Cytotoxic analogues of 2,6-bis(arylidene)cyclohexanones

Article abstract of DOI:10.1016/S0223-5234(02)01444-7

A series of 2,6-bis(arylidene)cycloalkanones (1) and related compounds containing one or two substituents at the four position of the cyclohexyl ring were prepared and shown to display cytotoxic activity towards murine P388 and L1210 cells as well as human Molt 4/C8 and CEM T-lymphocytes. In some of the series of compounds, positive correlations were noted between the potencies of the enones and the magnitude of the Hammett σ values of the aryl substituents. Four representative compounds were cytotoxic to a number of human tumours in vitro, particularly towards colon cancer and leukemic cells. A noteworthy feature of the compounds prepared in this study is that, in general, they were well tolerated when administered to rodents. A number of lead molecules emerged from this investigation as well as guidelines for future expansion of these series of compounds.

Full text of DOI:10.1016/S0223-5234(02)01444-7

European Journal of Medicinal Chemistry 38 (2003) 169Á177
/
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Original article
Cytotoxic analogues of 2,6-bis(arylidene)cyclohexanones
Jonathan R. Dimmock a,*, Maniyan P. Padmanilayam a, Gordon A. Zello a, Kurt
H. Nienaber b, Theresa M. Allen c, Cheryl L. Santos c, Erik De Clercq d, Jan Balzarini d,
Elias K. Manavathu e, James P. Stables f
a College of Pharmacy and Nutrition, University of Saskatchewan, 110 Science Place, Saskatoon, Sask., Canada S7N 5C9
b Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Sask., Canada S7N 5E5
c Department of Pharmacology, University of Alberta, Edmonton, Alta, Canada T6G 2H7
d Rega Institute for Medical Research, Katholieke Universiteit Leuven, 3000 Leuven, Belgium
e Department of Medicine, Wayne State University, Detroit, MI 48201-1908, USA
f National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-9020, USA
Received 18 July 2002; received in revised form 18 November 2002; accepted 20 November 2002
Abstract
A series of 2,6-bis(arylidene)cycloalkanones (1) and related compounds containing one or two substituents at the four position of
the cyclohexyl ring were prepared and shown to display cytotoxic activity towards murine P388 and L1210 cells as well as human
Molt 4/C8 and CEM T-lymphocytes. In some of the series of compounds, positive correlations were noted between the potencies of
the enones and the magnitude of the Hammett s values of the aryl substituents. Four representative compounds were cytotoxic to a
number of human tumours in vitro, particularly towards colon cancer and leukemic cells. A noteworthy feature of the compounds
prepared in this study is that, in general, they were well tolerated when administered to rodents. A number of lead molecules
emerged from this investigation as well as guidelines for future expansion of these series of compounds.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: a,b-Unsaturated ketones; Cytotoxicity; StructureÁactivity relationships; Bioscreens
/
1. Introduction
leading to 1bÁ
different electronic, hydrophobic and steric properties.
In fact, the groups in 1bÁe, g are found in three of the
/
g. The chosen aryl substituents had
A number of years ago the noteworthy cytotoxicity of
2,6-bis(phenylmethylene)cyclohexanone (1a) was dis-
closed [1]. The IC50 figure of this compound is 36.5
nM towards murine P388/MRI leukemic cells revealing
it possessed 25 times the potency of a reference drug
carmustine towards this cell line. This unsaturated
ketone did not bind to a synthetic DNA, namely
poly[dAT] and, as described in this study, 1a did not
induce mortality in mice which received 300 mg kgꢀ1 of
this compound (Figs. 1 and 2).
/
four quadrants of a Craig plot for para substituents [2]
which indicate the divergence of the sp and p values of
the chosen substituents. Second, many a,b-unsaturated
ketones exert their bioactivity, at least in part, by
reaction with cellular thiols [3]. In the present study,
the decision was made to explore the possibility of the
existence of an additional site of action with cellular
constituents that are distinct from the olefinic carbon
atoms Thus at position four of the cyclohexane ring
various groups were placed which had different steric,
electronic and hydrophobic properties, namely the
bromo, t-butyl, carboethoxymethylene, dimethylene-
The objective of the present investigation was to
develop structureÁactivity relationships from the lead
compound in the following ways. First, the placement of
various groups in the aryl rings of 1a was proposed
/
dioxy and acetoxy functions that gave rise to series 2Á
/
6, respectively. Where available, certain substituent
constants of the group at position four of the cycloali-
phatic ring supported the divergence of their physico-
chemical properties. Thus the solvent accessible surface
* Corresponding author.
E-mail address: dimmock@skyway.usask.ca (J.R. Dimmock).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(02)01444-7

170
J.R. Dimmock et al. / European Journal of Medicinal Chemistry 38 (2003) 169Á177
/
Fig. 1. Synthesis of the compounds in series 1Á
/
6. The nature of the substituents R1 ÁR3 are indicated in Table 1.
/
7b,c were synthesized with a view to comparing their
potencies with 5a, which possesses IC50 values lower
than melphalan when evaluated against the two human
T-lymphocytes vide infra. Previous work with certain
Mannich bases of conjugated arylidene ketones revealed
that these compounds demonstrated not only cytotoxi-
city [6] but also possessed antifungal properties [7].
However mice receiving either a single dose [8] or
multiple doses [6] of various members of this class of
compounds displayed toxicity including lethality. Hence
the evaluation of representative compounds for anti-
mycotic activity as well as toxicity to mice was planned.
2. Chemistry
Cyclohexanone, 4-t-butylcyclohexanone and 1,4-cy-
clohexanedione mono-ethylene ketal, which were used
in the synthesis of the compounds in series 1, 3 and 5,
respectively, were obtained from commercial sources.
The cycloaliphatic ketones required in the synthesis of
series 2, 4 and 6 were prepared as follows. Reaction of
1,4-cyclohexanediol with hydrobromic acid led to the
isolation of 4-bromocyclohexanol, which on treatment
with chromic oxide produced 4-bromocyclohexanone,
which was used in the synthesis of series 2. Reaction of
ethyl bromoacetate with triphenylphosphine and sub-
sequent treatment with sodium hydroxide led to the
corresponding Wittig reagent which was condensed with
1,4-cyclohexanedione to give 4-oxycyclohexylidene ace-
tic acid ethyl ester from which series 4 was prepared.
Chromic acid oxidation of 1,4-cyclohexanediol led to
the formation of 4-hydroxycyclohexanone, which was
acylated with acetic anhydride to give 4-acetoxycyclo-
hexanone, used in the preparation of series 6. Each of
these six cyclic ketones was condensed with the appro-
Fig. 2. Structures of compounds in series 7.
area (SASA) values of all of the substituents at position
four of the cyclohexane ring in series 1Á6 were 46.1,
/
2
˚
88.9, 169.4, 167.3, 129.4 and 145.7 A , respectively [4].
Furthermore various physicochemical constants are
available for the hydrogen, bromo, t-butyl and acetoxy
groups. For example, the field (F) effects for these four
substituents are 0.00, 0.44, ꢀ
while the fragment constants (f) are 0.23, 0.20, 2.22 and
0.72, respectively, and the molar refractivity (MR)
values are 1.03, 8.80, 19.62 and 11.85, respectively [5].
In addition, compounds 7aÁc were also synthesized in
/
0.07 and 0.41, respectively,
ꢀ
/
priate aryl aldehydes to give rise to the enones 1Á
7. 1H-
NMR spectroscopy revealed the compounds to be
isomerically pure. Previous X-ray crystallographic stu-
/
/
order to compare their cytotoxicities with those of
certain related congeners. Thus the structural isomer
of 1g, which displayed excellent potency in the P388
screen vide infra, namely 7a, was prepared. In addition,
dies of 1a [1] and related compounds [9Á/11] revealed
that the olefinic double bonds adopted the E config-

J.R. Dimmock et al. / European Journal of Medicinal Chemistry 38 (2003) 169Á
/
177
171
uration. Hence the compounds prepared in the present
investigation were considered to have the same stereo-
chemistry.
Table 2
Evaluation of 2b, 4c, 5a and 5f against human tumour cell lines
a
Compound
MG MID (mM)
All cell lines Colon cancer cells Leukemic cells
2b
4c
20.4
8.71
4.68
19.5
19.1
13.1
5.55
2.53
13.5
42.9
5.82
7.94
4.59
2.85
6.68
3.55
3. Bioevaluations
5a
5f
Melphalan
The a,b-unsaturated ketones 1Á
/7 were evaluated for
cytotoxicity towards murine P388 and L1210 leukemic
cells and human Molt 4/C8 and CEM T-lymphocytes.
These data are presented in Table 1. In addition, four
representative compounds, namely 2b, 4c, 5a and 5f,
were examined against a panel of human tumours and
5-Fluorouracil 29.5
52.9
a
The letters MG MID refer to the mean graph midpoint values
which are explained in the text.
Table 1
Cytotoxicity of the compounds in series 1Á/7 and melphalan against murine P388 and L1210 cells and human Molt 4/C8 and CEM T-lymphocytes
a
Compound
Aryl substituents
IC50 (mM)
R1
R2
R3
P388 cells
L1210 cells
Molt 4/C8 cells
CEM cells
1a
1b
1c
4.829
3.919
509
509
4.069
1.489
0.6399
0.8739
0.7319
3.859
2.679
1.049
5.429
2.199
21.09
10.49
2.669
2.459
0.2419
1.069
2.749
0.7869
1.459
22.39
2.809
3.949
12.69
50
7.719
5.479
1.389
0.9819
0.8359
2.269
0.5989
3.729
3.729
0.5109
0.2089
31.49
0.229
/
0.3
18.39
9.609
2489
2369
20.59
12.59
8.469
5.689
14.89
3129
11.69
6.309
9.059
10.69
1719
2229
2419
38.49
1.509
9.489
26.69
8.449
43.29
39.49
19.99
12.89
4029
500
500
15.99
13.69
40.19
2.649
14.29
8.969
13.39
41.19
2.019
0.8359
500
2.139
/
4.03
0.71
14.1
14.8
12.9
0.71
1.18
0.61
9.46
9
8.849
8.899
3039
2469
9.259
9.379
8.209
2.149
5.999
500
7.899
1.829
7.869
8.439
1219
2349
1729
33.29
0.329
29.29
40.29
10.89
38.79
41.49
41.19
1.869
500
500
500
81.29
8.529
7.949
1.429
2.889
1.969
8.209
10.59
1.519
0.3449
500
3.249
/
0.48
0.13
18.4
19.8
1.06
0.46
0.88
0.26
0.66
8.759
9.529
2629
1969
10.69
8.919
8.219
4.679
2.799
2259
7.669
1.769
7.669
9.329
1139
1849
1869
41.09
0.349
6.949
15.39
8.889
36.19
17.69
35.79
2.319
500
/
0.11
1.80
13.4
12.7
2.74
0.035
0.09
2.34
1.16
6
Cl
/0.8
/
/
/
CH3
OCH3
F
ꢀ
ꢀ/
/
/1.2
/
/
/
1d
1e
/2.3
/
/
/
/0.3
/
/
/
1f
Cl
Cl
NO2
/
0.03
0.04
0.06
0.1
/
/
/
1g
2a
2b
2c
/
/
/
/
/
/
/
/
Cl
/
/
/
/
CH3
OCH3
F
/0.1
/
]
/
/
2d
2e
/0.3
/
1.06
1.55
0.10
1.26
49.5
35
/
0.92
0.084
0.23
0.21
9.89
85
/
0.62
0.078
0.8
/
0.02
0.07
0.06
1.7
/
/
/
3a
3b
3c
/
/
/
/
Cl
/
/
/
/
0.20
7.07
48
CH3
OCH3
F
/
/
/
/
3d
3e
/0.5
/
/
/
/
0.08
0.03
0.05
0.08
0.2
/
29.6
5.72
0.38
0.29
10.8
0.11
1.1
/
20.5
2.19
0.0042
16.7
13.1
2.05
4.7
/0.0
3f
Cl
Cl
NO2
/
/
/
/
3.68
0.035
0.96
4.8
3g
4a
4b
4c
/
/
/
/
/
/
/
/
Cl
/
/
/
/
CH3
OCH3
F
/
0.03
0.06
1.4
/
/
/0.49
4d
4e
/
/
/
/2.3
/
/
3.5
/14.8
/6.1
4f
Cl
Cl
/0.1
/
8.3
/1.8
/0.9
5a
5b
5c
/0.13
/
2.3
/0.26
/0.06
Cl
/0.3
/
67
]
/
]/
CH3
OCH3
F
ꢀ/
ꢀ
ꢀ/
/
ꢀ
/
ꢀ
ꢀ/
/
500
500
5d
5e
/0.2
ꢀ/
/0.4
/
1.8
/
53.4
0.16
0.81
0.03
0.59
0.23
0.35
1.7
46.89
8.759
8.999
1.629
7.159
2.089
9.639
9.759
1.509
0.3219
500
2.479
/
26.2
0.72
0.13
0.01
0.014
0.21
2.16
2.04
0.08
0.028
5f
Cl
Cl
/0.2
/
0.8
/
/
5g
5h
6a
6b
6c
NO2
OCH3
/0.2
/
0.9
/
/
OCH3
OCH3
/0.1
/
0.26
2.82
0.80
4.60
1.3
/
/
/0.3
/
/
/
Cl
CH3
OCH3
/
0.01
0.09
0.08
0.02
0.01
5.0
/
/
/
/
/
/
/
6d
7a
7b
7c
/
/
/
/
Á/
Á/
Á/
Á/
Á/
Á/
Á/
Á/
Á/
Á/
Á/
Á/
/
/
0.40
0.049
/0.01
/
/
/
/0.011
/
/
ꢀ/
ꢀ/
ꢀ/
Melphalan
/0.01
/
0.03
/0.79
/0.79
a
For the sake of clarity, only non-hydrogen atoms are indicated.

172
J.R. Dimmock et al. / European Journal of Medicinal Chemistry 38 (2003) 169Á177
/
the results are portrayed in Table 2. Six representative
enones 1b, c, 3b, c, 6a and 6b were screened against three
isolates of Aspergillus fumigatus and one of Candida
albicans. Most of the compounds were evaluated for
murine toxicity.
thienyl analogue 7c had lower potency (or possibly
complete inactivity) in these assays. Thus the 2-nitro-
phenylmethylene and 2-pyridylmethylene groups may be
cytotoxic pharmacophores and future synthetic chemi-
cal strategies should incorporate these groups into
candidate cytotoxic and anticancer agents.
A further analysis of the biodata was undertaken in
order to determine whether correlations existed between
certain physicochemical constants of the aryl substitu-
ents and cytotoxicity. Thus linear, and semilogarithmic
plots were made between the s, p and MR values of the
4. Results and discussion
All of the compounds in series 1Á7 were evaluated
/
against two murine cell lines, namely P388 and L1210
neoplasms, as well as human Molt 4/C8 and CEM T-
lymphocytes. These data are presented in Table 1. The
R1Á
figures in each screen. The P values noted as B
0.05 and B0.01 are summarized in Section 6. The
/
R3 groups in each of the series 1Á
/6 with the IC50
/
0.1, B
/
compounds in series 1Á/7 which display cytotoxicity are
/
believed to do so by alkylation of various cellular
nucleophiles. Hence melphalan was included as the
reference compound in this screen since it is an
established anticancer alkylating agent.
following relationships were established. First, negative
correlations were observed between the Hammett s
values and the IC50 figures in series 1 and 3 (P388,
L1210, Molt 4/C8 and CEM screens), 2 (P388 test) and 6
(P388, Molt 4/C8 and CEM screens). Second, a negative
correlation was obtained between the MR values and
P388 cells in series 1. On the other hand, the MR values
positively correlated with the IC50 figures of series 2 and
4 in the Molt 4/C8 and CEM screens, respectively.
Third, positive correlations were found between the SR
values and the s constants in series 1 as well as the p
values in series 2. No other correlations were noted.
The value of establishing these relationships in drug
design includes the following considerations. The nega-
tive correlations with the s values in series 1, 2, 3 and 6
indicated that potency increased due to a rise in the
magnitude of the Hammett values. This observation is
consistent with the theory that these compounds exert
their bioactivity, at least in part, by thiolation. Thus by
increasing the fractional positive charge on the olefinic
carbon atoms attached at positions two and six of the
cyclohexyl ring, the compounds had an enhanced
electrophilicity for cellular thiols which led to greater
cytotoxicity. Future expansion of these groups of
compounds should incorporate multiple strongly elec-
tron-attracting substituents into the aryl rings. The sizes
and hydrophobicity of the aryl substituents are less of a
determinant factor in influencing cytotoxicity.
Previous studies from these laboratories revealed that
in general P388 cells are more sensitive to conjugated
styryl ketones than L1210, Molt 4/C8 and CEM cells
[12]. Hence the maximum concentration of compounds
used in the P388 test was 50 mM, whereas 500 mM was
the upper limit in the three other screens. Comparisons
between the IC50 values of the compounds in the P388
screen were made with the other three cell lines except
for 1c, d and 5c, since no IC50 figures were available in
the P388 screen for these three compounds. The lowest
IC50 values were invariably found in the P388 screen
except in the cases of 4e and 5a.. The percentage of
compounds with IC50 values less than 10 mM in the
P388, L1210, Molt 4/C8 and CEM screens were 80, 30,
53 and 58, respectively, revealing that an average of 55%
of these molecules display moderate potencies towards
these cell lines. In order to identify those compounds
with the greatest cytotoxicity towards all four cell lines,
the average IC50 values of the most potent compounds
were determined. The following enones were identified
as lead molecules based on their average IC50 values in
mM and their potency compared to the average IC50
value of melphalan, i.e. 2.02 mM, given, respectively, in
parentheses: 7b (0.43, 4.7), 3g (0.58, 3.5), 7a (1.39, 1.5),
5h (1.63, 1.2), 2e (2.73, 0.7), 2a (3.34, 0.6) and 6b (3.40,
0.6). Notably the three most potent compounds, namely
7b, 3g and 7a, have strongly electron-attracting groups
directly attached to the olefinic carbon atoms. These
molecular features would be expected to expedite attack
with cellular thiols.
The data in Table 1 reveal that when the 2,6-
bis(arylidene) groups are constant, cytotoxicity varied
depending on the nature of the R4 and R5 substituents.
The issue of whether the topography of the groups at
position 4 was correlated with cytotoxicity was ad-
dressed using the data of the unsubstituted compounds
1a, 2a, 3a, 4a, 5a and 6a. SASA figures are available for
all of the R4 and R5 groups of these compounds vide
supra and linear and semilogarithmic plots were con-
structed between these data and the IC50 values in each
A comparison of the cytotoxicities of 1g and 5a with
the closely related analogues in series 7 was undertaken.
First, changing the location of the nitro group from the
4 position in 1g to the ortho location as is present in 7a
led to increased cytotoxicity in all four screens. Second,
replacement of the phenylmethylene group of 5a by a 2-
pyridyl function led to 7b which possessed greater
potencies in all four tests. On the other hand, the 2-
of the cytotoxicity screens. No correlations (P ꢀ0.1)
/
were observed. In addition, molecular modeling of 1a,
2a, 3a, 4a, 5a and 6a revealed that when carbon atoms 1,
2 and 6 of the cyclohexyl ring were superimposed, the

J.R. Dimmock et al. / European Journal of Medicinal Chemistry 38 (2003) 169Á
/
177
173
rest of the molecules overlapped completely except when
the R4 and R5 groups were not hydrogen atoms. Thus
variation in the nature of the R4 and R5 groups do not
alter the relative positions of the rest of the molecules.
These observations reinforce the earlier conclusion that
the electron densities on the arylidene methine carbon
atoms are likely the most dominant effect influencing
cytotoxicity.
concentration (MIC) figures of 1b, c, 3b, c, 6a and 6b
towards three isolates of A. fumigatus and one of C.
albicans were in excess of 32 mg mLꢀ1 whereas the MIC
value of a reference antifungal agent voriconazole was
0.25 mg mLꢀ1 towards all four isolates. These repre-
sentative compounds are clearly bereft of marked
potencies towards these fungi. It is possible therefore
that the compounds in series 1Á
/
7 lack antifungal
In order to examine further the potential of 4-
substituted 2,6-bis(arylidene)cyclohexanones as cyto-
toxic agents, four compounds were evaluated by the
National Cancer Institute, USA against a panel of
approximately 56 human tumour cell lines from nine
different types of neoplasms, namely leukemia, mela-
noma and non-small cell lung, colon, central nervous
system, ovarian, renal, prostate and breast cancers [13].
The concentration range used in these experiments is
normally 10ꢀ4 to 10ꢀ8 M. The amount of compound
required to inhibit the growth of various cell lines by
50% is noted. On occasions, a 50% reduction of the
growth of the cells was not achieved when the maximum
concentration of a compound was employed, i.e. 10ꢀ4
M; however this figure of 10ꢀ4 M was used in
calculating the average cytotoxicity towards all cell
lines. Hence the term mean graph midpoint (MG
MID) rather than IC50 was employed.
The data in Table 2 reveal that the enones 2b, 4c, 5a
and 5f have 0.9, 2.2, 4.1 and 1.0 times the potencies of
melphalan, respectively, when cytotoxicity to all cell
lines was considered. An interest in these laboratories is
the discovery of new groups of compounds with potent
activities towards colon cancers [14] and leukemia [15].
Thus, the MG MID values of 2b, 4c, 5a and 5f towards
colon cancer and leukemic cell lines were calculated and
these data are presented in Table 2. These data are
presented in Table 2 along with the related information
for 5-fluorouracil, which is used in treating colorectal
cancers [16], and the antileukemic agent melphalan [17].
The potencies of 4c and 5a were 1.1 and 2.3 times,
respectively, that of 5-fluorouracil towards colon cancer
cells and they possessed 0.8 and 1.3 times, respectively,
the antileukemic activity of melphalan. Taking into
consideration the potencies towards all cell lines and,
in particular, colon and leukemic cells, 4c and 5a are
clearly useful lead molecules.
properties.
A number of alkylating agents used in cancer
chemotherapy have marked mammalian toxicity [21].
Previous work directed to determining murine toxicity
of various compounds containing the a,b-unsaturated
group involved the intraperitoneal administration of
single doses of 30, 100 and 300 mg kgꢀ1 to mice and
observations were made after 0.5 and 4 h [12,22]. A
number of the compounds examined under these con-
ditions proved to be lethal within these time frames and
other evidences of toxicity including neurotoxicity and
respiratory depression were observed. In the present
investigation, 1aÁ
/
g, 2a,cÁ
/
e, 3aÁ
/
g, 4a,b,dÁ
/
f, 5aÁh, 6a,b
/
and 7aÁc were examined in a similar fashion. No deaths
/
of the animals occurred.
Neurotoxicity, as determined by the rotorod test [23],
was absent in two-thirds of the compounds evaluated
and respiratory depression was not observed. In addi-
tion, the enones 1a,g, 5a,d and 7a were administered
orally to rats and at the doses utilized did not display
any overt toxic symptoms. In summary, the animal
experiments were encouraging insofar as at the max-
imum doses employed, the compounds were non-lethal
and in general were well tolerated.
5. Conclusions
A number of 2,6-bis(arylidene)cyclohexanones 1 and
related analogues 2Á7 have been prepared. In general,
/
these compounds displayed moderate potencies towards
P388, L1210, Molt 4/C8 and CEM cells. Four com-
pounds possessed greater or equal potencies as melpha-
lan when examined against a panel of human tumour
cell lines. Representative compounds did not display
significant antifungal activity nor marked murine toxi-
city. Guidelines for the future expansion of these
compounds included the following considerations. First,
positive correlations were noted between the Hammett s
values of the aryl substituents and potencies in a number
of cases. Second, various cytotoxic molecules were
found serving as prototypes for systematic molecular
modification. Thus from the P388, L1210, Molt 4/C8
and CEM assays, 2a,e, 3g, 5h, 6b, 7a and 7b were
identified as lead molecules. The enones 2b, 4c, 5a and 5f
displayed moderate potencies towards a panel of human
tumour cell lines. In addition, future work should be
directed to ascertaining the mode(s) of action of various
A final phase of the investigation sought to explore
further the therapeutic potential of representative com-
pounds. Accordingly evaluation for antifungal activity
and murine toxicity was undertaken.
Previous studies revealed that 5-dimethylamino-1-
phenyl-1-penten-3-one hydrochloride, which contains
an a,b-unsaturated keto group, displayed antifungal
activity [18] due, in part at least, to interference with
mitochondrial function [19]. More recently the antifun-
gal activity of an enone was associated with its inter-
ference with Hꢁ-ATPase [20]. The minimum inhibitory

174
J.R. Dimmock et al. / European Journal of Medicinal Chemistry 38 (2003) 169Á177
/
molecules and undertaking in vivo experiments for
anticancer activity.
hydrochloric acid (37% w/v, 0.5 g) was stirred at room
temperature for 16 h. Methanol was added and the
precipitate was collected and recrystallized from
chloroformÁ
chloroformÁ
The melting points and percentage yields were as
follows: 3a: 144 (lit. [26] mp 146), 85; 3b: 172Á173, 80;
3c: 155 (lit. [26] m.p. 164Á166, 76); 3d: 173Á175, 71; 3e:
173Á174, 72; 3f: 170Á172, 73; 3g: 208Á209, 68. The H-
/
methanol
(15:85),
except
that
6. Experimental protocols
/methanol (1:9) was used in the case of 3b.
6.1. Chemistry
/
/
/
1
Melting points are uncorrected and are in degrees
/
/
/
Celsius. Elemental analyses were carried out for 1aÁ
/
f,
NMR spectrum of a representative compound 3d was as
follows: d (CDCl3): 0.95 (s, 9H, t-C4H9), 1.46 (m, 1H, 4-
CH), 2.15 (m, 2H, 3-CH2), 3.15 (dd, 2H, 5-CH2), 3.83 (s,
2aÁe, 3aÁf, 4aÁf, 5aÁf, 6aÁd, 7c and 4Ábromo-2,6-
/
/
/
/
/
/
bis(3,4-dichlorophenylmethylene)cyclohexanone (C, H)
and 1g, 3g, 5g, 7a and 7b (C, H, N) by Mr. K. Thoms,
Department of Chemistry, University of Saskatchewan.
The results obtained were within 0.4% of the calculated
values except for 2b (calculated for H: 3.58%; found:
3.11%). 1H-NMR spectra were determined on all
compounds using a Bruker AM 500 FT-NMR instru-
ment (500 MHz).
6H, OCH3), 6.93 (d, 4H, Jꢂ/8.74 Hz, aryl H, 7.43 (d,
4H, Jꢂ8.71 Hz, aryl H), 7.71 (s, 2H, Ä
/
/CH).
6.1.4. Synthesis of series 4
Triphenylcarbethoxymethylenephosphorane (15.55 g,
44.68 mmol) which had been prepared by a literature
method [27] was added to a solution of 1,4-cyclohex-
anedione (5.0 g, 44.64 mmol) in benzene (100 mL). The
mixture was heated under reflux for 12 h. The residue
obtained after removal of the solvent in vacuo was
6.1.1. Synthesis of series 1 and 7a
Compounds 1aÁ
previously, were prepared by a literature procedure [24]
in yields of 61Á85%. All compounds were recrystallized
/g and 7a, which have been described
chromatographed on silica gel (60Á100 mesh) using a
/
/
solvent of ethyl acetate in hexane (5:95) to give ethyl 4-
1
oxocyclohexylideneacetate [28] in 71% yield. H-NMR:
from chloroform: methanol (2:8). The melting points
were in accord with those described in the literature. The
1H-NMR spectrum of a representative compound 1d
d (CDCl3): 1.26 (t, 3H, CH3), 2.42Á
6-CH2), 2.60Á2.66 (m, 2H, 3-CH2), 3.12Á
CH2), 4.12Á4.18 (q, 2H, CH2CH3), 5.86 (s, 1H, Ä
/
2.50 (m, 4H, 2-CH2,
3.20 (m, 2H, 5-
CH).
/
/
was as follows: d (CDCl3): 1.77Á
2.88Á2.91 (m, 4H, 3-CH2, 5-CH2), 3.82 (s, 6H, OCH3),
6.91 (d, 4H, Jꢂ9.70 Hz, aryl H), 7.43 (d, 4H, Jꢂ8.77
Hz, aryl H), 7.74 (s, 2H, ÄCH).
/1.79 (m, 2H, 4-CH2),
/
/
/
This ketone was reacted with the appropriate aldehyde
in the same manner as used in the preparation of series 2
to give the crude esters which were purified by recrys-
tallization from methanol (4a), chloroform: methanol
/
/
/
6.1.2. Synthesis of series 2
(15:85) (4b,dÁ
melting points and percentage yields were as follows: 4a:
107Á109, 60; 4b 122, 58; 4c: 121Á122, 51; 4d: 152Á153,
61; 4e: 112Á114, 50; 4f: 162Á
164, 56. The 1H-NMR
spectrum of a representative compound 4d was as
follows: d (CDCl3): 1.26 (t, 3H, CH3), 3.65 (s, 2H, 3-
CH2), 3.85 (s, 6H, OCH3), 4.20 (q, 2H, CH2CH3), 4.35
(s, 2H, 5-CH2), 5.86 (s, 1H, CH COOC2H5), 6.95 (d, 4H,
/
f) or chloroformÁ
/
methanol (2:8) (4c). The
A mixture of 4-bromocyclohexanone (1.0 g, 5.65
mmol), which was prepared in 68% yield by a literature
method [25], the appropriate aryl aldehyde (11.87 mmol)
and hydrochloric acid (37% w/v, 0.5 g) was stirred at
room temperature for 16 h. Methanol (24 mL) was
added and the precipitate was collected and recrystal-
/
/
/
/
/
lized from chloroformÁ
which was recrystallized from methanol. The melting
points and percentage yields were as follows: 2a: 157Á
158, 40; 2b: 192Á193, 35; 2c: 192Á194, 33; 2d: 182 (dec.),
30; 2e: 177Á
179, 45. The 3,4-dichloro analogue (2, R1ꢂ
R2ꢂCl, R3ꢂ
H), mp 189Á190, was prepared in 32%
/methanol (3:7) except for 2a,
Jꢂ
/
8.97 Hz, aryl H), 7.37 (d, 4H, Jꢂ8.64 Hz, aryl H),
/
/
7.69 (s, 1H, CH-aryl), 7.75 (s, 1H, CH-aryl).
/
/
/
/
6.1.5. Synthesis of series 5
/
/
/
A solution of sodium hydroxide (0.1 g, 2.5 mmol) in
water (1 mL) was added to a solution of 1,4-cyclohex-
anedione mono-ethylene ketal (0.7 g, 4.49 mmol) and
the appropriate aryl aldehyde (9.43 mmol) in ethanol
yield and purified by recrystallization from chloroformÁ
/
1
methanol (3:7). The H-NMR spectrum of a represen-
tative compound 2c was as follows: d (CDCl3): 2.35 (s,
6H, CH3), 3.32Á
5-CH2), 4.42-4.48 (m, 1H, 4-CH), 7.23 (d, 4H, Jꢂ
Hz, aryl H), 7.32 (d, 4H, Jꢂ
/
3.40 (m, 2H, 3-CH2), 3.46Á
/
3.54 (m, 2H,
8.92
8.03 Hz), 7.89 (s, 2H, Ä
(95% v/v, 20 mL) at 0Á5 8C. After the reaction mixture
/
/
had reached room temperature, it was stirred for 24 h.
Water (20 mL) was subsequently added and the
precipitate was collected, washed with ethanol (95% v/
v, 5 mL) and recrystallized from ethanol (95% v/v). The
melting points and percentage yields were as follows: 5a:
/
/
CH).
6.1.3. Synthesis of series 3
A mixture of 4-t-butylcyclohexanone (1.54 g, 10
mmol), the appropriate aryl aldehyde (21 mmol) and
165Á
/
166, 62; 5b: 210Á
/
212, 67; 5c: 238Á
/
240, 65; 5d: 224Á
/
226, 68; 5e: 208Á
/
210, 61; 5f: 206Á
/
208, 52; 5g: 244Á/246,

J.R. Dimmock et al. / European Journal of Medicinal Chemistry 38 (2003) 169Á
/
177
175
53; 5h: 162Á
representative compound 5g was as follows:
(CDCl3): 3.10 (s, 4H, 3-CH2, 5-CH2), 3.90 (s, 4H,
OCH2CH2O), 7.55 (d, 4H, Jꢂ8.61 Hz, aryl H), 7.86
(s, 2H, ÄCH), 8.25 (d, 4H, Jꢂ8.61 Hz, aryl H).
/
164, 52. The 1H-NMR spectrum of a
two aryl rings are present in series 1Á
/
6. The linear and
d
semilogarithmic plots were generated using a commer-
cial software package [32]. The following correlations
were noted [physicochemical constant, cytotoxicity
screen, linear (l) or semilogarithmic (sl) plots, [P value]
/
/
/
namely 1: s, P388, l, B
L1210, l, B0.1; 1: s, L1210, sl, B
l, B0.1; 1: s, Molt 4/C8, sl, B0.1; 1: s, CEM, l, B
1: s, CEM, sl, B0.1; 1: MR, P388, l, B0.1; 2: s, P388,
l, B0.1; 2: s, P388, sl, B0.05; 2: MR, Molt 4/C8, l, B
0.01; 2: MR, Molt 4/C8, sl, B0.01; 3: s, P388, l, B0.1;
3: s, P388, sl, B0.01; 3: s, L1210, l, B0.1; 3: s, L1210,
sl, B0.1; 3: s, Molt 4/C8, l, B0.1; 3: s, Molt 4/C8, sl,
0.05; 3: s, CEM, sl, B0.1; 4: MR, CEM, l, B0.1; 6:
s, P388, l, B0.05; 6: s, P388, sl, B0.05; 6: s, Molt 4/
C8, l, B0.1; 6: s, Molt 4/C8, sl, B0.05; 6: s, CEM, l,
0.05; 6: s, CEM, sl, B0.1.
/
0.01; 1: s, P388, sl, B
0.05; 1: s, Molt 4/C8,
0.1;
/
0.01; 1: s,
6.1.6. Synthesis of series 6
4-Hydroxycyclohexanone (4.0 g, 35.1 mmol), pre-
pared by a literature method [29], was dissolved in
/
/
/
/
/
/
/
chloroform (40 mL). The solution was cooled to 0Á
/
5 8C
/
/
/
to which was added pyridine (8.32 g, 105.3 mmol) and
acetic anhydride (7.16 g, 70.2 mmol). The mixture was
stirred at room temperature for 12 h and diluted with
water (50 mL). The organic phase was separated,
washed with hydrochloric acid (7.3% w/v, 50 mL) and
aqueous sodium bicarbonate solution (10% w/v, 50 mL),
and evaporation of the solvent afforded 4-acetoxycyclo-
/
/
/
/
/
/
B
/
/
/
/
/
/
/
B
/
/
hexanone (4.4 g, ꢁ/80%), which was used without
further purification.
6.1.9. Molecular modeling studies
A mixture of 4-acetoxycyclohexanone (1.0 g, 6.41
mmol), the appropriate aryl aldehyde (13.50 mmol) and
hydrochloric acid (36.5% w/v, 0.5 g) was stirred at room
temperature for 20 h. Methanol (20 mL) was added and
the precipitate was collected and recrystallized from
Molecular modeling was undertaken using the
MacroModel version 4.5 programme [4] for the calcula-
tion of the SASA figures, the minimization of the
structures of 1a, 2a, 3a, 4a, 5a and 6a and the overlap
of these six molecules.
methanol (6aÁ
methanol (6d). The melting points and percentage yields
were as follows: 6a: 164Á165 (lit. [30], m.p. 165 8C, 35;
169, 36. The
/
c) or a mixture of chloroform and
6.2. Bioevaluations
/
6b: 181Á
/
182, 40; 6c: 174Á
/
175, 40; 6d: 168Á
/
1H-NMR spectrum of a representative compound 6c
was as follows: d (CDCl3): 1.90 (s, 3H, COCH3), 2.35 (s,
6.2.1. Cytotoxicity determinations
The compounds were evaluated against murine
P388D1 cells using a reported procedure [33]. In brief,
solutions of at least three different concentrations of
compounds in suitable solvents were incubated at 37 8C
with the neoplastic cells and the percentage survival
noted after 48 h. Control experiments in which the
solvents were added to the leukemic cells were also
incubated at 37 8C for 48 h. All tests and control
experiments were carried out in triplicate at each
concentration of the compounds. A similar procedure
was employed for the L1210, Molt 4/C8 and CEM
assays [34].
6H, aryl CH3), 3.08Á
5.22 (m, 1H, 4-CH), 7.20 (d, 4H, Jꢂ
7.33 (d, 4H, Jꢂ8.01 Hz, aryl H), 7.86 (s, 2H, Ä
/
3.18 (m, 4H, 3-CH2, 5-CH2), 5.14Á
7.94 Hz, aryl H),
CH).
/
/
/
/
6.1.7. Synthesis of 7b and 7c
A solution of sodium hydroxide (0.1 g, 0.56 mmol) in
water (1 mL) was added to a solution of 1,4-cyclohex-
anedione mono-ethylene ketal (0.7 g, 4.49 mmol) and 2-
pyridinecarboxaldehyde (1.0 g, 9.5 mmol) in ethanol
(95% v/v, 15 mL) at 0Á5 8C. The mixture was stirred at
/
room temperature for 48 h, diluted with water (25 mL)
and the precipitate was collected and recrystallized from
Compounds 2b, 4c, 5a, 5f, melphalan and 5-fluorour-
methanolÁ
/
chloroform to give 7b, m.p. 164Á
/
165 8C in
181 8C
acil were evaluated against 56 (53Á59) human tumour
/
41% yield. The 2-thienyl analogue 7c, m.p. 180Á
/
cell lines using a previously described methodology [13].
In brief, compounds are evaluated using a minimum of
five concentrations at tenfold dilutions. The time of the
drug exposure to the cells was 48h and a protein assay
using sulphorhodamine B was used to measure cell
protein content and to estimate cell viability or growth
when compared to controls. IC50 values were obtained
for all cell lines except one ovarian tumour (2b), one
breast tumour (5a), two non-small cell lung, one
prostate and one breast neoplasms (5b) as well as one
non-small cell lung, one renal and one breast cancers in
the case of 5-fluorouracil. The number of cell lines in the
colon and leukemic subpanels were seven and six,
1
was prepared in a similar manner in 68% yield. The H-
NMR spectrum of a representative compound 7b was as
follows: d (CDCl3): 3.62 (s, 4H, 3-CH2, 5-CH2), 3.96 (s,
4H, OCH2CH2O), 7.16Á
7.42 (d, 2H, heteroaryl H), 7.67 (m, 4H, heteroaryl H),
8.67 (s, 2H, ÄCH).
/7.17 (m, 2H, heteroaryl H),
/
6.1.8. Statistical analyses
The s, p and MR values used in the statistical
analyses were taken from the literature [31]. The
individual physicochemical constants for the R1, R2
and R3 groups were added and multiplied by two since

176
J.R. Dimmock et al. / European Journal of Medicinal Chemistry 38 (2003) 169Á177
/
respectively, except five leukemic cell lines were used in
evaluating 5a.
References
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6.2.2. Antifungal evaluations
Deliv. 6 (1990) 183Á
/194.
The enones 1b, c, 3b, c, 6a and 6b were evaluated
against three isolates of A. fumigatus (ATCC 208995-
208997) and one isolate of C. albicans (ATCC 90028)
using the broth microdilution method [35]. In this assay,
the MIC of a reference compound voriconazole was 0.25
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.
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Compounds were evaluated for toxicity in mice or
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Doses of 30,100 and 300 mg/kg of 1a-g, 2a, cÁ
/
e, 3aÁg,
/
4a, b, dÁf, 5aÁh, 6a, b and 7aÁc were injected
/
/
/
/
intraperitoneally into mice and the animals observed
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number of animals displaying neurotoxicity/number of
animals in the experiment and the dose in mg/kg): 2a (2/
8, 100; 1/4, 300), 2c (4/8, 100; 4/4, 300); 3a (1/4, 30; 1/8,
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city was observed in the following cases, namely 2a (1/2,
300), 2c (1/4, 100), 3a (2/2, 300), 5b (1/2, 300), 5g (1/4,
100) and 7b (1/4, 100).
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Evaluation for neurotoxicity after oral dosing to rats
was also undertaken using 1a, g, 7a (30 mg/kg) and 5a, d
(50 mg/kg). The animals were observed after 0.25, 0.5, 1,
2 and 4 h, except in the case of 5d, where the 0.25 and 4
h times were omitted. No toxicity was observed. The
Anticonvulsant Screening Program of the National
Institute of Neurological Disorders and Stroke, USA
requires that all mice and rats be housed, fed and
handled in a manner consistent with the recommenda-
tions of the National Research Council Publication
‘Guide for the Care and Use of Laboratory Animals’.
All animals are euthanized in accordance with the
policies of the Institute of Laboratory Resources dealing
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/
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The following organizations are thanked for their
financial support of this study, namely Purdue Neu-
roscience Company, USA (J. R. D.), National Cancer
Institute of Canada (T. M. A.), Flemish Fonds Voor
Geneeskundig Weterschappelijk Onderzoek (E. D. C., J.
B.) and the National Institute for Neurological Dis-
orders and Stroke (J. P. S.). The National Cancer
Institute, USA, is thanked for the evaluations using a
panel of human tumour cell lines.
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