Cytotoxic N-[4-(3-aryl-3-oxo-1-propenyl)phenylcarbonyl]-3,5-bis(phenylmethylene)-4- piperidones and related compounds

Article abstract of DOI:10.1016/S0223-5234(02)01414-9

A series of 4-carboxychalcones 1 were prepared and coupled to 3,5-bis(phenylmethylene)-4-piperidone (2) giving rise to a novel series of N-[4-(3-aryl-3-oxo-1-propenyl)phenylcarbonyl]-3,5-bis(phenylmethylene)-4- piperidones (3). Molecular simplification of the amides 3 led to the formation of the corresponding N-(3-aryl-1-oxo-2-propenyl)-3,5-bis(phenylmethylene)-4-piperidones (4). A cytotoxic evaluation of the compounds in series 1-4 utilized murine P388 and L1210 cells as well as human Molt 4/C8 and CEM T-lymphocytes. In general, the compounds displayed significant toxicity; the IC₅₀ values of 54% of the enones were less than 10 μM when all four screens were considered and less than 1 μM for all members of series 3 in the P388 assay. Various correlations were established between the potencies of the compounds in series 1, 3 and 4 and the Hammett σ, Hansch π and molecular refractivity constants of the aryl substituents. Several torsion angles and interatomic distances of five representative compounds in series 3 and 4 were determined by X-ray crystallography, some of which contributed to the observed bioactivity. The marked cytotoxicity and lack of murine toxicity of most of the compounds described in this study, as well as their selective toxicity towards different tumour cell lines, revealed that development of the enones 2-4 as novel candidate antineoplastic agents should be pursued.

Full text of DOI:10.1016/S0223-5234(02)01414-9

European Journal of Medicinal Chemistry 37 (2002) 961ꢁ972
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Original article
Cytotoxic N-[4-(3-aryl-3-oxo-1-propenyl)phenylcarbonyl]-3,5-
bis(phenylmethylene)-4-piperidones and related compounds
Jonathan R. Dimmock a,ꢀ, Amitabh Jha a, Gordon A. Zello a, J. Wilson Quail b, Eliud
O. Oloo b, Kurt H. Nienaber c, Earl S. Kowalczyk c, Theresa M. Allen d, Cheryl
L. Santos d, Erik De Clercq e, Jan Balzarini e, Elias K. Manavathu f, James P. Stables g
a College of Pharmacy and Nutrition, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan, Canada S7N 5C9
b Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5C9
c Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5
d Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
e Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
f Department of Medicine, Wayne State University, Detroit, MI 48201-1908, USA
g National Institute of Neurological Disorders and Stroke, Bethesda, MD 20892-9020, USA
Received 23 May 2002; received in revised form 28 August 2002; accepted 9 September 2002
Abstract
A series of 4-carboxychalcones 1 were prepared and coupled to 3,5-bis(phenylmethylene)-4-piperidone (2) giving rise to a novel
series of N-[4-(3-aryl-3-oxo-1-propenyl)phenylcarbonyl]-3,5-bis(phenylmethylene)-4-piperidones (3). Molecular simplification of the
amides 3 led to the formation of the corresponding N-(3-aryl-1-oxo-2-propenyl)-3,5-bis(phenylmethylene)-4-piperidones (4). A
cytotoxic evaluation of the compounds in series 1ꢁ4 utilized murine P388 and L1210 cells as well as human Molt 4/C8 and CEM T-
/
lymphocytes. In general, the compounds displayed significant toxicity; the IC50 values of 54% of the enones were less than 10 mM
when all four screens were considered and less than 1 mM for all members of series 3 in the P388 assay. Various correlations were
established between the potencies of the compounds in series 1, 3 and 4 and the Hammett s, Hansch p and molecular refractivity
constants of the aryl substituents. Several torsion angles and interatomic distances of five representative compounds in series 3 and 4
were determined by X-ray crystallography, some of which contributed to the observed bioactivity. The marked cytotoxicity and lack
of murine toxicity of most of the compounds described in this study, as well as their selective toxicity towards different tumour cell
lines, revealed that development of the enones 2ꢁ
# 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
/
4 as novel candidate antineoplastic agents should be pursued.
´
Keywords: 4-Piperidones; Cytotoxicity; Murine toxicity; Structureꢁactivity relationships
/
1. Introduction
two functions are found in nucleic acids, conjugated
enones may lack the genotoxic properties associated
with currently used anticancer alkylating agents [11].
The objectives of the present study included the
syntheses of some novel compounds designed as thiol
alkylators and to evaluate their cytotoxicity. In addi-
tion, should significant potencies be demonstrated,
investigations would be undertaken in order to seek
correlations between the structures of the compounds
and the magnitude of their bioactivities. Such informa-
tion would be of value in a subsequent project aimed at
preparing analogues with increased potency and selec-
tivity for neoplastic rather than the corresponding
normal cells.
The principal aim of this laboratory is the preparation
of conjugated styryl ketones and related compounds as
candidate cytotoxic and anticancer agents [1,2]. These
compounds are a,b-unsaturated ketones which possess a
marked affinity towards thiols both in vitro [3,4] and in
vivo [5,6]. In addition, these enones display a capacity
for preferential electrophilic attack towards thiols rather
than amino or hydroxy groups [7ꢁ10]. Since these latter
/
ꢀ Correspondence and reprints
E-mail address: dimmock@skyway.usask.ca (J.R. Dimmock).
´
0223-5234/02/$ - see front matter # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 2 2 3 - 5 2 3 4 ( 0 2 ) 0 1 4 1 4 - 9

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J.R. Dimmock et al. / European Journal of Medicinal Chemistry 37 (2002) 961ꢁ972
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The specific aims of the present investigation were as
since no lethalities were noted after multiple daily doses
of 240 mg kgꢂ1 were administered [6]. Compound 2 has
greater potency towards two human T-lymphocytes
than the established anticancer alkylating agent mel-
phalan and has ca. 27% of the activity of melphalan
towards two murine cell lines (Table 1). N-Acylation of
2 with acryloyl chloride led to the corresponding amide
which possessed greater toxicity than 2 towards P388
cells and equal activity as 2 in the three other screens
[13]. In general, when analogues of 2 possessing different
aryl substituents were converted into the corresponding
N-acryloyl amides, increases in cytotoxicity were also
noted [13]. Hence, one of the reasons for preparing series
3 was to afford an insight into the structural require-
ments for cytotoxicity of the amidic group attached to
the piperidyl nitrogen atom of 2. Furthermore, if the
compounds in series 1 were cytotoxic, the joining of
these molecules to 2 would lead to molecular hybridiza-
tion, i.e. the linking of bioactive molecules via covalent
bond formation [14]. This approach may lead to
follows. First, the preparation of a series of prototypic
chalcones 1 was planned. Recently the antineoplastic
properties of various 1,3-diaryl-2-propen-1-ones or
chalcones have been reviewed [12] and the cytotoxicity
of different series of chalcones has been described [2]. A
carboxy group was attached to ring A in order that
facile conjugation procedures in vivo would lead to the
formation of water soluble glucuronides of promising
compounds that would expedite excretion of the mole-
cules. In this way amelioration of mammalian toxicity
and reduction or prevention of adsorption in lipoidal
tissues of the body may take place. Furthermore the aryl
carboxy group was chosen in order that molecular
hybridization could occur vide infra.
A second goal of this study was as follows. The
marked cytotoxicity of 3,5-bis(phenylmethylene)-4-pi-
peridone (2) has been described recently [13]; these data
are presented in Table 1. A previous study revealed that
the hydrochloride salt of 2 was well tolerated in mice
Table 1
Potencies of 1ꢁ/4 and melphalan towards murine P388 and L1210 leukemic cells and human Molt 4/C8 and CEM T-lymphocytes
a
b
Compound
IC50 (mM)
SR
P388 cells
L1210 cells
Molt 4/C8 cells
CEM cells
1a
1b
1c
1d
1e
1f
25.691.1
21.892.1
9.3391.6
20.090.9
6.6590.4
14.190.6
5.2190.4
32.692.7
21.390.2
0.7790.02
0.62390.05
0.75590.04
0.31290.02
0.37190.02
0.91990.03
0.47790.01
0.39990.1
0.22190.01
0.25090.01
1.2590.06
2.6390.02
2.0890.08
1.7490.04
1.9290.2
127945
11.593.7
9.0890.95
5.7090.75
11.390.6
7.7891.13
13.596.9
8.2491.05
40.093.5
54.0914.0
1.6790.15
1.9490.26
42.392.0
9.7192.39
32.198.8
29.2918.9
4.5693.20
1.6790.20
13.190.8
8.8990.17
7.8190.39
11.793.7
9.1090.03
15.590.1
8.5590.01
42.591.5
54.8913.1
1.7090.02
1.5890.26
32.393.8
6.3593.13
23.0916.7
30.0921.0
4.6091.81
1.9290.40
11.0
4.93
43.891.6
37.694.9
46.492.3
36.694.0
56.4912.7
17.096.6
185955
6.60
4.11
5.50
4.18
3.26
5.68
5.12
1g
1h
1i
109936
c
2
3a
7.9690.11
5.5694.88
94.1933.7
32.7910.0
44.293.9
57.1918.0
9.7990.30
9.6790.89
10.3
8.93
125
3b
3c
105
119
3d
3e
62.1
3f
3g
20.5
24.2
d
d
d
3h
ꢀ20
ꢀ20
ꢀ20
ꢀ91
3i
4a
5.3492.41
38.491.2
60.6913.5
39.892.8
8.4790.37
9.9493.62
8.9690.68
24.499.5
9.9090.28
112937
1.6090.06
33.1918.0
27.6910.7
66.2916.5
7.5092.08
3.6192.79
1.8990.11
9.0590.29
1.9790.10
39.393.7
1.6190.10
27.2915.3
32.492.2
63.097.1
6.6790.58
3.4592.69
2.0190.18
8.4490.06
2.0190.11
40.890.4
2.4790.30
21.4
30.7
23.0
31.8
4b
4c
4d
4e
4.87
5.18
11.2
37.7
8.54
9.66
14.7
4f
4g
0.80390.03
0.64890.02
1.1690.1
11.691.5
0.22090.01
4h
4i
Melphalan
e
2.1390.03
3.2490.79
a
The IC50 values indicate the concentration of compound required to inhibit cell growth by 50%.
Selectivity Ratio, i.e. or the ratio of the highest to the lowest IC50 values for each compound.
Reprinted in part from J. Med. Chem. 44 (2001) 586. Copyright 2001 American Chemical Society.
Insolubility at concentrations higher than 20 mM was noted.
b
c
d
e
´
Reprinted in part from Eur. J. Med. Chem. 35 (2000) 967. Copyright 2000 Editions scientifiques et me´dicales Elsevier.

J.R. Dimmock et al. / European Journal of Medicinal Chemistry 37 (2002) 961ꢁ
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972
963
increased cytotoxicity if the binding sites of the chal-
cones 1 and compound 2 are contiguous and various
portions of the molecules 3 are appropriately positioned
to elicit bioactivity.
crystallographic study had revealed that the 3,5-phenyl-
methylene groups of the hydrochloride salt of 2 also
possessed the E stereochemistry [10]. Various torsion
angles and interatomic distances of 3d and 4c,e,f,h were
determined by X-ray crystallography and are presented
in Table 3.
A third aspect of this study was the examination of
whether correlations existed between the cytotoxicities
and the electronic, hydrophobic and steric properties of
the aryl substituents in the series of prepared com-
pounds. The establishment of such relationships would
permit the expansion of clusters of compounds on a
rational basis. In addition, murine toxicity was evalu-
ated in tandem with the cytotoxicity evaluations in order
to guide the direction of further studies with this type of
compounds.
In summary, the objectives of the investigation were
to synthesize different series of prototypic molecules and
evaluate their cytotoxicity and murine toxicity with a
view to providing data enabling the design of further
candidate cytotoxic molecules.
3. Biological evaluations
The compounds in series 1, 3 and 4 were examined for
cytotoxic activity using murine P388 and L1210 cells as
well as human Molt 4/C8 and CEM T-lymphocytes.
These data are presented in Table 1. The enones 2, 3a,
3b and 4a were assessed against a panel of 5392 human
/
tumour cell lines and these results are summarized in
Table 4. Compounds 1a, e, 2, 3a and 3e were examined
for antifungal activity using three isolates of Aspergillus
fumigatus and one of Candida albicans. All compounds
were evaluated for murine toxicity using doses of 30, 100
and 300 mg kgꢂ1 as well as for possible penetration of
the central nervous system (CNS) using the maximum
electroshock seizure (MES) and subcutaneous pentyle-
netetrazole (scPTZ) anticonvulsant screens.
2. Chemistry
The preparation of the compounds in series 1 and 3
are outlined in Fig. 1. A Claisen-Schmidt condensation
between 4-carboxybenzaldehyde and various 1-aryl-1-
ethanones led to the formation of 1. Conversion of the
enones 1 into the corresponding acid chlorides, followed
by coupling with 3,5-bis(phenylmethylene)-4-piperidone
(2), led to the desired compounds 3. The compounds in
series 4, which are discussed subsequently, were pre-
pared by the route presented in Fig. 2. A Doebner
modification of a Knoevenagel reaction led to the
formation of the appropriate 3-aryl-2-propenoic acids,
which were converted into the corresponding acid
chlorides and condensed with 2 leading to the isolation
of the N-(3-aryl-1-oxo-2-propenyl)amides (4).
4. Results and discussion
The compounds in series 1ꢁ3 were evaluated for
/
cytotoxicity against murine P388 and L1210 cells, since
these cell lines have been claimed to be predictors of
clinically useful anticancer drugs [16]. The Molt 4/C8
and CEM T-lymphocytes were utilized in order to
observe whether activity towards human cell lines would
be demonstrated.
Initially, comparisons of the cytotoxicities of the
compounds in series 1ꢁ3 towards each cell line were
made. The average IC50 values of the compounds in
series 1 and 3 in the P388 screen were 17.4 and 0.481
mM, respectively, i.e. overall the compounds in series 3
/
1H-NMR spectroscopy revealed that the olefinic
double bonds of 1 and also in the N-acyl groups of 3
and 4 adopted the E configuration. A previous X-ray
Fig. 1. Synthesis of series 1 and 3. The reagents used were as follows, namely: (i) sodium hydroxide; (ii) thionyl chloride, dimethylformamide; (iii)
triethylamine. The aryl substitution pattern was as follows: a: R1ꢃR2ꢃH; b: R1ꢃCl, R2ꢃH; c: R1ꢃR2ꢃCl; d: R1ꢃCH3, R2ꢃH; e: R1ꢃR2ꢃ
CH3; f: R1ꢃOCH3, R2ꢃH, g: R1ꢃR2ꢃOCH3; h: R1ꢃNO2, R2ꢃH; i: R1ꢃOH; R2ꢃ
H.
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/

964
J.R. Dimmock et al. / European Journal of Medicinal Chemistry 37 (2002) 961ꢁ972
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Fig. 2. Synthesis of series 4. The reagents used were as follows: (i) pyridine, piperidine; (ii) thionyl chloride, dimethylformamide; (iii) 2, triethylamine.
The aryl substitution pattern was as follows: a: R1ꢃR2ꢃR3ꢃH; b: R1ꢃR3ꢃH, R2ꢃCl; c: R1ꢃR2ꢃCl, R3ꢃH; d: R1ꢃR3ꢃH, R2ꢃ
CH3;
eꢃ NO2.
R1ꢃR3ꢃH, R2ꢃOCH3; f: R1ꢃR2ꢃOCH3, R3ꢃH; g: R1ꢃR2ꢃR3ꢃOCH3; h: R1ꢃR2ꢃOCH2O, R3ꢃH; i: R1ꢃR3ꢃH, R2ꢃ
/
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/
/
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were 36 times more potent than the analogues in series 1
in this assay. All of the compounds in series 3 were more
potent than 2, except for 3b and 3e. The enone 3h
displayed the same cytotoxicity against P388 cells as
melphalan, while the remaining compounds in series 3
melphalan. On the other hand, 3a,g,i were more potent
than this reference drug in both the Molt 4/C8 and CEM
tests and 3f had activity equal to melphalan towards
Molt 4/C8 cells.
An important feature of promising cytotoxic agents is
the ability to display differential cytotoxicity towards
various cell lines [17]. In other words, compounds are
identified which possess markedly divergent potencies
towards various unrelated cell lines in contrast to those
molecules which exert similar biocidal or biostatic
effects to different cells. One noteworthy feature of
this differential cytotoxicity is that it opens up the
possibility that compounds displaying differential cell
growth inhibition may exert a selective toxicity for
malignant cells in contrast to the corresponding normal
tissue. For example, in the case of melphalan, there is a
14.7-fold difference between the potencies to P388 and
Molt 4/C8 cells. Thus in order to evaluate whether such
had 24ꢁ88% of the potency of this established alkylating
/
drug. In the case of the L1210, Molt 4/C8 and CEM
tests, the IC50 value for 3h was not determined due to its
insolubility when concentrations in excess of 20 mM
were employed. Therefore, when the potencies for the
compounds in series 1 were compared with series 3 in
these three tests, compound 3h as well as its analogue in
series 1, namely 1h, were omitted from consideration. In
contrast to the P388 screen, the differences between the
cytotoxicities of 1aꢁ
4/C8 and CEM tests were much smaller. Thus the
average IC50 values of 1aꢁg, i in the L1210, Molt 4/C8
and CEM screens were 59.2, 15.1 and 16.2 mM,
respectively, while for 3aꢁg, i the comparable values
/
g, i and 3aꢁ/g, i in the L1210, Molt
/
properties were displayed in the compounds in series 1ꢁ
/
/
3, the selectivity ratio (SR) values of each compound
were computed, SR being the ratio of the highest and
were 32.3, 15.4 and 12.7 mM, respectively. All of the
compounds in series 1 had lower potencies than
Table 2
a
Summary of the comparisons of potencies between different compounds in series 1ꢁ
/4
Series compared
Relationship evaluated
Percentage correlations
P388
L1210
Molt 4/C8
CEM
1, 3
2, 3
3ꢀ1
1ꢀ3
Equal potency
100
0
33
22
44
33
56
11
44
22
33
0
3ꢀ2
2ꢀ3
Equal potency
78
11
11
11
78
11
0
56
44
0
67
33
3aꢁ/d,fꢁ/h,4aꢁ/f,i
3aꢁ
4aꢁ
Equal potency
/
d,fꢁ
f,iꢀ3aꢁ
/
hꢀ4aꢁ
/
f,I
100
0
14
29
57
29
43
29
29
14
57
/
/d,fꢁh
/
0
a
In the case of compounds in series 1, 3 and 4, comparisons of potencies were made with analogues possessing identical aryl substituents.

J.R. Dimmock et al. / European Journal of Medicinal Chemistry 37 (2002) 961ꢁ
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972
965
lowest IC50 values observed in the four tests. These data
are presented in Table 1. A SR value of 10 was
arbitrarily chosen as an indication of promising selec-
tivity and the following compounds met this criterion,
synthesizing 4f, the close structural analogues 4g and 4h
should also be prepared.
The results obtained from evaluating the compounds
in series 4 against P388, L1210, Molt 4/C8 and CEM
cells are presented in Table 1. The data show that the
P388 cells were the most susceptible to the compounds; a
similar result was noted with the enones 3. In order to
determine the influence of the N-acyl group present in 3
namely 1a, 2 and 3bꢁ
tion of each molecule in series 1 with those in series 2,
yielding 3aꢁi, led to increases in the SR values with the
exception of the transformation of 1a into 3a. The SR
values of 3bꢁi were greater than the value obtained for
melphalan and the enormous differences in cytotoxicity
displayed by 3bꢁe and 3h are particularly noteworthy.
/i. Thus the molecular hybridiza-
/
and 4 upon cytotoxicity, the IC50 values of 4aꢁ
/
f,i were
h. The
/
compared with those of the analogues 3aꢁd,fꢁ
/
/
results, which are summarized in Table 2, revealed a
dependence upon the cell line. Thus, the N-(3-aryl-1-
/
The question of whether series 3, formed by molecular
hybridization between the compounds in series 1 and the
4-piperidone 2, would display greater potencies than the
compounds in both series 1 and 2 was addressed.
Comparisons were made between the IC50 values of
each of the compounds in series 1 with the analogue in
series 3 in the same screen, e.g. the IC50 value of 1a was
compared with 3a, 1b with 3b and so forth. In a similar
manner, the potency of 2 was contrasted with each of
the compounds in series 3. The data are summarized in
Table 2. The results indicate that the relative cytotoxi-
cities were dependent on the cell line. Thus, when the
P388 screen was considered, the compounds in series 3
were more potent than the analogues in series 1 and with
compound 2 in 100 and 78%, respectively, of the
comparisons made. On the other hand, comparisons
undertaken using L1210, Molt 4/C8 and CEM cells
revealed that the compounds in series 3 had greater
potencies than 1 in less than half of the cases. When the
IC50 values of the compounds in series 3 were compared
with those in 2 in the L1210, Molt 4/C8 and CEM tests,
greater activity of the compounds in series 3 was found
in 11% of the cases when L1210 cells were considered
and none in the assays using human T-lymphocytes.
Thus in the P388 screen, it is conceivable that the
binding sites for the chalcones 1 and compound 2 are in
close proximity and the topography of the molecules 3
enables the alignment of both portions of the com-
pounds to contribute to cytotoxicity. In the case of the
three other cell lines, the bulky N-acyl group may
impede alignment of the majority of the compounds in
series 3 at the binding site of the 4-piperidone 2, thereby
diminishing potencies.
oxo-2-propenyl)piperidines 4aꢁf,i had lower IC50 values
/
than the analogues in series 3 in the majority of the
comparisons made in L1210 and Molt 4/C8 cells. On the
other hand, the enones 3 were invariably more cytotoxic
towards P388 cells and on average had lower IC50 values
in the CEM screen. The general conclusion to be drawn
from the data in this study is that there is substantial
tolerance to variations in the acyl group attached to the
nitrogen atom of 2.
Two other features of the biodata pertaining to series
4 were noted as follows. First, the SR values of 4aꢁc,f,g
/
indicate substantial variations in potencies which, with
the exception of 4f, were greater than the SR value of
melphalan. Nevertheless, the enones 4aꢁ/f,i had lower
SR values than the analogues 3aꢁd,fꢁ
/
/h with the
exception of 4a which was higher than 3a. Second,
comparison of the potencies of 4f and 3g, which both
contain the 3,4-dimethoxyphenyl group, revealed that
the compounds were equiactive, except that 3g possessed
twice the potency of 4f in the P388 screen. A comparison
of the cytotoxicities between 4f and both of the closely
related congeners 4g and 4h was made. In the P388
screen, 4f was less cytotoxic than 4g but more potent
than 4h. Evaluation in the three other screens revealed
that 4f was more active than 4g and equipotent with 4h.
Thus, molecular modification of the 3,4-dimethoxy
group of 4f led to retention in cytotoxic properties and
in the case of 4g, a SR value of ca. 38 was achieved
which surpassed other compounds in series 4 and also
3g.
The evidence obtained from the four cytotoxicity
assays revealed that the compounds in both 3 and 4
represent novel series of antineoplastic agents, many of
which are promising lead molecules. The importance of
the electronic, hydrophobic and steric properties of the
aryl substituents in conferring cytotoxicity was exam-
ined with a view to establishing guidelines for the
subsequent development of the project. Compound 3h
was not included in this analysis since specific IC50
values were not available in three of the four screens. In
addition, 4h was not included in the plots involving
steric constants due to the apparent unavailability of the
molecular refractivity (MR) value of the methylenedioxy
substituent. Linear and semilogarithmic plots were
In order to gain further information on the structural
features of the N-acyl group that influence cytotoxicity,
the synthesis of 4aꢁi was carried out. The decision was
/
based on the following considerations. First, the process
of molecular simplification [18] was utilized whereby
one of the aryl rings and a carbonyl function in the N-
acyl groups of 3 were excised. Second, the aryl
substituents in 4aꢁ/f,i were made identical to those
found in 3aꢁd, fꢁh so that comparisons of the potencies
/
/
of these compounds could be made. Third, the excellent
bioactivity displayed by 3g suggested that, in addition to

966
J.R. Dimmock et al. / European Journal of Medicinal Chemistry 37 (2002) 961ꢁ972
/
made between the IC50 values in each cell line with the
Hammett s, Hansch p and MR values of the nuclear
groups. In addition, plots between the SR values in
series 1, 3 and 4 and the s, p and MR values of the aryl
groups were made in order to determine whether one or
more physicochemical constants influence the disparities
in toxicities between the cell lines. The P values obtained
two different measurements were made. First, the
distances between the three sites of thiolation were
obtained and designated d1ꢁd3 as indicated in Fig. 3.
/
Second, the elevation or depression of the C14 atom and
either the C28 (3d) or C23 (4c,e,f,h) atoms in relation to
the plane defined by the sp2 bonds of the C7 atom were
measured and designated d4 and d5, respectively. The
results are portrayed in Table 3.
as either B
/
0.01, B
/
0.05 or B0.1 are listed in Section 6
/
of this report.
The reported u1 and u2 values of 2 as the hydrochlo-
In the case of series 3, positive correlations were noted
between the Hansch p values and the IC50 values in the
L1210, Molt 4/C8 and CEM assays as well as in the SR
values. Positive correlations were also found with the p
constants in series 4 and the cytotoxicity towards Molt
4/C8 and CEM cells. In addition, positive correlations
were obtained between the s values of the enones 4 and
the IC50 values in the P388, L1210, Molt 4/C8 and CEM
tests. The compounds in series 1 displayed a negative
correlation between the Hansch p values in the L1210,
Molt 4/C8 and CEM assays and also negative relation-
ships between the MR values in the P388 and L1210
ride salt are 12.4 and ꢂ12.98 [15]. The lack of
/
coplanarity of the aryl rings with the adjacent olefinic
linkages in this compound was attributed, at least in
part, to non-bonded interactions between the equatorial
protons at positions 2 and 6 (C2He and C6He) with the
ortho hydrogen atoms attached to atoms 13 and 20 [6].
In the case of 3d, C2HeÃ
/
C13H, C12HÃ
/
C23H, C12HÃ
/
C24H and C13HÃ
/
C23H non-bonded interactions were
absent. However, the molecule was orientated to avoid
such interactions between rings A and D (Fig. 3) which
led to large u1ꢁu3 values. The u4 and u5 values reveal a
/
lack of coplanarity of rings A and B with the adjacent
olefinic and carbonyl groups, respectively. Non-bonded
interactions between the C2He and C13H atoms were
cells. No other correlations (P ꢀ0.1) were noted.
/
In general, the relationships were specific for each
group of compounds. The following guidelines pertain-
ing to amplification of these series of compounds
emerged from this analysis. For the development of
series 3, increased lipophilicity is suggested. This
approach should also increase the SRs. Amplification
of series 4 should include a major emphasis in preparing
compounds with strongly electron-withdrawing groups
in the aryl ring which should be lipophilic. The data
from the chalcone series 1 indicate that aryl substitution
with small hydrophilic groups should be prepared in
order to increase potency.
In order to seek correlations between cytotoxicity and
the shapes of representative molecules, X-ray crystal-
lography was employed. Suitable crystals of 3d and
4c,e,f,h were obtained. The numbering of various atoms
in these compounds are indicated in Fig. 3. The
noted in 4c,e,f,h but C12HÃ
C13HÃC22H interactions were absent. The lack of
coplanarity of ring E with the adjacent unsaturated
linkage in 4c,e,f,h (u6) was less than the u3ꢁu5 values in
3d. In general, u2ꢀu1, which was attributed to the steric
/
C22H, C12HÃ
/
C25H and
/
/
/
effect of the N-acyl group. The results in Table 3 reveal
that the distance d1 was virtually identical in 3d and
4c,e,f,h. On the other hand, while the larger d2 and d3
values of 3d compared to 4c,e,f,h were predictable, the
variation among 4c,e,f,h of the d2 and d3 values was
surprising. The d4 distances revealed that, in general, the
C7 and C14 atoms lay in the same plane. On the other
hand, in the case of 4c,e,f,h, the d5 values indicated that
˚
the C23 atoms were ca. 3.9 A out of the plane of the C7
atom.
Linear and logarithmic plots were made between the
IC50 values in all four screens with the magnitude of,
first, the u1 and u2 values of 2, 3d, 4c,e,f,h, second, the u6
numbering of the atoms 1ꢁ21 is equivalent in all five
/
compounds and hence most of these numbers are
omitted from the structures of 4c,e,f,h for the sake of
clarity. If the enones interact with cellular mercaptans,
then the steric environment of the olefinic carbon atoms
at which thiolation occurs likely influences bioactivity.
Furthermore, the aryl rings of the compounds in series 3
and 4 may form van der Waals bonds with similar
groups at a binding site and hence the spatial orienta-
tions of the aryl rings in these compounds would be
predicted to influence potency. Thus the torsion angles
values of 4c,e,f,h and finally, the distances d2ꢁd5 of 3d,
/
4c,e,f,h. Correlations were only noted using the biodata
obtained from the P388 screen. Cytotoxicity was nega-
tively correlated with the u6 values of 4c,e,f,h as well as
the d2 and d3 distances of these compounds plus 3d. The
P values are listed in Section 6. No other correlations
(P ꢀ0.1) were noted. On the basis of these results,
/
amplifications of these two series of compounds in the
following ways may lead to novel compounds with
increased potencies in the P388 screen. First, the absence
of ortho and meta substituents in ring E in series 4
would diminish the magnitude of the u6 values [19].
Second, the shortening of the d2 and d3 distances may be
accomplished by joining rings A or E to either ring D or
the piperidine ring with a short linker group.
u1ꢁu5 in 3d and u1, u2 and u6 in 4c,e,f,h were measured.
/
Furthermore, variations in potencies between these five
compounds could be due to differences in the relative
locations of the sites of thiolation which are believed to
be carbon atoms 7, 14 and 28 in 3d and 7, 14 and 23 in
the case of 4c,e,f,h. In order to examine this hypothesis,

J.R. Dimmock et al. / European Journal of Medicinal Chemistry 37 (2002) 961ꢁ
/
972
967
Fig. 3. The numbers assigned to some of the atoms, the torsion angles (u1 ꢁ
/
u6) and interatomic distances (d1 ꢁd3) of the X-ray crystallographic
/
structures of 3d and 4c,e,f,h are indicated.
The enones 2, 3a, b and 4a, as well as two reference
drugs, were evaluated for cytotoxicity towards a number
of human tumour cell lines representing different groups
of neoplasms [20]. In general, the compounds were
[21]. Hence the MG MID values of 3a,b and 4a towards
these groups of tumours were calculated and compared
to the data generated for 5-fluorouracil and melphalan,
which are used clinically to combat colon tumours and
leukemia, respectively [22,23]. The data in Table 4 reveal
that both 3a and 4a were more cytotoxic than either of
the reference drugs for these two groups of tumours. In
addition, a selectivity index (SI) was calculated for each
of the compounds in Table 4, i.e. the average MD MID
values obtained for all cell lines was divided by the MG
MID values of either colon cancer or leukemic cells. A
50% increase in selectivity was arbitrarily chosen as a
promising indicator of selectivity. The enones 3a and 4a
met this criterion when both colon cancer and leukemic
cells were considered and these compounds had similar
SI values as melphalan. Furthermore, the SR data for 3a
and 4a are noteworthy. The evaluation using a large
number of human tumour cell lines revealed that 3a and
4a are very useful prototypic cytotoxic molecules.
screened using a concentration range of 10ꢂ8ꢁ10ꢂ4 M.
/
The average cytotoxicity towards all cell lines was
expressed in terms of the mean graph midpoint (MG
MID) values rather than IC50 values since, if a
compound did not inhibit the growth of the tumour
cells by 50% at the highest concentration utilized namely
10ꢂ4 M, the value of 10ꢂ4 M was still included in
determining the average cytotoxicity towards all cell
lines. In the case of 2, 3a, 4a and melphalan, however,
the MG MID and IC50 values are the same. The data
generated are portrayed in Table 4.
The order of cytotoxicity of 3a,b and 4a towards
human tumour cell lines was 3aꢀ
/
4aꢀ3b. Both 3a and
/
4a were ca. 1.6 and 1.1 times, respectively, more potent
than melphalan. Furthermore there is an interest in this
laboratory in discovering novel compounds which have
marked potencies towards colon cancers and leukemias
The data presented so far revealed that the com-
pounds exerted differential cytotoxicity towards various
Table 3
The torsion angles u1 ꢁ
a
˚
d5 (A) in 3d and 4c,e,f,h determined by X-ray crystallography
/
u6 (8) and distances d1 ꢁ
/
Compound
u1
u2
u3
u4
u5
u6
d1
d2
d3
d4
d5
3d
4c
4e
4f
24.4
22.3
25.4
26.4
0.2
ꢂ39.9
ꢂ29.4
ꢂ41.6
ꢂ23.8
ꢂ36.8
48.5
ꢂ159.5
ꢁ/
ꢁ/
ꢁ/
ꢂ12.3
ꢁ/
ꢁ/
ꢁ/
ꢁ
/
4.86
4.85
4.85
4.87
4.88
6.72
4.76
5.26
5.39
5.23
10.1
0.21
0.06
ꢂ3.90
4.05
ꢁ/
ꢁ/
ꢁ/
ꢁ/
172.9
6.33
ꢂ170.7
178.0
176.3
7.01
6.65
7.18
ꢂ0.70
ꢂ0.012
0.094
ꢂ3.69
4.04
4h
ꢁ/
ꢁ/
ꢂ3.78
a
The positive and negative signs of the u1 ꢁu6 figures refer to clockwise and anticlockwise rotations, respectively. The positive and negative signs
/
of the d4 and d5 distances refer to the locations of various atoms as either above or below the plane of the C7 atom, respectively.

968
J.R. Dimmock et al. / European Journal of Medicinal Chemistry 37 (2002) 961ꢁ972
/
cell lines. A broader approach was instigated in order to
confirm that these compounds are not general biocidal
agents. First, the enones 1a,e, 2 (as the hydrochloride
salt) and 3a,e, which have widely divergent IC50 values
towards the four cell lines portrayed in Table 1, were
examined against three isolates of A. fumigatus and one
of C. albicans. The minimal inhibitory concentration
(MIC) values of the five enones towards the four fungal
pathogens were in excess of 32 mg mLꢂ1 and hence they
possess no significant activity towards these fungi. This
observation provides further evidence of the selective
tumour toxicity displayed by these compounds.
A second and more extensive investigation was
undertaken in order to determine whether the com-
pounds prepared in this study had the potential to spare
normal cells yet have lethal properties towards certain
tumours. All of the compounds were administered
intraperitoneally into mice using doses of 30, 100 and
300 mg kgꢂ1 and the animals were observed after 0.5
and 4 h. Neurotoxicity and other pathological symp-
toms were displayed by some of the compounds (Section
6). The only mortality was after 4 h in one out of two
mice that received 300 mg kgꢂ1 of 1a. In addition, no
toxicity to other tissues. In particular, the virtual
absence of murine toxicity displayed by series 3 is a
further indicator of this group of novel enones should be
considered to be lead molecules.
5. Conclusions
This study has revealed that N-[4-(3-aryl-3-oxo-1-
propenyl)phenylcarbonyl]-3,5-bis(phenylmethylene)-4-
piperidones (3) are novel cytotoxic agents. In particular,
significant potency towards P388 cells was demonstrated
with the IC50 values of all compounds in this series being
less than 1 mM. The SR values of various members of
series 3 are noteworthy. In general, the compounds in
series 3 were more potent than the analogues 1 and 4 but
less cytotoxic than the precursor 4-piperidone 2. The
absence of both antifungal activity of representative
enones and marked murine toxicity in virtually all of the
compounds, coupled to the variation in potencies
towards different tumours, suggest that those molecules
are selectively toxic to certain cells. Various guidelines
for amplification of the different series of compounds
have been made including those derived from structureꢁ
/
murine toxicity was displayed by 1c,e,h, 2, 3aꢁ
/
e,gꢁi and
/
activity relationships using the various physicochemical
constants of the aryl substituents as well as X-ray
crystallography. The evidence suggests that develop-
4a,c,h,i. Thus, in general, the compounds in series 3
possessed less murine toxicity than the chalcones 1 and
amides 4. Anticonvulsant activity was demonstrated by
1i and 3d in the MES screen and by 1a,b,g,i in the scPTZ
test, indicating that some CNS penetration occurred
with these compounds and possibly with related analo-
gues. Oral administration of three representative com-
pounds, namely 1b, 3d and 4h to rats did not reveal any
overt toxicity. Each of these three compounds displayed
marginal activity in the anticonvulsant screens indicative
of CNS penetration.
ment of the compounds in series 2ꢁ4 as candidate
/
antineoplastic agents is warranted.
6. Experimental
6.1. Chemistry
The conclusions to be drawn from the antifungal,
toxicity and anticonvulsant screens are that the com-
pounds described herein, while possessing marked
cytotoxic properties for tumour cells, displayed minimal
M.p. are in Celsius degrees and are uncorrected.
Elemental analyses, undertaken by K. Thoms, Depart-
ment of Chemistry, University of Saskatchewan, were
performed on 1aꢁ
/
i, 3aꢁ
/
i and 4aꢁ/i and were within 0.4%
Table 4
Evaluation of 2, 3a, 3b, 4a and reference compounds against various human tumour cell lines
c
Compound
All cell lines
Colon cancer cells
Leukemic cells
SR
a
a
b
a
b
MG MID (mM)
MG MID (mM)
SI
MG MID (mM)
SI
2
3a
1.62
5.01
53.7
7.25
7.94
29.5
0.658
3.19
60.8
4.09
7.87
6.47
2.46
1.57
0.88
1.77
1.01
4.56
0.455
1.79
27.9
2.35
2.61
3.63
3.56
2.80
1.93
3.09
3.04
8.13
93
85
3b
4a
14
62
Melphalan
5-Fluorouracil
87
ꢀ6456
a
The letters MG MID refer to the mean graph midpoint which is discussed in the text.
The letters SI refer to the selectivity index, i.e. the average MG MID values of all cell lines divided by the average MG MID figures for either the
b
colon cancer or leukemic cells.
c
The letters SR refer to the selectivity ratio which was obtained by dividing the IC50 figures of the compound towards the least susceptible and
most susceptible cell lines.

J.R. Dimmock et al. / European Journal of Medicinal Chemistry 37 (2002) 961ꢁ
/
972
969
1
of the calculated values. H-NMR spectra were deter-
quarter molecule of water, 3d,e and 3i with one half
molecule of water and 3h with one molecule of water of
crystallization. The m.p. and percentage yields of these
mined on all compounds using a Bruker AM 500 FT-
NMR instrument (500 MHz). TLC was carried out
using silica gel 60 F254-precoated plastic sheets and an
eluting solvent of CHCl3:CH3OH (9:1).
compounds were as follows: 3a: 190ꢁ
222, 67; 3c: 195ꢁ196, 61; 3d: 223, 56; 3e: 240ꢁ
186ꢁ
/
192, 65; 3b: 220ꢁ
242, 69; 3f:
260, 67; 3i: 240ꢁ
/
/
/
/
187, 62; 3g: 172ꢁ
/
174, 71; 3h: 255ꢁ
/
/
6.1.1. Synthesis of 1aꢁ
/
i
243, 45. The 1H-NMR spectrum of a representative
compound 3g is as follows: d (dmso-d6): 3.85 (s, 3H,
4-Carboxybenzaldehyde (0.03 mol) was added to a
solution of NaOH (0.05 mol) in water (25 mL) and
EtOH (10 mL) and the resultant mixture was stirred for
0.25 h with ice bath cooling. The appropriate 1-aryl-1-
ethanone (0.03 mol) was added and the solution stirred
at room temperature (r.t.) for 12 h. The reaction mixture
was cooled using an ice bath and acidified with HCl
(20% w/v). The precipitate was collected, washed with
cold water, dried and purified by recrystallization from
MeOH or digested in this solvent. The m.p. and
percentage yields of the compounds in series 1 were as
3?OCH3), 3.87 (s, 3H, 4?OCH3), 4.68 (bs, 2H, NCH ꢄ
2), 5.01 (bs, 2H, NCH ꢄ2), 7.11ꢁ7.91 (m, 2IH, aryl and
olefinic H).
/
/
/
6.1.3. Synthesis of 4aꢁ
The 3-aryl-2-propenoyl chlorides required in the
synthesis of 4aꢁi were obtained as follows. 3-Phenyl-2-
/
i
/
propenoyl chloride was purchased from the Aldrich
Chemical Company. The remaining acid chlorides were
synthesized by the following method. A mixture of the
appropriate 3-aryl-2-propenoic acid (0.003 mol), pre-
pared by a literature procedure [27], thionyl chloride
(0.007 mol), N,N-dimethylformamide (0.04 mL), and
dry dichloroethane (40 mL) was heated under reflux for
6 h. Evaporation in vacuo led to the isolation of the acid
chloride which was dissolved in dichloroethane (30 mL).
To this solution 2 (0.003 mol) was added and the
reaction mixture was cooled in an ice-bath for 20 min.
A solution of Et3N (0.006 mol) in dichloroethane (20
mL) was added dropwise over 0.5 h at ice-bath
temperature. The source of cooling was removed and
the reaction mixture gradually rose to r.t. The mixture
was stirred for 6 h after the addition of Et3N. After
evaporation in vacuo, HCl (2% w/v, 30 mL) was added
and the suspension stirred for 1 h at r.t. The precipitate
follows: 1a: 220ꢁ
266ꢁ267 (lit. [25] m.p. 267), 67; 1c: 259ꢁ
242ꢁ243, 54; 1e: 258, 63; 1f: 224ꢁ226, 68; 1g: 208 (lit.
[24] m.p. 208ꢁ209), 69; 1h: 274, 71; 1i: 282ꢁ283 (lit. [26]
m.p. 300ꢁ
301), 56. The 1H-NMR spectrum of a
representative compound 1c was as follows: d (dmso-
d6): 7.80 (d, 1H, CHÄCHCO, Jꢃ15.6 Hz), 7.84 (d, 1H,
C6?H, Jꢃ8.4 Hz, 7.98 (d, 2H, C3H, C5H, Jꢃ8.3 Hz),
8.03 (d, 2H, C2H, C6H, Jꢃ8.3 Hz), 8.07 (d, 1H, CH Ä
CHCO, Jꢃ15.6 Hz), 8.12 (m, 1H, C5?H), 8.43 (d, 1H,
C2?H, Jꢃ1.8 Hz), 13.15 (bs, 1H, COOH).
/
221 (lit. [24] m.p. 209ꢁ/211), 68; 1b:
/
/260, 65; 1d:
/
/
/
/
/
/
/
/
/
/
/
/
/
6.1.2. Synthesis of 3aꢁi
/
Thionyl chloride (0.007 mol) was added to a solution
of the appropriate 4-carboxychalcone (0.003 mol) in
CH2Cl2 (40 mL) which contained a catalytic quantity of
dimethylformamide (0.04 mL). The solution was heated
under reflux for 6 h. The formation of the acid chloride
was monitored by adding small quantities of the
reaction mixture to MeOH and noting the appearance
of the methyl ester by TLC. The solvents were removed
in vacuo and the residue was dissolved in dry CHCl3 (30
mL). A solution of 3,5-bis(phenylmethylene)-4-piperi-
done hydrochloride (0.003 mol), prepared by a literature
method [13], in dry CHCl3 (20 mL) and Et3N (0.006
mol) was added to a solution of the acid chloride under
ice-cold, dry conditions over the course of 10 min. For
the synthesis of 3i, the addition procedure was reversed
insofar as the acid chloride was added dropwise to a
solution of 2 over 0.5 h. The resultant mixture was
stirred at r.t. for 4 h; the reaction being monitored by
TLC. The solvents were evaporated and to the residue
was added MeOH (ca. 20 mL per gram of product) and
the suspension was heated at 60 8C for 4 h. The
precipitate was collected by filtration, washed with
cold MeOH and dried. Compounds 3a,e,g were subse-
quently recrystallized from CHCl3 and 3c was digested
with CHCl3. The amides 3a and 3g crystallized with one
was collected, washed with water (3ꢄ50 mL) and
/
MeOH (30 mL) and dried. The compounds were
recrystallized from a mixture of CH2Cl2 and MeOH
(3:2). The amides 4b,d,g crystallized with one quarter
mole of water and 4f was obtained as the monohydrate.
The m.p. and percentage yields were as follows: 4a: 197
(lit. [28] 194ꢁ
188, 75; 4e: 199, 69; 4f: 194ꢁ
178, 74; 4i: 237ꢁ238, 78. The H-NMR spectrum of a
representative compound 4b is as follows: d (CDCl3):
4.80 (bs, 2H, NÃCH2), 4.98 (bs, 2H, NÃCH2), 6.27 (d,
1H, COCH, Jꢃ15.4 Hz), 6.90 (d, 2H, C3H, C5H of N-
acyl ring, Jꢃ8.4 Hz), 7.17 (d, 2H, C2H, C6H of N-acyl
ring, Jꢃ8.4 Hz), 7.35ꢁ7.55 (m, 11H, 2ꢄC6H5,
COCHÄCH), 7.84 (s, 2H, 2ꢄCHÄ).
/
196), 77; 4b: 181, 78; 4c: 200ꢁ/201, 77; 4d:
/
195, 78; 4g: 191ꢁ
/193, 79; 4h:
1
/
/
/
/
/
/
/
/
/
/
/
6.1.4. Statistical analyses
The s and p constants of the aryl substituents in 1aꢁ
3aꢁg,i and 4aꢁi were culled from a reference source [29].
The MR figures at positions 3, 4 and 5 of aryl ring B of
1aꢁi, 3aꢁg,i and ring E in 4aꢁg,i were taken from the
/i,
/
/
/
/
/
literature [29]. The linear and semilogarithmic plots were
generated using a commercial software package [30].

970
J.R. Dimmock et al. / European Journal of Medicinal Chemistry 37 (2002) 961ꢁ972
/
The following correlations (i.e. Pearson’s correlation)
were noted [physicochemical constant, assay, linear (l)
or semilogarithmic (sl) plots, P value] namely 3: p,
dology [35] and the L1210, Molt 4/C8 and CEM tests
utilized a previously reported method [36]. The piper-
idones 2, 3a,b, 4a as well as melphalan and 5-fluorour-
L1210, l, B
0.05; p, CEM, sl, B
Molt 4/C8, l, B0.1; p, CEM, l, B
0.05; s, L1210, l and sl, B0.01; s, Molt 4/C8, l and
sl, B0.01; s, CEM, l and sl, B0.01; 1: p, L1210, sl, B
0.1; p, Molt 4/C8, l, B0.05; p, Molt 4/C8, sl, B0.01; p,
CEM, l and sl, B0.05; MR, P388, l, B0.05; MR, P388,
sl, B0.01 and MR, L1210, sl, B0.05.
The correlations obtained between the IC50 values in
the P388 screen and certain torsion angles and intera-
tomic distances obtained by X-ray crystallography were
as follows [physicochemical measurements, linear (l) or
/
0.1; p, L1210, sl, B
0.05; p, SR, l and sl, B
0.1; s, P388, l and sl,
/
0.05; p, Molt 4/C8, sl,
acil were evaluated against 55 (52ꢁ59) human tumour
/
B
/
/
/0.05; 4: p,
cell lines by a reported procedure [20]. Evaluation was
made using cells from the following neoplastic condi-
tions, namely leukemia, melanoma and non-small cell
lung, colon, CNS, ovarian, renal, prostate and breast
tumours, with the exception of 2 and 4a which were not
screened against prostate and breast neoplasms but were
evaluated using small cell lung tumour cell lines. In the
case of 2, 3a, 4a and melphalan, all of the log10 MG
MID values were IC50 values. However, specific values
were obtained for 34/55 and 51/57 cell lines for 3b and 5-
fluorouracil, respectively. In the evaluation of 3b, the
MG MID values of four of the seven colon cancer cell
lines were greater than 10ꢂ4; specific MG MID values
were obtained against this panel of tumours in the case
of 5-fluorouracil and towards leukemic cells for both
compounds.
/
/
B
/
/
/
/
/
/
/
/
/
/
/
logarithmic (ln) plots, P value]: u6, l, B
/
0.1; u6, ln, B
/
0.1, d2, l, B0.1; d2, ln, B0.05 and d3, ln, B
/
/
/
0.1.
6.1.5. X-ray crystallography
X-ray crystallographic data were collected for 3d and
4c,e,f,h using an EnrafꢁNonius CAD-4 diffractometer
/
with v scans. Data were processed using XTAL-3.6 [31].
The structures were solved using NRCVAX [32], while
SHELXL [33] was used for refinement. Atomic scattering
factors and anomalous dispersion corrections were
obtained from the literature [34]. Non-hydrogen atoms
were found in E-maps and were refined anisotropically.
Hydrogen atoms were placed on atoms by geometry
using a riding model and assigned temperature factors
6.2.2. Antifungal evaluations
Compounds 1a, e, 2, 3a and 3e were examined for
antifungal activity using three isolates of A. fumigatus
(ATCC 208995, 208996 and 208997) and one isolate of
C. albicans (ATCC 90028) by the broth microdilution
test [37]. The MIC values of all five compounds was in
excess of 32 mg mLꢂ1. In this assay, a reference drug
voriconazole possessed a MIC value of 0.25 mg mLꢂ1
.
derived from the attached atoms. The torsion angles u1ꢁ
u6 for 3d and 4c,e,f,h were obtained using the following
atoms: u1: C5ÃC14ÃC15ÃC20; u2: C3ÃC7ÃC8ÃC13; u3:
O2ÃC21ÃC22ÃC27; u4: C26ÃC25ÃC28ÃC29: u5: O3Ã
C30ÃC31ÃC36; u6: C22ÃC23ÃC24ÃC29. The intera-
tomic distances He(C2)ÃH(C13) for 4c,e,f,h were 2.133,
2.247, 2.028 and 2.182 A, respectively, while the
He(C2)ÃH(C13), H(C13)ÃH(C23), H(C12)ÃH(C23)
and H(C12)ÃH(C24) distances for 3d were 2.133,
2.247, 2.028 and 2.182 A and the H(C13)Ã
H(C12)ÃH(C25) and H(C12)ÃH(C22) distances for
4c,e,f,h were in excess of 2.4 A. There are very short
H(C22) distances in 4c,e,f,h, namely 1.76, 1.90,
˚
1.93 and 1.96 A, respectively. These distances are less
than the sum of the hydrogen atom van der Waals radii
˚
(2.2 A). Since these hydrogen atoms were placed by the
geometry of the non-hydrogen atoms and the data are
not of sufficient quality to refine the hydrogen atoms, it
is likely that their true positions are somewhat displaced
due to the collision of the hydrogen atoms, thus
resulting in some strain in the structures.
/
6.2.3. Toxicity, MES and scPTZ screens
/
/
/
/
/
/
Various compounds were administered to mice by the
intraperitoneal route and examined for neurotoxicity
[38] as well as anticonvulsant properties in the MES and
scPTZ tests [39].
/
/
/
/
/
/
/
/
/
/
/
/
/
˚
Compounds 1aꢁ
/
i, 2, 3aꢁ
/
i and 4aꢁi were examined in
/
/
/
/
the murine toxicity, MES and scPTZ screens. The
animals were observed 0.5 and 4 h after receiving doses
of 30, 100 and 300 mg kgꢂ1 of the enones. Neurotoxi-
city was noted with the following compounds (dose in
mg kgꢂ1, time in h, number of animals displaying
toxicity), namely 1a: 300, 4, 2/2; 1b: 300, 0.5, 2/4; 100, 4,
1/4; 300, 4, 2/2; 1d: 100, 0.5, 1/8; 300, 0.5, 1/4; 100, 4, 2/4;
300, 4, 2/2; 1f: 100, 0.5, 1/8; 300, 0.5, 1/4; 1g: 300, 0.5, 1/
4; 1i: 100, 0.5, 1/8; 300, 0.5, 2/4; 300, 4, 1/2; 3f: 100, 0.5,
2/8; 300, 0.5, 3/4; 100, 4, 1/4; 300, 4, 1/2; 4b: 100, 0.5, 2/8;
300, 0.5, 2/4; 300, 4, 1/2; 4d: 100, 0.5, 3/8; 300, 0.5, 1/4;
4e: 100, 0.5, 3/8; 300, 0.5, 1/4; 100, 4, 1/4; 300, 4, 2/2; 4f:
100, 0.5, 2/8; 300, 0.5, 3/4; 300, 4, 1/2; 4g: 300, 0.5, 2/4. In
addition, mice receiving a dose of 300 mg kgꢂ1 of
1a,b,d,g were unable to grasp a rotorod after 0.5 (1g) or
4 (1a,b,d) h after administration of the compounds.
Death was noted in one of two mice 4 h after
administration of 300 mg kgꢂ1 of 1a. In the MES
screen, a dose of 300 mg kgꢂ1 of 1i and 3d afforded
protection after 0.5 and 4 h, respectively. Protection in
/
˚
/H(C22),
/
/
˚
He(C2)Ã
/
6.2. Screening
6.2.1. Cytotoxicity evaluations
The evaluation of the compounds 1, 3 and 4 against
P388D1 cells was undertaken using a literature metho-

J.R. Dimmock et al. / European Journal of Medicinal Chemistry 37 (2002) 961ꢁ
/
972
971
the scPTZ test was displayed by the following com-
pounds (dose in mg kgꢂ1, time in h, number of animals
protected), namely 1a: 300, 0.5, 3/5; 1b: 300, 0.5, 4/5; 1g:
300, 0.5, 1/1; 1i: 300, 4, 1/1; 5c: 300, 0.5, 1/5. Tonic
extensions were noted in the scPTZ screen by 1b (300 mg
kgꢂ1 dose after 4 h) and 4d (100 mg kgꢂ1 dose after 0.5
h). In the same test, myoclonic jerks were noted 4 h after
administration of a 300 mg kgꢂ1 dose of 1i. No other
toxicities or protection against convulsions were noted
in these murine screens.
Acknowledgements
The authors thank the following sources for financial
support for this project namely Purdue Neuroscience
Company, USA (J.R.D.), Natural Sciences and Engi-
neering Research Council of Canada (J.W.Q.), Univer-
sity of Saskatchewan (E.O.O.), National Cancer
Institute of Canada (T.M.A.), Flemish Fonds voor
Geneeskundig Weterschappelijk Onderzoek (E.D.C.,
J.B.) and the National Institute of Neurological Dis-
orders and Stroke (J.P.S.). The US National Cancer
Institute is thanked for determining the bioevaluations
using a panel of human tumour cell lines.
Compounds 1f, 3d and 4h were administered to rats
per os using doses of 50, 30 and 300 mg kgꢂ1
,
respectively, and evaluated for neurotoxicity by obser-
ving the animals for overt evidence of ataxia as well as
abnormal gait and stance. Evaluations in the MES and
scPTZ screens were undertaken by a literature proce-
dure [39]. Observations were made 0.25, 0.5, 1, 2 and 4 h
after administration of the compounds except in the case
of 4h, where the 1 and 2 h observations were omitted.
No pathological symptoms were noted in the toxicity
screen. In addition, 3d and 4h were examined in the
MES screen and 1f and 4h in the scPTZ test using the
same doses and observation times as were employed in
the toxicity screen. In the MES test, 3d afforded
protection in 1/4 rats after 0.5, 1 and 4 h and in 3/4
animals after 2 h, while 4d displayed anticonvulsant
activity in 1/2 rats after 4 h. Evaluation in the scPTZ
screen revealed that 1f protected 1/4 animals against
seizures after 0.5 and 1 h and 4h after 0.25 and 0.5 h in 1/
2 rats. At other time intervals in the MES and scPTZ
screens, no protection was noted. In the case of 4h, one
of the two animals in the scPTZ screen died after 0.25 h.
A dose of 100 mg kgꢂ1 of 4h was administered
intraperitoneally to rats and observations were made
in the toxicity and MES screens after 0.25, 0.5, 1, 2 and 4
h. No toxicity was observed and 1/2 rats was protected
in the MES test after 0.25 h only. The Anticonvulsant
Screening Project of the National Institute of Neurolo-
gical Disorders and Stroke requires that all rats and
mice are housed, fed and handled in a manner consistent
with the recommendations in the National Research
Council Publication, ‘Guide for the Care and Use of
Laboratory Animals’. All animals were euthanized in
accordance with the Institute of Laboratory Resources
policies on the humane care of laboratory animals.
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