Curcumin-inspired cytotoxic 3,5-bis(arylmethylene)-1-(N-(ortho-substituted aryl)maleamoyl)-4-piperidones: A novel group of topoisomerase II alpha inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.08.023

Three series of novel 3,5-bis(arylmethylene)-1-(N-(ortho-substituted aryl)maleamoyl)-4-piperidones, designed as simplified analogs of curcumin with maleic diamide tether, were synthesized and bioevaluated. These compounds displayed potent cytotoxicity tow

Full text of DOI:10.1016/j.bmc.2015.08.023

Accepted Manuscript
Curcumin-inspired cytotoxic 3,5-bis(arylmethylene)-1-(N-(ortho-substituted
aryl)maleamoyl)-4-piperidones: A novel group of topoisomerase II alpha in-
hibitors
Amitabh Jha, Katherine M. Duffield, Matthew R. Ness, Sujatha Ravoori,
Gabrielle Andrews, Khushwant S. Bhullar, H.P. Vasantha Rupasinghe, Jan
Balzarini
PII:
S0968-0896(15)30005-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.08.023
BMC 12527
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 May 2015
10 August 2015
20 August 2015
Please cite this article as: Jha, A., Duffield, K.M., Ness, M.R., Ravoori, S., Andrews, G., Bhullar, K.S., Vasantha
Rupasinghe, H.P., Balzarini, J., Curcumin-inspired cytotoxic 3,5-bis(arylmethylene)-1-(N-(ortho-substituted
aryl)maleamoyl)-4-piperidones: A novel group of topoisomerase II alpha inhibitors, Bioorganic & Medicinal
Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.08.023
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Curcumin-inspired cytotoxic 3,5-bis(arylmethylene)-1-(N-(ortho-
substituted aryl)maleamoyl)-4-piperidones: A novel group of
topoisomerase II alpha inhibitors
Amitabh Jha,a,  Katherine M. Duffield,a Matthew R. Ness,a Sujatha Ravoori,a Gabrielle
Andrews,a Khushwant S. Bhullar,b H.P. Vasantha Rupasinghe,b and Jan Balzarinic
a Department of Chemistry, Acadia University, Wolfville, NS, Canada
b Department of Environmental Sciences, Faculty of Agriculture, Dalhousie University,
Truro, NS, Canada
c Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
ABSTRACT
Three series of novel 3,5-bis(arylmethylene)-1-(N-(ortho-substituted aryl)maleamoyl)-4-
piperidones, designed as simplified analogs of curcumin with maleic diamide tether, were
synthesized and bioevaluated. These compounds displayed potent cytotoxicity towards
human Molt 4/C8 and CEM T-lymphocytes as well as murine L1210 leukemic cells. In
contrast, the related N-arylmaleamic acids possessed little or no cytotoxicity in these
three screens. Design of these compounds was based on molecular modeling studies
performed on a related series of molecule in a previous study. Representative title
compounds were found to be significantly potent in inhibiting the activity of
topoisomerase II alpha indicating the possible mode of action of these compounds. These
compounds were also potent antioxidants in vitro and attenuated the AAPH triggered
peroxyl radical production in human fibroblasts. Various members of these series were
also well tolerated in both in vitro and in vivo toxicity analysis.
Keywords: curcumin analogs; 4-piperidones; maleamic acids; cytostatic activity; QSAR;
topoisomerase IIα inhibition.
 Corresponding author. Tel.: +1 902 585 1515; Fax +1 902 585 1414; E-mail: ajha@acadiau.ca.
1

1. Introduction
Curcumin (Figure 1) is a natural product isolated from Curcuma longa, commonly
known as turmeric, one of the most commonly used spices worldwide [1]. Scientists have
intensively studied this compound over the decades and a number of its pharmacological
aspects such as anti-inflammatory, antitumor, anti-angiogenic, antioxidant, antibacterial,
cardioprotective and neuroprotective properties have been documented [2-6]. More than
one hundred clinical trials have been/are being conducted on this natural compound [7],
including those 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 disease [13]. As commonly observed in dietary phytochemicals,
curcumin displays an excellent safety profile with negligible toxicological
manifestations; however, its clinical efficacy is marred with its poor bioavailability which
has prevented this molecule from culminating into an efficacious clinical drug [14].
Continuous efforts in our lab are focused on analogs of curcumin in attempts to
develop molecules with superior pharmacodynamic and pharmacokinetic profile than
those of curcumin [15-27]. Among our previous research, we have prepared and studied
curcumin analogs where the diarylheptanoid skeleton of curcumin was retained (Series 1-
3, Fig. 1) [15-18]. These studies involved evaluation of anticancer, antioxidant, anti-
hypertensive, and/or anti-HIV properties of these compounds. It was observed in these
investigations [15-18] that compounds retaining the oxygenation pattern similar to that of
curcumin were found to be more active in terms of their anticancer and antioxidant
potency in-vitro compared to the other analogs. Simultaneously, we have also studied a
large number of diarylpentanoids (Series 4-10, Figure 1) [19-24], which may be viewed
as simplified analogs of diarylheptanoid curcumin. Curcumin as well as its analogs (1-10)
contain α,β-unsaturated carbonyl group(s) and therefore are Michael acceptors with
selective affinity for protein thiols as opposed to O/N-based nuclephiles found on genetic
materials [25, 28]. Notably, the analogs from series 5-10 contain additional enone moiety
in their side chains bearing for interaction with auxiliary binding domain [20] and/or
protein thiolation [25, 28].
2

Figure 1: Molecular structures of curcumin and its previously studied analogs.
The parent diarylheptanoids, namely 3,5-bisarylmethylene-4-piperidones 4, and their
derivatives (5-10) (Figure 1) were consistently found to possess higher level of in vitro
cytotoxicity than diarylheptanoids 1-3. Most of these compounds displayed cytotoxicity
in single digit to sub-micromolar inhibitory concentrations against the cancer cell lines
tested.
We have identified human topoisomerase IIα as a potential molecular target for
3,5-bisarylmethylene-4-piperidone derivatives [22]. This thiol-rich enzyme is essential
for sustained cell proliferation [29-31]. This enzyme releases torsional strain in DNA
double helical structure through transient double strand breaks facilitating segregation of
the strands, allowing replication and transcription processes to occur [32]. Catalytic
inhibition of these processes halts replication and transcription, eventually arresting the
cell growth. Also, if the enzyme inhibition occurs by poisoning, the nicks in the DNA
strands created by the enzyme cannot re-ligate resulting in lethal lesions in the DNA
frame work, which then leads to apoptosis [29, 30]. Since the expression of
3

topoisomerase IIα is amplified in cancerous cells, targeting this enzyme by protein-
thiolators thus offers a viable pathway of selectivity targeting malignancies.
Of a particular relevance to this report are series 9 compounds ((Z)-4-((3E,5E)-
3,5-bisbenzylidene-4-oxopiperidin-1-yl)-N-aryl-4-oxobut-2-enamides, Figure 1) where
substituted anilines were conjugated to the parent 3,5-bisarylmethylene-4-piperidones via
maleamic diamide moiety [21]. The rationale behind their design was as follows: a)
incorporation of an additional enone functionality terminating with an aryl carbomoyl
group (as opposed to a proton) that will elicit higher electrophilicity to the enone towards
thiol group than the corresponding unsubstituted acryloyl group; b) the placement of
different substituents on the aryl ring adjacent to carbomoyl group would further alter the
polarity of the enone functionality; and c) the Z-geometry of the maleamic diamide
moiety will offer minimum impedance to the approach of the cellular thiol. All
compounds of series 9 were found to show remarkable in vitro cytostatic activity; the
most active of them all was found to be (Z)-4-((3E,5E)-3,5-dibenzylidene-4-
oxopiperidin-1-yl)-N-(2,6-dimethylphenyl)-4-oxobut-2-enamide with sub-micromolar
IC50 towards three out of four representative cancer cell lines tested. Among tested
compounds, this was the only compound with ortho- substitutions on the aniline ring
[21]. As a guided amplification of this project, we decided to synthesize a larger library
of series 9-type compounds with ortho- substituents on the aniline ring. The designed
compounds for this investigation can be broadly classified into three series (9Aa-i, 9Ba-i
and 9Ca-i; Scheme 1) based on substitution on the aryl ring (unsubstituted, 4-nitro- and
4-carboethoxy-) of the 3,5-bisarylmethylene-4-piperidone moiety. Since these
compounds are predicted to act, at least in part, via protein thiolation, electron
withdrawing groups like 4-nitro- and 4-carboethoxy- on the arylmethylene moiety should
impart higher electrophilicity to the enone system towards protein thiolation in
comparison to the corresponding phenylmethylene analogs. Overall, this study was
conducted with the aim of assessing the anti-cancer activity of these novel compounds in
comparison to parent molecules along with other concomitant pharmacological
properties. We herein report potent cytostatic, antioxidant, human topoisomerase IIα
inhibitory activities and toxicity of 27 such compounds.
4

2. Results and discussion
2.1. Chemistry
Synthesis of series 9 compounds was accomplished by following previously reported
procedure [23]. Reaction of 4-piperidone hydrate hydrochloride with two equivalents of
aromatic
aldehydes
(benzaldehyde,
4-nitrobenzaldehyde
and
4-
carboethoxybenzaldehyde) in acetic acid under the influence of dry HCl gas resulted in
formation of 3,5-diarylmethylenepiperidone acid salts (4A-C) as bright yellow or orange
precipitate. These compounds were then individually reacted with acryloyl chloride under
basic conditions to produce compounds 6A-C. Maleanilinic acids 11a-i were synthesized
by reacting maleic anhydride with corresponding substituted anilines in dichloromethane.
Maleanilinic acids 11a-i were activated by reacting them with ethyl chloroformate, and
triethylamine in THF. To these solutions, compounds 4A, 4B and 4C were then
individually added to yield compounds of series 9A, 9B and 9C, respectively (Scheme 1).
All products were purified by digestion in methanol or by column chromatography and
were then completely characterized by spectroscopic means. Compounds 6C, 9Aa-f,h,i,
9Ba-i and 9Ca-i are new to the chemical literature. Based on spectroscopic data, the
double bond geometry across the dibenzylidene system was found to be E, and that
across maleic diamide was found to be Z, as expected [21]. It was interesting to note that
all compounds of series 9 showed more peaks in the 13C NMR spectra than expected
based on C2 symmetry in the molecule along piperidone >C=O and N axis. We have
observed this phenomenon previously [33].
It meant that these compounds
predominantly occupy a non-symmetrical conformation due to the electron delocalization
on piperidone amide.
5

Scheme 1: Synthetic routes for the preparation of series 4, 6, 9 and 11 compounds.
Conditions: (i) HCl, AcOH; (ii) ClCOCH=CH2, N(C2H5)3, DCM; (iii) N(C2H5)3,
ClCOOC2H5/11a-i, THF.
2.2. Biological evaluations
2.2.1. Cytostatic activity
First, the cytostatic activities of compounds 4C, 6C, 9Aa-i, 9Ba-i, 9Ca-i and 11a-i were
+
evaluated against human CD4 T-lymphocyte Molt4/C8 and CEM cells as well as murine
L1210 lymphocytic leukemia cells following a literature procedure [34]. The results are
presented in Table 1 in four different groups for comparison purposes: 1) Group A: 3,5-
diphenylmethylene-4-piperidone (4A) and its derivatives (6A, 9Aa-i); 2) Group B: 3,5-
di(4-nitrophenylmethylene)-4-piperidone (4B) and its derivatives (6B, 9Ba-i); 3) Group
C: 3,5-di(4-carboethoxyphenylmethylene)-4-piperidone (4C) and its derivatives (6C,
9Ca-i); and 4) Group D: substituted maleanilinic acids (11a-i). Clinical anticancer drug
melphalan (alkylating agent) and curcumin were used as reference drugs.
With regards to the cytostatic activity, a few general observations will be stated
first. It is clearly evident from the data in Table 1 that nearly all the compounds tested
against three representative cancer cell lines are potent anticancer agents as well as
promising lead molecules for further development. Baring the parent compounds 4A-C,
all the other compounds tested have shown superior cytostatic activity than melphalan
and curcumin against each of the cancer cell lines. Approximately 75% of the IC50 data
reported in Table 1 are in submicromolar range. Human T-lymphocyte CEM cells turned
6

out to be the most sensitive cell line to the tested compounds (average IC50 0.27 µM for
series 9 compounds) while the least sensitive of the three was murine leukemic L1210
cells (average IC50 2.14 µM for series 9 compounds). Among the three groups, group B
compounds with 4B (R=NO2) as parent molecule emerged as most potent (average IC50
0.29 µM for three cell lines) but least selective (average selectivity ratio SI 2.6) series of
molecules. By the same token, group C compounds with 4C (R=CO2Et) as parent
molecule came out as most selective with average SI value of 15.3 and displayed
remarkably high potency against CEM cells (average IC50 0.17 µM). These observations
support the assertion that electron withdrawing groups on the aryl ring (e.g. 4-NO2 on 4B
and 4-CO2Et on 4C) improve the cytostatic activity presumably by increasing the
electrophilicity of the enone functionality towards cellular nucleophiles. Considering all
data reported in Table 1, compound 9Cb, formed by conjugation of 4C and 11b, showed
the lowest IC50 value of 89 nM against CEM cells.
Compounds 6A-C, the acryloyl amides of 4A-C, showed marked improvement in
their cytostatic activity as compared to their parent molecule, especially in group B and C
(Table 1). This gives confidence to the hypothesis that incorporation of an additional
enone functionality in the molecules improves the cytostatic activity. However, with the
replacement of one of the hydrogens from the terminal position of acryloyl group with N-
arylcarbamoyl group imparting Z geometry to the double bond (series 9A-C compounds)
has led to further increase in the cytostatic potential in most cases. As mentioned earlier,
series 9A-C were designed to include ortho-substituents on the arylamine rings as such a
modification appeared to improve the anticancer potential [21]. When the cytostatic
activities of series 9A is compared to that of series 9 compounds previously reported with
no, meta- and/or para-substitution(s) on the arylamine rings [21], a significant
improvement in the cytostatic activity was noticed. This gives positive support to our
hypothesis. As the ortho- substituents are known to impart electronic as well as steric
effects on the molecule, it is believed that the 3D topology of the resulting molecules is
significantly altered leading to better binding with the molecular target. This will be
discussed further in the QSAR section. Compounds of series 9 can be viewed as
diamides of maleic acid. One may argue that their cytostatic activity could be due to two
electrophilic molecules (the parent 4 and the corresponding N-arylmaleamic acid 11)
7

after the hydrolysis of the amide at the piperidyl nitrogen atom. This seems highly
unlikely for the following reasons. First, most of the series 9 compounds are significantly
more potent than the parent molecule 4. Secondly, the data in Table 1 reveals that N-
arylmaleamic acids 11a-i (Group D) are essentially inactive as cytostatic agents (IC50
value > 100 µM in all cases). Encouraged by these results, compounds 9Aa, 9Ad and
9Af were also evaluated against NCI’s 56 human tumor cell lines representing nine
different neoplastic conditions, namely breast, central nervous system, colon, leukemia,
melanoma, non-small cell lung, ovarian, prostate and renal [35]. The data generated are
summarized in Table 2.
Table 1. Evaluation of compounds from series 4, 6, 9 and 11 against human Molt-4/C8
and CEM T-lymphocytes and murine L1210 leukemic cells
IC50 (μM)
Compound
SRa
4.8
6.1
4.9
6.9
10.5
4.0
6.7
4.8
10.8
5.2
10.8
7.4
2.8
3.0
1.4
1.9
2.2
Molt 4/C8
1.67±0.15
1.42±0.27
0.53±0.18
0.82±0.56
0.42±0.13
0.43±0.05
0.90±0.63
0.44±0.06
0.40±0.04
0.44±0.05
0.43±0.07
8.28±0.75
0.15±0.06
0.12±0.06
0.27±0.02
0.26±0.15
0.27±0.13
0.37±0.05
0.28±0.09
0.21±0.13
0.15±0.06
0.19±0.08
1.1±0.7
CEM/0
1.70±0.02
1.48±0.34
0.48±0.07
0.59±0.22
0.46±0.06
0.42±0.01
0.54±0.18
0.40±0.01
0.39±0.05
0.42±0.03
0.37±0.06
4.47±2.28
0.26±0.02
0.14±0.09
0.23±0.20
0.22±0.20
0.15±0.09
0.37±0.07
0.18±0.12
0.11±0.05
0.10±0.04
0.12±0.07
1.0±0.7
L1210
7.96±0.11
8.69±0.73
2.1±0.3
4.1±3.0
4.4±3.0
1.7±0.1
3.6±0.6
1.9±0.1
4.2±2.8
4Ab
6Ab
9Aa
9Ab
9Ac
9Ad
9Ae
9Af
9Ag
9Ah
9Ai
2.2±0.3
4.0±2.4
4Bb
32.9±4.2
0.42±0.07
0.36±0.03
0.38±0.02
0.41±0.04
0.33±0.05
1.1±0.6
0.36±0.00
0.35±0.01
0.39±0.01
0.33±0.05
6.9±0.5
6Bb
9Ba
9Bb
9Bc
9Bd
9Be
9Bf
9Bg
9Bh
9Bi
3.0
2.0
3.2
3.9
2.8
6.9
4C
0.35±0.06
1.5±1.1
0.16±0.02
0.30±0.05
0.089±0.061
0.11±0.05
0.12±0.09
0.12±0.01
0.22±0.19
0.28±0.17
0.16±0.13
1.7±0.07
8.1±0.8
1.7±0.1
1.7±0.1
1.6±0.1
1.1±0.1
5.0±1.2
3.5±1.3
2.5±0.9
10.6
27.0
19.1
15.5
9.2
13.3
22.7
12.5
15.6
6C
9Ca
9Cb
9Cc
9Cd
9Ce
9Cf
0.59±0.38
0.67±0.40
0.36±0.08
0.29±0.03
1.1±0.6
0.95±0.47
0.67±0.39
9Cg
9Ch
8

0.95±0.47
365±0
228±27
>500
134±23
353±6
>500
0.28±0.17
480±29
279±3
>500
189±10
426±28
>500
>500
>500
>500
2.6±1.2
15 ± 5
3.5±1.3
333±13
251±23
>500
100±2
395±22
>500
>500
>500
>500
5.8±0.3
22 ± 1
12.5
NCc
NC
NC
NC
NC
NC
NC
NC
NC
3.9
9Ci
11a
11b
11c
11d
11e
11f
11g
11h
>500
>500
>500
11i
Melphalan
Curcumin
3.24±0.79
9.0±1.9
2.4
a SR indicates the selectivity ratio, i.e., the ratio between the highest and lowest IC50
values of either the T-lymphocytes or murine leukemic cells.
b Reprinted in part from literature [36].
c NC: Not calculated.
The results shown in Table 2 demonstrate that when all cell lines are considered,
each of the series 9A compounds tested are significantly more cytostatic than melphalan
and curcumin. An essential characteristic of a potential anticancer drug is selective
toxicity for certain tumor cell types rather than them being indiscriminately cytostatic.
The selectivity index (SI) values for compounds 9Aa, 9Ad and 9Af for all the tumor cell
lines reveal their wide differential sensitivity towards human tumor cell types; each of
these compounds boasted impressive selectivity (SI for 9Aa: >3236; SI for 9Ad: >1738;
SI for 9Af: 1318). A comparative review of the GI50 values with respect to various cell
line classes in Table 2 revealed that each of these three compounds exerted greater
toxicity against leukemic, colon and prostate cancer cell lines than against those
representing other cancers. Even among leukemic cell lines, compounds 9Aa and 9Ad
show GI50 in low nM range (60 and 20 nM respectively). Melphalan is used in
combination chemotherapy to treat chronic leukemias along with wide range of other
malignancies [37]. The results in Table 2 indicate that 9Aa and 9Ad are respectively, 93
and 280 times more potent than melphalan against the leukemic cell lines in vitro. The
novel findings that the compounds possess significant potencies and preferential
cytotoxicity for certain tumor cells suggests that compounds of series 9 studied here are
promising candidate anticancer agents with selective toxicity.
9

Table 2. Evaluation of compounds 9Aa, 9Ad and 9Af and reference compounds against
a panel of human tumor cell lines
Cell lines
Leukemia
No.a
3
Melphaland Curcumine
9Aa
0.06
6.61
2.04
3.86
3.60
5.66
2.97
2.40
4.43
2.47
9Ad
9Af
GI50 (µM)b
GI50 (µM)
GI50 (µM)
GI50 (µM)
GI50 (µM)
GI50 (µM)
GI50 (µM)
GI50 (µM)
GI50 (µM)
GI50 (µM)
SIc
0.02 0.24
3.70 1.80
0.67 0.43
2.00 1.30
1.60 1.20
3.30 1.40
2.30 1.70
0.63 0.30
1.50 0.88
1.20 1.00
5.6
25.6
48.9
27.4
33.0
41.1
32.2
35.5
33.5
24.6
79
3.7
9.2
4.7
5.8
5.9
9.1
8.9
11.2
8.1
6.7
10
Lung cancer
Colon cancer
CNS cancer
Melanoma
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
All cell lines
9
7
6
9
7
8
2
5
56
>3236 >1738 1318
a Number of individual cells lines tested representing the respective type of cancer.
c SI refers to the selectivity index. The SI figures for all cell lines were obtained by
dividing the GI50 values of the least and most sensitive cells.
d GI50 values for melphalan were obtained from online NCI database (COMPARE data
vector search, compound ID NSC 8806).
e GI50 values for curcumin were obtained from online NCI database (COMPARE data
vector search, compound ID NSC 32982).
2.2.2. Topoisomerase IIα inhibitory activity
Human topoisomerase IIα (topo IIα) has been validated as a clinically important target
for cancer drug development as it is a vital for chromosome segregation and cell division
in cancer cells [38]. Multiple topo IIα inhibitors have been developed as antitumor agents
and are already in clinical use for decades. The series of compounds 4A-C, 6A-C, 9Aa-i,
9Ba-i and 9Ca-i containing a pharmacophore similar to curcumin in the current
investigation were found to inhibit human topo IIα activity. Topo IIα inhibition assays
were performed using a commercial human DNA topoisomerase IIα inhibition kit,
according to the manufacturer's instructions (Topogen Inc, Columbus, OH). A
representative gel picture displaying the topoisomerase IIα inhibition potential of the
compounds 4A-C, 6A-C, 9Aa-i, 9Ba-i and 9Ca-i is presented in Figure 2 A-C.
Etoposide, sorafenib and cisplatin were used as reference drugs. Sorafenib, a multi-target
kinase inhibitor, is also known to inhibit topoisomerase IIα in-vitro [39, 40] and in-vivo
[41]. The results from this evaluation showed that all the assayed compounds, including
10

reference drugs (50 µM), exhibited strong inhibitory activity towards topo IIα in vitro.
The gel analysis showed that most compounds displayed a weak linear DNA band in gel,
thus exhibiting characteristics of interfacial topoisomerase poisons. Other compounds
exhibited catalytic inhibition of topo IIα in vitro. Additionally, the topo IIα inhibitory
effects of compounds 9Ad-9Ai, 4B, 6B were much stronger than the other compounds,
and that of the etoposide and the standard drugs at 50 µM concentrations. However,
compounds 9Cc-9Ch displayed a catalytic inhibitory activity as they blocked the activity
of topo IIα directly (no relaxed DNA but strong supercoiled plasmid, Fig. 2C). Overall,
the results revealed that all the tested compounds exhibited concrete influence on topo IIα
activity at low concentrations. In summary, we established that the compounds 4A-C,
6A-C, 9Aa-i, 9Ba-i and 9Ca-i demonstrated anticancer activity through the check on
topo IIα activity, possibly by weakening the enzyme activity via excessive supercoiling,
which proved detrimental for cancer cell proliferation.
A
B
Nicked Open DNA
Linear band
Supercoiled
4A 6A 9Aa 9Ab 9Ac 9Ad 9Ae 9Af 9Ag 9Ah 9Ai 4B 6B
9Ba 9Bb 9Bc 9Bd 9Be 9Bf 9Bg 9Bh 9Bi 4C 6C 9Ca 9Cb
D1 D2 E
L
K
D1 D2
E
L
K
C
9Cc 9Cd 9Ce 9Cf
9Cg 9Ch
9Ci
E
D1
Figure 2. Topo IIα inhibitory activities of 3,5-bis(arylmethylene)-1-(N-arylmaleamoyl)-4-
piperidones and related compounds. (A) The compounds were examined in a final
concentration of 50 µM, respectively, as designated. Lanes 4A-6B: pHOT1 DNA + 4 U
topo IIα + test compounds in designated concentrations; Lane D1: pHOT1 DNA+ 4 U
topo IIα + sorafenib; Lane D2: pHOT1 DNA + 4 U topo IIα + cisplatin; Lane E: pHOT1
DNA + 4 U topoII + etoposide; Lane L: Marker DNA, Linear pHOT1, Lane K: pHOT1
DNA, supercoiled. (B) The compounds were examined in a final concentration of 50 µM,
respectively, as designated. Lanes 9Ba-9Cb: pHOT1 DNA + 4 U topo IIα + test
11

compounds in described concentrations; Lane D1: pHOT1 DNA+ 4 U topo IIα +
sorafenib; Lane D2: pHOT1 DNA + 4 U topo IIα + cisplatin; Lane E: pHOT1 DNA + 4
U topoII + etoposide; Lane L: Marker DNA, Linear pHOT1, Lane K: pHOT1 DNA,
supercoiled. (C) The compounds were examined in a final concentration of 50 µM,
respectively, as designated. Lanes 9Cc-9Ci: pHOT1 DNA + 4 U topo IIα + test
compounds in described concentrations; Lane E: pHOT1 DNA + 4 U topoII + etoposide;
Lane D1: pHOT1 DNA + 4 U topo IIα + sorafenib.
2.2.3. Anti-oxidant activity
In order to evaluate compounds 4A-C, 6A-C, 9Aa-i, 9Ba-i and 9Ca-i as potential anti-
oxidant agents, we conducted in vitro tests using four antioxidant assays, viz. FRAP
(ferric-reducing ability of plasma), ORAC (oxygen radical absorbance capacity), DPPH
(2,2-diphenylpicrylhydrazyl anti-radical assay) and Cell-ROS assay (cellular reactive
oxygen species detection assay) as described in experimental section below. Table 3
summarizes the anti-oxidant and cytoprotective activity data obtained for these 33
compounds as well as curcumin at 100 µM concentrations. As observed before [33],
curcumin exhibited the highest antioxidant activity in all the four antioxidants assays
conducted in vitro (p≤0.05).
According to the FRAP assay results, all of the assayed compounds displayed
active FRAP activity and none of the compounds failed the FRAP activity test. Among
the synthesized compounds, 9Aa (476.8 µM TE/L) showed the strongest in-vitro
antioxidant activity in comparison to the other compounds (p≤0.05). Still, compared to
curcumin, ~3-fold lower anti-oxidant activity was exhibited by compound 9Aa in vitro. It
was closely followed by compounds 9Ab-9Ae in their ability to reduce ferric-
tripyridyltriazine (Fe3+-TPTZ) to its blue, ferrous form (Fe2+-TPTZ). On the contrary,
compound 4B exhibited the weakest anti-oxidant activity (19.2 µM TE/L) while the
compounds 4A (34.3 µM TE/L) and 9Bg (53.8 µM TE/L) closely preceded compound
4B in their in vitro anti-oxidant potential (p≤0.05). It was also observed that anti-oxidant
activities of the compounds 9Bd and 9Cf were ~three-fold weaker in comparison to the
active compound 9Aa (p≤0.05). Overall, the derivatives of the parent molecules indicated
stronger antioxidant activities, which indicate the positive influence of structural
modification on the biological properties of the compounds. The presence of higher
number of phenyl ring in compounds 9Aa improved its antioxidant activity in
comparison to the precursor molecule 4A. The peroxyl radical scavenging activities of
12

these compounds were also assessed by the ORAC assay using fluorescence as the
fluorophore, while curcumin was used as the reference index. Curcumin remained the
strongest inhibitor of fluorescence degradation by APPH injury in ORAC assay in vitro.
Interestingly, compounds 9Ad and 9Ae, which exhibited strong activity in FRAP
analysis, likewise displayed the strongest ORAC activity (215.3-222.9 µM TE/L) in
comparison to other compounds in vitro (p≤0.05). These were followed by compounds
9Cc, 6A, 9Ac, 9Ba, 9Bh and 9Cb (p≤0.05), which previously indicated active
antioxidant capacity in the FRAP analysis. Similar to the FRAP analysis, 9Bg
demonstrated the weakest antioxidant activity in vitro (p≤0.05). Conversely, weak FRAP
based anti-oxidant candidates like compounds 4A and 4B displayed much stronger anti-
oxidant potential in ORAC analysis (p≤0.05); on the other hand, in the same comparison,
compound 9Ai lost about 3-folds of its anti-oxidant potential in vitro. However, the
remaining compounds displayed similar anti-oxidant capacity to FRAP analysis in vitro
(p≤0.05). These results also supported the effect of structural modification as discussed in
FRAP analysis, particularly the compounds 9Ad and 9Ae with unique substituents at
positions 2 (R1= OCH3 and NO2) and/or 6 (R2=H).
Additionally, the in vitro anti-oxidant activity of these compounds was also
determined by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay (Table 3), which
estimates anti-oxidant activity by HAT mechanism. The compounds 9Bd and 9Bf
exhibited the strongest anti-radical scavenging activity, analogous to curcumin in vitro
(p≤0.05). The structure of these compounds was similar to 9Ad and 9Ae with similarly
substituents at positions 2 (R1= OCH3 and Cl) and/or 6 (R2=H). Likewise, assay of
compound 9Ae resulted in the smallest DPPH radical trapping potential, also in line with
FRAP results, but in contradiction with ORAC analysis in vitro (p≤0.05). Finally, the
ability of the test compounds to inhibit production of cellular ROS was assessed in a WI-
38 cell model system. The results showed that the strongest peroxyl radical scavenging
activity was displayed by compounds 9Bf, 9Bi and 9Ce in vitro (p≤0.05). These top
antioxidants were closely followed by multiple compounds in the ranges 9Be-i and 9Ca-
g, thus demonstrating strong cytoprotective activities of these series of assayed
compounds in vitro (p≤0.05). Weak ROS inhibitory activity was exhibited by compounds
of series 9A and compounds 9Ch and 9Ci, which showed ~half cytoprotective ability
13

compared to curcumin in vitro (p≤0.05). Such results can be related to the presence of
CH3 as key substituent substitutions at positions 2 (R1) and/or 6 (R2) in these
compounds. For compounds 9Aa, 9Ad, 9Ae and 9Bd, which topped the antioxidant
activity charts, their ROS scavenging ability was frail in cytoprotection analysis in vitro
(p≤0.05). Overall, the ROS inhibition results were generally similar to FRAP and DPPH
analysis with the exceptions of compounds 9Ae, 9Ag, 9Ah and 9Bc, which showed
<50% ROS inhibition in vitro. In summary, many compounds of these series were active
antioxidants but curcumin remained the strongest antioxidant compound in the current
investigation.
Table 3. Antioxidant activity evaluation of 3,5-bis(arylmethylene)-1-(N-
arylmaleamoyl)-4-piperidones (4A-C, 6A-C, 9Aa-i, 9Ba-i and 9Ca-i) and cytoprotective
agents under four assay systems.
Compound
FRAP
ORAC
DPPH
ROS
(µM TE/L)
34.3±2.3pq
149.2±5.7kl
476.8±6.8a
444.3±12.0b
432.4±13.2b
434.5±11.1b
441.4±4.5b
202.6±4.6ij
277.1±3.0e
259.7±2.6ef
302.1±5.7d
19.2±1.4q
(µM TE/L)
92.7±1.9ijkl
181.7±7.3bc
116.5±4.8fghij
134.6±3.3def
173.7±4.5bc
222.9±2.2a
(% inhibition)
52.5±2.6op
62.4±4.1jklmn
52.3±2.3op
53.3±1.0op
60.4±2.7lmno
54.9±1.8nop
49.5±2.8p
(% inhibition)
63.6±2.5cdefg
56.4±1.5fghi
58.2±0.9efgh
54.9±3.5fghij
52.2±2.9hij
54.5±0.8ghij
49.5±0.4hij
51.4±2.4hij
48.3±1.6ij
4A
6A
9Aa
9Ab
9Ac
9Ad
9Ae
9Af
9Ag
9Ah
9Ai
4B
215.3±6.4a
120.8±14.6efghi
161.8±9.4bcd
115.7±7.7fghijk
88.1±4.7jklm
104.2±3.6fghijk
95.3±2.0hijkl
176.0±5.3bc
92.9±4.5ijkl
92.3±4.1ijkl
70.10±3.2lm
105.6±6.6fghijk
68.3±0.8lm
69.1±3.3hijk
68.4±3.0hijkl
82.8±2.0cde
78.6±1.3defg
76.1±2.6efgh
76.4±4.6efgh
68.2±3.4hijklm
87.1±2.6bc
85.6±0.7bcd
96.8±1.5a
49.7±2.9hij
51.4±1.2hij
64.1±3.6cdef
61.7±2.3defg
65.9±2.6bcde
65.6±2.8bcde
63.5±0.4cdefg
69.1±2.4cbcd
71.4±4.1abc
77.7±4.7a
121.7±4.5mn
232.3±3.4gh
134.8±5.3lmn
195.8±2.7j
100.8±2.3o
120.5±2.4mno
140.5±5.8klm
53. 8±4.8p
245.4±7.4fg
153.7±4.4kl
160.7±2.9k
208.5±3.6ij
317.3±8.6d
191.3±8.8j
347.8±5.5c
239.9±3.8fgh
6B
9Ba
9Bb
9Bc
9Bd
9Be
9Bf
9Bg
9Bh
9Bi
4C
81.8±0.7cde
92.2±1.2ab
86.8±1.6bc
82.8±3.5cde
80.6±1.9cdef
68.6±1.8hijkl
54.7±2.0nop
59.1±1.1mno
68.4±1.2hijklm
57.5±3.9nop
66.8±5.2ijklm
60.5±5.4m
71.1±0.7abc
69.1±0.4abcd
77.9±4.2a
177.9±11.0bc
128.7±8.8efg
67.1±5.8lm
73.9±5.9ab
70.8±2.7abcd
69.1±2.6abcd
69.7±4.6abcd
67.8±3.6bcd
67.3±2.2bcde
150.4±8.0cde
180.5±16.5bc
124.2±10.9egfhi
191.4±9.8ab
119.1±2.1efghi
6C
9Ca
9Cb
9Cc
9Cd
14

305.1±9.7d
115.7±8.6no
220.9±4.8hi
245.1±6.7fg
251.5±3.6fg
1201.4±0.2
121.1±10.1efghi
73.8±4.4klm
125.9±4.3efgh
97.1±6.4ghijkl
66.9±8.3lm
56.6±2.0nop
70.7±2.8ghij
60.7±2.4klmno
71.4±1.3ghi
72.1±3.5fghi
96.8±1.3
77.7±4.5a
72.6±2.3abc
69.6±1.2abcd
46.9±1.5j
47.2±0.2j
98.7±0.4
9Ce
9Cf
9Cg
9Ch
9Ci
1581.9±28.4
Curcumin
Means ± standard deviation, followed by the different superscripted letters (a through q) within
column are significantly different, Tukey's multiple means comparison test, p≤0.05). The
compounds were assayed at 100 µM concentration.
2.2.4. In-vitro toxicology analysis
The in vitro cell culture model was used to screen the test compounds based on their
toxicity by assessing parameters such as cell viability and membrane damage, succeeding
incubation with the compounds for 12 h. These parameters were assessed by the MTS
cell viability and LDH cytotoxicity assays (Table 4), based on measuring the reduction of
tetrazolium to colored formazan, indicating the toxicity status. In line with the
voluminous literature, curcumin emerged as the safest tested compound with minimal
toxicological manifestations in the current study as well. Compounds 4A-C, 6A-C, 9Aa-
i, 9Ba-i and 9Ca-i, at the assayed concentration (100 µM) showed very low cytotoxic
activity in human fibroblasts, with an average cell survival >80% compared to a vehicle
control. However, among the tested compounds, compound 4B was the least innocuous
compound and exhibited highest toxicity to normal cells, as indicated by both MTS and
LDH assays in vitro (p≤0.05). 4B with highly electron withdrawing nature is the
precursor of the series and non-toxic manifestation of its derivatives indicated that the
modification of the compounds not only improved the cytostatic activity but augmented
the safety profile as well. Interestingly, compound 4B was also the weakest antioxidant
candidate in the FRAP analysis as well indicating its electron withdrawing nature
(p≤0.05). Following curcumin, the compounds 9Ad, 9Ag, 9Bb, 9Bc, 9Cd and 9Ch were
less deleterious, as the average cellular viability of fibroblasts in their presence was >90%
in vitro. These compounds were safer than their precursor molecule and the presence of
phenyl ring substitutions at positions 2 (R1) and/or 6 (R2) aided their tolerance in normal
cells. Likewise, the toxicity profile of these compounds in human fibroblasts was
supported by the LDH release assay in vitro (p≤0.05). Most of these compounds were
active inhibitors of cancer cell proliferation and topo IIα enzyme along with strong
15

antioxidant capacities. The non-toxic presence of these compounds in human fibroblast in
vitro and their robust action against the tumor cells certainly enhances and merits their
candidacy as potential anti-cancer agents.
Table 4. In vitro toxicity assessment of 3,5-bis(arylmethylene)-1-(N-arylmaleamoyl)-4-
piperidones (4A-C, 6A-C, 9Aa-i, 9Ba-i and 9Ca-i) using MTS cell viability and LDH
release cytotoxicity assay in WI-38 cells (human fibroblasts).
Compound Cell Viability Cytotoxicity Compound Cell Viability Cytotoxicity
81.7±2.8def
80.5±4.7ef
14.6±1.2efghi
18.5±1.2bcdefg
87.9±2.8abcdef
13.2±0.6ghij
4A
6A
9Be
9Bf
9Bg
9Bh
9Bi
4C
81.6±4.3abcdef 16.1±3.9defgh
87.2±7.1abcdef 14.6±1.7efghi
89.2±3.6abcdef 17.6±0.8cdefg
88.2±2.1abcdef 13.2±1.3fghij
82.9±4.2bcdef 18.4±0.6bcdefg
9Aa
9Ab
9Ac
9Ad
9Ae
9Af
9Ag
9Ah
9Ai
4B
84.1±3.5abcdef
81.4±3.1def
20.7±2.8bcde
19.7±2.1bcdef
90.3±1.5abcde
5.3±0.2k
87.2±2.6abcdef 16.3±1.6defgh
84.1±3.7abcdef 17.2±2.0cdefg
89.2±1.4abcdef 17.6±0.9bcdefg
6C
80.8±5.3ef
93.1±5.8abcd
90.8±3.5abcde
22.9±1.5bcd
13.5±0.7 fghi
15.8±3.6efghi
86.8±1.9abcdef
82.5±1.5cdef
86.1±3.7abcdef
93.8±1.8abc
23.1±6.3bc
21.7±1.6bcd
20.4±3.7bcde
18.4±0.5bcdefg
23.7±3.1b
9Ca
9Cb
9Cc
9Cd
9Ce
9Cf
9Cg
9Ch
9Ci
87.3±2.6abcdef 14.5±1.4efghi
78.8±6.2f
31.3±2.8a
87.1±2.6abcdef
89.6±7.0abcdef
87.7±1.8abcdef 19.8±2.5bcdefg
9.3±2.6hij
6B
90.8±2.3abcde
92.5±5.8ab
9.1±1.1
86.4±5.6abcdef 17.2±2.3cdefg
9Ba
9Bb
9Bc
9Bd
17.4±2.7bcdefg
92.2±4.3abcde
87.5±3.5abcdef
95.8±1.4a
9.5±1.7ijk
16.8±2.4defg
7.1±0.6jk
92.7±1.4abcde 19.3±1.6bcdefg
82.1±3.0cdef
13.2±0.6ghij
Curcumin
Different values followed by the different superscripted letters (a through k) within
column are significantly different; Tukey's multiple means comparison test, p≤0.05). The
compounds were assayed at 100 µM concentration.
2.2.5. In-vivo neurotoxicity studies
Twenty nine compounds (4C, 9Aa-g, 9Ai, 9Ba-i, 9Cb, 9cd and 11a-i) were evaluated for
murine toxicity in vivo following a literature procedure [42]. All of these compounds
were injected intraperitoneally into mice at doses of 30, 100 and 300 mg/kg. The animals
were then observed for 0.5 (4, 8 and 4 mice respectively for 30, 100 and 300 mg/kg
experiments) and 4 h (2, 4 and 2 mice respectively for 30, 100 and 300 mg/kg
experiments) post administration of the compounds. The injected compounds were found
to be well-tolerated. Following compounds did not show any signs of toxicity in the
experiments: 9Ae, 9Ag, 9Bd, 9Bf, 9Bg, 9Bi, 9Cb, 9Cd, 11a, 11c, 11g and 11h. Death of
one animal (out of two) was observed in the case of intermediate 11e at 300 mg/kg 4 h
16

post administration. Table 5 summarizes only those cases where some neurological
deficit was recorded at a particular dose either during 0.5 h or 4 h observation. These
results clearly indicate that the compounds studied here are well-tolerated in vivo and are
not general biocidal agents.
Table 5: Experimental data on compounds that displayed neurological deficit in the
neurotoxicity assay system.
Time
Dose
(mg/kg) tested
0.5 h
Animals
showing
4.0 h
Compound
Animals
Animals
tested
Animals
showing
Mortality
neurotoxicity
neurotoxicity
100
100
300
100
100
100
300
100
300
300
100
300
100
300
100
300
300
30
8
8
4
8
8
8
4
8
4
4
8
4
8
4
8
4
4
4
8
4
8
4
8
4
8
1
2
1
4
1
1
3
1
1
1
2
2
3
1
1
1
1
1
0
1
1
1
1
1
1
4
4
2
4
4
4
2
4
2
2
4
2
4
2
4
2
2
2
4
2
4
2
4
2
4
0
0
1
0
1
1
1
0
1
0
0
0
1
0
0
0
0
0
1
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4C
9Aa
9Ab
9Ac
9Ad
9Af
9Ai
9Ba
9Bb
9Bc
0
0
0
0
9Be
9Bh
100
300
100
300
100
300
100
11b
11d
11e
11f
0
1 (4 h)
0
0
0
11i
2.3. Quantitative structure-activity relationship studies
In an attempt to understand how the structural and electronic parameters effect the
cytostatic activity of series 9 compounds, a quantitative structure–activity relationship
17

(QSAR) study was undertaken using certain physicochemical parameters. The extent to
which the electrophilicity of the enone systems effected the bioactivity was evaluated by
comparing the electronic charge at various β-positions for the three enone systems
(calculated using a commercial software) [43] of the test compounds (Charge β1 and β2;
see Scheme 1 for these locations) with the IC50 values against the three cell lines (Table
1). Likewise, the molar refractivity (MRmol) values and the calculated partition coefficient
(logP) of series 9 compounds were also obtained using the same commercial software
[40] and utilized as a means of assessing the effect of steric properties and lipophilicity,
respectively, of the compounds with respect to their bioactivity. Physicochemical
parameters of the aromatic substituents [44] such as Taft Es steric constant for ortho-
substituents, Hansch π (a measure of the lipophilicity of the substituents), and molar
refractivity (MRsub, (a measure of the size of the substituents) were also used for statistical
correlation studies. Linear and semilogarithmic correlations were calculated using
statistical analysis [45] for the IC50 of each tumor cell line and the aforementioned
physicochemical parameters. Only the significant correlations obtained are included in
Table 6. All significant correlations were obtained in linear as well as semilogarithmic
calculations, so in the interest of space and clarity, only the linear correlations are
presented.
While several significant correlations were observed for compounds of series 9A
and 9B, series 9C compounds did not show any meaningful correlation with the
physicochemical properties employed. No significant correlation (P<0.01) was observed
between charge β2 with any of the biological activities for any molecule tested. The
correlations obtained for series 9A compounds are discussed first. A positive significant
correlation (P=0.005 or lower) between Taft Es and antiproliferative activity against
Molt4C/8 and CEM cells indicates that decrease in Taft Es has a decreasing effect
(desirable) on the Molt4C/8 and CEM IC50 values. A smaller Taft Es value means larger
steric bulk. Thus this correlation predicts that bulky substituent at the ortho- position of
the substituted aromatic amine moiety will lead to more potent compounds towards
Molt4C/8 and CEM cells.
18

Table 6. Linear Correlation constants between IC50 values of compounds of series 9A
and 9B, and their physicochemical parameters
Series
Independent
Dependent
Correlation coefficient ra
P valueb
Series 9A
Es
Es
Molt IC50
CEM IC50
CEM IC50
Molt IC50
CEM IC50
CEM IC50
Molt IC50
CEM IC50
CEM IC50
CEM IC50
0.809
0.852
0.005
0.002
0.023
0.089
0.020
0.096
0.063
0.053
0.032
0.069
MRsub
MRmol
MRmol
Hansch π
logP
-0.706
-0.565
-0.715
-0.555
-0.606
-0.626
0.711
logP
Es
logP
Series 9B
-0.630
a The magnitude of coefficient r shows the extent (the closer the values to 1, the better)
and the nature (sign positive or negative) of the correlation.
b The value of P in the range 0.05–0.1 indicates a trend toward significance, while values
<0.05 suggest significant correlation.
The same inference is drawn from the negative correlation with significance or trend
toward significance between MRsub with CEM IC50 values and MRmol with Molt4C/8 and
CEM IC50 values for series 9A compounds. This means that as the MR values (MRsub or
MRmol) increases, the anticancer IC50 value decreases (desirable). Larger MR values
signify larger steric bulk. Negative correlations with trend towards significance
(0.1>P>0.05) were observed between lipophilicity-related parameters Hansch π and logP
with Molt4C/8 and CEM IC50 values for series 9A compounds. This indicates that as
increased lipophilicity is leading to decreased (desirable) Molt4C/8 and CEM IC50 values.
Therefore, for future modifications in series 9A compounds, bulky and more lipophilic
substituents at the ortho- positions of the substituted aromatic amine moiety should be
considered.
For series 9B compounds, Taft Es is positively and significantly correlated with
CEM IC50 values. Thus, to produce more potent series 9B-based CEM cell growth
inhibitor, bulkier substituents (smaller Es value) at the ortho- positions of the substituted
aromatic amine moiety are desired. The negative correlation with trend towards
significance between logP and CEM IC50 values suggests that lipophilicity is desirable for
19

better anticancer activity. These correlations can be tremendously useful in designing
new analogs.
Having obtained the correlation between the cytostatic activities and various
physicochemical parameters, we decided to obtain a mathematical equation that describes
the relationship between the two using multiple linear regressions for the two series of
compounds [42]. For Series 9A analogs, where CEM IC50 correlated with Taft’s Es,
Hansch π and MRmol, and Molt IC50 correlated with Taft’s Es, logP and MRmol, two
equations were obtained:
[R square 77.96%]
[R square 75.91%]
For Series 9B compounds, where only CEM IC50 correlated with Taft’s Es and
logP, the following linear relationship was obtained:
[R square 59.30%]
These equations can be used to predict the cytostatic activities of new analogs and
future synthesis can be focused on preparing more potent analogs.
3. Conclusions
In conclusion, curcumin-derived three series of novel 3,5-bis(arylmethylene)-1-
(N-arylmaleamoyl)-4-piperidones inhibited proliferation of human Molt 4/C8 and CEM
T-lymphocytes as well as murine L1210 leukemic cells. The enhancement in anticancer
potency in comparison with melphalan and curcumin was 93-280 folds. It is apparent
from these data that curcumin-derived piperidones are potent cytostatic agents in vitro
with widespread efficacy in multiple cell lines at low concentrations. Several novel
compounds (such as 9Aa, 9Ad and 9Af) displayed excellent inhibition of cancer cell
proliferation in multiple cancer cell lines including breast cancer, central nervous system,
colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal cancer.
These may effectively serve as leads for further analysis and development of ‘Swiss army
20

knives’ for curcumin-inspired cancer research. The multifunctional curcumin-derived
compounds also displayed interesting properties as topoisomerase IIα inhibitory activity
and antioxidant action. Likewise, the safety profile of these compounds in both in vitro
and in vivo models make them it a safe candidate for further evaluation in animals and
subsequently in humans.
4. Experimental section
4.1. Materials and methods
All reagents and solvents were used as supplied by commercial sources without
further purification. Anhydrous solvent THF was obtained by distilling from sodium and
benzophenone. Oven dried glassware and an atmosphere of nitrogen was used for
moisture sensitive reactions. Precoated fluorescent silica gel TLC plates were used to
monitor the progress of the reactions. Silica gel 60 (SiliaFlash P60, Silicycle, Quebec
City, Canada) was used for flash chromatography. Melting points were measured using a
MEL-TEMP II apparatus and are uncorrected. IR spectra were recorded on a Nicolet
1
MAGNA-IR 560 Spectrophotometer. H and 13C NMR were recorded on a Bruker
AV500 spectrophotometer at 500 and 125 MHz, respectively in DMSO-d6 as the solvent.
Chemical shifts of the 1H NMR spectra are reported in parts per million (ppm) downfield
from tetramethylsilane. UV-Vis spectra were recorded on a LKB Biochrom Ultraspec
Plus 4054 UV/Visible spectrophotometer. ESI-HRMS were recorded on a microTOF
(Bruker Daltonics) spectrometer.
4.2. General synthesis
4.2.1. General procedure for the synthesis of 3,5-bisarylidene-piperidin-4-ones (4A-
C)
To a 250 mL round bottom oven dried flask, was added, 4-piperidone hydrate
hydrochloride (0.10 mol), appropriate aromatic aldehyde (0.20 mol), acetic acid (150 ml).
The reaction mixture was sealed and HCl (g) was passed through the reaction flask for 30
minutes while the reaction was stirred. During this time, the clear mixture yielded thick
yellow or orange precipitates. After the 30 minute period, the HCl gas was turned off and
the reaction was left to stir for 24 hours. The precipitate was then filtered using suction
filtration. The products as acid salts thus obtained were quite pure and were used for
21

subsequent reactions without further purification.
4.2.1.1. 3, 5-Bisbenzylidene-piperidin-4-one hydrochloride (4A)
Yellow power, yield 71%, m.p. 243-246 °C (lit. [46] m.p. 242-243). NMR
spectral data was found to be identical to those reported in the literature.[46]
4.2.1.2. 3, 5-Bis-4-nitrobenzylidene-piperidin-4-one hydrochloride (4B)
Orange powder, yield 65%, m.p. 286-289 °C (lit. [36] m.p. 242-243 free base).
NMR spectral data was found to be identical to those reported in the literature.[36]
4.2.1.3. 3,5-Bis-[1-(4-carboethoxy-phenyl)-meth-(E)-ylidenel-piperidin-4-one acetic
acid salt (4C)
1
Yellow powder, yield 60%, m.p. 225-221 °C. H NMR (500 MHz, DMSO-d6):
1.35 (t, J = 7.0 Hz, 6 H), 1.92 (s, 3H), 4.36 (q, J = 7.0 Hz, 4H), 4.50 (s, 4H), 7.68 (d, J =
13
8.0 Hz, 4H), 7.92 (s, 2H), 8.08 (d, J = 8.0 Hz, 4H), 10.03 (s, 2H). C NMR (125 MHz,
DMSO-d6): δ 14.1, 21.0, 43.6, 61.0, 129.4, 129.7, 130.5, 130.6, 137.9, 138.1, 165.1,
171.9, 182.2 (23/25). UV (λmax, EtOH): 217, 244, 315 nm. ESI-HRMS (amu): calcd.
C25H25NO5: 420.1811 [M+H]; found: 420.1824.
4.2.2. N-Acryloyl-3,5-bis-[1-(4-carboethoxy-phenyl)-meth-(E)-ylidenel-piperidin-4-
one (6C)
Compound 4C (0.006 mol) and triethylamine (0.014 mol) were dissolved in dry
o
DCM (50 mL) and cooled to 0 C using an ice bath. Under anhydrous conditions,
acryloyl chloride (0.007 mol) in dry DCM (30 mL) was added dropwise over a 10 minute
period. The mixture was stirred for 24 hours at room temperature. The solvents were then
evaporated under vacuum. Water (30 mL) was added to the residue and the product was
triturated and filtered. Under suction filtration, the solid product was washed with 10%
HCl, and then distilled water, and then a saturated potassium carbonate solution. The
solid product was washed once again with distilled water. The product was digested in
methanol to remove unwanted side products and unreacted starting materials. It was then
filtered and washed with cold methanol. The final product was filtered by suction
filtration and dried. Characterization data: Yellow powder, yield 62%, m.p. 120-123 °C.
1H NMR (500 MHz, DMSO-d6): 1.35 (t, J = 7.0 Hz, 6 H), 4.35 (q, J = 7.0 Hz, 4H), 4.92-
4.96 (m, 4H), 5.58 (d, J = 10.5 Hz, 1H), 6.00 (d, J = 17.0 Hz, 1H), 6.55 (dd, J = 17.0 &
22

11.0 Hz, 1H), 7.65-7.74 (m, 6H), 8.06 (d, J = 8.0 Hz, 4H). 13C NMR (125 MHz, DMSO-
d6): δ 14.6, 43.6, 61.5, 127.8, 128.9, 129.9, 130.8, 131.1, 134.7, 135.5, 165.4, 165.7,
168.8, 186.4 (23/25). ESI-HRMS (amu): calcd. C28H27NO6: 496.1736 [M+Na]; found:
496.1735.
4.2.3. General procedure to synthesize aryl maleamic acids (11a-i)
Maleic anhydride (0.004 mol) and the appropriate aryl amine (0.004 mol) were
dissolved in dichloromethane (100 mL) and were stirred for a time period that ranged
between 30 min to 48 h depending on the aryl amine used. Heat was also applied for
certain aryl amides to speed up the reaction. TLC plates were used to monitor the
progress of the reactions (5% methanol:dichloromethane). The solvents were removed by
vacuum, the precipitates were then washed with dichloromethane, filtered under suction
and dried. The aryl maleamic acids were obtained as powders and were very pure as was
evident by their melting points and their NMR spectra.
Deviations from procedure: Compounds 11d and 11f had to be stirred for 48 h
and heat had to be applied for one hour. The reactions still did not go all the way to
completion so the solvents were removed by suction filtration rather than vacuum.
Compound 11i had to be stirred for 12 h, compounds 11g and 11h needed stirring for 4 h
and reactions leading to compounds 11a, 11b, 11c and 11e were complete within 30 min.
4.2.3.1. (2-Flurophenyl)-maleamic acid (11a)
o
Yellow powder, 87%, m.p. 153-154°C (lit. [47] m.p. 163 C). Its NMR spectral
data was found to be identical to those reported in the literature [47].
4.2.3.2. (2-Chlorophenyl)-maleamic acid (11b)
Yellow powder, yield 84%, m.p. 129-132 °C (lit. [48] m.p. 134-136 oC). Its NMR
spectral data was found to be identical to those reported in the literature [48].
4.2.3.3. (2-Methylphenyl)-maleamic acid (11c)
o
White powder, yield 58%, m.p. 116-120 °C (lit. [49] m.p. 104-105 C). Its NMR
spectral data was found to be identical to those reported in the literature [49].
4.2.3.4. (2-Methyoxyphenyl)-maleamic acid (11d)
23

o
Green powder, yield 99%, m.p. 144-146 °C (lit. [49] m.p. 134-136 C). Its NMR
spectral data was found to be identical to those reported in the literature [49].
4.2.3.5. (2-Nitrophenyl)-maleamic acid (11e)
Yellow powder, yield 31%, m.p. 132-133 °C (lit. [50] m.p. 138-140 oC). 1H NMR
(300 MHz, DMSO-d6): δ 6.40 (d, 1H, J=12.0Hz), 6.50 (d, 1H, J=11.9Hz), 7.38-7.44 (m,
1H), 7.68-7.76 (m, 2H), 7.98 (d, 1H, J=8.2Hz), 10.60 (s, 1H). UV (λmax, EtOH): 217, 384
nm. IR (ν, KBr): 501, 695, 738, 912, 1214, 1279, 1353, 1520, 1587, 1712, 2599, 2950,
3016 cm-1.
4.2.3.6. (2,6-Dichlorophenyl)-maleamic acid (11f)
1
Yellow powder, 25%, m.p. 158-159 °C. H NMR (300 MHz, DMSO-d6): δ 6.44
(d, 2H), 6.60 (d, 2H), 7.38 (t, 1H, J=8.4Hz), 7.57 (d, 2H, J=8.2Hz), 10.39 (s, 1H). UV
(λmax, EtOH): 216 nm. IR (ν, KBr): 540, 790, 875, 1300, 1450, 1490, 1550, 1620, 1700,
2998, 3008, 3200 cm-1.
4.2.3.7. (2,6-Dimethylphenyl)maleamic acid (11g)
White powder, yield 87%, m.p. 173-175 °C. (lit. [51] m.p. 170-173 oC). Its NMR
spectral data was found to be identical to those reported in the literature [51].
4.2.3.8. (2,4,6-Trimethylphenyl)-maleamic acid (11h)
Yellow powder (76.4%, 0.71g); Rƒ 0.40 (5% CH3OH: CH2Cl2); m.p. 104-105 °C.
o
(lit. [52] m.p. 99-100 C). Its NMR spectral data was found to be identical to those
reported in the literature [52].
4.2.3.9. (2-Ethyl-6-methylphenyl)-maleamic acid (11i)
1
Grey powder, yield 66%, m.p. 150-152 °C. H NMR (300 MHz, DMSO-d6): δ
1.09 (t, 3H, J=7.5Hz), 2.16 (s, 3H), 2.60 (qt, 2H, J=7.6Hz), 6.32 (d, 1H, J=12.3Hz), 6.60
(d, 1H, J=12.3Hz), 7.10-7.15 (m, 3H), 10.00 (s, 1H). UV (λmax, EtOH): 241 nm. IR (ν,
KBr): 622, 862, 907, 980, 1249, 1309, 1520, 1632, 1660, 2964, 3254 cm-1.
4.2.4. General procedure for the synthesis of 4-(3,5-bisarylmethylene-4-oxo-
piperidin-1-yl)-4-oxo-but-2-enoic acid arylamides (9Aa-i, 9Ba-i and 9Ca-i)
The appropriate aryl maleamic acid (0.006 mol) and triethylamine (0.006 mol)
o
were dissolved in dry THF (50 mL) and cooled to 0 C using an ice bath. Under
anhydrous conditions, ethyl chloroformate (0.006 mol) in dry THF (40 mL) was added
24

dropwise over a 10 minute period. The mixture was stirred for 24 hours at room
temperature, cooled to 0 oC and appropriate 3,5-bisarylmethylene-piperidin-4-one
(0.0042 mol) was added. Subsequently triethylamine (0.006 mol) in dry THF (40 mL)
was added dropwise over 10 minutes. The reaction was then stirred for 24 hours during
which time the temperature slowly rose to room temperature. The solvents were then
evaporated under vacuum. Water (30 mL) was added to the residue and the product was
triturated and filtered. Under suction filtration, the solid product was washed with 10%
HCl, and then distilled water, and then a saturated potassium carbonate solution. The
solid product was washed once again with distilled water. The product was digested in
methanol to remove unwanted side products and unreacted starting materials. It was then
filtered and washed with cold methanol. The final product was filtered by suction
filtration and dried. The reactions were monitored by TLC (5% methanol:
dichloromethane).
Deviations from procedure: Compound 9Ah was completely soluble in methanol
so the unwanted side products had to be removed by column chromatography (0-3%
MeOH: Dichloromethane). The final product was obtained after vacuum evaporation.
4.2.4.1. (2Z)-4-(3E,5E)-(3,5-Bisbenzylidene-4-oxo-piperidine-4-oxo-piperidin-1-yl)-4-
oxo-but-2-enoic acid (2-flurophenyl)amide (9Aa):
1
Yellow powder, yield 64%, m.p. 171-173 °C. H NMR (500 MHz, DMSO-d6): δ
4.78 & 4.93 (s, 2H each), 6.25 & 6.49 (d, J = 11.5 Hz, 1H each), 7.18-7.62 (m, 14H),
13
7.77 (s, 1H), 7.95-7.98 (m, 1H), 9.89 (s, 1H). C NMR (125 MHz, DMSO-d6): δ 41.2,
46.7, 115.3 (d, J = 20 Hz), 124.3, 125.3 (d, J = 7 Hz), 125.6, 125.7, 125.8, 128.7, 128.8,
129.3, 129.5, 130.0, 130.5, 132.1, 134.3 (d, J = 20 Hz), 134.9, 135.4, 136.3, 153.0 (d, J =
244 Hz), 162.1, 166.3, 185.4. UV (λmax, EtOH): 326 nm. IR (ν, KBr): 501, 518, 561, 599,
666, 698, 753, 807, 886, 985, 1105, 1180, 1242, 1276, 1487, 1538, 1621, 1636, 1685,
2824, 3051, 3320 cm-1. ESI-HRMS (amu): calcd. C29H23FN2O3: 489.1590 [M+Na];
found: 489.1582.
4.2.4.2. (2Z)-4-(3E,5E)-(3,5-Bisbenzylidene-4-oxo-piperidine-4-oxo-piperidin-1-yl)-4-
oxo-but-2-enoic acid (2-chlorophenyl)amide (9Ab)
25

1
Yellow powder, yield 54%, m.p. 160-162 °C. H NMR (500 MHz, DMSO-d6): δ
4.78 & 4.91 (s, 2H each), 6.30 & 6.52 (d, J = 12.0 Hz), 7.22 (t, J = 7.5 Hz, 1H), 7.34-7.63
(m, 13H), 7.76 (s, 2H), 9.66 (brs, 1H). 13C NMR (125 MHz, DMSO-d6): δ 41.6, 46.8,
125.5, 125.6, 125.9, 126.4, 127.3 128.7, 128.8, 129.3, 129.4, 129.5, 130.1, 130.5, 132.0,
134.3, 134.3, 134.4, 135.1, 135.5, 136.2, 162.1, 166.3, 185.4. UV (λmax, EtOH): 325, 327
nm. IR (ν, KBr): 436, 517, 699, 759, 803, 884, 945, 1039, 1179, 1240, 1274, 1441, 1529,
1622, 1637, 1686, 2825, 3332 cm-1. ESI-HRMS (amu): calcd. C29H23ClN2O3: 505.1295
[M+Na]; found: 505.1285.
4.2.4.3. (2Z)-4-(3E,5E)-(3,5-Bisbenzylidene-4-oxo-piperidine-4-oxo-piperidin-1-yl)-4-
oxo-but-2-enoic acid (2-methylphenyl)amide (9Ac)
1
Yellow powder, yield 39%, m.p. 170-171 °C. H NMR (500 MHz, DMSO-d6): δ
2.15 (s, 3H), 4.80 & 4.90 (s, 2H each), 6.19 & 6.47 (d, J = 11.5 Hz, 1H each), 7.11 (t, J =
7.5 Hz, 1H), 7.17-7.24 (m, 2H), 7.41-7.60 (m, 11H), 7.63 & 7.75 (s, 1H each), 9.44 (brs,
1H). 13C NMR (125 MHz, DMSO-d6): δ 18.3, 42.1, 47.4, 125.0, 125.8, 126.4, 126.5,
127.3, 129.3, 129.9, 130.0, 130.7, 130.8, 131.0, 131.9, 132.6, 132.9, 134.7, 134.8, 134.9,
136.0, 136.2, 136.7, 162.3, 167.0, 185.9. UV (λmax,EtOH): 326 nm. IR (ν, KBr): 496, 517,
605, 697, 758, 803, 882, 946, 984, 1178, 1242, 1275, 1443, 1455, 1532, 1586, 1620,
1636, 1684, 1733, 2334, 2362, 2835, 3054, 3375 cm-1. ESI-HRMS (amu): calcd.
C30H26N2O3: 485.1841 [M+Na]; found: 485.1835.
4.2.4.4. (2Z)-4-(3E,5E)-(3,5-Bisbenzylidene-4-oxo-piperidine-4-oxo-piperidin-1-yl)-4-
oxo-but-2-enoic acid (2-methoxyphenyl)amide (9Ad)
1
Yellow powder, yield 55%, m.p. 179-181 °C. H NMR (500 MHz, DMSO-d6): δ
3.83 (s, 3H), 4.78 & 4.92 (s, 2H each), 6.36 & 6.42 (d, J = 11.5 Hz, 1H each), 6.94 (t, J =
7.5 Hz, 1H), 7.06 (d, J = 8.0 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 7.32 (brs, 2H), 7.41 (brs,
13
3H), 7.46-7.61 (m, 6H), 7.77 (s, 1H), 7.99 (d, J = 7.5 Hz, 1H), 9.31 (s, 1H). C NMR
(125 MHz, DMSO-d6): δ 42.1, 47.3, 56.1, 111.6, 120.7, 122.1, 125.1, 127.0, 127.4,
129.2, 129.3, 129.8, 130.0, 130.6, 131.0, 132.6, 132.9, 134.5, 134.8, 134.9, 136.0, 136.7,
149.9, 162.4, 167.1, 185.9. UV (λmax, EtOH): 204, 207, 249, 323 nm. IR (ν, KBr): 491,
522, 541, 630, 698, 755, 771, 809, 940, 991, 1021, 1045, 1114, 1177, 1249, 1284, 1331,
1524, 1681, 3397 cm-1. ESI-HRMS (amu): calcd. C30H26N2O4: 501.1790 [M+Na]; found:
501.1797.
26

4.2.4.5. (2Z)-4-(3E,5E)-(3,5-Bisbenzylidene-4-oxo-piperidine-4-oxo-piperidin-1-yl)-4-
oxo-but-2-enoic acid (2-nitrophenyl)amide (9Ae)
1
Yellow powder, yield 66%, m.p. 186-188 °C. H NMR (500 MHz, DMSO-d6): δ
4.76 & 4.89 (s, 2H each), 6.19 & 6.56 (d, J = 12.0 Hz, 1H each), 7.40-7.60 (m, 11H),
7.63 (s, 1H), 7.72-7.78 (m, 3H), 8.01 (d, J = 8.5 Hz, 1H), 10.42 (brs, 1H). 13C NMR (125
MHz, DMSO-d6): δ 41.6, 46.7, 125.0, 125.1, 125.3, 125.5, 128.7, 128.8, 129.4, 129.5,
130.2, 130.5, 130.8, 132.0, 134.1, 134.2, 134.3, 135.7, 135.9, 142.0, 162.0, 166.0, 185.4.
UV (λmax, EtOH): λmax: 204, 207, 327, 329 nm. IR (ν, KBr): 518, 599, 668, 694, 744,
802, 940, 983, 1174, 1280, 1341, 1436, 1498, 1652, 1699, 1772, 2338, 2362, 3374, 3853
cm-1. ESI-HRMS (amu): calcd. C29H23N3O5: 516.1535 [M+Na]; found: 516.1524.
4.2.4.6. (2Z)-4-(3E,5E)-3,5-Bisbenzylidene-4-oxo-piperidine-4-oxo-piperidin-1-yl)-4-
oxo-but-2-enoic acid (2,6-dichlorophenyl)amide (9Af)
1
Yellow powder, yield 47%, m.p. 197-200 °C. H NMR (500 MHz, DMSO-d6): δ
4.78 & 4.86 (s, 2H each), 6.21 & 6.55 (d, J = 12.0 Hz, 1H each), 7.37 (t, J = 8.5 Hz, 1H),
7.47-7.57 (m, 12H), 7.64 & 7.72 (s, 1H each), 10.13 (brs, 1H). 13C NMR (125 MHz,
DMSO-d6): δ 41.8, 47.3, 124.6, 129.0, 129.1, 129.3, 129.8, 130.0, 130.8, 130.9, 132.5,
132.6, 132.9, 134.0, 134.9, 136.3, 136.7, 162.2, 166.7, 185.9. UV (λmax, EtOH) 204, 227,
328 nm. IR (ν, KBr): 516, 537, 667, 695, 779, 943, 978, 1133, 1171, 1242, 1277, 1435,
1471, 1490, 1531, 1571, 1617, 1673, 2362, 3525 cm-1. ESI-HRMS (amu): calcd.
C29H22Cl2N2O3: 539.0905 [M+Na]; found: 539.0887.
4.2.4.7. (2Z)-4-(3E,5E)-(3,5-Bisbenzylidene-4-oxo-piperidine-4-oxo-piperidin-1-yl)-4-
oxo-but-2-enoic acid (2,6-dimethylphenyl)amide (9Ag)
1
Yellow powder, yield 83%, m.p. 204-206 °C. H NMR (500 MHz, DMSO-d6): δ
2.09 (s, 6H), 4.80 & 4.97 (s, 2H each), 6.19 & 6.49 (d, J = 11.5 Hz, 1H each), 7.06-7.08
(m, 3H), 7.46-7.57 (m, 10H), 7.64 & 7.72 (s, 1H each), 9.48 (brs, 1H). 13C NMR (125
MHz, DMSO-d6): δ 18.0, 41.4, 46.9, 125.3, 126.5, 127.6, 128.6, 128.8, 129.4, 130.2,
130.4, 132.0, 132.2, 134.3, 134.4, 134.5, 134.9, 135.6, 136.1, 161.5, 166.4, 185.3. UV
(λmax, EtOH) 204, 328 nm. IR (ν, KBr): 516, 526, 695, 770, 943, 977, 1170, 1241, 1442,
1469, 1527, 1617, 1663, 1733, 2339, 2362, 3235 cm-1. ESI-HRMS (amu): calcd.
C31H28N2O3: 499.1998 [M+Na]; found: 499.1989.
27

4.2.4.8. (2Z)-4-(3E,5E)-(3,5-Bisbenzylidene-4-oxo-piperidine-4-oxo-piperidin-1-yl)-4-
oxo-but-2-enoic acid (2,4,6-trimethylphenyl)amide (9Ah)
1
Yellow powder, yield 36%, m.p. 194-196 °C. H NMR (500 MHz, DMSO-d6): δ
2.04 (s, 6H), 2.23 (s, 3H), 4.79 & 4.86 (s, 2H each), 6.17 & 6.46 (d, J = 11.5 Hz, 1H
each), 6.88 (s, 2H), 7.45-7.57 (m, 10H), 7.64 & 7.72 (s, 1H each), 9.37 (brs, 1H). 13C
NMR (125 MHz, DMSO-d6): δ 18.4, 21.0, 41.9, 47.4, 125.8, 128.2, 128.7, 129.0, 129.3,
130.0, 130.7, 130.9, 132.3, 132.6, 132.7, 134.7, 134.8, 134.9, 135.1, 136.0, 136.6, 162.1,
167.0, 185.8. UV (λmax, EtOH): 219, 328 nm. IR (ν, KBr): 516, 671, 749, 808, 984, 1174,
1237, 1277, 1436, 1616, 1675, 1683,1733, 2915, 3675, 3780, 3853 cm-1. ESI-HRMS
(amu): calcd. C32H30N2O3: 513.2154 [M+Na]; found: 513.2138.
4.2.4.9. (2Z)-4-(3E,5E)-(3,5-Bisbenzylidene-4-oxo-piperidine-4-oxo-piperidin-1-yl)-4-
oxo-but-2-enoic acid (2-ethyl-6-methylphenyl)amide (9Ai)
1
Yellow powder, yield 28%, m.p. 181-184 °C. H NMR (500 MHz, DMSO-d6): δ
1.04 (t, J = 7.5 Hz, 3H), 2.08 (s, 3H), 2.45 (q, J = 7.5 Hz, 2H), 4.79 & 4.86 (s, 2H each),
6.20 & 6.49 (d, J = 11.5 Hz, 1H each) 7.09-7.57 (m, 13H), 7.64 & 7.72 (s, 1H each), 9.47
13
(brs, 1H). C NMR (125 MHz, DMSO-d6): δ 14.4, 18.0, 24.2, 41.4, 46.9, 125.2, 125.9,
126.7, 126.9, 127.0, 127.4, 127.7, 128.2, 128.5, 128.7, 129.2, 130.2, 130.4, 132.0, 133.8,
134.3, 134.5, 135.5, 136.1, 140.7, 162.0, 166.5, 185.3. UV (λmax, EtOH): 328 nm. IR (ν,
KBr): 516, 687, 760, 806, 938, 986, 1174, 1191, 1253, 1277, 1443, 1528, 1581, 1626,
1673, 2969, 3222 cm-1. ESI-HRMS (amu): calcd. C32H30N2O3: 513.2154 [M+Na]; found:
513.2135.
4.2.4.10. (2Z)-4-(3E,5E)-(3,5-Bis-4-nitrobenzylidene-4-oxo-piperidin-1-yl)-4-oxo-but-
2-enoic acid (2-flurophenyl)amide (9Ba)
1
Yellow powder, yield 47%, m.p. 192-194 °C. H NMR (500 MHz, DMSO-d6): δ
4.78 & 4.95 (s, 2H each), 6.21 & 6.47 (d, J = 11.5 Hz, 1H each), 7.19-7.21 (m, 2H), 7.26-
7.31 (m, 1H), 7.53 (d, J = 8.5 Hz, 1H ), 7.60 & 7.84 (s, 1H each), 7.87 (d, J = 8.5 Hz, 1H
), 7.93-7.96 (m, 1H), 8.22 & 8.34 (d, J = 8.5 Hz, 2H each), 9.82 (brs, 1H). 13C NMR (125
MHz, DMSO-d6): δ 42.2, 47.0, 115.9 (d, J = 20 Hz), 123.8, 124.1, 124.3, 124.9, 125.7 (d,
J = 6 Hz), 126.2, 126.3, 131.5, 132.0, 133.6, 134.8, 135.4, 135.8, 141.2, 141.3, 147.6,
147.8, 153.6 (d, J = 245 Hz), 162.5, 167.0, 185.6. UV (λmax, EtOH): 242, 331 nm. IR (ν,
KBr): 701, 756, 768, 854, 867, 982, 1107, 1174, 1243, 1255, 1282, 1345, 1457, 1511,
28

1537, 1596, 1621, 1645, 1686, 2362, 3310 cm-1. ESI-HRMS (amu): calcd. C29H21FN4O7:
579.1292 [M+Na]; found: 579.1264.
4.2.4.11. (2Z)-4-(3E,5E)-(3,5-Bis-4-nitrobenzylidene-4-oxo-piperidin-1-yl)-4-oxo-but-
2-enoic acid (2-chlorophenyl)amide (9Bb)
1
Yellow powder, yield 67%, m.p. 207-210 °C. H NMR (300 MHz, DMSO-d6): δ
4.78 & 4.93 (s, 2H each), 6.25 & 6.51 (d, J = 11.5 Hz, 1H each), 7.22 (t, J = 7.5 Hz, 1H),
7.36 (t, J = 7.5 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.66 (s, 1H),
7.75 (t, J = 8.5 Hz, 1H), 7.82 (s, 1H), 7.86, 8.26 & 8.33 (d, J = 8.5 Hz, 2H each), 9.61
(brs, 1H). 13C NMR (125 MHz, DMSO-d6): δ 42.2, 47.1, 124.1, 124.2, 124.3, 125.9,
126.2, 126.9, 127.9, 130.0, 131.5, 131.9, 132.0, 133.7, 134.7, 134.8, 135.4, 135.6, 135.8,
141.3, 147.7, 147.8, 162.5, 167.0, 185.6. UV (λmax, EtOH): 218, 329 nm. IR (ν, KBr):
618, 755, 767, 853, 983, 1107, 1174, 1345, 1510, 1524, 1593, 1620, 1685, 3365 cm-1.
ESI-HRMS (amu): calcd. C29H21ClN4O7: 595.0996 [M+Na]; found: 595.1014.
4.2.4.12. (2Z)-4-(3E,5E)-(3,5-Bis-4-nitrobenzylidene-4-oxo-piperidin-1-yl)-4-oxo-but-
2-enoic acid (2-methylphenyl)amide (9Bc)
1
Yellow powder, yield 74%, m.p. 195-198 °C. H NMR (500 MHz, DMSO-d6): δ
2.13 (s, 3H), 4.80 & 4.92 (s, 2H each), 6.16 & 6.46 (d, J = 11.5 Hz, 1H each), 7.12 (t, J =
7.5 Hz, 1H), 7.17-7.23 (m, 2H), 7.42 (d, J = 8.0 Hz, 1H), 7.64-7.68 (m, 3H), 7.81 (s, 1H),
13
7.86, 8.27 & 8.33 (d, J = 8.5 Hz, 2H each), 9.61 (brs, 1H). C NMR (125 MHz, DMSO-
d6): δ 18.3, 42.1, 47.2, 124.2, 124.3, 124.8, 125.8, 126.4, 130.8, 131.6, 131.7, 132.0,
133.7, 134.7, 135.4, 135.8, 136.2, 141.3, 147.7, 147.8, 162.3, 167.2, 185.6. UV (λmax
,
EtOH): 207, 209, 220 nm. IR (ν, KBr): 516, 592, 701, 755, 854, 866, 982, 1108, 1129,
1172, 1255, 1282, 1345, 1454, 1509, 1525, 1589, 1620, 1644, 1683, 3411 cm-1. ESI-
HRMS (amu): calcd. C30H24N4O7: 575.1543 [M+Na]; found: 575.1512.
4.2.4.13. (2Z)-4-(3E,5E)-(3,5-Bis-4-nitrobenzylidene-4-oxo-piperidin-1-yl)-4-oxo-but-
2-enoic acid (2-methoxyphenyl)amide (9Bd)
1
Yellow powder, yield 50%, m.p. 220-224 °C. H NMR (500 MHz, DMSO-d6): δ
3.84 (s, 3H), 4.77 & 4.94 (s, 2H each), 6.32 & 6.40 (d, J = 12.0 Hz, 1H each), 6.94 (t, J =
7.5 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.51 (d, J = 8.5 Hz, 2H),
7.60 & 7.84 (s, 1H each), 7.89 (d, J = 8.5 Hz, 2H), 7.97 (d, J = 8.0 Hz, 1H), 8.20 & 8.35
13
(d, J = 8.5 Hz, 2H each), 9.25 (brs, 1H). C NMR (125 MHz, DMSO-d6): δ 42.1, 47.1,
29