E,E-2-Benzylidene-6-(nitrobenzylidene)cyclohexanones: Syntheses, cytotoxicity and an examination of some of their electronic, steric, and hydrophobic properties

Article abstract of DOI:10.1016/j.bmc.2008.04.029

Three series of structurally isomeric 2-benzylidene-6-(nitrobenzylidene) cyclohexanones 1-3 were prepared and evaluated against human Molt/C8 and CEM T-lymphocytes as well as murine L1210 cells. The IC₅₀ values of the majority of compounds are

Full text of DOI:10.1016/j.bmc.2008.04.029

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 6261–6268
E,E-2-Benzylidene-6-(nitrobenzylidene)cyclohexanones:
Syntheses, cytotoxicity and an examination of some of their
electronic, steric, and hydrophobic properties
Umashankar Das,a Alireza Doroudi,a, Swagatika Das,a Brian Bandy,a Jan Balzarini,b
Erik De Clercqb and Jonathan R. Dimmocka,*
aCollege of Pharmacy and Nutrition, University of Saskatchewan, 110 Science Place, Saskatoon, Sask., Canada S7N 5C9
bRega Institute of Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received 27 December 2007; revised 9 April 2008; accepted 14 April 2008
Available online 16 April 2008
Abstract—Three series of structurally isomeric 2-benzylidene-6-(nitrobenzylidene) cyclohexanones 1–3 were prepared and evaluated
against human Molt/C8 and CEM T-lymphocytes as well as murine L1210 cells. The IC50 values of the majority of compounds are
less than 10 lM and in some assays, the figures for 1d and 1e are submicromolar. Correlations were discerned between cytotoxic
potencies and the atomic charges on one of the olefinic carbon atoms, the torsion angles between an aryl ring, and the adjacent
unsaturated group as well as logP values. Three representative compounds were examined for their effect on respiration in rat liver
mitochondria.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
substituents in each of the aryl rings differed in their
electronic properties.7,8 In these molecules, the charges
on the olefinic carbon atoms are predicted to be diver-
gent thereby enhancing sequential reactions.
The principal interest in our laboratory is the syntheses
of a variety of conjugated styryl ketones as candidate
antineoplastic agents. These compounds are thiol alky-
lators having little or no capacity to interact with amino
or hydroxy groups1,2; since these latter groups, but not
thiols, are found in nucleic acids, enones may be devoid
of the genotoxic problems displayed by a number of
anticancer drugs.3 Recently molecules have been de-
signed to enable successive alkylation of thiols to occur
since on occasion sequential reactions with cellular con-
stituents have been claimed to be more detrimental to
malignant cells than the corresponding normal tissues.4
These considerations led to the decision to prepare a
number of compounds which contain the 1,5-diaryl-3-
oxo-1,4-pentadienyl pharmacophore (ArCH@CR–CO–
CR@CHAr)5,6 thereby allowing stepwise alkylation of
cellular thiols. Recently a small number of 2,6-bis(ben-
zylidene) cyclohexanones were prepared in which the
The objectives of the present study were twofold. First,
an evaluation was planned of the hypotheses that cyto-
toxic potencies were correlated with both the charge
densities and the steric environment of the olefinic car-
bon atoms. Second, the original series consisted of a
small group of compounds which possessed widely dif-
fering potencies in the Molt 4/C8, CEM, and L1210 bio-
assays.8 Hence expansion of the cluster of compounds
was indicated in order to draw meaningful conclusions
pertaining to those structural features which contribute
to cytotoxicity.
In ring A of series 1, the strongly electron-attracting 2-
nitro group was proposed which should cause the ole-
finic carbon atoms, designated CA and CB as indicated
in Figure 1, to be electron deficient thereby enhancing
thiol alkylation. Substituents with varying Hammett sig-
ma (r) values were considered for insertion onto ring B,
including the 3,4,5-trimethoxy group due to our recent
disclosure of the cytotoxicity of compounds containing
the 3-(3,4,5-trimethoxyphenyl)-2-propenoyl substitu-
ent.9 In addition, the rate of electrophilic attack with
Keywords: Unsaturated ketones; Molecular modeling; Cytotoxicity;
Mitochondria.
*
Corresponding author. Tel.: +1 306 966 6331; fax: +1 306 966
6377; e-mail: jr.dimmock@usask.ca
Present address: School of Pharmacy, Ahwaz Jondishapur Univer-
sity of Medical Sciences, Ahwaz, Khouzestan, Iran.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.029

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U. Das et al. / Bioorg. Med. Chem. 16 (2008) 6261–6268
monobenzylidene analogs 4a–c were also proposed.
The general structures of these compounds are presented
in Figure 1.
θΑ
θΒ
O
CA
θΑ
CB
O
CA
R2
R3
R1
R1
A
B
A
R4
4
1-3
2. Results
1a-g: R1= 2-NO2
2a-g: R1= 3-NO2
3a-g: R1= 4-NO2
4a: R1= 2-NO2
4b: R1= 3-NO2
4c: R1= 4-NO2
The compounds in series 1–4 were prepared by the
synthetic routes outlined in Scheme 1. The majority
of the bis(benzylidene)cyclohexanones were prepared
by condensation of 4a, 4b, or 4c with various aryl
aldehydes under acidic conditions. However, attempts
to use this procedure in the preparation of 1a, 1c,
2a, and 3a led to the formation of dark polymeric
material from which no products were obtained. Un-
der basic conditions, 2-(4-dimethylaminobenzylidene)
cyclohexanone 5 and the related 4-methyl analog 6 re-
acted with the appropriate nitrobenzaldehyde to afford
1c and 2a. However under these conditions, reaction
of 5 with both 2-nitrobenzaldehyde and 4-nitroarylal-
dehydes led to the formation of multiple products
but under acidic conditions, both 1a and 3a were
formed. Initial attempts to prepare 4a–c from cyclo-
hexanone and the relevant nitrobenzaldehyde under
acidic conditions led to isolation of the corresponding
2,6-bis(nitrobenzylidene)cyclohexanones. In the pres-
ence of sodium hydroxide solution, the 2-nitro and
Figure 1. General structures of series 1–4. The R2, R3, and R4
substituents in series 1–3 are as follows, namely a: R2 = R4 = H,
R3 = N(CH3)2; b: R2 = R4 = H, R3 = OCH3; c: R2 = R4 = H,
R3 = CH3; d: R2 = R3 = R4 = OCH3; e: R2 = R3 = R4 = H; f:
R2 = R4 = H, R3 = F; g: R2 = R4 = H, R3 = Cl.
thiols will be influenced by the topography of the mole-
cules in the environment of the CA and CB atoms. Hence
the determination of the torsion angles hA and hB cre-
ated between the aryl rings A and B with the adjacent
olefinic carbon atoms was suggested. Such consider-
ations led to the decision to prepare series 1. In order
for these hypotheses to be examined further, the place-
ment of the nitro group in other locations of ring A
was planned leading to series 2 and 3. In addition, to as-
sist in the understanding of those structural features in
series 1–3 which contribute to cytotoxic potencies, the
O
O
CHO
O
R= N(CH3)2
i
R1
ii, iii
iv
+
N
N
R
5
1a: R1= 2-NO2
3a: R1= 4-NO2
R = CH3
O
NO2
O
O
v
O2N
CH3
CH3
N
1c
6
2a
O
O
CHO
R2
R3
vi
R1
R1
+
R4
R2
R4
R3
1b, d-g: R1= 2-NO2
2b-g: R1= 3-NO2
3b-g: R1= 4-NO2
4a-c
O
N
O
O
O
OH O
O2N
viii
i
v,vii
ix
R1
R1
4a: R1= 2-NO2
4c: R1= 4-NO2
4b
Scheme 1. Synthetic chemical routes in the preparation of the compounds in series 1–4. Reagents: (i) NaOH; (ii) HCl/2-NO2C6H4CHO; (iii) HCl/4-
NO2C6H4CHO; (iv) NaOH/3-NO 2C6H4CHO; (v) NaOH/2–NO2C6H4CHO; (vi) HCl; (vii) NaOH/4-NO2C6H4CHO; (viii) morpholine/4-
CH3C6H4SO2OH; (ix) 3-NO2C6H4CHO.

U. Das et al. / Bioorg. Med. Chem. 16 (2008) 6261–6268
6263
4-nitro benzaldehydes condensed with cyclohexanone
35
30
25
20
15
10
5
to produce the intermediate aldols which were dehy-
drated by acid to give 4a and 4c, respectively.
Under basic conditions, the 3-nitroaldehyde gave only
2,6-bis(3-nitrobenzylidene)cyclohexanone. Hence 4b
was prepared via the enamine route as illustrated in
Scheme 1. 1H NMR spectroscopy revealed that each
of the products in series 1–4 was isomerically pure.
The absorptions of the olefinic protons were in the
region of 7.47–7.97 ppm which is characteristic of E
isomers, since compounds possessing the Z configura-
tion absorb at higher fields.10 Furthermore, X-ray
crystallography revealed that the olefinic double bonds
adopted the E configuration in 3c11 and 3f12 as well as
1d
2d
3d
DMSO
0
1d
2d
3d
DMSO
a
related 2,6-bis(benzylidene)cyclohexanone.13 The
Compounds
assumption was made therefore that the olefinic bonds
in series 1–4 adopted the E configuration. Models of
the compounds in series 1–4 were built and the charge
densities of the CA and CB atoms as well as the tor-
sion angles hA and hB were determined and are pre-
sented in Table 2. In addition, the logP values of
all of the compounds were obtained and are portrayed
in Table 2.
Figure 2. The effect of 1d, 2d, 3d, and solvent control (dimethylsulf-
oxide 4 lL) on respiration in rat liver mitochondria. The figures for 1d,
2d, and 3d are different from each other and the solvent control taking
standard deviations into account.
3. Discussion
The bioevaluations of 1a–g, 2a–g, 3a–g, and 4a–c toward
three cell lines are presented in Table 1. The IC50 values
of 1d and 1e are submicromolar in some of the bioassays
and 58% of the IC50 values were less than 10 lM. In
view of these promising results, various studies were ini-
tiated to seek correlations between cytotoxic potencies
and different physicochemical and biochemical parame-
ters of these molecules with the aim of obtaining guide-
lines for expansion of this project.
All the compounds in series 1–4 were evaluated against
human Molt 4/C8 and CEM T-lymphocytes and murine
L1210 lymphoid leukemia cells. These data are summa-
rized in Table 1. The effect of representative compounds
on respiration in mitochondria isolated from rat liver
cells is presented in Figure 2.
Table 1. Cytotoxic properties of compounds 1–4
a
Compound
IC50 (lM)
Table 2. Some physicochemical properties of compounds 1–4
Molt 4/C8
122
CEM
L1210
164 27
Compound
Atomic chargesa
Torsion anglesb
logP
1a
1b
6
168 36
7.45 0.08
6.09 2.12
0.402 0.033
0.925 0.056
1.51 0.04
1.75 0.14
11.7 0.8
8.22 0.12
10.1 0.6
45.2 7.5
2.29 0.75
5.05 3.02
1.75 0.00
>500
qA
qB
hA
hB
8.90 0.20
7.52 0.45
0.702 0.22
1.48 0.11
1.87 0.06
3.86 1.00
10.9 0.8
7.98 0.54
9.53 1.23
44.0 2.7
1.70 0.42
5.12 2.31
2.02 0.28
>500
42.4 1.3
7.77 0.45
1.52 0.29
4.84 0.40
8.40 0.13
9.38 0.47
156 134
29.5 9.8
41.8 3.7
42.2 3.3
9.44 1.07
16.3 0.3
9.16 0.87
>500
1a
1b
1c
1d
1e
1f
À0.086
À0.081
À0.082
À0.092
À0.133
À0.130
À0.130
À0.093
À0.089
À0.083
À0.079
À0.087
À0.085
À0.084
À0.092
À0.096
À0.089
À0.086
À0.095
À0.092
À0.092
À0.092
À0.083
À0.090
À0.032
À0.041
À0.047
À0.053
À0.055
À0.058
À0.061
À0.028
À0.042
À0.045
À0.053
À0.053
À0.056
À0.059
À0.022
À0.039
À0.043
À0.053
À0.050
À0.053
À0.056
—
76.79
76.76
À51.12
À51.27
51.58
À51.53
À51.86
51.55
51.72
50.72
51.12
51.42
51.83
51.56
51.36
51.58
50.98
51.09
51.41
À51.86
51.56
51.37
51.57
—
4.80
4.76
5.15
4.33
4.70
4.86
5.38
5.01
4.96
5.35
4.53
4.90
5.07
5.58
5.03
4.98
5.38
4.56
4.93
5.09
5.61
2.92
3.12
3.14
1c
1d
À76.83
76.90
76.59
1e
1f
1g
À76.79
À76.84
À51.30
À51.28
À51.27
À51.32
À51.27
À51.25
À51.24
À51.14
À51.11
À51.14
51.17
2a
2b
1g
2a
2b
2c
2d
2e
2f
2c
2d
2e
2f
2g
3a
3bb
3c
300 54
250 6
240 8
2g
3a
3b
3c
3d
3e
3f
8.44 0.49
6.42 1.07
8.35 0.95
17.1 4.6
9.12 0.28
33.3 3.1
8.28 0.69
13.9 1.0
3.24 0.79
8.53 0.31
4.61 3.89
9.32 0.20
18.6 6.9
8.18 0.20
36.4 1.3
8.12 0.92
19.3 1.5
2.47 0.30
8.16 0.35
6.97 1.80
9.80 0.18
26.8 2.8
9.41 0.97
23.8 12.4
50.1 10.4
46.5 9.1
2.13 0.03
3db
3e
3f
3gb
4a
À51.14
À51.14
À51.16
À69.54
À51.50
À50.68
3g
4a
4b
4c
4b
4c
Melphalanb
—
—
—
a The IC50 values indicate the concentrations of compounds required
to inhibit the growth of the cells by 50%.
b The data were previously reported in Ref. 8 [copyright (2006) by
Elsevier].
a The atomic charges in esu are the electron densities on the carbon
atoms designated CA and CB in Figure 1.
b hA and hB refer to the torsion angles which are illustrated in Figure 1.

6264
U. Das et al. / Bioorg. Med. Chem. 16 (2008) 6261–6268
Interactions with cellular thiols are believed to occur at
the olefinic carbon atoms designated CA and CB. The
atomic charges on these atoms in the compounds 1–4
are presented in Table 2. The nitro group in ring A is
the most electron-attracting substituent having a Taft
r* value of 0.9714 (series 1 and 4a) and Hammett r val-
ues of 0.71 (series 2 and 4b) and 0.78 (series 3 and 4c).15
The r constants for the ring B substituents in a–g are
À0.83, À0.27, À0.17, À0.03, 0.00, 0.06, and 0.23, respec-
tively, 16 and are arranged in sequence with a bearing the
most electron-repelling group and g the most electron-
attracting substituent. For each compound in series 1–
3 , the electron densities are lower on the CB rather than
the CA atoms. Thus the polarization of the p electrons in
the conjugated 1,5-diaryl-3-oxo-1,4-pentadienyl group is
toward the nitro substituents, causing the CB atom to
have lower electron densities than CA. Hence thiol alkyl-
ation is predicted to take place initially at CB and subse-
quently at CA. In order to examine whether cytotoxic
potencies are correlated with the electron densities on
the CA and CB atoms, linear plots were made between
these values and the IC50 figures of 1a–g in each of the
three bioassays.This experiment was repeated with 2a–
g and finally with 3a–g. Positive correlations (p < 0.05)
were noted when considering the atomic charges on
the CB atoms except for the Molt 4/C8 and CEM bioda-
ta for series 2. No correlations were noted between the
IC50 figures and the charges on the CA atom (p >
0.05). This evaluation was repeated except that the
IC50 values were plotted against the r constants in ring
B. Negative correlations (p < 0.01) were obtained in all
cases except for 2a–g in the Molt 4/C8 and CEM tests
(p > 0.05). Thus potency increases (IC50 values dimin-
ish) as the electron densities on the CB atom are de-
creased (positive correlation) and the r constants are
elevated (negative correlation). This observation may
be rationalized by considering that attack of cellular thi-
ols at CB will be enhanced by a reduction in the atomic
charges on the CB atoms. Thus in the future, compounds
may be designed having substituents in ring B which
have large positive sigma values.
ene ring onto 4a–c leading to series 1–3, respectively,
caused only minimal changes in the CA and hA values
as the data in Table 2 indicate.
The biodata in Table 1 were examined further with a
view to discerning those structural features which influ-
ence cytotoxic potencies. First, the optimal position of
the nitro group in ring A was considered. A point system
of 3 (highest potency), 2, and 1 (lowest potency) was
used in comparing the IC50 values of compounds having
the same substituents in ring B. Thus in the Molt 4/C8
assay, the IC50 figures of 1a, 2a, and 3a were compared,
then 1b, 2b, and 3b and so forth. Standard deviations
were taken into account and when the IC50 values were
statistically indistinguishable, equal points were allo-
cated bearing in mind that for each comparison of three
compounds, a total of six points were invariably
awarded. Use of this methodology indicated that the fig-
ures for series 1, 2, and 3 are 16.5, 16.5, and 9, respec-
tively (Molt 4/C8 assay), 19.5, 13.5, and 9, respectively
(CEM test) and 18, 13, and 11, respectively (L1210
screen). Hence the optimal position of the nitro group
in ring A in terms of potency is the 2 position.
In order to assess whether the compounds in series 1–3,
which permit sequential alkylation to occur, have in-
`
creased cytotoxic potencies vis-a-vis the analogs in which
this process will not occur (series 4), the IC50 values of1a–g,
2a–g, and 3a–g were compared with those generated for
4a, 4b, and 4c, respectively. The results are summarized
in Table 3. In general, structural modification of 4a, 4b,
and 4c into series 1, 2, and 3, respectively, was accompa-
nied by increases in potencies in all three bioassays except
conversion of 4b into 2a–g did not lead to compounds
with lower IC50 values toward CEM cells.
The rate and extent of the ability of compounds to pen-
etrate the cell membranes of neoplastic and transformed
cells is dependent on a number of structural features
including the lipophilicity of the molecules. The logP
values of the compounds were calculated and are pre-
sented in Table 1. The average logP values for 1a–g,
2a–g, and 3a–g are 4.86, 5.06, and 5.08, respectively,
and hence the lower lipophilicity of the compounds in
series 1 may have contributed to the greater potencies
than the analogs in series 2 and 3. The generally lower
IC50 potencies displayed by the compounds in series 1–
3 than 4a–c also reflect a negative correlation with the
logP values.
Consideration was given to the possibility that the steric
environment at the olefinic carbon atoms influences the
extent of thiol alkylation and hence cytotoxic potencies.
Thiolation is believed to occur initially at CB and the hB
values recorded in Table 2 reveal that they are virtually
constant. Thus the average hB values for series 1, 2, and
3 are 51.5ꢁ, 51.4ꢁ, and 51.4ꢁ, respectively, and there are
very small variations in these torsion angles within each
series. Hence the differences in cytotoxic potencies are
unlikely to be influenced by the torsion angles hB. The
average hA angles in series 1, 2, and 3 are 76.8ꢁ, 51.3ꢁ,
and 51.1ꢁ, respectively, and minimal variation of these
torsion angles was noted within each series. Since the
cytotoxic potencies of the compounds in series 1 are
greater than the analogs in series 2 and 3 vide infra,
these torsion angles may influence the magnitude of
the cytostatic effect. Hence in the future, groups with
larger molecular refractivity values than nitro group
should be placed in the 2 position of ring A or two ortho
substituents should be employed which may lead to
more potent analogs. The insertion of a second arylid-
Table 3. Comparison between the potencies of the bisalkylators 1a–g,
2a–g, and 3a–g with the monobenzylidene analogs 4a, 4b, and 4c,
respectively
Bioassay
Comparison of potenciesa
1a–g 4a Equal 2a–g 4b Equal 3a–g 4c Equal
Molt 4/C8 86
14
14
29
0
0
0
43
29
57
29 29
43 29
57
57
71
29 14
29 14
CEM
L1210
86
71
0
43
29
0
Total
81
19
0
43
24 33
62
29
9
a The figures represent the percentage of compounds displaying greater
potency or were equipotent. The standard deviations of the IC50
figures were taken into account when making these comparisons.

U. Das et al. / Bioorg. Med. Chem. 16 (2008) 6261–6268
6265
Various compounds which are thiol reagents such as N-
ethylmaleimide and mersalyl interact with different mer-
capto groups in mitochondria.17 Furthermore, a Man-
nich base of a conjugated styryl ketone inhibited
respiration in rat liver mitochondria and the mode of ac-
tion, at least in part, was based on competition with the
conjugated unsaturated ketone coenzyme Q10.18 Thus
the decision was made to determine whether representa-
tive compounds interfered with respiration in mitochon-
dria isolated from rat liver cells, and if so whether the
magnitude of this effect correlated with cytotoxic poten-
cies. Three related compounds, namely, 1d, 2d, and 3d,
were chosen since they possessed markedly different
potencies having average IC50 figures of 0.88, 43.8,
and 6.00 lM, respectively, in the three cytotoxicity
screens. A concentration of 10 lM of each compound
was chosen which is in excess of the IC50 values of 1d
and 3d and substantially below that of 2d. The data in
Figure 2 reveal that 1d and 3d stimulated respiration.
However, the magnitude of the stimulatory effect was
negatively correlated with cytotoxic potencies. The least
potent of these three compounds, namely 2d, had virtu-
ally no effect on respiration. Increasing the concentra-
tion of 2d to 100 lM revealed no statistically
significant difference in stimulation of respiration from
the solvent control (data not shown). Nevertheless if
the causes for the relative cytotoxic potencies observed
in this study are multifactorial, the differences in the ef-
fects on mitochondrial function may have exerted some
contributions to the disparity of IC50 values.
130 ꢁC [lit.19] 127–127.5 ꢁC]. 1H NMR (CDCl3): 1.79
(p, 2H), 1.91 (p, 2H), 2.52 (t, 2H), 2.88 (t, 2H), 3.03
(s, 6H, 2· NCH3), 6.71 (d, 2H, Ar–H, J = 8.85 Hz),
7.41 (d, 2H, J = 8.83 Hz), 7.55 (s, 1H, @CH).
Dry hydrogen chloride was passed into a solution of 5
(0.005 mol) and 2-nitrobenzaldehyde (0.005 mol) in ace-
tic acid (15 mL) and the mixture stirred at room tempera-
ture overnight. Acetic acid was removed in vacuo and the
residue triturated with potassium carbonate solution
(10% w/v, 20 mL) and extracted with chloroform. The or-
ganic extract was washed with water and dried. Evapora-
tion of the solvent gave a semisolid which was purified by
chromatography using a column of silica gel 60 (70–
230 mesh) and an eluting solvent of 10–30% ethyl acetate
1
in hexane to produce 1a, mp 152 ꢁC in 41% yield. H
NMR (CDCl3): d 1.79 (p, 2H), 2.60 (t, 2H), 2.96 (t, 2H),
3.05 (s, 6H, 2· N(CH3)2), 6.73 (d, 2H, Ar–H, J = 8.80
Hz), 7.38 (d, 1H, Ar–H, J = 7.60 Hz), 7.49 (m, 3H, Ar–
H), 7.64 (t, 1H, Ar–H), 7.84 (s, 1H, @CH), 7.95 (s, 1H,
@CH), 8.13 (d, 1H, Ar–H, J = 8.20 Hz). Anal. Calcd
for C22H22N2O3: C, 72.91; H, 6.12; N, 7.73. Found: C,
72.62; H, 5.98; N, 7.53%.
Aqueous sodium hydroxide solution (20% w/v, 1 mL)
was added to a solution of 5 (0.005 mol) and 3-nitro-
benzaldehyde (0.005 mol) in ethanol (15 mL) at 8–
10 ꢁC. The solution was stirred at room temperature
for 0.5 h. The resultant precipitate was collected, washed
with water (3· 15 mL), dried and crystallized from chlo-
roform/ethanol (1:9) to give 2a, mp 169 ꢁC in 68% yield.
1H NMR (CDCl3): d 1.86 (p, 2H), 2.93 (t, 2H), 3.01 (t,
2H), 3.07 (s, 6H, 2· N(CH3)2), 6.75 (d, 2H, Ar–H,
J = 8.84 Hz), 7.50 (d, 2H, Ar–H, J = 8.82Hz), 7.60 (t,
1H, Ar–H), 7.76 (d, 1H, Ar–H, J = 7.7 Hz), 7.79 (s,
1H, @CH), 7.84 (s, 1H, @CH), 8.20 (d, 1H, Ar–H,
J = 8.18 Hz), 8.32 (s, 1H, Ar–H). Anal. Calcd for
C22H22N2O3: C, 72.91; H, 6.12; N, 7.73. Found: C,
72.87; H, 6.0; N, 7.46%.
4. Conclusions
A number of novel cytotoxic agents have been prepared,
many of which display good potencies toward Molt 4/
C8, CEM, and L1210 cells. The highest potencies were
displayed by the compounds in series 1 and in particular
1d and 1e are lead molecules having submicromolar IC50
values in some of the assays. Factors which influence
cytotoxic potencies in series 1–3 include the atomic
charges on the CB atoms, the torsion angle hA, and logP
values. Another factor which may have contributed to
the variation in IC50 values is the differences in the ef-
fects on respiration in rat liver mitochondria. A number
of guidelines for amplifying this project have been
proposed.
Dry hydrogen chloride was passed into a solution of 5
(0.005 mol) and 4-nitrobenzaldehyde (0.005 mol) in ace-
tic acid (15 mL) and the mixture was stirred overnight at
room temperature. The precipitate was collected, washed
with diethyl ether (2· 10 mL), and potassium carbonate
solution (10% w/v, 30 mL). The solid obtained was
washed with water (3· 10 mL), dried and crystallized
from chloroform/ethanol (1:9) to give 3a, mp 91–92 ꢁC
1
in 66% yield. H NMR (CDCl3): d 1.86 (p, 2H), 2.91 (t,
5. Experimental
2H), 3.0 (t, 2H), 3.07 (s, 6H, 2· N(CH3)2), 6.75 (d, 2H,
Ar–H, J = 8.84 Hz), 7.50 (d, 2H, Ar–H, J = 8.84 Hz),
7.60 (d, 2H, Ar–H, J = 8.63 Hz), 7.79 (s, 1H, @CH),
7.84 (s, 1H, @CH), 8.27 (d, 2H, Ar–H, J = 8.65 Hz).
Anal. Calcd for C22H22N2O3: C, 72.91; H, 6.12; N, 7.73.
Found: C, 72.94; H, 6.13; N, 7.52%.
5.1. Synthesis of compounds
Melting points in Celsius degrees were determined on a
Gallenkamp apparatus and are uncorrected. H NMR
1
spectra were recorded using a Bruker AMX 500 FT ma-
chine while elemental analyses were obtained using an
Elementer analyzer.
5.1.2. Synthesis of 1c. 2-(4-Methylbenzylidene)cyclohex-
anone 6 was prepared by a literature procedure20 and
crystallized from methanol to give 6, mp 71 ꢁC [lit.20
1
5.1.1. Syntheses of 1a, 2a, and 3a. 2-(4-Dimethylamino-
benzylidene)cyclohexanone 5 was prepared by a re-
ported procedure19 and crystallized from ethanol at 5–
6 ꢁC to give the desired product in 45% yield, mp
mp 60 ꢁC] in 40% yield. H NMR (CDCl3): 1.78 (p,
2H), 1.92 (m, 2H), 2.38 (s, 3H, CH3), 2.53 (t, 2H),
2.86 (t, 2H), 7.21 (d, 2H, Ar–H, J = 7.90 Hz), 7.32 (d,
2H, Ar–H, J = 7.96 Hz), 7.50 (s, 1H, @CH).

6266
U. Das et al. / Bioorg. Med. Chem. 16 (2008) 6261–6268
Aqueous sodium hydroxide solution (20% w/v, 1 mL)
was added to a solution of 6 (0.005 mol) and 2-nitro-
benzaldehyde (0.005 mol) in ethanol (15 mL) at 8–
10 ꢁC. The solution was stirred at room temperature
for 0.5 h. The reaction mixture was acidified with di-
lute hydrochloric acid and extracted with chloroform.
Evaporation of the organic solvent gave a viscous oil
which was purified by chromatography using a col-
umn of silica gel 60 (70–230 mesh) and an eluting sol-
vent of ethyl acetate/hexane (1:9) to give 1c, mp
120 ꢁC in 30% yield. 1H NMR(CDCl3): d 1.78 (p,
2H), 2.40 (s, 3H, CH3), 2.62 (p, 2H), 2.94 (t, 2H),
7.24 (d, 2H, Ar–H, J = 7.94 Hz), 7.39 (t, 3H, Ar–
H), 7.52 (t, 1H, Ar–H), 7.65 (t, 1H, Ar–H), 7.83 (s,
1H, @CH), 7.96 (s, 1H, @CH), 8.14 (d, 1H, Ar–H).
Anal. Calcd for C21H19NO3: C, 75.66; H, 5.74; N,
4.20. Found: C, 75.42; H, 5.71; N, 4.27%.
1H, Ar–H), 7.80 (s, 1H, @CH), 7.97 (s, 1H, @CH),
8.15 (d, 1H, Ar–H, J = 8.20 Hz). Anal. Calcd for
C20H16FNO3: C, 71.21; H, 4.78; N 4.15. Found: C,
70.93; H, 4.79; N 3.88%.
5.1.3.5. E,E-2-(4-Chlorobenzylidene)-6-(2-nitrobenzyl-
idene)cyclohexanone (1g). Mp 149 ꢁC; yield 41%. 1H
NMR (CDCl3): d 1.79 (p, 2H), 2.63 (t, 2H), 2.84 (t,
2H), 7.40 (m, 5H, Ar–H), 7.53 (t, 1H, Ar–H), 7.66 (t,
1H, Ar–H), 7.77 (s, 1H, @CH), 7.97 (s, 1H, @CH),
8.15 (d, 1H, Ar–H, J = 8.2 Hz). Anal. Calcd for
C20H16ClNO3: C, 67.90; H, 4.56; N, 3.96. Found: C,
67.70; H 4.61; N 3.78%.
5.1.3.6. E,E-2-(4-Methoxybenzylidene)-6-(3-nitroben-
1
zylidene)cyclohexanone (2b). Mp 114 ꢁC; yield 68%. H
NMR (CDCl3): d 1.85 (p, 2H), 2.94 (t, 2H), 2.98 (t,
2H), 6.96 (d, 2H, Ar–H, J = 8.50 Hz), 7.49 (d, 2H, Ar–
H, J = 8.45 Hz), 7.60 (t, 1H, Ar–H), 7.75 (d, 1H, Ar–
H, J = 7.7 Hz), 7.79 (s, 1H, @CH), 7.81 (s, 1H, @CH),
8.19 (d, 1H, Ar–H, J = 8.10 Hz), 8.32 (s, 1H, Ar–H).
Anal. Calcd for C21H19NO4: C, 72.19; H, 5.48; N 4.01.
Found: C, 71.91; H, 5.46; N, 3.90%.
5.1.3. Synthesis of 1b, d–g, 2b–g, and 3b–g. The enones
1b, d–g, 2b–g, and 3b–g were prepared by the following
general procedure. Dry hydrogen chloride was passed
into a solution of 4a , 4b, or 4c vide infra (0.005 mol)
and the appropriate aryl aldehyde (0.006 mol) in ether
(40 mL) and methanol (4 mL). The reaction mixture
was stirred at room temperature for 24 h and the resul-
tant solid was collected and crystallized from chloro-
form/methanol (1:3).
5.1.3.7. E,E-2-(4-Methylbenzylidene)-6-(3-nitrobenzyl-
idene)cyclohexanone (2c). Mp 156 ꢁC; yield 60%. 1H
NMR (CDCl3): d 1.84 (p, 2H), 2.94 (t, 2H), 2.98 (t,
2H), 7.25 (t, 2H, Ar–H), 7.41 (d, 2H, Ar–H, J = 7.85
Hz), 7.60 (t, 1H, Ar–H), 7.76 (d, 2H, Ar–H,
J = 7.65 Hz), 7.81 (s, 1H, @CH), 7.82 (s, 1H, @CH),
8.20 (d, 1H, Ar–H, J = 8.20 Hz), 8.32 (s, 1H, Ar–H).
Anal. Calcd for C21H19NO3: C, 75.66; H, 5.74; N,
4.20. Found: C, 75.58; H, 5.88; N, 3.92%.
5.1.3.1. E,E-2-(4-Methoxybenzylidene)-6-(2-nitroben-
1
zylidene)cyclohexanone (1b). Mp 157 ꢁC; yield 80%. H
NMR (CDCl3): d 1.79 (p, 2H), 2.61 (t, 2H), 2.94 (t,
2H), 3.86 (s, 3H, OCH3), 6.98 (d, 2H, Ar–H, J = 8.3
Hz), 7.38 (d, 1H, Ar–H, J = 7.65 Hz), 7.48 (d, 2H, Ar–
H, J = 8.45 Hz), 7.52 (t, 1H, Ar–H), 7.65 (t, 1H, Ar–
H), 7.82 (s, 1H, @CH), 7.96 (s, 1H, @CH), 8.14 (d,
1H, Ar–H, J = 8.20 Hz). Anal. Calcd for C21H19NO4:
C, 72.19; H, 5.48; N, 4.01. Found: C, 71.89; H, 5.53;
N, 3.90%.
5.1.3.8. E,E-2-(3,4,5-Trimethoxybenzylidene)-6-(3-
nitrobenzylidene)cyclohexanone (2d). Mp 172 ꢁC; yield
1
82%. H NMR (CDCl3): d 1.86 (p, 2H), 2.95 (t, 2H),
3.00 (t, 2H), 3.91 (s, 9H, 3· OCH3), 6.74 (s, 1H, Ar–
H), 7.61 (t, 1H), 7.76 (d, 2H, Ar–H, @CH, J = 7.70
Hz), 7.80 (s, 1H, @CH), 8.21 (d, 1H, Ar–H, J = 8.15
Hz), 8.29 (s, 1H, Ar–H). Anal. Calcd for
C23H23NO6: C, 67.47; H, 5.66; N, 3.42. Found: C,
67.29; H, 5.57; N, 3.30%.
5.1.3.2. E,E-2-(3,4,5-Trimethoxybenzylidene)-6-(2-
nitrobenzylidene)cyclohexanone (1d). Mp 159 ꢁC; yield
1
94%. H NMR (CDCl3): d 1.80 (p, 2H), 2.62 (t, 2H),
2.97 (t, 2H), 3.91 (s, 9H, 3· OCH3), 6.73 (s, 2H, Ar–
H), 7.39 (d, 1H, Ar–H, J = 7.60 Hz), 7.52 (t, 1H, Ar–
H), 7.66 (t, 1H, Ar–H), 7.77 (s, 1H, @CH), 7.96 (s,
1H, @CH), 8.14 (d, 1H, Ar–H, J = 8.15 Hz). Anal.
Calcd for C23H23NO6: C, 67.47; H, 5.66; N, 3.42.
Found: C, 67.60; H, 5.61; N, 3.58%.
5.1.3.9. E,E-2-(Benzylidene)-6-(3-nitrobenzylidene)-
1
cyclohexanone (2e). Mp 120 ꢁC; yield 22%. H NMR
(CDCl3): d 1.85 (p, 2H), 2.97 (m, 4H), 7.38 (t, 1H,
Ar–H), 7.44 (m, 2H, Ar–H), 7.49 (d, 2H, Ar–H,
J = 7.65 Hz), 7.61 (t, 1H, Ar–H), 7.76 (d, 1H, Ar–H,
J = 7.65 Hz), 7.80 (s, 1H, @CH), 7.84 (s, 1H, @CH),
8.21 (d, 1H, Ar–H, J = 8.25 Hz), 8.33 (s, 1H, Ar–H).
Anal. Calcd for C20H17NO3: C, 75.22, H 5.37; N,
4.39. Found: C, 75.28; H, 5.44; N, 4.28%.
5.1.3.3. E,E-2-(Benzylidene)-6-(2-nitrobenzylidene)-
1
cyclohexanone (1e). Mp 116 ꢁC; yield 47%. H NMR
(CDCl3): d 1.78 (p, 2H), 2.63 (t, 2H), 2.95 (t, 2H),
7.40 (m, 5H, Ar–H), 6.79 (d, 2H, Ar–H, J = 7.6 Hz),
7.52 (t, 1H, Ar–H), 7.65 (t, 1H, Ar–H), 7.85 (s, 1H,
@CH), 7.97 (s, 1H, @CH), 8.15 (d, 1H, Ar–H,
J = 8.20 Hz). Anal. Calcd for C20H17NO3: C, 75.22,
H, 5.37; N 4.39. Found: C, 74.82; H, 5.26; N, 4.04%.
5.1.3.10. E,E-2-(4-Fluorobenzylidene)-6-(3-nitroben-
1
zylidene)cyclohexanone (2f). Mp 126 ꢁC; yield 63%. H
NMR (CDCl3): d 1.85 (p, 2H), 2.81 (t, 2H), 7.13 (t,
2H, Ar–H), 7.48 (dd, 2H, Ar–H), 7.61 (t, 1H, Ar–H),
7.76 (d, 1H, Ar–H, J = 7.65 Hz), 7.80 (s, 2H, @CH),
8.21 (d, 1H, Ar–H, J = 8.15 Hz), 8.32 (s, 1H, Ar–H).
Anal. Calcd for C20H16FNO3: C, 71.21; H, 4.78; N,
4.15. Found: C, 70.97; H, 4.70; N, 4.08%.
5.1.3.4. E,E-2-(4-Fluorobenzylidene)-6-(2-nitrobenzyli-
dene)cyclohexanone (1f). Mp 136 ꢁC; yield 52%. 1H
NMR (CDCl3): d 1.79 (p, 2H), 2.63 (t, 2H), 2.91 (t,
2H), 7.12 (t, 2H, Ar–H), 7.39 (d, 1H, Ar–H, J = 7.65
Hz), 7.47 (q, 2H, Ar–H), 7.52 (t, 1H, Ar–H), 7.65 (t,

U. Das et al. / Bioorg. Med. Chem. 16 (2008) 6261–6268
6267
5.1.3.11. E,E-2-(4-Chlorobenzylidene)-6-(3-nitroben-
zylidene)cyclohexanone (2g). Mp 138 ꢁC; yield 68%. H
Calcd for C13H13NO3: C, 67.52; H, 5.67; N, 6.06.
Found: C, 67.40; H, 5.49; N, 6.29%.
1
NMR (CDCl3): d 1.99 (p, 2H), 2.96 (q, 4H), 7.42 (m,
4H), 7.62 (t, 1H), 7.77 (d, 2H, Ar–H & @CH,
J = 8.80 Hz), 7.81 (s, 1H, @CH), 7.84 (s, 1H, @CH),
8.16 (d, 1H, Ar–H, J = 8.16 Hz), 8.34 (s, 1H, Ar–H).
Anal. Calcd for C22H16ClNO3: C, 67.90; H, 4.56; N,
3.96. Found: C, 67.33; H, 4.58; N, 3.84%.
5.1.6. Synthesis of 4b. A solution of cyclohexanone
(4.9 g, 0.05 mol), morpholine (4.75 g, 0.055 mol), 4-tolu-
enesulfonic acid (0.02 g) in toluene (50 mL) was heated
under reflux using a Dean-Stark apparatus until the stoi-
chiometric amount of water separated (ꢀ8 h). 3-Nitro-
benzaldehyde (6.8 g, 0.045 mol) was added to the
reaction mixture and heating under reflux was continued
for 12 h. Water (25 mL) was added to the reaction mix-
ture which was heated at 50–55 ꢁC for ꢀ1 h. The organic
phase was separated, washed with hydrochloric acid
(5%, 20 mL) and water (3· 50 mL), and dried. Toluene
was removed in vacuo at 50–55 ꢁC to give a viscous oil
which was purified by chromatography using a column
of silica gel 60 (70–230 mesh) and an eluting solvent of
ethyl acetate/hexane (1:9) to give 4b, mp 51–52 ꢁC in
45% yield. 1H NMR(CDCl3): 1.82 (p, 2H), 1.98 (p,
2H), 2.59 (t, 2H), 2.86 (t, 2H), 7.52 (s, 1H, @CH), 7.58
(t, 1H, Ar–H), 7.69 (d, 1H, Ar–H, J = 7.6 Hz), 8.19
(d, 1H, Ar–H, J = 8.15 Hz), 8.25 (s, 1H, Ar–H). Anal.
Calcd for C13H13NO3: C, 67.52; H, 5.67; N, 6.06.
Found: C, 67.30; H, 5.48; N, 6.41%.
5.1.4. Compounds 3b–g. The synthesis of 3b, d, and g
has been reported previously.8
5.1.4.1. E,E-2-(4-Methylbenzylidene)-6-(4-nitrobenzyl-
idene)cyclohexanone (3c). Mp 136 ꢁC; yield 54%. 1H
NMR (CDCl3): d 1.84 (p, 2H), 2.41 (s, 3H, CH3), 2.92
(t, 2H), 2.98 (t, 2H), 7.25 (d, 2H, Ar–H, J = 7.8Hz),
7.41 (d, 2H, Ar–H, J = 7.80 Hz), 7.60 (d, 2H, Ar–H,
J = 8.35 Hz), 7.79 (s, 1H, @CH), 7.82 (s, 1H, @CH),
8.27 (d, 2H, Ar–H, J = 8.40 Hz). Anal. Calcd for
C21H19NO3: C, 75.66; H, 5.74; N, 4.20. Found: C,
75.45; H, 5.75; N, 4.13%.
5.1.4.2. E,E-2-(Benzylidene)-6-(4-nitrobenzylidene)cyclo-
hexanone (3e). Mp 141 ꢁC; yield 40%. 1H NMR (CDCl3):
d 1.84 (p, 2H), 2.93 (t, 2H), 2.98 (t, 2H), 7.38 (t, 1H, Ar–
H), 7.44 (t, 1H, Ar–H), 7.49 (d, 2H, Ar–H, J = 7.75
Hz), 7.60 (d, 2H, Ar–H, J = 8.30 Hz), 7.80 (s, 1H,
@CH), 7.84 (s, 1H, @CH), 8.27 (d, 2H, Ar–H,
J = 8.40 Hz). Anal. Calcd for C20H17NO3: C, 75.22; H,
5.37; N, 4.39. Found: C, 75.02; H, 5.29; N, 4.19%.
5.1.7. Synthesis of 4c. This compound was prepared by a
literature procedure21 to give 4c, mp 119 ꢁC [lit.21] mp
118–120 ꢁC] in 72% yield with respect to 4-nitrobenzal-
1
dehyde. H NMR(CDCl3): 1.82 (p, 2H), 1.98 (p, 2H),
2.59 (t, 2H), 2.83 (m, 2H), 7.47 (s, 1H), 7.53 (d, 2H),
8.25 (d, 2H).
5.1.4.3. E,E-2-(4-Fluorobenzylidene)-6-(4-nitrobenzyli-
dene)cyclohexanone (3f). Mp 167 ꢁC; yield 33%. 1H
NMR (CDCl3): d 1.85 (p, 2H), 2.94 (t, 4H), 7.13 (t,
2H), 7.48 (t, 2H), 7.60 (d, 2H), 7.79 (s, 2H, @CH),
8.27 (d, 2H, Ar–H, J = 8.5 Hz) Anal. Calcd for
C20H16FNO3: C, 71.21; H, 4.78; N, 4.18. Found: C,
70.88; H, 4.70; N, 4.10%.
5.2. Molecular modeling
Models of the compounds in series 1–4 were built using a
BioMedCache program.22 The lowest energy conformers
were generated using the CONFLEX program and opti-
mized by mechanics using augmented MM2 parameters.
5.1.5. Synthesis of 4a. A solution of sodium hydroxide
(0.6 g, 0.015 mol) in water (5 mL) was added dropwise
to a mixture of cyclohexanone (3.0 g, 0.02 mol) and 2-
nitrobenzaldehyde (5.85 g, 0.056 mol) at room temper-
ature for 0.25 h and the stirring was continued for
4 h. The solid was collected, dried, and recrystallized
from chloroform/methanol to yield 2-(a-hydroxy-2-
nitrobenzyl) cyclohexanone (7), mp 126 ꢁC in 28%
yield. 1H NMR (CDCl3): d 1.71 (m, 4H), 2.13 (m,
1H), 2.45 (m, 2H), 2.82 (m, 1H), 4.18 (d, 1H), 5.46
(t, 1H), 7.44 (t, 1H), 7.65 (t, 1H), 7.78 (d, 1H, J =
7.9 Hz), 7.86 (d, 1H, J = 8.15 Hz).
5.3. Determination of logP values
The logP values for enones 1–4 were generated with the
JME molecular editor.23
5.4. Cytotoxicity assays
A literature procedure was employed to examine the
cytotoxicity of 1a–g , 2a–g , 3a–g , and 4a–c toward hu-
man Molt 4/C8 and CEM T-lymphocytes as well as
murine L1210 cells.24 In brief, different concentrations
of compounds were incubated with the cells in RPMI
1640 medium at 37 ꢁC for 72 h (Molt 4/C8 and CEM
T-lymphocytes) or 48 h (L1210 cells). The correct IC50
values for 4c are presented in Table 1 which replaces
the figures quoted previously.25
Hydrochloric acid (2 mL) was added to a solution of 7
(10.5 g, 0.045 mol) in ethanol (30 mL) and the reaction
mixture was heated at 40–45 ꢁC for 4 h. The solvent
was removed in vacuo at 40–45 ꢁC and water (100 mL)
was added to the residue. The solid was collected and
5.5. Evaluation of 1d, 2d, and 3d on respiration in rat liver
mitochondria
1
dried to give 4a, mp 92 ꢁC in a yield of 64%. H NMR
(CDCl3): d 1.75 (p, 2H), 1.95 (p, 2H), 2.54 (t, 2H),
2.58 (t, 2H), 2.58 (t, 2H), 7.33 (d, 1H, Ar–H, J = 7.63
Hz), 7.51 (t, 1H, Ar–H), 7.60 (s, 1H, @CH), 7.64 (t,
2H, Ar–H), 8.12 (d, 1H, Ar–H, J = 8.21Hz). Anal.
Rats were anesthetized with isoflurane and decapitated.
A previously reported procedure was employed to iso-
late mitochondria from the liver.26 The consumption

6268
U. Das et al. / Bioorg. Med. Chem. 16 (2008) 6261–6268
10. Hassner, A.; Mead, T. C. Tetrahedron 1964, 20, 2201–
2210.
11. Quail, J. W.; Doroudi, A.; Pati, H. N.; Das, U.; Dimmock,
J. R. Acta Cryst. 2005, E61, 1795–1797.
12. Quail, J. W.; Doroudi, A.; Pati, H. N.; Das, U.; Dimmock,
J. R. Acta Cryst. 2005, E61, 1774–1776.
13. Quail, J. W.; Das, U.; Dimmock, J. R. Acta Cryst. 2005,
E61, 1150–1152.
of oxygen by mitochondria was determined by polarog-
raphy using a literature methodology.27
5.6. Statistical analyses
The linear, semilogarithmic and logarithmic plots were
constructed using a statistical software package.28
14. Taft, R. W., Jr. In Steric Effects in Medicinal Chemistry;
Newman, M. S., Ed.; John Wiley and Sons, Inc.: New
York, 1956; p 591.
References and notes
15. Hansch, C.; Leo, A. J. Substituent Constants for Correla-
tion Analysis in Chemistry and Biology; John Wiley and
Sons: New York, 1979, p 49.
16. Hansch, C.; Leo, A. J. Substituent Constants for Correla-
tion Analysis in Chemistry and Biology; John Wiley and
Sons: New York, 1979, pp 49–50.
1. Mutus, B.; Wagner, J. D.; Talpas, C. J.; Dimmock, J. R.;
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2. Baluja, G.; Municio, A. M.; Vega, S. Chem. Ind. 1964,
2053–2054.
17. Bryla, J. In Inhibition of Mitochondrial Function; Erec-
3. Benvenuto, J. A.; Connor, T. H.; Monteith, D. K.;
Laidlaw, J. A.; Adams, S. C.; Matney, T. S.; Theiss, J.
C. J. Pharm. Sci. 1993, 82, 988–991.
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inska, M., Wilson, D. F., Eds.; Pergamon Press: Oxford,
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18. Hamon, N. W.; Bassendowski, D. L.; Wright, D. E.;
Dimmock, J. R.; Noble, L. M. J. Pharm. Sci. 1978, 67,
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4. Dimmock, J. R.; Sidhu, K. K.; Chen, M.; Reid, R. S.;
Allen, T. M.; Kao, G. Y.; Truitt, G. A. Eur. J. Med. Chem.
1993, 28, 313–322.
19. Shriner, R. L.; Teeters, W. O. J. Am. Chem. Soc. 1938, 60,
936–939.
20. Colonge, J.; Sibeud, J. J. Bull. Soc. Chim. France 1952,
786–789.
5. Dimmock, J. R.; Padmanilayam, M. P.; Zello, G. A.;
Nienaber, K. H.; Allen, T. M.; Santos, C. L.; De Clercq,
E.; Balzarini, J.; Manavathu, E. K.; Stables, J. P. Eur. J.
Med. Chem. 2003, 38, 169–177.
21. Vieweg, H.; Wagner, G. Pharmazie 1979, 34, 785–788.
22. BioMedCache 6.1 Windows, BioMedCache, Fujitsu
America; Inc., 2003.
23. JME Molecular editor, April 2006 Version; http://
www.molinspiration.com.
24. Baraldi, P. B.; Nunez, M. Del. C.; Tabrizi, M. A.; De
6. Dimmock, J. R.; Padmanilayam, M. P.; Zello, G. A.;
Quail, J. W.; Oloo, E. O.; Prisciak, J. S.; Kraatz, H.-B.;
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