Sequential cytotoxicity: A theory evaluated using novel 2-[4-(3-aryl-2-propenoyloxy)phenylmethylene]cyclohexanones and related compounds

Article abstract of DOI:10.1021/jm000058o

Five series of novel compounds were synthesized in order to evaluate the theory of sequential cytotoxicity which seeks to exploit the view that various cancer cells are particularly susceptible to successive attacks by cytotoxic agents. The compounds prep

Full text of DOI:10.1021/jm000058o

J . Med. Chem. 2000, 43, 3933-3940
3933
Sequ en tia l Cytotoxicity: A Th eor y Eva lu a ted Usin g Novel
2-[4-(3-Ar yl-2-p r op en oyloxy)p h en ylm eth ylen e]cycloh exa n on es a n d Rela ted
Com p ou n d s
J onathan R. Dimmock,*,† N. Murthi Kandepu,† Adil J . Nazarali,† Narasimhan L. Motaganahalli,†
Travis P. Kowalchuk,† Uma Pugazhenthi,‡ J ared S. Prisciak,‡ J . Wilson Quail,§ Theresa M. Allen,|
Rene´e LeClerc,| Cheryl L. Santos,| Erik De Clercq,# and J an Balzarini#
College of Pharmacy and Nutrition and Departments of Biochemistry and Chemistry, University of Saskatchewan, Saskatoon,
Saskatchewan S7N 5C9, Canada, Department of Pharmacology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada,
and Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received February 15, 2000
Five series of novel compounds were synthesized in order to evaluate the theory of sequential
cytotoxicity which seeks to exploit the view that various cancer cells are particularly susceptible
to successive attacks by cytotoxic agents. The compounds prepared were various 2-[4-(3-aryl-
2-propenoyloxy)phenylmethylene]cyclohexanones 1 and the related Mannich bases 2. In addition
the analogues 3-5 lacking an olefinic bond in the ester group were also synthesized, which
were predicted to be less cytotoxic than the compounds of series 1 and 2. The atomic charges
at the potential sites for interaction with cellular constituents were determined by molecular
modeling calculations. The biodata obtained from murine and human neoplastic cells revealed
that the predictions made regarding the viability of the theory were fulfilled in approximately
two-thirds of the cases indicating that further investigation of this hypothesis is warranted.
In addition, the significant potencies of some of the Mannich bases toward human tumor cell
lines, in particular coupled to their selective toxicity toward human leukemic and colon cancer
cells, confirms their usefulness in serving as lead molecules for further development. A
preliminary investigation into the mode of action of representative compounds revealed their
ability to induce apoptosis and inhibit the biosyntheses of ribonucleic acid and proteins.
In tr od u ction
these drugs toward tumors relative to normal cells.3,4
Thus chemosensitization of malignant cells took place.
In addition, lonidamine, which is an inhibitor of cellular
energy metabolism, potentiated the activity of a number
of antineoplastic agents.5 Some of the thiol alkylators
prepared in this report are Mannich bases, representa-
tives of which inhibit respiration in isolated mitochon-
dria.6 On the basis of these arguments, sequential thiol
alkylation may lead to compounds with preferential
toxicity to certain malignant tissues.
The major emphasis in the present investigation was
the design of various groups of compounds as candidate
cytotoxics in order to examine a theory entitled sequen-
tial cytotoxicity. In addition, the aspiration was made
that novel prototypic molecules for further development
as candidate cytotoxic and anticancer agents would
emerge from this study. The theory of sequential cyto-
toxicity may be defined as the proposal that the suc-
cessive release of two or more cytotoxic agents will cause
greater toxicity to malignant tissue than to normal
cells.1 The arguments leading to the proposal of this
theory have been presented at length,1 some of which
are presented in this report vide infra.
The reasons for the design of the compounds prepared
in this study and their utility in evaluating this theory
are as follows. Antineoplastic agents that interact with
thiols (which are not found in nucleic acids) may be
devoid of the mutagenic and carcinogenic properties of
certain anticancer drugs which interact with the hy-
droxy and amino groups of DNA and RNA.2 In regard
to the theory of sequential cytotoxicity, on occasions the
lowering of cellular thiols prior to the administration
of various anticancer agents enhanced the toxicity of
To examine this hypothesis, compounds developed
from 2-arylmethylenecyclohexanones were considered
since previous reports from this laboratory revealed the
significant cytotoxicity of some of these compounds.7,8
Thus the preparation of the enones in series 1-5 as
indicated in Scheme 1 was proposed. These compounds
have different numbers of electrophilic centers. In
addition, the rates and extent of thiolation would be
expected to vary in those compounds having multiple
alkylation sites. The reasons for preparing each cluster
of R,â-unsaturated ketones are as follows. Compound
1a possesses two potential sites for thiolation which
were designated the R and â locations (Chart 1). Initial
attack will be predicted to be at the â site, since at this
portion of the molecule there will be electron-attracting
influences on the olefinic carbon atom by (i) the aryl ring
B, (ii) the adjacent ester group, and (iii), to a lesser
extent, the aryl ring A with an electron-withdrawing
group at the para position. On the other hand, the
electron-attracting influences at the R site are (i) the
* To whom inquiries should be directed. Tel: (306) 966-6331. Fax:
(306) 966-6377. E-mail: dimmock@skyway.usask.ca.
† College of Pharmacy and Nutrition, University of Saskatchewan.
‡ Department of Biochemistry, University of Saskatchewan.
§ Department of Chemistry, University of Saskatchewan.
| University of Alberta.
#
Katholieke Universiteit Leuven.
10.1021/jm000058o CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/30/2000

3934 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Dimmock et al.
Sch em e 1a
Reagents: (i) ClCOCHdCHArR1,R2; (ii) (CH3)2+NdCH2-Cl; (iii) ClCOArR1,R2; (iv) ClCOCH2CH2C6H5. The letters a -g indicate the
following aryl substitution pattern: a , R1 ) R2 ) H; b, R1 ) Cl, R2 ) H; c, R1 ) CH3, R2 ) H; d , R1 ) OCH3, R2 ) H; e, R1 ) F; R2 ) H;
f, R1 ) Br, R2 ) H; g, R1 ) R2 ) Cl.
a
Ch a r t 1
the compounds in series 4 have less capacity for thiola-
tion than the analogues 2.
In summary, therefore, a comparison of the cytotox-
icities of the compounds in series 1-5 may shed some
light on the viability or otherwise of the theory of
sequential cytotoxicity. In addition, one or more com-
pounds with significant antineoplastic activity may
emerge from this investigation serving as templates for
future molecular modifications.
keto group of the cyclohexane ring and (ii) the aryl ring
A to which is attached an electron-withdrawing ester
function. In addition, steric impedance to electrophilic
attack of cellular thiols would be expected to be greater
at the R site. The choice of an ester group was made
since on hydrolysis a phenol and carboxylic acid will be
liberated, both of which should be readily excreted either
per se or as polar metabolites. Hence the soft drug
principle9 will be exemplified whereby the compound
can exert its cytotoxic effect after which facile excretion
may take place thereby reducing unwanted toxicity.
Ch em istr y
The compounds in series 1-5 were prepared accord-
ing to the procedure indicated in Scheme 1. The stere-
ochemistry of the chiral axis adjacent to the cyclohexane
1
ring was assigned the E configuration based on H NMR
spectroscopic and X-ray crystallographic considerations.
Thus the methine protons, when separated from the
absorptions of the aryl hydrogen atoms, were noted at
1
7.4-7.8 ppm in the H NMR spectra. Previous studies
revealed that the methine protons of the E isomers of a
series of related 2-arylmethylene-1-cycloheptanones
absorbed in the region of 7.1-7.8 ppm.12 In addition,
while the methine proton of the E isomer of 2-phenyl-
methylene-6,6-diphenylcyclohexanone appeared in this
region, the corresponding absorption of the Z isomer was
located at 6.3 ppm.13 Furthermore, X-ray crystallogra-
phy of a representative compound (1b) revealed the E
configuration at the chiral axis which was in accord with
previous observations from this laboratory pertaining
to various 2-arylmethylenecycloalkanones14,15 and re-
lated 3,5-bis(arylidene)-4-piperidones and related com-
pounds.16 The double bond present in the 2-propenoyl
esters 1 and 2 was shown from the coupling constants
Molecular modification of 1a was planned as follows.
First, the placement of various substituents in aryl ring
B leading to compounds 1b-g would permit alterations
in the fractional positive charge at the â site which may
correlate with variations in cytotoxicity. It is conceivable
that small changes in the polarity at the R site may also
occur. Aryl substituents with both positive and negative
Hammett σ constants were therefore chosen. Second,
the conversion of the compounds in series 1 into the
corresponding Mannich bases 2 was predicated on the
basis not only of their potential to sensitize tumor cells
to chemical insults vide supra but also of the observation
that â-amino ketones may undergo deamination10 which
would lead to a third alkylation site. Third, to evaluate
further the theory of sequential cytotoxicity, a null
hypothesis11 was considered. In the case of series 3 and
compound 5 multiple alkylation is precluded. Similarly
1
of the olefinic protons of the H NMR spectra to have
the E configuration. The X-ray crystallographic data
generated for a representative compound (1b) supported
this conclusion.

Sequential Cytotoxicity
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3935
Ta ble 1. Atomic Charges on the R and â Carbon Atoms of the
Compounds in Series 1-5
Ta ble 3. Evaluation of the Theory of Sequential Cytotoxicity
Using the Biodata for Compounds in Series 1-5
charge densitiesa
observationa
compd
R carbon atom
â carbon atom
P388 L1210 Molt 4/C8 CEM/O
entry
prediction
cells
cells
cells
cells
1a -g
2a -g
3a -d
4a ,b,d
5
0.024 (0.023-0.025)
0.073 (0.070-0.074)
0.024 (0.023-0.025)
0.073 (0.072-0.073)
0.023
0.052 (0.047-0.057)
0.062 (0.057-0.068)
1
2
3
4
5
6
7
8
1a -d > 3a -d
1a > 5
2a > 5
+
+
+
+
-
+
-
+
-
-
+
-
+
-
+
+
+
+
-
+
2a -d > 3a -d
2a ,b,d > 4a ,b,d
2a -g > 1a -g
3a ) 5
- - - - - -
- - -
+
a
For the first four entries, the average value for the series of
compounds is presented while the range of charge densities is
given in parentheses.
+
N/A
+
+
N/A
+
N/A
+
4a ,b,d > 3a ,b,d
a
Ta ble 2. Cytotoxicity of 1-5 Against Murine P388 and L1210
Cells and Human Molt 4/C8 and CEM T-Lymphocytes
The designations + and - refer to verification and negation
of the hypothesis, respectively. Comparisons of most of the biodata
for 2a ,b,d with 4a ,b,d did not reveal in which group greater
activity resided; these observations are recorded as - - -. The
letters NA indicate that due to some of the cytotoxicity data for
3a being not available, no comparisons of bioactivity could be
made.
IC50 (µM)a
compd
P388 cells L1210 cells Molt 4/C8 cells CEM cells
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
2g
3a
3b
3c
3d
4a
4b
4d
5
20.3 ( 1.21 30.7 ( 0.7
30.7 ( 1.1
78.6 ( 27.3
156 ( 55
2.06 ( 1.69
26.6 ( 8.8
180 ( 10
47.3 ( 3.4
39.5 ( 1.6
2.65 ( 1.60
3.39 ( 2.00
26.0 ( 21.0
63.4 ( 32.1
230 ( 148
8.78 ( 2.15
14.4 ( 6.31
131 ( 82
63.8 ( 22.6
42.8 ( 3.0
3.56 ( 2.48
7.02 ( 2.60
>50
>50
64.3 ( 8.1
202 ( 11
Ta ble 4. Evaluation of Various Compounds Against a Panel of
Human Tumor Cell Lines
9.03 ( 0.83 10.2 ( 0.3
>50
>50
>50
30.9 ( 0.5
127 ( 38
64.8 ( 1.6
all cell lines leukemic cells colon cancer cells
MG MID
MG MID
MG MID
4.02 ( 0.12 32.8 ( 3.8
1.61 ( 0.24 1.74 ( 0.01
1.29 ( 0.03 2.43 ( 0.95
2.41 ( 0.05
compd
(µM)a
(µM)a
SIb
(µM)a
SIb
1a
1b
1d
1g
2a
2b
2g
4b
36.3
91.2
49.7
91.2
2.41
3.58
3.57
1.66
23.5
32.6
1.45
8.32
60.5
9.36
53.0
1.06
0.864
0.83
0.307
4.65
27.6
0.620
4.4
1.5
5.3
1.7
2.3
4.1
4.3
5.4
5.1
1.2
2.3
29.1
81.0
25.4
100
2.21
2.30
2.38
1.21
44.6
7.90
1.42
1.3
1.1
2.0
0.9
1.1
1.6
1.5
1.4
0.5
4.1
1.0
4.55 ( 0.02 1.84 ( 0.06
4.03 ( 0.53 1.78 ( 0.08
4.18 ( 0.43 3.31 ( 1.84
>50
3.78 ( 1.72
3.06 ( 2.21
4.57 ( 2.59
6.00 ( 4.52
4.44 ( 2.68
5.82 ( 3.38
25.50 ( 2.10 3.33 ( 10.1
38.0 ( 4.4
25.6 ( 4.9
34.0 ( 3.0
24.1 ( 18.2
5.11 ( 2.67
2.36 ( 1.23
31.1 ( 3.3
3.24 ( 0.79
23 ( 3.0
41.0 ( 7.4
22.7 ( 14.0
36.1 ( 10.9
29.4 ( 6.8
6.15 ( 0.92
5.07 ( 3.98
34.4 ( 2.9
2.47 ( 0.30
8.9 ( 0.43
>50
>50
44.8 ( 0.7
43.4 ( 1.3
melphalan
5-fluorouracil
helenalin
0.180 ( 0.02 23.4 ( 15.3
0.425 ( 0.04 5.61 ( 2.75
0.332 ( 0.06 2.87 ( 1.64
21.90 ( 0.97 52.4 ( 1.6
0.22 ( 0.01 2.13 ( 0.03
a
The letters MG MID refer to the mean graph midpoint values.
melphalan
b
This term is explained in the Discussion section. SI refers to the
5-fluorouracil 0.49 (0.01) 0.28 ( 0.14
selectivity index, i.e., the ratio between the MG MID value for all
cell lines and the MG MID value for either leukemic or colon cancer
cells.
a
Concentration of compound required to inhibit 50% of the
growth of the neoplasm.
Ta ble 5. Evaluation of Various Compounds in the Hollow
Molecular modeling was undertaken for the com-
pounds 1-5, and the atomic charges at the R and â
carbon atoms were determined using a HyperChem
program. The specific values generated are given in the
Experimental Section, while the average values for the
unsaturated ketones in series 1-4 as well as for 5 are
presented in Table 1. In addition the torsion angles
between the olefinic centers and the adjacent aryl rings
were measured for 1a , 2a , 3a , 4a , and 5.
Fiber Assaya
compd
ip score
sc score
net cell kill
2a
2b
4b
12
0
2
14
6
10
+
-
-
a
Different tumor cell lines in hollow fibers were implanted in
mice by the intraperitoneal (ip) and subcutaneous (sc) routes, while
the compounds were administered intraperitoneally to the animals
using two different doses (see Experimental Section for further
details). A total of 12 tumor cell lines were used, and a score of 2
was given when a 50% reduction or more of the viability of the
cancer cells was noted. A maximum score is therefore 96 (2 × 12
cell lines × 2 routes of implantation × 2 doses). Net cell kill
indicates whether cytotoxicity has occurred (+) or is absent (-)
at either implant site for one or more cell lines.
Cytotoxic a n d Bioch em ica l Eva lu a tion s
The R,â-unsaturated ketones 1-5 were examined
against murine P388 leukemic cells in vitro, while most
of the compounds were also evaluated for cytotoxicity
toward murine L1210 leukemic cells as well as human
Molt 4/C8 and CEM T-lymphocytes. These results are
presented in Table 2. Selected compounds were assessed
against a panel of human tumor cell lines, and the
overall cytotoxicity, as well as the potencies toward the
leukemic and colon cells, are given in Table 4. Three
compounds were also examined in the hollow fiber assay
(Table 5), while the results of determining the apoptotic
indices and effects on RNA and protein syntheses in
human J urkat leukemia cells are summarized in Table
6.
Discu ssion
The theory of sequential cytotoxicity requires that
candidate antineoplastic agents possess at least two
sites which react with cellular constituents at different
rates. To confirm that this situation prevails in the
compounds under review, the atomic charges at the R
and â carbon atoms were determined (Table 1). In the
case of series 1, electrophilicity at the â carbon atom
was greater than at the R site, as predicted. When
considering the R carbon atoms, the atomic charges in

3936 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Dimmock et al.
Ta ble 6. Cytotoxicity, Apoptotic Indices, and Inhibition of
RNA and Protein Syntheses of 1f, 2b,f, and 4b Using Human
J urkat T Cellsa
when comparisons were made using all four cell lines.
This observation suggests that the gradual formation
of an additional alkylating group after initial damage
to the cells has occurred may well lead to potent
antineoplastic agents. Further support for this view-
point is as follows. The Mannich bases 4a ,b,d have one
initial site for thiolation and one further center after
deamination has occurred. On the other hand, while
1a ,b,d are potentially bis-alkylators, their cytotoxicity
in all four screens is lower than 4a ,b,d . Second, the
prediction 1a > 5 was fulfilled in all four screens (entry
2). However 2a being substantially less cytotoxic than
the related Mannich bases in series 2 may explain why
the anticipated greater activity of 2a than 5 was noted
in only one-half of the comparisons made (entry 3).
Third, a review of entries 1 and 5 in Table 3 indicated
that in general the esters 3 and 4 were more cytotoxic
than the vinylogues 1 and 2. It is conceivable that the
2-(4-aroyloxyphenylmethylene) substituent in 3 and 4
is better accommodated at a vulnerable site on a
biological macromolecule than the related group in
series 1 and 2 which has an additional olefinic linkage.
% inhib of syntheses
IC50
(µM)
IC80
(µM)
apoptotic
index
RNA
(SD)
protein
(SD)
compd
1f
2b
2f
4b
19.8
31.7
23.3
87.0
59.2
73.3
63.2b
35.5
38.3
77.5 (7.3) 69.1 (3.5)
78.0 (1.9) 37.1 (7.4)
84.5 (1.9) 46.6 (2.4)
88.4 (4.4) 62.0 (0.6)
85.1b (4.7) 17.3 (4.9)
90.5 (2.2) 49.2 (1.5)
93.3b (3.6) 71.9b (3.5)
2.53
4.47
3.50
2.20b
4.04
7.63
5.60
3.52b
melphalan
actinomycin D 0.0045b 0.0072b
cycloheximide
0.93b
1.54b
a
The IC50 and IC80 values were obtained after exposure of the
cells to the compounds for 48 h. Apoptotic indices and inhibition
of RNA and protein syntheses were noted after 24, 8 and 8 h,
respectively. These latter three determinations were made using
the 80% inhibitory concentrations (IC80) of the compounds.
b
Reprinted in part from J . Med. Chem. 1999, 42, 1363. Copyright
1999 American Chemical Society.
series 2 were approximately 3 times the values obtained
for series 1 due to the electron-withdrawing influence
of the quadrivalent nitrogen atom in the Mannich bases
2. There was a small differential in the R and â atomic
charges in series 2. Similar atomic charges were noted
at the R sites between the compounds in series 1, 3, and
5 as well as between the Mannich bases 2 and 4. Thus
the compounds prepared permit an evaluation of the
hypothesis. Furthermore in order to evaluate the pre-
diction that steric impedance to nucleophilic attack is
greater at the R site than at the â location vide supra,
the torsion angles (θ) between the olefinic axis and the
adjacent aryl ring were measured for the unsubstituted
compounds 1a , 2a , 3a , 4a , and 5. The average θ value
at the R site for these five compounds was 53.8° (53.6-
54.0°), while at the â site of 1a and 2a the θ value was
23.3° in both cases. These data support the notion that
initial attack in series 1 will occur at the â site.
Virtually all of the compounds were examined against
two murine and two human tumor cell lines, and the
results are presented in Table 2. The murine cell lines
were chosen based on the claim of their being predictors
of clinically useful anticancer agents,17 while activity
against the Molt 4/C8 and CEM T-lymphocytes would
indicate the sensitivity of certain human cells to these
compounds. To evaluate the theory of sequential cyto-
toxicity, comparisons of potencies were made between
different groups of compounds by comparing the IC50
values of analogues having the same aryl substituents.
Thus, for example, when evaluating the prediction that
2a -g > 1a -g would support the hypothesis under
consideration, the potency of 2a was compared with 1a ,
2b with 1b, and so on. Verification of the theory was
recorded if the expectations were fulfilled in the majority
of the comparisons made, and vice versa for negation
of the hypothesis. The results are presented in Table 3.
Support for the theory of sequential cytotoxicity was
noted in 19 of the 29 comparisons made (66%). The
values generated indicated the verification of the theory
was somewhat higher for the murine cell lines (73%)
than for the human T-lymphocytes (57%). Some com-
ments on the anticipated and generated cytotoxicity will
be made (the number of the prediction indicated in
Table 3 is in parentheses). First the Mannich bases 2
and 4 were more potent than the analogous R,â-
unsaturated ketones in series 1 and 2 (entries 4, 6, 8)
To evaluate further the cytotoxicity data in reference
to the theory of sequential cytotoxicity, the biodata for
three series of compounds containing one (3), two (1),
and three (2) electrophilic centers were subjected to a
statistical analysis. These three groups of compounds
were chosen having the largest number of entries in
Table 2. A one-way analysis of variance followed by post
hoc analysis with a least-significant difference test18 was
used. In the P388 screen, the potencies of compounds
in series 2 were greater than the potencies of compounds
in series 1 and 3 (p ) 0.00). For L1210, Molt 4/C8, and
CEM cells, compounds 2 with three electophilic centers
were more active than analogues in series 1 (p ) 0.02,
0.03, and 0.05, respectively). No other significant dif-
ferences were noted: i.e., p > 0.05. Thus the evidence
revealed that compounds with the greatest number of
electrophilic centers, i.e., series 2, were more potent
than series 1 and 3 which had fewer sites for thiol
attack. In general therefore, the biodata generated
revealed that the theory of sequential cytotoxicity is
worthy of further consideration.
In view of the significant potencies of a number of
compounds prepared in this study, linear plots were
constructed between the Hammett σ values of the aryl
substituents of 1a -g, 2a -g, 3b-d , and 4a ,b,d and the
IC50 values of these compounds in each of the L1210,
Molt 4/C8, and CEM screens. In addition, similar plots
were generated with 2a -g and 4a ,b ,d using P388
cells. No correlations were noted (p > 0.1) suggesting
that cytotoxicity is mediated by factors other than solely
electronic considerations.
The remainder of this report is devoted to further
investigations of some of these lead molecules. The data
in Table 2 revealed that the greatest cytotoxicity was
noted among the Mannich bases 2 and 4. The average
IC50 values for the four cell lines employed for 2a -c,e-
g, 4a ,b,d , and melphalan (percentage potency of the
compound compared to melphalan in parentheses) were
as follows: 2a , 29.78 (7); 2b, 2.39 (85); 2c, 3.53 (57);
2e, 4.04 (50); 2f, 3.33 (61); 2g, 4.46 (45); 4a , 19.27 (11);
4b, 4.32 (47); 4d , 2.66 (76); melphalan, 2.02 (100) µM.
Thus with the exception of the unsubstituted compounds

Sequential Cytotoxicity
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3937
2a and 4a , compounds with potencies approaching that
of the reference drug melphalan were obtained in series
2 and 4.
average 2a ,b,g and 4b possessed 4 times the potency
of 5-fluorouracil toward colon cancers and 1d ,2b,g had
SI values of 1.5 or more. The remarkable selectivity of
1d against both leukemic and colon cancer cells is
noteworthy. In addition, since the compounds prepared
in this study are considered to interact with cellular
thiols, the data for helenalin, which is an established
thiol alkylator,23 was obtained and incorporated into
Table 4. Helenalin was highly cytotoxic toward the panel
of human tumor cells, and in concert with the results
for the enones described herein it displayed selective
toxicity toward leukemic cells.
Four Mannich bases (2a ,b,g and 4b) were evaluated
against a panel of 55 ( 5 human tumor cell lines. In
addition four related R,â-unsaturated ketones (1a ,b,d ,g)
were included in this screen, which were predicted to
be substantially less cytotoxic than 2a ,b,g and 4b. In
this assay, determination of the bioactivity of the
compounds toward cell lines from the neoplastic dis-
eases leukemia, melanoma, non-small-cell lung, colon,
central nervous system, ovarian, renal, prostate, and
breast cancers19 was undertaken. The concentrations of
compounds employed in this assay were generally log
10-4 to log 10-8 M, and the amounts required to inhibit
50% of the growth of each tumor were noted. On
occasion 50% of the growth of a cell line was not
inhibited at the maximum concentration utilized, e.g.,
log 10-4 M, although this number was incorporated into
the average value for all cell lines. Hence the term mean
graph midpoint (MG MID) was used since the numbers
generated were not invariably IC50 values. The number
of cell lines for which actual IC50 values were obtained
is given in the Experimental Section. In general, when
the MG MID values were less than 40 µM, IC50 values
were generated for most of the cell lines. On the other
hand, any MG MID value of 95-100 signifies that few
or none of the cell lines were inhibited by 50% at the
highest concentration of compound utilized. The MG
MID values of various compounds are presented in
Table 4.
The data in Table 4 revealed that the in vivo evalu-
ation of some of these compounds, particularly the
Mannich bases, was warranted. The hollow fiber assay24
was employed whereby different tumor cell lines were
placed in polyvinylidene fluoride fibers and inserted
intraperitoneally (ip) or subcutaneously (sc) into mice.
In the present investigation 2a ,b,4b were administered
intraperitoneally, and the results are summarized in
Table 5. Activity is denoted when a compound achieved
one of the following criteria for activity: namely a total
ip + sc score of more than 20, a sc value of greater than
8, and a net cell kill in one or more cell lines. Thus 2a
fulfilled all three criteria, while 4b possessed a sc score
of 10, revealing that both compounds are promising
candidates for further in vivo experimentation. In
addition, greater activity at the more remote implant
site (sc) relative to ip implantation is characteristic of
a compound being converted into a more potent product
in vivo.25 It is of interest to note that the sc scores of all
three compounds were higher than the ip results sug-
gesting that the Mannich bases were converted into
more toxic species in vivo.
The data in Table 4 revealed a similar cytotoxicity
pattern as the results obtained using the P388, L1210,
Molt 4/C8, and CEM cell lines. Thus a comparison of
the MG MID values between 2a ,b,g and 1a ,b,g indi-
cated greater cytotoxicity of the Mannich bases 2 as
predicted by the hypothesis assuming that deamination
occurred liberating a third olefinic center. On the other
hand, 4b was approximately twice as potent as its
vinylogue 2b in opposition to the theory under consid-
eration.
Finally in view of the promising cytotoxicity displayed
by various compounds described in this study, a pre-
liminary investigation into the possible mode(s) of action
of representative compounds was undertaken. A number
of anticancer agents induce apoptosis26 and/or inhibit
the syntheses of ribonucleic acid (RNA) and proteins.27
The compounds chosen were 1f, possessing very low
cytotoxicity, 2f, which is a potent analogue of 1f, 4b,
which displayed the highest activity against a panel of
human tumor cell lines, and its vinylogue 2b. A recent
investigation from this laboratory revealed that human
J urkat T cells were more sensitive to an R,â-unsaturated
ketone than other cell lines,28 and hence this neoplasm
was used in the present study. The results of examining
the modes of action of 1f, 2b,f, 4b, and melphalan as
well as actinomycin D and cycloheximide, which are
potent inhibitors of RNA and protein, respectively, are
summarized in Table 6.
Two other features pertaining to the data in Table 4
will be mentioned. First, while 1a ,b,d ,g were less active
than melphalan, 2a ,b,g and 4b possessed 10, 7, 7, and
14 times, respectively, the potency of this established
alkylating agent, suggesting their utilization as proto-
typic molecules for analogue development. Second, a
review of the mean graphs20 revealed that various
compounds displayed selective toxicity toward leukemic
and colon cancer cells. A comparison between the MG
MID values of each compound toward all neoplasms and
either leukemic or colon cancer cells gave rise to
selectivity index (SI) values which are indicated in Table
4. The arbitrary assumption was made that a SI value
of 1.5 was a noteworthy indication of selectivity for a
group of tumors: i.e., the particular tumors were 50%
more sensitive to the compound when compared to all
cell lines. The criterion for leukemic cells was met in
all eight compounds. In fact in the case of 1d and 4b,
the selectivity was greater than for the reference drug
melphalan which is used clinically in treating leuke-
mia.21 When bioevaluation toward colon cancer cells was
reviewed, comparisons were made with 5-fluorouracil
which is a drug used in treating colon cancer.22 On
A review of the IC50 values for 1b, 2b,f and 4b
presented in Table 2 revealed the far greater activity
of 2b,f and 4b than of 1f. The same observation was
made when J urkat T cells were employed as revealed
by the data in Table 6. To determine whether apoptosis
occurred, concentrations of compounds were used which
caused the death of the majority of the cells, and hence
IC80 values were generated. An apoptotic index [(the
number of cells with apoptotic nuclei/the number of cells
counted) × 100] was calculated for each compound.
Apoptosis was induced by all four compounds but to a
much greater extent by the more potent compounds 2b,f

3938 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Dimmock et al.
Syn th esis of 1a -g, 3a -d , a n d 5. The esters 1a -g were
prepared as follows. A solution of the appropriate 3-aryl-2-
propenoyl chloride (0.01 mol) in pyridine (0.5 mL) was added
to a solution of 1-(4-hydroxyphenylmethylene)cyclohexanone
(0.01 mol) in dry acetonitrile (40 mL) and the mixture was
heated under reflux for 5-6 h. Moisture was excluded by using
a calcium chloride guard tube on the reflux condenser. On
cooling, the precipitates were collected, washed successively
with hydrochloric acid (1 N), aqueous sodium carbonate
solution (10% w/v) and water and recrystallized from chloro-
form-diethyl ether (1a ), chloroform-acetonitrile (1b-f) or
chloroform (1g). The melting points (°C) and yields (%) were
as follows: 1a : 129-131, 74; 1b: 174-176, 75; 1c: 154-157,
65; 1d : 143-145, 60; 1e: 147-149, 75; 1f: 175-177, 80; 1g:
164-166, 72. The 1H NMR spectrum of a representative
compound 1c was as follows: δ (CDCl3) 1.75-1.79 (m, 2H,
4-CH2), 1.91-2.04 (m, 2H, 5-CH2), 2.39 (m, 3H, CH3), 2.54 (t,
2H, 3-CH2); 2.82-2.86 (m, 2H, 6-CH2), 6.58 (d, 1H, OCOCHd
CH, J ) 16.0 Hz), 7.18-7.25 (m, 4H, aryl H of ring B), 7.42-
7.50 (m, 5H, CH and aryl H of ring A), 7.85 (d, 1H, OCOCHd
CH, J ) 16.0 Hz).
Compounds 3a -d were prepared in a similar manner as
1a -g except that the time of heating under reflux was 7-8 h
after which time the reaction mixture was poured into water
(150 mL). The precipitates were collected after 1-2 h, washed
as described previously and purified from water-methanol
(3a ) or water-acetonitrile (3b-d ). The melting points (°C) and
yields (%) were as follows: 3a : 108-109, 64; 3b: 121-123,
70; 3c: 119-120, 55, 3d : 103-105, 68. The 1H NMR spectrum
of a representative compound 3d was as follows: δ (CDCl3)
1.74-1.79 (m, 2H, 4-CH2), 1.90-1.95 (m, 2H, 5-CH2), 2.53 (t,
2H, 6-CH2, J ) 6.7 Hz), 2.82-2.85 (m, 2H, 3-CH2), 3.86 (s,
3H, OCH3), 6.97 (d, 2H, 2 and 5 H of aryl ring B, J ) 8.7 Hz),
7.22 (d, 2H, 2 and 6 H of aryl ring A, J ) 8.5 Hz), 7.23 (d, 2H,
2 and 5 H of aryl ring A, J ) 8.5 Hz), 7.48 (s, 1H, dCH), 8.14
(d, 2H, 2 and 6 H of aryl ring B, J ) 8.7 Hz).
and 4b than 1f. All of the compounds markedly inhib-
ited RNA synthesis, while, in general, protein synthesis
was inhibited to a lesser extent. No correlations between
cytotoxicity and inhibition of the syntheses of either
RNA or protein were noted. One may conclude that the
compounds in this study likely exert their cytotoxicity
at least in part by inducing apoptosis and interfering
with the biosynthesis of important macromolecules.
Con clu sion s
This study describing the bioactivities of a selected
number of novel prototypic molecules revealed that the
theory of sequential cytotoxicity was fulfilled in ap-
proximately two-thirds of the evaluations made. Hence
this hypothesis is worthy of further consideration with
an expanded series of analogues as well as with other
groups of compounds. In particular various Mannich
bases displayed potent cytotoxicities toward murine
cells, human T-lymphocytes, and a panel of human
tumor cell lines, showed selective toxicity to human
leukemic and colon cells, and demonstrated activity in
vivo. These molecules may therefore be considered
prototypic molecules for further development as candi-
date cytotoxic and anticancer agents.
Exp er im en ta l Section
Melting points are uncorrected. Acetonitrile, used in the
synthesis of the Mannich bases, was dried over molecular
sieves. Cinnamoyl chloride used in the preparation of 1a , the
aroyl chlorides required for the synthesis of 3a -d and 3-phe-
nylpropionyl chloride required for the preparation of 5 were
obtained from Sigma-Aldrich Canada Ltd., Oakville, Ontario,
Canada. Elemental analyses were undertaken on 1a -g, 3a -
d , 5 (C,H) and 2a -g, 4a ,b,d (C,H,N) and were within 0.4% of
the calculated values. The Mannich bases 2f,g were obtained
as the hemihydrates and 4b as the monohydrate. 1H NMR
spectroscopy was carried out using a Bruker AMX 500 FT
NMR machine. A Nonius CAD-4 diffractometer was used for
the collection of X-ray crystallographic data. Thin-layer chro-
matography (TLC) was employed to monitor the progress of
the reactions and to determine the homogeneity of the reaction
products using silica gel 60 F254 precoated TLC plastic sheets
and a solvent system of hexane:ethyl acetate (7:3); observations
were made under ultraviolet light and/or in an iodine chamber.
Syn t h esis of 2-(4-H yd r oxyp h en ylm et h ylen e)cyclo-
h exa n on e. A solution of 4-hydroxybenzaldehyde (0.10 mol)
in aqueous sodium hydroxide solution (4.1% w/v, 150 mL) was
added slowly to a mixture of cyclohexanone (0.48 mol) in
aqueous sodium hydroxide solution (4.1% w/v, 50 mL). The
reaction mixture was stirred at room temperature for 36 h,
acidified with hydrochloric acid (1 N) and extracted with
chloroform (100 mL). The organic extract was washed with
water, dried (anhydrous magnesium sulfate) and evaporation
of the solvent gave an oil which was extracted while hot with
petroleum ether (bp 45-60 °C). Upon cooling a yellow pre-
cipitate was obtained and collected. Distillation of the product
using a Kugelrohr apparatus gave 2-(4-hydroxyphenylmeth-
ylene)cyclohexanone: bp 136-138 °C/10-1 mbar; mp 180-181
°C (lit.29 mp 172 °C); 40% yield.
The enone 5 was prepared in a similar manner as 1a -g
except that the time of heating under reflux was 4 h. The crude
product was recrystallized from acetonitrile to give 5: mp 97-
99 °C; 80% yield.
Syn th esis of 2a -g a n d 4a ,b,d . The Mannich bases 2a -g
were prepared as follows. A solution of the precursor ester in
series 1 (0.005 mol) in dry acetonitrile (40 mL) was prepared,
if necessary by warming, to which was added N,N-dimethyl-
methyleneammonium chloride (0.01 mol), which was synthe-
sized by a literature procedure.31 The mixture was stirred at
room temperature for 2-3 h and the resultant precipitate was
collected, washed with warm acetonitrile and recrystallized
from acetonitrile (2a ,d ) or chloroform (2b,c,e-g). The melting
points and yields were as follows: 2a : 120-122, 48; 2b: 165-
167, 60; 2c: 169-170, 50; 2d : 171-173, 62; 2e: 165-167, 60;
1
2f: 170-171, 60; 2g: 145-147, 52. The H NMR spectrum of
a representative compound 2e was as follows: δ (CDCl3) 1.73-
1.89 (m, 2H, 4-CH2), 1.94-2.02 (m, 2H, 5-CH2), 2.61-2.72 (m,
2H, 3-CH2), 2.71 [d, 3H, N(CH3), J ) 4.6 Hz], 2.85 [d, 3H,
N(CH3), J ) 4.5 Hz], 3.00-3.10 [m, 2H, CH2N(CH3)2], 3.62-
3.70 (m, 1H, 6-CH), 6.54 (d, 1H, OCOCHdCH, J ) 16.0 Hz),
7.10-7.57 (m, 8H, aryl H), 7.44 (s, 1H, CHd), 7.82 (d, 1H,
OCOCHdCH, J ) 16.0 Hz).
The Mannich bases 4a ,b,d were prepared in a similar
manner as 2a -g except 25 mL of acetonitrile was employed
and the times of stirring at room temperature were 3-6 h.
The products were recrystallized from methanol (4a ) or
acetonitrile (4b,d ). The melting points and yields were as
follows: 4a : 171-173, 70; 4b: 160-161, 75; 4d : 154-155,
65. The 1H NMR spectrum of a representative compound 4b
was as follows: δ (CDCl3) 1.75-1.86 (m, 2H, 4-CH2) 1.98-
2.00 (m, 2H, 5-CH2), 2.63-2.71 (m, 2H, 3-CH2), 2.79 [d, 3H,
N(CH3), J ) 4.8 Hz], 2.85 [d, 3H, N(CH3), J ) 4.7 Hz], 3.02-
3.10 [m, 2H, CH2N(CH3)2], 3.64-3.69 (m, 1H, 6-CH), 7.23-
7.25 (m, 2H, 3 and 5 H of aryl ring B), 7.42-7.47 (m, 5H, CHd,
2 and 6 aryl H of ring A, 3 and 5 aryl H of ring B), 8.11-8.13
(d, 2H, 2 and 6 aryl H of ring B).
Syn th esis of 3-Ar yl-2-p r op en oyl Ch lor id es. The 3-aryl-
2-propenoyl chlorides required in the synthesis of 1b-g were
obtained as follows. The appropriate 3-aryl-2-propenoic acid
(0.02 mol), prepared by a literature procedure,30 was mixed
with thionyl chloride (15 mL) using a condenser to which was
attached a calcium chloride guard tube. After heating under
reflux for 4 h, the mixture was evaporated under reduced
pressure to produce an oily residue which was dissolved in
petroleum ether (bp 35-45 °C). Removal of the organic solvent
gave the acid chloride which was used immediately in subse-
quent reactions.

Sequential Cytotoxicity
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3939
X-r a y Cr ysta llogr a p h y of 1b. Compound 1b was recrys-
tallized from ethanol-2-propanol by vapor diffusion. An ω scan
was used for data collection and the structure was solved by
direct methods using NRCVAX32 and refined using SHELXL
97.33 Atomic scattering factors were taken from the literature.34
All non-hydrogen atoms were found on the E-map and were
refined anistotropically. Hydrogen atom positions were calcu-
lated and not refined.
protein synthesis was undertaken by a previously published
methodology.42
Ack n ow led gm en t. Funding for this study was
provided by CoCensys, Inc. to J .R.D., the Natural
Sciences and Engineering Research Council of Canada
to A.J .N. and J .W.Q., the National Cancer Institute of
Canada to T.M.A., and the Fonds voor Wetenschappelijk
Onderzoek (FWO-Vlaanderen) to J .B. and E.D.C. The
technical assistance of Mrs. Lizette van Berckelaer is
greatly appreciated. The National Cancer Institute,
Bethesda, MD, evaluated various compounds against
the human tumor cell lines. Thanks are also extended
to Christa J amont and J ackie Huck who typed various
drafts of the manuscript.
Molecu la r Mod elin g. The structures of the compounds in
series 1-5 were built using the MacroModel version 4.5
program.35,36 A conformational search was undertaken using
the Monte Carlo method and MM2 force-field parameters in
order to obtain minimum energy conformations. The torsion
angles θ were obtained from these molecules. The θ values
formed between the olefinic group and the adjacent aryl ring
A in 1a , 2a , 3a , 4a and 5 were -53.7°, -53.7°, 53.8°, 54.0°
and -53.6°, respectively. The θ values obtained between the
ester olefinic group and aryl ring B were -23.3° and 23.3°,
respectively. The signs of the torsion angles are positive or
negative if the rotations are anticlockwise or clockwise,
respectively. A HyperChem molecular modeling program37 was
utilized to obtain the atomic charges on the R and â carbon
atoms. The atomic charges at the R and â sites (1, 2) and at
the R sites (3-5) are as follows: 1a : 0.024, 0.052; 1b: 0.024,
0.051; 1c: 0.025, 0.054; 1d : 0.025, 0.057; 1e: 0.024, 0.053;
1f: 0.025, 0.051; 1g: 0.023, 0.047; 2a : 0.070, 0.057; 2b: 0.073,
0.061; 2c: 0.074, 0.065; 2d : 0.074, 0.068; 2e: 0.073, 0.064;
2f: 0.074, 0.062; 2g: 0.073, 0.058; 3a : 0.023; 3b: 0.024; 3c:
0.025; 3d : 0.025; 4a : 0.073; 4b: 0.072; 4d : 0.073; 5: 0.023.
Su p p or tin g In for m a tion Ava ila ble: Details of the X-ray
crystallographic structure of 1b including the atomic coordi-
nates and equivalent isotropic displacement parameters,
anisotropic displacement parameters, hydrogen coordinates
and isotropic displacement parameters, and certain bond
lengths and bond angles. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Cytotoxicity Eva lu a tion s. Literature procedures were
followed when examining the compounds against murine P388
D1 cells38 as well as murine L1210 and human Molt 4/C8 and
CEM T-lymphocytes39 and human tumor cell lines.19 The
highest concentration of compound employed was log 10-4
M
except for 2b, melphalan and 5-fluorouracil in which cases the
maximum concentrations used were log 10-4.3, log 10-3.6 and
log 10-2.6 M, respectively. The number of cell lines in the
human tumor assay whose growth was inhibited by 50% or
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were conducted in duplicate (1d , 2a ,b,g) or triplicate (mel-
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7.91-10.8, 23.2-27.6; 2a : 2.00-2.82, 0.801-1.31, 1.64-2.78;
2b: 2.14-5.01, 0.648-1.08, 1.28-3.31; 2g: 3.24-3.89, 0.417-
1.24, 2.31-2.44; melphalan: 19.1-26.9, 2.82-7.16, 41.7-49.9;
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and 4b (50 and 75 mg/kg) were made daily for 4 days and the
fibers collected 1 day after the last treatment. The anticancer
effects were calculated by determining the viable cell mass
using the formazan dye (MTT) conversion assay.41 Net cell kill
was observed with the COLO 205 and MDA-MB-231 cell lines.
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