4-(β-arylvinyl)-3-(β-arylvinylketo)-1-ethyl-4-piperidinols and related compounds: A novel class of cytotoxic and anticancer agents

Article abstract of DOI:10.1021/jm9801455

The syntheses of a series of 1-aryl-5-diethylamino-1-penten-3-one hydrochlorides 1 and 1-aryl-3-diethylamino-1-propanone hydrochlorides 4 were accomplished. Attempts to prepare the corresponding bis(5-aryl-3-oxo-4- pentenyl)ethylamine hydrochlorides 2 and

Full text of DOI:10.1021/jm9801455

4012
J . Med. Chem. 1998, 41, 4012-4020
4-(â-Ar ylvin yl)-3-(â-a r ylvin ylk eto)-1-eth yl-4-p ip er id in ols a n d Rela ted
Com p ou n d s: A Novel Cla ss of Cytotoxic a n d An tica n cer Agen ts
J onathan R. Dimmock,*,† Sarvesh C. Vashishtha,† J . Wilson Quail,‡ Uma Pugazhenthi,‡ Zbigniew Zimpel,‡
Athena M. Sudom,§ Theresa M. Allen,| Grace Y. Kao,| J an Balzarini, and Erik De Clercq
College of Pharmacy and Nutrition, Department of Chemistry, and Department of Biochemistry, 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 March 11, 1998
The syntheses of a series of 1-aryl-5-diethylamino-1-penten-3-one hydrochlorides 1 and 1-aryl-
3-diethylamino-1-propanone hydrochlorides 4 were accomplished. Attempts to prepare the
corresponding bis(5-aryl-3-oxo-4-pentenyl)ethylamine hydrochlorides 2 and bis(3-aryl-3-oxo-
propyl)ethylamine hydrochlorides 5 led to the formation of a series of 4-(â-arylvinyl)-3-(â-
arylvinylketo)-1-ethyl-4-piperidinol hydrochlorides 9 and 4-aryl-3-arylketo-1-ethyl-4-piperidinol
hydrochlorides 11, most of which were converted subsequently into the corresponding
quaternary ammonium salts 10 and 12, respectively. The structures of these compounds were
determined by 1H NMR spectroscopy and confirmed by X-ray crystallography of representative
molecules. Most compounds displayed significant cytotoxicity toward murine P388 and L1210
cells as well as human tumors. In general, Mannich bases containing olefinic bonds were more
cytotoxic than the analogues without this functional group, while the piperidines 9 and 11
were more potent than the acyclic analogues 1 and 4, respectively. Correlations were noted
between various physicochemical constants in the aryl rings and cytotoxicity. Compound 9d
displayed promising in vivo activity against colon cancers. This study has revealed that the
piperidines 9 and 11 constitute new classses of cytotoxic agents.
In tr od u ction
sequential cytotoxicity further, the bis Mannich bases
2 were considered for bioevaluation. Initial thiol attack
could occur at one of the olefinic double bonds to be
followed by a second thiol interaction which could be
more damaging to neoplastic cells than normal tissues.
This assumption depends on nonequivalent charges at
the olefinic bonds in order to avoid synchronous attack
at both olefinic carbon atoms. The pKa values of the
acyclic Mannich base 7 and related analogues were
correlated with the magnititude of the Hammett σ
values in the aryl ring.9 Hence basicity and electron
densities of the â-arylvinyl group are interrelated.
When the nitrogen atoms are ionized, the electron
densities at the olefinic bonds will be reduced compared
to the corresponding free bases, and thus nucleophilic
attack by thiols will be enhanced. Hence the related
quaternary ammonium compounds 3 were predicted to
be more cytotoxic than 2 since the Mannich bases will
exist as a mixture of the free bases and protonated
hydrochloride salts. The use of a null hypothesis10
suggested the preparation of 4-6 which lacked olefinic
double bonds. These compounds would be predicted to
be less cytotoxic than the analogues 1-3; should any
bioactivity be displayed by these molecules, then one
may conclude that the structural features in 1-3 other
than the olefinic double bonds contributed to bioactivity.
The aryl substitution pattern in series 1-6, which has
been employed in a Topliss analysis,11 was chosen so
that atoms and groups with divergent electronic and
hydrophobic properties were used. In fact, the chloro,
methyl, and methoxy substituents are found in three
different quadrants of a two-dimensional Craig plot.12
Three related analogues were also considered for
cytotoxic evaluation. First, 7 (as the hydrobromide salt)
A number of Mannich bases derived from arylidene
alkyl ketones and related compounds possess significant
cytotoxic and anticancer properties.1 These compounds
were developed as thiol alkylators since R,â-unsaturated
ketones have a markedly greater affinity for thiols over
amino and hydroxy nucleophiles.2,3 Hence interactions
with nucleic acids may be avoided, and the disadvan-
tages of certain alkylating agents such as mutagenicity4
and carcinogenicity5 may be absent. Support for the
contention that these compounds have a different mode
of action than the widely used alkylating agent mel-
phalan was provided by noting that they displayed simi-
lar cytotoxicity toward melphalan-resistant and mel-
phalan-sensitive neoplastic cells, i.e., the melphalan-
resistant cell lines were free from cross-resistance to
these Mannich bases.6 In addition, several series of
Mannich bases have been prepared recently which were
designed using the concept of sequential cytoxicity.7,8
This theory may be defined as the successive chemical
attack by two or more cytotoxic agents whereby greater
toxicity to malignant rather than normal cells will be
displayed. The reasons for proposing this theory, as
well as the relevant literature, have been presented at
length.7
The original objectives of the present study included
the synthesis of series 1 (Scheme 1) for cytotoxic
evaluation. In addition, to evaluate the theory of
* To whom inquiries should be directed.
† College of Pharmacy and Nutrition.
‡ Department of Chemistry.
§ Department of Biochemistry.
| Department of Pharmacology.
Rega Institute for Medical Research.
S0022-2623(98)00145-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/19/1998

Novel Class of Cytotoxic and Anticancer Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4013
Sch em e 1a
a
Reagents: (i) HCHO, HN(C2H5)2HCl; (ii) HCHO, H2NC2H5HCl; (iii) CH3Br. The letters a -f reflect the aryl substitution pattern: a ,
R1 ) R2 ) H; b, R1 ) Cl, R2 ) H; c, R1 ) R2 ) Cl; d , R1 ) CH3, R2 ) H; e, R1 ) OCH3, R2 ) H; f, R1 ) OH, R2 ) H.
has 1.3 times the activity of 5-fluorouracil against the
human WiDr colon cancer in vitro,9 and a comparison
of its cytotoxicity with 1b using the screens employed
in the present study was of interest. Second, 8a ,b were
suggested in order to determine the effect of pKa on
cytotoxicity. Thus the pKa values of the nitrogen atoms
F igu r e 1. Numbering used in atoms 1a , 2a , 3a , 4a , 5a , and
6a for which electrostatic charges were computed.
of piperazine are 5.33 and 9.73, while the figure for
In summary therefore the aim of the present inves-
triethylamine is 10.75.13 Hence under biological condi-
tigation was the preparation of the acyclic molecules
1-8 as candidate cytotoxic and anticancer agents. The
results generated would enable evaluation of the predic-
tions, 1a should present a higher percentage of ionized
species than 8a and thus display greater toxicity. A
comparison of the screening data of 8a with 8b would
tions of relative bioactivity to be made and in particular
indicate the importance of olefinic bonds in this group
to examine the theory of sequential cytotoxicity.
of molecules.
Resu lts
To give theoretical support to these proposals, the
electrostatic charges on several of the atoms of the
unsubstituted compounds in series 1-6 were computed.
The atoms chosen and the results generated are indi-
cated in Figure 1 and Table 1, respectively.
Compounds 1a -e, 7, and 8a were prepared from the
appropriate â-arylvinyl alkyl ketone, formaldehyde, and
secondary amine hydrochloride. The Mannich bases
4a -e and 8b were prepared in a similar fashion from
the appropriate aryl methyl ketone. Attempts to pre-
pare the quaternary ammonium salts from the tertiary
amines 1 and 4 led to the isolation of impure products
only; these synthetic difficulties have also been noted
by other laboratories.14 However reaction of ethylamine
hydrochloride with excess of either â-arylvinyl methyl

4014 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Dimmock et al.
Ta ble 1. Electrostatic Charges on Certain Atoms of 1a , 2a ,
3a , 4a , 5a , and 6a
Ta ble 2. Electrostatic Charges on Some Carbon Atoms and
Cytotoxicity of 1a , 9a , 10a , and 14a
electrostatic charges of atomsa
IC50 (µM)
compd
C1
C2
N3
C4
C5
R6b
human
P388 L1210 tumor
screen screen screen
1a
2a
3a
4a
5a
6a
-0.296
0.017 -0.191
0.332
compd
C1
0.025 -0.576
0.057 -0.552 -0.403 -0.194
C2
C3
C4
-0.252 -0.151 -0.074 -0.173 -0.209 0.368
-0.280 -0.110
-0.112 -0.143 -0.112
0.364 -0.184 -0.202 0.108
1a
9a
1.7
1.4
2.3
25.7
5.3
9.8
2.8
8.3
0.368
-0.136 -0.040 -0.163 -0.087 -0.107 0.382
10a -0.001 -0.505 -0.370 -0.253
14
NAb
0.032 -0.192
0.269 -0.202
0.080 0.119
-0.055 -0.489 -0.354 -0.273
a
b
a
b
The designation of atoms is given in Figure 1. R ) H (1a ,
2a , 4a , 5a ) or CH3 (3a , 6a ).
The designation of atoms is given in Figure 3. Result not
available.
Ta ble 3. Cytotoxicity Data of Various Mannich Bases and
Related Compounds
IC50 (µM)
compda
1a
1b
1c
1d
1e
4a
4b
4c
4d
4e
7
8a
8b
9a
9b
P388 cells
L1210 cells
human tumors
9.77
1.7
0.3
0.39
1.1
0.67
2.6
3.2
0.31
2.2
1.6
2.2
0.62
2.84
1.4
1.1
0.4
25.69 ( 11.09
41.03 ( 2.32
61.18 ( 20.49
52.87 ( 26.61
44.32 ( 28.21
20.31 ( 3.10
36.93 ( 5.79
36.38 ( 3.86
21.54 ( 2.66
43.79 ( 27.96
6.39 ( 1.76
3.80
25.12
32.36
26.30
5.78 ( 1.94
12.02
9.71 ( 2.22
5.30 ( 2.44
2.75
25.72
0.38
5.01
8.31
8.31
2.19
F igu r e 2. ORTEP diagram of 10d .
6.96 ( 0.26
9c
9d
9e
2.1
5.0
6.08 ( 2.47
25.55 ( 14.85
10a
10c
10d
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
13a
13b
melphalan
2.27
0.64
0.62
1.07
5.14
3.11
8.6
4.17
1.4
1.3
1.5
2.3
2.1
0.98
1.89
2.59
0.22
21.44 ( 1.55
13.79 ( 6.89
20.07 ( 8.91
15.67 ( 8.56
30.19
83.18
16.59
25.12
19.49
20.89
12.88
12.88
9.54
38.24 ( 12.91
13.5 ( 5.19
9.75 ( 1.14
33.57 ( 6.17
50.11
10.23
2.46
F igu r e 3. Designations of atoms in 1a , 9a , 10a , and 14 for
which electrostatic charges were computed.
12.6 ( 10.4
20.9 ( 0.3
2.13 ( 0.02
ketones or aryl methyl ketones and formaldehyde led
to 9 and 11, respectively. The significant in vitro and
in vivo activity of 9d vide infra suggested the prepara-
tion of the analogues 13a ,b which were synthesized by
the same route. Reaction of the free bases of 9a ,c,d with
methyl bromide gave rise to 10a ,c,d , respectively while
the quaternary ammonium salts 12 were prepared from
the free bases of 11. The structures of the compounds
were confirmed by 1H NMR spectroscopy and elemental
analyses. X-ray crystallography was undertaken for 8b,
10d , 12d (as the iodide salt), and 13a ,b. The ORTEP
diagram of a representative compound, namely, 10d , is
displayed in Figure 2. The electrostatic charges on the
olefinic carbon atoms in 9a , 10a , the free base of 9a
referred to hereafter as 14, and 1a as indicated in
Figure 3 were calculated, and the results are given in
Table 2.
26.30
a
Hydrobromide salts obtained from the free bases of 4a and
11a -c,e were used in the human tumor screen. The hydrobromide
salt from the free base of 11d was employed in all screens.
tion against several human tumor xerografts, and these
data are presented in Table 5.
Discu ssion
The data provided in Table 1 supported the rationale
for the synthesis and bioevaluation of the compounds
in series 1-6. First, variation in the charges on carbon
atoms 1 and 5 in each of the bis compounds 2a and 3a
meant that in each molecule, a different electronic effect
will be exerted on the adjacent unsaturated keto group.
Thus initial nucleophilic attack by a cellular constituent
at one carbon atom would be followed by a subsequent
thiol alkylation as the theory of sequential cytotoxicity
requires. Second, in addition to thiol alkylation at the
olefinic bonds, Mannich bases can react by amino group
The cytotoxic properties of the compounds in series
1, 4, and 7-13 as well as the established anticancer
drug melphalan are given in Table 3. The promising
in vitro activity of 9d and 10a suggested their examina-

Novel Class of Cytotoxic and Anticancer Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4015
Ta ble 4. Correlations between the σ, π, and MR Constants in
the P388, L1210, and Human Tumor Screens
aryl
substituent
constant
corre-
screen
P388
series
plota
p valueb
lationc
1
4
9
9
1
1
4
4
MR
MR
σ
π
π
MR
MR
σ
lin, log <0.05, <0.1
lin, log <0.1, <0.05
lin, log <0.1, <0.005
lin, log <0.1, <0.05
lin, log <0.1, <0.1
lin, log <0.025, <0.025
+
+
+
+
-
-
-
-
L1210
log
log
<0.1
<0.1
human
tumors
4
4
9
π
MR
π
lin, log <0.1, <0.1
lin, log <0.1, <0.1
-
-
+
conformations. The piperazine analogues 8a ,b did not
undergo intramolecular cyclizations, the X-ray crystal-
lography of 8b confirming this conclusion. In these
cases, a cyclization process would require the formation
of a nine-membered ring system. Finally, a survey of
previous investigations revealed reports of this type of
intramolecular cyclization17-21 which probably resulted
from an acid-catalyzed aldol reaction of the bis Mannich
bases 2 or 5. In this process, the enol group of one
carbonyl function would attack the carbon atom of the
second (protonated) carbonyl group.
log
<0.1
a
Both linear (lin) and semilogarithmic (log) plots were made.
When two values are quoted, they refer to correlations obtained
b
from the linear and logarithmic plots, respectively. c Positive (+)
correlations indicate that cytotoxicity rose as the σ, π, and MR
values are increased, while negative (-) correlations reveal that
increased bioactivity occurred with diminishing σ, π, and MR
figures.
Ta ble 5. Effect of 9d and 10a on Various Human Tumor
Xenografts Passaged in Athymic Mice
% T/Ca
(dose in
mg/kg)
% ILSb
(dose in
mg/kg)
Predictions were made regarding the way in which
compounds containing olefinic groups could interact
with cellular thiols. Electrostatic charges of the olefinic
groups in 1a , 9a , 10a , and 14 (the free base of 9a ) as
indicated in Figure 3 were made. The data in Table 2
revealed that thiol alkylation would be predicted to
occur at carbon atoms C1 and C4 since they are more
electropositive than the adjacent atoms C2 and C3,
respectively. In the case of 9a , 10a , and 14, the C1
atoms are more electropositive than the C4 atoms. Thus
the data suggest that initial thiol alkylation will occur
at the C1 carbon atom and a subsequent thiolation will
take place at the C4 center thus exemplifying the
sequential cytotoxicity concept. Based on their electro-
static charges, the rates of thiol attack at the C1 and
C4 positions would be predicted to be 9a > 10a > 14.
The electrostatic charges for 1a revealed that nucleo-
philic attack will occur at the C1 position. If the initial
rate of thiol attack is the limiting factor in bioactivity,
then the order of cytotoxicity would be predicted to be
9a > 1a > 10a .
Figure 4 indicates three structural features of the
compounds in series 9 which could contribute to cyto-
toxicity. These piperidines are Mannich bases contain-
ing many of the structural features found in series 1 as
well as possessing an isolated â-arylvinyl group. In
addition, loss of the allylic hydroxy group could give rise
to a reactive carbonium ion stabilized by the presence
of the adjacent â-arylvinyl group. These features should
permit interaction with cellular thiols to occur which,
if a major contributor to bioactivity, permits the follow-
ing predictions pertaining to structure and cytotoxicity
to be made, namely, 1 > 4, 9 > 11, and 10a ,c,d , >
12a ,c,d . If quaternization increased chemical reactivity
of the olefinic centers, then 10a ,c,d > 9a ,c,d . Further-
more, addition of thiols to the â-arylvinyl double bond
has been shown to occur at a far greater rate with
Mannich bases of conjugated â-arylvinyl ketones than
the corresponding R,â-unsaturated ketones.22 Hence
thiol alkylation should occur at carbon atom C1 much
compd
tumor
classification
9d
COLO 205
SW-620
NCI-H522
colon
colon
57 (200)
32 (80)
23 (80)
28 (200)
9 (80)
-2 (80)
non-small-
cell lung
melanoma
colon
LOX IMVI
COLO 205
KM12
35 (80)
12 (80)
14 (16.8)
5 (25)
10a
20 (16.8)
45 (16.8)
43 (16.8)
colon
renal
CAKI-1
6 (16.8)
a
% T/C indicates the optimal value of the percentage reduction
of the median treated tumor weight compared to the median
control tumor weight. % ILS refers to the percentage increase
in the median time in days for the treated tumor to reach a certain
size compared to controls.
b
replacement by thiols at the 2 (and 4) atom(s). This
reaction may be an elimination-addition process or by
nucleophilic attack.15 The rate-determining step in an
elimination reaction is the loss of the proton adjacent
to the carbonyl group.16 Hence, if the elimination-
addition mechanism operates at the cellular level,
compounds 1-3 should be more active than the ana-
logues 4-6 since the negative charges on carbon atom
1 (and 5) are greater in 1a , 2a , and 3a , and hence the
proton is more acidic than in the analogues 4a , 5a , and
6a . Hence in comparing the olefinic versus the non-
olefinic analogues, the potency orders would be 1a >
4a , 2a > 5a , and 3a > 6a .
However while the acyclic mono Mannich bases 1 and
4 were prepared, only the 1,3,4-trisubstituted piperi-
dines 9-12 were isolated in the attempts to synthesize
2, 3, 5, and 6. The structures of 9-12 were proved by
1H NMR spectroscopy and confirmed by X-ray crystal-
lography of four representative compounds, namely, 10d
(Figure 2), 12d , and 13a ,b. The data from these two
physicochemical determinations revealed the trans ar-
rangement of the bulky groups at positions 3 and 4 and
olefinic double bonds displaying the E configuration. In
addition, the X-ray crystallographic data showed that
the piperidines adopted the chair conformations and the
larger groups at postions 3 and 4 were in the equatorial

4016 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Dimmock et al.
piperidinol in the colon subpanel of six human tumor
cell lines will be discussed. In addition, comparisons
will be made between 9d and 5-fluorouracil (5-FU) since
the latter compound is an established drug used clini-
cally against cancer of the colon. The six tumors (IC50
figures in µM of 9d and 5-FU in parentheses) were
COLO 205 (1.7, 7.4), HCC-2998 (2.5, 0.9), HCT-15 (3.7,
12.0), HT29 (1.9, 9.8), KM12 (1.8, 12.0), and SW-620
(2.0, 25.7) indicating that, with the exception of the
HCC-2998 neoplasm, 9d was more cytotoxic than 5-FU
to these tumors. In fact, the average IC50 figures for
9d and 5-FU were 2.3 and 11.3 µM, respectively,
revealing a 5-fold greater potency for 9d . Since the
average IC50 figures for all cell lines for 9d and 5-FU
were 5.0 and 24.6 µM, respectively, an approximately
2.2-fold selectivity for both 9d and 5-FU for colon
cancers was demonstrated.
F igu r e 4. Structural features of series 9 which may confer
antineoplastic activity.
more rapidly than at C4 (Figure 4), and the potential
of sequential cytotoxicity exists.
A comparison of the murine cytotoxicity data for 1b
and 7 was ambiguous pertaining to whether the re-
placement of the 4-methylene protons of 1b by two
methyl groups, as in 7, increased cytotoxicity or not. The
acyclic Mannich base 8a may be considered to be a bis
analogue of 1a . Hence if the theory of sequential
cytotoxicity is valid, 8a should possess more than twice
the activity of 1a . This speculation is valid when the
IC50 figures for the P388 and L1210 screens are con-
sidered but not for the human tumor test. As predicted,
8b lacking olefinic bonds was less cytotoxic than 8a .
To examine the viability of the predictions and
hypotheses discussed earlier, three cytotoxicity assays
were chosen for the following reasons. First, use of
murine P388 and L1210 leukemic cells was employed
since these tumors have been claimed to be good
predictors of clinically useful anticancer drugs.23 Sec-
ond, the human tumor assay employed approximately
55 tumor cell lines from different neoplastic diseases:
principally leukemia, melanoma, non-small-cell lung,
colon, central nervous system, ovarian, renal, prostate,
and breast cancers.24 If a 50% decrease in the growth
of cells was not achieved at the highest concentration,
i.e., 10-4 M, this figure of 10-4 M was still included in
the calculation of the average IC50 values. Hence the
figures are mean graph midpoint values rather than
true mean figures. Compounds which have a higher
toxicity to one or more of these neoplastic diseases may
display a greater activity toward these cancers than
normal tissues.
An overview of the biodata generated will be made,
followed by an evaluation of the predictions made
earlier. In the case of the P388 screen, all of the
compounds had IC50 figures of less than 10 µM, and for
27% of the compounds, these values were less than 1
µM. Four compounds, namely, 1b,c, 4c, and 9c, gave
IC50 values less than twice that of melphalan, and the
significant antileukemic activity of the compounds in
series 1 is noteworthy. The activity in the L1210 test
ranged from 5.3 (9a ) to 61.2 (1c) µM. In all cases the
compounds were less active toward the more rapidly
growing L1210 cells than the slower growing P388
leukemia cell line. In contrast to the results from P388
cells, greater cytotoxicity was found with the piperidines
9 and 11 than the analogous Mannich bases 1 and 4.
Most of the compounds prepared in this study were
assessed against a panel of human tumor cell lines. A
noteworthy feature was the fact that approximately 80%
of the compounds evaluated were more potent than
melphalan, and in particular 9c possessed 69 times the
potency of this widely used drug. Selective toxicity
toward leukemia was observed for four of the seven
quaternary ammonium salts examined in this screen,
namely, 10a and 12a ,b,e; this property was also noted
with 1a and 9e. In addition, 9d had preferential
cytotoxicity toward human colon cells.
The hypotheses outlined previously predicted that the
relative cytotoxicities of different groups of compounds
would be as follows: 1 > 4, 9 > 11, 10a ,c,d > 9a ,c,d ,
and 10a ,c,d > 12a ,c,d . To examine the viability of
these postulates, comparisons were made between the
cytotoxicities of pairs of compounds having the same
aryl substituents. For example, in contrasting the
compounds in series 1 and 4, 1a was compared with 4a ,
1b with 4b, and so forth. Comparisons were made if
data were available for at least three pairs of com-
pounds. A correlation was recorded if more than half
of the comparisons supported the theory. In the case
of the P388 screen, 1 > 4, 9 > 11, 10 < 9, and 10 > 12
indicating that three of the four predictions made were
established. The results in the L1210 test showed that
1 < 4 and 9 > 11 revealing that only the latter
relationship fulfilled the predictions made. The com-
pounds in series 9 were more cytotoxic than the ana-
logues in series 11 as predicted. In the remaining cases
there were insufficient data to make comparisons. Thus
in general the results support both the sequential
cytotoxicity concept and the importance of the presence
of olefinic bonds in these molecules. Hence design of
future analogues should incorporate unsaturated cen-
ters into their structures permitting alkylation of cel-
lular nuclephiles to occur.
To seek correlations between the cytotoxicity data and
the electronic, hydrophobic, and steric properties of the
aryl substituents, linear and semilogarithmic plots
between the IC50 values and the Hammett σ, Hansch
π, and molar refractivity (MR) constants in each of the
series 1, 4, and 9-12 were made, providing that
screening results were available for at least three
members of a particular series. The test for zero
correlation25 was applied at the 95% and 90% signifi-
cance levels. In cases where good correlations were
In view of the in vivo activity of 9d toward colon
tumors vide infra, the impressive cytotoxicity of this

Novel Class of Cytotoxic and Anticancer Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4017
noted, the data was further evaluated revealing p values
of less than 0.05. The significant relationships which
were obtained are summarized in Table 4.
had greater cytotoxicity than analogues bereft of this
functional group; however, these latter compounds
displayed cytotoxicity, and therefore structural features
other than the presence of chemically reactive double
bonds contributed to bioactivity. A number of prototypic
molecules emerged from this study, and in addition, the
promising in vivo activity of 9d toward colon cancers
was noteworthy. Thus development of this series of
compounds is warranted with a view to discovering
novel clinically useful antineoplastic drugs.
The data in Table 4 revealed that 11 correlations
between cytotoxicity and the σ, π, and MR constants
were noted in both series of acyclic Mannich bases 1
and 4 as well as the piperidines of series 9. No
correlations were discerned in the other three series of
compounds (10-12). The relationships between cyto-
toxicity and the MR, π, and σ values of the aryl
substituents were noted in five, four, and two cases,
respectively. Thus, where correlations were detected,
differences in the sizes and hydrophobic properties of
the aryl groups influenced activity more than their
chemical reactivity.
Exp er im en ta l Section
A. Ch em istr y. Melting points are uncorrected. Com-
pounds 1a ,d ,e, 4a -e, and 11a have been reported previously
and, in general, had melting points similar to those recorded
in the literature.27-33 Elemental analyses (C,H,N) were un-
dertaken on 1a -e, 4a -e, 7, 8a ,b, 9a -e, 10a ,c,d , 11a -c,e,f,
12a -e, and 13a ,b, as well as the hydrobromide salts of the
free bases obtained from 4a and 11a -e, by Mr. K. Thoms,
Department of Chemistry, University of Saskatchewan, and
were within 0.4% of the calculated values. 1H NMR spectra
were determined using a Bruker AM 500 FT NMR machine
(500 MHz), while a Varian T-60 (60 MHz) instrument was used
to confirm the structures of intermediate R,â-unsaturated
ketones. A Nonius CAD-4 diffractometer was used for the
collection of X-ray crystallographic data. TLC was undertaken
using silica gel plastic-backed sheets. All compounds were
homogeneous using solvent systems of hexane-methanol (7:
3) for the intermediate R,â-unsaturated ketones, chloroform-
methanol (7:3) for the Mannich bases, and chloroform-
methanol-ammonium hydroxide (7:3:0.08) for the quaternary
ammonium salts. Compounds 8b, 11f, and 12c,d were ob-
tained as the hemihydrates and 12a as the monohydrate. The
percentage yields of the Mannich bases were calculated on the
basis of the lowest molar fraction of the reactants used. For
example, in the case of 4a the molar ratios of diethylamine
hydrochloride, acetophenone, and paraformaldehyde were 0.01,
0.04, and 0.03, respectively, and the 55% yield recorded was
based on the premise that a maximum yield would be 0.01
mol of pure product.
Syn t h esis of In t er m ed ia t e r,â-Un sa t u r a t ed Ket on es
Requ ir ed in th e P r ep a r a tion of 1, 7, 8a , 9, 10, a n d 13.
4-Phenyl-3-buten-2-one was obtained from the Aldrich Chemi-
cal Co. The remaining arylvinyl ketones were prepared by a
literature method14 and purified by recrystallization or distil-
lation. The products had melting points or boiling points
consistent with literature values. The structures were con-
firmed by 1H NMR spectroscopy (60 MHz, CDCl3), and the
spectrum of a representative compound, 4-(4-methoxyphenyl)-
3-buten-2-one, was as follows. δ: 2.30 (s, 3H, COCH3), 3.80
(s, 3H, OCH3), 6.40-6.60 (d, 1H, CHdCHCO, J ) 18 Hz),
6.70-7.50 (m, 5H, aryl H, CH)CHCO).
For the purpose of subsequent drug design, develop-
ment of each of the series 1, 4, and 9 will use these
correlations. However the test system being utilized
needs to be considered since, as Table 4 indicates,
positive correlations were noted with the P388 screen,
negative relationships were found in the L1210 test, and
both positive and negative correlations were obtained
using the human tumor assay. For example, for future
expansion of series 4, an increase in the size of the aryl
substituent would be predicted to increase cytotoxicity
in the P388 screen. On the other hand, a reduction in
the MR value of the aryl group is expected to increase
activity in the L1210 and human tumor assays. Simi-
larly for series 1, while increases in the size of the aryl
substituents would be expected to increase activity in
the P388 screen, compounds containing aryl substitu-
ents with smaller MR values would be predicted to
display increased cytotoxicity in the L1210 test.
As indicated previously, several compounds such as
9d and 10a displayed selective toxicity in the human
tumor screen toward colon and leukemia cells, respec-
tively. To evaluate whether these promising results
could be translated into in vivo activity, both compounds
were examined in the murine P388 screen and against
certain human tumors in athymic mice. Evaluation in
the P388 screen revealed that 9d was inactive and 10a
displayed marginal potency, whereby an increase in the
life span of the mice by 20% was noted. The activity of
these two compounds toward several xenografts is
summarized in Table 5. Reductions in the sizes of the
tumors were observed with both compounds, and the
potency of 9d against the COLO 205 tumor is of
particular interest. A very recent mode of action study
with this lead molecule revealed that 9d caused apop-
tosis in human J urkat leukemia cells.26
Syn th esis of Ser ies 1 a n d 4 a n d Com p ou n d 7.
A
mixture of the appropriate 4-aryl-3-buten-2-one, 1-aryl-1-
ethanone, or 1-aryl-4-methyl-1-penten-3-one, paraformalde-
hyde, diethylamine hydrochloride, trifluoroacetic acid (0.04
mL; 1a -e, 7), or hydrochloric acid (37% w/v, 0.04 mL; 4a -e),
and acetonitrile (100 mL; 1a -e, 4a ,c, 7) or 2-propanol (100
mL; 4b,d ,e) was heated under reflux for different periods of
time. After removal of the solvent in vacuo, the resultant oil
was triturated with anhydrous ether and subsequently with
acetone. The solid obtained was washed with ether and
recrystallized from ether-methanol (1c-e, 4a ,d , 7), acetone
(1a , 4b), acetonitrile (4c), ethanol-acetone (4e), or methanol
(1b). A constant quantity of diethylamine hydrochloride was
used throughout, namely, 0.01 mol. The molar ratios of ketone
and paraformaldehyde, times of heating under reflux (h), yields
(%), and melting points (°C) were as follows. 1a : 0.03:0.03,
36, 61, 130-132. 1b: 0.03:0.03, 24, 68, 150-152. 1c: 0.03:
0.03, 24, 54, 158-160. 1d : 0.03:0.03, 48, 48, 156-158. 1e:
0.03:0.03, 48, 89, 148-150. 4a : 0.04:0.03, 30, 55, 109-111.
4b: 0.025:0.03, 17, 61, 138-140. 4c: 0.03:0.025, 24, 67, 131-
The promising in vitro and in vivo activity of 9d
suggested that analogues containing one or two aro-
matic methyl groups may also display selective toxicity
to malignant cells. The data in Table 3 revealed that
13a ,b had comparable cytotoxicity to 9d , although
neither 13a ,b displayed selective toxicity for colon
cancers (or any other neoplastic disease) in the human
tumor screen.
Con clu sion s
This study outlined the preparation of various acyclic
Mannich bases and the formation of some 1,3,4-trisub-
stituted piperidines and related quaternary ammonium
salts. In general, compounds containing olefinic bonds

4018 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Dimmock et al.
133. 4d : 0.04:0.03, 42, 53, 118-120. 4e: 0.04:0.03, 42, 63,
119-121. 7: 0.05:0.05, 48, 61, 160-162. The 1H NMR (500
MHz) spectra of representative compounds were as follows.
1e: δ(CDCl3) 7.64 (1H, d, CH)CHCO, J ) 16.2 Hz), 7.51 (2H,
d, 2,6 aryl H, J ) 8.8 Hz), 6.90 (2H, d, 3,5 aryl H, J ) 8.8 Hz),
6.62 (1H, d, CHdCHCO, J ) 16.1 Hz), 3.83 (3H, s, OCH3),
3.43 (2H, def t, COCH2), 3.35 [2H, def t, CH2N(C2H5)2], 3.10
[def q, 4H, CH2N(CH2CH3)2, J ) 7.1 Hz], 1.39 [6H, t,
N(CH2CH3)2, J ) 7.4 Hz]. 4e: δ(CDCl3) 7.98 (2H, d, 2,6 aryl
H, J ) 8.9 Hz), 6.92 (2H, d, 3,5 aryl H, J ) 8.9 Hz), 3.86 (3H,
s, OCH3), 3.70 (2H, t, COCH2, J ) 7.3 Hz), 3.42 [2H, t, CH2N-
(C2H5)2, J ) 7.3 Hz], 3.12 [4H, def q, N(CH2,CH3)2, J ) 7.1],
1.40 (6H, t, 2 CH3, J ) 7.3 Hz).
extracts were combined and dried (anhydrous magnesium
sulfate), and removal of the solvent gave a residue which was
dissolved in anhydrous ether (100 mL). Excess of dry hydro-
gen bromide was passed into the ethereal solution at 0 °C,
and the precipitate was collected, washed with anhydrous
ether and chilled ethanol, and dried. The reaction products
were recrystallized from 2-propanol to give the hydrobromide
salts of the free bases from the following compounds: 11a , mp
183-184 °C; 11c, mp 182-184 °C; 11e, mp 176-178 °C. The
free bases of 11b,d were obtained using the method described
for the preparation of series 10 and 12 vide infra. Addition of
dry hydrogen bromide gas to an ice-cooled solution of the
Mannich base (0.001 mol) in ether (50 mL) led to precipitates
which were collected, dried, and recrystallized from 2-propanol
to give 11b, mp 196-198 °C, or from ether-methanol leading
to 11d , mp 192-194 °C.
Syn th esis of 10a ,c,d a n d Ser ies 12. A stirring solution
of the piperidinols 9a ,c,d and 11a -e (0.001 mol) in aqueous
methanol (20% v/v, 25 mL) was cooled and maintained at less
than 10 °C while basified with aqueous sodium bicarbonate
solution (10% w/v). The mixture was extracted with ether (5
× 25 mL) and dried (anhydrous magnesium sulfate). Removal
of the solvent under vaccuum gave an oil which was dissolved
in anhydrous ether (50 mL) to which was added a 2.0 M
solution of methyl bromide (0.01 mol) in tertiary butyl methyl
ether at 0 °C. The reaction mixture was stirred at 0 °C for 6
h. The precipitates were collected, washed with dry ether,
dried, and recrystallized from ethanol (95% v/v, 10a ,d ) or
ether-methanol (10c, 12a -e). The yields (%) and melting
points (°C) were as follows. 10a : 77, 178-179. 10c: 70, 202-
204. 10d : 78, 222-224. 12a : 86, 192-194. 12b: 73, 164-
166. 12c: 79, 168-170. 12d : 80, 152-154. 12e: 76, 188-
190. TLC of the reaction products obtained in a similar
manner from 9b,e revealed the presence of an impurity.
Recrystallization and column chromatography did not lead to
the isolation of a pure compound.
The methiodide of the free base of 12d was prepared in an
identical manner except methyl iodide was used in place of
methyl bromide and the reaction mixture was stirred at 0 °C
overnight. The precipitate was collected and recrystallized
from alcohol (95%) to give the desired compound, mp 177-
178 °C, in 85% yield.
X-r a y Cr ysta llogr a p h y of 10d . Compound 10d was
recrystallized from diethyl ether-methanol by vapor diffusion.
A Nonius CAD-4 diffractometer with a ω scan was used for
data collection, and the structure was solved by direct methods
using NRCVAX34 and refined using SHELX93.35 Atomic
scattering factors were taken from the literature.36 All non-
hydrogen atoms were found on the E-map and refined aniso-
tropically. Hydrogen atom positions were calculated and not
refined.
Utilization of a literature procedure for preparing a series
of 3-dimethylamino-1-aryl-1-propanone hydrobromides3 led to
the synthesis of the hydrobromide salt of the free base of 4a
in 15% yield. It was recrystallized from acetone-methanol,
mp 104-107 °C.
Syn th esis of Ser ies 8, 9, 11, a n d 13. A mixture of the
4-aryl-3-buten-2-one or 1-aryl-1-ethanone, paraformaldehyde,
piperazine dihydrochloride (8a ,b), ethylamine hydrochloride
(9a -e, 11a -c,e,f, 13a ,b), or ethylamine hydrobromide (11d ),
hydrochloric acid (37% w/v, 0.04 mL; 8b, 9a -e, 11b,c, 13a ,b)
or trifluoroacetic acid (0.04 mL; 11a ,d ,e) (3 mL; 11f) in
acetonitrile (100 mL; 8a , 11a ,d ,e,), and ethanol (95% v/v, 100
mL; 8b, 9a -e, 13a ,b) or 2-propanol (100 mL; 11b,c,f) was
heated under reflux for varying lengths of time. In the case
of 11b, the product which deposited from the reaction mixture
was collected and washed with 2-propanol. For the other
compounds, the solvent was removed in vacuo leading to oils
which were washed with ether and dissolved in ethanol (10
mL) to which ether was added to induce precipitation. After
refrigerating the solution at 4 °C for 2-3 days, the deposited
solids were collected and recrystallized from 60% ethanol (8a ),
70% ethanol (8b), 95% ethanol (9a ,c, 11a ,b), ether-methanol
(9b,d ,e, 11d ,e, 13b), or methanol (11c,f, 13a ). A constant
quantity of amine hydrohalide was used (0.01 mol). The molar
ratios of ketone to paraformaldehyde, times of heating (h),
yields (%), and melting points (°C) were as follows. 8a : 0.04:
0.03, 4, 61, 234 dec. 8b: 0.06:0.06, 17, 41, 198 dec. 9a : 0.06:
0.04, 36, 57, 194-196. 9b: 0.06:0.04, 30, 24, 210-212. 9c:
0.08:0.08, 24, 63, 192-194. 9d : 0.06:0.04, 42, 21, 190-192.
9e: 0.05:0.05, 45, 25, 180-182. 11a : 0.04:0.04, 20, 47, 208-
210. 11b: 0.03:0.025, 48, 71, 202-203. 11c: 0.03:0.025, 24,
70, 185-187. 11d : 0.04:0.04, 20, 50, 198-200. 11e: 0.04:
0.04, 48, 56, 178-179. 11f: 0.05:0.05, 48, 51, 178-180. 13a :
0.06:0.04, 36, 30, 164-166. 13b: 0.06:0.04, 72, 28, 172-174.
The 1H NMR (500 MHz) spectra of two representative com-
pounds (9e and 11e) are given below. In the case of 9e, the
aryl rings adjacent to carbon atoms C1 and C4 (Figure 4) are
referred to as aryl A and aryl B, respectively. 9e: δ(CDCl3)
8.05 (1H, d, CHdCHCO, J ) 16.3 Hz), 7.59 (2H, d, 2,6 aryl A
H, J ) 8.8 Hz), 7.21 (2H, d, 2,6 aryl B H, J ) 8.8 Hz), 6.89
(2H, d, 3,5 aryl A H, J ) 8.8 Hz), 6.77 (2H, d, 3,5 aryl B H, J
) 8.8 Hz), 6.63 (1H, d, CHdCH aryl B, J ) 15.9 Hz), 6.53
(1H, d, CHdCHCO, J ) 16.3 Hz), 6.07 (1H, d, CHdCH aryl
B, J ) 15.9 Hz), 4.67 (1H, br d, OH, J ) 1.5 Hz), 4.51 (1H, br
d, C3Ha, J ) 8.3 Hz), 3.83 (3H, s, aryl A OCH3), 3.75 (3H, s,
aryl A OCH3), 3.34-3.41 (2H, m, C2He, C6He), 3.00-3.25 (4H,
m, C2Ha, C6Ha, N-CH2CH3), 2.43-2.53 (1H, m, C5Ha), 1.86
(1H, d, C5He, J ) 14.7 Hz), 1.48 (3H, t, CH2CH3, J ) 7.3 Hz).
11e: δ(CDCl3) 8.12 (2H, d, 2,6 aroyl H, J ) 8.9 Hz), 7.42 (2H,
def d, 2,6 aryl H, J ) 8.8 Hz), 6.91 (2H, def d, 3,5 aroyl H, J
) 9.0 Hz), 6.74 (2H, def d, 3,5 aryl H, J ) 8.8 Hz), 5.50 (1H,
dd, C3Ha, J 3a/2a ) 12.1 Hz, J 3a/2e ) 3.6 Hz), 5.24 (1H, d, OH, J
) 2.6 Hz), 3.83 (3H, s, aroyl OCH3), 3.68 (3H, s, aryl OCH3),
3.41-3.48 (2H, m, C2He, C6He), 3.29-3.39 (2H, m, C2Ha,
C6Ha), 3.04-3.18 (2H, m, NCH2CH3), 2.74-2.83 (1H, m,
C5Ha), 1.92 (1H, br d, C5He, J ) 14.9 Hz), 1.50 (3H, t,
NCH2CH3, J ) 7.3 Hz).
The data for 10d were as follows:
C27H33BrNO2, Mr )
483.45; a ) 8.0240(9), b ) 10.253(2), c ) 15.594(5) Å; R )
74.398(23)°, â ) 83.549(16)°, γ ) 86.847(14)°; Z ) 2; space
group ) P1, triclinic; Dx ) 1.308 g cm-3, λ(Mo KR) ) 0.7093
Å; T ) 123 K. Merging R is based on intensities of 0.015 for
447 replicate reflections. Refinement on F2: R[F2 > 2σ(F2)] )
0.0598 (2951 reflections), wR(F2) ) 0.1797 (all data), S ) 1.52.
A total of 4790 reflections were measured of which 4343 were
independent and used in the refinement of the structure.
Parameters refined ) 280, [w ) 1[σ2(Fo2) + (0.1222P)2
+
2
0.000P], where P ) (Fo + 2Fc2)/3. ∆F in the final difference
map within +2.094 and -0.469 eÅ-3
.
Ca lcu la tion s of Atom ic Ch a r ges. The molecular confor-
mations were optimized using the semiempirical AM1 method.
The electrostatic potential-derived charges were calculated
using the CHELPG scheme at the RHF/3-21G level (restricted
Hartree-Fock method with the 3-21G standard basis set). All
computations were carried out using the Gaussian 92 pro-
gram.37
The hydrobromide salts of the free bases from 11a ,c,e were
prepared as follows. A solution of 11a ,c,e (0.01 mol) in water
(50 mL) was basified with sodium bicarbonate solution (10%
w/v) and extracted with ether (5 × 25 mL). The organic
B. Bioeva lu a tion . Cytotoxicity Assa ys. Evaluation of
the compounds using P388 D1 cells was undertaken by a
literature procedure,38 and the examination with L1210 cells
was achieved using a previously reported method.39 The assay

Novel Class of Cytotoxic and Anticancer Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4019
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system, melanoma, ovarian, renal, prostate, and breast can-
cers. Compounds 9a ,b, 10a , 11d , and 12a ,e were not evalu-
ated against prostate and breast cancers, but they were tested
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examined by the Developmental Therapeutics Program, Na-
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lymphocytic leukemia assay was conducted by a reported
method,41 and the maximum ILS figures for 9d and 10a were
5% and 20%, respectively, using doses of 54 and 6.7 mg/kg,
respectively. An increase of 20% or more in the life spans is
considered to be statistically significant. The reference drug
5-fluorouracil has an ILS of >35 using a dose of 20 mg/kg when
given intraperitoneally for 5 days.42 Passage of human tumors
in athymic mice was undertaken by a published method.43 No
definitions of activity are available, but as a general rule
compounds causing a 60% reduction in tumor weights in one
of these screens would be evaluated further. For example,
cyclophosphamide, while inactive toward COLO 205, SW-620,
and NCI-H522 xenografts, reduced the growth of the LOX
IMVI and CAKI-1 tumors by 100-150% and 60-100%,
respectively.
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Ack n ow led gm en t . The following agencies are
thanked for providing financial support for this study.
The Medical Research Council of Canada and the
National Cancer Institute of Canada provided grants
to J . R. Dimmock and T. M. Allen, respectively. S. C.
Vashishtha was the recipient of a University of Sas-
katchewan Graduate Scholarship. E. De Clercq and J .
Balzarini received funding from the Biomedical Re-
search Programme of the European Community and the
Belgian Fonds voor Geneeskundig Wetenschappelijk
Onderzoek (FGWO). The National Cancer Institute,
Bethesda, MD, evaluated a number of the compounds
against various human tumor cell lines. In addition, Dr.
K. Shyam prepared the hydrobromide salt of 4a , while
Dr. V. K. Arora synthesized the hydrobromide salts of
11a ,c,e; these activities are gratefully acknowledged.
Appreciation is extended to Dr. G. D. Abrams, Plant
Biotechnology Institute, Saskatoon, for assistance in the
1
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thank Dr. J . A. Weil, Department of Chemistry, Uni-
versity of Saskatchewan, for allowing the computation
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