Novel 3,5-bis(arylidene)-4-piperidone dimers: Potent cytotoxins against colon cancer cells

Article abstract of DOI:10.1016/j.ejmech.2013.03.055

Two novel series of dimeric 3,5-bis(arylidene)-4-piperidones 7 and 8 were prepared as cytotoxic agents. A specific objective of this study was the discovery of novel compounds displaying potent anti-proliferative activities against colon cancers. Most of

Full text of DOI:10.1016/j.ejmech.2013.03.055

Accepted Manuscript
Novel 3,5-bis(arylidene)-4-piperidone dimers: Potent cytotoxins against colon cancer
cells
Swagatika Das, Umashankar Das, Deborah Michel, Dennis K.J. Gorecki, Jonathan R.
Dimmock
PII:
S0223-5234(13)00215-8
10.1016/j.ejmech.2013.03.055
EJMECH 6093
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 December 2012
Revised Date: 24 March 2013
Accepted Date: 26 March 2013
Please cite this article as: S. Das, U. Das, D. Michel, D.K.J. Gorecki, J.R. Dimmock, Novel 3,5-
bis(arylidene)-4-piperidone dimers: Potent cytotoxins against colon cancer cells, European Journal of
Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.03.055.
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GRAPHICAL ABSTRACT

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Novel 3,5-bis(arylidene)-4-piperidone dimers: Potent cytotoxins against colon cancer cells
Swagatika Das, Umashankar Das*, Deborah Michel, Dennis K. J. Gorecki, Jonathan R.
Dimmock*
Drug Discovery and Development Research Group, College of Pharmacy and Nutrition,
University of Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada
*Corresponding authors: Tel.: +1 306 966 6358; Fax: +1 306 966 6377.
E-mail addresses: umashankar.das@usask.ca (U. Das), jr.dimmock@usask.ca (J.R. Dimmock).
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Abstract
Two novel series of dimeric 3,5-bis(arylidene)-4-piperidones 7 and 8 were prepared as cytotoxic
agents. A specific objective of this study was the discovery of novel compounds displaying
potent anti-proliferative activities against colon cancers. Most of the compounds demonstrate
potent cytotoxicity against HCT116 and HT29 colon cancer cell lines in which the IC50 values
range from low micromolar to nanomolar values. In general, the majority of the compounds
showed greater cytotoxicity and some degree of selectivity towards HCT116 cells compared to
HT29 cells. Compound 9 in which the amidic carbonyl groups were excised was substantially
less potent than 8a in both cell lines suggested that the amide groups are important components
of the molecules for exhibiting cytotoxicity. Virtually all the compounds were more potent than
a reference drug 5-fluorouracil which is used in treating colon cancers as well as a related enone
curcumin. QSAR studies were undertaken and some guidelines for amplification of the project
have been formulated. Flow cytometry analysis of a representative potent compound 7f revealed
that it induces apoptosis in HCT116 cells.
Keywords: piperidone, α,β-unsaturated ketone, cytotoxicity, selective toxicity
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1. Introduction
Colorectal cancer is the second leading cause of death in men and women among all
cancers [1]. The low incidence of colorectal cancer in Asian countries has been suggested to be
due inter alia to the regular use of curcumin in the diet [2, 3]. Curcumin 1 produces a profound
cytotoxic effect on colon carcinogenesis in rats and mice, reduces a number of colon tumours in
a mouse model with a mutation in the APC gene [4] and decreases the multiplicity of colon
adenomas [5]. Curcumin was found to protect against the development of colon cancer induced
by azoxymethane during both the initiation and the promotion stages in both rats and mice [6,7]
and also demonstrates potent anticancer effects on intestinal cancer, stomach cancer, and
hepatocellular carcinoma [8]. The α,β-unsaturated keto motif is considered to contribute
significantly to the bioactivity observed. Subsequently a large number of curcuminoids have
been described in the literature, which display potent cytotoxic potencies against various cancers
including colon cancers [9-11]. One of the reasons for the interest in these compounds is that
α,β-unsaturated ketones display a selective affinity for thiols compared to hydroxyl and amino
groups [12] which are present in nucleic acids. Hence these molecules should not elicit genotoxic
properties associated with a number of alkylating agents used in cancer chemotherapy [13].
Cytotoxic α,β-unsaturated ketones are thiol alkylators [12] and the importance of multitargeted
ligands has been documented very recently [14-16].
[PLEASE INSERT FIGURE 1]
Our major interest is the development of conjugated unsaturated ketones as candidate
cytotoxins with special emphasis on those compounds which inhibit the growth of colon cancers
[17,18]. In view of these considerations, a bifunctional 1,5-diaryl-3-oxo-1,4-pentadienyl
pharmacophore was incorporated into a variety of cyclic and acyclic analogs that led to the
discovery of a number of potent cytotoxins possessing the general structure 2 [14]. In particular,
some 3,5-bis(arylidene)-4-piperidones demonstrate IC50 values in the low micromolar to
submicromolar range towards a number of malignant cell lines [19,20]. These compounds were
designed based on the hypothesis of sequential cytotoxicity which states that successive chemical
attacks may lead to greater damage in cancer cells compared to normal cells [19]. This theory
was formulated on the observations that an initial lowering of the concentrations of cellular thiols
followed by a second chemical attack would cause a greater deleterious effect to neoplasms than
normal cells [21,22]. A N-acyl-3,5-bis(benzylidene)-4-piperidone 4a displayed more than 8-fold
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greater cytotoxic potency than its precursor 3 towards P388/MRI cells [23]. An investigation of
the effect of these two compounds 3 and 4a on hepatic glutathione levels in mice revealed
reductions in the glutathione concentrations of 19 and 29%, respectively, suggesting that an
increase in the number of thiol alkylating sites in the molecule contributed to an improvement in
cytotoxic potency [23]. Based on this observation, the N-acryloyl-3,5-bis(arylidene)-4-
piperidone 4b and a number of aryl substituted analogs were developed which possess greater
cytotoxic potencies over their 3,5-bis(arylidene)-4-piperidone precursors [19]. These
observations unequivocally demonstrate the value of incorporating additional thiol alkylating
groups in the molecule and led to a decision to prepare series 5. In these compounds two
molecules of 3 are attached by a linker with varying carbon chain lengths keeping in view that
the relative location of one of the 1,5-diaryl-3-oxo-1,4-pentadienyl groups in series 5 with
respect to the other one may be important for binding to the active sites of the proteins and may
influence cytotoxicity substantially. The compounds in series 5 displayed potent cytotoxicity
against human Molt4/C8 and CEM T-lymphocytes as well as murine L1210 lymphoid leukemic
cells [24] while further investigations revealed the greater toxicity of these compounds to
neoplasms than non-malignant cells [25]. From these studies two prototypic molecules 7a and 8a
emerged which displayed remarkable cytotoxic potencies having average IC50 values of 0.17 and
0.11 µM, respectively, against a panel of seven different colon cancer cell lines [25]. These two
compounds are clearly lead molecules for further analog development.
The aims of the present study are fourfold namely (i) examining the compounds in series
7 and 8 as candidate cytotoxins which are particularly effective against colon cancers, (ii) to
evaluate the influence of the electronic, hydrophobic and steric properties of the aryl ring
substituents in 7a-i and 8a-j on cytotoxic potencies and if possible to develop structure-activity
relationships which will be helpful for designing further potent cytotoxic molecules, (iii) to
establish the role of the amidic groups in governing cytotoxic properties of the molecules, and
(iv) to investigate the mode action of a representative molecule whereby the cytotoxicity of this
class of compounds is mediated.
[PLEASE INSERT SCHEME 1]
2. Chemistry
The synthesis of compounds in series 6-9 is outlined in Scheme 1. Briefly, the 3,5-
bis(benzylidene)-4-piperidones 6a-j were synthesized by acid-catalyzed condensation of 4-
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piperidone hydrochloride with aryl aldehydes [19]. The dimers in series 7 and 8 were obtained
from their corresponding 3,5-bis(benzylidene)-4-piperidones 6 by reacting with oxaloyl chloride
or malonyl chloride, respectively. The synthesis of 9 was accomplished by a base catalyzed
condensation of two mole equivalents of 6a with one mole equivalent of 1,3-dibromopropane.
The structures of all the compounds were established by 1H NMR and elemental analysis.
1
The H NMR spectra of 7a-i, 8a-j and 9 revealed that the compounds are isomerically
pure and all four olefinic double bonds exhibit the E-configuration. The E-stereochemistry of
these compounds was indicated based on the following observations. First, the olefinic protons
of the dimers 7-9 appear at 7.39-7.79 ppm in the 1H NMR spectra, which is an indicative of the E
geometry [19]. Second, a number of 3,5-bis(benzylidene)-4-piperidones 6 adopt the E-
stereochemistry as established by X-ray crystallography [19, 23]. In addition, recently an X-ray
crystal structure of the 2-fluoro analog of 7 was shown to possess the E- geometry [26]. In order
to evaluate the hypothesis that the amidic groups in the series 7-8 are essential for cytotoxicity, a
representative molecule 9 was prepared which is bereft of both amidic carbonyl groups. Various
physicochemical properties of the aryl substituents and the topography of the molecules were
examined for correlations with the IC50 values.
3. Bioevaluations
The compounds in series 7-9 as well as two reference compounds 5-fluorouracil (5-FU)
and curcumin were evaluated against two human colon cancer cell lines HCT116 and HT29.
These biodata are reported in Table 1. Solubility considerations mandated that the highest
concentration of 8i that could be used is 25 µM, which was insufficient to generate an IC50 value.
A representative lead cytotoxin 7f was examined for its ability to cause apoptosis in HCT116 cell
lines. This result is presented in Figure 2.
4. Results and Discussion
The compounds 7a-i and 8a-j were evaluated against human HCT116 and HT29 colon
cancer cells. The biodata presented in Table 1 show remarkable cytotoxic potencies of all the
compounds except 7d,i and 8d,i. The majority of the IC50 values were in the submicromolar to
nanomolar range. The compounds displaying IC50 values < 1µM are considered to be highly
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cytotoxic. In series 7, 78% of the IC50 values were <1 µM against both the cell lines and in
series 8, IC50 values of <1µM were observed in 80% of the compounds against HCT116 and
HT29 cells.
[PLEASE INSERT TABLE 1]
In particular, 7a,e and 8a,c,h have IC50 values in double digit nanomolar range except in
the case of 8a, the IC50 value is 3 nM in the HT29 bioassay. The importance of the marked
cytotoxic potencies of most of these compounds was confirmed when the IC50 values of 7-9 are
compared with that of 5-FU which is a drug used in treating colon cancers. Relative potency
(RP) figures were generated by comparing the IC50 figures of the compounds in series 7-9 with
that of 5-FU in these two screens. These data are presented in Table 1 which reveals that in
general the compounds are far more toxic to these two neoplasms than the clinically useful drug.
In addition, the RP figures indicate that in general the HCT116 cells are more sensitive to these
compounds than the HT29 neoplasms. This difference between the RP values for each
compound may reflect a capacity to display greater toxic effects to malignant cells than normal
tissues. This possibility is enhanced by the variation in efficacy of the compounds to inhibit the
growth of the HCT116 and HT29 cells, e.g. the 304-fold difference between 7b and 7i in the
HCT116 screen. In other words, the compounds in series 7 and 8 are not general biocidal agents
and possibly are tumour-selective cytotoxins. Virtually all of the compounds in series 7-9 are
substantially more potent than curcumin.
The following correlations were observed between the nature of the aryl substituents in
series 7 and 8 and their cytotoxic potencies in both screens. First, the placement of a methyl
(7b,8b), chloro (7c,8c), fluoro (7e,8e), or methoxy (7f,8f) group into the 4 position of the aryl
rings led to compounds having similar potencies in the range of 0.02-0.78 µM. However, the
introduction of a 4-dimethylamino substituent (7i,8i) into the aryl rings caused potency to
plummet considerably. Second, the addition of a 3-chloro group to the 4-chloro analogs leading
to the 7d and 8d causes a dramatic reduction in potency, e.g., there is a 668-fold difference
between the IC50 values of 7c and 7d in the HCT116 screen.
[PLEASE INSERT TABLE 2]
The next part of the analysis of the biodata involved a comparison of the potencies of
some of the compounds in series 7 and 8. These comparisons are presented as ꢀ8/7 values which
were obtained by dividing the IC50 value of a compound in 8a-i by that of the analog in series 7
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which possesses the same aryl substituent. In this way one may be able to ascertain whether an
oxalyl [-C(O)-C(O)-] or malonyl [-C(O)-CH2-C(O)-] linker between the piperidinyl nitrogen
atoms is optimal. Taking standard deviations into account, in the HCT116 bioassay 7b-g are
more potent than 8b-g while 8h has a lower IC50 value than 7h. The unsubstituted analogs 7a
and 8a are equipotent. In the case of the HT29 screen, 7b,e,f are more potent than 8b,e,f while
8a,c,d,h have lower IC50 figures than 7a,c,d,h and 7g and 8g have equal potency. Thus the
oxalyl and malonyl linkers are favoured in 56% and 31%, respectively, of the enones 7a-h and
8a-h while equipotency was observed in 13% of the comparisons made. A quantitative estimate
of the differences in potencies between 7a-h and 8a-h, which are dependent on the nature of the
spacer group, is presented as ∆8/7 values in Table 2. Thus the oxalyl group is marginally
preferable to the malonyl linker although excellent cytotoxic potencies were observed in each
series of compounds.
A question to be addressed is whether the substituted dimers 7b-i and 8b-i display greater
cytotoxic potencies than the unsubtituted analogs 7a and 8a, respectively. For series 7, the enone
7a has greater potency than 7d,h,i and was equipotent with 7b,c,e-g in the HCT116 assay. For
the HT29 screen, 7a was more potent than 7b-d,f-i and was equipotent with 7e. In the case of
series 8, the unsubstituted analog 8a has lower IC50 values than 8b-g,i and displays equal
potency as 8h in the HCT116 screen while against HT29 cells, 8a is more potent than 8b-i. Thus
overall 7a and 8a are more potent than the substituted dimers 7b-i and 8b-i in 78% of the
comparisons made while the same potency was noted in 22% of the cases. These comparisons
are presented in quantitative form in Table 2 where, in general, the ∆7b-i/7a figures are smaller
than the ∆8b-i/8a values. Hence substitution in the aryl rings in series 7 rather than 8 may lead to
potency increases over the substituted components. Further, the importance of the amidic groups
and their contributions towards the cytotoxicity of the dimers were suggested by comparing the
IC50 values of 8a and 9 against HCT116 and HT29 cells. Compound 9 was found to be a potent
cytotoxin compared to 5-FU and curcumin towards both cell lines. However, it displayed more
than 30-fold and 700-fold lower cytotoxic potencies in comparison to 8a against HCT116 and
HT29 cells, respectively. These observations suggest that the amidic carbonyl groups contribute
significantly to cytotoxic potencies. Therefore, further analog development of these classes of
compounds should retain the amidic groups.
In order to evaluate whether the magnitude of some of the physicochemical properties of
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the aryl substituents such as electronic (σ), hydrophobic(π), and steric properties (MR) influence
the cytotoxic potencies in series 7 and 8, linear and semilogarithmic plots were made between
these constants and the IC50 values of 7a-i and 8a-j in the HCT116 and HT29 screens.
Statistically significant negative correlations (p<0.05) were noted between σ and the IC50 values
of 7a-i and 8a-j in the HCT116 screen and a trend to a negative correlation (p=0.06) was noted
between the σ constants and the IC50 values of 7a-i in the HT29 bioassay. These results suggest
that in general further development of this class of compounds should include placing stronger
electron-withdrawing groups in the aryl rings. Such an approach will reduce the electron density
on the olefinic carbon atoms which will enhance electrophilic attack with cellular thiols.
However there are exceptions to this generalization. For example, analogs containing the 4-
fluoro (an electron-withdrawing atom) and 4-methoxy (an electron-repelling group) substituents
are at variance with this generalization in the HCT116 screen. Thus 7e and 7f are equipotent
while 8f is 10 times more potent than 8e revealing the need for further exploration in this area.
No correlations were noted between the π and MR constants of the aryl substituents and the IC50
values.
[PLEASE INSERT FIGURE 2]
A final issue to be investigated is the way in which these classes of compounds exert their
cytotoxicity. A number of anticancer agents exert their activity by inducing apoptotic cell death
in cancer cells. Compound 7a was shown to trigger apoptosis, not necrosis, in a number of T-cell
and B-cell lymphomas [24]. In order to examine whether the substituted dimers also induce
apoptotic cell death in colon cancer cells, HCT116 cells was treated with a potent representative
compound 7f using a concentration of 1µM. The results are presented in Figure 2. After 24 h,
34.9% apoptotic cell death was observed in the treated cells compared to 2.82% in untreated
cells. After 48 h, the percentage of apoptotic cells was increased slightly to 38.5%. This
observation suggests that apoptosis is one of the ways by which 7f displays cytotoxicity in
HCT116 colon cells. Due to its structural similarity to other analogs in series 7 and 8, it is
possible that these compounds also follow the same mode of action.
A number of cytotoxic agents are known to interfere at different phases of cell cycle
progression [27-30]. To evaluate this phenomenon, HCT116 and HT29 cells were treated with 1
µM concentration of 7f for 48 h and the effect of 7f on cell cycle was investigated using flow
cytotometry. Curcumin, a potent cytotoxic agent [31] that possesses some structural similarities
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with 7f and is also known to induce G2/M phase arrest in HCT116 [29] and HT29 cells [30] was
used as a positive control for this study. When compared to untreated cells, the treatment of 7f
did not display any significant changes in the cell population in the S or G2/M phases of the cell
cycle, either in HCT116 or in HT29 cells (data not given). These results suggest that 7f causes
cytotoxicity and apoptosis in HCT116 and HT29 cells via mechanisms other than cell cycle
arrest. In future, further modes of studies of the lead cytotoxic agent 7f are warranted to gain
some insight of the ways this class of compounds display cytotoxicity.
5. Conclusions
This study reveals that in general the dimers in series 7-9 are potent cytotoxic agents
towards HCT116 and HT29 colon cancer cells. In general, these molecules are substantially
more potent than either 5-FU or curcumin. Evidence was obtained which suggested that the
amidic groups in series 7 and 8 contributed to the cytotoxic potencies. In particular, 7a, e and 8a,
c, h emerged as lead molecules for further development. QSAR revealed that the placement of
strong electron withdrawing groups into the aryl rings may enhance cytotoxic potencies still
further. A lead representative compound 7f induced apoptosis in HCT116 cells. In conclusion,
this investigation has provided adequate evidence for initiating further studies of these novel
cytotoxins with a view to obtaining a clinical candidate. However structural modifications to
improve a variety of physicochemical properties will be required. For example, the clogP values
of 7a and 8a are 5.40 and 5.34, respectively [25]. Hence aryl substitution with hydrophilic
moieties, such as a glucosidic group on one or more of the aryl rings, should be undertaken.
6. Experimental protocols
6.1. Chemistry
Melting points were determined on a Gallenkamp instrument and are uncorrected. 1H and
13C NMR were recorded using a Bruker Avance 500 MHz spectrometer equipped with a 5mm
BBO probe. Chemical shifts (δ) are reported in ppm. Elemental analyses were undertaken using
1
13
an Elementer CHNS analyzer. The H and C NMR spectra of three representative compounds
namely 7b, 8b and 9 are presented as supplementary material.
6.1.1. Synthesis of 3,5-bis(arylidene)-4-piperidones (6a-j)
The synthesis of 6a-j was reported previously [18,19].
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6.1.2. General procedure for the synthesis of 3,5-bis(arylidene)-4-piperidone dimers (7)
Oxaloyl chloride (0.003 mol, 0.39 gm) in DCE (5 ml) was added dropwise to a stirred
suspension of a 3,5-bis(benzylidene)-4-piperidone 6 (0.006 mol) in DCE (20 ml) containing
triethylamine (0.006 mol, 0.61 gm) at ~20 oC for a period of 30 min. The reaction was stirred at
room temperature 8-12 h. The solvent was removed under reduced pressure at 45 oC. An aqueous
solution of potassium carbonate (25 ml, 5 % w/v) was added to the crude mass and stirred for 2
h. The solid obtained was filtered, dried, and crystallized from a suitable solvent to yield pure
products.
6.1.2.1. 1,2-bis[3,5-bis(Benzylidene)-4-oxo-piperidin-1-yl]ethane-1,2-dione (7a)
The synthesis of 7a was reported previously [24].
6.1.2.2. 1,2-bis[3,5-bis(4-Methylbenzylidene)-4-oxo-piperidin-1-yl]ethane-1,2-dione (7b)
o
1
Yield: 53%; mp (chloroform/methanol) 275 C; H NMR (500 MHz, CDCl3): δ 7.79(s, 2H,
2×=CH), 7.65 (s, 2H, 2×=CH), 7.29 (d, J=7.70 Hz, 8H, Ar-H), 7.18(q, 8H, Ar-H), 4.53 (d,
J=15.61 Hz, 8H, 4×NCH2), 2.44 (s, 6H, 2×CH3), 2.28 (s, 6H, 2×CH3). 13C NMR (125 MHz,
CDCl3): δ 185.04(CO), 162.73(CON), 140.31(C=C-Ph), 140.22(C=C-Ph), 139.15(C=C-Ph),
138.29(C=C-Ph), 131.57(Ar-C),131.07(Ar-C), 130.67(Ar-C), 130.33(Ar-C), 129.72(Ar-C),
129.62(Ar-C), 129.46(Ar-C), 129.00(Ar-C), 46.07(CH2NCO), 42.05(CH2NCO), 21.53(CH3),
21.43(CH3). Anal.calcd for C44H40N2O4.1.5 H2O: C 76.76; H 6.30; N 4.07 %, found: C 76.46; H
6.39; N 3.93%.
6.1.2.3. 1,2-bis[3,5-bis(4-Chlorobenzylidene)-4-oxo-piperidin-1-yl]ethane-1,2-dione (7c)
Yield: 61%; mp (methanol) 289 oC; 1H NMR (500 MHz, CDCl3): δ 7.72(s, 2H, 2×=CH), 7.54 (s,
2H, 2×=CH), 7.46 (d, J=8.40 Hz, 2H, Ar-H), 7.37(d, J=8.37 Hz, 2H, Ar-H ), 7.31(d, J=8.42 Hz,
4H, Ar-H), 7.20(d, J=8.38 Hz, 4H, Ar-H), 4.62 (s, 4H, 2×NCH2), 4.52 (s, 4H, 2×NCH2).
Anal.calcd for C40H28Cl4N2O4.3 H2O: C 60.26; H 3.54; N 3.51 %, found: C 60.35; H 3.70; N
3.15%.
6.1.2.4. 1,2-bis[3,5-bis(3,4-Dichlorobenzylidene)-4-oxo-piperidin-1-yl]ethane-1,2-dione (7d)
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o
1
Yield: 64%; mp (chloroform/methanol) 271 C; H NMR (500 MHz, CDCl3): δ 7.68 (s, 2H,
2×=CH), 7.55 (s, 1H, =CH), 7.53 (s, 1H, =CH), 7.46 (d, J=8.15 Hz, 6H, Ar-H), 7.41(d, J=1.71
Hz, 2H, Ar-H), 7.20 (dd, J=1.67Hz, J=1.71Hz, 2H, Ar-H), 7.05 (dd, J=1.71 Hz, J=1.77 Hz, 2H,
Ar-H), 4.67 (s, 4H, 2×NCH2), 4.56 (s, 4H, 2×NCH2). Anal.calcd for C40H24Cl8N2O4: C 54.58; H
2.75; N 3.18 %, found: C 54.52; H 2.90; N 2.95 %.
6.1.2.5. 1,2-bis[3,5-bis(4-Fluorobenzylidene)-4-oxo-piperidin-1-yl]ethane-1,2-dione (7e)
o
1
Yield: 67%; mp (chloroform/methanol) 253 C; H NMR (500 MHz, CDCl3): δ 7.73(s, 2H,
2×=CH), 7.57 (s, 2H, 2×=CH), 7.37(q, 4H, Ar-H), 7.25 (q, 4H, Ar-H), 7.18 (t, 4H, Ar-H),
7.098(t, 4H, Ar-H), 4.63 (s, 4H, 2×NCH2), 4.51 (s, 4H, 2×NCH2). Anal.calcd for
C41H26Cl8N2O4.H2O: C 53.93; H 3.09; N 3.07 %, found: C 53.84; H 2.98; N 2.74%.
6.1.2.6. 1,2-bis[3,5-bis(4-Methoxybenzylidene)-4-oxo-piperidin-1-yl]ethane-1,2-dione (7f)
o
1
Yield: 71%; mp (chloroform/methanol) >300 C; H NMR (500 MHz, CDCl3): δ 7.74(s, 2H,
2×=CH), 7.58 (s, 2H, 2×=CH), 7.35 (d, J=8.65 Hz, 4H, Ar-H), 7.22 (d, J=8.63 Hz, 4H, Ar-H),
6.98 (d, J=8.71 Hz, 4H, Ar-H), 6.91 (d, J=8.67 Hz, 4H, Ar-H), 4.60 (s, 4H, 2×NCH2), 4.53 (s,
4H, 2×NCH2), 3.90 (s, 6H, 2×OCH3), 3.76 (s, 6H, 2×OCH3). Anal.calcd for C44H40N2O8 : C
72.91; H 5.56; N 3.86 %, found: C 72.57; H 5.94; N 3.75%.
6.1.2.7. 1,2-bis[3,5-bis(3,4-Dimethoxybenzylidene)-4-oxo-piperidin-1-yl]ethane-1,2-dione (7g)
o
1
Yield: 60%; mp (chloroform/methanol) 282 C; H NMR (500 MHz, CDCl3): δ 7.71(s, 2H,
2×=CH), 7.42 (s, 2H, 2×=CH), 6.99 (d, J=8.47 Hz, 4H, Ar-H), 6.94(d, J=8.08 Hz, 2H, Ar-H),
6.80(m, 6H, Ar-H), 4.70 (s, 4H, 2×NCH2), 4.64 (s, 4H, 2×NCH2), 4.00 (s, 6H, 2×OCH3), 3.96 (d,
12H, 4×OCH3), 3.74 (s, 6H, 2×OCH3). Anal.calcd for C48H48N2O12.H2O: C 66.75; H 5.84; N
3.24 %, found: C 66.31; H 5.62; N 3.06%.
6.1.2.8.1,2-bis[3,5-bis(3,4,5-Trimethoxybenzylidene)-4-oxo-piperidin-1-yl]ethane-1,2-dione (7h)
o
1
Yield: 57%; mp (ethanol) 273 C; H NMR (500 MHz, CDCl3): δ 7.63(s, 2H, 2×=CH), 7.44 (s,
2H, 2×=CH), 6.66 (s, 4H, Ar-H), 6.49 (s, 4H, Ar-H), 4.75 (s, 4H, 4×NCH2), 4.68 (s, 4H,
4×NCH2), 3.96 (s, 12H, 4×OCH3), 3.94 (s, 9H, 3×OCH3), 3.88 (s, 9H, 3×OCH3), 3.84 (s, 6H,
2×OCH3). Anal.calcd for C52H56N2O16 : C 64.72; H 5.85; N 2.90 %, found: C 64.95; H 6.16; N
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2.78%.
6.1.2.9.1,2-bis[3,5-bis{4-(N,N-Dimethylamino)benzylidene}-4-oxo-piperidin-1-yl]ethane-1,2-
dione (7i)
o
1
Yield: 46%; mp (chloroform/methanol) >300 C; H NMR (500 MHz, CDCl3): δ 7.75(s, 2H,
2×=CH), 7.65 (s, 2H, 2×=CH), 7.33 (d, J=8.75 Hz, 4H, Ar-H), 7.21(d, J=8.71 Hz, 4H, Ar-H),
6.73 (d, J=16.93 Hz, 8H, Ar-H), 4.58 (s, 4H, 2×NCH2), 4.54 (s, 4H, 2×NCH2), 3.08 (s, 12H,
4×NCH3), 2.95 (s, 12H, 4×NCH3). Anal.calcd for C44H40N2O4.1.5 H2O: C 76.76; H 6.30; N 4.07
%, found: C 76.46; H 6.39; N 3.93%.
6.1.3. General procedure for the synthesis of 3,5-bis(arylidene)-4-piperidone dimers (8a-j)
Malonyl chloride (0.01 mol, 1.4 gm) in DCE (5 ml) was added dropwise to a stirred
suspension of a 3,5-bis(arylidene)-4-piperidone 6 (0.02 mol) in DCE (20 ml) containing
o
triethylamine (0.02 mol, 2.02 gm) at ~20 C for a period of 30 min. The reaction was stirred at
o
room temperature 8-12 h. The solvent was removed under reduced pressure at 45 C. An
aqueous solution of potassium carbonate (25 ml, 5% w/v) was added to the crude mass and
stirred for 2 h. The solid obtained was filtered, dried, and crystallized from a suitable solvent to
yield pure products.
6.1.3.1. 1,3-bis-[3,5-bis(Benzylidene)-4-oxo-piperidin-1yl]propane-1,3-dione (8a)
The synthesis of 8a was reported previously[24].
6.1.3.2. 1,3-bis-[3,5-bis(4-Methylbenzylidene)-4-oxo-piperidin-1yl]propane-1,3-dione (8b)
o
1
Yield: 43%; mp (chloroform/methanol) 251 C; H NMR (500 MHz, CDCl3): δ 7.81 (s, 2H,
2×=CH), 7.74 (s, 2H, 2×=CH), 7.40 (d, J=7.90 Hz, 4H, Ar-H), 7.27 (d, J=7.60 Hz, 4H, Ar-H),
7.21(q, 8H, Ar-H), 4.89 (s, 4H, 2×NCH2), 4.65 (s, 4H, 2×NCH2), 3.15 (s, 2H, CH2), 2.43 (s, 6H,
2×CH3), 2.41(s, 6H, 2×CH3). 13C NMR (125MHz, CDCl3): δ 186.39(CO), 165.30(CON),
140.31(C=C-Ph), 140.17(C=C-Ph), 138.62(C=C-Ph), 137.34(C=C-Ph), 131.86(Ar-C),
131.39(Ar-C), 130.88(Ar-C), 130.53(Ar-C), 130.45(Ar-C), 130.39(Ar-C), 130.33(Ar-C),
129.68(Ar-C), 129.61(Ar-C), 128.77(Ar-C), 46.79(CH2NCO), 43.85(CH2NCO), 40.54
(COCH2CO), 21.53(CH3). Anal.calcd for C45H42N2O4.H2O: C 77.94; H 6.40; N 4.04 %, found: C
12

ACCEPTED MANUSCRIPT
77.94; H 6.63; N 3.98%.
6.1.3.3. 1,3-bis-[3,5-bis(4-Chlorobenzylidene)-4-oxo-piperidin-1yl]propane-1,3-dione (8c)
o
1
Yield: 58%; mp (chloroform/methanol) 260 C; H NMR (500 MHz, DMSO-d6): δ 7.64 (s, 2H,
2×=CH), 7.60 (s, 2H, 2×=CH), 7.57 (d, J=11.46 Hz, 4H, Ar-H), 7.54 (d, J=7.60 Hz, 4H, Ar-H),
7.49 (q, 8H, Ar-H), 4.62 (d, 8H, J=10.15 Hz, 4×NCH2), 3.51 (s, 2H, CH2). Anal.calcd for
C41H30Cl4N2O4.2.5 H2O: C 61.38; H 3.77; N 3.49 %, found: C 61.10; H 3.69; N 3.30%.
6.1.3.4. 1,3-bis-[3,5-bis(3,4-Dichlorobenzylidene)-4-oxo-piperidin-1yl]propane-1,3-dione (8d)
o
1
Yield: 65%; mp (chloroform/methanol) 236 C; H NMR (500 MHz, CDCl3): δ 7.71 (s, 2H,
2×=CH), 7.63 (s, 2H, 2×=CH), 7.56 (q, 8H, Ar-H), 7.51 (d, J=8.29 Hz, 2H, Ar-H), 7.30 (d,
J=1.45 Hz, 2H, Ar-H), 7.31 (dd, J=1.45 Hz, J=1.53 Hz, Ar-H), 7.15 (dd, J=1.48 Hz, 2H, Ar-H),
4.78 (d, 8H, J=6.72 Hz, 4×NCH2), 3.30 (s, 2H, CH2). Anal.calcd for C41H26Cl8N2O4. H2O: C
53.93; H 3.09; N 3.07 %, found: C 53.84; H 2.98; N 2.74%.
6.1.3.5. 1,3-bis-[3,5-bis(4-Fluorobenzylidene)-4-oxo-piperidin-1yl]propane-1,3-dione (8e)
o
1
Yield: 56%; mp (chloroform/methanol) >300 C; H NMR (500 MHz, CDCl3): δ 7.81 (s, 2H,
2×=CH), 7.75 (s, 2H, 2×=CH), 7.49(q, 4H, Ar-H), 7.33 (q, 4H, Ar-H), 7.18 (t, 4H, Ar-H), 7.12
(t, 4H, Ar-H), 4.84 (s, 4H, 2×NCH2), 4.79 (s, 4H, 2×NCH2), 3.22 (s, 2H, CH2). Anal.calcd for
C41H30F4N2O4.H2O: C 69.42; H 4.27; N 3.95 %, found: C 69.58; H 4.30; N 3.71%.
6.1.3.6. 1,3-bis-[3,5-bis(4-Methoxybenzylidene)-4-oxo-piperidin-1yl]propane-1,3-dione (8f)
o
1
Yield: 61%; mp (chloroform/methanol) 245 C; H NMR (500 MHz, CDCl3): δ 7.80 (s, 2H,
2×=CH), 7.73 (s, 2H, 2×=CH), 7.48 (d, J=8.6 Hz, 4H, Ar-H), 7.29 (d, J=10.17 Hz, 2H, Ar-H),
6.98 (d, J=8.49 Hz, 4H, Ar-H), 4.85(s, 4H, 2×NCH2), 4.72 (s, 4H, 2×NCH2), 3.88 (d, J=7.72 Hz,
12H, 4×OCH3), 3.22 (s, 2H, CH2). Anal.calcd for C45H42N2O8.4 H2O: C 66.59; H 5.22; N 3.45
%, found: C 66.68; H 5.38; N 3.27%.
6.1.3.7. 1,3-bis-[3,5-bis(3,4-Dimethoxybenzylidene)-4-oxo-piperidin-1yl]propane-1,3-dione (8g)
Yield: 65 %; mp (chloroform/methanol) >300 oC; 1H NMR (500 MHz, DMSO-d6): δ 7.86 (s, 4H,
4×=CH), 7.17 (s, 4H, Ar-H), 7.13 (s, 8H, Ar-H), 4.54 (s, 8H, 4×NCH2), 3.84 (d, J=7.27 Hz,
13

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24H, 8×OCH3), 3.48 (s, 2H, CH2). Anal.calcd for C49H50N2O12.7 H2O: C 59.69; H 6.54; N 2.84
%, found: C 59.82; H 6.18; N 2.71%.
6.1.3.8. 1,3-bis-[3,5-bis(3,4,5-Trimethoxybenzylidene)-4-oxo-piperidin-1yl]propane-1,3-dione
(8h)
o
1
Yield: 53%; mp (chloroform/methanol) 115 C; H NMR (500 MHz, CDCl3): δ 7.78 (s, 2H,
2×=CH), 7.74 (s, 2H, 2×=CH), 6.72 (s, 4H, Ar-H), 6.58 (s, 4H, Ar-H), 4.96 (s, 4H, 2×NCH2),
4.90 (s, 4H, 2×NCH2), 3.92 (d, J=15.37 Hz, 30H, 10×OCH3), 3.86 (s, 6H, 2×OCH3), 3.37 (s, 2H,
CH2). Anal.calcd for C53H58N2O16.H2O: C 63.83; H 6.07; N 2.81 %, found: C 63.84; H 6.27; N
2.51%.
6.1.3.9. 1, 3-bis-[3, 5-bis{4-(N,N-Dimethylamino)benzylidene}-4-oxo-piperidin-1yl]propane-1,3-
dione (8i)
o
1
Yield: 42%; mp (chloroform/methanol) >300 C; H NMR (500 MHz, CDCl3): δ 7.78 (s, 4H,
4×=CH), 7.36 (d, J=8.64 Hz, 8H, Ar-H), 6.73 (d, J=8.70 Hz, 8H, Ar-H), 4.21(s, 8H, 4×NCH2),
3.05 (s, 26H, 8×NCH3 and CH2). Anal.calcd for C49H54N6O4.1.5 H2O: C 71.88; H 7.02; N 10.61
%, found: C 71.59; H 7.28; N 10.78 %.
6.1.3.10. 1,3-bis-[3,5-bis(4-Hydroxybenzylidene)-4-oxo-piperidin-1yl]propane-1,3-dione (8j)
o
1
Yield: 56%; mp (chloroform/ethanol) >300 C; H NMR (500 MHz, DMSO-d6): δ 7.64 (s, 2H,
2×=CH), 7.60 (s, 2H, 2×=CH), 7.57 (d, J=11.46 Hz, 4H, Ar-H), 7.54 (d, J=7.60 Hz, 4H, Ar-H),
7.49 (q, 8H, Ar-H), 4.62 (d, 8H, J=10.15 Hz, 4×NCH2), 3.51 (s, 2H, CH2). Anal.calcd for
C41H34N2O8.4.5 H2O: C 64.46; H 5.68; N 3.67 %, found: C 64.59; H 5.31; N 3.59%.
6.1.4. Synthesis of 1,3-bis-[3,5-bis(benzylidene)-4-oxo-piperidin-1yl]propane (9)
A mixture of 3,5-bis(benzylidene)-4-piperidone 6a (0.004 mol, 1 gm), 1,3-
dibromopropane (0.002 mol, 0.41 gm), potassium carbonate (0.002, 0.28 gm) and a catalytic
amount of potassium iodide (10 mg) in acetonitrile (30 ml) was heated at reflux temperature for
6-7 h. Further quantities of 1,3-dibromopropane (0.001 mol, 0.2 gm) were added and the reaction
continued for another 3-4 h. The completion of the reaction was monitored by TLC (solvent:
chloroform-methanol 98:2). After the reaction was complete, the solvent was removed under
o
reduced pressure at 45 C. An aqueous solution of potassium carbonate (25 ml, 10% w/v) was
14

ACCEPTED MANUSCRIPT
added to the crude mass and stirred for 2 h. The solid obtained was filtered, dried, and
o
1
crystallized from acetone to yield pure product. Yield: 63 %; mp (acetone) 135 C; H NMR
(500 MHz, CDCl3): δ 7.81(s, 4H, 4×=CH), 7.39 (m, 20H, Ar-H), 3.78 (s, 8H, 4×NCH2), 2.54 (t,
4H, 2×CH2), 1.60 (p, 2H, CH2). 13C NMR (125 MHz, CDCl3): δ 187.34 (CO), 136.57 (C=C-Ph),
135.20 (C=C-Ph), 133.11 (C=C-Ph), 130.40 (Ar-C), 129.05 (Ar-C), 128.60(Ar-C), 55.38
(NCH2CH2), 54.97 (NCH2-piperidone), 25.91 (CH2). Anal.calcd for C41H38N2O2: C 83.36; H
6.48; N 4.74 %, found: C 83.74; H 6.79; N 5.05%.
6.2. Biology
6.2.1. Cell culture
Phosphate buffered saline (PBS), pH 7.8, versene, and trypsin (2.5%) was obtained from
Invitrogen Inc. (Burlington, ON, Canada). Fetal bovine serum (FBS) was purchased from Fisher
Scientific (Toronto, ON, Canada). McCoy’s 5A medium (ATCC cat No: 30-2007) was
purchased from the ATCC (American Type Culture Collection, Rockville, USA). The penicillin-
streptomycin antibiotic solution and all other chemicals unless otherwise indicated were
purchased from Sigma (Oakville, ON, Canada). MilliQ water was obtained from a MilliQ water
purification system (Millipore, MA, USA). Dr. Keith Bonham, Saskatoon Cancer Center,
Saskatoon kindly gifted the HCT116 and HT29 colon cancer cell lines. The HCT116 and HT29
cell lines were subcultured in McCoy’s 5A medium (ATCC cat No: 30-2007) supplemented with
10% fetal bovine serum and 1% penicillin-streptomycin antibiotic solution. The cells were
grown in an atmosphere of 95% O2 and 5% CO2 with 95% humidity.
6.2.2. Cytotoxicity assay
The cytotoxicity of the compounds 7-9, 5-fluorouracil and curcumin were determined using the
sulforhodamine B assay as reported previously [32,33]. The cells were grown in McCoy’s 5A
medium supplemented with 10% FBS. The cells were harvested using 0.25 % trypsin in versene
and the cell count was determined using the trypan blue exclusion method. In a 96-well plate,
about 5×103 cells in 100 ꢁl of complete media were plated per well and incubated for 24 h. The
test compounds were serially diluted in DMSO and added to media for a final concentration of
1% DMSO. From this solution an aliquot of 100 µl was added to each well and incubated for 48
h. The cells were fixed by adding 50 ꢁl of 50% w/v aqueous trichloroacetic acid to each well and
15

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incubated at 4°C for 1 h. To obtain the cell numbers at zero time point (Tz), a plate was fixed at
the time of treatment of the cells. The plates were rinsed with tap water four times and air dried.
The fixed cells were stained with 0.4% w/v sulforhodamine in 1% v/v acetic acid for 10 min and
washed four times with 1% v/v acetic acid and air dried. The dye was re-dissolved in 200 ꢁl of
10 mM Trizma base and the absorbance read at 515 nm. The percent growth was calculated as
per the equation below. The IC50 values were derived by fitting a four-parameter curve into the
percent cell growth versus log concentration data using GraphPad Prism 5.0 for Windows
(GraphPad Software, San Diego, California, USA).
6.2.3. Apoptosis assay
Apoptosis assay was conducted using Annexin V-FITC apoptosis detection kit (Biovision;
Catalogue No: ALX-850-250-KI02) as per the suggested protocol. HCT116 cells were grown in
McCoys’5A Modified media supplemented with 10% FBS and 1% antibiotic-antimycotic
solution in a humidified incubator at 37°C with 5% CO2. About 1.5×103 cells per well were
o
seeded in 6 well plates and incubated for 24 h at 37 C following which the cells were treated
with 1µM conc. of 7f in media with a final conc. of 1% DMSO. Apoptotic cell death was
examined at different time points 1, 5, 11, 24 and 48 h and compared against untreated cells. The
cells were collected by trypsinization (0.2% Trypsin-EDTA) followed by centrifugation for 5
min at 1000g at 4 °C. The pellet was washed with 1ml of 1X PBS, resuspended in 500
microlitres of 1X binding buffer and treated with 5 µl of Annexin V-FITC (fluorescein
isothiocyanate labeled annexin V) and 5 µl of propidium iodide. After incubating the samples in
the dark for 5 min at room temperature, apoptotic cell death was detected by a fluorescence-
activated cell sorter (FACS) and analyzed by CellQuest (Becton-Dickinson) software.
6.2.4. Cell cycle analysis
Cell cycle analysis was carried out by modifying a literature procedure [34]. HCT116 and HT29
cells were maintained in McCoys’5A Modified media supplemented with 10 % FBS and 1%
antibiotic-antimycotic solution. About 1×106 cells were seeded in 75 cm2 flasks and incubated
for 24 h in a humidified incubator at 37 °C with 5% CO2. The cells were treated with 1µM of 7f
in media having 1% DMSO. The treated cells and untreated control were incubated in a
16

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humidified incubator for 48 h at 37° C with 5 % CO2. Floating cells were collected and adherent
cells were harvested with trypsin-EDTA (0.2%) and pooled. The samples were washed with cold
PBS, fixed in 70 % ethanol and left on ice for 2 h. Further samples were washed after 2 h with
PBS and resuspended in PBS containing RNase (300 ꢁg/ml). The samples were incubated for 20
min in the dark with propidium iodide (20 ꢁg/ml) and RNase (300 ꢁg/ml). The samples were
analyzed by a FACScalibur (BD) flow cytotmeter and data were analyzed using Modfit LT free
trial version 3.3 available from Variety software house. Cells were gated to include G0/G1, S-
phase, and G2/M populations.
Acknowledgements
This study was funded by a CIHR-RPP Saskatchewan grant to J. R Dimmock and U. Das.
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Captions for the figures and schemes
Figure 1. Structures of the compounds in series 1-5.
Figure 2. Analysis of the effect of 7f on HCT116 cells after incubation for 24 h and 48 h by flow
cytometry. The experiments were carried out in duplicate and the % of live, dead and apoptotic
cells are the average values of two independent experiments.
Scheme 1. Syntheses of the compounds in series 7-9. i = acetic acid/dry HCl; ii = oxalyl
chloride (for 7a-i) and malonyl chloride (for 8a-j); iii = 1,3-dibromopropane/K2CO3/KI. The
aryl substituents are presented in Table 1.
20

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Table 1: Evaluation of 7a-i, 8a-j and 9 against HCT116 and HT29 colon cancer cell lines.
Compound
Aryl substituents
IC50 (µM)
HCT116 HT29
RPa
R1
H
H
H
H
H
H
H
R2
H
R3
H
H
H
Cl
H
HCT116 HT29
0.04±0.03
0.09±0.02
0.03±0.01
0.014±0.002 113
0.129±0.035 50
0.78±0.13
454
49
8
0.13
212
37
11
24
0.34
2117
32
71
1.1
35
8
20
159
<1
14
3
1.00
0.48
7a
7b
7c
7d
7e
7f
7g
7h
7i
8a
8b
8c
8d
8e
8f
8g
8h
CH3
Cl
Cl
151
20.05±1.01 49.46±4.50 0.23
F
0.02±0.01
0.03±0.001 226
0.17±0.04 226
0.568±0.265 151
0.27±0.15 17
27.37±3.68 18.56±2.30 0.17
OCH3
H
0.02±0.01
OCH3 OCH3 0.03±0.01
OCH3 OCH3 OCH3 0.27±0.06
H
H
H
H
H
H
H
H
N(CH3)2
H
H
H
H
H
Cl
H
H
0.02±0.01
0.41±0.25
0.003±0.002 226
0.20±0.02
CH3
Cl
Cl
11
90
0.17
8
75
25
226
<1
6
0.05±0.001 0.09±0.05
26.73±2.35 6.05±0.10
F
0.60±0.01
0.06±0.01
0.18±0.08
0.77±0.05
0.31±0.12
OCH3
OCH3 OCH3 0.18±0.07
OCH3 OCH3 OCH3 0.02±0.003 0.04±0.02
H
H
-
-
-
N(CH3)2
H
H
-
-
-
>25
>25
8i
8j
9
OH
0.72±0.10
0.68±0.19
4.52±0.54
0.46±0.21
2.15±0.65
6.35±1.12
-
-
-
7
1.00
5-FU
Curcumin
11.54±4.45 13.20±2.08 0.39
aThe relative potency (RP) of the compounds against a reference drug, 5-fluorouracil was
calculated by dividing the IC50 value of 5-fluorouracil by the IC50 value of the compound against
a particular cell line. Only the mean IC50 values were considered for calculating RP values.
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Table 2. Comparisons between some of the potencies of the compounds in series 7 and 8.
a
b
c
Aryl
∆
8/7
∆
7b-i/7a
∆
8b-i/8a
Series 7,
substituent
HCT116
HT29
HCT116
HT29
HCT116
HT29
8
a
b
c
d
e
f
g
h
i
H
4-CH3
4-Cl
3,4-Cl2
4-F
0.50
4.56
1.67
1.33
30.0
3.00
6.00
0.07
>0.19
0.21
1.55
0.12
0.12
6.00
4.53
0.54
0.15
>1.35
---
---
---
20.5
2.50
1337
30.0
3.00
9.00
1.00
>1250
---
66.7
30.0
2017
60.0
257
103
13.3
>8333
2.25
0.75
501
0.50
0.50
0.75
6.75
684
9.21
55.7
353
2.14
12.1
40.7
19.3
1326
4-OCH3
3,4-(OCH3)2
3,4,5-(OCH3)3
4-N(CH3)2
aThe ∆8/7 values are the quotients of the IC50 values in series 8 divided by the analog in series 7
which has the same aryl substituent.
bThe ∆7b-i/7a values are the quotients of the IC50 values of 7b-i divided by the figure for the
unsubstituted compound 7a.
cThe ∆8b-i/8a values are the quotients of the IC50 values of 8b-i divided by the figure of the
unsubstituted compound 8a.
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O
X
O
O
R1
R2
R1
R2
H3CO
HO
OCH3
OH
1
2
X=CH2, NH, NCH3
O
N
O
N
H
O
R
3
4
O
a: R=COOH; b : R= H
1,5ꢂdiarylꢂ3ꢂoxoꢂ1,4ꢂpentadienyl
pharmacophore
(primary alkylating site)
N
O
1,5ꢂdiarylꢂ3ꢂoxoꢂ1,4ꢂpentadienyl
pharmacophore
O
X
(secondary alkylating site)
N
O
5
X= (CH2)n, ꢂCH=CHꢂ , ꢂC=Cꢂ
y
n= 0,1, 2,3,6
y=1,2
Figure 1
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Figure 2
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Scheme 1
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Highlights
-
-
-
-
-
Novel 3,5-bis(benzylidene)-4-piperidones were developed as cytotoxic agents
The compounds were effective against HCT116 and HT29 colon cancer cells
The IC50 values of most of the compounds are in the 10-7 to 10-9 M range
QSAR studies were undertaken
-A lead compound demonstrated apoptosis in HCT116 cells

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SUPPLIMENTARY MATERIALS
Novel 3,5-bis(arylidene)-4-piperidone dimers: Potent cytotoxins against colon cancer cells
Swagatika Das, Umashankar Das*, Deborah Michel, Dennis K. J. Gorecki, Jonathan R.
Dimmock*
1H and 13C NMR spectra for representative compounds

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