The cytotoxic properties and preferential toxicity to tumour cells displayed by some 2,4-bis(benzylidene)-8-methyl-8-azabicyclo[3.2.1] octan-3-ones and 3,5-bis(benzylidene)-1-methyl-4-piperidones

Article abstract of DOI:10.1016/j.ejmech.2008.03.015

This study demonstrated that replacement of the axial protons on the C2 and C6 atoms of various 1-methyl-3,5-bis(benzylidene)-4-piperidones 3 by a dimethylene bridge leading to series 2 lowered cytotoxic potencies. Four compounds 2a and 3a-c emerged as lead molecules based on their toxicity towards different neoplasms and their selective toxicity for malignant rather than normal cells. Some possible reasons for the disparity between the IC₅₀ values in the two series of compounds are presented based on molecular modeling, log P values and respiration in rat liver mitochondria.

Full text of DOI:10.1016/j.ejmech.2008.03.015

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 54e62
http://www.elsevier.com/locate/ejmech
Original article
The cytotoxic properties and preferential toxicity to tumour cells
displayed by some 2,4-bis(benzylidene)-8-methyl-8-azabicyclo[3.2.1]
octan-3-ones and 3,5-bis(benzylidene)-1-methyl-4-piperidones
Hari N. Pati a, Umashankar Das a, Swagatika Das a, Brian Bandy a, Erik De Clercq b,
Jan Balzarini b, Masami Kawase c, Hiroshi Sakagami d, J. Wilson Quail e,
James P. Stables f, Jonathan R. Dimmock a,
*
a College of Pharmacy and Nutrition, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan S7N 5C9, Canada
b Rega Institute of Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
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
e Saskatoon Structural Sciences Centre, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan S7N 5C9, Canada
f National Institute of Neurological Disorders and Stroke, 6001 Executive Boulevard, Rockville, MD 20852, USA
Received 31 October 2007; received in revised form 5 March 2008; accepted 6 March 2008
Available online 29 March 2008
Abstract
This study demonstrated that replacement of the axial protons on the C2 and C6 atoms of various 1-methyl-3,5-bis(benzylidene)-4-piperidones 3
by a dimethylene bridge leading to series 2 lowered cytotoxic potencies. Four compounds 2a and 3aec emerged as lead molecules based on their
toxicity towards different neoplasms and their selective toxicity for malignant rather than normal cells. Some possible reasons for the disparity
between the IC50 values in the two series of compounds are presented based on molecular modeling, log P values and respiration in rat liver mi-
tochondria.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Tropinones; 4-Piperidones; Cytotoxicity; Molecular modeling; X-ray crystallography; Mitochondria
1. Introduction
drugs [4]. There are a number of critical biochemical pro-
cesses which involve thiols and the importance of compounds
which interact with multiple molecular targets has been em-
phasized recently [5,6].
The major interest in these laboratories is the development
of antineoplastic agents which are structurally divergent from
contemporary anticancer drugs. These novel compounds are
principally conjugated unsaturated ketones which are known
to react with thiols [1] but have low or nonexistent affinities
for amino and hydroxy groups [2,3]. Since thiols, in contrast
to amino or hydroxy groups, are not found in nucleic acids,
a,b-unsaturated ketones may be bereft of the carcinogenic
and mutagenic properties displayed by various anticancer
The 1,5-diaryl-3-oxo-1,4-pentadienyl group has been
mounted on a variety of cyclic scaffolds leading to the discov-
ery of a number of potent cytotoxins [7,8]. This group is con-
sidered to react at a primary binding site. However, the
magnitude of the bioactivity observed will be influenced by
the presence of other structural units in the molecule which
align at an auxiliary site. These possibilities are illustrated in
Fig. 1A. In order to probe as to the nature of the groups in
the vicinity of the pharmacophore which affect cytotoxic po-
tencies, various compounds possessing the general structure
1 were prepared (Fig. 1B). Several studies revealed that
* 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 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.03.015

H.N. Pati et al. / European Journal of Medicinal Chemistry 44 (2009) 54e62
The 1,5-diaryl-3-oxo-1,4-pentadienyl
55
pharmacophore which may interact
at a primary binding site
O
O
3
θ1
θ2
5
1
R1
R1
B
R1
4
2
A
R1
6
2
N
R2
1
The portion of the molecule which is capable
of enhancing or reducing cytotoxic potencies
A possible auxiliary binding site
A
B
Fig. 1. A) The possible interactions of series 1e3 at binding sites. (B) The general structure 1.
compounds in which R2 is an acyl group have increased cyto-
toxic potencies compared to the analogs when R2 is a hydrogen
atom [9,10]. In fact a number of N-acyl compounds have sub-
micromolar IC50 values and displayed selective toxicity to
neoplasms than normal cells [11]. Thus by expanding the
size of the molecules, there is the possibility of additional
binding of the ligand at a receptor which results in the lower-
ing of the IC50 values. The hypothesis formulated in this study
is that by increasing the size of the heterocyclic scaffold,
cytotoxic potencies will be elevated compared to the analogs
lacking this additional structural unit. In the present case, a di-
methylene bridge was placed between carbon atoms 2 and 6 of
the piperidine ring to give series 2 with a view to comparing
cytotoxic potencies with the analogs 3 which lack this struc-
tural feature.
Previous studies revealed that the lack of coplanarity of rings
A and B with the adjacent unsaturated linkages in 1 was caused,
inter alia, by nonbonded interactions between one of the ortho
protons of each aryl ring with the equatorial hydrogen atoms
at C2 and C6 [12]. The decision was made, therefore, to replace
the axial and not equatorial protons on the C2 and C6 atoms by
substituents. In this way, changes in the cytotoxic potencies be-
tween 1 and various analogs could be attributed to the topo-
graphical, physicochemical and chemical properties of the
groups at C2 and C6 per se and the interpretation of the results
would not be complicated by changes in the torsion angles q1
and q2. X-ray crystallography revealed that the displacement
of the C2 and C6 axial hydrogen atoms of various piperidines
by a dimethylene bridge afforded 8-azabicyclo[3.2.1]octanes
[13,14]. Hence the aim of the present investigation was to pre-
pare a small cluster of prototypic molecules related to 1 which
bear C2 and C6 substituents, namely series 2, and to compare
their cytotoxic properties with the analogs having both axial
protons intact on the C2 and C6 atoms viz series 3. In particular,
the information gained from this study may contribute to an
understanding of those structural features which lead to marked
cytotoxic properties.
this compound is displayed in Fig. 4. Molecular models of
2aee and 3aee were built and the torsion angles q1 and q2
are recorded in Table 1.
3. Bioevaluations
All of the compounds in series 2 and 3 were evaluated against
human Molt 4/C8 and CEM T-lymphocytes and murine L1210
lymphoid leukemia cells. These results are portrayed in Table 1.
In addition, these compounds were assayed for inhibitory ef-
fects towards human HSC-2, HSC-3 and HSC-4 oral squamous
cell carcinomas and human HL-60 promyelocytic leukemia
cells. Three normal human cell lines were also used, namely
HGF gingival fibroblasts, HPC pulp cells and HPLF periodontal
ligament fibroblasts. The data from these determinations are pre-
sented in Table 2. The effects of 2a and 2d on respiration and
swelling of rat liver mitochondria are presented in Figs. 5 and 6,
respectively. Doses of 30, 100 and 300 mg/kg of 2aee and
3aee were injected intraperitoneally into mice and the animals
were observed after 0.5 and 4 h for any mortalities. In addition,
using these doses and time intervals, the mice were examined
for neurotoxicity by the rotorod test [16]. Two representative
compounds, namely 2e and 3c, were administered orally to
rats using a dose of 50 mg/kg and the animals were examined
for mortalities and neurotoxicity after 0.25, 0.5, 1 and 2 h and
in the case of 2e after 4 h also.
4. Results and discussion
1H NMR spectroscopy revealed that the compounds in series
2 and 3 are isomerically pure. X-ray crystallography of 8-
methyl-2,4-bis(3-thienylmethylene)-8-azabicyclo[3.2.1]octan-
3-one [17] as well as 2e revealed that the olefinic double bonds
adopted the E configuration. In addition, the same stereochem-
istry was noted with various 3,5-bis(benzylidene)-1-methyl-4-
piperidones [18,19]. Hence the assumption was made that all
of the compounds in series 2 and 3 are the E,E isomers.
All of the compounds in series 2 and 3 were evaluated
against human Molt 4/C8 and CEM T-lymphocytes in order
to ascertain the toxicity of these compounds towards human
neoplastic cells. In addition, 2aee and 3aee were examined
towards murine L1210 cells since a number of anticancer
drugs are cytotoxic to this cell line [20] and hence it serves
2. Chemistry
The compounds in series 2 and 3 were prepared by the syn-
thetic chemical route presented in Scheme 1. X-ray crystallog-
raphy was undertaken on 2e and an ORTEP diagram [15] of

56
H.N. Pati et al. / European Journal of Medicinal Chemistry 44 (2009) 54e62
O
N
O
CHO
3
i
2
1
4
5
+
+
2
2
8
N
R1
R1
6
7
R1
2a-e
O
O
N
CHO
4
ii
5
3
2
R1
N
1
R1
6
R1
3a-e
Scheme 1. Synthetic routes employed in the synthesis of series 2 and 3 in which i ¼ NaOH and ii ¼ HCl/CH3COOH. The R1 substituents are as follows, namely a:
R1 ¼ H; b: R1 ¼ Cl; c: R1 ¼ NO2; d: R1 ¼ CH3; e: R1 ¼ OCH3.
as an indicator of compounds having potential clinical utility.
These data are presented in Table 1.
parameter is in effect [21]; i.e., the 4-substituent in 2bee and
3bee may cause an unfavourable steric impedance to the align-
ment of the molecules at critical binding sites. A second factor
which may influence cytotoxic potencies in series 3 is the elec-
tronic contributions of the nuclear substituents. Thus in 3aec,
the s values of the R1 group are 0.00e0.78 [22] while in the sub-
stantially less potent molecules 3d,e, the s constants are ꢀ0.17
to ꢀ0.27 [22]. Thus in the latter two compounds the methyl and
methoxy aryl groups lower the fractional positive charge on the
olefinic protons relative to 3aec thereby reducing electrophilic-
ity towards cellular thiols.
The biodata presented in Table 1 reveal very clearly that the
substitution of a dimethylene bridge for the axial protons at-
tached to the C3 and C5 atoms of series 3 which generated
2aee leads to a reduction in cytotoxic potency. This conclu-
sion may be drawn by noting that when the same substituents
are present in the aryl rings, the compounds in series 3 are
more potent than the analogs 2aee with the exception that
2e is more cytotoxic than 3e in the L1210 screen.
The results indicate that among the tropinone derivatives,
only 2a displayed noteworthy cytotoxicity having an IC50 value
of approximately 10 mM and on average possessing approxi-
mately one-quarter of the potency of melphalan. The 4-methoxy
analog 2e exhibited moderate potency towards L1210 cells but
not to the T-lymphocytes while the remaining compounds in se-
ries 2 have IC50 values considerably in excess of 100 mM. On
the other hand, in the 4-piperidone series, both 3a and 3c are po-
tent cytotoxins especially towards the T-lymphocytes. Com-
pound 3a is 1.6 times more potent than melphalan towards
Molt 4/C8 cells and is equipotent with this drug in the CEM as-
say. The 4-nitro analog 3c is equipotent with melphalan in both
the Molt 4/C8 and CEM tests. Clearly both 3a and 3c are useful
lead molecules. Compound 3b demonstrated modest potencies
while the IC50 values of 3c and 3d are in excess of 100 mM. The
unsubstituted compound in both series 2 and 3 possesses the
lowest IC50 values which may indicate that an E4 operating
Table 1
Some cytotoxic and physicochemical properties of 2aee and 3aee
Compound
IC50 (mM)a
Torsion angles
c
c
q2
Molt 4/C8
CEM
L1210
Averageb
q1
log Pd
TPSAd
2a
2b
8.51 ꢁ 0.60
>500
>500
8.99 ꢁ 0.54
>500
11.8 ꢁ 2.0
>500
9.77
>500
>361
>500
e
47.4
121.7
120.2
47.1
46.2
45.6
45.5
46.7
45.5
43.2
e
ꢀ47.4
ꢀ45.3
ꢀ120.5
ꢀ47.1
ꢀ46.3
ꢀ46.0
ꢀ45.9
ꢀ47.3
ꢀ46.0
ꢀ43.7
e
4.33
5.68
4.25
5.22
4.44
3.95
5.31
3.87
4.85
4.06
e
20.3
20.3
112.0
20.3
38.8
20.3
20.3
112.0
20.3
38.8
e
2c
2d
247 ꢁ 112
336 ꢁ 3
>500
>500
>500
ee
>500
2e
3a
40.0 ꢁ 18.4
8.77 ꢁ 0.28
96.8 ꢁ 3.5
14.0 ꢁ 1.8
305 ꢁ 10
281 ꢁ 15
2.13 ꢁ 0.02
1.98 ꢁ 0.27
36.9 ꢁ 8.0
2.42 ꢁ 0.38
277 ꢁ 6
3.32 ꢁ 2.30
33.9 ꢁ 12.9
5.21 ꢁ 3.06
233 ꢁ 27
172 ꢁ 6
4.69
55.9
7.21
171
3b
3c
3d
3e
230 ꢁ 1
228
Melphalanf
3.24 ꢁ 0.56
2.47 ꢁ 0.21
2.61
a
The IC50 values represent the concentrations of compounds required to inhibit the growth of the cells by 50%.
These figures indicate the average of the IC50 figures towards the three cell lines.
b
c
d
e
f
The q values refer to the torsion angles between the aryl rings and the adjacent olefinic linkage.
The letters log P and TPSA indicate the calculated log P and total polar surface area values, respectively, of the molecules.
The percentage inhibition of CEM cells by 2e was inconsistent viz 61 ꢁ 7, 46 ꢁ 3, 64 ꢁ 4 and 12 ꢁ 4 at concentrations of 500, 100, 20 and 4 mM, respectively.
The data for melphalan was taken from Pharmazie 52 (1997) 182e186 with the permission of the copyright owner.

H.N. Pati et al. / European Journal of Medicinal Chemistry 44 (2009) 54e62
57
Table 2
Examination of 2aee, 3aee and melphalan against some human malignant and normal cells
Compound
Human tumour cells CC50 (mM)a
Human normal cells CC50 (mM)a
HSC-2
HSC-3
HSC-4
HL-60
8.3
Averageb
HGF
HPC
HPLF
SIc
2a
2b
21
>400
308
>400
300
4.2
44
>400
>400
>400
380
23
>400
>400
>400
250
24
>400
>377
>400
324
298
>400
>400
>400
162
>400
>400
>400
>400
>400
64
>400
>400
>400
>400
366
>15
>400
>400
>400
364
w1.0
w1.1
w1.0
w1.0
9.2
2c
2d
2e
3a
7.9
7.4
2.0
5.4
45
323
40
>400
326
3b
3c
7.4
16
47
39
22
14
20
19
26
369
>19
>11
16
170
>400
>400
>400
>200
3d
3e
276
344
35
>400
>400
115
338
>400
81
>400
>400
6
>354
>386
59
>400
>400
>200
>400
>400
>200
w1.1
w1.0
>3.4
Melphaland
a
The CC50 values are the concentrations of the compounds required to kill 50% of the cells. Determinations were carried out in duplicate and the variation
between experiments was less than 5%.
b
The average values reflect the mean of the CC50 figures for the compounds generated using HSC-2, HSC-3, HSC-4 and HL-60 cells.
The letters SI refer to the selectivity index (SI) values which are the quotients of the average CC50 figures of the compounds towards normal cells divided by the
c
average CC50 data for the malignant cell lines.
d
Solubility considerations precluded the use of concentrations higher than 200 mM. The data for melphalan are taken from Bioorganic and Medicinal Chemistry
2007; 15:3373e3380 and is reproduced with the permission of Elsevier.
Attempts were made to determine the reasons for the low-
ering of potencies when substituents were placed on the C2
and C6 atoms in series 3. The information generated may as-
sist in gleaning further knowledge of those structural features
which influence cytotoxicity. First, molecular models of 2aee
and 3aee were made and the torsion angles q1 and q2 are listed
in Table 1. Apart from the anomolous behaviour of 2b and 2c,
these angles are all in the range of 43e47ꢂ. Thus, in general,
substitution of the C2 and C6 axial protons in both series of
compounds does not affect the magnitude of the deviation
from coplanarity between rings A and B and the adjacent ole-
finic linkages. The changes in bioactivity between series 2 and
3 are, therefore, likely due to factors in the loci of the carbon
atoms attached to the nitrogen atom. The biodata in Table 1
reveal that in all three assays, 3a possessed greater cytotoxic-
ity potencies than 2a. Models of both compounds are pre-
sented in Fig. 2 and the greater steric bulk in the vicinity of
the basic centre and the adjacent carbon atoms in 2a are appar-
ent. Furthermore in order to provide some quantitative infor-
mation pertaining to the steric bulk in the vicinity of the C1/
C5 and C2/C6 atoms in 2a and 3a, respectively, the inter-
atomic distances d1 and d2 as indicated in Fig. 3 were mea-
X-ray crystallography of a representative compound in se-
ries 2, namely 2e, was undertaken to confirm the E stereo-
chemistry of the olefinic double bonds and to compare the
q1 and q2 values with the published X-ray crystallographic
data for 3e. In the case of 2e there are two molecules, desig-
nated 2e1 and 2e2, in the asymmetric unit of the centrosym-
metric space group P21/c. These molecules are very similar
in shape and an ORTEP-3 diagram [15] of 2e1 is presented
in Fig. 4. Both 2e1 and 2e2 are the E,E isomers. The C4-
C9-C10-C11 (q1) and C2-C16-C17-C18 (q2) values for 2e1
are 22.1ꢂ and ꢀ14.0ꢂ, respectively, while the comparable q1
and q2 figures for 2e2 are 13.9ꢂ and ꢀ15.4ꢂ, respectively.
The q1 and q2 values for 3e are 18.4ꢂ and ꢀ26.8ꢂ, respectively
[18]. In general, therefore, the X-ray crystallographic data sup-
port the concept that replacement of the axial protons in series
3 by a dimethylene bridge does not change the torsion angles
q1 and q2 to an appreciable extent.
Since the hydrophobicity of molecules may influence the
extent of bioactivity significantly [23], the log P values of
2aee and 3aee were computed. These data are presented in
Table 1. The lower log P values of the compounds in series
3 than 2 when comparing pairs of compounds having identical
aryl substituents may have contributed to the generally greater
cytotoxic potencies of the 1-methyl-4-piperidones 3. Com-
pounds in series 2 and 3 which have the same R1 substituents
have identical total polar surface area (TPSA) values as indi-
cated in Table 1; consequently TPSA values do not contribute
to potency differences between the two series of compounds.
In order to seek possible correlations between cytotoxic poten-
cies in series 3 and both the log P and TPSA values, linear,
semilogarithmic and logarithmic plots were made between
these physicochemical parameters and the IC50 values of
3aee in each of the three assays. However, no correlations
were observed ( p > 0.1).
˚
sured. The d1 values of 2a and 3a are 2.21 and 1.11 A,
˚
respectively, while the d2 figures are 2.25 and 2.38 A, respec-
tively. Thus while both the C1/C5 and C2/C6 atoms in 2a and
3a, respectively, could align at the same portion of a binding
site, the dimethylene bridge likely exerts a significant steric re-
pulsion. The areas occupied by the axial substituents in 2a and
˚
3a, i.e., d1 ꢃ d2, are 4.97 and 2.64 sq A, respectively. Thus, in
future, the design of compounds occupying an area in between
these two values may afford further information as to the ef-
fect of substituents on the C2 and C6 atoms in series 3, e.g.,
a methyl or trifluoromethyl group could be placed on one of
the C2 or C6 atoms in series 3.

58
H.N. Pati et al. / European Journal of Medicinal Chemistry 44 (2009) 54e62
Fig. 2. Molecular models of 2a and 3a.
Various acyclic 3-aminoketones inhibit or stimulate respira-
A difference in electrophilicity may explain the difference in
the ability of 2a and 2d to cause mitochondrial swelling. The
opening of the mitochondrial permeability transition pore in-
volves alkylation or cross-linking of a critical thiol on a protein
of the permeability transition pore complex [32,33], and as
noted earlier these conjugated unsaturated ketones are known
to react with thiols [1]. In the less potent 2d, the R1 methoxy
substituents are less electronegative (s value ¼ ꢀ0.17) than
the R1 protons (s value ¼ 0.00) of 2a and would thereby de-
crease the electrophilicity of 2d towards thiols compared to 2a.
All of the compounds in series 2 and 3 were evaluated fur-
ther using human HSC-2, HSC-3, HSC-4 and HL-60 neo-
plasms. These data are presented in Table 2. The results are
similar to the biodata generated using Molt 4/C8, CEM and
L1210 cells, namely in series 2 only 2a displays noteworthy
cytotoxicity while 3aec are substantially more potent than
3d,e. Taking into consideration the average CC50 values to-
wards these four cell lines, 2a and 3aec possess 2.5, 10.9,
3.1 and 2.3 times the potency of melphalan and are clearly
lead molecules. The CC50 values of 2a and 3aec towards hu-
man HGF, HPC and HPLF normal cells reveal their excellent
selectivity (SI) figures demonstrating the preferential toxicity
of these compounds for neoplastic cells, which further con-
firms their importance as templates for further development.
tion in mitochondria isolated from rat and mouse liver cells
[24,25]. Since the compounds prepared in this study are cyclic
3-aminoketones, the question arises whether different effects
on mitochondria may explain the variation in cytotoxic poten-
cies. Accordingly two compounds displaying markedly diver-
gent cytotoxic properties, namely 2a and 2d, were examined.
Fig. 5 shows that after a delay both compounds exert a strong
stimulating effect on mitochondrial respiration, with com-
pound 2a giving
a significantly shorter latent period
(1.96 ꢁ 0.05 min versus 4.45 ꢁ 0.13 min). Measurements
shown in Fig. 6 reveal that 2a produces rapid mitochondrial
swelling, while 2d does so only more slowly. Mitochondrial
swelling involves opening of the mitochondrial permeability
transition pore and collapse of the mitochondrial membrane
potential [26]. This collapse of the membrane potential de-
creases the resistance to electron flow in the respiratory chain
and increases mitochondrial respiration [27], which accounts
for the increase in respiration observed in Fig. 5. The mito-
chondrial permeability transition is a critical trigger for apo-
ptosis [28] and has been identified as a target for cancer
therapy [29e31]. The greater ability of 2a to induce mitochon-
drial swelling, therefore, may have contributed to its higher
cytotoxic potency in the cancer cell lines.
AXIS 2
H
H
H
H
H
H
d1
C6
C7
d1
AXIS 1
C2
C6
C5
C1
AXIS 1
d2
d2
H
H
H
H
A
B
Fig. 3. A comparison of the steric bulk of portions of the structures of 2a (A) and 3a (B).

H.N. Pati et al. / European Journal of Medicinal Chemistry 44 (2009) 54e62
59
Fig. 4. An ORTEP-3 diagram of 2e1 determined by X-ray crystallography.
A number of acylic 3-aminoketones or Mannich bases are le-
thal to mice at low doses, e.g., 30 mg/kg, and also display neu-
rotoxicity [34]. Since 4-piperidones may be considered cyclic
3-aminoketones, the evaluation of the compounds in series 2
and 3 with regard to mortality and neurological deficit was un-
dertaken. Doses up to and including 300 mg/kg of 2aee and
3aee were administered intraperitoneally to mice and after
4 h, no deaths of the animals were noted. Minimal neurotoxicity
was observed with 2e, 3a,c,e after 0.5 h and with 3a after 4 h. A
dose of 300 mg/kg of 2e caused neurotoxicity in all of the ani-
mals. No neurological disturbances were caused by the other
compounds. A dose of 50 mg/kg of two representative com-
pounds 2e and 3c was administered orally to rats and the ani-
mals were examined at different time intervals up to 4 (2e)
and 2 (3c) h. No mortalities or neurological deficit was noted.
The conclusion drawn from this short-term toxicity evaluation
is that the compounds in series 2 and 3 are well tolerated in ro-
dents thereby enhancing their potential for future development.
to 2aee is accompanied by a reduction in cytotoxic potencies.
Thus development of the cytotoxic 3,5-bis(benzylidene)-4-pi-
peridones in which two protons are present on the carbon atoms
attached to the basic centre appears to be a prudent decision.
However, limited molecular modifications whereby groups of
varying sizes are placed on the 2 and 6 carbon atoms of series
3 may establish the generality or otherwise that such structural
changes are disadvantageous in terms of cytotoxic potencies.
The reasons for the disparity in cytotoxic potencies between
series 2 and 3 may have been due to the dimethylene bridge in
2aee exerting a steric impedance to alignment at one or more
binding sites as well as variation in hydrophobic properties and
possibly differential effects on mitochondrial respiration.
In terms of cytotoxic potencies, the data in Table 1 reveal
that 2a, 3a and 3c are lead molecules while moderate potency
was displayed by 3b. The assessment of these four compounds
against human tumour cell lines as revealed in Table 2 con-
firmed their cytotoxic properties which in these assays are
on average more potent than a reference drug melphalan.
The importance of these four compounds as templates for fu-
ture development was enhanced by two additional observa-
tions. First, 2a and 3aec display preferential cytotoxicity for
malignant rather than normal cells as revealed by the SI
5. Conclusions
This study has revealed clearly that in general replacement of
the 2a and 6a protons in series 3 by a dimethylene bridge leading
300
Compound
280
260
240
220
200
180
160
140
120
100
1.2
Compound
1.1
1.0
0.9
0.8
0.7
0.6
0
1
2
3 4
Time (min)
5
6
7
0
1
2
Time (min)
3
4
Fig. 5. Effects of 2a and 2d on respiration of rat liver mitochondria. Where
indicated by the arrow, compounds were added to a respiring suspension of
rat liver mitochondria to a concentration of 250 mM: d 2a, e e e 2d.
Fig. 6. Effects of 2a and 2d on swelling of rat liver mitochondria. Where in-
dicated by the arrow, compounds were added to a respiring suspension of rat
liver mitochondria to a concentration of 250 mM: d 2a, e e e 2d.

60
H.N. Pati et al. / European Journal of Medicinal Chemistry 44 (2009) 54e62
figures in Table 2. Second, these compounds and their analogs
in series 2 and 3 are well tolerated in mice.
(s, 3H), 2.62 (p, 2H), 3.87 (s, 6H), 4.41 (dd, 2H), 6.98 (d,
4H, J ¼ 8.64 Hz), 7.40 (d, 4H, J ¼ 8.68 Hz), 7.82 (s, 2H).
Anal. calcd. for C24H25NO3: C, 76.77; H, 6.71; N, 3.73.
Found: C, 76.89; H, 6.54; N, 3.77%.
6. Experimental protocols
6.1. Chemistry
6.1.2. Synthesis of 3,5-bis(benzylidene)-1-
methyl-4-piperidones (3aee)
Melting points which are uncorrected were determined us-
ing a Gallenkamp instrument. 1H NMR spectra were recorded
using a Bruker AMX 500 FT machine while elemental analy-
ses (C, H, N) were obtained using an Elementer analyzer and
were within 0.4% of the calculated values. 4-Piperidones
3a,c,d were crystallized with 0.25, 0.75 and 0.25 mol of water
of crystallization, respectively. X-ray crystallography was
undertaken using a Nonius instrument.
Dry hydrogen chloride was passed into a solution of 1-
methyl-4-piperidone (0.05 mol) and the appropriate aryl alde-
hyde (0.10 mol) in acetic acid (25 ml) at room temperature.
The mixture was stirred at room temperature for 6e8 h and
the precipitate was collected, washed with acetone (20 ml)
and added to a solution of aqueous potassium carbonate solu-
tion (5% w/v). The free base was collected, dried under vac-
uum at 45e50 ꢂC and crystallized from ethanol (3a),
chloroformemethanol (3b,d,e) or chloroformeethanol (3c).
6.1.1. Synthesis of 2,4-bis(benzylidene)-8-methyl-
8-aza-bicyclo[3.2.1]octan-3-ones (2aee)
6.1.2.1. 3,5-Bis(benzylidene)-1-methyl-4-piperidone (3a).
Yield: 71%, m.p. 110 ꢂC (lit. [36] m.p. 115e117 ꢂC). 1H
NMR (CDCl3) d: 2.49 (s, 3H), 3.80 (s, 4H), 7.42 (m, 10H),
7.85 (s, 2H). Anal. calcd. for C20H19NO.0.25H2O: C, 81.66;
H, 6.46; N, 4.76%. Found: C, 81.90; H, 6.36; N, 4.69%.
Sodium hydroxide solution (5N, 1 ml) was added dropwise
to a solution of 8-methyl-8-aza-bicyclo[3.2.1]octan-3-one
(0.5g, 0.0036 mol) and the appropriate aryl aldehyde
(0.0072 mol) in ethanol (20 ml) at room temperature. The re-
action mixture was stirred under nitrogen for 2 h at room tem-
perature and then water (15 ml) was added. The precipitate
was collected and recrystallized from ethanol.
6.1.2.2. 3,5-Bis(4-chlorobenzylidene)-1-methyl-4-piperidone
(3b). Yield: 88%, m.p. 183 ꢂC (lit. [36] m.p. 174e176 ꢂC).
1H NMR (CDCl3) d: 2.49 (s, 3H), 3.75 (s, 4H), 7.35 (d, 4H,
J ¼ 8.44 Hz), 7.43 (d, 4H, J ¼ 8.46 Hz), 7.77 (s, 2H). Anal.
calcd. for C20H17Cl2NO: C, 67.05; H, 4.78; N, 3.91. Found:
C, 66.89; H, 4.67; N, 3.81%.
6.1.1.1. 2,4-Bis(benzylidene)-8-methyl-8-aza-bicyclo[3.2.1]oc-
tan-3-one (2a). Yield: 70%; m.p. 148 ꢂC, (lit. [35] m.p.
150e151 ꢂC). 1H NMR (CDCl3) d: 2.06 (q, 2H), 2.33
(s, 3H), 2.61 (p, 2H), 4.43 (dd, 2H), 7.40 (m, 10H), 7.86
(s, 2H). Anal. calcd. for C22H21NO: C, 83.78; H, 6.74; N,
4.44%. Found: C, 83.57; H, 6.54; N, 4.40%.
6.1.2.3.
3.5-Bis(4-nitrobenzylidene)-1-methyl-4-piperidone
(3c). Yield: 81%, m.p. 223 ꢂC (lit. [36] m.p. 229e231 ꢂC).
1H NMR (CDCl3) d: 2.89 (s, 3H), 3.56 (br s, 4H), 7.52 (d,
4H, J ¼ 8.62 Hz), 7.76 (s, 2H), 8.23 (d, 4H, J ¼ 8.61 Hz).
Anal. calcd. for C20H17N3O5 0.75 H2O: C, 61.08; H, 4.32;
N, 10.69. Found: C, 61.05; H, 4.42; N, 11.07%.
6.1.1.2. 2,4-Bis(4-chlorobenzylidene)-8-methyl-8-aza-bicyclo
[3.2.1]octan-3-one (2b). Yield: 85%, m.p. 183 ꢂC. H NMR
(CDCl3) d: 2.02 (q, 2H), 2.32 (s, 3H), 2.62 (p, 2H), 4.35 (p,
2H), 7.34 (d, 4H, J ¼ 8.38 Hz), 7.43 (d, 4H, J ¼ 8.61 Hz),
7.78 (s, 2H). Anal. calcd. for C22H19Cl2NO: C, 68.76; H,
4.98; N, 3.64. Found: C, 68.71; H, 4.98; N, 3.77%.
1
6.1.2.4. 3,5-Bis(4-methylbenzylidene)-1-methyl-4-piperidone
(3d). Yield: 76%, m.p. 201 ꢂC (lit. [36] m.p. 192e195 ꢂC).
1H NMR (CDCl3) d: 2.42 (s, 6H), 2.49 (s, 3H), 3.79 (s, 4H),
7.26 (d, 4H, J ¼ 7.95 Hz), 7.33 (d, 4H, J ¼ 8.0 Hz), 7.82 (s,
2H). Anal. calcd. for C22H23NO 0.25 H2O: C, 82.00; H,
7.14; N, 4.35. Found: C, 81.92; H, 7.19; N, 4.25%.
6.1.1.3.
8-Methyl-2,4-bis(4-nitrobenzylidene)-8-aza-bicyclo
1
[3.2.1]octan-3-one (2c). Yield: 73%, m.p. 247 ꢂC. H NMR
(CDCl3) d: 2.06 (q, 2H), 2.34 (s, 3H), 2.68 (p, 2H), 4.34
(dd, 2H), 7.55(d, 4H, J ¼ 8.60 Hz), 7.83 (s,2H), 8.33 (d, 4H,
J ¼ 8.67 Hz). Anal. calcd. for C22H19N3O5: C, 65.18; H,
4.72; N, 10.37. Found: C, 64.95; H, 4.67; N, 10.60%.
6.1.2.5. 3,5-Bis(4-methoxybenzylidene)-1-methyl-4-piperidone
(3e). Yield: 83%, m.p. 186 ꢂC (lit. [36] m.p. 199e201 ꢂC).
1H NMR (CDCl3) d: 2.51 (s, 3H), 3.79 (s, 4H), 3.88 (s, 6H),
6.98 (d, 4H, J ¼ 8.71 Hz), 7.40 (d, 4H, J ¼ 8.67 Hz), 7.80 (s,
2H). Anal. calcd. for C22H23NO3: C, 75.62; H, 6.63; N,
4.01. Found: C, 75.72; H, 6.71; N, 4.22%.
6.1.1.4. 8-Methyl-2,4-bis(4-methylbenzylidene)-8-aza-bicyclo
[3.2.1]octan-3-one(2d). Yield: 80%, m.p. 172 ꢂC. H NMR
(CDCl3) d: 2.05 (q, 2H), 2.33 (s, 3H), 2.42 (s, 6H), 2.63 (p,
2H), 4.43 (dd, 2H), 7.26 (d, 4H, J ¼ 7.92 Hz), 7.33 (d, 4H,
J ¼ 7.99 Hz), 7.84 (s, 2H). Anal. calcd. for C24H25NO: C,
83.93; H, 7.34; N, 4.08. Found: C, 84.13; H, 7.02; N 4.20%.
1
6.1.3. X-ray crystallography of 2,
4-bis(4-methoxybenzylidene)-8-methyl-8-
aza-bicyclo[3.2.1]octan-3-one (2e)
6.1.1.5. 2,4-Bis(4-methoxybenzylidene)-8-methyl-8-aza-bicyclo
With the exception of the structure factors, data pertaining
to the X-ray crystallographic determination of 2e have been
deposited with the Cambridge Crystallographic Data Centre
[3.2.1]octan-3-one (2e). Yield: 82%, m.p. 160 ꢂC (lit. [35]
1
m.p. 162e163 ꢂC). H NMR (CDCl3) d: 2.03 (q, 2H), 2.34

H.N. Pati et al. / European Journal of Medicinal Chemistry 44 (2009) 54e62
61
as supplementary publication no. CCDC 656960. This infor-
mation may be obtained without cost by applying to CCDC,
12 Union Road, Cambridge CB2 1EZ, UK, (fax: þ44 (0)
1223 336033 or e-mail deposit@ccdc.cam.ac.uk).
6.2.3. Toxicity and neurotoxicity evaluations in rodents
The toxicity and neurotoxicity evaluations were undertaken
by the National Institute of Neurological Disorders and Stroke,
USA according to their protocols [44]. Doses of 30, 100 and
300 mg/kg of 2aee and 3aee were injected intraperitoneally
into mice and observed for both mortalities and neurotoxicity
at the end of 0.5 and 4 h. No deaths were observed. Neurotox-
icity was observed in the following cases (dose in mg/kg, time
of observation in hours, number of animals displaying neuro-
logical deficit/total number of animals in the test) viz 2d (300,
4, 2/2), 2e (100, 0.5, 1/8), 3a (100, 0.5, 2/8; 100, 4, 1/4), 3c
(100, 0.5, 1/8) and 3e (300, 0.5, 1/4). In addition, doses of
50 mg/kg of 2e and 3c were administered orally to rats. At
the end of 0.25, 0.5, 1 and 2 h (also 4 h in the case of 2e),
no deaths or neurotoxicity was observed.
6.1.4. Molecular modeling
The molecular models of 2aee and 3aee were built using
the BioMedCache program [37]. MOPAC optimized geometry
calculations using AM1 parameters were employed in order to
produce the lowest energy conformations.
6.1.5. Determination of the calculated log P and total polar
surface area values of 2aee and 3aee
The log P and TPSA data were generated using the JME
molecular editor [38].
Acknowledgments
6.1.6. Statistical calculations
The linear, semilogarithmic and logarithmic plots between
the IC50 values of 3aee in different bioassays and the c log P
and TPSA data were made using a software package [39].
The authors thank the following agencies and individuals
who enabled this study to be undertaken. The Canadian Insti-
tutes of Health Research provided operating grants to J.R.
Dimmock and B. Bandy. The Molt 4/C8, CEM and L1210 as-
says were undertaken by Mrs. Lizette van Berckelaer and
funded by the Flemish Fonds voor Wetenschappelijk Onder-
zoek (FWO). A Grant-in-Aid was provided by the Ministry
of Education, Science, Sports and Culture of Japan to H. Sa-
kagami (No. 19592156). The Canadian Foundation for Innova-
tion and the Government of Saskatchewan provided funding
for the X-ray crystallography laboratory. The National Insti-
tute of Neurological Disorders and Stroke undertook the ro-
dent toxicity studies while Ms. B. McCullough typed various
drafts of the manuscript.
6.2. Bioassays
6.2.1. Cytotoxicity evaluations
The assays using human Molt 4/C8 and CEM T-lympho-
cytes and murine L1210 cells have been described previously
[40]. In brief, the cells are incubated with different concentra-
tions of compounds in RPMI 1640 medium at 37 ꢂC for 72 h
(Molt 4/C8 and CEM cells) and 48 h (L1210 cells).
A literature procedure was utilized for the bioevaluation of
2aee and 3aee towards HSC-2, HSC-3, HSC-4, HL-60, HGF,
HPC and HPLF cells [41]. In brief, with the exception of as-
says using HL-60 cells, different concentrations of compounds
were incubated with the cell lines in DMEM medium supple-
mented with 10% heat-inactivated fetal bovine serum. Cell vi-
ability was determined by the MTT method after 24 h
incubation at 37 ꢂC. A similar procedure was followed for
the HL-60 cells except RPMI 1640 media containing 10%
fetal bovine serum was used and cytotoxicity was assessed
by the trypan blue exclusion procedure.
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