Cytotoxic 3,5-bis(benzylidene)piperidin-4-ones and N-acyl analogs displaying selective toxicity for malignant cells

Article abstract of DOI:10.1016/j.ejmech.2007.03.010

A series of 3,5-bis(benzylidene)piperidin-4-ones 1, 1-acryloyl-3,5-bis(benzylidene)piperidin-4-ones 2 and adducts of 2 with sodium 2-mercaptoethanesulfonate (mesna), namely series 3, were prepared as candidate cytotoxic agents. These compounds were examin

Full text of DOI:10.1016/j.ejmech.2007.03.010

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1e7
http://www.elsevier.com/locate/ejmech
Original article
Cytotoxic 3,5-bis(benzylidene)piperidin-4-ones and N-acyl analogs
displaying selective toxicity for malignant cells
Hari N. Pati a, Umashankar Das a, J. Wilson Quail b, Masami Kawase c,
Hiroshi Sakagami d, Jonathan R. Dimmock a,
*
a College of Pharmacy and Nutrition, University of Saskatchewan, 110 Science Place, Saskatoon Saskatchewan S7N 5C9, Canada
b Saskatchewan Structural Sciences Centre, University of Saskatchewan, 110 Science Place, Saskatoon SK S7N 5C9, Canada
c Faculty of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime 790 8578, Japan
d Division of Pharmacology, Department of Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry, Saitama 350 0283, Japan
Received 3 January 2007; received in revised form 6 March 2007; accepted 8 March 2007
Available online 3 April 2007
Abstract
A series of 3,5-bis(benzylidene)piperidin-4-ones 1, 1-acryloyl-3,5-bis(benzylidene)piperidin-4-ones 2 and adducts of 2 with sodium 2-mer-
captoethanesulfonate (mesna), namely series 3, were prepared as candidate cytotoxic agents. These compounds were examined against neoplas-
tic HSC-2, HSC-4 and HL-60 cells as well as HGF, HPC and HPLF normal cell lines and many of the compounds displayed selective toxicity for
malignant cells. The CC50 values of the analogs in series 2 towards the cancer cell lines were mainly submicromolar. The relative potencies,
selectivity and log P values were in the order of 2 > 1 > 3. The sulfonic acid group of a representative compound in series 3 was replaced
by a thiol function to produce 4 leading to substantial increases in cytotoxic potencies and hydrophobicity indicating that the presence of a hy-
drophilic sulfonic acid group was disadvantageous in terms of potency. Molecular modeling suggested that the superior cytotoxicity of various
members of series 1e3 over an acyclic analog 5 may have been due to the greater torsion angles q1 and q2 created between the arylidene aryl
rings and the adjacent olefinic groups in series 1e3.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Piperidin-4-ones; Enones; Sodium 2-mercaptoethanesulfonate; Mesna; Cytotoxicity; Molecular modeling; X-ray crystallography; Selective cytotoxicity
1. Introduction
permitting two successive alkylations of thiols to occur
[5e7]. This sequential attack with cellular thiols is illustrated
in Fig. 1. The rationale behind this latter molecular design is
as follows. A number of studies have demonstrated that de-
pletion of thiol concentrations prior to treatment with various
anticancer drugs has increased cell killing compared to the
use of the drug alone [8,9]. In addition, various conjugated
enones inhibit the p isozyme of glutathione-S-transferase
[10] and such inhibition has been shown to enhance the
cytotoxicity of the cancer chemotherapeutic alkylating agent
thiotepa [11]. Hence the initial alkylation may create a selec-
tive chemosensitivity in the malignant cells to a further inter-
action with cellular thiols.
One of the major interests in this laboratory is the study
of the antineoplastic properties of conjugated arylidene
ketones. This decision is based on their preferential affinity
towards thiols rather than hydroxy or amino groups [1e3].
Hence such molecules may not interact with nucleic acids
and thus be bereft of the genotoxic effects of many antican-
cer drugs used today [4]. Originally only one conjugated
arylidene keto group was present in the molecules. However,
recently the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore
has been incorporated into various cyclic systems thereby
However, while these previous studies [5e7] demonstrated
the cytotoxic properties of such molecules, the bioevaluations
* 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 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.03.010

2
H.N. Pati et al. / European Journal of Medicinal Chemistry 43 (2008) 1e7
O
3
preparation of series 1 which on treatment with acryloyl chlo-
1
5
R
R
ride produced the corresponding amides 2aee. Acylation of
4-piperidone with acryloyl chloride produced 1-acryloylpiper-
idin-4-one which condensed with mesna to provide the sodium
salt of the thiol adduct which, in the presence of acid, was
condensed with various aryl aldehydes to give the desired
products 3aee. As a result of the marked variation in the cy-
totoxic properties of the compounds in series 1e3 vide infra,
two analogs 4 and 5 were synthesized in order to assist in the
interpretation of the biodata. The preparation of 4 is indicated
in Scheme 1 whereby 1-acryloylpiperidin-4-one was con-
densed with 1,2-ethanedithiol which gave rise to 1-[3-(2-mer-
captoethylsulfanyl)-propionyl]piperidin-4-one which reacted
with 4-methoxybenzaldehyde to form the desired product.
The reaction of 4-nitrobenzaldehyde with acetone leading to
the isolation of 5 is portrayed in Scheme 2.
R1SH
R1
4
2
S
O
R
R
R1
R2
S
O
S
R
R2SH
R
Fig. 1. Sequential interaction of the cellular thiols HSR1 and HSR2 with the
1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore.
1H NMR spectroscopy indicated that the compounds in
series 1e5 were stereoisomerically pure. Previous X-ray
crystallographic studies revealed the E,E configuration of the
3,5-arylidene groups in representative compounds in series 1
[16] and 2 [6]. Additionally, in this study the X-ray crystallo-
graphic structure of 4 showed that the compounds adopted
the E,E configuration. In the case of 5, the J values of the
olefinic protons confirmed the E stereochemistry of the ole-
finic groups.
used neoplastic and transformed cells. Hence little or no indi-
cation was gleaned as to whether these molecules possessed
selective toxicity to malignant cells. In order to address this
issue, the decision was reached to examine various cyclic
compounds containing the 1,5-diaryl-3-oxo-1,4-pentadienyl
pharmacophore against both malignant and normal cells. An
initial study using alicyclic scaffolds demonstrated clearly
that selectivity and potency were both dependent on the nature
of the scaffold [12].
The objectives of the present investigation were as follows.
First, an evaluation of whether the incorporation of the 1,5-di-
aryl-3-oxo-1,4-pentadienyl group into a heterocyclic rather
than alicyclic ring would lead to compounds possessing selec-
tive toxicity to malignant cells was planned. Previous studies
from our laboratories demonstrated that a series of 3,5-bis
(benzylidene)piperidin-4-ones 1 possessed excellent cytotox-
icity towards human Molt 4/C8 and CEM T-lymphocytes as
well as murine L1210 lymphoid leukemia cells [6]. In order
to increase the extent of interactions with cellular thiols, the
N-acryloyl analogs 2 were prepared and in general they pos-
sessed greater potencies than 1 [6]. Second, selective toxicity
may be achieved by the use of chemoprotectants of normal
cells. In particular, the administration of sodium 2-mercaptoe-
thanesulfonate (mesna) with various anticancer drugs led to re-
tention of the drug’s neoplastic properties but amelioration of
its side effects by the thiol [13e15]. The potential exists for
the compounds in series 3, to undergo a retro-Michael reaction
liberating the analogs 2 plus a cytoprotective thiol.
3. Bioevaluations
All of the compounds 1aee, 2aee, 3aee, 4 and 5 as well as
mesna, 1,2-ethanedithiol and the anticancer alkylating agent
melphalan were evaluated for cytotoxic properties. The assays
used the following human malignant cell lines, namely two
squamous cell carcinomas (HSC-2, HSC-4) and a promyelo-
cytic leukemia neoplasm (HL-60). The three normal human
cells used were a gingival fibroblast (HGF), pulp cells
(HPC) and a peridontal ligament fibroblast (HPLF). These
data are presented in Table 1.
4. Results and discussion
The biodata in Table 1 reveal that most of the compounds in
series 2 are potent inhibitors of the growth of various malig-
nant cells. The N-acyl analogs 2 have submicromolar IC50
values in most cases and, on average, are 23 times more potent
to HSC-2, HSC-4 and HL-60 cells than 1aee. On the other
hand, the mesna adducts 3aee have approximately one-third
of the potencies of the compounds in series 1. In order to iden-
tify novel compounds possessing a preferential toxicity for
neoplasms, selectivity index (SI) figures were calculated for
the compounds in series 1e3 and are presented in Table 1.
A SI value of 10 was arbitrarily chosen as an indication of
noteworthy selectivity and from the results obtained, the per-
centage of compounds meeting this criterion in series 1, 2
and 3 were 60, 100 and 0, respectively. The conclusions to
be drawn from these observations are that the N-acryloylpiper-
idines 2 are potent cytotoxins which exert a selective toxicity
for malignant cells. In general, these properties are lower in
In summary, the initial experimentation, aimed at the dis-
covery of candidate cytotoxics with selective toxicity for neo-
plastic cells, involved the synthesis and bioevaluation of the
compounds in series 1e3 towards both malignant and normal
cell lines. If this quest was successful, attempts to ascertain
why this phenomenon occurred were planned.
2. Chemistry
The compounds in series 1e3 were prepared by the syn-
thetic chemical routes presented in Scheme 1. Acid catalyzed
reaction of various aryl aldehydes with 4-piperidone led to the

H.N. Pati et al. / European Journal of Medicinal Chemistry 43 (2008) 1e7
3
O
O
O
N
i
ii
N
H
R
N
H
R
R
R
O
1a-e
iii
2a-e
O
N
O
N
O
N
iv
i
R
R
SO3H
SO3- Na+
O
O
S
O
O
S
3a-e
v
O
O
vi
N
H3CO
N
OCH3
SH
SH
O
S
S
4
Scheme 1. Synthetic chemical pathway to the compounds in series 1e4. The reagents used in the syntheses were as follows: (i) 2RC6H4CHO/CH3COOH/HCl; (ii)
CH2]COCl/K2CO3; (iii) CH2]CHCOCl/K2CO3/tetrabutyl ammonium bromide; (iv) HSCH2CH2SO3Na/N(C2H5)3; (v) HSCH2CH2SH/N(C2H5)3; (vi) 2C6H4(4-
OCH3)CHO/CH3COOH/HCl. The aryl substituents in series 1e3 were as follows: a: R ¼ H; b: R ¼ Cl; c: R ¼ NO2; d: R ¼ CH3; e: R ¼ OCH3.
series 1 while the mesna adducts are substantially less prom-
ising than series 1 and 2 in terms of both potency and
selectivity.
refractivity (MR) values in order to provide variation in the
electronic, hydrophobic and steric properties, respectively, of
these groups. Thus in addition to the unsubstituted compounds
1a, 2a and 3a, substituents with both positive (þ) and negative
(ꢀ) s and p figures were utilized, namely the 4-chloro (þ, þ),
4-nitro (þ, ꢀ), 4-methyl (ꢀ, þ) and 4-methoxy (ꢀ, ꢀ) groups.
Furthermore, the MR values ranged from 1.03 for hydrogen to
7.87 for the 4-methoxy group. In order to ascertain whether
one or more of these physicochemical constants correlated
with the SI values, linear and semilogarithmic plots were
made between the SI values and the s, p and MR constants
of the aryl substituents in 1aed, 2aee and 3aee. However,
no correlations were noted ( p > 0.05). In addition, the calcu-
lated log P values of the compounds in series 1e5 are pre-
sented in Table 1. The average figures for 1aee, 2aee and
3aee are 3.81, 4.37 and 1.61, respectively, indicating that
the relative hydrophobicities are 2 > 1 > 3. In general, this se-
quence is the same as noted for cytotoxic potencies (the aver-
age CC50 figures against human tumors for 1, 2 and 3 are 29.4,
1.27 and 81.3 mM, respectively), and SI values (the average SI
values are >11.8, 17.6 and 3.88 for 1, 2 and 3, respectively).
The substantially lower log P values of series 3 compared to
1aee and 2aee indicates clearly that the polar sulfonic acid
group in 3 diminishes the hydrophobic properties of the mol-
ecules (e.g., substitution of the sulfonic acid group by a mer-
capto function increased the log P value by 3.23) which likely
has an adverse effect on membrane permeability and cytotoxic
potencies.
Further experimentation was undertaken with a view to de-
termine some of the factors which contributed to the variation
in potencies and especially to the differing SI values. In terms
of potencies, the greater toxicity of series 2 than 1 was attrib-
uted to the additional electrophilic group in 2aee enabling
these compounds to act as trifunctional alkylating agents.
The lower potencies of 3aee may have been due to a number
of factors including the presence of the highly polar sulfonic
acid group which could impede transport via cell membranes.
In order to examine this possibility, compound 4 was prepared
which is identical to 3e except that the sulfonic acid group was
replaced by a mercapto function. The data in Table 1
strengthen this hypothesis insofar as the substantially greater
potency of 4 compared to 3e is revealed. Second, the 4-nitro
analogs 1c, 2c and 3c possess the highest potencies in each
of the series 1e3. In order to examine whether the compres-
sion of part of the 1,5-bis(4-nitrophenyl)-3-oxo-1,4-penta-
dienyl group into a piperidine ring influenced cytotoxicity,
the open chain analog 5 was assayed towards the same cell
lines. The data in Table 1 reveal that the average potencies
of 1c, 2c and 3c range from 20 to 362 times that of 5, confirm-
ing the important contribution that the rigidity of the 4-piper-
idones makes to potent cytotoxic properties.
In order to discover some of the factors that influence SI
values, the following determinations were undertaken. First,
the physicochemical properties of the aryl substituents in
series 1e3 were considered. These groups were chosen with
different Hammett sigma (s), Hansch pi (p) and molar
Second, the SI values of the compounds in series 1 and 2
are impressive in general but they are substantially lower in
3aee. In order to investigate whether this observation

4
H.N. Pati et al. / European Journal of Medicinal Chemistry 43 (2008) 1e7
CHO
olefinic linkages (Fig. 2A). A comparison of the locations of
O
rings A and B was made by measuring the distances d1ed3
which are the spans between the centres of the aryl rings
and the keto oxygen atoms (Fig. 2B). In addition, the bond an-
gles j1 and j2 were obtained. These data for q1, q2, d1ed3, j1
and j2 are presented in Table 2.
O
i
+
H3C
CH3
O2N
NO2
5
NO2
Scheme 2. Synthesis of compound 5; (i) NaOH.
The data in Table 2 indicate that in the case of the 4-piper-
idones 1c, 2c and 3c, the torsion angles q1 and q2 vary between
42ꢁ and 61ꢁ. The lack of coplanarity between aryl rings and
the adjacent olefinic linkages in 4-piperidones in which two
arylidene rings are conjugated with a keto function has been
attributed to nonbonded interactions between one of the ortho
hydrogen atoms of the aryl rings with the equatorial protons at
positions 3 and 5 of the piperidine ring [6,16]. In contrast, the
aryl rings in 5 are virtually coplanar with the adjacent unsatu-
rated groups. The other parameters of 1c, 2c and 3c, namely
the interatomic distances d1ed3 as well as the bond angles
j1 and j2, are similar to the figures obtained for 5. Thus
one reason for the marked disparity in cytotoxic potencies be-
tween the piperidones in series 1e3 and the acyclic analog 5
may be that the marked noncoplanarity of rings A and B in se-
ries 1e3 which permits a favorable topography for alignment
at a binding site.
pertaining to series 3 was associated with the stabilities of the
compounds, a solution of a representative compound 3c was
incubated at 37 ꢁC for 24 h which were the temperature and
time of the cytotoxicity assays. The compound was stable un-
der these conditions. Thus the probability exists that 3c does
not release 2c and 2-mercaptoethanesulfonic acid, which
may account for the lower cytotoxic potency and selective tox-
icity of 3c compared to 2c. Presumably other members of
series 3 were also stable which accounts for their generally
lower cytotoxicity than is found in series 1 and 2.
Third, the lower potency of 5 compared to 1c, 2c and 3c
may have been due to differences in the relative positions of
important atoms and groups of the 1,5-bis(4-nitrophenyl)-3-
oxo-1,4-pentadienyl toxophore. In particular, possible differ-
ences between the orientation and relative positions of the
aryl rings were considered and in order to address this issue,
molecular models of these four compounds were built. The
aryl rings were designated A and B. Ring B and the substituent
R in 2c and 3c are located on the same side of axis 1 as indi-
cated in Fig. 2A. The torsion angles q1 and q2 reflect the lack
of coplanarity of rings A and B, respectively, with the adjacent
The X-ray crystallographic structure of 4 revealed that this
molecule crystallized with two nonequivalent conformations
in the asymmetric unit designated 4A and 4B. The ORTEP-3
diagram [17] of 4A is presented in Fig. 3. The principal differ-
ence between the two conformers 4A and 4B is the relative lo-
cations of the terminal 2-mercaptoethyl group. As indicated in
Table 1
The cytotoxicity and calculated log P values of the compounds in series 1e5
SIb
log P
a
CC50 (mM)
Compound
Human tumor cells
Human normal cells
HGF HPC
20 38
HSC-2
HSC-4
4.4
20
HL-60
1.5
22
3.6
4.5
170
Average
HPLF
Average
1a
1b
2.2
18
2.7
20
24
56
27
85
10
4.3
3.36
4.71
3.27
4.25
3.47
3.92
5.27
3.84
4.81
4.03
1.15
2.51
1.07
2.05
1.27
4.50
4.10
e
69
13
130
88
1c
1d
0.90
3.9
100
6.1
9.3
3.5
5.9
39
47
13
29
150
>400
200
>400
15
170
160
173
>320
9.7
1e
2a
75
115
0.86
0.72
0.26
0.71
3.8
>2.8
11
0.63
0.63
0.094
0.57
1.5
1.2
0.74
0.55
0.13
0.81
1.9
6.0
13
4.6
5.6
100
8.2
2b
2c
0.99
0.56
0.75
8.1
6.0
3.9
18
6.3
3.6
8.4
4.0
12
15
2d
2e
11
11.5
129
290
163
36
16
34
190
290
140
48
97
250
3a
3b
200
32
5.0
71
24
5.2
190
33
4.0
154
30
330
200
31
1.9
5.4
150
30
3c
3d
4.7
7.7
1.9
150
88
89
57
140
130
126
92
300
250
210
270
220
180
243
233
11.5
>400
>400
>400
>200
3e
4
2.5
3.4
4.0
3.5
2.6
3.4
8.1
9.4
17
5
Mesna
41
>400
>400
35
83
>400
385
158
>400
221
94
>400
>400
>400
>200
>400
>400
>400
>200
>400
>400
>400
>200
>4.3
w1.0
w1.2
>4.9
>400
>335
40.7
1,2-Ethanediol
Melphalan
a
e
e
81
6.0
The CC50 figure indicates the concentration of compound required to reduce the number of viable cells by 50%. Data were obtained from dose response curves
while the CC50 values are the mean figures from duplicate determinations. The differences between two determinations were within 5%. The maximum concen-
tration of the compounds was 400 mM except in the case of melphalan in which solubility problems necessitated a higher concentration of 200 mM.
b
SI indicate the selectivity index which is the ratio of the average CC50 figures for the normal cells and tumor cells.

H.N. Pati et al. / European Journal of Medicinal Chemistry 43 (2008) 1e7
5
O
O
N
d1
d2
A
B
d3
R
R
R
R
R
Axis 1
A
B
Fig. 2. (A) Designation of the torsion angles q1 and q2 in representative com-
pounds. (B) The positions of rings A and B in relation to the keto oxygen atom
determined by measurements of the interatomic distances d1ed3 and bond an-
gles j1 and j2. The distance d4 is the span between the centre of ring B and
axis 2 which is the plane of the centre of ring A and the keto oxygen atom.
Fig. 3. ORTEP-3 diagram of 4A.
of increasing potency and selectivity. For example, the design
of further analogs of series 1e3 should be undertaken in order
to determine the importance of hydrophobicity. In addition,
substituents of varying sizes should be placed in the ortho po-
sitions of the arylidene rings to find whether the magnitude of
the q1 and q2 values correlate with potency and selectivity.
Furthermore, the sulfonic acid group in series 3 should be
esterified which may facilitate penetration of the cell wall
which, on subsequent hydrolysis and dethiolation, could lead
to mesna and 1-acryloyl-3,5-bis(benzylidene)piperidin-4-one.
Future studies will also be directed to design compounds
which will liberate the cytoprotective thiol and antineoplastic
agent rapidly and completely. Since high levels of thiol are re-
quired to protect normal cells [15], the liberated a,b-unsatu-
rated ketones must be well tolerated in animals and hence in
vivo evaluations will need to be undertaken.
Fig. 3, the C26eC27eS2 substituent is approximately perpen-
dicular to ring B. On the other hand, this group in 4B is twisted
towards ring B. The shape of 4 determined by molecular mod-
eling was similar to 4A and not 4B. The X-ray determination
was undertaken for two reasons. In the first place, the data
confirm the proposed structure for 4 in terms of the integrity
of the olefinic double bonds, i.e., while a free mercapto group
is present in 4, no intramolecular thiolation at the olefinic car-
bon atoms occurred. In addition, as noted with analogs of 4,
the olefinic double bonds in this compound adopted the E con-
figuration. Furthermore, X-ray crystallography confirmed the
marked lack of coplanarity between rings A and B and the
adjacent olefinic groups. The C5eC14eC15eC20 (q1) and
C3eC7eC8eC13 (q2) torsion angles are ꢀ24.7ꢁ and 38.0ꢁ,
˚
respectively. Interatomic distances of less than 2.5 A are indic-
ative of nonbonded interactions. In the case of 4A, the C6Hee
˚
˚
C20H and C2HeeC13H spans are 2.271 A and 2.353 A,
respectively, which are believed to account for the q1 and q2
torsion angles being greater than 0ꢁ.
6. Experimental protocols
6.1. Chemistry
5. Conclusions
Melting points were determined using a Gallenkamp instru-
ment and are uncorrected. Elemental analyses (C, H, N) were
undertaken using an Elementer analyzer and were within 0.4%
This study revealed that a number of 3,5-bis(benzylidene)-
piperidin-4-ones and N-acyl analogs display selective toxicity
for malignant cells. In particular, series 2 are potent cytotoxins
with noteworthy SI values. The additional site for thiolation in
series 2, compared to series 1 and 3, for example, permits
a greater number of sequential interactions with cellular nucle-
ophiles which may contribute significantly to these favorable
properties. The data generated in this project enable sugges-
tions to be made for further avenues to pursue with a view
1
of the calculated values. H and 13C NMR spectra were re-
corded on a Bruker AMX 500 FT machine while the mass
spectrum was obtained with a VG 7OSEa instrument. The
X-ray crystallographic diffractions were determined using
a Nonius machine.
6.1.1. Synthesis of 3,5-bis(benzylidene)piperidin-4-ones
(1aee) and 1-acryloyl-3,5-bis(benzylidene)piperidin-
4-ones (2aee)
Table 2
Some torsion angles (q1, q2), interatomic distances (d1ed3) and bond angles
(j1, j2) present in 1c, 2c, 3c, 4 and 5 determined by molecular modeling
The synthesis of the 4-piperidones 1aee and 2aec, e has
been described previously [6]. Compound 2d was prepared
by a reported method [6] and recrystallized from 95% ethanol.
Compound
q1
q2
d1
d2
d3
j1
j2
1
Yield: 88%; m.p. 147 ꢁC; H NMR (CDCl3) d: 2.42 (s, 6H),
1c
2c
3c
4
60.1
ꢀ61.1
41.8
43.6
7.12
7.02
7.10
7.05
7.13
7.12
7.04
7.08
7.07
7.13
12.16
12.90
12.44
12.59
12.12
31.4
23.4
28.7
26.9
31.7
31.4
23.3
28.7
26.9
31.7
4.68 (s, 2H), 4.82 (s, 2H), 5.57 (d, 1H, J ¼ 10.30 Hz), 6.19
(d, 1H, J ¼ 16.75 Hz), 6.28 (m, 1H), 7.28 (m, 8H), 7.84 (s,
2H). Anal. calcd. for C24H23NO2: C, 80.64; H, 6.49; N,
3.92%. Found: C, 80.79; H, 6.30; N, 4.06%.
ꢀ43.8
ꢀ41.5
ꢀ39.2
ꢀ0.1
61.5
ꢀ0.3
5

6
H.N. Pati et al. / European Journal of Medicinal Chemistry 43 (2008) 1e7
6.1.2. General method for the synthesis of 2-{3-
[3,5-bis(benzylidene)-4-oxopiperidin-1-yl]-3-
for C24 H23N3O9S2$2H2O: C, 48.19; H, 3.84; N, 7.02%.
Found: C, 48.29; H, 3.81; N, 7.00%.
oxopropylsulfanyl}ethanesulfonic acids (3aee)
A mixture of 4-piperidone (0.01 mol), acryloyl chloride
(0.012 mol), potassium carbonate (0.02 mol) and tetrabutyl
ammonium bromide (0.001 mol) in acetone (75 ml) was
stirred at room temperature overnight. The reaction mixture
was filtered and after evaporation of the solvent, the residue
was dissolved in chloroform and washed with water
(100 ml). The organic layer was dried over sodium sulfate, fil-
tered and removal of the solvent in vacuo gave 1-acryloylpi-
peridin-4-one as viscous oil which was used without any
purification.
6.1.2.4. 2-{3-[3,5-Bis(4-methylbenzylidene)-4-oxopiperidin-1-
yl]-3-oxopropylsulfanyl}ethanesulfonic
acid
(3d). Yield:
80%; m.p. 143 ꢁC; 1H NMR (CDCl3) d: 2.42 (d, 6H,
J ¼ 12.20 Hz), 2.67 (t, 4H), 2.82 (t, 2H), 3.22 (t, 2H), 4.87
(s, 2H), 4.98 (s, 2H), 7.29 (m, 8H), 7.95 (s, 1H), 8.01 (s,
1H). Anal. calcd. for C26H29NO5S2$1.5H2O: C, 59.24; H,
5.52; N, 2.85%. Found C, 59.36; H, 5.52; N, 2.85%.
6.1.2.5. 2-{3-[3,5-Bis(4-methoxybenzylidene)-4-oxopiperidin-
1-yl]-3-oxopropylsulfanyl}ethanesulfonic acid (3e). Yield:
1H NMR (CDCl3) d: 2.49 (br s, 4H), 3.75 (br s, 2H), 3.91
(br s, 2H), 5.75 (dd, 1H), 6.31 (dd, 1H), 6.66 (dd, 1H).
A mixture of 1-acryloylpiperidin-4-one vide supra, sodium
mercaptoethanesulfonate (0.005 mol), triethylamine (0.001
mol), chloroform (25 ml) and methanol (25 ml) was stirred
at room temperature overnight. The precipitate was collected,
washed with a mixture of chloroform and methanol (1:1,
10 ml, previously cooled to 5 ꢁC) and dried to produce so-
dium 2-[3-(4-oxopiperidin-1-yl)-3-oxo- propylsulfanyl]etha-
nesulfonate. The crude product, which was prepared in
55% yield with respect to 4-piperidone, was used without
82%; m.p. 157 ꢁC; H NMR (D2O) d: 2.17 (br s, 2H), 2.31
1
(br s, 2H), 2.43 (t, 2H), 2.74 (t, 2H), 3.49 (s, 3H), 3.57 (s,
3H), 4.29 (s, 2H), 4.36 (s, 2H), 6.58 (d, 2H, J ¼ 7.71 Hz),
6.70 (d, 2H, J ¼ 7.79 Hz), 7.01 (m, 4H), 7.32 (s, 1H), 7.41
(s, 1H). 13C NMR (D2O): d 26.46, 27.22, 33.27, 43.80,
46.58, 51.38, 55.59, 55.74, 114.67, 114.85, 126.91, 127.21,
129.03, 129.36, 133.18, 133.55, 137.65, 138.34, 160.72,
160.88, 171.79, 186.35. Anal. calcd. for C26H29NO7S2$2H2O:
C, 54.96; H, 5.10; N, 2.46%. Found C, 55.22; H, 4.63; N,
2.41%.
1
purification in the synthesis of series 3. H NMR (D2O) d:
2.78 (m, 8H), 3.06 (t, 2H), 3.52 (t, 2H), 3.77 (t, 2H), 3.81
(t, 2H).
6.1.3. Synthesis of 1-[3-(2-mercaptoethylsulfanyl)
propionyl]-3,5-bis(4-methoxybenzylidene)piperidin-4-
one (4)
A
mixture of sodium 2-[3-(4-oxopiperidin-1-yl)-3-
This piperidone was prepared from 1-acryloylpiperidin-4-
one by the same methodology employed for the synthesis of
the compounds in series 3 except that 1,2-ethanedithiol was
used in place of sodium 2-mercaptoethanesulfonate. The crude
product was crystallized from ethanol. Yield: 82%; m.p.
128 ꢁC; 1H NMR (CDCl3) d: 2.47 (t, 2H), 2.73 (m, 4H),
3.52 (m, 2H), 3.85 (d, 6H, J ¼ 12.89 Hz), 4.72 (s, 2H), 4.94
(s, 2H), 6.90 (m, 4H), 7.36 (d, 2H, J ¼ 8.41 Hz), 7.46 (d,
2H, J ¼ 8.45 Hz), 7.80 (s, 1H), 7.85 (s, 1H). Mass (LCMS):
(M þ 2þ 1): 486. Anal. calcd. for C26H29NO4: C, 64.57; H,
6.04; N, 2.90%. Found: C, 64.31; H, 5.92; N, 3.09%.
oxopropylsulfanyl]ethanesulfonate (0.0032 mol) and aryl
aldehyde (0.0066 mol) in glacial acetic acid (15 ml) was
acidified with dry hydrogen chloride and stirred at room
temperature for 8 h. The precipitate was collected, washed
with glacial acetic acid (5 ml) and dried under vacuum
overnight at 45 ꢁC. The product obtained was crystallized
from ethanol.
6.1.2.1.
2-{3-[3,5-Bis(benzylidene)-4-oxopiperidin-1-yl]-3-
oxopropylsulfanyl}ethanesulfonic acid (3a). Yield: 73%; m.p.
125 ꢁC; 1H NMR (CDCl3) d: 2.27 (br s, 2H), 2.58 (br s,
2H), 2.68 (br s, 2H), 3.12 (br s, 2H), 4.51 (s, 2H), 4.81 (s,
2H), 7.11 (m, 10H), 7.75 (s, 1H), 7.89 (s, 1H). Anal. calcd.
for C24H25NO5S2$2H2O: C, 56.75; H, 4.92; N, 2.96%. Found:
C, 56.75; H, 5.00; N, 2.50%.
6.1.4. Synthesis of 1,5-bis-(4-nitrophenyl)-1,4-pentadien-3-
one (5)
A solution of sodium hydroxide (10% w/v, 1 ml) was added
to a solution of acetone (0.01 mol) and 4-nitrobenzaldehyde
(0.02 mol) in ethanol (20 ml). The mixture was stirred at
room temperature for 10 min. The precipitate was collected
and crystallized from acetonitrile to give 5. Yield: 62%; m.p.
145e146 ꢁC; 1H NMR (DMSO-d6) d: 7.56 (d, 1H,
J ¼ 16.10 Hz), 7.93 (d, 1H, J ¼ 16.10 Hz), 8.07 (d, 2H,
J ¼ 8.55 Hz), 8.31 (d, 2H, J ¼ 8.55 Hz). Anal. calcd. for
C17H12N2O5: C, 62.96; H, 3.73; N, 8.64%. Found: C, 62.84;
H, 3.71; N, 8.72%.
6.1.2.2. 2-{3-[3,5-Bis(4-chlorobenzylidene)-4-oxopiperidin-1-
yl]-3-oxopropylsulfanyl}ethanesulfonic
acid
(3b). Yield:
1
85%; m.p. 142 ꢁC; H NMR (CDCl3) d: 2.59 (t, 2H), 2.68
(t, 2H), 2.78 (t, 2H), 3.15 (t, 2H), 4.79 (s, 2H), 4.91 (s, 2H),
7.29 (m, 8H), 7.87 (s, 1H), 7.94 (s, 1H). Anal. calcd. for
C24H23Cl2NO5S2$1.5 H2O: C, 50.75; H, 4.05; N, 2.46%.
Found: C, 50.76; H, 4.23; N, 2.32%.
6.1.2.3. 2-{3-[3,5-Bis(4-nitrobenzylidene)-4-oxopiperidin-1-yl]-
6.1.5. X-ray crystallographic determination
3-oxopropylsulfanyl}ethanesulfonic acid (3c). Yield: 75%;
of 1-[3-(2-mercaptoethylsulfanyl)propionyl]-3,5-bis
(4-methoxybenzylidene)piperidin-4-one (4)
1
m.p.135 ꢁC; H NMR (CDCl3) d: 2.65 (br s, 2H), 2.76 (m,
4H), 3.23 (br s, 2H), 4.76 (s, 2H), 4.89 (s, 2H), 7.57 (m,
4H), 8.00 (s, 1H), 8.06 (s, 1H), 8.34 (m, 4H). Anal. calcd.
Crystallographic data (excluding structure factors) for the
structure in this paper have been deposited with the Cambridge

H.N. Pati et al. / European Journal of Medicinal Chemistry 43 (2008) 1e7
7
Crystallographic Data Centre as supplementary publication
no. CCDC 630874. Copies of the data can be obtained, free
of charge, on application to CCDC, 12 Union Road, Cam-
bridge CB2 1EZ, UK, (fax: þ44 (0)1223 336033 or email
deposit@ccdc.cam.ac.uk).
Sakagami from the Ministry of Education, Science, Sports
and Culture of Japan (No. 14370607). The constructive com-
ments of two reviewers are recorded with gratitude.
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´ ´
´
The authors thank the Canadian Institutes of Health Re-
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Saskatchewan for funding the X-ray crystallography labora-
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