Design, synthesis and cytotoxic properties of novel 1-[4-(2-alkylaminoethoxy)phenylcarbonyl]-3,5-bis(arylidene)-4-piperidones and related compounds

Article abstract of DOI:10.1016/j.ejmech.2006.08.002

The 3,5-bis(arylidene)-4-piperidones 1 contain the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore which is considered to interact at a complementary binding site in susceptible neoplasms. The hypothesis was formulated that the presence of an acyl group attached to the piperidyl nitrogen atom in series 1 may interact with an additional binding site thereby enhancing cytotoxic potencies. This concept led to the synthesis of various N-acyl-3,5-bis(arylidene)-4-piperidones 3-7 many of which displayed significant cytotoxicity towards a variety of cancer cell lines. A comparison of the potencies between the compounds in series 1 and the related nonquaternary analogues 3-6 revealed that in approximately half of the comparisons made, the N-acyl analogues had increased potencies.

Full text of DOI:10.1016/j.ejmech.2006.08.002

European Journal of Medicinal Chemistry 42 (2007) 71e80
http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and cytotoxic properties of novel
-[4-(2-alkylaminoethoxy)phenylcarbonyl]-3,5-bis(arylidene)-4-
piperidones and related compounds
1
a
a
b
Umashankar Das , Jane Alcorn , Anuraag Shrivastav , Rajendra K. Sharma ,
b
c
Erik De Clercq , Jan Balzarini , Jonathan R. Dimmock
c
a,
*
a
College of Pharmacy and Nutrition, University of Saskatchewan, 110 Science Crescent, Saskatoon, Saskatchewan S7N 5C9, Canada
b
Department of Pathology, College of Medicine and Cancer Research Unit, Saskatoon Cancer Centre, 20 Campus Drive,
Saskatoon, Saskatchewan S7N 4H4, Canada
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
c
Received 8 February 2006; received in revised form 24 July 2006; accepted 11 August 2006
Available online 22 September 2006
Abstract
The 3,5-bis(arylidene)-4-piperidones 1 contain the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore which is considered to interact at a com-
plementary binding site in susceptible neoplasms. The hypothesis was formulated that the presence of an acyl group attached to the piperidyl
nitrogen atom in series 1 may interact with an additional binding site thereby enhancing cytotoxic potencies. This concept led to the synthesis of
various N-acyl-3,5-bis(arylidene)-4-piperidones 3e7 many of which displayed significant cytotoxicity towards a variety of cancer cell lines. A
comparison of the potencies between the compounds in series 1 and the related nonquaternary analogues 3e6 revealed that in approximately half
of the comparisons made, the N-acyl analogues had increased potencies.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: N-Acyl-4-piperidones; Structureecytotoxicity relationships; a,b-Unsaturated ketones; Apoptosis
1
. Introduction
but subsequently two of these groups have been utilized
thereby creating bifunctional alkylating agents for the follow-
ing reason. On a number of occasions, chemosensitization is
greater in tumours than in normal cells [5,6]; when this occurs,
the tumours are more vulnerable to a subsequent chemical in-
sult. In this manner, greater toxicity for malignant cells may
occur. Hence the potential for two sequential electrophilic at-
tacks with cellular thiols was incorporated into the design of
the candidate cytotoxins. It is likely that enones exert their bio-
activities by interacting with a number of different molecular
targets. Since there are a variety of dysregulated processes oc-
curring in neoplasia, such pleiotropy may be a distinct advan-
tage. In fact, the benefits of promiscuous binding of a ligand at
multiple sites have recently been articulated [7].
One of the principal aims of these laboratories is to dis-
cover novel cytotoxic and anticancer agents. In many cases,
the design of the target compounds has incorporated one or
more conjugated unsaturated keto moieties into the molecules.
These groups have a greater and, on occasions, exclusive affin-
ity for thiols in contrast to amino and hydroxy groups [1e3].
Since thiols are absent in nucleic acids, the genotoxic effects
associated with a number of currently available anticancer
drugs [4] may be absent in these enones. Initial studies incor-
porated one a,b-unsaturated keto function into the molecules
Recently the cytotoxic properties of 3,5-bis(arylmethy-
lene)-4-piperidones 1aec and related compounds towards
*
Corresponding author. Tel.: þ1 306 966 6331; fax: þ1 306 966 6377.
E-mail address: jr.dimmock@usask.ca (J.R. Dimmock).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.08.002

72
U. Das et al. / European Journal of Medicinal Chemistry 42 (2007) 71e80
human Molt 4/C8 T-lymphocytes and other cell lines were de-
scribed [8]. In addition, the hydrochloride salt of 1a was well
tolerated in mice whereby five daily doses up to and including
1,5-diaryl-3-oxo-1,4-pentadienyl
pharmacophore
primary binding site A
O
N
2
40 mg/kg did not cause mortalities [9]. This compound low-
ered hepatic glutathione concentrations in mice [9] confirm-
ing, at least in part, that this compound acted as a thiol
alkylator. The development of series 1 seemed therefore to
be judicious and the following approach was considered.
The 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore has
been incorporated not only into piperidine rings but also into
alicyclic compounds with retention of cytotoxic properties
R1
R1
auxiliary binding site B
substituent on piperidyl nitrogen atom
[
10,11] and thus a reasonable hypothesis is that this group in-
Fig. 1. Design of potential cytotoxins to interact at binding sites A and B.
teracts with cellular constituents at a specific binding site re-
ferred to subsequently as site A (Fig. 1). However,
alignment of the 1,5-diaryl-3-oxo-1,4-pentadienyl group at
site A may be influenced by the nature of the group on the het-
erocyclic nitrogen atom. This group could lead to increases in
cytotoxic potencies due to either additional binding with cellu-
lar constituents at an auxiliary binding site (site B, Fig. 1) or
alternatively by facilitating the interaction of the cytotoxin at
site A. On the other hand, the group on the nitrogen atom
may lead to a reduction in potency due to repulsion between
this group and site B thereby preventing interaction between
the 1,5-diaryl-3-oxo-1,4-pentadienyl moiety and site A.
In order to evaluate this hypothesis, various model com-
pounds were designed based on the following considerations.
One of the potential problems with 1a which has a basic centre
is that under physiological conditions, the molecule exists as
an equilibrium between the ionized molecule and the free
base. The charged molecules may be unable to penetrate cell
membranes and exert a cytotoxic effect. Hence N-acylation
was considered a route to follow rendering the nitrogen
atom in 1a nonbasic. An aryl ring in the N-acyl function
was chosen since it may allow van der Waal bonding with
site B and hence the synthesis and bioevaluation of 2a was
suggested. The cytotoxicity of the initial compounds was mon-
itored using the Molt 4/C8 assay and as Fig. 2 reveals, there
was no difference in potency between 1a and 2a.
directions. First, various substituents were placed in rings A
and B which should influence the rate and extent of thiolation
at the adjacent olefinic linkages. Second, the terminal basic cen-
tre of 2a was altered in order to determine whether the pK and
a
size of these groups would afford some idea of the structural re-
quirements at a binding site. Such considerations led to the de-
cision to prepare series 3e7. With a view to identifying any
general trends, all compounds were planned to be evaluated
not only in the Molt 4/C8 screen but also against human CEM
T-lymphocytes and murine L1210 cells.
2. Chemistry
The synthetic chemical routes employed in producing se-
ries 1e7 are portrayed in Scheme 1. The compounds in series
1 were prepared by acid-catalyzed condensation between var-
ious aryl aldehydes and 4-piperidone. The reaction of 1a with
different aroyl chlorides led to the isolation of 2a,b. Alkylation
of methyl 4-hydroxybenzoate with 2-dimethylaminoethyl hy-
drochloride gave rise to the corresponding ether which upon
hydrolysis produced 4-(2-dimethylaminoethyloxy)benzoic
acid isolated as the hydrochloride salt. This compound was
converted to the corresponding acid chloride which condensed
with 1aed leading to the formation of 3aed, respectively. A
similar synthetic pathway was employed using related N-(2-
chloroethyl)amine hydrochlorides in place of 2-dimethylami-
noethyl hydrochloride leading to the formation of 4aed,
5aed and 6aed. Reaction of the free bases derived from
4aed with methyl iodide gave rise to the corresponding qua-
ternary ammonium salts 7aed. In addition, the preparation of
4-(2-diethylaminoethoxy)benzoic acid 8, which is structurally
related to the compounds in series 4, was synthesized by the
procedure indicated in Scheme 2.
In order to explore whether alkoxy or aminoalkoxy groups
placed in ring C would interact at a complementary area at
a binding site, 2b and 3a were examined for cytotoxic potencies.
The result indicated in Fig. 2 reveals that while 2b had statisti-
cally indistinguishable IC50 figures in the Molt 4/C8 screen
from 1a and 2a, the presence of the dimethylaminoethoxy group
found in 3a led to increased potencies. Thus 3a became the lead
molecule in this study and development was planned in two
O
O
N
O
N
O
N
A
B
N
H
O
O
O
C
.HCl
N
OCH3
O
1a
2a
2b
(2.3ꢀ ± 0.ꢁ39
3a
(
1.6ꢁ ± 0.159
(1.ꢁ0 ± 0.0ꢁ9
(0.58 ± 0.0ꢀ9
Fig. 2. Structures of 1a, 2a, 2b and 3a with IC50 values in mM towards Molt 4/C8 lymphocytes in parentheses.

U. Das et al. / European Journal of Medicinal Chemistry 42 (2007) 71e80
73
O
CHO
O
O
N
i
ii
+
2
R1
R1
N
H
N
H
R1
O
R2
1a-d
2a,b
COOCH3
OH
COOCH3
O
COOH
O
COCl
iii
iv
v
O
vi
R3 . HCl
R3
R3 . HCl
O
N
O
N
vii
R1
R1
R1
R1
CH3
CH3
O
O
I
R3
N
. HCl
O
O
CH3
7
a-d
3
3
a-d: R = N(CH 9
3
2
3
4
a-d: R = N(C H 9
2
5
2
3
5
a-d: R =
N
3
6
a-d: R =
N
O
1
1
1
1
1
Scheme 1. Synthesis of the compounds 1e7. The substituents R in series 1, 3e7 were as follows: a ¼ R ¼ H; b: R ¼ Cl; c: R ¼ NO
2
; d: R ¼ CH
3
. The nature
2
2
2
2
R ;
of the R moieties in series 2 were a: R ¼ H; b: R ¼ OCH
3
. The following reagents were used in the synthetic routes viz i ¼ HCl/CH
3
COOH; ii ¼ ClCOC
6
H
4
3
R .HCl/K
iii ¼ ClCH CH
2 2
2
CO
3
; iv ¼ NaOH/HCl; v ¼ SOCl
2
; vi ¼ 1aed; vii ¼ CH I/K CO
3
2
3
.
3
. Bioevaluations
All the compounds in series 1e7 were evaluated against
4. Results and discussion
The evaluation of the compounds in series 1e8 towards hu-
man Molt 4/C8 and CEM T-lymphocytes was undertaken in
order to determine whether the compounds were cytotoxic to
human transformed cells. In addition, a number of clinically
useful anticancer drugs inhibit the growth of murine L1210
cells [12] and consequently assays using this cell line were
also undertaken. The data are presented in Table 1 which is
so formatted that variation in potencies within different series,
between series and in relation to melphalan may be rapidly
perceived. Furthermore this presentation reveals the sensitivity
of the different cell lines to the compounds.
human Molt 4/C8 and CEM T-lymphocytes as well as murine
L1210 cells. In addition, in order to assess the possible contri-
bution of the N-acyl-group per se to cytotoxicity, compound 8
was included in these assays. These data are presented in Table 1.
The cytotoxic potencies of various compounds towards a panel
of approximately 49 human tumour cell lines are given in Ta-
ble 2. A TUNEL assay of a representative compound 6b re-
vealed that at a concentration of 20 mM, apoptosis occurred
in human HepG2 liver cancer cells.
The following comments will be made in reference to first
the potencies of the molecules, second whether the data sup-
port the concept of a supplementary binding site B and third
whether the nature of either the basic group in series 3e7 or
the aryl substituents in rings A and B influence cytotoxic
potencies.
The potencies of the compounds in series 1e7 were com-
pared to the values generated from melphalan which is an an-
ticancer drug exerting its action by alkylating cellular
constituents [13]. Compounds 1a,d, 2a, 3aed, 4a,c,d, 5a,c,d
and 6c,d were more potent than melphalan in both the Molt
4/C8 and CEM assays. In particular, the IC50 values of the
COOCH3
OH
COOCH3
COOH
i
ii
iii
O
O
N
N
HCl
8
Scheme 2. Synthesis of compound 8. The reagents used in preparing this com-
)
pound were as follows: i ¼ ClCH
2
CH
2
N(C
2
H
5
2
. HCl/K CO
2
3
/KI; ii ¼ NaOH;
iii ¼ HCl.

74
U. Das et al. / European Journal of Medicinal Chemistry 42 (2007) 71e80
4-nitro analogues 3c, 4c, 5c and 6c (all in the submicromolar
Table 1
Potencies of the compounds in series 1e8 towards human Molt 4/C8 and CEM
range) towards both cell lines were, on average, nine times
lower than the reference drug. The data, therefore, provide am-
ple evidence for pursuing these novel antineoplastic agents.
The second phase of evaluating the biodata involved com-
paring the potencies of the compounds in series 1 with those ob-
tained for the N-acyl analogues 2e7. The aim was to determine
whether a favourable alignment of the N-acyl group occurred or
not. Compounds 1a, 2a and 2b were equipotent in both the Molt
a
T-lymphocytes and murine L1210 cells
b
IC50 (µM)
Compound Molt 4/C8 cells SD
CEM cells SD L1210 cells SD
1
ac
bc
cc
1.67
13.4
8.28
1.69
0.15
4.0
1.70
8.63
4.47
1.69
1.64
5.00
1.24
1.09
0.02
0.48
2.28
0.00
0.03
3.33
0.57
0.67
7.96
41.4
32.9
8.47
12.0
37.8
10.2
7.18
5.19
7.14
8.23
47.3
3.63
2.28
8.40
103
0.11
0.3
1
4/C8 and CEM assays indicating that the N-aroyl groups of 2a,b
1
0.75
0.09
0.07
0.73
0.09
0.57
0.035
0.21
0.15
14.9
0.01
0.45
0.12
19.5
0.011
0.45
0.16
10.1
0.013
0.21
82
4.2
had no effect on potencies. However, both 2a and 2b had greater
IC50 values than 1a in the L1210 screen, implying that the aroyl
group in 2a and 2b exerted a dystherapeutic effect.
1
d
0.14
0.0
1
.70
2.39
.58
The promising effect of 3a in the Molt 4/C8 screen predi-
cated that a more thorough study should be conducted on an-
alogues of this compound whereby variation of the basic group
could occur as well as the placement of different substituents
in the arylidene aryl rings A and B (Fig. 2). The basic groups
studied varied in size and electronic properties while the aryl
substituents were chosen from three of the four quadrants of
a Craig scatter diagram [14]. The first analysis examined the
effect on cytotoxic potencies of converting 1aed into the am-
ides bearing the same aryl substituent in series 3e6, e.g., the
potency of 1a was compared with 3a, then 4a, 5a and 6a in
each of the three cell lines. The potencies of the amides in se-
ries 3e6 compared to the analogues in series 1 were either in-
creased, equipotent or diminished in 48%, 35% and 17%,
respectively, of the comparisons made. The increases in poten-
cies were most noticeable in 3c, 4c, 5c and 6c which contained
a 4-nitro group. In all cases, a comparison of the cytotoxicity
of the quaternary ammonium salts 7aed with the data gener-
ated for 1aed revealed reductions in cytotoxic potencies.
While a number of quaternary ammonium compounds possess
antineoplastic properties [15], the positively charged nitrogen
atom in series 7 may have impeded, although not prevented,
the penetration of the molecules via the cell membranes to
one or more sites of action inside the cell. Thus, in general,
potency was either increased or retained when the 4-piperi-
dones 1aed were converted into the nonquaternary amides
in series 3e6. As mentioned previously, the compounds pre-
pared in this study likely interact with different molecular tar-
gets. Hence the structural requirements of ligands for
alignment at various binding sites will vary. However, the
data obtained in this study suggest that for some of the molec-
ular targets, an auxiliary binding site exists with which the
2
a
2
b
3.7
0
0.8
3
a
3
b
1.44
0.314
1.39
1.28
18.3
0.31
1.32
1.48
34.0
0.330
0.91
8.42
18.8
0.343
1.72
124
1.30
0.71
1.33
0.43
4.7
3
c
0.319 0.013
0.884 0.532
3
d
4
a
1.65
12.3
0.34
1.33
1.16
9.98
0.15
5.9
4
b
4
c
0.01
0.30
0.64
2.99
0.20
0.08
0.22
47
4
d
5
5
a
b
5
c
0.315 0.002
4.74
5.14
222
2.47
1.64
13
5
d
1.14
6.84
8.26
0.73
0.09
1.00
6
a
6
b
81.4
6.34
21.8
160
54.6
1.08
9.6
6
c
0.379 0.032
6
d
1.36
110
0.13
12
7
a
30
7
b
55.8
40.2
20.6
2.9
71.2
39.3
23.7
> 500
2.47
43.6
5.2
729
230
0.9
7
c
65.0
18.7
> 500
2.13
2-alkylaminoethoxyphenylcarbonyl group of 3e6 interacts.
A further point to consider in assessing the biodata pre-
1
2.4
2.6
4.3
1.5
7d
8
> 500
3.24
n/a
n/a
n/a
sented in Table 1 is as follows. In the case of those amides
in series 3e6 which were more potent than the analogues in
series 1, the possibility exists that the N-acyl group per se pos-
sesses cytotoxic properties. In addition, if hydrolysis of the
amides occurs, a cytotoxic carboxylic acid could be liberated.
In order to assess this issue, 4-(2-diethylaminoethyloxy)ben-
zoic acid hydrochloride 8 was prepared. The IC50 values of
8 in the Molt 4/C8, CEM and L1210 bioassays are presented
in Table 1 and its lack of potent cytotoxic properties suggests
that the N-acyl groups in 3e6 exert an anchoring effect
c
Melphalan
0.56
0.21
0.02
a
The background colors reflect the potencies expressed as IC50 values, namely
black (<1 µM), dark grey (1–10 µM), grey (11–99 µM) and light grey (>100
µM).
b
c
The IC50 value is the concentration of a compound which is required to inhibit
the growth of the cells by 50
.
Data reproduced from J. Med. Chem. 2001, 44, 586. Copyright American
Chemical Society.

U. Das et al. / European Journal of Medicinal Chemistry 42 (2007) 71e80
75
Table 2
Cytotoxicity of representative compounds towards human tumour cell lines
Compound
All cell lines
Colon cancer cells, IC50 (mM)
Leukemic cells, IC50 (mM)
a
IC50 (mM) S.I.
COLO 205 HCT-116 HCT-15 KM12 SW-620 HCC-2998 K-562 RPMI-8226 HL-60 (TB) SR
b
1
3
3
4
5
5
6
6
6
7
7
5
a
b
d
a
b
d
a
b
d
a
d
1.62
1.91
93.3
141
1.55
1.70
1.02
0.182
6.03
1.38
0.631
0.295
0.912
1.17
0.794
1.74
0.776
1.74
19.1
0.148
0.129
2.29
1.12
43.7
9.33
7.94
43.7
0.575
0.209
0.562
1.35
2.00
0.263
0.229
e
0.490
0.331
1.82
0.275
0.098
0.224
<0.01
0.759
0.123
0.123
0.132
0.115
17.4
0.363
1.41
1.32
e
0.209
0.126
0.479
e
1.15
39.8
<1.38
>11.5
1.38
>2455 2.63
<0.01
47.9
1.58
0.043
4.37
>66.1
93.3
15.8
6.92
22.9
e
49.0
1.07
3.55
1.58
e
20.4
0.933
0.562
5.50
1.10
>50.1
11.8
85.1
2.04
2.29
e
0.339
0.191
1.45
0.589
0.158
6.76
>2.63
<0.562
<0.708
>41.7
7.76
>457
>2188
0.339
7.25
2.45
1.45
2.19
e
e
e
<0.005
e
>1738 1.15
0.832
>50.1
9.33
1.02
30.2
0.275
43.7
1.35
0.245
38.0
22.9
60.3
1.86
>4.47
166
20.4
8.91
>50.1
14.1
2.04
28.2
6.17
5.75
41.7
10.5
18.6
1.26
3.55
-Fluorouracil >29.5
26.9
>4365 3.39
118 66.1
6.61
36.3
126
43.7
Melphalan
30.2
38.9
66.1
a
The letters S.I. refer to the selectivity index, i.e., the ratio of the IC50 figures for the least and most sensitive cell lines to the compound.
The hydrochloride salt of 1a was used in this assay.
b
thereby enhancing the critical interactions of the 1,5-diaryl-3-
and aryl substituents are that future developments should in-
volve the placement of strongly basic groups at the end of
the 4-ethoxyphenylcarbonyl chain and inserting nitro or
methyl substituents in the arylidene aryl rings.
oxo-1,4-pentadienyl group with cellular constituents. In addi-
tion, hydrolysis of the amides 4aed to a cytotoxic carboxylic
acid does not appear to take place.
In order to obtain guidelines for expanding this study, re-
views of the biodata summarized in Table 1 for the compounds
in series 3e7 were made in order to find the terminal group of
the N-acyl side chain and the aryl substituent which were op-
timal in terms of cytotoxic potencies. In regard to the five ter-
minal groups of the N-acyl side chains in 3e7, a scale of 5
The data in Table 1 revealed that the human transformed
cells (Molt 4/C8 and CEM T-lymphocytes) were particularly
vulnerable to many of the compounds prepared in this
study. Hence an evaluation of selected compounds against
a substantially greater number of human malignant cell lines
was undertaken. The results of this determination are sum-
marized in Table 2. In this assay, an average of 49 cell lines
from nine different neoplastic diseases, namely leukemia,
melanoma, non-small cell lung, colon, central nervous sys-
tem, ovarian, renal, prostate and breast cancers were em-
ployed [16].
(
lowest IC50 values), 4, 3, 2 and 1 (lowest potency) was em-
ployed which is described in greater detail in Section 6. The
total scores for the four analogues in the three bioassays in
each of the series 3e7 were 49, 45, 44, 28.5 and 13.5, respec-
tively. The disparity in potencies between the series of com-
pounds could have been due to variations in the sizes and
electronic properties of the terminal group of the N-acyl side
chain. Consequently linear, semilogarithmic and logarithmic
plots were made between the potency scores in 3e6 and
both the solvent accessible surface area (SASA) figures and
The average IC50 values against all cell lines were lower
than that of melphalan except for 7a. In particular, the data
for 6b and 6d were particularly noteworthy since in the case
of both compounds, the IC50 values against some of the cell
lines were lower than the minimum concentration used, viz.
ꢀ8.3
the pK values of the basic groups. In addition, plots were
a
10
38 times, respectively, the potency of melphalan.
M. Hence 6b and 6d possessed in excess of 48 and
made between the potency scores of series 3e7 and the
SASA figures of the protonated basic groups of series 3e6
plus the quaternary ammonium function in 7. However, no
correlations were found ( p > 0.1).
The relative contributions of the arylidene aryl substituents
in series 3e7 were examined using a sliding scale of 4, 3, 2
and 1. The combined scores for the unsubstituted, 4-chloro,
An important feature of novel antineoplastic agents is the
display of greater toxicity for malignant cells rather than the
corresponding normal tissues. A differentiation in cytotoxicity
towards the cell lines would reveal that the compound displayed
selective toxicity in contrast to being a general biocidal agent;
such selectivity may translate into a preferential lethality for tu-
mour cells rather than related non-malignant cells. Accordingly,
a selective index (SI) figure for each compound was calculated
being the ratios of the IC50 values between the least potent and
most sensitive cell lines. A figure of 100 was arbitrarily chosen
as significant since it approximated to the data for melphalan.
The amides 3b, 4a, 6a,b,d and 7d met this criterion and, in par-
ticular, the high selectivity of 4a, 6b and 6d indicated the utility
of these three molecules as lead compounds.
4
-nitro and 4-methyl compounds in the Molt 4/C8, CEM
and L1210 tests were 29.5, 22, 52 and 46.5, respectively. In
order to determine whether the electronic, hydrophobic or ste-
ric properties of the aryl substituents were responsible for the
marked influences of the substituents on potencies, linear and
semilogarithmic plots between the potency scores and the
Hammett sigma, Hansch pi and molar refractivity values of
the aryl substituents were made but no correlations were found
( p > 0.1). The conclusions to be drawn from evaluating the
contributions of the terminal group of the N-acyl side chain
A review of the mean graphs [17] revealed that in general
the IC50 values of the compounds were lower towards colon

76
U. Das et al. / European Journal of Medicinal Chemistry 42 (2007) 71e80
and leukemic neoplasms than the other cell lines. The selective
toxicity for these groups of cancers is revealed in Table 2 since
the IC50 figures of many of the colon and leukemic cell lines
were lower than the values for all cell lines. An effective drug
against colon tumours is 5-fluorouracil [18]. The data in Table
(500 MHz) of all of the compounds in series 1e8 and the
1
3
C NMR spectra (125 MHz) of representative compounds
were determined in deuterated solvents using a Bruker AM
500 FT NMR machine.
2
reveal that the IC50 figures for the compounds prepared in
6.1.1. Synthesis of 1aed
this study towards the six colon cancer cell lines indicated
in Table 2 were lower than that of 5-fluorouracil in 67% of
the cases. Melphalan is used in combination chemotherapy
to treat chronic leukemias [19] and in 80% of the comparisons
made for the K-562, RPMI-8226, HL-60 (TB) and SR cell
lines, the compounds in series 1, 3e7 had lower IC50 figures
than melphalan. A general conclusion to be drawn from the
evaluation of representative compounds against a panel of ap-
proximately 49 human tumour cell lines is that their potencies
are substantially greater than certain clinically used drugs and
that they have a particular toxicity towards colon cancer and
leukemic cells.
Compounds 1aec were prepared by a literature procedure
[8]. The same procedure was used to prepare 1d which was
purified by recrystallization from chloroformeethanol to
give the desired product.
6.1.1.1. 3,5-Bis(4-methylphenylmethylene)-4-piperidone(1d). M.p.
ꢁ
1
180e181 C. Yield: 79%. H NMR (CDCl ): 2.40 (s, 6H), 4.16 (s,
3
4H), 7.24 (d, 2H, J ¼ 7.75 Hz), 7.31 (d, 2H, J ¼ 7.85 Hz), 7.80 (s,
2H). Found C, 82.87; H, 6.94; N, 4.60%. Anal. (C H NO ) re-
21
26
2
quires C, 83.13; H, 6.98; N, 4.62%.
6.1.2. General procedure for the synthesis of 2a,b
The possible way in which these compounds exert their cy-
totoxic action was addressed. In a previous study, a cytotoxic
piperidine bearing pendant conjugated arylvinylketo and aryl-
vinyl groups was found to cause apoptosis in human Jurkat T
leukemic cells [20]. Furthermore a number of anticancer drugs
induce apoptosis [21]. Hence a representative compound was
evaluated for apoptosis-inducing properties. A TUNEL assay
revealed that at a concentration of 20 mM, 6b induced apopto-
sis in human HepG2 liver cancer cells, indicating that, at least
in part, this mechanism of action accounts for the cytotoxicity
of 6b and likely for the related analogs as well.
A solution of the aroyl chloride (0.015 mol) in 1,2-dichlo-
roethane (50 ml) was added over 0.5 h to a solution of 1a
(0.01 mol) and triethylamine (0.03 mol) in 1,2-dichloroethane
ꢁ
(30 ml) at w5e6 C. After stirring at room temperature for
6 h, the solvent was removed in vacuo. An aqueous solution
of potassium carbonate (10% w/v, 50 ml) was added to the res-
idue and the resultant slurry was stirred for 4 h at room tem-
perature. The precipitate was collected, washed with water
and dried. The products were purified by crystallization from
chloroformemethanol.
6
.1.2.1. 1-Benzoyl-3,5-bis(phenylmethylene)-4-piperidone (2a). M.p.
ꢁ
165e166 C. Yield: 85%. H NMR (CDCl ): 4.72 (br s, 2H), 5.04
3
1
5
. Conclusions
(br s, 2H), 7.06 (t, 2H), 7.22 (d, 4H), 7.38 (br s, 9H), 7.92 (s, 2H). Found
C, 82.02; H, 5.49; N, 3.56%. Anal. (C H NO ) requires C, 82.30; H,
5.58; N, 3.69%.
The 1-[4-(2-alkylaminoethoxy)phenylcarbonyl]-3,5-bis(ar-
26
21
2
ylidene)-4-piperidones are a novel group of cytotoxins.
Many of these compounds exhibit significantly greater poten-
cies than two established anticancer drugs in different
bioassays. The attachment of the N-acyl group to the 3,5-
bis(arylidene)-4-piperidones 1 leading to series 3e6 was
accompanied by increases in potencies in approximately half
of the comparisons made with the analogues in series 1.
This observation suggests that in the case of various members
of series 3e6, alignment of the N-acyl groups with auxiliary
binding sites takes place reinforcing the interaction of the
6.1.2.2. 1-(4-Methoxybenzoyl)-3,5-bis(phenylmethylene)-4-pi-
ꢁ
1
peridone (2b). M.p. 177e178 C. Yield: 72%. H NMR
(CDCl ): 3.70 (s, 3H), 4.87 (br s, 4H), 6.51 (d, 2H,
3
J ¼ 8.62 Hz), 7.17 (d, 2H, J ¼ 8.69 Hz), 7.40 (br s, 10H),
8.07 (s, 2H). Found C, 79.19; H, 5.72; N, 3.25%. Anal.
(C H NO ) requires C, 79.19; H, 5.66; N, 3.42%.
2
7
23
3
6.1.3. General procedure for the synthesis of series 3e6
The general method employed for the preparation of the
compounds in series 3e6 is exemplified by the synthesis of
1
,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore with a pri-
mary binding site.
4a. A mixture of 8 vide infra (4.10 g, 0.015 mol), thionyl
6
. Experimental protocols
chloride (23.8 g, 2 mol) and dimethylformamide (0.01 ml)
was heated under reflux for 3 h. Evaporation in vacuo led
to the isolation of the corresponding acid chloride which
was dissolved in 1,2-dichloroethane (50 ml) and added over
a period of 0.5 h to a solution of 1a (2.75 g, 0.01 mol) and
triethylamine (3.03 g, 0.03 mol) in 1,2-dichloroethane
6
.1. Chemistry
Melting points are uncorrected. Elemental analyses (C, H,
N) were undertaken on 1d and 2e7 by the Microanalytical
Laboratory, Department of Chemistry, University of Alberta
and were within 0.4% of the calculated values. Compounds
3
drates, 7b as the monohydrate and 5c was obtained with
ꢁ
(30 ml) at w5e6 C. After stirring at room temperature for
6 h, the solvent was removed in vacuo. The solid obtained
was suspended in a solution of potassium carbonate (10%
w/v, 50 ml) and stirred at room temperature for 4 h. The pre-
cipitate was collected, washed with water and dried to give
bed, 4a, 5a,d, 6a,c,d and 7a,d were isolated as the hemihy-
1
.5 mol of water of crystallization. The H NMR spectra
1

U. Das et al. / European Journal of Medicinal Chemistry 42 (2007) 71e80
77
1
-[4-(2-diethylaminoethyloxy)phenylcarbonyl]-3,5-bis(phenyl-
methylene)-4-piperidone, i.e., the free base corresponding to
a. The hydrochloride salt was prepared by dissolving the
free base in a mixture of isopropanol and chloroform (3:2;
0 ml) and after the addition of activated charcoal (0.5 g),
10H), 7.88 (s, 2H), 13.08 (br s, 1H). Found C, 71.38; H,
6.75; N, 5.21%. Anal. (C H ClN O $0.5H O) requires C,
71.16; H, 6.53; N, 5.18%.
3
2
35
2
3
2
4
5
6.1.3.5. 1-[4-(2-Diethylaminoethoxy)phenylcarbonyl]-3,5-bi-
the mixture was stirred at room temperature for 1 h. The so-
lution was acidified with dry hydrogen chloride gas and the
mixture was stirred at room temperature for 1 h. The solvent
was removed in vacuo to yield a viscous oil to which was
added acetone (50 ml) and the mixture was heated under
reflux for 0.5 h. On cooling to room temperature, the precip-
itate was collected and recrystallized from acetone to give
s(phenylmethylene)-4-piperidone hydrochloride hemihydrate
ꢁ
1
(
4a). M.p. 184e185 C. Yield: 72%. H NMR (CDCl3):
1
2
2
.45 (t, 6H, 2 ꢂ CH ), 3.22 (m, 4H, 2 ꢂ NCH CH ), 3.40 (t,
3
2
3
H, OCH CH N), 4.41 (t, 2H, OCH CH N), 4.85 (br s, 4H,
2 2 2 2
ꢂ piperidyl NCH ), 6.50 (d, 2H, aryl H, J ¼ 8.1 Hz), 7.17
2
(
(
d, 2H, aryl H, J ¼ 9.05 Hz), 7.44 (br s, 10H, aryl H), 7.92
13
s, 2H, ]CH), 12.70 (s, 1H, NH). C NMR (CDCl ):
3
4
a. The analogs 4bed were prepared using the same meth-
187.41 (CO), 170.14(eCOeNe), 158.77, 134.88, 132.21,
odology except that isopropanol was used as the recrystalli-
zation solvent for 4b and 4c. Series 3, 5 and 6 were
obtained in a similar manner from methyl 4-hydroxybenzoate
and the appropriate 2-chloro-N-substituted-ethylamine hydro-
chloride. The solvent used in recrystallizing 3aed, 5a,b and
6aed was isopropanol except that diethyl etheremethanol
was used to purify 5c, while 5d was recrystallized from
acetone.
1
30.77, 130.08, 129.70, 129.25, 127.99, 114.19, 63.05
(OCH ), 50.95 (OCH eCH eN), 47.39 (NCH ), 8.89 (CH ).
2 2 2 2 3
Found C, 71.22; H, 6.41; N, 4.79%. Anal.
(C H ClN O $0.5H O) requires C, 71.16; H, 6.71; N,
32 35 2 3 2
5.18%.
6.1.3.6. 3,5-Bis(4-chlorophenylmethylene)-1-[4-(2-diethylaminoe-
thoxy)phenylcarbonyl]-4-piperidone hydrochloride (4b). M.p.
ꢁ
10e211 C. Yield: 68%. H NMR (CDCl ): 1.45 (t, 6H), 3.22
3
1
2
6
.1.3.1. [4-(2-Dimethylaminoethoxy)phenylcarbonyl]-3,5-bis
(br s, 4H), 3.41 (t, 2H), 4.45 (t, 2H), 4.81 (br s, 4H), 6.57 (d,
2H, J ¼ 8.34 Hz), 7.17 (d, 2H, J ¼ 8.30 Hz), 7.31 (m, 4H),
7.41 (d, 2H, J ¼ 6.54 Hz), 7.84 (s, 2H), 12.70 (br s, 1H). Found
C, 64.28; H, 5.26; N, 4.98%. Anal. (C H Cl N O ) requires C,
(
phenylmethylene)-4-piperidone hydrochloride (3a). M.p.
ꢁ
1
2
6
44e245 C. Yield: 76%. H NMR (DMSO-d ): 2.83 (s,
6
H), 3.46 (t, 2H), 4.22 (t, 2H), 4.80 (br s, 4H), 6.55 (d, 2H,
32
33
3 2 3
J ¼ 8.55 Hz), 7.08 (d, 2H, J ¼ 8.65 Hz), 7.37 (br s, 10H),
64.06; H, 5.54; N, 4.67%.
7
5
5
.76 (s, 2H), 11.08 (br s, 1H). Found C, 71.80; H, 5.90; N,
.50%. Anal. (C H ClN O ) requires C, 71.63; H, 6.21; N,
3
0
31
2 3
6.1.3.7. 1-[4-(2-Diethylaminoethoxy)phenylcarbonyl]-3,5-bis(4-
.57%.
nitrophenylmethylene)-4-piperidone hydrochloride (4c). M.p.
ꢁ
97e198 C. Yield: 61%. H NMR (CDCl ): 1.46 (t, 6H), 3.19
3
1
1
6
.1.3.2. 3,5-Bis(4-chlorophenylmethylene)-1-[4-(2-dimethyla-
(br s, 2H), 3.26 (br s, 2H), 4.45 (t, 2H), 4.45 (t, 2H), 4.82 (br
s, 4H), 6.60 (d, 2H, J ¼ 8.25 Hz), 7.14 (d, 2H, J ¼ 8.25 Hz),
7.51 (br s, 4H), 7.89 (s, 2H), 8.26 (d, 2H, J ¼ 6.35 Hz), 12.57
(br s, 1H). Found C, 62.06; H, 5.38; N, 9.19%. Anal.
(C H ClN O ) requires C, 61.88; H, 5.36; N, 9.02%.
minoethoxy)phenylcarbonyl]-4-piperidone hydrochloride hemi-
ꢁ
1
hydrate (3b). M.p. 250e251 C. Yield: 71%. H NMR
DMSO-d ): 2.92 (s, 6H), 3.47 (t, 2H), 4.22 (t, 2H), 4.81 (br
(
6
s, 4H), 6.58 (d, 2H, J ¼ 7.61 Hz), 7.14 (d, 2H, J ¼ 7.65 Hz),
.39 (m, 8H), 7.82 (d, 2H, J ¼ 8.75 Hz), 12.84 (br s, 1H).
Found C, 62.45; H, 5.15; N, 4.86%. Anal.
32
33
4
7
7
6.1.3.8.
1-[4-(2-Diethylaminoethoxy)phenylcarbonyl]-3,5-
(
C H Cl N O $0.5H O) requires C, 62.02; H, 5.03; N,
30 29 3 2 3 2
bis(4-methylphenylmethylene)-4-piperidone
hydrochloride
4d). M.p. 204e205 C. Yield: 62%. H NMR (CDCl3):
.45 (t, 6H), 2.40 (s, 6H), 3.22 (m, 4H), 3.40 (t, 2H), 4.46
4
.82%.
ꢁ
1
(
1
6
.1.3.3.
1-[4-(2-Dimethylaminoethoxy)phenylcarbonyl]-3,5-
(t, 2H), 4.87 (br s, 4H), 6.57 (d, 2H, J ¼ 8.53 Hz), 7.23 (d,
2H, J ¼ 8.37 Hz), 7.32 (m, 4H), 7.88 (s, 2H), 12.62 (br s,
1H). Found C, 73.34; H, 6.86; N, 5.36%. Anal.
(C H ClN O ) requires C, 73.04; H, 7.03; N, 5.01%.
bis(4-nitrophenylmethylene)4-piperidone hydrochloride hemihy-
drate (3c). M.p. 215e217 C. Yield: 62%. H NMR (DMSO-
d6): 2.78 (s, 6H), 3.42 (t, 2H), 4.21 (t, 2H), 4.85 (br s, 4H),
ꢁ
1
34
39
2 3
6
4
5
.70 (d, 2H, J ¼ 8.28 Hz), 7.15 (d, 2H, J ¼ 8.25 Hz), 7.72 (m,
H), 7.82 (s, 2H), 8.27 (br s, 4H), 11.08 (br s, 1H). Found C,
9.45; H, 4.93; N, 9.08%. Anal. (C H ClN O $0.5H O) re-
6.1.3.9. 1-[4-{2-(1-Piperidinyl)ethoxy}phenylcarbonyl]-3,5-bi-
3
0
29
4
7
2
s(phenylmethylene)-4-piperidone hydrochloride hemihydrate
quires C, 59.85; H, 4.85; N, 9.30%.
ꢁ
1
(
5a). M.p. 214e215 C. Yield: 52%. H NMR (CDCl3):
.43 (t, 1H), 1.88 (t, 3H), 2.28 (q, 2H), 2.76 (t, 2H), 3.34 (br
1
6
.1.3.4.
1-[4-(2-Dimethylaminoethoxy)phenylcarbonyl]-3,5-
s, 2H), 3.60 (t, 2H), 4.44 (br s, 2H), 4.86 (br s, 4H), 6.50 (d,
2H, J ¼ 7.75 Hz), 7.16 (d, 2H, J ¼ 7.2 Hz), 7.38 (br s, 10H),
7.90 (s, 2H), 12.61 (br s, 1H). Found C, 71.48; H, 6.38; N,
5.04%. Anal. (C H ClN O $0.5H O) requires C, 71.79; H,
bis(4-methylphenylmethylene)-4-piperidone
hemihydrate (3d). M.p. 224e226 C. Yield: 64%. H NMR
hydrochloride
1
ꢁ
(
CDCl ): 2.40 (s, 6H), 2.90 (t, 2H), 3.43 (t, 2H), 4.42 (t,
3
33 35
2
3
2
2
H), 4.87 (br s, 4H), 6.58 (d, 2H, J ¼ 8.10 Hz), 7.23 (m,
6.38; N, 5.07%.

78
U. Das et al. / European Journal of Medicinal Chemistry 42 (2007) 71e80
J ¼ 12.02 Hz), 4.02 (dd, 2H), 4.30 (t, 2H), 4.47 (br s, 2H),
.86 (br s, 4H), 6.51 (d, 2H, J ¼ 8.56 Hz), 7.19 (d, 2H,
J ¼ 8.50 Hz), 7.44 (br s, 10H), 7.92 (s, 2H), 13.59 (br s,
H). Found C, 59.52; H, 4.72; N, 8.51%. Anal.
C H ClN O $0.5H O) requires C, 59.67; H, 4.85; N,
6
.1.3.10. 3,5-Bis(4-chlorophenylmethylene)-1-[4-{2-(1-piperi-
4
dinyl)ethoxy}phenylcarbonyl]-4-piperidone
hydrochloride
ꢁ
1
(
1
5b). M.p. 257e258 C. Yield: 75%. H NMR (CDCl3):
.44 (q, 1H), 1.92 (t, 3H), 2.32 (q, 2H), 2.80 (q, 2H), 3.38
1
(
3
2
31
4
8
2
(
(
br s, 2H), 3.63 (d, 2H, J ¼ 10.23 Hz), 4.54 (br s, 2H), 4.80
8.69%.
br s, 4H), 6.57 (d, 2H, J ¼ 7.84 Hz), 7.17 (d, 2H,
J ¼ 7.99 Hz), 7.34 (m, 8H), 7.90 (s, 2H), 12.68 (br s, 1H).
6.1.3.16. 3,5-Bis(4-methylphenylmethylene)-1-[4-{2-(4-mor-
Found C, 64.35; H, 5.39; N, 4.45%. Anal. (C H Cl N O )
3
3
33
3 2 3
pholinyl)ethoxy}phenylcarbonyl]-4-piperidone hydrochloride
requires C, 64.77; H, 5.44; N, 4.58%.
ꢁ
1
hemihydrate (6d). M.p. 235e236 C. Yield: 66%. H NMR
(
(
CDCl ): 2.40 (s, 3H), 3.03 (m, 2H), 3.40 (br s, 2H), 3.54
3
d, 2H, J ¼ 11.98 Hz), 4.02 (dd, 2H), 4.29 (t, 2H), 4.50 (br
6
.1.3.11. 3,5-Bis(4-nitrophenylmethylene)-1-[4-{2-(1-piperidi-
nyl)ethoxy}phenylcarbonyl]-4-piperidone hydrochloride 1.5
s, 2H), 4.87 (s, 4H), 6.58 (d, 2H, J ¼ 8.63 Hz), 7.24 (m,
ꢁ
1
hydrate (5c). M.p. 224e226 C. Yield: 64%. H NMR
DMSO-d ): 1.42 (t, 1H), 1.81 (t, 3H), 2.04 (q, 2H), 2.86 (q,
1
6
7
0H), 7.88 (s, 2H), 13.68 (br s, 1H). Found C, 70.39; H,
.69; N, 4.79%. Anal. (C H ClN O $0.5H O) requires C,
(
6
3
4
37
2
4
2
2H), 3.36 (d, 2H), 3.48 (m, 2H), 4.32 (br s, 2H), 4.75 (br s,
4H), 6.56 (d, 2H, J ¼ 8.25 Hz), 7.07 (d, 2H, J ¼ 8.10 Hz),
7.60 (m, 4H), 7.78 (s, 2H), 8.16 (br s, 4H), 11.53 (s, 1H). Found
0.15; H, 6.57; N, 4.81%.
6
.1.4. General procedure for the synthesis of 7aed
Activated charcoal (0.5 g) was added to a solution of the
C, 71.38; H, 6.75; N, 5.21%. Anal. (C H ClN O $1.5H O)
3
requires C, 71.16; H, 6.53; N, 5.18%.
2
35
2
3
2
free base of 4a vide supra (4.95 g, 0.0l mol) in acetone
25 ml) and the mixture was stirred at room temperature for
h. The suspension was filtered via celite and the celite bed
was washed with acetone (10 ml). Methyl iodide (2.13 g,
.015 mol) was added to the combined filtrates and the mix-
(
1
6
.1.3.12. 3,5-Bis(4-methylphenylmethylene)-1-[4-{1-piperidi-
nyl)ethoxy}phenylcarbonyl]-4-piperidone hydrochloride hemi-
ꢁ
1
hydrate (5d). M.p. 105 C (dec). Yield: 65%. H NMR
CDCl ): 1.42 (q, 1H), 1.89 (t, 3H), 2.22 (q, 2H), 2.80 (br s,
0
(
3
ture was stirred at room temperature for 4e5 h. The resultant
precipitate was collected, dried and recrystallized from diethyl
ether/methanol to give the desired products.
2
2
H), 3.40 (br s, 2H), 3.65 (d, 2H, J ¼ 9.56 Hz), 4.48 (br s,
H), 4.88 (br s, 4H), 5.44 (br s, 2H), 6.56 (d, 2H,
J ¼ 7.91 Hz), 7.23 (m, 10H), 7.99 (s, 2H), 11.80 (br s, 1H).
Found C, 71.38; H, 6.75; N, 5.21%. Anal.
(
6
.1.4.1.
1-[4-(2-Diethylaminoethoxy)phenylcarbonyl]-3,5-bi-
C H ClN O $0.5H O) requires C, 71.16; H, 6.53; N,
32 35 2 3 2
s(phenylmethylene)-4-piperidone methoiodide (7a). M.p. 161e
5
.18%.
1
62 C. Yield: 85%. H NMR (CDCl ): 1.45 (t, 6H,
ꢁ
1
2
4
3
ꢂ CH ), 3.34 (s, 3H, NeCH ), 3.70 (m, 4H, 2 ꢂ NCH CH ),
3
3
2
3
6
.1.3.13. 1-[4-{2-(4-Morpholinyl)ethoxy}phenylcarbonyl]-3,5-
.17 (br s, 2H, OCH CH N), 4.39 (br s, 2H, OCH CH N), 4.86
2
2
2
2
bis(phenylmethylene)-4-piperidone hydrochloride hemihydrate
(
br s, 4H, piperidyl H), 6.56 (d, 2H, aryl H, J ¼ 8.58 Hz), 7.19
(d, 2H, aryl H, J ¼ 8.03 Hz), 7.39 (br s, 12H, aryl H), 7.91 (s,
H, olefinic H). Found C, 61.11; H, 5.64; N, 4.23%. Anal.
C H IN O $0.5H O) requires C, 61.39; H, 5.77; N, 4.33%.
ꢁ
1
(
6a). M.p. 208e209 C. Yield: 73%. H NMR (CDCl ): 3.05
3
(
2
q, 2H), 3.40 (br s, 2H), 3.53 (d, 2H, J ¼ 12.02 Hz), 4.02 (dd,
2
(
H), 4.30 (t, 2H), 4.47 (br s, 2H), 4.86 (br s, 4H), 6.51 (d, 2H,
3
3
37
2
3
2
J ¼ 8.56 Hz), 7.19 (d, 2H, J ¼ 8.50 Hz), 7.44 (br s, 10H), 7.92
s, 2H), 13.69 (br s, 1H). Found C, 69.54; H, 5.95; N, 4.96%.
Anal. (C H ClN O $0.5H O) requires C, 69.36; H, 6.00; N,
(
6
.1.4.2. 3,5-Bis(4-chlorophenylmethylene)-1-[4-(2-diethylami-
3
2
33
2
4
2
noethoxy)phenylcarbonyl]-4-piperidone methoiodide (7b). M.p.
2
5
.05%.
ꢁ
1
20e223 C. Yield: 69%. H NMR (DMSO-d ): 1.32 (t, 6H),
6
3
.07 (d, 3H, J ¼ 7.99 Hz), 3.45 (t, 4H), 3.74 (t, 2H), 4.30 (br
6
.1.3.14. 3,5-Bis(4-chlorophenylmethylene)-1-[4-{2-(4-mor-
s, 1H), 4.39 (br s, 1H), 4.61 (br s, 4H), 6.60 (d, 1H,
J ¼ 8.22 Hz), 7.11 (d, 1H, J ¼ 8.35 Hz), 7.23 (d, 1H,
J ¼ 8.36 Hz), 7.71 (br s, 1H), 7.89 (s, 2H). Found C, 54.44;
H, 4.79; N, 3.75%. Anal. (C H Cl IN O $0.5H O) requires
pholinyl)ethoxy}phenylcarbonyl-4-piperidone hydrochloride
ꢁ
1
(
3
6b). M.p. 240e241 C. Yield: 74%. H NMR (CDCl3):
.06 (br s, 2H), 3.40 (br s, 2H), 3.55 (t, 2H), 4.04 (d, 2H,
J ¼ 11.97 Hz), 4.29 (t, 2H), 4.30 (t, 2H), 4.50 (br s, 2H),
.81 (br s, 4H), 6.59 (d, 2H, J ¼ 7.30 Hz), 7.14 (d, 2H,
J ¼ 7.47 Hz), 7.51 (m, 4H), 7.90 (s, 2H), 8.26 (br s, 4H),
3.59 (s, 1H). Found C, 62.39; H, 4.94; N, 4.47%. Anal.
C H Cl N O ) requires C, 62.60; H, 5.09; N, 4.56%.
3
3
35
2
2
3
2
C, 54.78; H, 4.87; N, 3.92%.
4
6.1.4.3. 1-[4-(2-Diethylaminoethoxy)phenylcarbonyl]-3,5-bis(4-
1
(
nitrophenylmethylene)-4-piperidone methoiodide (7c). M.p.
ꢁ
193e195 C (dec). Yield: 74%. H NMR (DMSO-d ): 1.24 (t,
6
1
3
2
31
3
2
4
6H), 3.00 (s, 3H), 3.38 (q, 4H), 3.64 (t, 2H), 4.27 (t, 2H),
6
.1.3.15. 1-[4-{2-(4-Morpholinyl)ethoxy}phenylcarbonyl]-3,5-
4.40 (br s, 4H), 6.70 (d, 2H, J ¼ 8.41 Hz), 7.16 (d, 2H,
J ¼ 8.39 Hz), 7.74 (br s, 4H), 7.83 (s, 2H), 8.27 (br s, 4H).
Found C, 54.41; H, 4.71; N, 7.60%. Anal. (C H IN O ) re-
bis(4-nitrophenylmethylene)-4-piperidone hydrochloride hemi-
ꢁ
1
hydrate (6c). M.p. 224e226 C. Yield: 64%. H NMR
CDCl ): 3.05 (q, 2H), 3.40 (br s, 2H), 3.53 (d, 2H,
3
3
35
4 7
(
quires C, 54.55; H, 4.86; N, 7.71%.
3

U. Das et al. / European Journal of Medicinal Chemistry 42 (2007) 71e80
79
6
.1.4.4.
1-[4-(2-Diethylaminoethoxy)phenylcarbonyl]-3,5-
methoiodide
7d). M.p. 212e213 C. Yield: 72%. H NMR (DMSO-d ):
5-fluorouracil and melphalan were evaluated towards 57 cell
lines. In this assay, compounds were incubated for 48 h using
five different concentrations at series tenfold dilutions of
bis(4-methylphenylmethylene)-4-piperidone
(
1
2
7
ꢁ
1
6
ꢀ8.3
ꢀ4.3
ꢀ8
ꢀ4
.24 (t, 6H), 2.34 (s, 6H), 3.01 (s, 3H), 3.39 (q, 4H), 3.68 (t,
H), 4.33 (t, 2H), 4.87 (br s, 4H), 6.75 (d, 2H, J ¼ 8.34 Hz),
.22 (d, 2H, J ¼ 8.30 Hz), 7.28 (m, 8H), 7.72 (s, 2H). Found
10
fluorouracil (10
10
Me10
M except for 1a, 4a (10 Me10 M), 5-
6.6
ꢀ
ꢀ2.6
ꢀ7.6
Me10
M) and melphalan (10 Me
M). The MG MID figures for 1a, 3b,d, 5d, 7d and
ꢀ
3.6
C, 62.65; H, 6.25; N, 4.10%. Anal. (C H IN O $0.5H O) re-
quires C, 62.40; H, 6.13; N, 4.15%.
melphalan were IC50 values. In the following cases, the num-
ber of cell lines compared to the total number of cell lines for
which an IC50 figure were not obtained were as follows [max-
imum or minimum concentrations (molar) in parentheses],
3
5
41
2
3
2
6
.1.5. Synthesis of 8
A mixture of methyl 4-hydroxybenzoate (15.21 g, 0.1 mol),
ꢀ
8
ꢀ4.3
namely 4a: 2/53 (<10 ), 5b: 2/50 (>10 ), 6a: 3/48
ꢀ8.3
ꢀ
4.3
(>10
52 (>10
ꢀ8.3
), 6d: 3/44 (<10 ), 7a: 36/
anhydrous potassium carbonate (34.55 g, 0.25 mol), 2-chloro-
N,N-diethylamine hydrochloride (25.81 g, 0.15 mol), potas-
sium iodide (0.166 g, 0.001 mol) and acetone (75 ml) was
heated under reflux for 8e9 h. On cooling, the solid was re-
moved by filtration and the solvent was evaporated. The resi-
due was dissolved in toluene (75 ml) and the solution was
washed with sodium hydroxide solution (2% w/v, 30 ml) and
deionized water (2 ꢂ 30 ml). Removal of the solvent afforded
methyl 4-(2-diethylaminoethyloxy)benzoate as an oil which
was dissolved in ethanol (50 ml) and added to a solution of so-
dium hydroxide (8.0 g, 0.2 mol) in water (50 ml). The mixture
was heated under reflux for 2 h. The ethanol was removed in
vacuo and the aqueous solution was acidified with hydro-
), 6b: 11/38 (<10
4.3
) and 5-fluorouracil 3/57 (>10 ).
ꢀ
ꢀ2.6
6.3. Rankings of potencies of the compounds in
series 3e7
The first investigation involved comparing the IC50 values
of those compounds in series 3e7 which had the same sub-
stituents in the arylidene aryl ring. The scores for the com-
pounds in each of the Molt 4/C8, CEM and L1210 cell
lines were obtained as follows. The compound having the
highest potency was assigned a value of 5, the compound
with the next lowest IC50 figures was given a rating of
ꢁ
chloric acid (12 N) at 5e6 C. The solid was collected, tritu-
ꢁ
4
and so forth; the amide with lowest potency was given
rated with water (previously cooled to 5e6 C), filtered and
ꢁ
a figure of 1. The standard deviations for each IC50 value
were taken into consideration. The scores available for equi-
potent compounds were divided equally and a total of 15
points were awarded for each of the comparisons. The sec-
ond analysis was designed to find the optimal substitution
pattern in the arylidene aryl rings in the three cell lines.
Thus in each of the series 3e7, the most potent compound
was given a rating of 4, the second most potent analog 3
and so forth. A total of 10 points were used in each compar-
ison. Standard deviations were taken into consideration and
the scores for compounds having the same potencies were
divided equally.
dried at 55e60 C in vacuo and recrystallized from water to
give 8.
6
.1.5.1. 4-(2-Diethylaminoethoxy)benzoic acid hydrochloride
ꢁ
ꢁ
(
8). M.p. 170e172 C [lit. [22], m.p. 171e174 C]. Yield:
1
7
3
4
7
3%. H NMR (D O): 1.17e1.20 (t, 6H, 2 ꢂ CH ), 3.13e
2
3
.24 (m, 4H, 2 ꢂ CH ), 3.47e3.48 (t, 2H, OCH CH N),
2 2 2
.26e4.28 (t, 2H, OCH CH N), 6.89e6.91 (d, 2H, aryl H),
2
2
.80e7.82 (d, 2H, aryl H).
6
.1.6. Statistical evaluations
The SASA figures of the dimethylamino, diethylamino, pi-
peridino and morpholino groups (free bases and protonated
species) as well as the diethylmethylammonium substituent
were obtained from MacroModel 7.1 [23], while the pKa
values of the basic groups in series 3e6 were culled from
the literature [24]. The Hammett sigma, Hansch pi and molar
refractivity values of the hydro, 4-chloro, 4-nitro and 4-methyl
substituents were taken from a reference source [25].
6.4. Apoptosis assay
HepG2 cells obtained from the ATCC were grown in
Dulbecco’s modified eagle medium supplemented with a 1%
antibiotic solution containing penicillin G sodium, streptomy-
cin sulfate and amphoterin B (GIBCO, Burlington, Canada) as
well as 10% fetal calf serum. An environment of humidified
ꢁ
6
.2. Cytotoxic evaluations
air containing 5% carbon dioxide was maintained at 37 C.
The HepG2 cells were cultured in chambered slides using
20 mM of 6b while control experiments with the cells in the
absence and presence of dimethylsulfoxide were undertaken.
The cells were fixed and permeabilized after 48 h and apopto-
tic cells were detected by the TUNEL assay. This assay
employed the Apoptosis Detection System, Fluorescein
(Promega, Madison) and followed the protocol of the manu-
facturer. Apoptosis cells having fluorescently labelled DNA
were visualized under a fluorescent microscope. The percent-
age of apoptotic cells was 29.25 ꢃ 7.6.
The compounds in series 1e8 and melphalan were evalu-
ated against Molt 4/C8, CEM and L1210 cells by a literature
procedure [26]. In these assays, at least three different concen-
trations of compounds were incubated with the neoplastic cells
ꢁ
at 37 for 48 h after which time inhibition of growth was
determined.
Selected 4-piperidones were examined for antineoplastic
properties towards a panel of 49 (38e59) human tumour
cell lines using a literature method [16]. In addition, both

8
0
U. Das et al. / European Journal of Medicinal Chemistry 42 (2007) 71e80
[11] J.R. Dimmock, P. Kumar, A.J. Nazarali, N.L. Motaganahalli,
Acknowledgements
T.P. Kowalchuk, M.A. Beazely, J.W. Quail, E.O. Oloo, T.M. Allen,
J. Szydowski, E. De Clercq, J. Balzarini, Eur. J. Med. Chem. 35
The authors thank the Canadian Institutes of Health Re-
search for awards of operating grants to J.R.D. and R.K.S.,
the Flemish Fonds voor Wetenschappelijk Onderzoek
(
2000) 967e977.
[
12] W.B. Pratt, R.W. Ruddon, The Anticancer Drugs, Oxford University
Press, New York, 1979 p. 273.
[13] G. Thomas, Medicinal Chemistry: An Introduction, John Wiley and
(
FWO) enabling the Molt 4/C8, CEM and L1210 assays
Sons, Ltd., Chichester, U.K., 2000 pp. 408e409.
14] P.N. Craig, J. Med. Chem. 14 (1971) 680e684.
15] F. Civoli, L.W. Daniel, Cancer Chemother. Pharmacol. 42 (1998)
to be performed (E.D.C., J.B.), and the National Cancer In-
stitute who generated the data in Table 2. We thank Lizette
van Berckelaer for proficient help with the Molt 4/C8, CEM
and L1210 assays and Jason Jobse for assistance with the
formatting of Table 1.
[
[
3
[16] M.R. Boyd, K.D. Paull, Drug. Dev. Res. 34 (1995) 91e109.
19e326.
[17] M.R. Grever, S.A. Schepartz, B.A. Chabner, Semin. Oncol. 19 (1992)
22e638.
6
[
18] W.A. Remers, in: J.N. Delgado, W.A. Remers (Eds.), Wilson and
Gisvold’s Textbook of Organic Medicinal and Pharmaceutical Chemistry,
tenth ed. Lippincott-Raven Publishers, Philadelphia, 1998, p. 367.
References
[
1] B. Mutus, J.D. Wagner, C.J. Talpas, J.R. Dimmock, O.A. Phillips,
R.S. Reid, Anal. Biochem. 177 (1989) 237e243.
[19] B. Althaus, in: E.T. Herfindal, D.R. Gourley (Eds.), Textbook of Thera-
peutics Drug and Disease Management, seventh ed. Lippincott Williams
and Wilkins, Philadelphia, 2000, p. 1724.
[
2] G. Baluja, A.M. Municio, S. Vega, Chem. Ind. (1964) 2053e2054.
3] J.R. Dimmock, S.K. Raghavan, B.M. Logan, G.E. Bigam, Eur. J. Med.
Chem. 18 (1983) 248e254.
[
[20] S.C. Vashishtha, A.J. Nazarali, J.R. Dimmock, Cell. Mol. Neurobiol. 18
(1998) 437e445.
[
4] J.A. Benvenuto, T.A. Connor, D.K. Monteith, J.W. Laidlaw, S.C. Adams,
T.S. Matney, J.C. Theiss, J. Pharm. Sci. 82 (1993) 988e991.
5] G.W. Xu, J.S. Mymryk, J.G. Cavincross, Int. J. Cancer 116 (2005) 187e192.
6] S. Fulda, K.-M. Debatin, Neoplasia (New York) 7 (2005) 162e170.
7] L.M. Espinoza-Fonseca, Bioorg. Med. Chem. 14 (2006) 896e897.
8] J.R. Dimmock, M.P. Padmanilayam, R.N. Puthucode, A.J. Nazarali,
N.L. Motaganahalli, G.A. Zello, J.W. Quail, E.O. Oloo, H.-B. Kraatz,
J.S. Prisciak, T.M. Allen, C.L. Santos, J. Balzarini, E. De Clercq,
E.K. Manavathu, J. Med. Chem. 44 (2001) 586e593.
[21] M. Tsurusawa, K. Saeki, T. Fujimoto, Int. J. Hematol. 66 (1997)
79e88.
[
[
[
[
[22] C.D. Jones, M.G. Jevnikar, A.J. Pike, M.K. Peters, L.J. Black,
A.R. Thompson, J.F. Falcone, J.A. Clemens, J. Med. Chem. 27 (1984)
1057e1066.
[23] MacroModel 7.1, Department of Chemistry, Columbia University, New
York, 2000.
[24] A. Albert, E.P. Serjeant, The Determination of Ionization Constants, third
ed. Chapman and Hall, London, 1984 pp. 151e152.
[25] C. Hansch, A.J. Leo, Substituent Constants for Correlation Analysis in
Chemistry and Biology, John Wiley and Sons, New York, 1979 p. 49.
[26] J. Balzarini, E. De Clercq, M.P. Mertes, D. Shugar, P.F. Torrence,
Biochem. Pharmacol. 31 (1982) 3673e3682.
[
9] J.R. Dimmock, V.K. Arora, S.L. Wonko, N.W. Hamon, J.W. Quail, Z. Jia,
R.C. Warrington, W.D. Fang, J.S. Lee, Drug Des. Deliv. 6 (1990) 183e194.
[
10] J.R. Dimmock, U. Das, H.I. Gul, M. Kawase, H. Sakagami, Z. Bar a´ th,
I. Ocsovsky, J. Moln a´ r, Bioorg. Med. Chem. Lett. 15 (2005) 1633e1636.