Bioorganic & Medicinal Chemistry 23 (2015) 411–421

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Investigation of fatty acid conjugates of 3,5-bisarylmethylene-

4-piperidone derivatives as antitumor agents and human
topoisomerase-IIa inhibitors
Elizabeth Potter a, Mamta Jha a, Khushwant S. Bhullar b, H. P. Vasantha Rupasinghe b, Jan Balzarini c,
Amitabh Jha a,⇑
a
Department of Chemistry, Acadia University, Wolfville, Nova Scotia B4P 2R6, Canada
b
Department of Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, Nova Scotia B2N 5E3, Canada
c
Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: A series of five 3,5-bisarylidene-4-piperidones designed as analogs of curcumin and their twenty five fatty
Received 22 September 2014 acid conjugates were synthesized as candidate anticancer agents. The fatty acid conjugates were designed
Revised 8 December 2014 for efficient delivery of these compounds at the targeted cancer sites. The cytostatic potential of these com-
Accepted 17 December 2014
pounds was evaluated against three representative cancer cell lines namely murine leukemic L1210 cells,
Available online 26 December 2014
and human T-lymphocyte CEM cells and cervical HeLa cells. Most compounds were found to exhibit signif-
icant anti-cancer activity in vitro. QSAR studies indicated electrophilicity of these compounds towards cel-
Keywords:
lular nucleophiles may have a key role to play in their cytostatic activity. Representative compounds were
Curcumin analogs
Fatty acid amides
also tested for topoisomerase IIa inhibitory potential, which indicated strong catalytic inhibition of the
3,5-Bisarylmethylene-4-piperidones enzyme in vitro. The data showed that the fatty acid conjugates also possessed robust antioxidant activity
Michael acceptor in multiple analyses. This study also indicated that these compounds prompted significantly lower cellular
Anti-oxidant damage in human fibroblasts than a currently used cancer drug sorafenib in vitro. The wide spectrum of
Cytostatic activity anticancer action, supplemented with antioxidant potential along with non-toxic manifestations, certainly
Topoisomerase IIa inhibitors augment the anticancer candidacy of the novel fatty acid conjugates.
QSAR Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Our research group is actively working on making analogs of

Curcumin (Fig. 1) is a dietary phytochemical isolated from Cur-
cuma longa, commonly known as turmeric, a widely used spice in
South East Asia.1 Biological activities of this compound have been
a subject of intensive research worldwide and over the years a
number of its pharmacological characteristics such as antiinflam-
matory, antitumor, antiangiogentic, antioxidant, antibacterial, car-
dioprotective and neuroprotective properties have been
unravelled.2–6 Curcumin is being/has been subjected to a large
number of clinical trials,7 including for the treatment of metastatic
breast cancer,8 colon cancer,9 advanced pancreatic cancer,10 cystic
fibrosis,11 inflammatory bowel disease,12 and Alzheimer’s dis-
ease.13 As expected from a commonly used dietary phytochemical,
curcumin displays excellent safety profile but its clinical efficacy is
marred with it poor bioavailability which has kept this molecule
from culminating into a successful clinical drug.14
⇑ Corresponding author. Tel.: +1 902 585 1515; fax: +1 902 585 1414.
E-mail address: ajha@acadiau.ca (A. Jha).
curcumin in attempts to develop molecules with improved phar-
macokinetic as well as pharmacodynamic profile than those of cur-
cumin.15–27 We previously prepared and studied curcumin analogs
where the diarylheptanoid framework of curcumin was retained
(series 1–3, Fig. 1).15–18 These studies involved anticancer cyto-
static, anti-melanoma, antioxidant, anti-hypertensive, and/or
anti-HIV properties of these compounds. Overall, several com-
pounds from series 1 and 2, which retained most of the structural
features of curcumin were found to be most active in terms of their
anticancer and antioxidant potency in vitro. We have also worked
considerably on simplified diarylpentanoid analogs of curcumin,
viz. 3,5-bisarylmethylene-4-piperidones, where the central keto-
enol moiety was replaced by a ketone functionality (series 4–
10).19–24 Curcumin as well as its various series of analogs (1–10)
contain enone functional group(s) and therefore are Michael
acceptors with selective affinity for cellular thiols as opposed to
nucleophiles found on genetic materials.25,28 It should be noted
that analogs from series 5–10 contain side chains bearing addi-
tional enone functionality for interaction with auxiliary binding

http://dx.doi.org/10.1016/j.bmc.2014.12.042
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
412 E. Potter et al. / Bioorg. Med. Chem. 23 (2015) 411–421

O OH O OH
O O
R R
HO Curcumin OH
n
Series 1, n=0
Series 2, n=1
O O

R
R N R
H
Series 3 Series 4
O O

R N R N

O O
5 R'=H
6 E, R' = Ar R' R'
7 E, R' = CONHAr
8 Z, R' = CONHAr 10
9 Z, R' = CONH-amino acid esters O

Figure 1. Molecular structures of curcumin and its previously studied analogs.

domain20 and/or protein thiolation.25,28 The parent 3,5-bisarylm-
ethylene-4-piperidones 4 and there derivatives (5–10) (Fig. 1)
were consistently found to possess higher level of antiproliferative
activity than diarylheptanoids 1–3. Of particular relevance to this
report are series 9 compounds where micronutrient amino acids
were conjugated to the parent 3,5-bisarylmethylene-4-piperidones
via maleamic diamide moiety to selectively target cancer cells
where the constant cell division leads to increased requirement
of cellular micronutrients. Most of these compounds displayed
cytotoxicity in sub-micromolar inhibitory concentrations against
cancer cell lines tested and were identified as potent human topo-
isomerase IIa inhibitors.22
As stated, human topoisomerase IIa has been identified as a
potential molecular target for 3,5-bisarylmethylene-4-piperidone
derivatives in a previous investigation in our laboratory.22 This
sulfhydryl-rich enzyme is essential for cell proliferation and sur-
vival.29–31 It releases torsional strain in DNA double helix through
transient double stranded nicks facilitating segregation of the
strands, allowing replication and transcription processes to
occur.29,30,32–34 Inhibition of these processes brings replication
and transcription to halt, thus impeding cell growth. Alternatively,
if topoisomerase poisoning occurs, the breaks in the DNA strands
created by the enzyme cannot ligate back resulting in lethal lesions
in the DNA frame work, which then leads to apoptosis.29,30 Since
the expression of topoisomerase IIa is heightened in rapidly divid-
ing cancerous cells, targeting this enzyme by protein-thiolators
offers a crucial pathway of selectivity targeting malignant cells to
inhibit cancer growth.
In the present investigation, we aimed to design and develop
fatty acid conjugates of the established pharmacophore 3,5-bisa-
rylmethylene-4-piperidones (Fig. 2) as therapeutic agents with
dual mode of action: (a) protein thiolation by the enone function-
alities, and (b) targeted delivery at the cancer site. The conjugation
of an essential fatty acid with the 3,5-bisarylmethylene-4-piperi-
done core is hypothesized to both increase site specificity and
the ability of the compounds to penetrate the extracellular mem-
brane. This may increase the bioavailability of the drug resulting
in lower clinical dose requirements. Fatty acids are known to be
taken up more rapidly by tumour cell, as they are vital in the syn-
thesis of biochemical precursors, as well as an energy source for
rapid biosynthesis.35–37 Furthermore, fatty acids are also readily
incorporated into the lipid bilayer, thus disrupting the fluidity

Ar = R= H 1
A
O
2
O O
B
EtO2 C Ar Ar 3
O 9
N
C 4
N R 11
O

N A1-A7 5
B1, B3, B6, B7 13
D C1, C3-C7 O
D1, D3-D7 6
E1-E7 15
O
E 7
O
6 6

Figure 2. Novel amides of 3,5-bisarylmethylene-4-piperidones synthesised and investigated.

 E. Potter et al. / Bioorg. Med. Chem. 23 (2015) 411–421
and structure of the cancer cell membrane.35 It is suggested that
this results in improved chemosensitivity facilitating antitumor
targeting. Conjugates of paclitaxel, an antitumor agent, with essen-
tial polyunsaturated fatty acids led to the development of Taxopr-
exinÒ, the conjugate formed with docosahexaenoic acid.35–37 This
conjugation leads to an increase in antitumor activity, as well as
a decrease in toxicity relative to the parent molecule, paclitaxel.35
Butyric, lauric, myristic, palmitic, stearic and oleic acids were cho-
sen to represent fatty acids (Fig. 2). Butyric acid is a short chain
fatty acid known to have strong anticancer properties.38,39
The aryl rings in the designed compounds are represented by
phenyl, 4-carboethoxyphenyl, 2-furyl, 2-pyridyl and 4-pyridyl
rings. The rationales behind the selection of these aryl group are
as follows: (a) In terms of substitution, the unsubstituted phenyl
ring offers the reference point; (b) since protein thiolation is being
considered as the primary mode of action, electron withdrawing
groups on the phenyl ring are expected increase the electrophilic-
ity of dienone functionality; carboethoxy group at position 4 of the
phenyl was introduced as an electron withdrawing group; (c) 2-
furyl group, which happens to be a polar electron-rich heteroaro-
matic ring, was chosen to understand its effect on bioactivity due
to reduced electrophilicity of the dienone functionality and altered
solubility; and (d) the selection of 2-pyridyl and 4-pyridyl aromatic
substituents was based on their ability to form salts, increasing the
solubility of the compound and facilitating drug delivery. Also, the
pyridyl rings are electron-deficient and therefore make the die-
none system more electrophilic towards protein thiolation. This
coupled with the rigidity imparted to the system by the 4-piperi-
done ring appear to be essential in achieving desirable antitumor
properties.40 The compounds in Figure 2 may be viewed as pro-
drugs of 3,5-bisarylmethylene-4-piperidone molecules but they
can also be viewed as active compounds in their own right as they
bear the pharmacophore required for the activity. Also, as these
compounds are structurally divergent from known human topoiso-
merase-IIa inhibitors currently available, their development may
be of value in treating drug-resistant tumours.
2. Results and discussion
2.1. Chemistry
The synthetic strategy followed to synthesize five 3,5-bisaryl-
idenepiperidones and their twenty five fatty acid amides is shown
in Scheme 1. Aldol condensations between 2 equiv of aromatic/
heteroaromatic aldehydes with 4-piperidone hydrochloride
hydrate were carried out according to literature procedures under
O O
AcOH
H HCl (g)
Ar
Ar
O N N N
2 eq. H H
.H 2O .HCl A1 .HCl (Ar=Phenyl)
Ar = Phenyl B1 .AcOH (Ar=4-(CO2 Et)Ph)
C1 .3HCl (Ar=4-Pyridyl)
4-(CO2 Et)Ph
D1 (Ar=2-Pyridyl)
4-Pyridyl E1 (Ar=2-Furyl)
O Cl(CH 2) 2Cl, SOCl2 O A1-E1, Cl(CH 2) 2Cl
R OH 0o C to reflux, 3h R Cl Et3N (2-5 eq), RT, 17 h
Scheme 1. Synthetic strategy followed to synthesize 3,5-bisarylmethylene-4-piperidone fatty acid amides in the present investigation. Please refer to Figure 2 for the
structure of various R-groups.
413
acidic41 or basic conditions42 and the products were isolated as
acid salts or free bases, respectively. The subsequent conversion
to fatty acid amides were carried out by converting the fatty acids
to corresponding acid chlorides and their conjugation with 3,5-bis-
arylidenepiperidones in presence of appropriate amount of trieth-
ylamine (Scheme 1). All fatty acid amides were purified by column
chromatography and were conclusively characterized by spectro-
scopic means. It was interesting to note that the fatty acid conju-
gates acquired non-symmetrical conformation in the 3,5-
bisarylidenepiperidone moiety as evident from the peaks in the
NMR spectra. All compounds, except A143 and C1,44 studied in this
investigation are new to chemical literature.
2.2. Biological evaluation
2.2.1. Cytostatic activity
All compounds synthesized under this investigation were eval-
uated for cytostatic activity against murine L1210 leukemia cells,
as well as human T-lymphocyte CEM and HeLa cells, following a
literature procedure.45 The choice of cell lines was determined on
the basis that the murine cell lines such as L1210 have been
claimed to be good predictors of clinical anticancer agents46 while
activity towards the T-lymphocyte CEM and HeLa cells would indi-
cate a capacity to inhibit the growth of human tumours. The results
are presented in Table 1.
Some general observations with regards to the structure and the
cytostatic activity will be made first. As evident from Table 1, nearly
50% of the compounds tested displayed high cytostatic activity
against three representative cancer cell lines with IC50 values in
low micromolar or submicromolar range. This provides strong evi-
dence that these molecules are potent anti-tumour agents as well
as promising lead molecules for further development. Precursor
3,5-bisarylmethylene-4-piperidones A1–D1 showed potent cyto-
static activity against each of the three cell lines and, on an average,
were found to be more potent than the reference drugs melphalan
and curcumin in vitro. In general, the majority of the fatty acid con-
jugates of series A–D were found to be significantly cytostatic with
no or small loss in magnitude of the activity with increasing chain
length. Interestingly, conjugation of stearic acid (A6–E6) consis-
tently led to significant or complete loss of activity in each series
of molecules tested. However, conjugation with the corresponding
monounsaturated oleic acid (A7–E7), led to restoration of the cyto-
static activity.
Compound E1 and its fatty acid amides (E2–E7) were found to be
inactive or moderately active in the cytostatic assays. Interestingly,
the precursor E1 was completely inactive (IC50 >250 lM against
O
10% Aq KOH
H
Ar EtOH
Ar
O
H 2 eq.
.H 2O .HCl
Ar = 2-Pyridyl
2-Furyl
O
Ar Ar
A2-A7
N
B3, B6, B7
O R C3-C7
D3-D7
E2-E7
414 E. Potter et al. / Bioorg. Med. Chem. 23 (2015) 411–421

Table 1 each of the three cell lines) but five of its fatty acid conjugates (E2–
Evaluation of cytostatic potential of compounds of series A–D against human CEM T- E5, E7) were found to be moderately active (average IC50 51 lM) in
lymphocytes, HeLa and murine L1210 leukemic cells
the same assay system.
Compound IC50 (lM) SRa Poor cytostatic activity of compounds of series E was expected
CEM HeLa L1210 due to electron-rich nature of furan ring which renders the corre-
A1 0.9 ± 0.0 2.6 ± 0.7 7.0 ± 2.3 7.7
sponding bisarylmethylene-4-piperidone Michael acceptor system
A2 3.7 ± 2.3 3.9 ± 0.6 14.0 ± 7.0 3.8 much less electrophilic. This in turn adversely impacts the protein
A3 5.7 ± 0.7 2.6 ± 1.8 5.6 ± 0.2 2.2 thiolating capacity of these compounds retarding anticancer
A4 7.2 ± 3.4 4.8 ± 0.4 18.0 ± 6.0 3.8 effect by this mechanism. We have consistently observed this in
A5 140.0 ± 43.0 32.0 ± 6.0 109.0 ± 4.0 4.4
past19–25,27,28,47,48 and have confirmed the thiolation capacity of
A6 >250 >250 >250 —
A7 70.0 ± 47.0 25.0 ± 3.0 58.0 ± 25.0 2.8 the enone system.25,28 By the same token, series B–D were
B1 1.0 ± 0.7 0.8 ± 0.0 6.9 ± 0.5 8.7 expected to show superior cytostatic activity than series A (or
B3 13.0 ± 1.0 12.0 ± 1.0 3.5 ± 2.1 3.7 E). Series B–D bear electron deficient aromatic rings which make
B6 >125 >125 >125 —
the corresponding dienone systems more thiophilic. The manifes-
B7 29.0 ± 9.0 26.0 ± 2.0 29.0 ± 2.0 1.1
C1 1.7 ± 0.0 0.8 ± 0.1 8.9 ± 0.4 10.9
tation of this characteristic in the cytostatic activities of the cor-
C3 1.1 ± 0.3 2.6 ± 1.8 2.8 ± 0.7 2.5 responding analogs (barring the stearic acid conjugates, vide
C4 1.2 ± 0.0 0.9 ± 0.3 3.0 ± 0.3 3.3 supra) was indeed observed (Table 1). This effect was dramatic
C5 2.1 ± 1.0 3.0 ± 0.5 7.0 ± 1.1 3.3 in series C and D where nearly all compounds were found to pos-
C6 9.7 ± 6.3 2.2 ± 1.8 14.0 ± 9.0 6.4
sess potent cytostatic activity with low or sub-micromolar IC50
C7 8.5 ± 4.2 4.7 ± 0.2 6.6 ± 2.2 1.8
D1 1.9 ± 0.2 0.2 ± 0.0 1.0 ± 0.7 11.2 values against each of the three cell lines tested. All compounds
D3 2.5 ± 1.6 0.9 ± 0.1 2.8 ± 0.4 3.1 of series C were found to be more potent than curcumin while
D4 0.9 ± 0.1 0.7 ± 0.0 0.6 ± 0.0 1.5 most of the series D analogs (D1, D3–D5) were more potent than
D5 1.4 ± 0.6 0.7 ± 0.0 0.9 ± 0.1 2.0 both melphalan and curcumin in vitro.
D6 63.0 ± 5.6 100.0 ± 14.0 26.0 ± 6.0 3.9
D7 6.2 ± 0.6 15.0 ± 7.0 7.6 ± 5.1 2.4
Analysis of selective toxicity of these compounds towards the
E1 >500 >250 >250 — cell lines tested revealed moderate selectivity in the parent bisa-
E2 47.0 ± 40.0 95.0 ± 19.0 65.0 ± 40.0 2.0 rylmethylene-4-piperidones A1–E1 (SR range 4.7–11.2, Table 1);
E3 19.0 ± 9.0 84.0 ± 21.0 21.0 ± 2.0 4.4 the fatty acid conjugate, albeit potent, generally showed poor
E4 24.0 ± 7.0 71.0 ± 2.0 26.0 ± 10.0 3.0
selectivity (SR range 1.1–6.4, Table 1).
E5 26.0 ± 1.0 71.0 ± 35.0 34.0 ± 20.0 2.7
E6 >250 >250 >250 —
E7 53.0 ± 8.0 74.0 ± 35.0 62.0 ± 20.0 1.4 2.2.1.1. Quantitative structure–activity relationship stud-
Melphalan 2.6 ± 1.2 2.7 ± 2.4 5.8 ± 0.3 2.2 ies. In an attempt to investigate the effect of structural and
Curcumin 15.0 ± 5.0 18.0 ± 3.0 22.0 ± 1.0 1.5 electronic parameters on the cytostatic activity of the five series
The SR values in bold highlight compounds that displayed significant selective of compounds investigated, a quantitative structure–activity rela-
cytostatic activity against the tested cell lines. tionship (QSAR) study was undertaken using certain physicochem-
a
SR indicates the selectivity ratio, i.e., the ratio between the highest and lowest ical parameters of the bioactivity. The extent to which the
IC50 values of either the T-lymphocytes or murine leukemic cells.
electrophilicity of the dienone system affected the bioactivity
was evaluated by comparing the electronic charge at the b-position
Table 2 (calculated using a commercial software)49 of the symmetrical
Correlation constants between IC50 values of compounds under investigation and
enone structure with the IC50 values against the three cell lines.
their physicochemical parameters
Likewise, the calculated partition coefficient (log P) and molar
Independent Dependent Type Correlation coefficient ra p valueb refractivity (MR) values of the test compounds were also obtained
Charge b L1210 IC50 Semi-Log 0.479 0.0077 using a commercial software46 and utilized as a means of assessing
CEM IC50 Semi-Log 0.473 0.0084 the effect of the lipophilicity and steric properties of the com-
HeLa IC50 Semi-Log 0.638 0.0003 pounds with respect to their bioactivity. Linear and semilogarith-
a
The magnitude of coefficient r shows the extent (the closer the values to 1, the mic correlations were calculated using statistical analysis50 for
better) and the nature (sign positive or negative) of the correlation. the IC50 of each tumor cell line and the aforementioned physico-
b
The value of p <0.01 suggest significant correlation. chemical parameters. Only the significant correlations obtained
are included in Table 3.
Table 3 No significant correlation (p <0.05) was observed between log P
Antioxidant capacity measured by FRAP, ORAC and DPPH radical assays for 3,5-
and molar refractivities of the molecule (MR) with any of the bio-
bisarylidene-4-piperidones and curcumin
logical activities. Negative and significant correlations (p <0.05)
Compound FRAP ORAC DPPH ROS between charge b and antiproliferative activity against each of
(lmol TE/L) (lmol TE/L) (% inhibition) (% inhibition)
the three cell lines indicated that an increase in charge b led to a
A1 34.3 ± 2.2h 92.77 ± 1.9f 52.5 ± 2.6e 63.6 ± 2.5e decrease in the inhibitory concentration (desirable) on the cell
A3 91.1 ± 6.0g 288.4 ± 7.2b 83.5 ± 1.1b 84.6 ± 1.2b,c lines assayed. This was anticipated (vide supra) as the molecules
B1 160.8 ± 2.9e 67.07 ± 5.8f 68.6 ± 1.8c,d 73.9 ± 5.9d
B3 206.9 ± 18.8d 256.5 ± 2.7c 85.7 ± 1.1b 84.0 ± 1.9b,c
were expected to exert their cytostatic effect via protein thiolation.
C1 28.0 ± 2.7h 204.6 ± 16.5d 67.5 ± 1.6c,d 59.9 ± 3.6e This result also suggest that analogs with higher charge b than the
C4 127.0 ± 2.6f 170.2 ± 3.1e 83.5 ± 1.0b 86.4 ± 2.1b,c current compounds (more electrophilic) should be made and
D1 209.2 ± 9.2d 88.1 ± 5.6f 73.2 ± 1.2c 57.9 ± 1.1e tested to fine-tune the anticancer potential of these molecules.
D4 651.9 ± 9.5b 205.7 ± 4.2d 74.0 ± 6.4c 89.5 ± 0.5b
E1 38.2 ± 0.3h 245.4 ± 3.0c 7.2 ± 2.1f 61.2 ± 3.1e
E3 621.2 ± 19.3c 234.3 ± 1.1c 63.7 ± 4.2d 81.0 ± 1.5c,d 2.2.2. Topoisomerase inhibitory activity
Curcumin 1201.4 ± 0.2a 1581.9 ± 28.4a 96.8 ± 1.3a 98.7 ± 0.4a Based on the cytostatic activity displayed by the compounds
(Table 1), the precursor 3,5-bisarylidenepiperidones A1–E1 and
Antioxidant capacity measured by FRAP, ORAC, DPPH and ROS radical assays for the
test compounds 6–17 at 100 lM concentrations. Means ± standard deviation, fol- one of their potent fatty acid (dodecanoic or tetradecanoic acid)
lowed by the different letters within column is significantly different (Tukey’s analogues A3, B3, C4, D4 and E3 were selected for detailed biolog-
multiple means comparison test, p <0.05), ND: Not detected. ical activity analysis to discern possible mechanism of action.
 E. Potter et al. / Bioorg. Med. Chem. 23 (2015) 411–421 415

Nicked Open

Linear band
Supercoiled

Figure 3. Topoisomerase IIa inhibitory activities of of 3,5-bisarylidene-4-piperidones derivatives. All the compounds were examined at a final concentration of 10 lM,
respectively, as designated. Lane K: pHOT1 DNA, supercoiled, Lane L: Marker DNA, Linear pHOT1, Lanes A1–E3: pHOT1 DNA + 4 U topoisomerase IIa + test compounds, Lane
SOR: pHOT1 DNA + 4 U topoisomerase IIa + Sorafenib.

Sorafenib, an orally bioavailable multi-target kinase inhibitor, also 50

known to inhibit topoisomerase IIa in vitro51 and in vivo,52 was a
used as the reference drug. The topoisomerase IIa inhibitory activ-

Percentage LDH Release

40
ity of these compounds (10 lM) is shown in Figure 3. From the
results of Figure 3, it is clear that the parent compounds and their 30
analogs (except E1) showed strong catalytic inhibitory action
towards topoisomerase IIa like the reference drug, sorafenib. 20
b
Decreased topoisomerase IIa activity indicates the fewer DNA b
b
strand breaks in gel, indicating potentially strong cytotoxicity in 10 cd
c
cd cd d
c
d
cancer cells. The results were in agreement with the cytostatic e

activity (IC50 values, Table 1) as compounds A1 and A3, C1 and 0

C3 exhibited similar topoisomerase IIa inhibition to their cyto- A1 A3 B1 B3 C1 C4 D1 D4 E1 E3 CUR SOR
static activity in vitro. These groups of compounds displayed sim- Compounds

ilar linear bands between nicked open DNA and supercoiled DNA,
Figure 5. In vitro toxicity assessment of of 3,5-bisarylidene-4-piperidones deriv-
indicating active catalytic inhibitory activity against topoisomer- atives using WI-38 cells (human fibroblasts) as measured by LDH release assay.
ase IIa action in vitro. The gel analysis showed that the compound Different letters (a through d) on columns designate a statistical significant
B3 displayed weaker topoisomerase IIa activity than its parent difference of p <0.05 between the assayed compounds using a Tukey’s test (n = 3),
CUR: Curcumin; SOR: Sorafenib.
molecule B1, as indicated by their band intensity. This observation
was also in agreement with IC50 values of both compounds, which
indicated weaker anti-cancer potential of compound B3 in vitro
logues, A3, B3, C4, D4 and E3 showed a more active antioxidant
(p <0.05). Compound E1, with presence of massive relaxed bands,
activity than their precursor compounds, as indicated by their
indicating weakest topoisomerase IIa activity among all com-
FRAP values (p <0.05). Especially, compound D4, with fatty acid
pounds examined in vitro. Its weak topoisomerase IIa activity
moiety, showed highest antioxidant activity among all tested
was also in agreement with its weak cytostatic activity, as its
derivatives (p <0.05). Moreover, compound E3, with 16 times
IC50 values range between 250 and 500 lM in vitro. However, its
higher antioxidant potential than its precursor molecule, closely
analog E3 exhibited potent topoisomerase IIa inhibitory activity
followed the leading compound D4 in vitro (p <0.05). Furthermore,
via catalytic inhibition of topoisomerase enzyme in vitro. Its activ-
among precursor compounds, A1, C1 and E1 exhibited the weakest
ity was comparable to analogs A1, A3, B1 and C1. Overall the
antioxidant activity, as measured by FRAP assay (p <0.05). Conse-
results were consistent with the anticancer drug, sorafenib, at least
quently, the structural modification emerged as a potent factor
in part, as potent topoisomerase IIa inhibitor.
towards change in antioxidant activity as compared to parent com-
pounds in vitro. The current study also revealed the peroxy radical
2.2.3. Antioxidant activity
scavenging activity of compounds using ORAC analysis. Interest-
The antioxidant properties of precursor 3,5-bisarylidene-4-pip-
ingly, compounds A3, B3, and D4, altered by structural modifica-
eridones and their selected fatty acid analogs an were investigated
tions exhibited better antioxidant activities than their
using multiple assays and compared with that of known antioxi-
corresponding parent compounds, as indicated by ORAC values
dant, curcumin. Among all the compounds, curcumin exhibited
(p <0.05). On the other hand, the antioxidant activity of compounds
the highest antioxidant activity in all the four antioxidants assays
of E1 and E3 had no statistical difference (p <0.05). Likewise, par-
conducted in vitro (p <0.05). As shown on Table 3, all novel ana-
ent compounds A1, B1 and D1 exhibited weakest peroxy radical
scavenging activity, possibly due to the absence any functionality
that could enable such effect. The antioxidant activity of these
120
compounds can also be associated with their ability to release elec-
a tron or hydrogen radical, resulting in stable DPPH in vitro. The
100 a a
Percentage Inhibition

DPPH assay confirmed the FRAP and ORAC based in vitro antioxi-
80 dant activity findings. The fatty acid amides, A3, B3, C4, and E3
b
exhibited relatively stronger anti-radical activity in vitro
60 b
bc (p <0.05). Among all modified compounds, A3, B3 and C4 exhibited
40 strongest activity while compound E1 was weakest anti-radical
c
d compound in vitro (p <0.05). These widespread variations in anti-
20 oxidant activities may be related to the variations in the electron
e e
f or hydrogen transfer abilities of these compounds, possibly due
0
A1 A3 B1 B3 C1 C4 D1 D4 E1 E3 KA to hydrolysis of amides resulting in in situ formation of fatty acids.
Compounds To get a more quantitative understanding for the possible effect of
these compounds on the cellular ROS, WI-38 cells cultured in 96-
Figure 4. Anti-tyrosinase activity of 3,5-bisarylidene-4-piperidones derivatives in
well plates were pre-exposed to all compounds, followed by per-
comparison to Kojic Acid (KA). Different letters (a through d) on columns designate
a statistical significant difference of p <0.05 between the assayed compounds using
oxy radical insult (AAPH induced) before measuring DCF fluores-
a Tukey’s test (n = 3), KA: Kojic acid. cence. All the modified compounds displayed higher ROS
416 E. Potter et al. / Bioorg. Med. Chem. 23 (2015) 411–421
inhibition and induced significant changes in DCF fluorescence as
compared to parent compounds and assay controls (p <0.05). As
indicated in earlier assays, all analogs A3, B3, C4, D4 and E3
showed strong inhibition of cellular ROS in vitro (p <0.05). Regard-
ing the low ROS inhibitory activity, all parent compounds followed
the earlier trend as compounds A1, C1, D1 and E1 exhibited statis-
tically weak inhibition of cellular ROS in vitro (p <0.05).
2.2.4. Tyrosinase inhibition
Tyrosinase is a crucial enzyme involved in the initial stages of
melanin biosynthesis and its overproduction is observed in mela-
noma cells.53 Investigations were undertaken to examine the inhib-
itory potential of the studied compounds against tyrosinase activity
in vitro with kojic acid (as positive control). All of the ten compounds
exhibited wide range of inhibitory effects against tyrosinase activity
in vitro. The dramatic decrease in enzyme activities was observed
with the structural modification of parent compounds increasing.
All tested analogs (A3, B3, C4, D4 and E3) showed strong inhibition
of tyrosinase in vitro (p <0.05) compared to parent molecules (A1–
E1). Among these, E3 and D4 displayed very high inhibition of tyros-
inase while C1 was the weakest tyrosinase inhibitor (p <0.05). For
the parent molecules the tyrosinase inhibitory effects followed the
sequence: C1 < A1 < B1 < E1 < D1 and the inhibitory effects were
amplified with the structural modification as fatty acid conjugates.
2.2.5. In vitro-toxicity analysis
The in vitro cytotoxicity of the selected novel analogues was
measured using normal fibroblasts cell line, WI-38. Toxicity was
evaluated by LDH release assay with sorafenib and curcumin as
the positive controls, after exposure of cells to the tested com-
pounds for 24 h. As shown in Figure 5, these compounds exhibited
low toxic effects towards normal cells and were significantly safer
as compared to sorafenib in vitro (p <0.05). Most of compounds
exhibited <10% LDH release as compared to vehicle and curcumin
emerged as safest compound in vitro (p <0.05). The membrane
damage due to curcumin was 8 times lower than sorafenib
in vitro (p <0.05). Among the newly synthesized derivatives, com-
pound A1, B3 and E3 exhibited highest membrane damage while
C4 and D4 emerged as least cytotoxic towards normal cells
in vitro (p 6 0.05).
3. Conclusion
In conclusion, 25 novel derivatives conjugating fatty acids of
varying chain lengths to 3,5-bisarylmethylene-4-piperidones
through the amide bond were synthesised and their preliminary
bioevaluations were performed. C-4, C-12, C-14 and C-16 fatty acid
amides of highly electron deficient pyridine ring-containing 3,5-
bisarylmethylene-4-piperidones were found to possess significant
cytostatic activity. The compounds A3, C3, D4 and E3 emerged as
potent catalytic inhibitors of topoisomerase IIa in vitro. The data
also indicated that most analogues exhibited potent antioxidants
and exhibited tyrosinase inhibitory activities, indicating anti-mel-
anoma potential. Furthermore, the results indicated that all the
analogs were less toxic towards human lung fibroblasts as com-
pared to a widely used anticancer drug, sorafenib. Overall these
results show that these compounds were effective non-toxic anti-
cancer agents with strong antioxidant ability, which may act as
auxiliary therapy in cancer.
4. Experimental
4.1. Chemistry
All reagents were purchased from Aldrich Chemical Co and
were used without further purification. Precoated fluorescent silica
gel TLC plates were used to monitor the progress of the reactions.
1
H NMR and 13C NMR were recorded on a Brucker AV300 spectro-
photometer at 300 MHz and 75 MHz, respectively. Melting points
were recorded on a MEL-TEMP II apparatus and are uncorrected.
UV–Vis and IR spectra were recorded on Pharmacia Biotech Ultro-
spec 4000 and Nicolet Avatar 330FT-IT spectrophotometers respec-
tively. HR-MS spectra were recorded at the Department of
Chemistry, Dalhousie University by Mr Xiao Feng.
4.1.1. Synthesis of (3E,5E)-3,5-diarylidenepiperidin-4-ones (A1–
E1)
Method A: To the appropriate aromatic aldehyde (20 mmol),
acetic acid (125 mL) was added and the reaction mixture cooled
on ice. 4-Piperidone hydrochloride monohydrate (10 mmol) was
then added and dry HCl(g) passed briskly through the solution for
3 h. The reaction mixture was then sealed and allowed to stir at
room temperature overnight, or until the product crystallized from
solution. The resultant precipitate was filtered and washed with
ice-cold methanol.
4.1.1.1. (3E,5E)-3,5-Dibenzylidenepiperidin-4-one hydrochlo-
ride (A1). Yellow powder; yield: 67%; mp 174–176 °C (lit.43
1
177–178 °C). H NMR (300 MHz; DMSO-d6): d 2.79 (s, 1H, NH),
4.01 (s, 4H, NCH2), 7.45–7.49 (m, 10H, ArH), 7.60 (s, 2H, ArH).
The spectral data were found to be in agreement with those
reported.43
4.1.1.2. Diethyl 4,40 -(1E,10 E)-(4-oxopiperidine-3,5-diylidene)bis
(methan-1-yl-1-ylidene)dibenzoate acetic acid salt (B1). Yellow
powder; yield: 78%; mp 230–232 °C. 1H NMR (300 MHz; DMSO-d6): d
1.35 (t, J = 4.2 Hz, 6H, 2  CH3), 1.92 (s, 3H, CH3CO), 4.36 (q, 4H,
J = 4.2 Hz, 2  OCH2), 4.50 (s, 4H, 2  NCH2), 7.68 (d, J = 5.1 Hz, 2H,
ArH), 7.92 (s, 2H, 2  vinylic-H), 8.08 (d, J = 5.1 Hz, 2H, ArH), 10.03
(br s, 2H, N+H2). 13C NMR (75 MHz; DMSO-d6): d 14.09, 21.01,
43.75, 61.00, 129.42, 129.67, 130.57, 130.62, 137.88, 138.09, 165.13,
171.91, 182.24. ESI-MS (amu): calcd C25H26NO5 [M+H]: 420.1811;
found [M+H]: 420.1824.
4.1.1.3. (3E,5E)-3,5-Bis(pyridin-4-ylmethylene)piperidin-4-one
trihydrochloride (C1). Yellow powder; yield: 80%; mp 202–
204 °C. 1H NMR (300 MHz; DMSO-d6): d 4.00 (s, 4H, 2  NCH2),
7.44 (d, J = 5.1 Hz, 4H, ArH), 7.51 (s, 2H, 2  vinylic-H), 8.65 (d,
J = 5.1 Hz, 4H, ArH). 13C NMR (75 MHz; CDCl3): d 48.18, 124.10,
124.43, 133.72, 138.22, 142.64, 150.58, 150.84, 187.35. ESI-HRMS
(amu): calcd C17H15N3O [M+]: 277.1215; found [M+]: 277.1221.
Method B: To an ice-cold ethanolic solution of KOH (10 g in
30 mL), the appropriate aromatic aldehyde (20 mmol) was added.
While maintaining a reaction temperature of approximately 0 °C,
4-piperidone hydrochloride monohydrate (10 mmol) was added
slowly in portions over 5 min. The reaction mixture was allowed
to stir for 4 h at room temperature and then cooled on ice. The pre-
cipitated product was filtered and washed with ice-cold ethanol
yielding the product as a yellow or orange powder.
4.1.1.4. (3E,5E)-3,5-Bis(pyridin-2-ylmethylene)piperidin-4-one
(D1). Yellow powder; yield: 77%; mp 193–194 °C. 1H NMR
(300 MHz; DMSO-d6): d 4.32 (s, 4H, 2  NCH2), 7.16 (br s, 2H,
ArH), 7.35 (br s, 2H, ArH), 7.66 (d, J = 7.7 Hz, 2H, ArH), 7.84 (br
s, 2H, 2  vinylic-H), 8.70 (br s, 2H, ArH). 13C NMR (75 MHz;
DMSO-d6): d 49.00, 123.97, 128.45, 132.01, 137.72, 140.20,
150.50, 155.19, 189.91. ESI-HRMS (amu): calcd C17H15N3O
[M+H]: 278.1293; found [M+H]: 278.1287.
4.1.1.5. (3E,5E)-3,5-Bis(furan-2-ylmethylene)piperidin-4-one
(E1). Orange powder; yield: 97%; mp 227–228 °C. 1H NMR
(300 MHz; CDCl3): d 4.30 (s, 4H, 2  NCH2), 6.55 (br s, 2H,
 E. Potter et al. / Bioorg. Med. Chem. 23 (2015) 411–421
ArH), 6.68 (d, J = 3.30 Hz, 2H, ArH), 7.52 (s, 2H, 2  vinylic-H),
7.69 (br s, 2H, ArH). 13C NMR (75 MHz; CDCl3): d 44.94, 47.47,
113.87, 118.46, 121.30, 132.04, 147.17, 151.94, 152.15, 186.56.
ESI-HRMS (amu): calcd C15H13NO3 [M+H]: 256.0974; found
[M+H]: 256.0949.
4.1.2. General procedure for the synthesis of (3E,5E)-N-acyl-3,5-
diarylidenepiperidin-4-ones
To an ice-cold mixture of the appropriate fatty acid (1 mmol) in
dry DCE (10 mL), SOCl2 (2.4 mmol) was added dropwise maintain-
ing a temperature of 5 °C. The reaction mixture was then brought
to room temperature and then gradually heated to the reflux tem-
perature of 85 °C and maintained there for 3 h. The remaining
SOCl2 and DCE were removed by rotary evaporation, yielding the
acid chloride. The prepared acid chloride was dissolved in dry
DCE (5 mL) and added dropwise to a mixture of the appropriate
3,5-bisarylmethylene-4-pipridone (A1–E1, 1 mmol) and triethyl-
amine (3 mmol for A1 and B1, 5 mmol for C1 and 2 mmol for D1
and E1) in dry DCE. The reaction mixture was sealed and allowed
to stir at room temperature overnight. An aqueous extraction
was then carried out to remove triethylamine HCl. The crude com-
pound was then purified via column chromatography (12.5–25%
ethyl acetate/hexanes) and dried on vacuum.
4.1.2.1. (3E,5E)-3,5-Dibenzylidene-1-butyrylpiperidin-4-one
(A2). Yellow powder; yield: 60%; mp 116–119 °C. 1H NMR
(300 MHz; CDCl3): d 0.77 (t, J = 7.4 Hz, 3H, CH3), 1.46–1.55 (m,
2H, CH2), 2.12 (t, J = 7.5 Hz, 2H, COCH2), 4.73 and 4.96 (s, 2H
each, 2  NCH2), 7.47 (m, 10H, ArH), 7.89 (br s, 2H, 2  vinylic-
H). 13C NMR (75 MHz; CDCl3): d 13.74, 18.49, 34.92, 43.55,
46.23, 128.83, 129.59, 130.07, 130.68, 131.87, 132.03, 134.61,
137.18, 138.49, 171.90, 186.88. IR (KBr; mmax): 2953, 2912,
2835, 1655, 1603, 1578, 1445, 1427, 1174, 984, 772, 694 cm1.
UV (CHCl3; kmax): 330 nm. ESI-HRMS (amu): calcd C 23H23NO2
[M+Na]: 368.1626; found [M+Na]: 368.1593.
4.1.2.2. (3E,5E)-3,5-Dibenzylidene-1-dodecanoylpiperidin-4-one
(A3). Yellow powder; yield: 46%; mp 66–68 °C. 1H NMR
(300 MHz; CDCl3): d 0.90 (t, J = 6.6 Hz, 3H, CH3), 1.10–1.28 (m,
16H, CH2), 1.44 (m, 2H, CH2), 2.14 (t, J = 7.6 Hz, 2H, COCH2), 4.72
and 4.95 (s, 2H each, 2  NCH2), 7.40–7.60 (m, 10H, ArH), 7.85
and 7.90 (s, 1H each, 2  vinylic-H). 13C NMR (75 MHz; CDCl3): d
14.09, 22.67, 25.11, 29.18, 29.28, 29.31, 29.42, 29.55, 29.58,
31.89, 33.17, 43.58, 46.26, 127.02, 127.98, 128.81, 129.09, 129.30,
129.56, 129.92, 130.04, 130.61, 131.88, 134.54, 137.10, 138.49,
172.07, 186.89. IR (KBr; mmax): 2952, 1919, 1848, 1675, 1637,
1613, 1579, 1457, 1446, 1273, 1168, 984, 937, 769, 690 cm1. UV
(CHCl3; kmax): 330 nm. ESI-HRMS (amu): calcd C31H39NO2 [M+H]:
458.3059; found [M+H]: 458.3011.
4.1.2.3. (3E,5E)-3,5-Dibenzylidene-1-tetradecanoylpiperidin-4-
one (A4). Yellow powder; yield: 19%; mp 68–69 °C. 1H NMR
(300 MHz; CDCl3): d 0.91 (t, J = 6.2 Hz, 3H, CH3), 1.07–1.50 (m,
22H,11  CH2), 2.14 (t, J = 7.5 Hz, 2H, COCH2), 4.73 and 4.95 (s,
2H each, 2  NCH2), 7.40–7.51 (m, 10H, ArH), 7.85 and 7.90 (s,
1H each, 2  vinylic-H). 13C NMR (75 MHz; CDCl3): d 14.14,
22.70, 25.12, 29.21, 29.30, 29.37, 29.45, 29.5, 29.65, 29.69, 31.93,
33.19, 43.56, 46.24, 128.79, 129.58, 130.07, 130.69, 131.86,
132.06, 134.56, 137.12, 138.52, 172.04, 186.90. IR (KBr; mmax):
2956, 2918, 1847, 1674, 1634, 1611, 1578, 1469, 1456, 1438,
1276, 1266, 1169, 984, 937, 768, 690 cm1. UV (CHCl3; kmax):
333 nm. ESI-HRMS (amu): calcd C33H43NO2 [M+H]: 486.3372;
found [M+H]: 486.3323.
4.1.2.4. (3E,5E)-3,5-Dibenzylidene-1-palmitoylpiperidin-4-one
(A5). Yellow powder; yield: 57%; mp 76–78 °C. 1H NMR
417
(300 MHz; CDCl3): d 0.92 (t, J = 6.2 Hz, 3H, CH3), 1.06–1.48 (m,
26H, 13  CH2), 2.14 (t, J = 7.5 Hz, 2H, COCH2), 4.72 and 4.95 (s,
2H each, 2  NCH2), 7.40–7.51 (m, 10H, Ar-H), 7.86 and 7.90 (s,
1 h each, 2H, 2  vinylic-H). 13C NMR (75 MHz; CDCl3): d 14.10,
22.68, 25.12, 29.19, 29.29, 29.35, 29.43, 29.56, 29.65, 29.68,
31.92, 33.15, 43.59, 46.26, 128.81, 129.55, 130.07, 130.64, 132.00,
134.63, 137.09, 138.49, 172.07, 186.83. IR (KBr; mmax): 2953,
2917, 2848, 1666, 1635, 1612, 1578, 1470, 1457, 1439, 1269,
1096, 985, 938, 769, 691 cm1. UV (CHCl3; kmax): 330 nm. ESI-
HRMS (amu): calcd C35H47NO2 [M+H]: 514.3685; found [M+H]:
514.3641.
4.1.2.5. (3E,5E)-3,5-Dibenzylidene-1-stearoylpiperidin-4-one
(A6). Waxy yellow powder; yield: 56%; mp 71–73 °C. 1H
NMR (300 MHz; CDCl3): d 0.90 (t, J = 6.6 Hz, 3H, CH3), 1.06–
1.49 (m, 30H, 15  CH2), 2.14 (t, J = 5.0 Hz, 2H, COCH2), 4.73
and 4.96 (s, 2H each, 2  NCH2), 7.42–7.59 (m, 10H, ArH), 7.86
and 7.90 (s, 1H each, 2H, 2  vinylic-H). 13C NMR (75 MHz;
CDCl3): d 14.13, 22.70, 25.12, 25.62, 29.21, 29.30, 29.37, 29.45,
29.58, 29.67, 29.71, 31.93, 33.19, 43.57, 46.23, 128.10, 128.80,
129.57, 130.05, 130.68, 134.53, 137.12, 138.52, 172.04, 186.91.
IR (KBr; mmax): 2949, 2917, 1848, 1635, 1612, 1580, 1470,
1456, 1438, 1269, 1169, 1095, 985, 768, 690 cm1. UV (CHCl3;
kmax): 330 nm. ESI-HRMS (amu): calcd C37H51NO 2 [M+Na]:
564.3817; found [M+Na]: 564.3758.
4.1.2.6. (3E,5E)-3,5-Dibenzylidene-1-oleoylpiperidin-4-one
(A7). Reddish-orange low melting solid; yield: 56%; 1 H
NMR (300 MHz; CDCl 3): d 0.89–0.95 (dist. t, 3H, CH 3), 1.00–
1.48 (m, 22H, 11  CH2 ), 2.01–2.14 (m, 6H, 3  CH 2), 4.72 and
4.95 (s, 2H each, 2  NCH2 ), 5.36 (br s, 2H, 2  Z-vinylic-H),
7.43–7.47 (m, 10H, ArH), 7.85 and 7.90 (s, 1H each,
2  vinylic-H). 13 C NMR (75 MHz; CDCl 3): d 14.13, 22.69,
25.10, 27.19, 27.24, 28.99, 29.11, 29.10, 29.26, 29.33, 29.45,
29.53, 29.68, 29.78, 31.79, 31.91, 32.62, 33.16, 43.57, 46.25,
128.83, 129.58, 129.75, 129.98, 130.07, 130.69, 131.86,
132.07, 134.57, 134.68, 137.10, 138.51, 172.01, 186.86. IR
(KBr; m max ): 3002, 2921, 2852, 1636, 1612, 1579, 1446, 1272,
1170, 985, 769, 692 cm 1 . UV (CHCl 3; kmax): 332 nm. ESI-HRMS
(amu): calcd C37 H49 NO 2 [M+Na]: 562.3661; found [M+Na]:
562.3609.
4.1.2.7. Diethyl 4,40 -(1E,10 E)-(1-dodecanoyl-4-oxopiperidine-3,5-
diylidene)bis(methan-1-yl-1-ylidene)dibenzoate (B3). Yellow
powder; yield: 55%; mp 88–91 °C. 1H NMR (300 MHz; CDCl3): d
0.89 (t, J = 6.2 Hz, 3H, CH3); 1.00–1.70 (m, 24H, 11  CH2;
2  CH3), 2.33 (t, J = 7.4 Hz, 2H, COCH2), 4.31 (dist. q, J = 6.6 Hz,
4H, 2  OCH2), 4.73 and 4.94 (s, 2H each, 2  NCH2), 7.46 and
7.56 (d, 2H each, J = 7.2 Hz, ArH), 7.86 and 7.91 (s, 2H,
2  vinylic-H), 8.11–8.14 (m, 4H, ArH). 13C NMR (75 MHz; CDCl3):
d 14.12, 14.32, 22.68, 24.75, 25.04, 29.09, 29.23, 29.26, 29.31, 29.41,
29.54, 29.59, 31.91, 33.05, 33.69, 43.47, 46.30, 61.26, 61.36, 129.47,
129.90, 130.35, 131.06, 131.24, 133.29, 136.20, 137.47, 138.63,
165.81, 165.94, 172.02. IR (KBr; mmax): 2955, 2925, 2851, 1715,
1672, 1645, 1605, 1581, 1562, 1467, 1438, 1412, 1288, 1258,
1183, 1172, 1105, 1020, 990, 862, 774 cm1. UV (CHCl3; kmax):
334 nm. ESI-HRMS (amu): calcd C37H47NO6 [M+Na]: 624.3301;
found [M+Na]: 624.3257.
4.1.2.8. Diethyl 4,40 -(1E,10 E)-(4-oxo-1-stearoylpiperidine-3,
5-diylidene)bis(methan-1-yl-1-ylidene)dibenzoate (B6). Yellow
powder; yield: 87%; mp 76–78 °C. 1H NMR (300 MHz; CDCl3): d
0.89 (t, J = 6.1 Hz, 3H, CH3); 1.05–1.48 (m, 30H, 13  CH2; 2  CH3),
2.12 (t, J = 7.3 Hz, 2H, COCH2), 4.43 (dist. q, J = 6.2 Hz, 4H, 2  OCH2),
4.73 and 4.94 (s, 2H each, 2  NCH2), 7.47–7.57 (m, 4H, ArH),
7.85 and 7.91 (s, 1H each, 2  vinylic-H), 8.10–8.20 (m, 4H, ArH).
418 E. Potter et al. / Bioorg. Med. Chem. 23 (2015) 411–421
13
C NMR (75 MHz; CDCl3): d 14.13, 14.32, 22.69, 25.03, 29.24, 29.27,
29.36, 29.42, 29.56, 29.66, 29.70, 31.92, 33.04, 43.46, 46.30, 61.25,
129.90, 130.34, 131.23, 133.31, 136.17, 137.44, 138.63, 165.80,
171.96, 186.39. IR (KBr; mmax): 2979, 2922, 2851, 1708, 1674, 1629,
1610, 1577, 1453, 1414, 1306, 1285, 1228, 1185, 1185, 1109, 1023,
943, 857, 771 cm1. UV (CHCl3; kmax): 336 nm. ESI-HRMS (amu):
calcd C43H59NO6 [M+Na]: 708.4240; found [M+Na]: 708.4198.
4.1.2.9. Diethyl 4,40 -(1E,10 E)-(1-oleoyl-4-oxopiperidine-3,5-diy-
lidene)bis(methan-1-yl-1-ylidene)dibenzoate (B7). Yellow
low melting solid; yield: 55%; 1H NMR (300 MHz; CDCl3): d 0.89
(t, J = 6.5 Hz, 3H, CH3); 1.10–1.27 (m, 22H, 11  CH2), 1.44 (t,
J = 6.9 Hz, 6H, 2  CH3), 2.00 (m, 2H, CH2), 2.13 (t, J = 7.5 Hz, 2H,
COCH2), 4.43 (dist. q, J = 6.8 Hz, 4H, 2  OCH2), 4.73 and 4.94 (s,
2H each, NCH2), 5.30–5.36 (m, 2H, 2  Z-vinylic-H), 7.46 and 7.56
(d, 2H each, J = 7.2 Hz, ArH), 7.86 and 7.91 (s, 2H, 2  vinylic-H),
8.11–8.18 (m, 4H, ArH). 13C NMR (75 MHz; CDCl3): d 14.12,
14.32, 22.68, 25.01, 27.15, 27.22, 28.98, 29.06, 29.15, 29.24,
29.32, 29.52, 29.64, 29.76, 31.78, 31.90, 33.02, 43.47, 46.30,
61.35, 129.70, 129.91, 129.99, 130.34, 131.21, 133.29, 136.19,
137.46, 138.69, 165.92, 171.94, 186.37. IR (KBr; mmax): 2923,
2851, 1710, 1626, 1610, 1577, 1456, 1414, 1366, 1305, 1284,
1185, 1108, 1021, 857, 771 cm1. UV (CHCl3; kmax): 335 nm. ESI-
HRMS (amu): calcd C43H57NO6 [M+Na]: 706.4084; found [M+Na]:
706.4013.
4.1.2.10. (3E,5E)-1-Dodecanoyl-3,5-bis(pyridin-4-ylmethylene)
piperidin-4-one (C3). Yellow powder; yield: 89%; mp 66–
70 °C. 1H NMR (300 MHz; CDCl3): d 0.87 (br s, 3H, CH3); 1.12–
1.66 (m, 18H, 9  CH2), 2.13 (t, J = 6.9 Hz, 2H, COCH2), 4.70 and
4.88 (s, 2H each, 2  NCH2), 7.26–7.36 (m, 4H, ArH), 7.70 and
7.76 (s, 1H each, 2  vinylic-H), 8.70–8.75 (m, 4H, ArH). 13C
NMR (75 MHz; CDCl3): d 14.10, 22.66, 24.93, 25.00, 29.21,
29.24, 29.29, 29.32, 29.37, 29.47, 29.53, 29.60, 31.88, 32.91,
34.30, 43.10, 46.22, 123.76, 124.03, 134.56, 134.95, 135.54,
141.97, 150.01, 150.16, 171.97, 177.19, 185.68. IR (KBr; mmax):
3019, 2920, 2849, 1723, 1678, 1639, 1619, 1593, 1465, 1417,
1234, 1216, 1189, 1168, 995, 848, 824 cm1. UV (CHCl3; kmax):
304 nm. ESI-HRMS (amu): calcd C29H37N3O2 [M+H]: 460.2964;
found [M+H]: 460.2939.
4.1.2.11. (3E,5E)-3,5-Bis(pyridin-4-ylmethylene)-1-tetradecanoyl-
piperidin-4-one (C4). Yellow powder; yield: 53%; mp
98–100 °C. 1H NMR (300 MHz; CDCl3): d 0.89 (br s, 3H, CH3);
1.14–1.50 (m, 22H, 11  CH2), 2.14 (br s, 2H, COCH2), 4.72 and
4.90 (s, 2H each, 2  NCH2), 7.26–7.36 (m, 4H, ArH), 7.73 and 7.78
(s, 1H each, 2  vinylic-H), 8.75 (br s, 4H, ArH). 13C NMR (75 MHz;
CDCl3): d 14.12, 22.68, 24.95, 25.07, 29.25, 29.34, 29.39, 29.55,
29.63, 31.90, 32.94, 43.12, 46.22, 123.65, 123.92, 134.64, 134.87,
135.69, 141.69, 150.42, 171.92, 176.41, 185.77. IR (KBr; mmax):
3035, 3019, 2917, 1848, 1676, 1637, 1619, 1592, 1544, 1458, 1430,
1416, 1275, 1257, 1232, 1215, 1167, 996, 930, 831, 759, 717 cm1.
UV (CHCl3; kmax): 304 nm. ESI-HRMS (amu): calcd C31H41N3O2
[M+H]: 488.3277; found [M+H]: 488.3286.
4.1.2.12. (3E,5E)-1-Palmitoyl-3,5-bis(pyridin-4-ylmethylene)
piperidin-4-oneone (C5). Yellow powder; yield: 55%; mp
96–98 °C. 1H NMR (300 MHz; CDCl3): d 0.92–1.49 (m, 29H,
1  CH3; 13  CH3), 2.16 (br s, 2H, COCH2), 4.75 and 4.92 (s,
2H each, 2  NCH2), 7.10–7.38 (m, 4H, ArH), 7.74 and 7.79 (s,
1H each, 2  vinylic-H), 8.74–8.78 (br s, 4H, ArH). 13C NMR
(75 MHz; CDCl3): d 14.13, 22.69, 24.96, 29.27, 29.36, 29.67,
31.92, 32.95, 43.14, 46.22, 123.65, 123.91, 134.67, 134.87,
135.71, 141.66, 150.48, 171.92, 185.77. IR (KBr; mmax): 2917,
2849, 1635, 1619, 1592, 1465, 1416, 1260, 1226, 1167,
996, 929, 841, 820, 755, 714 cm1. UV (CHCl3; kmax): 302 nm.
ESI-HRMS (amu): calcd C33H45N3O2 [M+H]: 516.3590; found
[M+H]: 516.3539.
4.1.2.13. (3E,5E)-3,5-Bis(pyridin-4-ylmethylene)-1-stearoylpiperi-
din-4-one (C6). Yellow powder; yield: 90%; mp 88–90 °C. 1H
NMR (300 MHz; CDCl3): d 0.90 (t, J = 6.4 Hz, 3H, CH3); 1.14–.48 (m,
30H, 15  CH2), 2.15 (t, J = 7.5 Hz, 2H, COCH2), 4.72 and 4.90 (s, 2H
each, 2  NCH2), 7.28–7.38 (m, 4H, ArH), 7.74 and 7.79 (s, 1H each,
2  vinylic-H), 8.74–8.78 (m, 4H, ArH). 13C NMR (75 MHz; CDCl3): d
14.13, 22.70, 24.96, 29.28, 29.37, 29.42, 29.48, 29.58, 29.70, 29.92,
31.93, 32.96, 33.97, 43.14, 46.22, 123.67, 123.94, 134.75, 141.75,
150.35, 150.35, 171.95, 185.78. IR (KBr; mmax): 3035, 3023, 2916,
2848, 1723, 1679, 1637, 1619, 1593, 1545, 1469, 1416, 1274, 1234,
1218, 1167, 996, 929, 840, 824 cm1. UV (CHCl3; kmax): 325 nm.
ESI-HRMS (amu): calcd C35H49N3O2 [M+H]: 544.3903; found
[M+H]: 544.3845.
4.1.2.14. (3E,5E)-1-Oleoyl-3,5-bis(pyridin-4-ylmethylene)piperi-
din-4-one (C7). Yellow low melting solid; yield: 36%; 1H NMR
(300 MHz; CDCl3): d 0.89 (t, J = 6.1 Hz, 3H, CH3); 1.10–1.49 (m,
18H, 9  CH2), 2.00 (t, J = 5.3 Hz, 4H, 2  CH2), 2.14 (t, J = 7.5 Hz,
2H, COCH2), 4.71 and 4.90 (s, 2H each, 2  NCH2), 5.35 (m, 2H,
2  Z-vinylic-H), 7.20–7.35 (m, 4H, ArH), 7.73 and 7.79 (s, 1H each,
2  vinylic-H), 8.75 (d, J = 9.3 Hz, 4H, ArH). 13C NMR (75 MHz;
CDCl3): d 14.13, 22.69, 24.93, 27.15, 27.22, 29.05, 29.20, 29.32,
29.52, 29.66, 29.76, 31.78, 31.90, 32.93, 43.13, 46.21, 123.63,
123.93, 134.69, 134.83, 135.75, 141.63, 150.55, 171.88, 185.79. IR
(KBr; mmax): 2923, 2852, 1680, 1634, 1619, 1592, 1544, 1457,
1429, 1416, 1259, 1232, 1167, 996, 929, 820 cm1. UV (CHCl3;
kmax): 302 nm. ESI-HRMS (amu): calcd C35H47N3O2 [M+H]:
542.3747; found [M+H]: 542.3703.
4.1.2.15. (3E,5E)-1-Dodecanoyl-3,5-bis(pyridin-2-ylmethylene)
piperidin-4-one (D3). Yellow powder; yield: 35%; mp 102–
104 °C. 1H NMR (300 MHz; CDCl3): d 0.90 (br s, 3H, CH3); 1.21–
1.25 (m, 16H, 8  CH2), 1.58 (br s, 2H, CH2), 2.39 (br s, 2H, COCH2),
5.32 and 5.39 (s, 2H each, 2  NCH2), 7.29–7.77 (m, 8H, ArH,
2  vinylic-H), 8.76 (s, 2H, ArH). 13C NMR (75 MHz; CDCl3): d
14.09, 22.66, 25.39, 29.30, 29.45, 29.57, 31.89, 33.55, 44.66,
46.61, 123.08, 123.29, 127.59, 128.23, 132.68, 134.43, 135.77,
136.39, 136.64, 136.72, 149.54, 149.62, 154.17, 154.41, 172.52,
188.01. IR (KBr; mmax): 2955, 2923, 2849, 1676, 1640, 1583, 1560,
1466, 1428, 1304, 1273, 1162, 986, 939, 790 cm1. UV (CHCl3;
kmax): 333 nm. ESI-HRMS (amu): calcd C29H39N3O2 [M+H]:
460.2964; found [M+H]: 460.2923.
4.1.2.16. (3E,5E)-3,5-Bis(pyridin-2-ylmethylene)-1-tetradecanoyl-
piperidin-4-one (D4). Waxy yellow powder; yield: 30%; mp
108–110 °C. 1H NMR (300 MHz; CDCl3): d 0.89 (t, J = 6.4 Hz, 3H,
CH3); 1.17–1.28 (m, 20H, 10  CH2), 1.56 (quintet, J = 7.6 Hz, 2H,
CH2), 2.38 (t, J = 7.8 Hz, 2H, COCH2), 5.31 and 5.38 (s, 2H each,
2  NCH2), 7.21–7.25 (m, 2H, ArH), 7.50 (t, J = 6.8 Hz, 2H, ArH),
7.65–7.77 (m, 4H, ArH, 2  vinylic-H), 8.74 (br s, 2H, ArH). 13C
NMR (75 MHz; CDCl3): d 14.10, 22.67, 24.87, 25.41, 29.15, 29.10,
29.15, 29.30, 29.33, 29.46, 29.59, 29.63, 29.66, 31.90, 33.56, 34.05,
44.72, 46.57, 122.99, 123.29, 127.57, 128.22, 132.63, 134.87,
135.76, 136.10, 136.29, 136.71, 149.63, 149.83, 154.40, 172.60,
188.09. IR (KBr; mmax): 2956, 2920, 2850, 1639, 1582, 1469, 1427,
1269, 1163, 1098, 986, 790 cm1. UV (CHCl3; kmax): 334 nm. ESI-
HRMS (amu): calcd C31H41N3O2 [M+H]: 488.3277; found [M+H]:
488.3232.
4.1.2.17. (3E,5E)-1-Palmitoyl-3,5-bis(pyridin-2-ylmethylene)
piperidin-4-one (D5). Yellow powder; yield: 53%; mp
98–100 °C. 1H NMR (300 MHz; CDCl3): d 0.89 (br s, 3H, CH3);
1.19–1.27 (m, 24H, 12  CH2), 1.57 (br s, 2H, CH2), 2.38 (t,
 E. Potter et al. / Bioorg. Med. Chem. 23 (2015) 411–421
J = 7.1 Hz, 2H, COCH2), 5.32 and 5.39 (s, 2H each, 2  NCH2),
7.22–7.78 (m, 8H, ArH; 2  vinylic-H), 8.75 (s, 2H, ArH). 13C
NMR (75 MHz; CDCl 3): d 14.10, 22.67, 24.94, 25.41, 29.34,
29.47, 29.68, 31.91, 33.57, 34.01, 44.73, 46.58, 122.98, 123.28,
127.58, 128.22, 132.60, 134.86, 135.79, 136.13, 136.27, 136.71,
149.64, 149.84, 154.43, 172.55, 188.11. IR (KBr; mmax): 3056,
2955, 2918, 1849, 1670, 1650, 1621, 1588, 1578, 1560, 1471,
1427, 1274, 1167, 987, 787 cm1. UV (CHCl3; kmax): 334 nm.
ESI-HRMS (amu): calcd C33H45N3O 2 [M+H]: 516.3590; found
[M+H]: 516.3560.
4.1.2.18. (3E,5E)-3,5-Bis(pyridin-2-ylmethylene)-1-stearoylpi-
peridin-4-one (D6). Yellow powder; yield: 25%; mp 102–
104 °C. 1H NMR (300 MHz; CDCl3): d 0.90 (t, J = 6.3 Hz, 3H, CH3),
1.19–1.40 (m, 28H, 14  CH2), 1.53–1.58 (m, 2H, CH2), 2.38 (t,
J = 7.5 Hz, 2H, COCH2), 5.33 and 5.39 (s, 2H each, 2  NCH2),
7.24–7.28 (m, 2H, ArH), 7.52 (t, J = 7.1 Hz, 2H, ArH), 7.66–7.79
(m, 4H, ArH; 2  vinylic-H), 8.76 (br s, 2H, ArH). 13C NMR
(75 MHz; CDCl3): d 14.13, 22.70, 25.42, 29.34, 29.27, 29.49, 29.62,
29.66, 29.71, 31.93, 33.59, 44.72, 46.59, 122.98, 123.29, 127.61,
128.25, 132.60, 134.88, 135.81, 136.15, 136.27, 136.72, 149.65,
149.87, 154.44, 172.49, 188.18. IR (KBr; v): 3056, 2955, 2918,
1849, 1650, 1622, 1588, 1578, 1560, 1471, 1427, 1275, 1167,
987, 787 cm1. UV (CHCl3; kmax): 334 nm. ESI-HRMS (amu): calcd
C35H49N3O2 [M+H]: 544.3903; found [M+H]: 544.3860.
4.1.2.19. (3E,5E)-1-Oleoyl-3,5-bis(pyridin-2-ylmethylene)piperi-
din-4-one (D7). Orange solid; yield: 45%; mp 72–74 °C. 1H
NMR (300 MHz; CDCl3): d 0.88 (dist. t, 3H, CH3), 1.18–1.40 (m,
22H, 11  CH2), 1.49–1.57 (m, 2H, CH2), 1.99–2.05 (m, 4H,
2  CH2), 2.38 (t, J = 7.7 Hz, 2H, COCH2), 5.31–5.38 (m, 6H,
2  NCH2; 2  Z-vinylic), 7.24–7.28 (m, 2H, ArH), 7.51 (t,
J = 6.4 Hz, 2H, ArH), 7.65–7.77 (m, 4H, ArH; 2  vinylic-H), 8.74
(br s, 2H, ArH). 13C NMR (75 MHz; CDCl3): d 14.17, 22.65, 25.38,
27.20, 28.96, 29.11, 29.21, 29.42, 29.46, 29.50, 29.68, 29.75, 31.88,
32.58, 33.53, 44.72, 46.57, 122.97, 123.27, 127.57, 128.21, 129.76,
129.93, 132.60, 134.82, 135.79, 136.14, 136.29, 136.70, 149.63,
149.80, 154.41, 172.50, 188.08. IR (KBr; mmax): 2990, 2923, 2851,
1676, 1640, 1582, 1560, 1465, 1428, 1273, 1164, 986, 939, 790,
746 cm1. UV (CHCl3; kmax): 334 nm. ESI-HRMS (amu): calcd
C35H47N3O2 [M+H]: 542.3747; found [M+H]: 542.3703.
4.1.2.20. (3E,5E)-1-Butyryl-3,5-bis(furan-2-ylmethylene)piperi-
din-4-one (E2). Yellow powder; yield: 66%; mp 116–118 °C.
1
H NMR (300 MHz; CDCl3): d 0.95 (t, J = 7.4 Hz, 3H, CH3), 1.68 (sex-
tet, J = 7.4 Hz, 2H, CH2), 2.43 (t, J = 7.5 Hz, 2H, COCH2), 4.98 and 5.07
(br s, 2H each, 2  NCH2), 6.54–6.60 (m, 2H, ArH), 6.78 (br s, 2H,
ArH), 7.52 and 7.55 (s, 1H each, 2  vinylic-H), 7.65 (br s, 2H,
ArH). 13C NMR (75 MHz; CDCl3): d 13.95, 18.69, 35.20, 43.44,
46.11, 112.66, 112.79, 118.05, 118.44, 121.87, 123.53, 128.13,
128.46, 145.59, 145.87, 151.77, 171.96, 185.92. IR (KBr; mmax):
3109, 1962, 1921, 2872, 2847, 1645, 1609, 1576, 1540, 1464,
1440, 1263, 1244, 1200, 1174, 1024, 977, 753, 621 cm1. UV (CHCl3;
kmax): 383 nm. ESI-HRMS (amu): calcd C19H19NO4 [M+Na]:
348.1212; found [M+Na]: 348.1189.
4.1.2.21. (3E,5E)-1-Dodecanoyl-3,5-bis(furan-2-ylmethylene)
piperidin-4-one (E3). Orange powder; yield: 57%; mp 78–
81 °C. 1H NMR (300 MHz; CDCl3): d 0.90 (t, J = 6.1 Hz, 3H, CH3),
1.10–1.40 (m, 16H, 8  CH2), 1.62 (quintet, J = 6.7 Hz, 2H, CH2),
2.44 (t, J = 7.6 Hz, 2H, COCH2), 4.98 and 5.06 (s, 2H each,
2  NCH2), 6.55–6.60 (m, 2H, ArH), 6.77–6.78 (m, 2H, ArH),
7.52 and 7.55 (s, 1H each, 2  vinylic-H), 7.65 (br s, 2H, ArH).
13
C NMR (75 MHz; CDCl3): d 14.10, 22.67, 25.34, 29.32, 29.35,
29.47, 29.59, 31.90, 33.40, 43.52, 46.14, 112.72, 118.02, 118.43,
419
121.87, 123.58, 128.18, 145.58, 145.82, 151.76, 172.26, 185.86.
IR (KBr; mmax): 3133, 2921, 2848, 1647, 1613, 1567, 1546, 1466,
1246, 1168, 1092, 992, 882, 773, 743, 621 cm1. UV (CHCl3;
kmax): 382 nm. ESI-HRMS (amu): calcd C27H35NO4 [M+Na]:
460.2464; found [M+Na]: 460.2436.
4.1.2.22. (3E,5E)-3,5-Bis(furan-2-ylmethylene)-1-tetradecanoyl-
piperidin-4-one (E4). Yellow powder; yield: 74%; mp 74–76 °C.
1
H NMR (300 MHz; CDCl3): d 0.90 (br s, 3H, CH3); 1.10–1.40 (m,
20H, 10  CH2), 1.62 (m, 2H, CH2), 2.44 (t, J = 6.9 Hz, 2H, COCH2),
4.98 and 5.06 (s, 2H each, 2  NCH2), 6.54–6.60 (m, 2H, ArH),
6.77 (br s, 2H, ArH), 7.51 and 7.55 (s, 1H each, 2  vinylic-H),
7.65 (br s, 2H, ArH). 13C NMR (75 MHz; CDCl3): d 14.12, 22.69,
24.84, 25.34, 29.13, 29.36, 29.48, 29.64, 31.92, 33.42, 33.87,
43.50, 46.14, 112.67, 112.79, 118.05, 118.46, 121.85, 123.57,
128.13, 128.44, 145.56, 145.87, 151.76, 172.26, 185.86. IR (KBr;
mmax): 2949, 2921, 2947, 1643, 1617, 1575, 1542, 1464, 1443,
1255, 1203, 1171, 982, 884, 757, 620 cm1. UV (CHCl3; kmax):
383 nm. ESI-HRMS (amu): calcd C29H39NO4 [M+Na]: 488.2777;
found [M+Na]: 488.2751.
4.1.2.23. (3E,5E)-3,5-Bis(furan-2-ylmethylene)-1-palmitoylpi-
peridin-4-one (E5). Yellow powder; yield: 51%; mp 84–
85 °C. 1H NMR (300 MHz; CDCl3): d 0.90 (t, J = 6.2 Hz, 3H, CH3);
1.24–1.27 (m, 24H, 12  CH2), 1.62 (quintet, J = 6.2 Hz, 2H, CH2),
2.45 (t, J = 6.4 Hz, 2H, COCH2), 5.00 and 5.05 (s, 2H each, 2  NCH2),
6.58 (br s, 2H, ArH), 6.77–6.78 (m, 2H, ArH), 7.54 (br s, 2H,
2  vinylic-H), 7.65 (br s, 2H, ArH). 13C NMR (75 MHz; CDCl3): d
14.09, 22.67, 25.34, 29.34, 29.47, 29.64, 29.67, 31.91, 33.38,
43.54, 46.12, 112.71, 118.31, 121.89, 123.59, 128.33, 145.69,
151.77, 172.28, 185.82. IR (KBr; mmax): 2949, 2920, 2848, 1647,
1613, 1567, 1469, 1256, 1245, 1169, 1093, 992, 774, 744,
622 cm1. UV (CHCl3; kmax): 385 nm. ESI-HRMS (amu): calcd
C31H43NO4 [M+Na]: 516.3090; found [M+Na]: 516.3047.
4.1.2.24. (3E,5E)-3,5-Bis(furan-2-ylmethylene)-1-stearoylpiperi-
din-4-one (E6). Yellow powder; yield: 86%; mp 82–84 °C. 1H
NMR (300 MHz; CDCl3): d 0.90 (t, J = 5.9 Hz, 3H, CH3); 1.25–1.28
(m, 28H, 14  CH2), 1.63 (quintet, J = 7.0 Hz, 2H, CH2), 2.44 (t,
J = 7.5 Hz, 2H, COCH2), 4.98 and 5.07 (s, 2H each, 2  NCH2),
6.55–6.60 (m, 2H, ArH), 6.78 (br s, 2H, ArH), 7.52 and 7.56 (s, 1H
each, vinylic-H), 7.65 (br s, 2H, ArH). 13C NMR (75 MHz; CDCl3): d
14.12, 22.69, 25.34, 29.37, 29.49, 29.66, 29.70, 31.93, 33.43,
43.49, 46.14, 112.66, 112.79, 118.03, 118.44, 121.84, 123.57,
128.17, 128.47, 145.55, 145.86, 151.78, 172.21, 185.91. IR (KBr;
mmax): 2952, 2920, 2847, 1643, 1617, 1577, 1543, 1463, 1444,
1254, 1198, 1171, 982, 757, 620 cm1. UV (CHCl3; kmax): 382 nm.
ESI-HRMS (amu): calcd C33H47NO4 [M+Na]: 544.3403; found
[M+Na]: 544.3359.
4.1.2.25. (3E,5E)-3,5-Bis(furan-2-ylmethylene)-1-oleoylpiperi-
din-4-one (E7). Brownish/yellow low melting solid; yield:
28%; 1H NMR (300 MHz; CDCl3): d 0.90 (t, J = 6.6 Hz, 3H, CH3),
1.10–1.40 (m, 22H, 11  CH2), 1.63 (m, 2H, CH2), 2.02 (m, 4H,
CH2), 2.44 (t, J = 7.6 Hz, 2H, COCH2), 4.98 and 5.07 (s, 2H each,
2  NCH2), 5.36 (m, 2H, Z vinylic-H), 6.58 (m, 2H, ArH), 6.78–6.79
(m, 2H, ArH), 7.52 and 7.56 (s, 1H each, 2  vinylic-H), 7.65 (br s,
2H, ArH). 13C NMR (75 MHz; CDCl3): d 14.12, 22.68, 23.49, 27.22,
27.79, 28.95, 29.00, 29.06, 29.33, 29.53, 29.65, 29.72, 29.77,
31.91, 39.69, 44.08, 46.33, 112.70, 112.91, 118.36, 118.66, 122.31,
123.75, 127.40, 127.85, 129.77, 129.97, 145.82, 145.99, 151.48,
151.70, 168.48, 185.35. IR (KBr; mmax): 3105, 2924, 2852, 1644,
1611, 1569, 1541, 1465, 1283, 1258, 1166, 1020, 988, 758,
622 cm1. UV (CHCl3; kmax): 383 nm. ESI-HRMS (amu): calcd
C33H43NO4 [M+Na]: 542.3246; found [M+Na]: 542.3198.
420 E. Potter et al. / Bioorg. Med. Chem. 23 (2015) 411–421
4.2. Biological evaluations
4.2.1. Cytotoxicity evaluations
The Molt 4/C8, CEM and L1210 assays were conducted using a
literature methodology.45 The data is reported in Table 1.
4.2.2. Cell culture
Human diploid lung fibroblast WI-38 cells were obtained from
Cederlane Labs (Burlington, ON, Canada) and cultured according
to the supplier’s instructions. The WI-38 cells were maintained in
75 cm2 culture flasks in a humidified incubator (VWR Interna-
tional, Mississauga, ON, Canada) at temperature of 37 °C. WI-38
cells were supplied with DMEM medium, supplemented with
10% fetal bovine serum (FBS) and 100 mg/L of penicillin and strep-
tomycin. The cells were used between passages 2–8, and the
growth medium of cultured cells was changed every 48 h.
4.2.3. Ferric reducing ability of plasma (FRAP) assay
The FRAP assay was performed according to the method
described earlier.15 The test compounds (20 lL) and FRAP working
reagent (180 lL) were added to 96-well microplates and absor-
bance was read at 590 nm using the FLUOstar OPTIMA plate reader
(BMG Labtech, Durham, NC, USA). The antioxidant capacity results
were expressed as lM TE L1 of solution. The data is reported in
Table 3.
4.2.4. Oxygen radical absorbance capacity (ORAC) assay
The ORAC assay was performed according to the method
described earlier with appropriate controls.15 The fluorescence sig-
nal was read at excitation and emission wavelength of 485 nm and
510 nm using the FLUOstar OPTIMA plate reader (BMG Labtech,
Durham, NC, USA). The antioxidant capacity results were expressed
as lM TE L1 of solution. The data is reported in Table 3.
4.2.5. The 2,2-diphenylpicrylhydrazyl (DPPH) assay
The DPPH assay was performed according to the method
described earlier.15 The test compounds were challenged against
0.2 mM DPPH solutions in 1:1 ratio in 96 well plates and the absor-
bance was measured using FLUOstar OPTIMA plate reader (BMG
Labtech, Durham, NC, USA) at 520 nm. The data is reported in
Table 3.
4.2.6. Cellular reactive oxygen species (ROS) detection assay
Cellular ROS was analyzed using Abcam’s cellular reactive oxy-
gen species detection assay kit (Abcam Inc, Toronto, ON, Canada).
The assay uses the cell permeant reagent 20 ,70 -dichlorofluorescin
diacetate (DCFDA), a fluorogenic dye that measures hydroxyl, per-
oxyl and other reactive oxygen species (ROS) activity within the
cell. The results were expressed as percentage inhibition of ROS
with respect to control cell using FLUOstar OPTIMA plate reader
(BMG Labtech, Durham, NC, USA) with maximum excitation and
emission spectra of 490 nm and 510 nm, respectively. The data is
reported in Table 3.
4.2.7. Tyrosinase inhibitor assay
Tyrosinase inhibition potential was determined using the mod-
ified dopachrome method with L-DOPA as substrate.15 Assay was
conducted in a 96-well microtitre plate using test compounds
and kojic acid (100 lM) as the reference compound. The results
were expressed as percentage inhibition of tyrosinase with respect
to the assay control. The data is reported in Figure 4.
4.2.8. In vitro toxicity analysis
In vitro toxicity analysis was conducted using the CytoTox-
ONE™ homogeneous membrane integrity assay (Promega Corpora-
tion, Madison, WI, USA). Briefly, 2  104 WI-38 cells were seeded in
a 96-well tissue culture plate. Cells were pretreated with the test
compounds for 24 h (100 lM) and placed in a humidified incubator
(VWR International, Mississauga, ON, Canada) for 24 h. Plates were
removed from incubator and LDH release assessment was per-
formed according to the manufacturer’s instructions. The mem-
brane damage was expressed as the percentage release of the
enzyme lactate dehydrogenase (LDH) with respect to the positive
control. The absorbance was measured using BioTek, Power wave
XS2 spectrophotometer (BioTek, Winooski, VT, USA) at emission
spectra of 490 nm. The data is reported in Figure 5.
4.2.9. Topoisomerase IIa activity assay
Topoisomerase IIa inhibitory activity was performed using a
commercial kit, (Topogen Inc, Columbus, OH, USA). The com-
pounds were challenged against 4 units of topoisomerase enzyme
using pHOT1 DNA as the substrate. All the preparatory and analysis
steps were conducted as per the manufacturer’s instructions and
all samples (10 lM) were loaded directly onto a 2% agarose gel
in 1 TBE (Tris/Borate/EDTA) running buffer. In addition to the test
compounds, sorafenib was also used as a standard drug in the
assay. The DNA bands were visualized by placing gels on a UV sam-
ple tray, followed by exposure to UV light using gel Doc™ EZ Sys-
tem (Biorad, ON, Canada).
4.2.10. Biostatistics
All the biological analysis was conducted using completely ran-
domized design (CRD) of experiments. One way analysis of vari-
ance (ANOVA) was performed to analyse all data from biological
assays using a commercial software package.47 Data was consid-
ered significant at p60.05 and the means were compared using
Tukey’s multiple means comparison test. All the analyses were per-
formed in triplicates.
4.2.11. Statistical analyses using electronic and physicochemical
parameters
Electronic and physicochemical properties such as charge b,
partition coefficient (log P), and molar refractivity of the molecule
were calculated using a commercial software.46 The correlations
between these molecular descriptors and the IC50 values of the
three cell lines (Table 1) were generated using a commercial soft-
ware package.47 All significant correlations are reported in Table 2.
Acknowledgments
The authors thank the following agencies for financial support,
namely the Nova Scotia Health Research Foundation (A.J., E.P.),
Natural Sciences and Engineering Research Council (NSERC) of
Canada (A.J.), Fonds voor Wetenschappelijk Onderzoek-Vlaanderen
(J.B.). Appreciation is extended to Mrs. Lizette van Berckelaer for
conducting the HeLa, CEM and L1210 assays. H.P.V.R. gratefully
acknowledges funding from the Canada Research Chair program
and the Discovery Grant program of the Natural Sciences and Engi-
neering Research Council (NSERC) of Canada.
Supplementary data
Supplementary data (spectroscopic data) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmc.2014.12.042.
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