Tumour-specific cytotoxicity and structure-activity relationships of novel 1-[3-(2-methoxyethylthio)propionyl]-3,5-bis(benzylidene)-4-piperidones

Article abstract of DOI:10.1016/j.bmc.2016.03.056

A series of 1-acyl-3,5-bis(benzylidene)-4-piperidones 3-7 were designed and synthesized as novel cytotoxic agents. These compounds displayed potent cytotoxic properties towards human Molt4/C8, CEM, HSC-2, HSC-3 and HSC-4 neoplasms and also to murine L1210

Full text of DOI:10.1016/j.bmc.2016.03.056

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Tumour-specific cytotoxicity and structure–activity relationships
of novel 1-[3-(2-methoxyethylthio)propionyl]-3,5-bis(benzylidene)-
4-piperidones
b
b
c
Mohammad Hossain a,y, Umashankar Das a, , Naoki Umemura , Hiroshi Sakagami , Jan Balzarini ,
⇑
Erik De Clercq c, Masami Kawase d, Jonathan R. Dimmock a,
⇑
a Drug Discovery and Development Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan S7N 5C9, Canada
b Division of Pharmacology, Mekai University School of Dentistry, Saitama 350-0238, Japan
c Rega Institute of Medical Research, Katholieke Universiteit Leuven, 3000 Leuven, Belgium
d Faculty of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime 790-8578, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1-acyl-3,5-bis(benzylidene)-4-piperidones 3–7 were designed and synthesized as novel
cytotoxic agents. These compounds displayed potent cytotoxic properties towards human Molt4/C8,
CEM, HSC-2, HSC-3 and HSC-4 neoplasms and also to murine L1210 cells. The majority of the compounds
have sub-micromolar or very low micromolar IC50 and CC50 values and are significantly more potent than
the reference alkylating drug melphalan. Evaluation of these compounds against non-malignant HGF and
HPLF cells revealed the tumour-specific toxicity. In particular, 3e emerged as a promising lead cytotoxic
agent which caused apoptosis and PARP1 cleavage in HSC-2 cells.
Received 29 January 2016
Revised 17 March 2016
Accepted 27 March 2016
Available online xxxx
Keywords:
4-Piperidone
Ó 2016 Elsevier Ltd. All rights reserved.
a,b-Unsaturated ketone
Cytotoxicity
Structure–activity relationships
Tumour-specific toxicity
1. Introduction
The 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore has been
mounted on heterocyclic scaffolds such as series 1 (Fig. 1A). In the
The major interest in this laboratory is the design of conjugated
a
,
case of 1a, c–f, the cytotoxic potencies towards a number of
neoplastic and transformed cells are in the low micromolar range
in general.8 The cytotoxic potencies were attributed to the interac-
tions between the pharmacophoric group in the ligand and
primary binding sites of biomacromolecules. It is possible however
that the groups on the piperidyl nitrogen atom may be added in
series 1 which react at auxiliary binding sites thereby enhancing
cytotoxic potency (Fig. 1B). On the other hand, this group may
prevent the ligand interacting at primary binding sites and potency
declines relative to the analogs in series 1.
The addition of N-acyl groups to series 1 led to 2a–e which has
the capacity to form hydrogen bonding and hydrophobic
interactions at putative auxiliary binding sites (Fig. 1C). Compar-
isons between the IC50 values of 2a–e and the analogs in series 1
possessing the same aryl substituent towards human Molt4/C8
and CEM T-lymphocytes as well as murine L1210 leukemic cells
revealed that in 93% of the comparisons made, greater potency
was found in series 2 while equipotency was observed in 7% of
the comparisons.9 These results provide support for the hypothesis
of the existence of both auxiliary and primary binding sites.
b-unsaturated ketones as candidate antineoplastic agents. These
compounds have an exclusive or preferential affinity for reacting
with thiols rather than with hydroxyl groups and amines.1 Since
the latter two groups but not thiols are found in nucleic acids,
a
,b-unsaturated ketones may be devoid of the genotoxic side effects
of various anticancer drugs.2 In particular, recent emphasis has been
placed on developing compounds containing the 1,5-diaryl-3-
oxo-1,4-pentadienyl pharmacophore [ArCH@CHC(O)CH@CHAr].3–5
This group permits sequential alkylation of thiols at the olefinic
carbon atoms. This successive attack may lead to tumour-selective
toxicity since various studies revealed that after an initial chemical
interaction, some tumours are more sensitive to a further toxic effect
than various nonmalignant cells.6,7
⇑
Corresponding authors. Tel.: +1 306 966 6358; fax: +1 306 966 6377 (U.D.);
tel.: +1 306 966 6331; fax: +1 306 966 6377 (J.R.D.).
E-mail addresses: umashankar.das@usask.ca (U. Das), jr.dimmock@usask.ca
(J.R. Dimmock).
y
Current address: Department of Chemistry and Physics, St. Cloud State University,
St. Cloud, MN 56301, USA.
http://dx.doi.org/10.1016/j.bmc.2016.03.056
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Hossain, M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.056

2
M. Hossain et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 1. (A) General structure of 3,5-bis(benzylidene)-4-piperidones 1. (B) Postulated mode of interactions of 1-substituted-3,5-bis(benzylidene)-4-piperidones at binding
sites. (C) Series 2. The substituents in the aryl rings in series 1 and 2 are as follows: a: R1@R2@H; b: R1@CH3, R2@H; c: R1@OCH3, R2@H; d: R1@Cl, R2@H; e: R1@NO2, R2@H; f:
R1@F, R2@H; g: R1@R2@OCH3.
The objective of the present study is to glean some appreciation
of the structural requirements of some N-acyl ligands which
influence the rate and extent of interactions at the auxiliary bind-
ing sites in neoplastic cells. It is possible that the N-acyl ligands
interact differently with neoplastic and non-malignant cells which
may lead to tumour-selective toxicity. The emphasis in this inves-
tigation is given to elucidate structure–activity relationships upon
changing two portions of the N-acyl group in series 2. First, the ter-
minal hydroxyl group in 2a–e may enhance cytotoxic potency by
forming hydrogen bonds with a group of auxiliary binding sites.
On the other hand, the hydroxyl group being polar in nature may
impede cellular penetration. Hence series 3 was designed in which
the hydroxyl group in N-acyl side chain in series 2 is converted to
the corresponding methyl ether. Second, the sulfur atom in
representative molecules in series 3 was replaced by an isosteric
oxygen atom (4) and by groups having different sizes, electronic
properties and hydrogen bonding capacities (series 5–7). In
summary, the targeted compounds 3–7 were designed to gain an
insight of the structural requirement of the N-acyl groups which
enhance interactions of the 1,5-diaryl-3-oxo-1,4-pentadienyl
group at the primary binding sites.
2.2. Biological activity and the structure–activity relationships
All of the compounds is series 3–7 were evaluated against
human Molt4/C8 and CEM T-lymphocytes in order to determine
whether these molecules are cytotoxic to human malignant and
transformed cells. In addition, the dienones were examined for
their toxicity towards murine L1210 cells since a number of
anticancer drugs are effective against this cell line.10 Hence this
bioassay may reveal compounds with clinical potential. These
biological data are presented in Table 1.
Some general comments regarding the biological data pre-
sented in Table 1 are as follows. First, in general the compounds
in series 3–7 are potent cytotoxic agents. The average IC50 values
of these dienones against Molt4/C8, CEM and L1210 cells are
2.04, 1.73 and 4.40 lM, respectively. Thus Molt4/C8 and CEM
T-lymphocytes are more sensitive to these compounds than are
L1210 leukemic cells. In fact, 89% of the IC50 values of 3–7 towards
Molt4/C8 and CEM cells are less than 2 lM and 5f and 6f have
submicromolar IC50 figures towards CEM cells. Second, these
compounds may be viewed chemically as alkylating agents.
Consequently an alkylating agent used in cancer chemotherapy
namely melphalan was chosen as a reference standard. The follow-
ing compounds have statistically significant lower IC50 values than
melphalan, namely 3a,d–g, 4, 5a,f, 6a,f, 7 in the Molt4/C8 assay, 3a,
d,e, 5a,f, 6a,f, 7 in the CEM screen and 3e,g, 6a,f in the L1210 bioas-
say. Thus in 59% of these determinations, the novel dienones are
more potent than melphalan.
2. Results and discussion
2.1. Chemistry
The compounds in series 1 were prepared by the reaction of
4-piperidone hydrochloride hydrate with an appropriate aryl
aldehyde in the presence of dry hydrogen chloride gas in acetic
acid. 3-(2-Methoxyethylthio)propionyl chloride (8) was reacted
with 1a–g to produce the corresponding N-acyl derivatives 3a–g
(Scheme 1). Compound 4 which is an oxo analog of 3a in series 3
was prepared from 1a by condensing with 3-(2-methoxyethy-
loxy)propionyl chloride (9). The intermediates 8 and 9 were
synthesized by the route outlined in Scheme 2. Representative
compounds in series 3 were modified in order to guide future
development of these molecules. The reaction of 3a and 3f with
peracetic acid led to the formation of sulfoxides 5a and 5f,
respectively. Treatment of 3a,f with 3-chloroperoxybenzoic acid
led to oxidation of the sulfur atom to produce the corresponding
sulfones 6a,f. The olefinic groups in 3a,f remained intact during
this oxidation process as revealed by 1H NMR and the J values of
the olefinic protons of 6a,f. Compound 7 was prepared by the
reaction of 3a with trimethyloxonium tetrafluoroborate.
Analysis of the biological data was carried out with two aims.
The first objective is to find the structural modifications of the
1-acyl-3,5-bis(benzylidene)-4-piperidones 2 which govern cyto-
toxic potencies. Second, an examination was undertaken to evalu-
ate whether the results support the concept of auxiliary binding
sites. In order to achieve these objectives, four comparisons were
undertaken of the data in Table 1 (a similar method was used for
the evaluation of the results portrayed in Tables 3 and 4 vide infra).
(1) The IC50 values of 3a–g were compared with the data available
for 1a,c–f,8 1b11 and 1g12 in which the aryl substituents are iden-
tical i.e., the potencies in the Molt4/C8 bioassay of 3a and 1a were
compared, then 3b with 1b and so forth. These comparisons are
portrayed as
D1 figures in Table 2. These comparisons should
reveal whether the addition of a N-acyl group in series 3 leads to
potency increases. (2) The IC50 figures of 3a–e were also compared
with the data available for 2a–e9 when the aryl groups are the
same. These comparisons are indicated as
D2 figures in Table 2.
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M. Hossain et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
O
O
O
N
CHO
R1
R2
R1
R2
R1
i,ii
9
/iii
+
N
H
R2
N
H
1a-g
OCH3
O
O
4
8/iii
O
N
O
R2
R1
R2
R2
R1
R2
iv
R1
N
R1
OCH3
OCH3
O
S
O
S
O
3a-g
5a f
,
v
vi
O
N
O
R2
R1
R2
R1
N
O
S
O
OCH3
-
OCH3
O
BF4
O
S
CH3
6a,f
7
Scheme 1. Synthesis of compounds in series 1, 3–7. The aryl substituents in series 3–7 are the same as indicated in Figure 1. Reagents and conditions: (i) HCl (gas)/acetic acid;
(ii) K2CO3 (20% w/v aq solution); (iii) Et3N; (iv) peracetic acid (10% w/v in acetic acid); (v) 3-chloroperoxybenzoic acid; (vi) trimethyloxonium tetrafluoroborate.
i,ii
CH3OCCH2CH2SH
O
+
ClCH2CH2OCH3
Table 2
HOCCH2CH2SCH2CH2OCH3
O
Comparison of IC50 values of the compounds in series 3–7 against human Molt 4/C8
and CEM T-lymphocytes and murine leukemia L1210 cells
iii
Compound
Molt 4/C8
1a 2b
0.49
CEM
L1210
ClCCH2CH2SCH2CH2OCH3
D
D
D
1a
D
2b
D
1a
D
2b
O
8
3a
3b
3c
3d
3e
3f
3g
4
5a
5f
6a
6f
7
1.25c
0.64
40.7
7.75
6.04
2.91
0.14
0.68
1.03
3.91
1.01
4.46
1.25
1.38
0.87
26.0
6.96
3.47
1.28c
0.20
0.97
1.32
2.47
1.49
3.36
1.21
0.46
0.56
0.56c
0.56c
0.37
2.65
1.73
18.5
12.1
15.5
6.19
0.85c
1.28
1.62
10.9
2.98
14.8
2.31
1.27c
1.21c
1.49c
1.88c
1.84
O
0.70c
0.24
0.24
0.36
iv,ii
HOCCH2CH2OCH2CH2OCH3
O
HOCH2CH2OCH3
CH3CH2OCCH=CH2
+
iii
—
—
—
—
—
—
ClCCH2CH2OCH2CH2OCH3
O
0.73
0.70
0.48
9
0.83c
1.34
0.95c
1.93
0.61c
1.76
Scheme 2. Synthesis of the intermediates 8 and 9. Reagents and conditions: (i)
0.81c
1.54
1.08c
2.62
1.12c
2.39
NaOH/ethanol; (ii) aq NaOH; (iii) SOCl2; (iv) K2CO3.
1.00c
0.88c
0.87c
Table 1
a
The
D1 figures are the quotients of IC50 values of (i) 1a–g and 3a–g when the
Evaluation of series 3–7 and melphalan against human Molt 4/C8 and CEM T-
lymphocytes and murine leukemia L1210 cells
aryl substituents are identical and (ii) 1a with 4, 5a, 6a, 7 as well as 1f with 5f and
6f.
b
a
The D2 figures are the quotients of IC50 values of (i) 2a–e with 3a–e which have
Compound
IC50
(
lM)
clogP
the same aryl substituent and (ii) 3a with 4, 5a, 6a, 7 as well as 3f with 5f and 6f.
c
Molt 4/C8
CEM
L1210
The figure is not statistically significant when standard deviations are taken
into account.
3a
3b
3c
3d
3e
3f
3g
4
5a
5f
6a
6f
1.34 0.21
2.66 0.59
7.08 0.33
1.73 0.01
1.37 0.04
1.72 0.11
1.81 0.06
1.84 0.08
1.62 0.11
1.28 0.04
1.66 0.34
1.12 0.21
1.34 0.21
3.48 1.23
1.23 0.06
1.95 0.13
6.31 1.60
1.24 0.35
1.29 0.22
1.60 0.18
1.84 0.08
1.76 0.03
1.29 0.05
0.83 0.14
1.14 0.48
0.61 0.20
1.40 0.14
1.74 0.08
3.00 1.12
4.90 1.89
13.2 4.1
4.05
4.95
4.17
5.41
3.97
4.38
3.35
3.32
2.53
2.85
2.93
3.26
1.07
0.08
3.44 1.82
2.13 0.58
5.85 0.05
1.57 0.10
6.24 1.60
4.91 3.00
3.33 1.83
2.67 1.23
2.45 0.97
3.45 2.64
5.94 2.01
1a which lacks a N-acyl group. In addition, the potency of the
4-fluoro analog 3f was compared with 5f and 6f. These compar-
isons are indicated as
one or more of the N-acyl substituents enhance potencies. (4)
The IC50 figures of 4, 5a, 6a and 7 were compared with 3a. In
addition the potencies of 3f were compared with 5f and 6f. These
figures are indicated as
should reveal whether modifications of the N-acyl group of 3a
affects cytotoxic potencies.
D1 figures in Table 2 and should reveal if
D2 values in Table 2. The comparisons
7
Melphalan
a
First, the potencies of the dienones 3a–g were compared with
The concentration of compound required to inhibit tumour cell proliferation by
the IC50 values of 1a–g. The
D1 figures in Table 2 reveal that the
50%.
compounds in series 3 are more potent than the analogs in series
1 in 67% of the comparisons made. Equal potency was noted in
14% of the comparisons. Hence support for the concept of potency
increases by increased ligand interactions at auxiliary binding sites
appears to take place in the majority of cases. Second, the data for
This information should disclose whether a terminal methoxy or
hydroxyl group of the N-acyl substituent affects cytotoxic poten-
cies. (3) The IC50 values of 4, 5a, 6a and 7 were compared with
Please cite this article in press as: Hossain, M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.056

4
M. Hossain et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 3
Evaluation of 3a–g, 4, 5a, f, 6a and 7 against human tumour cell lines
Compd
HSC-2
HSC-3
HSC-4
Ave
PSEe
a
a
a
a
CC50
SIb
D
1c
D
2d
CC50
SIb
D
1c
D
2d
CC50
SIb
7.07
8.12
10.1
6.93
10.2
9.16
10.4
7.41
8.92
6.46
6.93
6.02
6.99
D
1c
D
2d
CC50
SIb
6.53
6.86
8.55
5.84
8.57
6.74
3a
1.30
1.45
1.97
1.09
0.40
0.76
0.40
1.01
0.58
0.69
1.46
1.56
6.64
1.69
2.69
50.8
16.5
2.25
—
—
2.18
3.79
1.23
0.61
1.22
0.43
0.85
—
—
1.29
2.24
—
1.47
1.70
2.27
1.03
0.39
1.19
1.22
1.32
2.10
1.01
1.19
1.43
9.90
5.87
5.73
7.23
5.45
7.85
4.31
7.34
6.85
4.33
4.48
7.57
5.72
7.84
—
—
—
—
—
—
—
—
—
—
—
—
—
1.91
1.00
1.45
0.94
1.77
—
—
1.11
0.70
—
1.22
1.20
1.63
0.81
0.30
0.56
0.86
1.22
1.02
0.70
1.30
1.36
3.61
7.75
46.0
24.7
20.3
—
—
3.61
4.31
—
3.39
3.24
1.07
1.00
1.47
0.63
1.27
—
—
1.00
1.20
—
1.33
1.45
1.96
0.98
0.36
0.84
0.83
1.18
1.23
0.80
1.32
1.45
4.91
3b
3c
3d
6.72
8.33
5.15
7.65
6.75
22.4
8.95
15.7
6.55
6.17
5.24
4.65
4.73
4.36
5.96
3e
3f
3g
23.8
8.02
16.2
13.4
4
5a
5f
6a
7.74
9.65
5.83
6.89
5.66
6.49
6.56
7.85
7.29
5.22
3.90
0.52
—
1.51
1.41
0.89
0.83
—
1.24
1.03
—
0.94
0.90
—
7
Melphalan
16.7
—
11.1
—
12.6
a
The CC50 values in lM are the concentrations of the compounds required to kill 50% of the cells. The bioassays were undertaken in duplicate and the CC50 values differed
by less than 10% at each concentration.
b
The letters SI refer to the selectivity index which is the quotient of the average CC50 values towards nonmalignant HGF and HPLF cells (the data is presented in Table 4)
and the CC50 figure towards a particular cell line.
c
The
The
D
D
1 figures are the quotients of the CC50 values of (i) 1a–e and 3a–e in which the aryl substituents are identical and (ii) 1a with 4, 5a, 6a, 7.
2 values are the quotients of the CC50 figures of (i) 2a–e and 3a–e in which the aryl substituents are the same and (ii) 3a with 4, 5a, 6a, 7.
d
e
The letters PSE refer to the potency-selectivity expression. These values are the products of the reciprocal of the average CC50 value towards HSC-2, HSC-3 and HSC-4 cells
and the average SI figures.
Table 4
especially towards L1210 cells. Fourth, comparisons of the poten-
cies of 4, 5a, 6a and 7 with 3a were made and are indicated in
Evaluation of 3a–g, 4, 5a, f, 6a and 7 against nonmalignant HGF and HPLF cells
Table 2 as
D2 values. Of the 12 comparisons made, equipotency
Compd
HGF
1b
HPLF
1b
Ave CC50
of 3a with 5a, 6a and 7 was noted while the oxo isostere 4 was sig-
nificantly less potent. In addition, 5f and 6f are approximately
twice as potent as 3f as revealed by the
2c
CC50
D
D
2c
a
a
CC50
9.67
10.3
17.2
5.24
D
D
3a
3b
3c
3d
3e
3f
3g
4
5a
5f
6a
7
0.48
0.07
<0.04
0.08
0.27
—
0.57
1.13
1.01
0.63
0.65
—
—
0.96
0.94
—
7.58
9.17
0.32
0.05
0.10
0.11
0.07
—
—
0.37
0.38
—
0.58
1.09
0.97
0.69
0.41
—
—
1.17
1.20
—
8.63
9.74
16.4
5.61
3.06
5.13
8.95
9.04
9.10
4.52
9.01
8.18
77.6
D2 values in Table 2. Thus
in general the sulfur atom in 3a and 3f may be replaced by groups
with larger size, lower hydrophobicity and greater electron-
attracting properties leading to increases or retention of cytotoxic
potencies. The analyses of the biological data presented in Tables 1
and 2 reveal that in general the addition of N-acyl groups to series
1 leads to increases in potency. In addition, the replacement of the
terminal methoxy group in 3a–e by a hydroxy substituent leads to
a lowering of the IC50 values.
The logP values of the compounds in series 3–7 were obtained
using a software package.13 These data are presented in Table 1.
Part of the rule of five14 stipulates that for satisfactory absorption,
a compound should have a logP value of less than five. This crite-
rion is met by all of the compounds except 3d. The optimum logP
values for oral bioavailability are considered to be in the range of
0-315 and 5a, f, 6a and 7 fall into this category. Hence further ana-
log development should include the incorporation of sulfinyl and
sulfonium moieties into the N-acyl groups. Linear, semilogarithmic
and logarithmic plots between the IC50 values of 3–7 in the
Molt4/C8, CEM and L1210 bioassays and the logP figures were
made with a view to finding if cytotoxic potencies correlate with
the lipophilicity of the molecules. However neither correlations
(p <0.05) nor trends to a correlation (p <0.1) were observed.
In a quest to find guidelines for developing series 3, the possibil-
ity of the electronic, hydrophobic and/or steric properties of the
aryl substituents controlling cytotoxic potencies was examined.
Hence linear and semilogarithmic plots were made between the
15.5
5.97
2.59
4.99
8.07
8.84
9.10
4.21
8.43
6.25
3.52
5.27
9.83
9.24
9.09
4.83
9.59
—
0.46
0.46
—
0.48
0.51
—
0.99
1.05
—
0.35
0.26
—
1.11
0.82
—
10.1
78.0
Melphlan
77.1
a
The CC50 values in
50% of the cells.
lM are the concentrations of the compounds required to kill
b
The
D1 figures are the quotients of the CC50 values of (i) 3a–e and 1a–e in which
the aryl substituents are the same and (ii) 4, 5a, 6a, 7 with 1a.
c
The
D2 values are the quotients of the CC50 figures of (i) 3a–e and 2a–e in which
the aryl substituents are identical and (ii) 4, 5a, 6a, 7 with 3a.
3a–e were compared with the results generated for 2a–e in which
the same substituents are present in the aryl rings. The
D2 figures
in Table 2 reveal that of the 15 comparisons in potency, 7 compar-
isons revealed greater potency in the analogs in series 2, in 7 cases
equal potency was noted and among series 3, only 3e in the L1210
screen displayed a lower IC50 value than 2e. Hence one may con-
clude that the terminal hydroxyl group in series 2 likely forms
hydrogen bonds at auxiliary binding sites which enhances cyto-
toxic potencies. Third, in order to evaluate the contribution of
the sulfur atom in representative members of series 3, several ana-
logs were prepared which have varying electronic, hydrophobic
and steric properties. Thus the sulfur atom of 3a was replaced by
oxygen (4), sulfinyl (5a), sulfonyl (6a) and methylthio (7) groups.
In addition, the sulfur atom of the 4-fluoro analog 3f was replaced
by sulfinyl and sulfonyl groups leading to 5f and 6f, respectively. A
comparison between the IC50 values of 1a with 4, 5a, 6a and 7 is
Hammett sigma (r), Hansch pi (p) and molar refractivity (MR)
values of the aryl groups against the IC50 values generated in the
Molt4/C8, CEM and L1210 bioassays, but no correlations were
noted (p >0.1).
The dienones 3a–g, 4, 5a,f, 6a and 7 were further evaluated
against human HSC-2, HSC-3 and HSC-4 squamous cancer cells
and the results are presented in Table 3. These biological data con-
firm that these molecules are potent cytotoxic agents in which 94%
presented as
D1 values in Table 2 and revealed that the addition
of N-acyl groups to 1a generally led to modest increases in potency.
The 4-fluoro analogs 5f and 6f are appreciably more potent than 1f
of the CC50 values are less than 2 lM and 31% are submicromolar.
Please cite this article in press as: Hossain, M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.056

M. Hossain et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
All of these compounds are considerably more potent than mel-
phalan, especially 3e having an average CC50 value which is 35
times lower than the average CC50 value for melphalan. The data
in Table 3 were examined in a similar way as the results obtained
in the Molt4/C8, CEM and L1210 bioassays. First, 1a–e have been
evaluated previously against HSC-2 and HSC-4 cells16 and a com-
parison of the CC50 values of these compounds and 3a–e against
average D2 figures generated using HGF and HPLF cells are 0.80
and 0.75, respectively, revealing that in general the compounds
with terminal hydroxyl groups in series 2 are less toxic than the
analogs in series 3. Third, the CC50 values of 4, 5a, 6a and 7 were
compared with that of 1a and the results are indicated as
D1 figures
in Table 4. The data reveal the increase in toxicity when the N-acyl
groups are added to the basic centre of the compounds in series 1.
Fourth, the toxicity data of 4, 5a, 6a and 7 were compared with that
HSC-2 and HSC-4 cells are presented as
D1 figures in Table 3.
The results indicate that the addition of the 3-(2-methox-
yethylthio)propionyl group to 1a–e invariably leads to potency
increases. This observation is particularly noteworthy in the case
of 3c whose potency is much higher than 1c. Second a comparison
of the CC50 values of 3a–e and 2a–e9 in the HSC-2, HSC-3 and
HSC-4 bioassays in which the aryl substituents are the same was
of 3a and these results are indicated as D2 values. The average D2
figures generated in the HGF and HPLF bioassays are 0.99
(0.94–1.15) and 1.08 (0.82–1.20), respectively, indicating that
modifications of the 3-(2-methoxyethylthio)propionyl group of 3a
leading to 4, 5a, 6a and 7 causes little or no change in toxicity.
In summary, the results obtained in Table 3 reveal that the
compounds in series 2 which have a terminal hydroxyl substituent
in the N-acyl group are less toxic than the homo analogs 3a–e.
Introduction of N-acyl group leading to 3–7 was accompanied with
lower selective cytotoxicity as compared to series 1.
There are a number of different ways in which cytotoxic com-
pounds can exert their lethal effects. In order to examine if a rep-
resentative compound exerts its cytotoxic effect at least in part by
apoptosis, the most potent compound 3e (CC50: 0.36 lM, SI: 8.57)
was incubated with HSC-2 cells for 24 h and analysed by a FACS
undertaken and the results are indicated as
D2 figures in Table 3.
The 2 figures are very small ranging from 0.43 to 1.91 which sug-
D
gests that in the growth inhibition of HSC-2, HSC-3 and HSC-4 cells,
the terminal group of the N-acyl substituent can be either hydroxyl
(series 2) or methoxy (series 3). Third, a comparison between the
CC50 value of 1a and the N-acyl analogs 4, 5a, 6a and 7 in the
HSC-2 and HSC-4 screens was made and are expressed as
ures in Table 3. In virtually all cases, the amides are more potent
towards HSC-2 and HSC-4 cells than 1a. Fourth, the small 2 val-
D1 fig-
D
ues in Table 2 generated when the potencies of 4, 5a, 6a and 7 were
compared with 3a suggest that the sulfur atom of 3a can be
replaced by related groups with only minor changes in potencies.
In summary, the data in Table 2 confirm the marked potencies of
the novel cytotoxic agents in series 3–7. The addition of various
N-acyl groups to 3,5-bis(benzylidene)-4-piperidones in series 1
led to potency increases.
Annex in V/PI procedure. The percentage of apoptotic cell death
was measured using concentrations of 0.5, 1.0 and 2.0 lM of 3e
and the biological data are presented in Figure 2. The result shows
dose-dependent increase in the induction of apoptotic cell death.
The cleavage of poly(ADP-ribose)polymerase 1 (PARP1) is con-
sidered a hallmark of apoptosis. This nuclear enzyme causes repair
of DNA single-stranded breaks and thus compounds which cleave
PARP1 may have a pivotal role to play in cancer chemotherapy.
The next phase of the investigation was directed to determine
whether the compounds in series 3–7 display greater toxicity to
neoplasms than nonmalignant cells. The dienones 3–7 were
screened against human oral HGF and HPLF nonmalignant cells
(Table 4). Under clinical conditions, a tumour will be surrounded
by different normal cells. Thus selectivity index (SI) figures were
computed which are the quotients of the average CC50 values
towards HGF and HPLF cells (biological data presented in Table 4
vide infra) and a specific cell line such as HSC-2, HSC-3 and
HSC-4. The SI values are presented in Table 3. All of the compounds
in series 3–7 are tumour-specific cytotoxic agents with SI values
greater than 1. A SI value of 10 was arbitrarily chosen as indicative
of lead molecules and this criterion was achieved by 3g and 5a
(HSC-2) and 3c,e,g (HSC-4 assay). In general the average SI figures
are better than, or similar to, melphalan. In addition to permitting
SI values to be calculated vide supra, these determinations were
undertaken in order to gain some idea of the toxicity of these die-
nones and in particular to attempt to correlate structural features
with toxicity. The highest average CC50 values are the 4-methyl
and 4-methoxy analogs in series 3 namely 3b and 3c. Thus future
development of these dienones should include the preparation of
analogs in series 3 with one or more methyl and methoxy groups
in the aryl rings, especially in view of the encouraging SI values
of the 3,4-dimethoxy analog 3g as indicated in Table 3. The
introduction of a 4-fluoro substituent into the aryl rings of 3a
and 5a leading to 3f and 5f, respectively, was accompanied by
increase in toxicity to both HGF and HPLF cells. All of the
compounds in series 3–7 are more toxic than melphalan.
Using concentrations of 0.1, 0.2, 0.3 and 0.4
concentration-dependent PARP1 cleavage while 3g caused PARP1
cleavage only at the highest concentration of 0.4
(Fig. 3).
lM, 3e demonstrated
lM in HSC-2 cells
3. Conclusions
The series 3–7 were disclosed as novel cytotoxic agents. The
study was aimed at exploring the hypothesis that a suitable acyl
group attached to the nitrogen atom of series would align at aux-
iliary binding sites which could enhance the interaction of the
1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore at
a primary
binding site. The results obtained support this theory and have
led to a series of novel antineoplastic agents which display potent
tumour-selective toxicity. In particular 3e demonstrated excellent
selective cytotoxicity against HSC cell lines (ave CC50: 0.36 lM, ave
SI: 8.57 and ave PSE: 23.8) as compared to normal cells which
warrants its further investigation as a candidate antineoplastic
agent. In the future, one or more fluoro or methoxy groups should
be placed in the aryl rings with the aim of increasing potencies.
Incorporation of aryl methyl and methoxy substituents into new
analogs may lower toxicity towards non-malignant cells and hence
increase the tumour-selective properties of these compounds.
4. Experimental methods
4.1. Chemistry
The toxicity data presented in Table 4 was analyzed in the same
manner as undertaken previously. First, the CC50 values of 3a–e
towards HGF and HPLF cells were compared with the figures for
1a–e which have been disclosed previously.16 These data presented
4-Piperidone hydrochloride hydrate and arylaldehydes were
purchased from Sigma–Aldrich. All other chemicals were analytical
grade and obtained from commercial suppliers. These chemicals
were used without further purification. The 1H NMR and 13C
NMR spectra were obtained using a Bruker Avance AMX 500 FT
spectrometer equipped with a BBO probe. The chemical shifts are
as
D1 figures in Table 4. Clearly the addition of the 1-acyl group to
the compounds in series 1 increases toxicity markedly. Second, the
toxicity of 3a–e was compared with the O-demethylated analogs
2a–e9 and the results are indicated as
D2 figures in Table 4. The
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6
M. Hossain et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7.47–7.37 (m, 10H, Ar–H), 4.93 (s, 2H, NCH2), 4.72 (s, 2H, NCH2),
3.45 (t, 2H, OCH2), 3.29 (s, 3H, OCH3), 2.73 (t, 2H, SCH2), 2.56
(t, 2H, SCH2), 2.44 (t, 2H, COCH2). 13C NMR (125 MHz, CDCl3): d
ppm 186.67, 169.95, 138.63, 137.54, 134.61, 134.38, 131.57,
130.65, 130.16, 129.76, 129.65, 129.01, 128.81, 71.99, 58.70,
46.23, 43.60, 33.45, 31.87, 27.58. HRMS (EI): m/z: 421.1722 (M)+,
calcd 421.1712 for C25H27NO3S. Anal. (C25H27NO3S) requires C,
71.23; H, 6.47; N, 3.32; S, 7.61; found C, 71.14; H, 6.63; N, 3.02;
S, 7.75.
4.1.2.2. 1-[3-(2-Methoxyethylthio)propionyl]-3,5-bis(4-methyl-
Figure 2. The dose-dependent induction of apoptotic cell death by 3e in HSC-2
cells.
benzylidene)-4-piperidone (3b).
Yield 78%. Mp 90–92 °C.
1H NMR (500 MHz, CDCl3): d ppm 7.84 (s, 1H, @CH), 7.80 (s, 1H,
@CH), 7.37 (d, 2H, Ar–H, J = 7.6 Hz), 7.27–7.26 (m, 4H), 7.23 (d,
2H, Ar–H, J = 7.6 Hz), 4.92 (s, 2H, NCH2), 4.71 (s, 2H, NCH2), 3.45
(t, 2H, OCH2), 3.30 (s, 3H, OCH3), 2.73 (t, 2H, SCH2), 2.56 (t, 2H,
SCH2), 2.45 (t, 2H, COCH2), 2.38 (s, 3H, Ar–CH3), 2.34 (s, 3H,
Ar–CH3). 13C NMR (CDCl3, 125 MHz): d ppm 186.69, 169.92,
140.19, 140.09, 138.54, 137.45, 131.90, 131.61, 130.81, 130.29,
129.75, 129.56, 71.98, 58.69, 46.23, 43.67, 33.47, 31.87, 27.60,
21.51. HRMS (EI): m/z 449.2017 (M)+, calcd 449.2025 for
C27H31NO3S. Anal. (C27H31NO3S) requires C, 72.12; H, 6.96; N,
3.12; S, 7.13; found C, 72.38; H, 7.56; N, 3.07; S, 7.06.
4.1.2.3. 3,5-Bis(4-methoxybenzylidene)-1-[3-(2-methoxyethyl
thio)propionyl]-4-piperidone (3c).
Yield 80%. Mp 92–95 °C.
1H NMR (500 MHz, CDCl3): d ppm 7.82 (s, 1H, @CH), 7.78 (s, 1H,
@CH), 7.45 (d, 2H, Ar–H, J = 8.3 Hz), 7.35 (d, 2H, Ar–H, J = 8.2 Hz),
6.97 (d, 2H, Ar–H, J = 8.2 Hz), 6.94 (d, 2H, Ar–H, J = 8.2 Hz), 4.92
(s, 2H, NCH2), 4.72 (s, 2H, NCH2), 3.87 (s, 3H, ArOCH3), 3.85 (s,
3H, ArOCH3), 3.46 (t, 2H, OCH2), 3.30 (s, 3H, OCH3), 2.75 (t, 2H,
SCH2), 2.58 (t, 2H, SCH2), 2.49 (t, 2H, COCH2). 13C NMR (125 MHz,
CDCl3): d ppm 186.49, 169.91, 160.80, 160.77, 138.07, 137.04,
132.72, 132.18, 129.70, 129.58, 127.46, 127.05, 114.53, 114.33,
71.97, 58.69, 55.42, 46.32, 43.62, 33.51, 31.90, 27.63. HRMS (EI):
m/z 481.1916 (M)+, calcd 481.1923 for C27H31NO5S. Anal.
(C27H31NO5S) requires C, 67.34; H, 6.49; N, 2.91; S, 6.66; found C,
67.08; H, 6.78; N, 2.81; S, 6.48.
Figure 3. Cleavage of PARP1 by 3e and 3g in HSC-2 cells.
reported in ppm. Mass spectra were taken using a QSTAR XL
Hybrid LC/MS/MS System. Elemental analyses were undertaken
using an Elementer CHNS analyser which revealed that the com-
pounds have >95% purity. Melting points in degrees Celsius are
uncorrected which were determined with a Gallenkamp apparatus.
4.1.1. Synthesis of 3,5-bis(benzylidene)-4-piperidones 1a–g
The methodologies for the preparation of 1a,8 1b,11 c–f8 and
1g12 in series 1 have been described previously.
4.1.2.4. 3,5-Bis(4-chlorobenzylidene)-1-[3-(2-methoxyethylthio)
propionyl]-4-piperidone (3d).
Yield 73%. Mp 110–113 °C. 1H
NMR (500 MHz, CDCl3): d ppm 7.81 (s, 1H, @CH), 7.76 (s, 1H,
@CH), 7.45 (d, 2H, Ar–H, J = 7.7 Hz), 7.40 (s, 4H, Ar–H), 7.32
(d, 2H, Ar–H, J = 7.2 Hz), 4.88 (s, 2H, NCH2), 4.69 (s, 2H, NCH2),
3.47 (t, 2H, OCH2), 3.30 (s, 3H, OCH3), 2.74 (t, 2H, SCH2), 2.58
(t, 2H, SCH2), 2.45 (t, 2H, COCH2). 13C NMR (125 MHz, CDCl3): d
ppm 186.17, 169.97, 137.32, 136.37, 136.02, 135.83, 132.95,
132.70, 131.80, 131.39, 129.37, 129.14, 72.01, 58.72, 46.23, 43.42,
33.43, 31.97, 27.59. HRMS (EI) m/z: 489.0918 (M)+, calcd
489.0932 for C25H25Cl2NO3S. Anal. (C25H25Cl2NO3S) requires C,
61.22; H, 5.15; N, 2.86; S, 6.54; found C, 61.21; H, 5.21; N, 2.77;
S, 6.85.
4.1.2. General synthetic procedure for compounds 3a–g
A mixture of 3-(2-methoxyethylthio)propionic acid (1.05 g,
6.4 mmol), which was prepared by a literature method,17 thionyl
chloride (5 ml) and dimethylformamide (0.02 ml) was stirred at
room temperature for 3 h. Excess thionyl chloride was removed in
vacuo. The residue was dissolved in dichloromethane (5 ml) and
added to a solution of the appropriate dienone in series 1 (4.0 mmol)
in dichloromethane (70 ml) at 0 °C under argon. Subsequently tri-
ethylamine (1.1 ml, 8.0 mmol) was added and the mixture stirred
at room temperature for 24 h. The precipitate was collected and
the solvent removed in vacuo to yield a residue. The precipitate
and residue were combined and suspended in a solution of potas-
sium carbonate in water (5% w/v, 25 ml) and stirred vigorously over-
night. On occasions a solid was formed which was collected by
filtration and washed with methanol. Alternatively if no precipitate
was formed, the mixture was extracted with chloroform (2 ꢀ 30 ml),
dried over anhydrous magnesium sulfate and the solvent removed.
The crude products were purified by silica gel column chromatogra-
phy using an eluting solvent of a mixture of ethyl acetate and hexane
(1:1) to yield the desired products 3a–g.
4.1.2.5. 1-[3-(2-Methoxyethylthio)propionyl]-3,5-bis(4-nitroben-
zylidene)-4-piperidone (3e).
Yield 70%. Mp 136–138 °C. 1H
NMR (500 MHz, CDCl3): d ppm 8.35 (d, 2H, Ar–H, J = 7.7 Hz), 8.30 (d,
2H, Ar–H, J = 7.8 Hz), 7.90 (s, 1H, @CH), 7.85 (s, 1H, @CH), 7.63 (d,
2H, Ar–H, J = 7.7 Hz), 7.56 (d, 2H, Ar–H, J = 7.7 Hz), 4.90 (s, 2H,
NCH2), 4.75 (s, 2H, NCH2), 3.46 (t, 2H, OCH2), 3.30 (s, 3H, OCH3),
2.75 (t, 2H, SCH2), 2.58 (t, 2H, SCH2), 2.45 (t, 2H, COCH2). 13C NMR
(125 MHz, CDCl3): d ppm 185.58, 170.03, 148.12, 147.93, 140.59,
140.34, 136.19, 135.52, 134.08, 133.92, 130.97, 130.75, 124.26,
124.05, 72.00, 58.73, 46.37, 43.24, 33.35, 32.09, 27.58. TOF MS: m/z
512.1525 (M+1)+ and 534.1332 (M+Na)+, calcd 511.1413 for
4.1.2.1. 1-[2-Methoxyethylthio)propionyl]-3,5-bis(benzylidene)
-4-piperidones (3a).
(500 MHz, CDCl3): d ppm 7.88 (s, 1H, @CH), 7.83 (s, 1H, @CH),
Yield 83%. Mp 76–78 °C. 1H NMR
C25H25N3O7S. Anal. (C25H25N3O7S) requires C, 58.69; H, 4.94; N,
8.22; S, 6.27; found C, 58.56; H, 5.24; N, 7.95; S, 6.17.
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M. Hossain et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
4.1.2.6. 3,5-Bis(4-fluorobenzylidene)-1-[3-(2-methoxyethylthio)
propionyl]-4-piperidone (3f).
Yield 78%. Mp 110–112 °C. 1H
Ar–H), 4.92 (s, 2H, NCH2), 4.77 (s, 2H, NCH2), 3.68 (t, 2H, OCH2),
3.50 (m, 2H, OCH2), 3.45 (m, 2H, OCH2), 3.32(s, 3H, OCH3), 2.50
NMR (500 MHz, CDCl3): d ppm 7.82 (s, 1H,=CH), 7.78 (s, 1H,=CH),
7.45 (dd, 2H, Ar–H, J = 5.7 Hz), 7.38 (dd, 2H, Ar–H, J = 6.0 Hz),
7.17 (dd, 2H, Ar–H, J = 8.1 Hz), 7.11 (dd, 2H, Ar–H, J = 8.2 Hz),
4.88 (s, 2H, NCH2), 4.69 (s, 2H, NCH2), 3.45 (t, 2H, OCH2), 3.30 (s,
3H, OCH3), 2.74 (t, 2H, SCH2), 2.58 (t, 2H, SCH2), 2.45 (t, 2H, COCH2).
13C NMR (125 MHz, CDCl3): d 186.30, 169.95, 164.32, 162.32,
137.41, 136.44, 132.70, 132.63, 132.25, 132.18, 130.76, 130.50,
116.39, 116.22, 116.13, 115.96, 72.00, 58.70, 46.22, 43.40, 33.43,
31.95, 27.59. HRMS (EI): m/z 457.1515 (M)+, calcd 457.1523 for
(t, 2H, COCH2). 13C NMR (125 MHz, CDCl3):
d ppm 186.70,
169.68, 138.35, 137.52, 134.66, 134.42, 131.63, 131.54, 130.61,
130.29, 129.73, 129.57, 128.97, 128.79, 71.74, 70.33, 67.07, 59.00,
46.52, 43.28, 33.26. HRMS (EI): m/z 405.1940 (M)+, calcd
405.1940 for C25H27NO4. Anal. (C25H27NO4) requires C, 74.05; H,
6.73; N, 3.46; found C, 73.85; H, 6.75; N, 3.15.
4.1.4. General synthetic procedure for compounds 5a, 5f
Peracetic acid (10% w/v in acetic acid, 2 ml) was added slowly to
a solution of 3a (0.8 g, 1.9 mmol) or 3f (0.87 g, 1.9 mmol) in
dichloromethane (5 ml) at 0 °C and the mixture stirred at 0 °C for
1 h. The reaction was quenched with aqueous sodium bicarbonate
solution (10% w/v, 20 ml) and the mixture extracted with chloro-
form (2 ꢀ 20 ml). The organic extract was dried over anhydrous
sodium sulfate and evaporation of the solvent in vacuo led to the
isolation of the crude product which was purified by silica gel
column chromatography using an eluting solvent of chloroform/
methanol (99:1) leading to the desired product.
C25H25F2NO3S. Anal. (C25H25F2NO3S) requires C, 65.62; H, 5.52; N,
3.06; S, 7.01; found C, 65.75; H, 6.02; N, 3.07; S, 6.95.
4.1.2.7. 3,5-Bis(3,4-dimethoxybenzylidene)-1-[3-(2-methoxyeth
ylthio)propionyl]-4-piperidone
(3g).
Yield
79%.
Mp
122–125 °C. 1H NMR (500 MHz, CDCl3): d ppm 7.80 (s, 1H,=CH),
7.76 (s, 1H,=CH), 7.08 (d, 1H, Ar–H, J = 7.1 Hz), 7.00 (s, 1H, Ar–H),
6.95 (s, 2H, Ar–H), 6.91 (s, 2H, Ar–H), 4.92 (s, 2H, NCH2), 4.73 (s,
2H, NCH2), 3.93–3.91 (12H, ArOCH3), 3.45 (t, 2H, OCH2), 3.28 (s,
3H, OCH3), 2.74 (t, 2H, SCH2), 2.57 (t, 2H, SCH2), 2.49 (t, 2H, COCH2).
13C NMR (CDCl3): d ppm 186.33, 169.93, 150.52, 149.15, 148.93,
138.35, 137.34, 129.97, 129.83, 127.68, 127.33, 124.43, 123.46,
113.86, 113.69, 111.24, 111.16, 71.96, 58.69, 56.00, 46.39, 43.58,
33.49, 31.92, 27.65. HRMS (EI): m/z 541.2134 (M)+, calcd
541.2132 for C29H35NO7S. Anal. (C29H35NO7S) requires C, 64.30;
H, 6.53; N, 2.59; S, 5.92; found C, 64.49; H, 7.05; N, 2.52; S, 5.92.
4.1.4.1. 3,5-Bis(benzylidene)-1-[3-(2-methoxyethylsulfinyl)pro-
pionyl-4-piperidone (5a).
Yield 75%. Mp 82–85 °C. 1H NMR
(500 MHz, CDCl3): d ppm 7.89 (s, 1H, @CH), 7.84 (s, 1H, @CH),
7.49–7.34 (m, 10H, Ar–H), 5.05 (d, 1H, NCH2, J = 6.5 Hz), 4.82 (d,
2H, NCH2, J = 18.1 Hz), 4.72 (d, 1H, NCH2, J = 16.5 Hz), 3.74 (t, 2H,
OCH2), 3.33 (s, 3H, OCH3), 3.11–3.05 (m, 1H), 2.91–2.70 (m, 4H),
2.65–2.61 (m, 1H). 13C NMR (125 MHz, CDCl3): d ppm 186.45,
168.83, 138.67, 137.95, 134.54, 134.19, 131.32, 131.17, 130.62,
130.21, 129.91, 129.68, 129.08, 128.83 64.90, 59.00, 52.97, 47.68,
46.19, 43.71, 25.58. HRMS (ESI): m/z 438.1678 (M+1)+, calcd
437.1661 for C25H27NO4S. Anal. (C25H27NO4S) requires C, 68.62;
H, 6.23; N, 3.20; S, 7.33; found C, 68.67; H, 6.14; N, 3.18; S, 7.09.
4.1.3. 3,5-Bis(benzylidene)-1-[3-(2-methoxyethyloxy)propionyl]-
4-piperidone (4)
2-Methoxyethanol (4.6 g, 0.06 mol) and finely ground anhy-
drous potassium carbonate (20.7 g, 0.15 mol) were added to a solu-
tion of ethyl acrylate (9.0 g, 0.09 mol) in acetonitrile (100 ml). The
mixture was heated under reflux at 90 °C for 24 h and then filtered.
The solvent was evaporated from the filtrate to give a residue
which was suspended in ethanol (10 ml) to which a solution of
sodium hydroxide (3.6 g) in water (30 ml) was added. The mixture
was heated under reflux at 90 °C for 1 h. After removing the etha-
nol in vacuo, the aqueous solution was washed with chloroform
(2 ꢀ 15 ml). The aqueous phase was acidified with hydrochloric
acid to pH 2 at 0 °C and then extracted with ethyl acetate
(3 ꢀ 20 ml). The organic phase was dried with anhydrous sodium
sulfate and removal of the solvent afforded 3-(2-methoxyethy-
loxy)propionic acid (4.3 g, 48% yield). 1H NMR (500 MHz, CDCl3):
d ppm 3.74 (t, 2H), 3.60 (m, 2H), 3.52 (m, 2H), 3.36 (s, 3H), 2.63
(t, 2H). This compound was used in the next reaction step without
any further purification.
A mixture of 3-(2-methoxyethyloxy)propionic acid (0.95 g,
6.4 mmol), thionyl chloride (5 ml) and dimethylformamide
(0.04 ml) was stirred at room temperature for 3 h. Unreacted thio-
nyl chloride was removed in vacuo and the residue was dissolved
in dichloromethane (5 ml) and added to a solution of 1a (1.1 g,
4.0 mmol) in dichloromethane (70 ml) at 0 °C under argon.
Triethylamine (1.1 ml, 8.0 mmol) was added and the mixture stir-
red at room temperature for 24 h. The precipitate was collected
by filtration and the filtrate evaporated to dryness to give a residue
which was combined with the precipitate, suspended in aqueous
potassium carbonate solution (5% w/v, 25 ml) and stirred vigor-
ously overnight. The mixture was extracted with chloroform
(2 ꢀ 30 ml) and the organic phase was dried over anhydrous
sodium sulfate and the solvent removed in vacuo. The crude pro-
duct was purified by silica gel column chromatography using a
mixture of ethyl acetate/hexane (3:2) as an eluting solvent to give
a yellow oil which subsequently solidified and was identified as
the dienone 4. Yield 90%. Mp 80–82 °C. 1H NMR (500 MHz, CDCl3):
d ppm 7.86 (s, 1H, @CH), 7.83 (s, 1H, @CH), 7.48–7.37 (m, 10H,
4.1.4.2. 3,5-Bis(4-fluorobenzylidene)-1-[3-(2-methoxyethylsulfi-
nyl)propionyl-4-piperidone
(5f).
Yield
65%.
Mp
118–120 °C. 1H NMR (500 MHz, CDCl3): d ppm 7.83 (s, 1H, @CH),
7.79 (s, 1H,=CH), 7.45 (t, 2H, Ar–H, J = 6.6 Hz), 7.38 (t, 2H, Ar–H,
J = 6.6 Hz), 7.17 (t, 2H, Ar–H, J = 8.8 Hz), 7.13 (t, 2H, Ar–H,
J = 8.8 Hz), 5.01 (d, 1H, NCH2, J = 16 Hz), 4.79 (d, 2H, NCH2,
J = 16 Hz), 4.70 (d, 1H, NCH2, J = 16 Hz), 3.75 (t, 2H, OCH2), 3.34 (s,
3H, OCH3), 3.14–3.08 (m, 1H), 2.98–2.84 (m, 3H), 2.80–2.74 (m,
1H), 2.70–2.64 (m, 1H). 13C NMR (125 MHz, CDCl3): d ppm 186.09,
168.81, 164.42, 164.33, 162.41, 162.33, 137.47, 136.94, 132.66,
132.59, 132.36, 132.29, 130.84, 130.69, 130.33, 116.50, 116.33,
116.17, 115.99, 64.85, 59.03, 52.89, 47.57, 46.23, 43.50, 25.74. TOF
MS: m/z 496.1357 (M+Na)+, calcd 473.1472 for C25H25F2NO4S. Anal.
(C25H25F2NO4S) requires C, 63.41; H 5.33; N, 2.96; S, 6.77; found C,
63.70; H, 5.48; N, 2.93; S, 6.78.
4.1.5. General synthetic procedure for compounds 6a, 6f
3-Chloroperoxybenzoic acid (77%, 1.1 g, 4.75 mmol) was added
in portions to a solution of 3a (0.80 g, 1.9 mmol) or 3f (0.87 g,
1.9 mmol) in dichloromethane (15 ml) at 0 °C and the mixture
stirred at 0 °C for 0.5 h. The reaction was quenched with aqueous
sodium bicarbonate solution (10% w/v, 20 ml) and the mixture
extracted with chloroform (2 ꢀ 20 ml). The organic extract was
dried over anhydrous sodium sulfate and removal of the
solvent in vacuo gave the crude product which was purified by sil-
ica gel column chromatography and an eluting solvent of ethyl
acetate/hexane (3:2) to give the desired products.
4.1.5.1. 3,5-Bis(benzylidene)-1-[3-(2-methoxyethylsulfonyl)pro-
pionyl-4-piperidone (6a).
Yield 93%. Mp 117–120 °C. 1H
NMR (500 MHz, CDCl3): d ppm 7.89 (s, 1H, @CH), 7.85 (s, 1H,
@CH), 7.48–7.38 (m, 10H, Ar–H), 4.93 (s, 2H, NCH2), 4.74 (s, 2H,
Please cite this article in press as: Hossain, M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.056

8
M. Hossain et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
NCH2), 3.73 (t, 2H, OCH2), 3.36 (t, 2H, SO2CH2), 3.29 (s, 3H, OCH3),
3.10 (t, 2H, SO2CH2), 2.69 (t, 2H, COCH2). 13C NMR (CDCl3): d ppm
186.46, 168.00, 138.72, 138.02, 134.51, 134.20, 131.25, 131.15,
130.62, 130.18, 129.92, 129.71, 129.09, 128.84, 65.96, 59.04,
54.10, 50.42, 46.23, 43.81. HRMS (EI) m/z: 453.1609 (M)+, calcd
453.1610 for C25H27NO5S. Anal. (C25H27NO5S) requires C, 66.20;
H, 6.01; N, 3.09; S, 7.07; found C, 66.05; H, 6.10; N, 2.96; S, 6.78.
cells except the time of incubation was extended from 24 h to
48 h.21 In brief, different concentrations of the compounds were
incubated at 37 °C with the cells in DMEM medium supplemented
with 10% heat-inactivated fetal bovine serum. The MTT method
was used in determining cell viability.
4.4.2. Mode of action studies
Compound 3e was incubated for 24 h at 37 °C with HSC-2 cells
in RPMI media. The percentage of cells undergoing early and late
apoptosis were determined by flow cytometry using Annexin-V-
FITC and propidium iodide.
The evaluation of whether 3e and 3g caused cleavage of PARP1
in HSC-2 cells was undertaken using a Promega cleaved PARP (Asp
214) human specific antibody assay22 details of which have been
described recently.23 In brief, HSC-2 cells were cultured in 6-well
4.1.5.2. 3,5-Bis(4-fluorobenzylidene)-1-[3-(2-methoxyethylsul-
fonyl)propionyl]-4-piperidone (6f).
Yield 59%. Mp 135–
137 °C. 1H NMR (500 MHz, CDCl3): d ppm 7.84 (s, 1H, @CH), 7.80
(s, 1H, @CH), 7.45 (t, 2H, Ar–H, J = 6. 6 Hz), 7.40 (t, 2H, Ar–H,
J = 6.6 Hz), 7.18 (t, 2H, Ar–H, J = 8. 8 Hz), 7.13 (t, 2H, Ar–H,
J = 8.8 Hz), 4.89 (s, 2H, NCH2), 4.73 (s, 2H, NCH2), 3.75 (t, 2H,
OCH2), 3.38 (t, 2H, SO2CH2), 3.31 (s, 3H, OCH3), 3.15 (t, 2H, SO2CH2),
2.73 (t, 2H, COCH2). HRMS (EI): m/z: 489.1430 (M)+, calcd 489.1422
for C25H25F2NO5S. Anal. (C25H25F2NO5S) requires C, 61.33; H, 5.16;
N, 2.86; S, 6.55; found C, 61.07; H, 5.29; N, 2.78; S, 6.57.
plates for 24 h and then treated with 0.1, 0.2, 0.3 and 0.4 lM of
either 3e or 3g for 24 h. Subsequently the cells were scraped, col-
lected in a lysis buffer and subjected to 8% SDS–polyacrylamide
gel electrophoresis and then transferred to a polyvinylidene diflu-
oride membrane which was exposed to X-ray films.
4.1.6. {3-[3,5-Bis(benzylidene)-4-oxopiperidin-1-yl)]}-3-(oxo-
propyl)(2-methoxyethyl)(methyl)sulfonium tetrafluoroborate
(7)
Acknowledgements
A solution of 3a (0.745 g, 1.8 mmol) in dichloromethane (4 ml)
was added to a suspension of trimethyloxonium tetrafluoroborate
(0.26 g, 1.8 mmol) in dichloromethane (5 ml) and the mixture was
stored at room temperature for 1 h. The solvent was removed in
vacuo and the residue was purified by silica gel column chro-
matography using an eluting solvent of chloroform/methanol
(95:5) to yield 7 as viscous oil. Yield 79%. 1H NMR (500 MHz,
CDCl3): d ppm 7.86–7.85 (2H, @CH), 7.50–7.40 (m, 10H, Ar–H),
4.88 (s, 2H, NCH2), 4.77 (s, 2H, NCH2), 3.79–3.73 (m, 2H, OCH2),
3.66–3.61 (m, 1H, CH2S+), 3.53–3.49 (m, 2H, CH2S+), 3.35–3.30
(m, 1H, CH2S+), 3.28 (s, 3H, OCH3), 2.93 (s, 3H, CH3S+), 2.90 (t,
2H, COCH2). 13C NMR (CDCl3): d 186.22, 168.38, 138.60, 138.38,
134.47, 133.93, 130.95, 130.61, 130.58, 130.53, 130.09, 129.74,
129.23, 128.89, 66.91, 59.12, 46.34, 44.14, 43.51, 40.58, 28.63,
25.75. Anal. (C26H30 BF4NO3S) requires C, 52.44; H, 5.08; N, 2.35;
S, 5.37; found C, 52.02; H, 5.10; N, 2.08; S, 4.98.
The Canadian Institutes of Health Research (CIHR), Canada and
the Saskatchewan Health Research Foundation (SHRF), Canada
awarded a grant to J.R.D. and U.D. The Ministry of Education,
Science, Sports and Culture, Japan provided support for a Grant-
in-Aid (No. 19592156) to H.S. The Geconcerteerde Onderoeksac-
ties, Belgium (GoA 05/19) provided funds for J.B. Financial support
was provided to M.H. by the Professional Development Fund of the
Asian University for Women, Chittagong, Bangladesh. The Univer-
sity of Saskatchewan, Canada is thanked for awarding a Visiting
Scholar position to M. H. L. Van Berckelaer, Rega Institute, Belgium
carried out the Molt4/C8, CEM and L1210 bioassays.
References and notes
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4.3. Statistical analyses
The
r, p and MR constants of the aryl substituents in series 3
were obtained from the literature.18 Since 3g is a disubstituted
derivative, the other compounds in series 3 were treated as having
two aryl substituents. The MR value of the hydrogen atom is 1.03
not 0.00 and this figure was added to the MR values of the R1
groups in 3b–f. The MR figure for the unsubstituted compound
3a is 2.06, i.e., 1.03 ꢀ 2.
The linear and semilogarithmic plots were made using a
statistical package.19
4.4. Biological evaluations
4.4.1. Cytotoxicity assays
The methodology used in evaluating the compounds in series 3–
7 against Molt4/C8, CEM and L1210 cells has been described previ-
ously.20 In brief, different concentrations of the compounds were
added to the cells in RPMI 1640 medium and incubated at 37 °C
for 72 h (Molt4/C8 and CEM cells) or 48 h (L1210 cells).
A literature procedure was employed when examining the
lethal effects of series 3–7 on HSC-2, HSC-3, HSC-4, HGF and HPLF
18. Hansch, C.; Leo, A. J. Substituent Constants for Correlation Analysis in Chemistry
and Biology; John Wiley and Sons Inc: New York, 1979; p p. 49.
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9
19. Statistical Package for Social Sciences, SPSS for Windows, Release 17.0; SPSS:
Chicago, 2008.
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Kawase, M.; Sohara, Y.; Tani, S.; Shirataki, Y.; Sakagami, H.; Satoh, K.;
22. Distributed by Cell Signalling Technology Inc, Beverly, MA.
23. Das, S.; Das, U.; Sakagami, H.; Umemura, N.; Iwamoto, S.; Matsuta, T.; Kawase,
M.; Molnar, J.; Serly, J.; Gorecki, D. K. J.; Dimmock, J. R. Eur. J. Med. Chem. 2012,
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Please cite this article in press as: Hossain, M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.056