Correlations between cytotoxicity and topography of some 2-arylidenebenzocycloalkanones determined by X-ray crystallography

Article abstract of DOI:10.1021/jm010559p

Three series of 2-arylidenebenzocycloalkanones 1-3 were prepared in order to compare the topography of the molecules with cytotoxicity. These compounds contain two aryl rings whose spatial relationships to each other were influenced by the size of the alicyclic ring and the nature of the substituents in the arylidene aryl rings. All compounds were evaluated against murine P388 and L1210 cells as well as human Molt 4/C8 and CEM T-lymphocytes. From these results, 1l and 2c,l emerged as useful lead molecules and 11 was shown to significantly inhibit macromolecular DNA, RNA, and protein syntheses in L1210 cells. Various interatomic distances, bond angles, and a torsion angle of 19 representative compounds were determined by X-ray crystallography, and correlations between these data and the cytotoxicity were noted in nearly 40% of the cases examined. Structure-activity relationships revealed that in general, the steric properties of the groups in the arylidene aryl ring, as revealed by measurements of the molar refractivity values, contributed more to bioactivity than the electronic and hydrophobic properties of the aryl substituents. The compounds displayed little murine toxicity, which favors the decision to develop these molecules as cytotoxic and anticancer agents.

Full text of DOI:10.1021/jm010559p

J . Med. Chem. 2002, 45, 3103-3111
3103
Cor r ela tion s betw een Cytotoxicity a n d Top ogr a p h y of Som e
2-Ar ylid en eben zocycloa lk a n on es Deter m in ed by X-r a y Cr ysta llogr a p h y
J onathan R. Dimmock,*,† Gordon A. Zello,† Eliud O. Oloo,‡ J . Wilson Quail,‡ Heinz-Bernhard Kraatz,‡
Pa´l Perje´si,*,§ Ferenc Aradi,| Krisztina Taka´cs-Nova´k, Theresa M. Allen,# Cheryl L. Santos,# J an Balzarini,∇
Erik De Clercq,∇ and J ames P. StablesO
College of Pharmacy and Nutrition and Department of Chemistry, University of Saskatchewan,
Saskatoon, Saskatchewan S7N 5C9, Canada, Department of Medical Chemistry and Central Research Laboratory,
University of Pe´cs, Department of Pharmaceutical Chemistry, Semmelweis University, Budapest, Hungary, Department of
Pharmacology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada, Rega Institute for Medical Research,
Katholieke Universiteit Leuven, B-3000 Leuven, Belgium, National Institute of Neurological Disorders and Stroke,
National Institutes of Health, Bethesda, Maryland 20892-9020
Received December 10, 2001
Three series of 2-arylidenebenzocycloalkanones 1-3 were prepared in order to compare the
topography of the molecules with cytotoxicity. These compounds contain two aryl rings whose
spatial relationships to each other were influenced by the size of the alicyclic ring and the
nature of the substituents in the arylidene aryl rings. All compounds were evaluated against
murine P388 and L1210 cells as well as human Molt 4/C8 and CEM T-lymphocytes. From
these results, 1l and 2c,l emerged as useful lead molecules and 1l was shown to significantly
inhibit macromolecular DNA, RNA, and protein syntheses in L1210 cells. Various interatomic
distances, bond angles, and a torsion angle of 19 representative compounds were determined
by X-ray crystallography, and correlations between these data and the cytotoxicity were noted
in nearly 40% of the cases examined. Structure-activity relationships revealed that in general,
the steric properties of the groups in the arylidene aryl ring, as revealed by measurements of
the molar refractivity values, contributed more to bioactivity than the electronic and hydrophobic
properties of the aryl substituents. The compounds displayed little murine toxicity, which favors
the decision to develop these molecules as cytotoxic and anticancer agents.
In tr od u ction
The principal objective of the present study was to
prepare three series of 2-arylidenebenzocycloalkanones
1-3 with a view to discern those structural features
that influence cytotoxicity (Chart 1).
The design of the compounds in series 1-3 was based
on the following considerations. First, chalcones or 1,3-
diaryl-2-propenones have a variety of bioactivities in-
cluding cytotoxicity.1 However, for these molecules, a
number of spatial orientations can be adopted and the
shapes that elicit bioactivity are therefore difficult to
discern. Thus, the restriction of such mobility may be
achieved by incorporating the chalcone motif into the
2-arylidenebenzocycloalkanones 1-3. In this way, the
location of potentially important groups such as the aryl
rings designated A and B (Figure 1), which contribute
to cytotoxicity relative to one another, may be identified.
The changes in topography in 1-3 were anticipated to
F igu r e 1. Axes 1 and 2 in relation to a representative
compound 3a .
Ch a r t 1. General Structure of Series 1 (n ) 0),
2 (n ) 1), and 3 (n ) 2).a
* To whom correspondence should be addressed. J .R.D.: Tel.:
306-966-6331. Fax:306-966-6377. E-mail: dimmock@skyway.usask.ca.
P.P.:
Tel.:
36(72)536-222.
Fax:
36(72)536-225.
E-mail:
a
pal.perjesi@oak.pte.hu.
The nature of the R1-R4 atoms and groups are indicated in
† College of Pharmacy and Nutrition, University of Saskatchewan.
‡ Department of Chemistry, University of Saskatchewan.
§ Department of Medical Chemistry, University of Pe´cs.
| Central Research Laboratory, University of Pe´cs.
Table 1.
be achieved in two ways. In the first place, variation in
the size of the cycloaliphatic ring would be expected to
alter the relative positions of different functional groups,
particularly rings A and B. Second, the introduction of
ortho substituents having different sizes into the
arylidene aryl ring would be predicted to lead to
variation in the torsion angles between the aryl ring
Department of Pharmaceutical Chemistry, Semmelweis Univer-
sity.
#
Department of Pharmacology, University of Alberta.
∇ Rega Institute for Medical Research, Katholieke Universiteit
Leuven.
National Institute of Neurological Disorders and Stroke, National
Institutes of Health.
O
10.1021/jm010559p CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/04/2002

3104 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Ch a r t 2. General Structures of Series 4.a
Dimmock et al.
Bioeva lu a tion s
All of the compounds in series 1-4 were evaluated
against murine leukemia P388 and L1210 cells as well
as human Molt 4/C8 and CEM T-lymphocytes. These
data are presented in Table 1. The inhibitory effects of
1a ,l on the biosynthesis of DNA, RNA, and protein in
L1210 cells are summarized in Table 5. Compounds 1-4
were examined for anticonvulsant activity and neuro-
toxicity in mice using doses of either 30 100 or 300 mg/
kg.
a
The nature of the R1-R3 groups are indicated in Table 1.
and the adjacent olefinic group. These structural changes
may profoundly affect bioactivity.
A second aspect of the design of these molecules was
based on a previous observation from these laboratories
that 2-(4-methoxyphenylmethylene)benzosuberone pos-
sessed 11 times the potency of melphalan when exam-
ined against a number of neoplastic cell lines.2 Hence,
the methoxy group was incorporated into different
positions of the arylidene aryl ring as displayed in 1a -
e, 2a -e, and 3a -e, while different ortho substituents
were incorporated into most of the remainder of the
molecules.
Furthermore, the evaluation of the compounds out-
lined in this study for anticonvulsant activity and
neurotoxicity in mice was planned for two reasons. First,
compounds affording protection against convulsions do
so by penetrating the central nervous system (CNS);
hence, they may find use in treating CNS tumors.
Second, the absence of neurotoxicity and other overt side
effects coupled with their cytotoxic activity would
enhance the usefulness of these compounds as antican-
cer agents.
In summary, the decision was made to prepare the
compounds 1-3, in which the aryl substitution pattern
in ring B was the same in all three series of compounds,
and to examine their potential as cytotoxins. Correla-
tions were sought between the topography of represen-
tative compounds and the cytotoxicity. A comparison of
the cytotoxicity of the 2-arylidenebenzocycloalkanones
with the representative acyclic analogues 4a -f (Chart
2) was also proposed.
Discu ssion
The cytotoxicity evaluation of the compounds 1-4
utilized murine P388 and L1210 cells, which are claimed
to be good predictors of clinically useful anticancer
drugs.4 In addition, evaluation vs human Molt 4/C8 and
CEM T-lymphocytes was undertaken in order to discern
whether the compounds would display cytotoxicity
toward human tumors. Previous work undertaken with
conjugated aryl R,â-unsaturated ketones revealed that
the P388 cells were generally more sensitive to enones
than other cell lines.5 Consequently, while a maximum
concentration of 50 µM was used in the P388 test, a
higher concentration of 500 µM was employed in the
L1210, Molt 4/C8, and CEM assays. The data generated
are presented in Table 1.
A review of the biodata in Table 1 indicated that in
general, the P388 cells were the most sensitive to the
enones 1-4. Thus, in those cases where IC50 figures
were available for all four cell lines, the lowest IC50
figures toward P388 cells were noted in 66% of the
comparisons made. The average IC50 values of 1l, 2c,l,
and melphalan were 3.61, 3.54, 9.69, and 2.02 µM,
respectively, when all four cell lines were taken into
consideration, revealing that these three enones are
clearly useful lead molecules. The promising activity of
1l and 2l may have been due to the molecules per se
and/or to reduction of the nitro group to toxic species.6
A number of cytotoxic compounds possess two aryl rings,
which are separated by one or two carbon atoms in
which methoxy groups are present on at least one of
the aromatic rings. For example, combretastatin A-4
[Z-1-(3-hydroxy-4-methoxyphenyl)-2-(3′,4′,5′-trimethox-
y)stilbene] interacts at the colchicine site of â-tubulin,7
and the importance of the presence of a 4-methoxyaryl
group for strong interactions has been suggested.8 It is
conceivable, therefore, that the promising activity of 2c
and analogues containing methoxy groups is due, at
least in part, to a similar mode of action.
A comparison of the bioactivity between series 1-3
was undertaken with a view to determining which
spatial orientations of the molecules favored cytotoxic-
ity. The means of the IC50 figures of compounds 1-3
were compared in order to find if there were differences
in the relative potencies between the compounds in
series 1-3. One way analysis of variance (ANOVA) tests
using equal numbers in groups followed by a series of
post-hoc tests, e.g., least significant differences, revealed
that the potencies of the compounds in series 1 were
significantly lower than the analogues in series 2 and
3 across all screens (p < 0.1). In this approach, however,
compounds having IC50 values of >50 µM in the P388
screen or >20, >100, and >500 µM in the other screens
were removed from the analysis along with the ana-
Ch em istr y
The compounds in series 1-3 were synthesized by the
condensation between the appropriate aryl aldehydes
and the benzocycloalkanones. Several representative
acyclic chalcones 4a -f were also prepared by the same
1
route. H Nuclear magnetic resonance (NMR) spectra
were obtained for all compounds. In cases where the
olefinic and aryl protons did not overlap, the olefinic
protons of the compounds 1-3 were detected in the
region of 7.2-8.0 ppm indicating the E configuration of
the chiral axis.2 The shapes of 1a ,d ,h ,k ,l, 2a ,d -f,h ,j,k ,
and 3a ,d ,e,h -j,l were determined by X-ray crystal-
lography; in all cases, the E stereochemistry was noted.
The assumption was made, therefore, that all of the
compounds 1-3 were the E isomers. In the case of the
chalcones 4, the coupling constants of the protons on
1
the olefinic group determined by H NMR spectroscopy
revealed the E configuration, an observation that has
been noted previously.3 Molecular modeling was under-
taken with 1a ,l, 2a , and 3a ,l. The redox potentials of
three representative compounds 1c, 2c, and 3c were
measured. The log P figures of 3a -l determined by a
reverse phase thin-layer chromatography (TLC) method
are summarized in Table 4.

2-Arylidenebenzocycloalkanones
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3105
Ta ble 1. Evaluation of the Compounds in Series 1-4 against Murine P388 and L1210 Leukemic Cells and Human Molt 4/C8 and
CEM T-Lymphocytesa
IC50 (µM)
cmpd
R1
R2
R3
R4
P388 cells
L1210 cells
Molt 4/C8 cells
CEM cells
1a
1b
1c
1d
1e
1f
1g
1h
1i
OCH3
OCH3
OCH3
28.4 ( 3.5
28.7 ( 0.7
13.0 ( 0.4
23.9 ( 1.8
>50 ( 1.3
23.9 ( 1.1
31.7 ( 0.81
26.5 ( 1.63
>50 ( 0.8
25.3 ( 1.6
>50 ( 5.6
0.74 ( 0.04
17.5 ( 0.88
12.3 ( 0.6
2.61 ( 0.06
5.69 ( 0.42
6.72 ( 0.6
17.7 ( 1.0
21.8 ( 0.73
17.5 ( 0.3
17.2 ( 0.5
13.1 ( 2.3
19.7 ( 1.4
12.6 ( 1.6
18.0 ( 0.75
8.58 ( 0.7
7.82 ( 0.9
8.19 ( 1.03
8.68 ( 0.6
15.2 ( 0.5
13.2 ( 1.6
12.7 ( 0.93
14.0 ( 1.09
12.2 ( 0.1
19.8 ( 1.5
36.5 ( 3.6
15.9 ( 1.6
9.75 ( 0.7
10.63 (1.3
3.74 ( 0.2
15.3 ( 0.5
7.58 ( 0.8
0.22 ( 0.01
170 ( 32
214 ( 12
14.0 ( 0.7
264 ( 6
308 ( 42
53.0 ( 3.3
48.5 ( 37.0
468 ( 45
>500
58.2 ( 9.7
37.6 ( 7.6
87.1 ( 60.7
286 ( 120
>500
56.5 ( 6.1
47.1 ( 7.6
57.0 ( 10.6
>500
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
>500
CH3
F
Cl
208 ( 94
150 ( 38
152 ( 11
>500
154 ( 34
54.2 ( 3.5
128 ( 44
>500
Cl
Cl
Cl
1j
1k
1l
Cl
>500
>500
397 ( 146
46.8 ( 12.4
3.53 ( 2.07
12.6 ( 1.6
28.7 ( 8.7
1.88 ( 0.68
8.55 ( 0.66
7.04 (0.58
43.2 ( 1.8
20.8 ( 7.8
8.80 ( 0.86
8.73 ( 1.24
9.77 ( 0.74
25.4 ( 5.9
7.22 ( 1.60
28.9 ( 8.2
142 ( 64
48.4 ( 13.4
28.0 ( 10.6
11.7 ( 1.2
43.2 ( 1.5
28.2 ( 2.4
18.4 ( 5.0
407 ( 131
11.8 ( 2.0
32.1 ( 5.6
16.2 ( 2.6
8.56 ( 0.35
8.55 ( 0.04
33.5 ( 5.5
8.60 ( 0.30
8.90 ( 0.80
13.8 ( 1.6
2.47 ( 0.30
Br
NO2
OCH3
OCH3
OCH3
64.2 ( 36.8
7.61 ( 1.17
40.5 ( 2.9
38.2 ( 2.3
7.98 ( 1.86
42.0 ( 1.3
31.2 ( 0.8
50.2 ( 7.2
22.8 ( 23.5
30.0 ( 3.7
34.4 ( 5.4
210 ( 18
33.4 ( 2.0
10.8 ( 2.0
46.1 ( 2.5
g500
95.4 ( 4.5
2.54 ( 0.61
31.3 ( 1.1
41.3 ( 1.4
1.69 ( 0.20
11.6 ( 0.4
8.28 ( 0.51
41.4 ( 1.5
36.0 ( 2.3
29.6 ( 5.6
28.4 ( 7.6
28.6 ( 17.0
36.8 ( 1.3
8.12 ( 0.44
37.1 ( 1.8
g500
2a
2b
2c
2d
2e
2f
2g
2h
2i
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
CH3
F
Cl
Cl
Cl
Cl
2j
2k
2l
Cl
Br
NO2
OCH3
OCH3
OCH3
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
4a
4b
4c
4d
4e
4f
OCH3
OCH3
OCH3
OCH3
12.4 ( 0.78
71.4 ( 13.8
OCH3
OCH3
g100b
g20b
OCH3
35.8 ( 3.8
61.2 ( 9.8
60.4 ( 26.9
42.3 ( 4.2
g500
45.7 ( 4.5
37.0 ( 2.0
47.0 ( 34.2
64.4 ( 2.3
45.1 ( 4.9
50.4 ( 7.9
30.4 ( 7.2
82.9 ( 41.1
59.1 ( 61.2
2.13 ( 0.03
23.1 ( 8.2
36.2 ( 4.9
42.1 ( 0.8
37.1 ( 0.7
>500
18.2 ( 1.1
38.4 ( 2.2
40.0 ( 15.5
8.45 ( 0.37
7.94 ( 0.72
30.0 ( 0.04
8.38 ( 0.20
8.53 ( 0.20
8.32 ( 0.11
3.24 ( 0.79
CH3
F
Cl
Cl
Cl
Cl
Cl
Br
NO2
OCH3
-
-
-
-
-
-
OCH3
NO2
OCH3
NO2
NO2
melphalanc
a
For the sake of clarity, only the nonhydrogen substituents in series 1-4 are indicated. The dashes indicate the absence of the R4
group in series 4. This compound was soluble at 40 µM but insoluble at 200 µM. c Reprinted in part from J . Med. Chem. 2000, 43, 3935.
Copyright 2000 American Chemical Society.
b
logues in the other series having the same aryl substit-
uents. For example, in the P388 screen, 1e,i,k had IC50
figures of >50 µM; hence, 1e,i,k , 2e,i,k , and 3e,i,k were
removed from evaluation in these comparisons. In the
case of the CEM screen, for example, 1e,i, 2e,i, and 3e,i
were not used in the analysis. Therefore, small sample
sizes were used in these comparisons.
To include more data, comparisons of the IC50 values
were made in each series when the aryl substituents
were held constant, e.g., the cytotoxicities of 1a , 2a , and
3a in the P388 screen were compared. This approach
enabled comparisons to be made when not more than
one compound among the three analogues under review
did not have a specific IC50 value, e.g., >50 µM. The
values obtained (Table 1) were ranked with scores of 3,
2, and 1 given to the compounds displaying the highest,
intermediate, and lowest potencies, respectively. In
assigning scores, the standard deviations of the IC50
figures were taken into consideration and the sum of
the scores of the relative potencies of each group of three
compounds was invariably six. Thus, in the case of 1a ,
2a , and 3a in the P388 test, scores of 1, 2.5, or 2.5,
respectively, were assigned, while in the L1210 screen
the figures were 1, 3, or 2, respectively. No comparisons
were possible for 1d , 2d , and 3d or between 1i, 2i, and
3i in both the L1210 or the Molt 4/C8 screens due to a
lack of specific IC50 values for some of the compounds.
The total scores for series 1 in the P388, L1210, Molt
4/C8, and CEM screens were 14, 13, 13.5, and 15.5,
respectively; for series 2, the figures were 28.5, 28, 27,
and 33.5, respectively; in the case of series 3, the values
were 29.5, 19, 19.5, and 23, respectively. Taking into
consideration all four test systems, the average scores
for 1-3 were 14.0, 29.3, and 22.8. Thus, in three of the
four screens, the molecules in series 2 displayed the
greater potencies, and in all four tests, series 1 was the
least active. Furthermore, the significant differences
apparent using ANOVA were confirmed by a nonpara-
metric statistical approach (i.e., Kruskal-Wallis test)
using the ranked data, which revealed differences
between the three cell lines (p < 0.001). One may
conclude from the statistical methods that the potencies

3106 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Dimmock et al.
F igu r e 2. Measurements d4, ψ1-ψ3, and θ1 in a representative
compound 3a .
F igu r e 4. Measurements d7-d13 and ψ5.
rings were reviewed. The lengths of the C1-C2, C2-
C12, and C12-C13 bonds as well as the C2-C13
distances were designated d1-d4, respectively (Figure
2). In addition, the distances between the CA and C12
atoms (d5) and the C12 atoms and axis 2 (d6) were
recorded (Figure 3). The bond angles ψ1-ψ3 and torsion
angle θ1 are illustrated in Figure 2, and the angle
between the line CA-C12 and axis 2 is referred to as ψ4
(Figure 3).
To discern the relative positions of the two aryl rings
in the benzocycloalkanones, the following data were
obtained. Most of these determinations are illustrated
in Figure 4. The span between CA and CB was desig-
nated d7. The distance of the CB atoms above or below
the plane of ring A is referred to as d8. In Figure 4, the
CB atom lies above the plane of ring A, as is the case of
3a , for example. The negative d8 values indicate those
compounds in which CB is below the plane of ring A. It
was obtained by creating a perpendicular from CB to
the plane of ring A at a point X. The perpendicular
distance between axis 2 and point X is referred to as
d9. The figures for d10 represent the distance between
CA and point X while d11 is the span between CA and
the point on axis 2 at right angles to X. To obtain an
understanding of the tilt of ring B in relation to ring A,
the distances of the C12 and C15 atoms from the plane
F igu r e 3. Measurements d6 and ψ4 in a representative
compound 3a .
of the compounds in series 2 and 3 were greater than
the analogues in series 1. Overall, the data indicated
that cytotoxicity was in the order of 2 > 3 > 1.
X-ray crystallographic data were obtained on 19
compounds, namely, 1a ,d ,h ,k ,l, 2a ,d -f,h ,j,k , and
3a ,d ,e,h -j,l. The next phase of the study was to
determine whether correlations existed between various
interatomic distances and bond and torsion angles
culled from the X-ray crystallographic data and cyto-
toxicity. While the shapes of compounds determined by
X-ray crystallography may or may not be identical to
the topography of the bioactive species, it is an excellent
tool for determining the geometry of molecules. A
previous study revealed the importance of the aryl ring
attached to the cycloheptanone moiety in eliciting
cytotoxicity.2 Thus, a number of measurements obtained
from the X-ray crystallographic data were made in
reference to this aryl ring in all three series of com-
pounds. In particular, a major emphasis in this inves-
tigation was the consideration that cytotoxicity could
be correlated with the spatial relationships between the
two aryl rings. To investigate this possibility, the centers
of aryl rings A and B were designated CA and CB,
respectively. This procedure is illustrated for a repre-
sentative compound 3a in Figure 1. Axis 1 was con-
structed through C7, CA and C10, and axis 2 was
formed at right angles to axis 1 and in the same plane
(Figure 1).
of ring A were measured and designated d12 and d13
,
respectively. The positive and negative figures pre-
sented in Table 2 reflect whether C12 and C15 were above
or below the plane of ring A. Two other portions of these
molecules that may influence cytotoxicity were also
considered. In the first place, the distance between CA
and the center of the alkylene portion of the cy-
cloaliphatic ring d14 was recorded (in the case of 3a , this
part of the molecule comprises carbon atoms 3-5 and
the attached protons). Second, the distance d15 is the
span between CA and the carbonyl oxygen atom.
The X-ray crystallographic data revealed that there
was little variation between the d1-d4 data. The average
d1-d4 values were 1.50 (1.49-1.52), 1.34 (1.32-1.36),
1.47 (1.46-1.48), and 2.54 (2.51-2.57) Å, respectively,
indicating that these interatomic distances are unlikely
to contribute to the differences in cytotoxicity displayed
by these compounds. On the other hand, disparate d5-
The specific measurements made for all of the com-
pounds whose structures had been determined by X-ray
crystallography were as follows. For the sake of clarity,
the nature of some of these determinations is illustrated
for 3a (Figures 2 and 3). Initially, some of the structural
characteristics of the linker group between the two aryl
d
15, ψ1-ψ5, and θ1 values were noted and these data
are presented in Table 2.
To discern whether correlations existed between the
cytotoxicity and the interatomic distances d5-d15, the

2-Arylidenebenzocycloalkanones
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3107
Ta ble 2. Interatomic Distances d5-d15 (Å), Bond Angles ψ1-ψ5 (deg), and a Torsion Angle θ1 (deg) of Various Compounds
Determined by X-ray Crystallographya
compd
d5
d6
d7
d8
d9
d10
d11
d12
d13
d14
d15
ψ1
ψ2
ψ3
ψ4
ψ5
θ1
1a
1d
1h
1k
1l
4.80 0.24 7.23 -0.18 1.53 7.23 7.07 -0.12 -0.25 2.52 3.76 106.5 118.7 132.0
4.81 0.20 7.17 -0.23 1.68 7.16 6.96 -0.14 -0.34 2.51 3.76 106.8 119.9 130.4
2.9 12.3 -178.7
2.4 13.7
2.3 13.6
178.8
177.5
4.79 0.19 7.19
4.80 0.21 7.19
4.80 0.23 7.18
0.07 1.69 7.19 6.98
0.05 1.71 7.18 6.98
0.21 1.67 7.18 6.98
0.01
0.08
0.05
0.12 2.52 3.77 107.6 119.3 131.2
0.03 2.50 3.76 106.0 118.5 130.0
0.38 2.52 3.76 106.4 119.0 129.7
2.5 13.8 -176.9
2.7 13.6
174.6
2a
2d
2e
2f
2h
2j
2k
3a
3d
3e
3h
3i
5.06 1.42 7.66 -0.18 0.64 7.66 7.63 -0.16 -0.19 2.84 3.65 117.9 116.2 129.9 16.3
5.09 1.44 7.69 -0.48 0.73 7.68 7.64 -0.31 -0.64 2.84 3.65 117.9 116.4 130.3 16.4
5.07 1.38 7.62 -0.51 0.59 7.61 7.58 -0.30 -0.73 2.83 3.64 118.6 117.9 130.1 15.8
5.08 1.29 7.55
5.08 1.32 7.57 -0.41 0.23 7.56 7.56 -0.21 -0.59 2.83 3.65 117.5 118.4 127.1 15.0
5.10 1.50 7.65 -1.34 0.71 7.54 7.50 -0.87 -1.78 2.84 3.66 117.7 117.1 128.5 17.1 11.4
5.0 -149.0
6.5
5.9
147.4
147.3
0.28 0.20 7.55 7.55
0.07
0.48 2.83 3.65 117.7 118.9 127.0 14.7
2.6 -135.6
3.5
137.6
152.4
147.7
138.2
150.2
164.0
132.4
132.4
5.08 1.42 7.67
5.11 1.75 7.67
5.13 1.75 7.75
5.07 1.47 7.71
5.13 1.99 7.72
5.14 2.07 7.72
0.09 0.62 7.67 7.65
1.83 1.21 7.45 7.35
1.48 1.41 7.61 7.47
0.56 1.02 7.69 7.62
1.91 1.65 7.48 7.29
2.07 1.62 7.43 7.25
0.23 -0.04 2.84 3.64 118.0 116.6 128.7 16.3
4.7
2.42 2.94 3.61 119.0 117.3 128.3 20.0 16.6
2.00 2.97 3.61 119.2 116.8 130.0 20.0 15.3
0.83 2.98 3.60 119.7 116.4 132.4 16.9 8.7
1.22
0.98
0.31
1.30
1.47
2.51 2.97 3.61 119.5 116.5 127.5 22.8 19.1
2.63 2.97 3.62 119.2 116.6 127.0 23.8 19.9
3j
3l
5.08 1.49 7.72 -0.67 1.04 7.69 7.62 -0.36 -0.99 2.98 3.61 119.1 116.9 131.6 17.0
9.2 -166.7
5.12 1.78 7.67 2.05 1.10 7.39 7.30 -1.40 2.70 2.93 3.69 118.5 116.6 126.9 20.4 17.7
139.3
a
The positive and negative interatomic distance figures indicate that the locations of one of the atoms is above or below the plane of
aryl ring A, respectively. The positive and negative θ1 values signify that the torsion angle is clockwise and anticlockwise, respectively.
Ta ble 3. Correlations Noted between Various Interatomic
Distances and Bond Angles Obtained by X-ray Crystallography
and Cytotoxicity
and d11), and (iv) ring A and the alkylene portion of the
molecules (d14). In addition, potency was increased as
the magnitude of the ψ1 and ψ4 angles was reduced. On
distance
or bond
angle
distance
or bond
angle
the other hand, lengthening both the distances between
ring B and axis 2 (d9) and the span between ring A and
the carbonyl oxygen atom (d15) led to increased cyto-
toxicity. Bioactivity was increased as the ψ2 and ψ5
angles rose. These correlations established the impor-
tant observation that the spatial orientations of the aryl
rings in the 2-arylidene-1-benzocycloalkanones contrib-
uted to bioactivity on a number of occasions. Of par-
ticular note was the observation that the d10 and d15
distances correlated with cytotoxicity in three of the four
screens. Thus, in the future, the preparation and
bioevaluation of different series of compounds that take
into consideration these correlations may lead to ana-
logues with greater cytotoxicity.
screensa
screensa
d5
d6
d7
d9
d10
L1210b (-0.464)
Molt 4/C8c (-0.631)
L1210b (-0.473)
Molt 4/C8c (-0.622)
L1210c (-0.482)
Molt 4/C8c (-0.640)
Molt 4/C8b (0.481)
CEMb (0.412)
d14
d15
L1210c (-0.498)
Molt 4/C8c (-0.623)
P388b (0.404)
L1210c (0.514)
Molt 4/C8c (0.610)
L1210c (-0.488)
Molt 4/C8c (-0.642)
L1210b (0.479)
ψ1
ψ2
ψ4
ψ5
P388b (-0.405)
Molt 4/C8c (0.607)
L1210b (-0.473)
Molt 4/C8c (-0.621)
CEMb (0.418)
L1210c (-0.488)
Molt 4/C8c (-0.613)
L1210b (-0.469)
Molt 4/C8c (-0.603)
d11
a
b
Pearson’s r value is in parentheses. p < 0.1. c p < 0.05.
A second physicochemical method was employed in
order to confirm the variation in topography displayed
by the compounds in series 1-3. Molecular modeling
was undertaken with 1a ,l, 2a , and 3a ,l, and the d1-
d15, ψ1-ψ5, and θ1 values were obtained. The percentage
differences between the X-ray crystallographic and
molecular modeling data were less than 6.1% in the case
of the d1-d5, d7, d10, d11, d14, d15, and ψ1-ψ3 determina-
tions; in fact, these differences were less than 3% in the
majority of the comparisons made. The d6 and ψ4 data,
determined by X-ray crystallography and molecular
modeling, revealed that the percentage differences for
1a ,l were 31 and 52; in contrast, the figures for 2a , and
3a ,l were less than 6%. However, differences of up to
97% were noted in virtually all of the comparisons when
d8, d9, d12, d13, ψ5, and θ1 determinations were consid-
ered.
Linear and semilogarithmic plots were undertaken
between the d1-d15, ψ1-ψ5, and θ1 values of 1a ,l, 2a ,
and 3a ,l, generated by molecular modeling, and the IC50
figures in each of the four cytotoxicity screens. Correla-
tions were noted between the d2 and ψ2 figures and
potency in the P388 screen and the d2-d4 and ψ3 values
in the L1210, Molt 4/C8, and CEM assays. Thus,
correlations were noted in five of the 17 measurements
angles ψ1-ψ5, and the torsion angle θ1, which had been
determined by X-ray crystallography, bivariate correla-
tion analysis was undertaken. In this process, linear
plots of the IC50 values of the compounds determined
in each cell line were made against the individual
distances and angles. Semilogarithmic plots were also
determined but did not lead to any changes in the
statistically significant correlations obtained using the
linear plots. Hence, only the latter are presented in
Table 3. Correlations were noted in 37% of the cases.
These data revealed that of the 17 physicochemical
measurements, i.e., d5-d15, ψ1-ψ5, and θ1, 12 of these
determinations exerted statistically significant effects
on cytotoxicity in at least one cell line. The extent of
influence upon bioactivity was dependent on the screen
under consideration since the percentage of correlations
noted in the P388, L1210, Molt 4/C8, or CEM screens
was 8, 40, 44, or 8, respectively.
The correlations were negative, except for the d9 and
d15 distances as well as the ψ2 and ψ5 angles. Thus,
elevation of potencies was achieved by decreasing the
distances between the following groups, namely, (i) ring
A and the olefinic carbon atom (d5), (ii) the olefinic
carbon atom and axis 2 (d6), (iii) the aryl rings (d7, d10
,

3108 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Ta ble 4. Log P Values of 3a -l
Dimmock et al.
Ta ble 5. Effect of 1a ,l on DNA, RNA, and Protein
Biosyntheses in L1210 Cellsa
compd
log Pa
compd
log Pa
IC50 (µM)
protein
3a
3b
3c
3d
3e
3f
4.69 ( 0.09
4.54 ( 0.06
5.01 ( 0.08
4.13 ( 0.07
4.29 ( 0.05
4.92 ( 0.11
3g
3h
3i
3j
3k
3l
4.55 ( 0.06
5.04 ( 0.04
5.65 ( 0.01
5.71 ( 0.05
5.04 ( 0.02
4.07 ( 0.07
DNA
RNA
L1210
compd
synthesis
synthesis
synthesis proliferation
1a
1l
407
14.7 (27.7)
292
167 170
11.7 (25.0) 17.2 (9.71)
7.61 (22.3)
2.13
melphalanb >100
>100
94.6
a
a
The data for 3a ,f-h ,k ,l are taken from J . Planar Chromatogr.
2001, 14, 42. Reproduction with permission of the copyright owner.
The figures in parentheses indicate the fold increase in potency
b
of 1l as compared to 1a . Reprinted in part from J . Med. Chem.
2001, 44, 586. Copyright 2001 American Chemical Society.
and in 21% of the evaluations made. These relationships
were all positive except for the ψ2 data. This evidence
suggests that the bioactivity observed is correlated with
various interatomic distances and bond angles to a
greater extent using the data generated by X-ray
crystallography rather than molecular modeling.
Linear and semilogarithmic plots were made between
specific IC50 values of the compounds in each of the
series 1-3, where available, and the electronic (Ham-
mett σ and/or Taft σ* values), hydrophobic (π constants),
and steric [molar refractivity (MR) figures] properties
of the aryl substituents. The following correlations were
noted. The σ and/or σ* constants of the compounds in
series 3 influenced cytotoxicity significantly in the Molt
4/C8 screen, while the π values of the aryl substituents
in series 2 correlated with the IC50 figures in the L1210
screen. The MR constants of the series 2 compounds
were correlated with cytotoxicity in the P388 and Molt
4/C8 screens, while in series 3 these constants correlated
with the cytotoxic properties in the P388 and L1210
series. Negative correlations were noted in all cases
except for the π/L1210 relationship in series 2. One may
conclude that in general, steric factors in series 2 and
3, as determined using MR values that are related to
London dispersion forces, influenced cytotoxicity more
than the electronic and hydrophobic properties of the
molecules. Hence, insertion of small groups into the
arylidene aryl ring should be borne in mind when
considering the development of this cluster of com-
pounds.
The log P values of the compounds in series 3 were
determined, and the results are portrayed in Table 4.
Linear and semilogarithmic plots between these figures
and the π values of the aryl substituents in 3a -l
revealed an excellent correlation (p < 0.001). However,
no correlations were noted when, in a similar fashion,
the log P figures were plotted against the IC50 data
generated in each of the four screens (>20, >100, and
>500 µM figures were ignored). These data confirm that
the relative hydrophobicities of the molecules in series
3 were not correlated with cytotoxicity.
The acyclic analogues of 1a , 2a , and 3a and 1l, 2l,
and 3l were prepared; namely, 4a ,d , respectively. These
compounds were evaluated for cytotoxicity, and the
results are presented in Table 1. The chalcone 4a had
lower IC50 figures than the rigid analogues 1a , 2a , and
3a , when three of the four cell lines were examined.
However, in general, the IC50 values of 1l and 2l were
lower than that of 4d ; hence, one cannot deduce whether
cytotoxicity is enhanced by the relative rigidity or
flexibility of these molecules. In general, the placement
of the methoxy and nitro substituents in the ortho
position of the aryl ring favored cytotoxicity in contrast
to the meta and para locations.
Correlations have been noted previously between the
redox potentials of R,â-unsaturated ketones and the
cytotoxicity.1 In the present investigation, in each series,
compounds with comparatively high cytotoxicity (1c, 2c,
and 3c) were chosen as well as analogues possessing
significantly lower bioactivity (1i, 2i, and 3i). The Ep
values of 1c, 2c, and 3c were 1571, 1560, and 1513 mV,
respectively, while no peak potentials were obtained for
1i, 2i, and 3i. The combined Taft σ* and Hammett σ
values for the 2,4-dimethoxy and 2,4-dichloro substit-
uents were -0.50 and 0.61, respectively. Thus, while
cytotoxicity is undoubtedly influenced by many consid-
erations, a contributing factor is the extent to which a
compound receives or donates electrons in the cells. This
observation suggests that the incorporation of strongly
electron-donating substituents into aryl ring B may lead
to compounds with significant cytotoxicity.
To gain more insight into the possible mechanisms
of action of these compounds, two representative enones
with markedly different cytotoxicities were chosen. The
ortho nitro indanone 1l was 22 times more potent than
the methoxy analogue 1a in the L1210 screen (Table
1). The data in Table 5 reveal that this disparity in
bioactivities is positively correlated with the inhibition
of the biosyntheses of DNA, RNA, and protein. While
1l possessed approximately one-third of the potency of
melphalan, its mode of action with regard to the effects
on macromolecular biosyntheses is different from the
established cytotoxic alkylating agent. Therefore, these
compounds may find use against melphalan-resistant
tumor cells, an observation that has been made for the
Mannich bases of certain R,â-unsaturated ketones.9
To address the question of whether the R,â-unsatur-
ated ketones described in this study either penetrated
the CNS, displayed neurotoxicity, or were lethal to mice
using doses up to and including 300 mg/kg, the following
experiments were conducted. The compounds 1a -l, 2a -
l, 3a -l, and 4a -f were administered intraperitonally
to mice and were examined in the maximal electroshock
(MES) and subcutaneous pentylenetetrazole (scPTZ)
screens, which are claimed to be predictors of antiepi-
leptic drugs.10 In addition, the neurotoxicity of these
compounds was determined.11 Specific details are sum-
marized in the Experimental Section. The enones 2f and
4a ,d afforded protection in the MES test, and anticon-
vulsant activity was demonstrated in less than half of
the animals in the scPTZ screen by 2j, 3e, and 4a . One-
third of the compounds displayed neurotoxicity; how-
ever, in general, these results were noted using the
higher doses of 100 and 300 mg/kg and observed in less
than half of the animals. No mortalities were noted in
the neurotoxicity screen. To examine whether CNS
activity or neurotoxicity would be demonstrated when

2-Arylidenebenzocycloalkanones
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3109
6.80-8.35 (m, 11H, aryl and olefinic H). The infrared spectra
of 1f, 2f, 3f, and 4a determined in chloroform revealed carbonyl
frequencies of 1696, 1663, 1664, and 1661 cm -1, respectively.
X-r a y Cr ysta llogr a p h y of 1a ,d ,h ,k ,l, 2a ,d -f,h ,j,k , a n d
3a ,d ,e,h -j,l. Suitable crystals were obtained from the com-
pounds produced by our syntheses, with the exception of 1a ,
which was crystallized from hexane-dimethyl sulfoxide by
vapor diffusion at 4 °C. An ω scan was used for data collection.
The structures of 1a ,h ,k ,l, 2a ,d , and 3a ,d ,l were solved by
XTAL 3.414 while NRCVAX15 was used to determine the
structures of 1d , 2e,j, and 3e,h . The SHELX-97 program16 was
used to solve the structures of 2f,h ,k , and 3i,j. Refinement
was undertaken using SHELX-97. Atomic scattering factors
were taken from the literature.17 All nonhydrogen atoms were
found on the E-map and were refined anisotropically. Hydro-
gen atom positions were calculated and not refined. The figures
were generated by ORTEP18 as implemented in Xtal 3.4.
The distances d1-d3, bond angles ψ1-ψ3, and torsion angle
θ1 were obtained directly from the X-ray crystallographic data.
X-ray crystallographic coordinates were loaded into a Macro-
Model 4.5 program19 in order to facilitate the determination
of the distances d4-d7, d14, and d15 and angles ψ4 and ψ5. The
distances d8, d12, and d13 were obtained using the LSQPL
program in XTAL 3.4. The d9-d11 figures were calculated as
the compounds were administered orally, representative
compounds that were active (2f and 4a ) or inactive (1h ,i
and 2i) in the mouse intraperitoneal MES screen were
administered orally to rats and activity (or lack thereof)
in the MES and neurotoxicity screens was observed at
various time intervals. Using a dose of 30 mg/kg,
protection against seizures in 25% of the animals was
demonstrated in the case of 1h , 2f, and 4a , while none
of the five compounds demonstrated neurotoxicity. One
may conclude that it is likely that both CNS penetration
and neurotoxicity are minimal in these compounds and
the absence of deaths at the highest dose of 300 mg/kg
enhances their potential as candidate anticancer drugs.
Con clu sion s
The cytotoxic evaluation of three series of 2-aryli-
denebenzocycloalkanones revealed that the presence of
a six- and seven-membered alicyclic ring favored bio-
activity. From the biodata generated, 1l, and 2c,l
emerged as lead molecules, a viewpoint strengthened
by the lack of marked murine toxicity in this group of
compounds. The future development of these compounds
should bear in mind the correlations between cytotox-
icity and those physicochemical parameters established
by X-ray crystallography and other structure-activity
relationships.
follows. The distance d9 was obtained from the relationship
2
d9 ) (d102 - d11
)
1/2, while for d10 the figure was calculated from
2
2
the equation d10 ) (d7 - d8 )
1/2. The distance d11 was derived
from the relationship d11 ) d7cosψ5.
Molecu la r Mod elin g of 1a ,l, 2a , a n d 3a ,l. Models of 1a ,l,
2a , and 3a ,l were built using the MacroModel 4.5 program,19
which was followed by a Monte Carlo search for the lowest
energy conformations using an amber force field on 2000 initial
conformations of each compound. Interatomic distances and
angles were obtained from the lowest energy conformations
by direct measurements or standard geometrical procedures.
Evaluation of the relationships between the d1-d15, ψ1-ψ5,
and θ1 figures and cytotoxicity revealed the following correla-
tions [screen, linear (l), or semilogarithmic (sl) plots, p value,
Pearson’s r value]. d2: P388, l, <0.1, 0.810; P388, sl, <0.05,
0.981; L1210, sl, <0.1, 0.849; Molt 4/C8, sl, <0.1, 0.836; CEM,
sl, <0.1, 0.825. d3: L1210, sl, <0.05, 0.966; Molt 4/C8, sl, <0.05,
0.933; CEM, l, <0.05, 0.903. d4: L1210, l, <0.05, 0.966; L1210,
sl, <0.1, 0.794; Molt 4/C8, l, <0.05, 0.969; Molt 4/C8, sl, <0.1,
0.805; CEM, l, <0.05, 0.981; CEM, sl, <0.1, 0.814. ψ2: P388,
sl, <0.1, -0.826. ψ3: L1210, l, <0.05, 0.983; L1210, sl, <0.1,
0.832; Molt 4/C8, l, <0.05, 0.980; Molt 4/C8, sl, <0.1, 0.842;
CEM, l, <0.05, 0.986; CEM, sl, <0.1, 0.835.
Sta tistica l An a lyses Usin g σ/σ*, π, a n d MR Va lu es. The
Hammett σ values were taken from the literature,20 and the
Taft σ* figures were from a reference source.21 The Hansch π
values were obtained from published data.22 The MR figures,
which were taken from the literature,22 of all of the R1-R4
groups were combined prior to the statistical analysis. For
example, in computing the figure for the 3,4-dimethoxy
analogue, the MR values of the two methoxy groups plus two
hydrogen atoms were added. The following p values were noted
when correlations between the physicochemical constants of
the aryl substituents and cytotoxicity were obtained. In series
2, the following relationships were noted (the p values from
the linear and semilogarithmic plots, respectively, are in
parentheses), namely, π/L1210 (0.042, 0.035), MR/P388 (0.002,
0.010), and MR/Molt 4/C8 (0.071, 0.096). In the case of series
3, the correlations observed were in the following cases,
namely, σ,σ*/Molt 4/C8 (0.042, 0.044), MR/P388 (0.141, 0.044),
and MR/L1210 (0.045, 0.101).
Exp er im en ta l Section
Melting points were determined on a Boetius apparatus at
a heating rate of 4 °C/min and are uncorrected. Elemental
analyses (C, H) were undertaken at the Department of Organic
Chemistry, Eo¨tvo¨s Lora´nd University, Budapest, Hungary, on
unreported compounds 1c,k , 2c,i,k , and 3b,d ,e,h as well as
those compounds whose melting points differed from literature
values, namely, 1j, 2b,e, 3c, and 4d ,e and were within 0.3%
of the calculated figures. Column chromatography was under-
taken using kieselgel (Reanal, Hungary) and an eluting solvent
of toluene. The purity of compounds was evaluated by TLC
using Merck silica gel 60 F254 aluminum sheets and developing
solvents of either toluene or toluene:ethanol (4:1). 1H NMR
spectroscopy was recorded using Perkin-Elmer R12A (60 MHz)
or J EOL FX90Q (90 MHz) instruments on all compounds in
deuteriochloroform (0.4 M solutions, except for saturated
solutions used in the case of 1i,j). Tetramethylsilane was used
as the internal standard. IR spectra were determined using a
Nicolet Impact 400 FTIR spectrometer on all compounds as
potassium bromide disks, while 0.2 M chloroform solutions of
3a -l were also recorded.
Syn th eses of Com p ou n d s. The compounds in series 1-4
were prepared by a base-catalyzed condensation between the
appropriate ketone and the aryl aldehyde using a literature
method,12 except for 1g,l, 2g,l, and 3l, which were prepared
under acidic conditions.13 The compounds were purified by
column chromatography and subsequently by recrystallization
from methanol expect for 2f,g and 3f,g, which were recrystal-
lized from hexane. The melting points (°C) of unreported
compounds were as follows: 1c, 126-128; 1k , 137-139; 2c,
125-127; 2i, 108-110; 2k , 78-80; 3b, 105-107; 3d , 146-148;
3e, 137-139; and 3h , 76-78. The melting points of several
compounds that were different from reported values and those
obtained in the present study were as follows: 1j, 206-208;
2b, 79-81; 2e, 109-111; 3c, 122-124; 4d , 118-120; and 4e,
141-143. The remaining compounds had melting points in
accord with literature values. The 1H NMR spectra of repre-
sentative compounds were as follows: 1f: 2.48 (s, 3H, CH3),
3.80-4.00 (m, 2H, CH2), 6.95-8.10 (m, 9H, aryl and olefinic
H); 2f: 2.37 (s, 3H, CH3), 2.98 (m, 4H, CH2CH2), 6.95-8.35
(m, 8H, aryl H), 7.97 (quasi s, 1H, olefinic H); 3f: 1.60-3.10
(m, 6H, CH2CH2CH2), 2.34 (s, 3H, CH3), 7.05-7.95 (m. 8H,
aryl H), 7.97 (quasi s, 1H, olefinic H); 4a : 3.90 (s, 3H, OCH3),
Deter m in a tion of log P Va lu es of 3a -l. The log P figures
were obtained by a literature procedure.23
Mea su r em en t of Red ox P oten tia ls. The Ep values of 1c,i,
2c,i, and 3c,i were determined by a literature procedure.5
These oxidation potentials were irreversible and are referenced
to the silver/silver chloride electrode potential.
Cytotoxicity Eva lu a tion s. Compounds were evaluated
against murine P388D1 lymphocytic leukemic cells using a
literature procedure.24 A reported methodology was employed

3110 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Dimmock et al.
to evaluate the cytotoxic potential of the compounds using
murine L1210 lymphoid leukemic cells as well as human Molt
4/C8 and CEM T-lymphocytes.25
d1-d4 values determined by X-ray crystallography, compari-
sons between the d1-d15, θ1, and ψ1-ψ5 figures obtained by
X-ray crystallography and molecular modeling for these com-
pounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Deter m in a tion of th e Effect of 1a ,l on Ma cr om olecu la r
Biosyn th eses. The following method was used for determin-
ing the inhibitory effects of 1a ,l on DNA, RNA, and protein
biosyntheses in L1210 cells. The tumor cells were seeded in
96 well microtiter plates using ∼2 × 105 cells per well (200
µL) in the presence of different concentrations of 1a ,l and 0.25
µCi of [methyl-3H]deoxythymidine, [5-3H]uridine, or [3,4-3H]-
leucine. After incubation at 37 °C for 20 h, the trichloroacetic
acid-insoluble fractions of the cell cultures were analyzed for
radioactivity using a liquid scintillation counter.
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Eva lu a tion of Com p ou n d s for An ticon vu lsa n t Activity
a n d Neu r otoxicity. The enones 1a -l, 2a -l, 3a -l, and 4a -f
were examined for anticonvulsant activity in the MES and
scPTZ screens as well as neurotoxicity using a literature
procedure.10 In brief, the compounds were administered in-
traperitoneally in mice using doses of 30, 100, and 300 mg/
kg. After 0.5 and 4 h, any protection against seizures or
neurotoxicity was recorded. Using a dose of 300 mg/kg, activity
was noted by three compounds in the MES screen, namely, 2f
and 4a after 0.5 h and 4d at the end of 4 h. Use of the scPTZ
screen revealed that the number of animals protected at 0.5 h
after administration were as follows: 2j, 2/5 at 100 mg/kg, and
3e and 4a , 1/5 using 300 mg/kg. Various pathological effects
were noted in this screen for the following compounds (dose
in mg/kg and time of observation in h in parentheses), namely,
continuous seizure activity in the case of 1e (300, 0.5; 300, 4),
1k (100,4), and 2e (300,4), tonic extension with 1k (30, 0.5)
and 2g (100, 0.5), death following clonic seizure in the case of
2d (30, 0.5), and myoclonic jerks with 2j (100, 0.5). The
following compounds displayed neurotoxicity (the figures in
parentheses indicate the number of animals demonstrating
neurological deficit as compared to the total number of mice
used, the dose at which neurotoxicity was found, and the time
after administration of the compound when toxicity was
noted): 1e (1/8, 100, 0.5); 1f (1/8, 100, 0.5; 1/4, 300, 0.5); 1g
(2/8, 100, 0.5; 1/4, 100, 4); 1k (1/8, 100, 0.5; 1/4, 300, 0.5); 1l
(1/4, 30, 0.5; 1/4, 300, 0.5); 2a (1/4, 30, 0.5; 1/8, 100, 0.5); 2g
(1/4, 300, 0.5); 2l (1/4, 300, 0.5); 3a (2/4, 30, 0.5); 3d (1/8, 100,
0.5; 1/4, 300, 0.5); 3j (1/4, 300, 0.5; 1/2, 300, 4); 3k (3/8, 100,
0.5; 1/4, 300, 0.5; 1/4, 100, 4; 1/2, 300, 4); 4e (1/4, 100, 4; 1/2,
300, 4); and 4f (1/4, 300, 0.5).
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Reactions; Cope, A. C., Ed.; J ohn Wiley and Sons: New York,
1968; Vol. 16, pp 44-47.
Compounds 1h ,i, 2f,i, and 4a were administered orally to
rats using a dose of 30 mg/kg and examined at the end of 0.25,
0.5, 1, 2, and 4 h in the MES and neurotoxicity screens. The
following compounds afforded protection in one of four animals
(time in h): 1h (4), 2f (0.5, 2, 4), and 2i (1, 4). No protection
was noted at any of the times with 1i and 4a . None of the
compounds demonstrated neurological deficit.
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IV.
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Ack n ow led gm en t. We thank the following agencies
for financial support, namely, the Natural Sciences and
Engineering Research Council of Canada (E.O.O., J.W.Q.,
and H.-B.K.), the Hungarian Ministry of Health (P.P.,
Grant ETT 390/2000), the National Cancer Institute of
Canada (T.M.A.), and Fonds voor Wetenschappelijk
Onderzoek-Vlaanderen (J .B. and E.D.C.). Thanks are
also extended to Mrs. Lizette van Berckelaer for excel-
lent technical assistance (L1210, Molt 4/C8, and CEM
assays), The National Cancer Institute (human tumor
cell lines screen), and Ms. Beryl McCullough who typed
various drafts of the manuscript.
Su p p or tin g In for m a tion Ava ila ble: Details of the X-ray
crystallographic structures of 1a ,d ,h ,k ,l, 2a ,d -f,h ,j,k , and
3a ,d ,e,h -j,l including the atomic coordinates and equivalent
isotropic displacement parameters, anistropic displacement
parameters, hydrogen coordinates and isotropic displacement
parameters, and certain bond lengths and bond angles. The
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