Bis[3,5-bis(benzylidene)-4-oxo-1-piperidinyl]amides: A Novel Class of Potent Cytotoxins

Article abstract of DOI:10.1002/cmdc.201100199

The principal objective of this study was the examination of the theory of cytotoxic synergism. In this exploratory study, we tested the hypothesis that doubling the number of sites available for thiol alkylation in a series of candidate cytotoxins increases potency more than two-fold. This concept was verified in one-third of our comparisons using human Molt 4/C8 and CEM T-lymphocytes and murine L1210 cells. In addition, the significant potencies of various members of our compound series justified further studies. Molecular modeling revealed that relative locations of the amidic groups correlate with cytotoxicity. A potent cytotoxic compound, 1,2-bis(3,5-dibenzylidene-4-oxo-piperidin-1-yl)ethane-1,2-dione (1a) inhibited the growth of a large number of human tumor cell lines and displayed greater toxicity toward certain non-adherent cells than toward adherent neoplasms or fibroblasts. The mode of action of 1a includes induction of apoptosis and necrosis.

Full text of DOI:10.1002/cmdc.201100199

MED
DOI: 10.1002/cmdc.201100199
Bis[3,5-bis(benzylidene)-4-oxo-1-piperidinyl]amides: A
Novel Class of Potent Cytotoxins
Swagatika Das,[a] Umashankar Das,[a] Armando Varela-Ramꢀrez,[b] Carolina Lema,[b]
Renato J. Aguilera,[b] Jan Balzarini,[c] Erik De Clercq,[c] Stephen G. Dimmock,[d] Dennis
K. J. Gorecki,[a] and Jonathan R. Dimmock*[a]
The principal objective of this study was the examination of
the theory of cytotoxic synergism. In this exploratory study, we
tested the hypothesis that doubling the number of sites avail-
able for thiol alkylation in a series of candidate cytotoxins in-
creases potency more than two-fold. This concept was verified
in one-third of our comparisons using human Molt 4/C8 and
CEM T-lymphocytes and murine L1210 cells. In addition, the
significant potencies of various members of our compound
series justified further studies. Molecular modeling revealed
that relative locations of the amidic groups correlate with cyto-
toxicity. A potent cytotoxic compound, 1,2-bis(3,5-dibenzyli-
dene-4-oxo-piperidin-1-yl)ethane-1,2-dione (1a) inhibited the
growth of a large number of human tumor cell lines and dis-
played greater toxicity toward certain non-adherent cells than
toward adherent neoplasms or fibroblasts. The mode of action
of 1a includes induction of apoptosis and necrosis.
Introduction
The principal aim of this work was the design, syntheses, and
biological evaluation of conjugated styryl ketones as candidate
antineoplastic agents. A number of studies have revealed that
these enones react readily with thiols.[1,2] In particular, conju-
gated styryl ketones react only with the thiol group in a variety
of compounds containing other functional groups such as
amino,[3] and hydroxy groups,[4] as well as with proteins con-
taining one or more mercapto substituents.[5] This affinity of
conjugated arylidene ketones for thiols, in contrast to other
functional groups present in nucleic acids, indicates that the
genotoxic properties displayed by various contemporary anti-
cancer drugs[6] should be absent in these compounds. Further-
more, a number of different proteins contain thiol groups,
leading to the possibility that these compounds have multiple
molecular targets. The importance of such pleiotropy has re-
cently been discussed.[7–9] Currently, emphasis has been placed
on the inclusion of a 1,5-diaryl-3-oxo-1,4-pentadienyl group
(ARCH=CHCOCH=CHAR), referred to hereafter as a dienone
moiety, into candidate cytotoxins. This pharmacophore pres-
ents the possibility that sequential thiol alkylation can occur
with the olefinic carbon atoms. Multiple studies have revealed
that an initial lowering of the concentration of cellular thiols,
followed by a second chemical attack, is more detrimental to
malignant cells than normal tissues.[10,11] In addition, if this in-
teraction occurs at two different sites, the effect may be far
more detrimental to the neoplasm than reaction at only one
site.[12]
Figure 1. General structure of series 1. indicates the olefinic carbon atoms
that are capable of interacting with different thiol groups of a protein.
*
sion of two such groups into a molecule may more than
double the potency of a related compound containing only
one dienone moiety. Series 1 was designed to include com-
pounds in which the locations of the dienone groups vary in
[a] S. Das, Dr. U. Das, Prof. Dr. D. K. J. Gorecki, Prof. Dr. J. R. Dimmock
Drug Design & Discovery Research Group, College of Pharmacy & Nutrition,
University of Saskatchewan
216 Thorvaldson, 110 Science Place, Saskatoon, Saskatchewan S7N 5C9
(Canada)
E-mail: jr.dimmock@usask.ca
[b] Dr. A. Varela-Ramꢀrez, Dr. C. Lema, Prof. Dr. R. J. Aguilera
Cell Culture & High-Throughput Screening Facility, Border Biomedical Re-
search Center&Department of Biological Sciences, University of Texas at El
Paso, 500 West University Avenue, El Paso, Texas 79968-0519 (USA)
[c] Prof. Dr. J. Balzarini, Prof. Dr. E. D. Clercq
Rega Institute of Medical Research, Katholieke Universiteit Leuven
Minderbroedersstraat 10, 3000 Leuven (Belgium)
The aim of the present investigation was to evaluate the hy-
pothesis of cytotoxic synergism in cancer cells, which suggests
that compounds capable of multiple cellular interactions in ne-
oplasms exert a synergistic effect. In order to probe the viabili-
ty of this theory, we designed a series of compounds (1) con-
taining two dienone groups (Figure 1). In this case, the inclu-
[d] Prof. Dr. S. G. Dimmock
Department of Finance, Nanyang Technological University
S3-B1A-02, 100 Nanyang Avenue, 639798 (Singapore)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100199.
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Novel and Potent Cytotoxins
relation to each other. Molecular modeling was used as a
means to determine the spatial relationship between the phar-
macophores. Biological evaluations were undertaken using
multiple cell lines in order to explore the generality of any
trends in the relative potencies of the compounds.
that these compounds adopt the E stereochemistry.[13,14] While
this study was in progress, syntheses of 2-fluoro analogues of
1a and 1 f were described, and X-ray crystallography of the 2-
fluoro analogue of 1a showed that the four olefinic double
bonds possess an E configuration.[15]
The compounds in series 1 were evaluated against human
Molt 4/C8 and CEM T-lymphocytes in order to determine
whether they demonstrate cytotoxic properties towards
human transformed cells. A murine L1210 assay was employed,
as a number of anticancer drugs display potency in this
screen,[16] and it may, therefore, identify compounds of poten-
tial clinical value. The results presented in Table 1 show that a
number of compounds in series
Results
Compounds were synthesized using the procedure indicated
in Scheme 1. All of the compounds in series 1 were evaluated
against human Molt 4/C8 and CEM T-lymphocytes, as well as
murine L1210 cells (Table 1). Various structural features of 1a–j
1 are potent cytotoxins. Howev-
er, compound 1k is virtually in-
soluble in multiple solvents, and
the observed IC50 values of
greater than 500 mm in the three
assays is likely due to the insuffi-
cient solubility of 1k in the
media preventing penetration of
the malignant cells. Hence, this
compound has been removed
from further discussion of the
correlations between series
1
compounds and cytotoxic po-
tencies. With regard to the IC50
values of 1a–j, 47% are more
potent than melphalan, 60% are
below 5 mm, and six are in the
sub-micromolar range. Of partic-
Scheme 1. Synthesis of series 1. Reagents and conditions: a) AcOH, dry HCl(g), 10% aq K2CO3, RT, 12h, 80%;
b) SOCl2, 60–658C, 4–5h, 90%; c) Et3N, ~208C, 12h, 48–72%.
were examined by molecular modeling. The most active com-
pounds, 1a and 1b, were evaluated against a large number of
human tumor cell lines, and selected biological data from
these studies is presented in Figure 4. In general, lead com-
pound 1a displayed greater potency toward non-adherent
cells than toward either an adherent neoplasm or two fibro-
blasts. These results are illustrat-
ular interest are 1a and 1b, which have average IC50 values to-
wards Molt 4/C8 and CEM T-lymphocytes of 0.61 and 0.14 mm,
respectively, and clearly emerge as lead molecules. The follow-
ing compounds have IC50 values that indicate higher potency
than melphalan, which is an alkylating agent used in cancer
chemotherapy (the fold increase in relative potency as com-
ed in Figures 5 and
6 and
Table 1. Evaluation of 1a–j against Molt 4/C8, CEM, and L1210 cells, as well as a comparison of their potencies
with 2.[a]
Table 3. Cell-cycle analysis of 1a
revealed that this compound re-
sulted in apoptosis in four neo-
plastic cell lines, and necrosis
was also observed (Figure 7).
Compd
Molt 4/C8 cells
IC50 [mm]
CEM cells
IC50 [mm]
L1210 cells
IC50 [mm]
RP[b]
RP[b]
RP[b]
1a
1b
1c
1d
1e
1 f
1g
1h
0.46ꢀ0.11
0.07ꢀ0.01
0.57ꢀ0.16
1.61ꢀ0.05
1.53ꢀ0.54
4.40ꢀ2.95
1.50ꢀ0.10
25.1ꢀ15.8
0.85ꢀ0.41
12.5ꢀ1.8
18
115
14
0.75ꢀ0.16
0.20ꢀ0.17
1.21ꢀ0.87
2.03ꢀ0.48
2.28ꢀ0.27
7.75ꢀ0.07
3.35ꢀ1.27
39.2ꢀ5.4
2.5
9.3
1.5
0.9
0.8
0.2
0.6
0.1
1.3
0.1
–
4.46ꢀ0.23
1.23ꢀ0.38
14.0ꢀ1.2
11.5ꢀ0.3
1.8
6.5
0.6
0.7
0.5
0.3
0.8
0.1
1.9
0.1
–
Discussion
5.0
5.3
1.8
5.4
0.3
9.5
0.7
–
15.3ꢀ4.2
28.9ꢀ2.0
9.86ꢀ0.76
79.0ꢀ10.2
4.19ꢀ2.19
150ꢀ65
1H NMR spectra of 1a–k indicate
that the compounds are stereo-
isomerically pure, and absorb-
ance of the olefinic protons in
the region from 7.63–7.90 ppm
indicates that the compounds
have an E configuration.[13] In ad-
dition, X-ray crystallography of a
number of 3,5-bis(benzylidene)-
4-piperidones also confirmed
1i
1j
2
1.45ꢀ0.85
36.0ꢀ7.6
8.07ꢀ0.45
3.24ꢀ0.79
1.86ꢀ0.08
2.47ꢀ0.30
7.97ꢀ0.75
2.13ꢀ0.03
Melphalan
–
–
–
[a] IC50 values were determined using a literature procedure and are the average of three independent experi-
[24]
ments ꢀSD.
Cells were exposed to the compounds for 3 d (Molt 4/C8 and CEM screens) or 2 d (L1210
assay). [b] Relative potencies (RP) were obtained by dividing the IC50 values of 2 by the values for each of the
compounds in series 1.
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pared with melphalan is given in parentheses): 1a (7.0), 1b
(44), 1c (5.7), 1d (2.1), 1g (2.2), and 1i (3.8) in the Molt 4/C8
screen; 1a (3.3), 1b (12), and 1c (2.0) in the CEM test; 1b (1.7)
towards L1210 cells.
The hypothesis that synergism may occur with the presence
of two dienone groups was examined by comparing the IC50
values of 1a–j with that of piperidone 2. This dienone was
chosen because, like the compounds in series 1, 2 is a 1-acyl-
3,5-bis(benzylidene)-4-piperidone. Additionally, the hydropho-
bic 1-tetradecanoyl group ensures that 2 resembles series 1
with regard to their markedly lipophilic nature. For example,
the logP values of 1e and 2 are 8.58 and 9.02, respectively. If
the hypothesis of cytotoxic synergism is valid, then the cyto-
toxic potencies of 1a–j should be greater than twice the fig-
ures generated for 2. Therefore, the IC50 value of 2 was divided
by the corresponding value for each of analogues 1a–j in the
Molt 4/C8, CEM, and L1210 assays to give the relative potency
(RP) figures (Table 1). RP values greater than two were ob-
tained for 1a–e, g, and i in the Molt 4/C8 screen, 1a and b in
the CEM assay, and 1b in the L1210 test, that is, in one-third of
the comparisons made. The RP figures for the Molt 4/C8
screen are particularly encouraging, and these results warrant
further evaluation of the hypothesis.
For 1a–j, IC50 values are lowest in the Molt 4/C8 screen and
highest in the L1210 assay. This observation is confirmed by
average IC50 values for 1a–j in the Molt 4/C8, CEM, and L1210
tests of 4.86, 9.42, and 31.8 mm, respectively (Table 1). Varia-
tions in potency were displayed by each compound 1a–j to-
wards Molt 4/C8, CEM, and L1210 cells. For example, Molt 4/C8
T-lymphocytes are 25-times more sensitive to treatment with
1c than are L1210 cells, and this differential toxicity may be
also be observed between malignant and non-malignant cells,
leading to greater adverse effects toward neoplasms.
The following observations were made pertaining to the
effect of the nature of the spacer group (X in structure 1) on
average IC50 values of 1a–j towards T-lymphocytes (these
values are given in parentheses). The most potent compound
is 1b (0.14 mm) which has a single methylene group spacer. Re-
moval of the spacer, leading to 1a (0.61 mm), or insertion of ad-
ditional methylene groups to 1b, giving rise to 1c (0.89 mm),
1d (1.82 mm), and 1e (1.91 mm), led to reduced potencies. A
comparison of IC50 values for three compounds which have a
two carbon atom spacer showed that 1 f (6.08 mm) and 1g
(2.43 mm) are less potent than 1c (0.89). The addition of a fur-
ther olefinic linkage to 1 f (6.08 mm), creating 1h (32.2 mm), re-
duced potency five-fold. Upon introduction of an aryl ring
spacer, the relative location of the substituents influences po-
tency considerably, as observed by the substantial difference in
IC50 values for 1c (1.15 mm) and 1j (24.3 mm).
Previous studies by our group involved the evaluation of
several series of structurally related 1-acyl-3,5-bis(benzylidene)-
4-piperidones for cytotoxic properties, including assessment
using Molt 4/C8, CEM, and L1210 cells. Series 3–5 have, respec-
tively, aroyl,[18] acryloyl,[13] and phosphono[19] groups attached
to the nitrogen atom (Figure 2). In general, series 1 exhibits
If the hypothesis is valid that synergism occurs when each
compound interacts at a different binding site, then one
would expect to observe similar relative potencies using vari-
ous biological assays. In order to evaluate this possibility, Ken-
dall’s coefficient of concordance[17] was applied to data gener-
ated in our three assays. Equation (1) used in this determina-
tion is given below, where W is Kendall’s coefficient of con-
cordance, i denotes the individual compound (i=1 for 1a, i=
2 for 1b, etc.), n is the number of compounds, Ri is the aver-
age rank given to cell line i, m is the number of cell lines, T is
the correction factor for ties and j refers to the individual cell
line. Kendall’s coefficient of concordance is a test for assessing
the similarity of rankings. If the relative potency rank for each
assay is identical, Kendall’s coefficient of concordance will be 1.
If there is no agreement in rankings, Kendall’s coefficient of
concordance will be zero. The coefficient of concordance for
our biological data was found to be 0.924 (p=0.03), providing
very strong evidence that the relative potency rankings are
similar across the assays. One may conclude, therefore, that de-
spite the difference in sensitivity of the three cell lines to 1a–j,
the molecular shapes may influence cytotoxic potencies in a
similar fashion.
Figure 2. Structures of compounds in series 2–5.
slightly weaker activities than 3–5; however, removal of the
two outliers (1h and 1j) shows that the observed IC50 values
for the remaining members of the series are comparable to
series 3–5 (Table 2).
Further evaluation of the biological data presented in
Table 1 was undertaken in order to examine the theory that
the topography of 1a–j controls cytotoxicity. While a substan-
tial number of interatomic distances as well as various bond
Table 2. Potencies of series 1 compounds as compared to series 3–5.
Compd
IC50[a] [mm]
Molt 4/C8
CEM
L1210
3
2.64
1.42
0.91
4.86
1.37
2.92
1.48
1.70
9.42
2.38
49.8
8.69
7.33
31.8
11.2
4[b]
5
1a–j
1a–g,i
ꢀ
ꢁ
P
n
i¼1
2
12
R2i ꢁ 3m2nðn þ 1Þ
ꢀ ꢁ
P
W ¼
ð1Þ
m
m2nðn2 ꢁ 1Þ ꢁ m
Tj
[a] Values shown are the average of three independent experiments.
[b] Data reported previously.[13]
j¼1
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Novel and Potent Cytotoxins
and torsion angles could be determined, we focused primarily
on the relative positions of the olefinic carbon atoms of both
pharmacophores, followed by the piperidyl nitrogen atoms
and the spacer group oxygen atoms. The generated figures
are presented in table S1 of the Supporting Information.
and y were measured. Linear and semi-logarithmic plots were
generated using the average IC50 values of 1a–j towards Molt
4/C8 and CEM T-lymphocytes and their d1, d2, and y data. No
correlations (p>0.05) nor trends to significance (p>0.1) were
found, although a negative trend to significance was nearly at-
tained when using y values (p=0.11). Thus, the possibility
exists that the preparation of analogues of series 1 in which y
values are increased may lead to molecules with greater cyto-
toxicity. Linear and semi-logarithmic plots were constructed
using the average IC50 values of 1a–j towards the two T-lym-
phocyte cell lines and the interatomic distances d3 (N1ꢁN2), d4
(N1ꢁO2) and d5 (O2ꢁO4) (Figure 3c). Positive correlations were
noted between the IC50 values and d3 (p=0.04), d4 (p=0.04),
and d5 (p=0.02), indicating that potency increases as the d3–
d5 spans diminish. Expansion of this project should involve the
design of molecules in which the spacer group (X) is either
small or eliminated entirely. In addition, these results highlight
the importance of the proximity of the piperidyl nitrogen
atoms to the oxygen atoms. Further experimentation should
be pursued, such as incorporating the COꢁXꢁCO group into
rigid heterocyclic rings with the goal of finding the optimal
topography of these molecules to maximize cytotoxic proper-
ties.
The relative positions of the olefinic carbon atoms in both
pharmacophoric groups were determined as follows: The four
olefinic carbon atoms were designated CA, CB, CC, and CD (Fig-
ure 3a). Axes 1 and 2 were constructed (Figure 3b), and d1, d2,
In light of the encouraging biological data displayed by 1a–
g, and i, we undertook further evaluations. First, it was neces-
sary to select a lead compound. As shown in Table 1, both 1a
and 1b exhibited sub-micromolar IC50 values toward human
cancer cells. These molecules were, therefore, evaluated
against a substantial number of human tumor cell lines,[20] with
the cytotoxic effect of 1a and 1b against some of these neo-
plasms presented in Figure 4. The data presented shows that
1a generally displays lower IC50 values than 1b; consequently,
1a was chosen for further studies. An additional noteworthy
feature of 1a and 1b is the differential potencies both com-
pounds display towards the cell lines. This observation
strengthens the view expressed earlier that these compounds
and analogues in series 1 may possess greater toxicity towards
neoplasms than normal cells.
Figure 3. Various structural features of 1a–j as determined by molecular
modeling: a) olefinic carbon atoms CA–CD, piperidyl nitrogen atoms N1 and
N2, and carbonyl oxygen atoms O1–O4; b) interatomic distances d1 and d2,
and bond angle y; and c) interatomic distances d3–d5.
Figure 4. Growth inhibitory effect of 10 mm 1a or 1b toward a number of human tumor cell lines. Cells were exposed to 1a (&) and 1b (&) for 48 h using a
previously described procedure.[20]
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The ability of non-adherent cells to diffuse in vivo, leading
to metastasis, led us to next address the discovery of novel
prototypic molecules that inhibit the growth of non-adherent
neoplasms. The potential of such compounds would be en-
hanced even further if greater toxicity could be demonstrated
towards non-adherent neoplasms over either adherent or non-
malignant cells. Compound 1a was examined against the fol-
lowing non-adherent cell lines: human CEM, JURKAT, and SUP-
T1 and murine EL-4 T-cell lymphomas, as well as human BJAB,
Nalm-6, and Ramos B-cell lymphomas. This compound was
also assessed against adherent human HeLa ovarian cancer
cells, as well as two adherent non-malignant cell lines: human
foreskin Hs27 and murine N1H-3T3 fibroblasts.
a common property of the more potent compounds in series
1. Accordingly, 1b–g, and i were evaluated against JURKAT and
SUP-T1 non-adherent cells, as well as against normal (Hs27)
and malignant (HeLa) adherent cell lines (Figure 6). The various
Figure 5 shows that, in general, greater toxicity was demon-
strated toward non-adherent cells than toward either the ad-
herent HeLa cell line or the two fibroblasts. CC50 values were
Figure 5. Cytocidal effects of 1a (5 mm) as determined by flow cytometry
using a previously reported method.[25] Cells are non-adherent with the ex-
ception of the adherent HeLa, Hs27, and N1H-3T3 cell lines. Each bar repre-
sents the average value of triple measurements with error bars showing
standard deviations. Cells were exposed to 1a for 22 h.
Figure 6. Evaluation of 1b–g, and 1i against non-adherent (JURKAT, SUP-T1)
and adherent (Hs27, HeLa) cells. Concentrations of compounds used were
2.5 mm (1 f, g, and i), 5 mm (1e), and 10 mm (1b–d). Cells were exposed to
compounds for 22 h as previously described.[25]
also determined in order to garner an appreciation of the dif-
ferential toxicity between several non-adherent cells and the
adherent NIH-3T3 fibroblast (Table 3). Selectivity index values
are impressive and establish 1a as an important lead molecule.
Further experimentation was initiated to determine whether
higher toxicity toward non-adherent over adherent cell lines is
concentrations of 1b–g and i were chosen to emphasize the
consistently greater cytotoxicity of these compounds in adher-
ent versus non-adherent cells. The results provide unequivocal
evidence that 1b–g and i, similar to 1a, display preferential
toxicity against non-adherent cells. This observation strength-
ens the need for development of these compounds, which dis-
play antimetastatic potential in addition to their cytotoxic
properties.
Table 3. Evaluation of 1a against non-adherent cell lines and NIH-3T3 fi-
broblasts.
Cell line
CC50[a] [mm]
SI[b]
CEM
EL-4
JURKAT
Nalm6
Sup-T1
NIH-3T3
0.034
0.031
0.029
0.022
0.384
12.78
376
412
441
581
33
Flow cytometry was pursued, using four non-adherent cell
lines, in order to gain an understanding of the ways in which
1a mediates its cytotoxic properties (Figure 7). After 8 h incu-
bation, an average of 19% of the cells were apoptotic, with
this percentage doubling after 20 h. Necrosis was virtually
absent after 8 h, while on an average of 9% of the cells were
necrotic after 20 h. One may therefore conclude that 1a
causes cell death inter alia by apoptosis and, to a lesser
–
[a] Cells were incubated with 1a for 22 h as previously described.[25]
Values shown are the average of three independent experiments. [b] Se-
lectivity index (SI) figures are quotients of the CC50 values of 1a toward
NIH-3T3 fibroblasts and the data for each of the non-adherent cell lines.
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Novel and Potent Cytotoxins
Figure 7. Flow cytometry analysis of the cytotoxic effect of 1a on four non-adherent cell lines following incubation for 8 and 24 h.[25] The exact percentage of
apoptotic (&), necrotic (&), and viable (&) cells is indicated at the top of each bar graph. Two concentrations of 1a are shown on the x-axis. Note that two-
fold higher concentrations were utilized for the most resistant cell lines, SUP-T and CEM.
Experimental Section
degree, by necrosis. In a previous study, a cytotoxic 1-acyl-3,5-
bis(benzylidene)-4-piperidone activated caspase-3, with the
extent of activation dependent on the cell line under investiga-
tion.[21] Since caspases are involved in most apoptosis, it is
likely that one mechanism by which 1a–j may exert their cyto-
toxic properties is the activation of one or more caspases.
Chemistry
Synthesis of 1a–k: Melting points were determined on a Gallen-
kamp instrument and are uncorrected. 1H and 13C NMR spectra
were obtained using a Bruker Avance AMX 500 spectrometer
equipped with a BBO probe. Chemical shifts (d) are reported in
ppm. Mass spectra were obtained using a quad tandem 4000
QTRAP mass analyzer. Elemental analyses were undertaken using a
CHNS elemental analyzer (Vario EL III microanalyzer).
Conclusions
This study reveals that the unsaturated ketones in series 1 are,
in general, potent cytotoxins. The theory that increasing the
number of sites for thiol alkylation two-fold should more than
double cytotoxic potency was validated in approximately half
of the biological data generated using Molt 4/C8 and CEM T-
lymphocytes, although there was little support when utilizing
the murine L1210 screen. Extensions of this study should con-
sider the design and evaluation of analogues of 1a–j that con-
tain more than two identical phamacophores. In addition, the
synthesis of heterodimers should be pursued in which different
substituents are appended to the aryl rings, thereby creating
molecules with varying atomic charges on the olefinic carbon
atoms. For compounds such as these, stepwise reactions with
thiols should be enhanced, which may lead to greater increas-
es in toxicity to neoplasms than to normal cells.[10,11] Molecular
modeling was also used to emphasize the importance of the
relative positions of the amide groups.
General procedure for the synthesis of 3,5-bis(benzylidene)-4-pi-
peridone dimers (1a–k): A mixture of the corresponding dicarbox-
ylic acid (0.005 mol) and thionyl chloride (0.02 mol, 2.4 g) was
heated at 60–658C for 4–5 h. Excess thionyl chloride was removed
at 458C in vacuo and moisture-free conditions. The resulting acid
chloride was used for further reaction without purification.
The previously prepared acid chloride in 1,2-dichloroethane (DCE;
5 mL) was added slowly over a period of 30 min to a stirred sus-
pension of 3,5-bis(benzylidene)-4-piperidone (0.009 mol, 2.75 g)
prepared according to a literature method[13] in DCE (20 mL) con-
taining Et3N (0.11 mol, 1.12 g) at ꢂ208C. The reaction stirred at RT
overnight, then the solvent was removed in vacuo at 458C. Aq
K2CO3 (25 mL, 10% w/v) was added to the crude material and
stirred for 2 h. The resulting solid was filtered, dried, and crystal-
lized from a suitable solvent to yield pure product. In the case of
1a, b, d, and e, the appropriate acid chlorides were procured from
commercial sources.
1,2-Bis(3,5-dibenzylidene-4-oxo-piperidin-1-yl)ethane-1,2-dione
(1a): Yield: 62%; mp: 2468C (CHCl3/MeOH); 1H NMR (500 MHz,
[D6]DMSO): d=7.72 (s, 2H, 2ꢂ=CH), 7.56 (s, 2H, 2ꢂ=CH), 7.53 (t,
4H, Ar-H), 7.49 (d, J=7.07 Hz, 2H, Ar-H), 7.45 (m, 6H, Ar-H), 7.39
(m, 8H, Ar-H), 4.48 ppm (d, J=23.28 Hz, 8H, 4ꢂNCH2); 13C NMR
(125 MHz, [D6]DMSO): d=184.7, 162.6, 137.9, 137.5, 134.4, 134.1,
131.4, 131.0, 130.9, 130.7, 130.2, 130.1, 129.3, 129.2, 46.4, 41.6 ppm;
MS (ESI) m/z: 627 [M+Na]+; Anal. calcd for C40H32N2O4·H2O: C
77.17; H 5.14; N 4.50, found: C 77.05; H 4.87; N 4.42.
From this initial investigation, 1a and 1b emerged as lead
molecules, displaying potent cytotoxicity against a wide range
of human tumor cell lines. Further evaluations using 1a re-
vealed its increased toxicity toward non-adherent cell lines
than toward either an adherent neoplasm or fibroblasts. Com-
pound 1a was shown to exert its toxic effects against certain
cancer cells through apoptosis and necrosis. Sufficient evi-
dence has been presented through these studies to warrant
rapid expansion of evaluation of this compound series in order
to further investigate their potential as candidate anticancer
agents.
1,3-Bis-(3,5-dibenzylidene-4-oxo-piperidin-1yl)propane-1,3-dione
(1b): Yield: 65%; mp: 2018C (acetone); 1H NMR (500 MHz,
[D6]DMSO): d=7.72 (s, 2H, 2ꢂ=CH), 7.57 (s, 2H, 2ꢂ=CH), 7.53 (d,
ChemMedChem 2011, 6, 1892 – 1899
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J. R. Dimmock et al.
MED
J=4.18 Hz, 8H, Ar-H), 7.47 (m, 12H, Ar-H), 4.62 (d, J=21.13 Hz, 8H,
4ꢂNCH2), 3.46 ppm (s, 2H, CH2); 13C NMR (125 MHz, [D6]DMSO):
d=186.3, 165.9,136.6, 136.5, 134.7, 134.5, 132.6, 132.5, 131.0, 130.9,
130.1, 130.0, 129.3, 129.2, 47.0, 42.4 ppm; MS (ESI) m/z: 641 [M+
Na]+; Anal. calcd for C41H34N2O4·H2O: C 77.27; H 5.65; N 4.39,
found: C 77.31; H 5.50; N 4.47.
1,2-Bis-[(3,5-dibenzylidene-4-oxo-piperidin-1-yl)-1-carbonyl]ben-
zene (1i): Yield: 68%; mp: 2408C (CHCl3/MeOH; dec.); 1H NMR
(500 MHz, [D6]DMSO): d=7.77 (s, 2H, 2ꢂ=CH), 7.69 (s, 2H, 2ꢂ=
CH), 7.57 (t, 10H, Ar-H), 7.27 (brs, 5H, Ar-H), 7.18 (brs, 5H, Ar-H),
6.89 (m, 2H, Ar-H), 6.78 (m, 2H, Ar-H), 4.92 (brs, 4H, 2ꢂNCH2),
4.49 ppm (s, 4H, 2ꢂNCH2); 13C NMR (125 MHz, [D6]DMSO): d=
186.1, 167.9, 137.3, 136.5, 134.8, 133.8, 132.6, 131.1, 130.3, 129.8,
129.4, 129.0, 126.4, 47.8, 43.4 ppm; MS (ESI) m/z: 703 [M+Na]+;
Anal.calcd for C46H36N2O4·H2O: C 78.99; H 5.43; N 4.0, found: C
79.09; H 5.42; N 4.02.
1,4-Bis-(3,5-dibenzylidene-4-oxo-piperidin-1-yl)butane-1,4-dione
(1c): Yield: 58%; mp: 1888C (CHCl3/MeOH); 1H NMR (500 MHz,
[D6]DMSO): d=7.68 (s, 4H, 4ꢂ =CH), 7.49 (m, 20H, Ar-H), 4.78 (d,
J=10.95 Hz, 8H, 4ꢂNCH2), 2.29 ppm (s, 4H, 2ꢂCH2); 13C NMR
(125 MHz, [D6]DMSO): d=186.5, 170.4, 136.6, 136.5, 134.8, 134.5,
133.0, 132.8, 131.0, 130.0, 129.3, 46.3, 42.9, 27.16 ppm; MS (ESI)
m/z: 655 [M+Na]+; Anal. calcd for C42H36N2O4·0.5H2O: C 78.53; H
5.76; N 4.36, found: C 78.16; H 5.71; N 4.11.
1,3-Bis-[(3,5-dibenzylidene-4-oxo-piperidin-1-yl)-1-carbonyl]ben-
zene (1j): Yield: 63%; mp: 2208C (CHCl3/MeOH; dec.); 1H NMR
(500 MHz, [D6]DMSO): d=7.78 (s, 4H, 4ꢂ =CH), 7.53 (m, 10H, Ar-
H), 7.31 (m, 10H, Ar-H), 7.11 (s, 1H, Ar-H), 7.06 (d, J=8.93 Hz, 2H,
Ar-H), 6.75 (t,1H, Ar-H), 4.97 (brs, 4H, 2ꢂNCH2), 4.57 ppm (brs, 4H,
2ꢂNCH2); 13C NMR (125 MHz, [D6]DMSO): d=186.03, 168.0, 137.2,
134.51, 132.6, 131.0, 130.1, 129.2, 128.9, 128.3, 125.7, 48.9,
45.6 ppm; MS (ESI) m/z: 703 [M+Na]+; Anal.calcd for
C46H36N2O4·0.5H2O: C 80.02; H 5.36; N 4.01, found: C 80.31; H 5.29;
N 4.01.
1,5-Bis-(3,5-dibenzylidene-4-oxo-piperidin-1-yl)pentane-1,5-
dione (1d): Yield: 43%; mp: 1708C (CHCl3/MeOH); 1H NMR
(500 MHz, [D6]DMSO): d=7.71 (s, 2H, 2ꢂ=CH), 7.66 (s, 2H, 2ꢂ=
CH), 7.54 (m, 14H, Ar-H), 7.42 (m, 6H, Ar-H), 4.76 (d, 8H, 4ꢂNCH2,
J=28.18 Hz), 2.02 (t, 4H, 2ꢂCH2), 1.42 ppm (p, 2H, CH2); 13C NMR
(125 MHz, [D6]DMSO): d=186.6, 171.0, 136.6, 134.8, 134.5, 133.1,
133.0, 131.0, 130.3, 129.3, 46.4, 42.8, 31.3, 20.4 ppm; MS (ESI) m/z:
627 [M+Na]+; Anal.calcd for C43H38N2O4·0.25H2O: C 79.22; H 5.91;
N 4.30, found: C 79.18; H 5.64; N 4.08.
1,4-Bis-[(3,5-dibenzylidene-4-oxo-piperidin-1-yl)-1-carbonyl]ben-
zene (1k): Yield: 72%; mp: 2208C (AcOH/H2O; dec.); 1H NMR
(500 MHz, CF3COOD): d=10.44 (s, 2H, 2ꢂ=CH), 10.38 (s, 2H, 2ꢂ=
CH), 9.81 (d, J=8.4 Hz, 10H, Ar-H), 9.63 (brs, 6H, Ar-H), 9.49 (brs,
4H, Ar-H), 9.16 (s, 4H, Ar-H), 7.47 (s, 4H, 2ꢂNCH2), 6.86 ppm (s, 4H,
2ꢂNCH2); 13C NMR (125 MHz, CF3COOD): d=192.9, 174.1, 146.6,
144.2, 135.9, 135.4, 135.3, 133.4, 133.0, 132.6, 132.2, 132.0, 131.0,
130.9, 130.8, 129.0, 49.5, 47.12 ppm; Anal. calcd for
C46H36N2O4·0.5H2O: C 80.02; H 5.36; N 4.01, found: C 79.67; H 5.25;
N 3.99.
1,8-Bis-(3,5-dibenzylidene-4-oxo-piperidin-1-yl)octane-1,8-dione
(1e): Yield: 64%; mp: 1608C (CHCl3/MeOH); 1H NMR (500 MHz,
[D6]DMSO): d=7.71 (s, 4H, 4ꢂ =CH), 7.51 (m, 20H, Ar-H), 4.81 (d,
8H, J=29.13 Hz, 4ꢂNCH2), 2.00 (t, 4H, 2ꢂCH2), 1.20 (m, 4H, 2ꢂ
CH2), 0.76 ppm (m, 4H, 2ꢂCH2); 13C NMR (125 MHz, [D6]DMSO):
d=186.6, 171.4, 136.8, 136.4, 134.8, 134.6, 133.2, 131.0, 130.0,
129.3, 46.5, 43.1, 32.4, 28.5, 24.7 ppm; MS (ESI) m/z: 711 [M+Na]+;
Anal.calcd for C46H44N2O4·0.5H2O: C 79.10; H 6.30; N 4.01, found: C
78.85; H 6.32; N 4.04.
3,5-Bis(benzylidene)-1-tetradecanoyl-4-piperidone (2): Myristoyl
chloride (0.011 mol, 2.7 g) in DCE (5 mL) was added slowly over a
period of ꢂ30 min to a suspension of 3,5-bis(benzylidene)-4-piperi-
1,4-Bis-(3,5-dibenzylidene-4-oxo-piperidin-1-yl)but-2-ene-1,4-
dione (1 f): Yield: 71%; mp: 2208C (EtOH); 1H NMR (500 MHz,
[D6]DMSO): d=7.74 (s, 2H, 2ꢂ=CH), 7.66 (s, 2H, 2ꢂ=CH), 7.51 (m,
20H, Ar-H), 6.92 (s, 2H, 2ꢂ=CH), 4.83 ppm (d, J=8.54 Hz, 8H, 4ꢂ
NCH2); 13C NMR (125 MHz, [D6]DMSO): d=186.2, 163.9, 136.9,
136.7, 134.7, 134.3, 132.6, 131.0, 130.1, 129.3, 46.9, 43.1 ppm; MS
(ESI) m/z: 653 [M+Na]+; Anal.calcd for C42H34N2O4·5H2O: C 69.92; H
4.71; N 3.88, found: C 69.85; H 4.79; N 3.57.
done (0.007 mol, 2 g), prepared according to
a literature
method,[13] in DCE (15 mL) containing Et3N (0.016 mol, 1.7 gm) at
ꢂ158C. The reaction stirred at RT overnight, then the solvent was
removed in vacuo at 458C. Aq K2CO3 (25 mL, 10% w/v) was added
to the crude, and the mixture was stirred for 2 h. The resulting
solid was filtered, dried, and crystallized from EtOH. Yield: 90%;
1
mp: 828C; H NMR (500 MHz, CDCl3): d=7.91 (s, 1H, =CH), 7.85 (s,
1H, =CH), 7.53–7.40 (m, 10H, Ar-H), 4.96 (s, 2H, NCH2), 4.87 (s, 2H,
NCH2), 2.14 (t, 2H, COCH2), 1.45 (p, 2H, CH2), 1.26 (m, 16H, Ar-H),
1.09 (m, 4H, 2ꢂCH2), 0.91 ppm (t, 3H, CH3); 13C NMR (125 MHz,
CDCl3): d=186.9, 172.1, 138.6, 137.1, 134.7, 134.6, 132.1, 131.9,
130.7, 130.1, 129.6, 128.9, 128.8, 46.3, 43.6, 33.2, 32.0, 29.7, 29.7,
29.6, 29.5, 29.4, 29.3, 29.2, 25.1, 22.7, 14.2 ppm; Anal.calcd for
C31H39NO2: C 81.60; H 8.92; N 2.88, found: C 81.27; H 9.29; N 2.83.
1,4-Bis-(3,5-dibezylidene-4-oxo-piperidin-1-yl)-but-2-yne-1,4-
dione (1g): Yield: 48%; mp: 2208C (CHCl3/MeOH; dec.); 1H NMR
(500 MHz, [D6]DMSO): d=7.81 (s, 1H, =CH), 7.75 (d, 2H, 2ꢂ=CH,
J=17.63 Hz), 7.68 (s,1H, =CH), 7.56 (m,20H, Ar-H), 4.79 (d, J=
18.80 Hz, 4H, 2ꢂNCH2), 4.64 ppm (d, J=16.96 Hz, 4H, 2ꢂNCH2);
13C NMR (125 MHz, [D6]DMSO): d=186.0, 185.7, 185.4, 162.4, 161.9,
149.9, 137.2, 134.5, 132.3, 131.8, 131.5, 131.0, 130.7, 130.4, 129.1,
128.7, 125.25, 95.25, 46.8, 42.7, 42.2 ppm; MS (ESI) m/z: 651 [M+
Na]+; Anal.calcd for C42H32N2O4·2H2O: C 75.82; H 4.81; N 4.21,
found: C 75.48; H 4.74; N 4.01.
Computational experiments
Molecular modeling: Models were built using the SYBYL 8.0 pro-
gram[22] on a Lenovo workstation with the RHEL 4.0 operating
system. Energy minimizations were performed with the conjugate
gradient method using the Tripos force field and Gasteiger–Huckel
charges with a convergence criterion of 0.001 kcalmolꢁ1 ꢃ. Each
structure was further subjected to simulated annealing for identify-
ing the lowest energy conformation. The system was heated at
1000 K for 1 ps, and then cooled at 200 K for 1 ps. The exponential
annealing function was used, and ten such cycles were run. The
lowest energy conformer was used to calculate the distance be-
tween two points and bond angles as depicted in Figure 3.
1,6-Bis-(3,5-dibenzylidene-4-oxo-piperidin-1-yl)-hexa-2,4-diene-
1,6-dione (1h): Yield: 56%; mp: 1998C (CHCl3/MeOH); 1H NMR
(500 MHz, CDCl3): d=7.87 (s, 4H, 4ꢂ =CH), 7.42 (m, 20H, Ar-H),
6.94 (m, 2H, 2ꢂ=CH), 6.16 (m, 2H, 2ꢂ=CH), 4.98 (s, 4H, 2ꢂNCH2),
4.77 ppm (s, 4H, 2ꢂNCH2); 13C NMR (125 MHz, [D6]DMSO): d=
186.6, 164.7, 140.3, 137.5, 134.5, 131.5, 130.6, 130.0, 125.9, 125.6,
46.3, 44.1 ppm; MS (ESI) m/z: 679 [M+Na]+; Anal.calcd for
C44H36N2O4·2.5H2O: C 75.23; H 5.12; N 3.98, found: C 75.23; H 5.02;
N 3.91.
1898
www.chemmedchem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1892 – 1899

Novel and Potent Cytotoxins
Determination of logP values: The predicted logP values for 1e and
2 were obtained using Molinspiration Chemoinformatics software
online.[23]
compounds 1a and 1b against a number of human tumor cell
lines. This work was also supported by the Border Biomedical Re-
search Center at the University of Texas at El Paso (USA)
(5G12RR008124–16A1). B. McCullough and C. Sutton are thanked
for their efforts in the preparation of this manuscript.
Biology
Cytotoxicity assays: The compounds in series 1 were evaluated
against Molt 4/C8, CEM, and L1210 cells using a previously report-
ed procedure.[24] Briefly, various concentrations of the compounds
were incubated with the appropriate cell line in RPMI 1640
medium at 378C for 72 h (Molt 4/C8 and CEM assays) or 48 h
(L1210 screen). Numbers of cells were determined using a Coulter
counter. The IC50 value given is the concentration required to in-
hibit cell proliferation by 50%. Data are expressed as the mean ꢀ
SD from the dose–response curves of at least three independent
experiments.
Keywords: apoptosis · cytotoxic synergism · cytotoxicity ·
molecular modeling · unsaturated ketones
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Acknowledgements
The authors thank the Canadian Institutes of Health for an oper-
ating grant to J.R.D. Appreciation is extended to the Belgian Con-
certed Research Actions (GoA 10/014) for providing funds to J.B.
for the Molt 4/C8, CEM, and L1210 screens undertaken by L. van
Berckelaer, and to the US National Cancer Institute for evaluating
Received: April 18, 2011
Published online on August 8, 2011
ChemMedChem 2011, 6, 1892 – 1899
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1899