in MDR-reversing properties between 1 which is an ac-
tive compound and 3 and 4 which are devoid of this
property. Figure 3 reveals the differences in the relative
locations of not only the aryl rings A and B but the ole-
finic carbon atoms as well, suggesting that the topogra-
phy of these molecules impacts considerably on the
magnitude of the biological responses. Third, both com-
pounds containing a sole methoxy group in aryl ring A
viz. 2 and 5 were devoid of MDR-reversing properties.
Fourth, all three bis compounds 6–8 reverse MDR. In
particular, the remarkable potency of 8 establishes it
as a very useful lead compound which is likely due, at
least in part, to the favorable spatial arrangement of
In addition, an assessment of whether the analogs of 1
would demonstrate cytotoxic activity was planned and,
if so, whether selective toxicity to malignant cells oc-
curred. The final phase of the study was the evaluation
of the biodata in the light of molecular modeling tech-
niques as well as probing for the mode of action of rep-
resentative compounds.
2. Results
Compounds 3–10 were prepared by the synthetic chem-
ical pathways indicated in Figure 2. The ability of these
compounds to reverse MDR was assessed using murine
lymphoma L-5178 cells transfected with the human
MDR1 gene and the data are presented in Table 1. In
addition, two representative compounds 7 and 8 were
evaluated for reversal of MDR using human colo 320

U. Das et al. / Bioorg. Med. Chem. 15 (2007) 3373–3380
3375
O
O
H3CO
H3CO
R
A
B
A
B
NO2
H3CO
NO2
H3CO
R
3
i, ii
iii, ii
4
5
R = OCH3
R = H
R
R
H3CO
CHO
iv,i,v
iii
i
O
O
H3CO
H3CO
OCH3
OCH3
H3CO
H3CO
OCH3
OCH3
OCH3
O
H3CO
H3CO
10
n = 2
n = 3
6
7
H3CO
8 n = 1
9
Figure 2. The synthetic chemical scheme used in preparing 3–10. The reagents used were as follows, i, cycloheptanone/NaOH; ii,
4-nitrobenzaldehyde/NaOH; iii, acetone/NaOH; iv, cyclohexanone/HCl; v, cyclopentanone/HCl.
Table 2. Evaluation of 1–10 for reversal of MDR and cytotoxic properties
a
Compound
CC50 (lM)
HSC-2
HSC-3
HSC-4
HL-60
Aveb
HGF
HPLF
HPC
SIc
1d
0.76
>400
116
<3.1
6.3
4.7
>400
279
5.7
16
1.5
>400
100
3.8
12
1.1
>400
266
6.4
25
< 2.0
>400
190
<4.8
15
336
>400
256
16
378
>400
370
347
>400
198
94
>176
ꢀ1.0
1.4
2d
3
4
106
193
>15
9.3
5
81
141
285
276
247
277
188
>200
6
34
62
36
67
46
46
45
90
335
229
240
262
159
>200
388
250
7.5
2.8
7
113
19
132
89
8
58
21
31
47
281
291
5.5
1.6
9
10
214
55
200
72
269
41
179
54
47
6
263
>200
3.8
>3.4
Melphalane
35
115
81
59
a The CC50 figure is the concentration of compound required to kill 50% of the cells and is the average of two independent determinations.
b Average CC50 value of the compound toward HSC-2, HSC-3, HSC-4, and HL-60 neoplastic cells.
c The letters SI refer to the selectivity index.
d With the exception of the figure of HSC-4 cells, the data were previously reported in Ref. 5 copyright (2005) with permission of Elsevier.
e Solubility problems of melphalan in dimethylsulfoxide precluded the use of concentrations above 200 lM.
the 3,4,5-trimethoxyphenylmethylene groups. Fifth,
removal of one of the 3,4,5-trimethoxyphenylmethylene
groups of 7 yielding 9 led to an increase in potency sug-
gesting that only one of these structural moieties may be
required to confer MDR reversing properties in these
compounds. Finally in considering the possible struc-
tural features which may influence the magnitude of
MDR reversal in mouse L-5178 cells, two other physico-
chemical parameters were considered. The ability of the
3-(3,4,5-trimethoxyphenyl)-2-propenoyl group to align
at a binding site will likely be influenced by the torsion
angle h between aryl ring A and the adjacent enone
Figure 3. Overlap of ring A in 1 (pink), 3 (green), and 4 (blue).
Hydrogen atoms were removed for clarity.

3376
U. Das et al. / Bioorg. Med. Chem. 15 (2007) 3373–3380
group. Hence the torsion angles of 1–10 were obtained
and are listed in Table 3. In addition, the olefinic carbon
atom adjacent to ring A may well undergo electrophilic
attack with cellular nucleophiles and hence the varying
charges on these atoms may influence bioactivity. These
atomic charges were obtained and are presented in
Table 2. Linear and semilogarithmic plots were made
between the FAR figures generated using 40 lg/ml of
the compounds 1–10 and both the h values and atomic
charges. No significant correlation was noted between
the FAR values and either the torsion angles or atomic
charges (p > 0.05).
Studies were undertaken to determine the manner
whereby cytotoxicity was caused by two of the lead mol-
ecules 1 and 4. Most if not all cytotoxic agents induce
apoptosis.19 Incubation of 1 and 4 revealed that DNA
laddering, a hallmark of apoptosis, occurred only in
HL-60 cells, while they did not induce DNA laddering
in HSC-2 cells, possibly due to the lack of activation
of endonuclease or change in the chromatin structure
(Fig. 5). Death stimuli activate two principal pathways
which lead to apoptosis; both biochemical mechanisms
are dependent on various cysteine proteases or caspases.
First, the mitochondrial pathway involves the activation
of caspase-9 while second, the production of caspase-8 is
increased in the death receptor pathway.20 Both pro-
cesses lead to activation of downstream effector caspases
such as caspase-3.21 Hence the ability of 1 and 4 to acti-
vate caspases-3, -8, and -9 in HL-60 and HSC-2 cells was
investigated.
The potential of these compounds as MDR revertants
will be enhanced if similar results were observed using
a different species and another neoplastic disease. In
order to explore this avenue, two representative com-
pounds 7 and 8 with substantially different potencies
in the murine L-5178 lymphoma assay were examined
using human colo 320 colon cancer cells. When a con-
centration of 4 lg/ml was employed, the FAR values
of 7 and 8 are 3.65 and 8.26, respectively, which com-
pares favorably with verapamil (a concentration of
10 lg/ml of this compound in this assay led to a FAR
value of 3.84). These results provide additional evidence
of the value of exploring further the 3-(3,4,5-trimethoxy-
phenyl)-2-propenoyl pharmacophore in the design of
MDR revertants.
These data are presented in Figure 4. In the case of
HL-60 cells, at the lowest concentration utilized viz.
4 lM, caspase-3 was activated markedly by 1, while 4 in-
creased the activity of caspases-3, -8, and -9 above 8 lM.
Some increases in the activations of caspases-8 and -9
were noted when 8 lM of 1 was employed. It should
be noted that the extent of caspase activation was com-
parable with that achieved with the positive control acti-
nomycin D. These data reveal that the mode of action of
1 and 4 in this cell line is due, at least in part, to activa-
tion of caspases-3, -8, and -9. On the other hand, while 1
and 4 were more cytotoxic toward HSC-2 cells than the
HL-60 neoplasm, activation of caspases was appreciably
less noticeable as compared with that attained by actino-
mycin D. Thus while 4 activated caspase-3 at 8 lM, only
at the highest concentration of 32 lM were there in-
creases in the activation of both caspases-3 and -8 by
1 and in caspase-8 and -9 by 4, while 1 failed to activate
caspase-9. Thus the modes of action of 1–10 likely vary
depending on the cell line. These compounds may in-
duce apoptosis in HL-60 cells while they may induce
incomplete apoptosis in other type of cells. This capacity
to cause toxicity by different biochemical mechanisms
depending on the cells under consideration may contrib-
ute to the selective damage to malignant cells observed
on occasions vide supra thereby enhancing the potential
of these compounds as lead molecules in developing
agents for use in cancer chemotherapy.
The evaluation of compounds 1–10 for cytotoxic prop-
erties and selective toxicity to malignant cells is summa-
rized in Table 2. Comments will be made on the new
biodata generated for 3–10. All of the enones display
cytotoxic properties to the malignant HSC-2, HSC-3,
HSC-4, and HL-60 cells. Apart from 2, 3, and 9, the
average CC50 values were lower than that of melphalan
and the most potent compounds are the acyclic enones 4
and 5. In order to identify those compounds displaying
selective toxicity to neoplastic cells, selective index (SI)
figures were calculated. These values reveal that the
compounds demonstrate greater cytotoxicity toward
tumorous cells, particularly 4 and 5. Thus in terms of
both cytotoxic potencies and selective toxicity for neo-
plastic cells, both 4 and 5 emerged as lead molecules.
Table 3. Torsion angles and charge densities in compounds 1–10
Compound
ha (ꢁ)
Atomic chargeb (esu)
4. Conclusions
1
2
ꢁ42.5
ꢁ42.9
ꢁ54.0
ꢁ0.01
ꢁ0.11
ꢁ0.02
ꢁ52.1
8.15
ꢁ0.052
ꢁ0.037
ꢁ0.098
ꢁ0.030
ꢁ0.015
ꢁ0.050
ꢁ0.101
ꢁ0.053
ꢁ0.053
ꢁ0.029
Compounds 6–10 are novel MDR-reversing agents
which have been developed from the lead compound
1. In particular the remarkable potency of 8 was discov-
ered in this study. Future work will involve placing one
or more 3-(3,4,5-trimethoxyphenyl)-2-propenoyl groups
onto a variety of acyclic, alicyclic, aryl, and heteroaryl
scaffolds in an attempt to find correlations between the
topography of these molecules and potencies in this as-
say. Most of the compounds possess noteworthy cyto-
toxic potencies and display greater lethality to some
neoplasms than normal cells. Activation of different
caspases which induced apoptosis was shown in repre-
3
4
5
6
7
8
9
10
ꢁ51.8
ꢁ0.05
a h is the torsion angle between aryl ring A and the adjacent olefinic
group.
b The atomic charge is the electron density on the olefinic carbon atom
adjacent to ring A.

U. Das et al. / Bioorg. Med. Chem. 15 (2007) 3373–3380
3377
0.4
0.3
0.2
0.1
0
0.4
Compound 1
HL-60
Compound 4
HL-60
0.3
0.2
0.1
0
0
4
8
16
32
ActD
0
4
8
16
32
ActD
Concentration ( M)
μ
Concentration( M)
μ
0.5
0.4
0.3
0.2
0.1
0
0.5
0.4
0.3
0.2
0.1
0
Compound 1
HSC-2
Compound 4
HSC-2
0
4
8
16
32
ActD
0
4
8
16
32
ActD
Concentration ( M)
μ
Concentration ( M)
μ
Caspase 3
Caspase 8
Caspase 9
Figure 4. The effect of 1 and 4 on the activation of caspases-3, -8, and -9 in HL-60 and HSC-2 cells. HL-60 cells (4 · 106 cells) and HSC-2 cells
(2 · 106 cells) were incubated for 4 h with the indicated concentrations of 1 or 4 or actinomycin D (1 lg/mL), and caspase activity was measured by
the substrate cleavage assay. Each value represents the mean from 2 to 3 experiments.
sentative molecules to be at least one way whereby cyto-
toxicity was mediated.
washed with water, dried, and crystallized from chloro-
form/ethanol to give 3. Mp 157 ꢁC; yield 61%. 1H NMR
(CDCl3): 2.04 (br s, 4H, 2 · CH2), 2.70 (s, 2H, CH2),
2.76 (s, 2H, CH2), 3.90 (s, 9H, 3 · OCH3), 6.71 (s, 2H,
Ar–H), 7.38 (s, 1H, @CH), 7.42 (s, 1H, @CH), 7.59
(d, 2H, Ar–H, J = 8.50 Hz), 8.28 (d, 2H, Ar–H,
J = 8.55 Hz). Anal. Calcd for C24H25NO6: C, 68.07; H,
5.95; N, 3.31. Found C, 68.21; H, 5.82; N, 2.99%.
5. Experimental
The melting point (mp) of each compound was deter-
mined on a Gallenkamp melting point apparatus and
are uncorrected. 1H (500 MHz) and 13C (125 MHz)
NMR spectra were recorded on a Bruker AM 500 FT
NMR instrument. The letters s, br s, d, t, and q refer
to singlet, broad singlet, doublet, triplet, and quartet,
respectively. Elemental analyses were undertaken on
3–5 (C, H, N) and 6–10 (C, H) by Mr. K. Thoms,
Department of Chemistry, University of Saskatchewan.
Column chromatography was carried out using silica gel
60 (70–230 mesh).
5.1.2. 1-(4-Nitrophenyl)-5-(3,4,5-trimethoxyphenyl)-1,4-
pentadien-3-one (4). Aqueous sodium hydroxide solution
(10%, 3 ml) was added dropwise over a period of 10 min
to a cooled solution of 10 (2.36 g, 0.01 mol) vide infra
and 4-nitrobenzaldehyde (1.51 g, 0.01 mol) in ethanol
(25 ml). A yellow solid separated after the addition of
sodium hydroxide was completed. The mixture was stirred
for additional 1 h, filtered, washed with water, and dried.
The crude compound was purified by column chroma-
tography [eluent/ethyl acetate and hexane (2:8)] and
finally crystallized from ethanol to give 4. Mp 169 ꢁC;
5.1. Synthesis of 3–10
1
5.1.1. 2-(4-Nitrophenylmethylene)-7-(3,4,5-trimethoxy-
phenylmethylene)-cycloheptanone (3). Aqueous sodium
hydroxide solution (10%, 3 ml) was added dropwise over
a period of 10 min to a cooled solution of 9 (2.9 g,
0.01 mol) vide infra and 4-nitrobenzaldehyde (1.51 g,
0.01 mol) in ethanol (25 ml). The mixture was stirred
for 8 h. The yellow solid separated out was filtered,
yield 42%. H NMR (CDCl3): 3.93 (s, 3H, OCH3), 3.94
(s, 6H, 2 · OCH3), 6.87 (s, 2H, Ar–H), 6.97 (d, 1H,
@CH, J = 15.85 Hz), 7.23 (d, 1H, @CH, J = 15.87 Hz),
7.71 (d, 1H, @CH, J = 15.88 Hz), 7.76 (d, 1H,
J = 16.15 Hz), 7.78 (d, 2H, J = 8.68 Hz), 8.29 (d, 2H,
J = 8.7 Hz). Anal. Calcd for C20H19NO6: C, 65.03; H,
5.18; N, 3.79. Found C, 64.82; H, 4.98; N, 3.61%.

3378
U. Das et al. / Bioorg. Med. Chem. 15 (2007) 3373–3380
Figure 5. HL-60 or HSC-2 cells (5 · 105 cells) were incubated for 6 hours with the indicated concentrations of 1 or 4, or actinomycin D (1 lg/mL).
DNA fragmentation was detected above 4 lM in HL-60 cells. No DNA fragmentation was observed in HSC-2 cells. M is the molecular weight
marker of DNA. The arrow indicates a large DNA fragment. Reproducible results were obtained in another experiment.
5.1.3. 1-(4-Methoxyphenyl)-5-(4-nitrophenyl)-1,4-penta-
dien-3-one (5). 4-Methoxybenzaldehyde was condensed
with acetone using the same method employed in the
presence of 10 to give 4-(4-methoxyphenyl)-3-buten-2-
one. Mp 73 ꢁC (lit.22 72 ꢁC); yield 62%. 1H NMR
(CDCl3): d 2.37 (s, 3H, COCH3), 3.86 (s, 3H,
OCH3), 6.63 (d, 2H, Ar–H, J = 8.64 Hz), 7.48 (d, 1H,
@CH, J = 15.0 Hz), 7.51 (d, 2H, Ar–H, J = 8.08 Hz).
This intermediate was reacted with 4-nitrobenzalde-
hyde using the same procedure as employed in the syn-
thesis of 4 to give the desired product which was
purified by crystallization from chloroform/ethanol.
Mp 182 ꢁC; yield 56%. 1H NMR (CDCl3): 3.87 (s,
3H, OCH3), 6.96 (m, 3H, @CH and Ar–H), 7.20 (d,
1H, @CH, J = 15.85 Hz), 7.61 (d, 1H, J = 6.9 Hz),
7.75 (m, 4H, 2 · @CH and Ar–H), 8.28 (d, 2H, Ar–
H, J = 8.75 Hz). Anal. Calcd for C18H15NO4: C,
69.89; H, 4.89; N, 4.53. Found C, 69.74; H, 4.97; N,
4.49%.
18H, 6 · OCH3), 6.72 (s, 4H, Ar–H), 7.35 (s, 2H,
2 · =CH). Anal. Calcd for C26H30O7: C, 68.71; H
6.65. Found C, 68.58; H 6.67%.
5.1.5. 2,7-Bis-(3,4,5-trimethoxyphenylmethylene)-cyclo-
heptanone (7). A mixture of cycloheptanone (1.12 g,
0.01 mol), 3,4,5-trimethoxybenzaldehyde(3.92 g, 0.02 mol),
and aqueous sodium hydroxide solution (35%, 2 ml)
in ethanol (30 ml) was stirred at room temperature
for 3 h and then heated under reflux for 4 h.
The reaction mixture was cooled to room temperature
and stirred overnight. The solid obtained was
filtered, washed with water, and crystallized from chlo-
roform/ethanol to give 7. Mp 182 ꢁC; yield 63%. 1H
NMR (CDCl3):
d 1.84 (m, 2H, CH2), 2.97 (t,
4H, 2 · CH2), 3.90 (s, 18H, 6 · OCH3), 6.73 (s, 4H,
Ar–H), 7.74 (s, 2H, @CH). Anal. Calcd for
C27H32O7: C, 69.21; H 6.88. Found C, 69.21; H
7.10%.
5.1.4. 2,6-Bis-(3,4,5-trimethoxyphenylmethylene)-cyclo-
hexanone (6). Dry hydrogen chloride was bubbled
through a solution of cyclohexanone (1.96 g, 0.01 mol)
and 3,4,5-trimethoxybenzaldehyde (3.92 g, 0.02 mol) in
ether (30 ml) for 10 min. The solution was stirred at
room temperature for 2 h. The solid obtained was fil-
tered and crystallized from chloroform/ethanol to give
6. Mp 208 ꢁC; yield 68%. 1H NMR (CDCl3): d 2.05
(br s, 4H, 2 · CH2), 2.75 (br s, 4H, 2 · CH2), 3.88 (s,
5.1.6. 2,5-Bis-(3,4,5-trimethoxyphenylmethylene)-cyclo-
pentanone (8). Compound 8 was prepared from cyclo-
pentanone by the same procedure which was employed
in synthesizing 6. The crude product was recrystallized
from chloroform/ethanol. Mp 205 ꢁC; yield 71%. 1H
NMR (CDCl3): d 3.16 (s, 4H, 2 · CH2), 3.92 (s, 18H,
6 · OCH3), 6.87 (s, 4H, Ar–H), 7.54 (s, 2H, 2 · @CH).
Anal. Calcd for C25H28O7: C, 68.17; H, 6.41. Found
C, 68.12; H 6.45%.

U. Das et al. / Bioorg. Med. Chem. 15 (2007) 3373–3380
3379
5.1.7. 2-(3,4,5-Trimethoxyphenylmethylene)-cyclohepta-
A
Verapamil was employed asa positive control using a con-
centration intermediate between 4 and 40 lg/ml, namely
10 lg/ml. A similar method was followed when evaluating
MDR in human colo 320 colon cancer cells, except that
before flow cytometry was employed, the cells which form
a monolayer were treated with 0.25% trypsin solution for
2–3 min in order to produce a suspension of individual
cells.
none (9).
0.03 mol),
solution of cycloheptanone (3.36 g,
3,4,5-trimethoxybenzaldehyde (3.92 g,
0.02 mol), and aqueous sodium hydroxide solution
(10%, 3 ml) in ethanol (30 ml) was stirred at room tem-
perature for 12 h. The solution was acidified with dilute
hydrochloric acid and the solvents removed in vacuo.
The residue remaining was extracted with chloroform,
the organic extracts were washed with water, dried over
sodium sulfate, and evaporated under vacuum. The res-
idue obtained was purified by column chromatography
using ethylacetate/hexane(1:9) as eluent. Mp 88 ꢁC; yield
5.4. Evaluation against cancerous and malignant cells
The procedure utilized for evaluating the cytotoxic
properties of the enones 3–10 using both neoplastic
and non-malignant cell lines has been described previ-
ously.25 In brief, the cells were incubated in DMEM
supplemented with 10% heat-activated FBS in a humid-
ified 5% carbon dioxide atmosphere for 24 h. Cells in the
presence of various concentrations of compounds as
well as untreated cells were washed once with phos-
phate-buffered saline and incubated for 4 h in fresh med-
ia containing MTT. The cells were isolated and lysed
with dimethylsulfoxide and the relative number of viable
cells was determined at 540 nm using a Microplate read-
er. In the case of HL-60 cells, a slightly different strategy
was employed insofar as this cell line was cultured in
RPMI1640 media plus 10% FBS and viable cells were
measured by trypan blue exclusion. The CC50 values
were determined from dose–response curves and the fig-
ures in Table 1 are means from duplicate experiments.
The selectivity index figures were obtained from the fol-
1
61%. H NMR (CDCl3): d 1.80 (m, 6H, 3 · CH2), 2.73
(t, 4H, 2 · CH2), 3.87 (s, 6H, 2 · OCH3), 3.89 (s, 3H,
OCH3), 6.60 (s, 2H, Ar–H), 7.48 (s, 1H, @CH). Anal.
Calcd for C17H22O4: C, 70.32; H, 7.64. Found C,
70.59; H, 7.63%.
5.1.8. 4-(3,4,5-Trimethoxyphenyl)-3-buten-2-one (10). A
solution of acetone (40 ml), 3,4,5-trimethoxybenzalde-
hyde (4.9 g, 0.025 mol), and aqueous sodium hydroxide
solution (4%, 10 ml) was stirred at room temperature for
12 h. The solution was neutralized with dilute hydro-
chloric acid, extracted with chloroform, the combined
organic extracts were dried over sodium sulfate and
evaporated to dryness. The residue obtained was puri-
fied by column chromatography (ethyl acetate/hexane
1:9) to obtain the desired product. Mp 91 ꢁC; yield
1
54%. H NMR (CDCl3): d 2.39 (s, 3H, COCH3), 3.90
(s, 9H, 3 · OCH3), 6.63 (d, 1H, @CH, J = 16.16 Hz),
6.81 (s, 2H, Ar–H),7.44 (d,1H, @CH, J = 16.17 Hz).
Anal. Calcd for C13H16O4: C, 66.09; H, 6.83. Found
C, 66.23; H, 6.97%.
lowing
(HPLF) + CC50 (HPC)]/[CC50 (HSC-2) + CC50 (HSC-
equation,
namely
[CC50(HGF) + CC50
3) + CC50 (HSC-4) + CC50(HL-60)] · 4/3.
5.2. Molecular modeling
5.5. Effect of 1 and 4 in causing apoptosis and activating
caspases in HL-60 and HSC-2 cells
Models of the compounds 1–10 were built using Bio-
MedCache 6.1 for Windows. The lowest energy confor-
mations were obtained from optimized geometry
calculations in MOPAC using AM1 parameters. All
six carbon atoms comprising ring A in 1, 3, and 4 were
overlapped when constructing Figure 3.
The procedure for determining apoptosis and measuring
caspase activation has been described previously.26 In
brief, the cells were cultured in either RPMI1640 media
supplemented with 10% heat-inactivated FBS (HL-60
cells) or in DMEM supplemented with 10% FBS
(HSC-2 cells) in a humidified 5% carbon dioxide
atmosphere. For the DNA fragmentation assay, the cells
were incubated at 37 ꢁC for 6 h with varying concentra-
tions of compounds as well as untreated cells. The cells
were then removed, lysed, and assayed for internucleos-
omal DNA fragmentation by agarose gel electrophore-
sis. The fragmentation pattern was examined in
photographs taken under UV illumination. The caspase
activation was determined by the substrate cleavage
method. Cells were incubated for 4 h with test samples,
and then removed and lysed. The lysate (200 lg pro-
teins) was mixed with the substrates for caspase-3
(DEVD-pNA(p-nitroanilide)), caspase-8 (IETD-pNA)
or caspase-9 (LEHD-paNA). After incubation for 2 h
at 37 ꢁC, the absorbance at 405 nm of the liberated chro-
mophore pNA was measured using a microplate read-
er.27 The concentrations of compounds used in the
DNA fragmentation assay were 0.5, 1, 2, 4, 8, and
16 lM and for caspase activation 4, 8, 16, and 32 lM
were employed. In all of these assays, actinomycin D
(1 lg/mL) was used as the positive control.
5.3. MDR-reversal assay
This assay has been reported previously.23 In brief,
L-5178 mouse T-cell lymphoma cells were transfected
with the pHa MDR1/A retrovirus using a literature meth-
odology24 and cultured in the presence of colchicine in or-
der to maintain the expression of the MDR phenotype.
Two concentrations of the test compound in dimethyl-
sulfoxide viz. 4 and 40 lg/mL were added to both the par-
ent L-5178 cells and the MDR1 transfected subline. After
incubation at room temperature for 10 min, a solution of
rhodamine 123 was added to the samples and incubated at
37 ꢁC for 20 min. The fluorescence of the parental cells
(designated F1 and F2 for the treated and untreated cells,
respectively) and the MDRl subline (designated F3 and
F4 for the treated and untreated cells, respectively) was
measured bya Beckton–Dickinson FACScan flow cytom-
eter. The FAR value was calculated from the following
equation, namely FAR ¼ ½F3=F4ꢂ=½F1=F2ꢂ. The FAR
value for dimethylsulfoxide ranged from 1.02 to 2.33.

3380
U. Das et al. / Bioorg. Med. Chem. 15 (2007) 3373–3380
E.; Balzarini, J.; Manavathu, E. K.; Stables, J. P. Eur. J.
Med. Chem. 2003, 38, 169–177.
Acknowledgments
12. Dimmock, J. R.; Chamankhah, M.; Das, U.; Zello, G. A.;
Quail, J. W.; Yang, J.; Nienaber, K. H.; Sharma, R. K.;
Selvakumar, P.; Balzarini, J.; De Clercq, E.; Stables, J. P.
J. Enzyme Inhib. Med. Chem. 2004, 19, 1–10.
13. Kevill, D. N.; Weiler, E. D.; Cromwell, N. H. J. Org.
Chem. 1964, 29, 1276–1278.
14. Dimmock, J. R.; Kandepu, N. M.; Hetherington, M.;
Quail, J. W.; Pugazhenthi, U.; Sudom, A. M.; Chaman-
khah, M.; Rose, P.; Pass, E.; Allen, T. M.; Halleran, S.;
Szydlowski, J.; Mutus, B.; Tannous, M.; Manavathu, E.
K.; Myers, T. G.; De Clercq, E.; Balzarini, J. J. Med.
Chem. 1998, 41, 1014–1026.
The authors thank the Canadian Institutes of Health
Research for an operating grant to J. R. Dimmock
and a Grant-in-Aid to H. Sakagami from the Ministry
of Education, Science, Sports and Culture of Japan
(No. 14370607). Support was also provided by the Sze-
ged Foundation of Cancer Research, Hungary to J.
´
Molnar.
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