Curcumin analogues as possible anti-proliferative & anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2011.03.060

A series of novel curcumin analogues has been designed, synthesized and tested in vitro/in vivo as potential multi-target agents. Their anti-proliferative and anti-inflammatory activities were studied. Compounds 1b and 2b were stronger inhibitors of soybean lipoxygenase (LOX) than curcumin. Analogue 1b was also the most potent aldose reductase (ALR2) inhibitor. Two compounds, (1a and 1f) exhibited in vivo anti-inflammatory activity comparable to that of indomethacin, whereas derivative 1i exhibited even higher activity. The derivatives were also tested for their anti-proliferative activity using three different human cancer cell lines. Compounds 1a, 1b, 1d and 2b exhibited significant growth inhibitory activity as compared to curcumin, against all three cancer cell lines. Lipophilicity was determined as R_M values using RPTLC and theoretically. The results are discussed in terms of the structural characteristics of the compounds. Docking simulations were performed on LOX and ALR2 inhibitor 1b and curcumin. Compound 1b is well fitted in the active site of ALR2, binding to the ALR2 enzyme in a similar way to curcumin. Allosteric interactions may govern the LOX-inhibitor binding.

Full text of DOI:10.1016/j.ejmech.2011.03.060

European Journal of Medicinal Chemistry 46 (2011) 2722e2735
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Curcumin analogues as possible anti-proliferative & anti-inflammatory agentsq
,
,
A.-M. Katsori a, M. Chatzopoulou a 1, K. Dimas b 1, C. Kontogiorgis c, A. Patsilinakos d, T. Trangas e,
,
D. Hadjipavlou-Litina a
*
a Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
b Department of Pharmacology, School of Medicine, University of Thessaly, Larissa, Greece
c Institute of Pharmaceutical Sciences, School of Biomedical and Health Services, King’s College of London, London, UK
d Rome Center for Molecular Design, Dipartimento di Chimica e Tecnologie del Farmaco, University of Rome “Sapienza”, Rome, Italy
e Department of Biological Applications and Technologies, University of Ioannina, Ioannina, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel curcumin analogues has been designed, synthesized and tested in vitro/in vivo as
potential multi-target agents. Their anti-proliferative and anti-inflammatory activities were studied.
Compounds 1b and 2b were stronger inhibitors of soybean lipoxygenase (LOX) than curcumin. Analogue
1b was also the most potent aldose reductase (ALR2) inhibitor. Two compounds, (1a and 1f) exhibited in
vivo anti-inflammatory activity comparable to that of indomethacin, whereas derivative 1i exhibited
even higher activity. The derivatives were also tested for their anti-proliferative activity using three
different human cancer cell lines. Compounds 1a, 1b, 1d and 2b exhibited significant growth inhibitory
activity as compared to curcumin, against all three cancer cell lines. Lipophilicity was determined as RM
values using RPTLC and theoretically. The results are discussed in terms of the structural characteristics of
the compounds. Docking simulations were performed on LOX and ALR2 inhibitor 1b and curcumin.
Compound 1b is well fitted in the active site of ALR2, binding to the ALR2 enzyme in a similar way to
curcumin. Allosteric interactions may govern the LOX-inhibitor binding.
Received 21 January 2011
Received in revised form
21 March 2011
Accepted 29 March 2011
Available online 5 April 2011
Keywords:
Curcumin analogues
Lipoxygenase
Aldose reductase
Carrageenin
Anti-inflammatory activity
Anti-proliferative activity
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
It is generally accepted that there is a close association between
cancer and chronic inflammation [7e9]. Inflammation and carci-
In the past years, targeted therapies towards a specific molec-
ular target, reducing toxic effects and damage of normal tissues,
have been applied in the treatment of cancer. Despite the initial
enthusiasm for the efficacy of these treatments, cancer patients
often develop drug resistance due to the activation of alternative
pathways. In the meanwhile, multi-target drug strategies have
emerged as a therapeutic approach to treat diseases that stem from
a combination of factors, leading to the final pathology, such as
cancer [1e6]. Using this strategy, a single molecule hits multiple
targets, which participate in pathways implicated to a given
disease, leading to a more efficacious therapy, minimizing the
emergence of resistance.
nogenesis have been extensively studied at the molecular level,
revealing potential novel targets for the treatment and chemo-
prevention of a number of different types of cancers [10,11].
Epidemiological studies have also shown that chronic inflammation
preexists in some types of cancer and various inflammatory medi-
ators like cytokines and chemokines, are present in tumour sites.
It is therefore evident that the use of multi-target ligands, that
interact with multiple targets, could be valuable for the treatment
of the above mentioned pathophysiological conditions [12]. It has
already been proven for a number of commercially available non-
steroidal anti-inflammatory drugs (NSAIDs) that they possess
a combination of these properties. Namely, AspirinÒ and other non-
steroidal anti-inflammatory drugs may prevent cancer, through
their action as cycloxygenase-2 (COX-2) inhibitors [13,14].
Furthermore, Zileuton (ZyfloÒ), a 5-lipoxygenase inhibitor, prevents
lung tumorigenesis [15].
q
Part of these results has been presented at: 14th Panhellenic Pharmaceutical
Congress, May 9e11, 2009, Athens, Greece and XXth International Symposium on
Medicinal Chemistry, August 31eSeptember 4, 2008, Vienna, Austria.
* Correspondence author. Tel.: þ302310997627; fax: þ302310997679.
E-mail address: hadjipav@pharm.auth.gr (D. Hadjipavlou-Litina).
Curcumin or diferuloylmethane (Fig. 1) is a polyphenolic natural
derivative, coloured yellow, isolated from the dried rhizome of the
herb Curcuma longa Linn (Turmeric). Turmeric has been used as
a dietary pigment, spice and in traditional medicine, in India and
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.060

A.-M. Katsori et al. / European Journal of Medicinal Chemistry 46 (2011) 2722e2735
2723
2. Results and discussion
2.1. Chemistry
For the synthesis of the desired curcumin analogues 1aei two
different synthetic approaches were followed: In Method A, a Clai-
seneSchmidt condensation was used [47] between the 4-piperidine
hydrochloride monohydrate and the appropriate aryl aldehyde at
a molar ratio 1:2, in acetic acid by passing through the mixture dry
HCl gas. In Method B, an aldol condensation was recruited [48]
between 4-piperidine hydrochloride monohydrate and the appro-
priate aryl aldehyde at a molar ratio 1:2 respectively, in an alcoholic
solution of NaOH (10% w/v) (Scheme 1). All the synthesized
analogues along with their physicochemical properties are pre-
sented in Table 1. Both methods were applied for the synthesis of
compounds 1a, 1c, 1d, 1h and 1i. For compounds 1a and 1c both
methods led to the same products (they were identified spectro-
photometrically and were found to be identical). Interestingly,
attempts to synthesize 1d, according to Method B resulted in the
formation of a mixture of bis- and mono derivatives, whereas for 1h
and 1i the same method produced the desired product more effi-
ciently. Our attempt to synthesize the pyrrolyl analogue using both
methods did not succeed. When Method B was used, a mixture of
several different unidentified products was obtained. Using Method
A, the reaction product was polymerized under acidic conditions.
Condensation of the free base 1b with the acryloyl chloride led
to the formation of 2b (Scheme 2). All the synthesized compounds
were characterized spectrophotometrically. Elemental analysis of C,
H, and N, and melting points are given. Representative carbon
nuclear magnetic resonance spectra were obtained. For compounds
1aei, the NH absorptions were not observed in the 1H NMR spectra
of the compounds, as it has already been refined by other
researchers [40,41,43,44,47]. The X-ray crystallography studies of
a number of previously synthesized analogues confirmed the
isolation of E, E isomers [36,47], hence the assumption was made
that the olefinic double bond possesses E stereochemistry.
Fig. 1. Curcumin and curcumin analogues.
China, as an antiseptic, wound healing and anti-inflammatory
agent. According to recent reviews, curcumin is a multi-target
pleiotropic agent, exhibiting a broad range of biological activities.
It interacts with multiple cellular targets implicated in cancer and
inflammation [16e23]. Past and ongoing clinical trials have
demonstrated that curcumin is safe at high doses and it seems to
respond in various inflammatory and cancer-related diseases
[16,23e27]. Although, its potential use as a therapeutic agent is
severely affected by its low water solubility, rapid metabolism and
poor bioavailability [20,28,29].
Thus, numerous approaches have been undertaken to synthe-
size new analogues and derivatives in order to enhance curcumin’s
bioavailability along with its anti-inflammatory or/and anticancer
properties [20,30,31]. Recent reports have demonstrated that the
b
-diketone moiety of curcumin is a specific substrate for liver aldo-
keto reductases. The presence of -diketone may contribute to the
b
rapid metabolism of curcumin in vivo [32].
In previous attempts to improve curcumin’s poor pharmacoki-
netics a series of curcumin analogues containing a monocarbonyl
moiety incorporated in a piperidone ring have been reported
(Fig. 1). These analogues possess high anticancer activity [33e39].
Moreover, they induce apoptosis in human HL-60 promyelocytic
leukaemia, HSC-2 squamous cell carcinoma and in human HepG2
liver cell lines [40], activate caspases ꢀ3, ꢀ8, and ꢀ9 [41], inhibit
NF-kB [42], and stimulate fyn kinases [43], indicating that they can
act upon a variety of cellular processes. All the above, support
a multiplicity in biological targets, whereas no toxicity effects were
observed in in vivo assays in mice [35,44]. Furthermore, evidence
from a degradation degree and a pharmacokinetic in vivo study
demonstrated that cycloexanone analogues have enhanced
stability and activity compared to curcumin [45].
The presence of heteroaromatic cores is correlated with potent
anti-proliferative and anti-inflammatory activities [33,45,46]. Thus,
we decided to synthesize a series of curcumin analogues containing
a variety of heterocyclic rings, (more specifically the indolyl, imi-
dazolyl, thienyl) and the naphthyl ring in order to investigate their
behaviour not only as proliferation inhibitors but as anti-
inflammatory agents too. To evaluate the biological activity of the
synthesized compounds, their ability to inhibit the inflammation-
related enzymes Lipoxygenase (LOX) and Aldose Reductase (ALR2)
was tested, as well as their anti-proliferative activity upon three
different cancer cell lines (NCI-H460, MCF7 and SF268).
Compounds structurally similar to 1a have marked affinities for
thiols but not for amino or hydroxy groups [47a], which are found in
nucleic acids. Thus, these compounds may be free from the muta-
genicity/carcinogenicity, which is associated with a number of
alkylating agents. The known “parent” molecule 1a [44,47a] and the
novel N-acryloyl derivative 2b were synthesized and included in the
biological study. Previous findings support the fact that N-acryloyl
derivatives are more potent than N-acetyl or N-methyl derivatives
[47b]. Since steric impedance to attack by thiols would be less in the
case of 2b than N-acetyl or N-methyl derivatives, it is conceivable
that the N-acryloyl derivative would have greater potency.
Scheme 1. General methods used for the synthesis of 1aei curcumin analogues.

2724
A.-M. Katsori et al. / European Journal of Medicinal Chemistry 46 (2011) 2722e2735
Table 1
Lipophilicity values: theoretically calculated (clog P) and RM values; theoretically calculated molecular refractivity values of Ar (MR-Ar) [72].
a/a
Ar
clog P
RM ꢁ SD
MR-Ar
2.59
1a
3.71
0.135 ꢁ 0.00
1b
1c
6.06
3.00
0.463 ꢁ 0.00
0.176 ꢁ 0.00
4.27
2.40
1d
1e
4.15
3.69
0.597 ꢁ 0.08
0.166 ꢁ 0.025
3.70
3.70
1f
8.81
0.076 ꢁ 0.0016
6.49
1g
1h
ꢀ0.63
ꢀ0.552 ꢁ 0.042
ꢀ0.030 ꢁ 0.0017
1.80
1.86
4.00
1i
4.00
6.37
0.031 ꢁ 0.0018
ꢀ0.050 ꢁ 0.001
1.87
4.27
2b
2.2. In vitro assays
membrane phospholipids and can be converted into leukotrienes
(LTs) through 5-lipoxygenase (5-LOX). Leukotriene B4 (LTB4) is
a potent mediator of inflammation, enhancing recruitment and
activation of inflammatory cells. LTB4 generation is considered to
be important in the pathogenesis of neutrophil-mediated inflam-
matory diseases [49] with a marked relation to the severity of
2.2.1. In vitro inhibition of soybean lipoxygenase
Eicosanoids are oxygenated metabolites of arachidonic acid (AA)
with broad implications in a variety of diseases. Upon appropriate
stimulation of neutrophils, arachidonic acid is cleaved from
Scheme 2. Synthesis of the N-acryloyl derivative 2b.

A.-M. Katsori et al. / European Journal of Medicinal Chemistry 46 (2011) 2722e2735
2725
Table 2
found that the presence of a bromo-benzyloxy template diminishes
LOX activity [61]. Herein, compound 1f, containing this scaffold, did
not confirm this finding.
Soybean lipoxygenase (LOX) % at 100 mM and IC50 (mM) inhibitory activity; Aldose
reductase (ALR2)% at 100
carrageenin rat paw oedema (CPE%) at 0.01 mmol/kg body weight.
mM inhibitory activity data; Inhibition % of induced
Lipophilicity is referred as an important physicochemical
property for LOX inhibitors [62] and in our case the higher lip-
ophilicity value (clog P) supports the higher activity [clog
P 2b ¼ 6.37, clog P 1b ¼ 6.06] (Table 1). The theoretically calculated
clog P value of 1f is too high to be realistic and thus, it was not taken
under consideration in the discussion of the results.
Most of the LOX inhibitors are antioxidants or free radical scav-
engers [63]. In our case preliminary antioxidant studies did not
demonstrate a significant antioxidant effect for the tested analogues
(datanot shown). Theinhibitoryactivityof thecompounds, which do
notcontain aneasilyoxidisablemoiety, might beattributedmainlyto
their ability to attach to the active site of the enzyme or to act in an
allosteric manner and to a lesser degree to their electron donating
ability. It seems that replacement of H (NeH) by the acryloyl moiety
(2b) does not significantly alter the inhibition pattern.
Compound
LOX Inhibition
ALR2 Inhibitiona
%CPE
(C ¼ 100
m
M)/IC50
(
m
M)
(C ¼ 100
m
M)/IC50
(m
M)
Inhibitionb
1a
1b
1c
1d
1e
1f
1g
1h
1i
2b
Curcumin
Sorbinil
IMAe
8%
37 mM
49.0%
70.6%
45.5%
27.8%
nac
43.0*
nac
330
280
410
18%
36%
nac
m
m
m
M
M
M
nac
33.0*
36.0*
49.0*
11.5*
27.0*
62.0*
27.0*
nac
nac
56.0%
nac
nac
47
m
M
37.5%
12.0%
38%
0.25 m
Md
47.0*
Each experiment was performed at least in triplicate and the standard deviation of
absorbance was less than 10% of the mean.
We further examined the possibility of compound 2b acting as
a prodrug of 1b, though incubation of 2b, under our experimental
conditions, did not result in the production of 1b.
*p < 0.01.
a
n < 3.
b
Statistical studies were done with student’s T-test.
No activity under the experimental conditions.
c
d
Reported IC50 ¼ 0.25
mM [78].
2.2.2. In vitro inhibition of aldose reductase
e
Indomethacin.
Aldose reductase (ALR2) is the first enzyme of the polyol
pathway and catalyzes the conversion of glucose to sorbitol. A
number of studies, in the past years, demonstrated the implication
of ALR2 in long-term diabetic complications. Nowadays, it has been
found that ALR2 up-regulates a series of factors, involved in various
pathological conditions [64]. Recently, Ramana et al. reviewed the
implication of ALR2 in inflammatory pathologies, indicating the
multiple roles of ALR2 inhibitors [65]. Curcumin has already been
described as a specific ALR2 inhibitor [66,67]. It has also been
published that curcumin analogues with structural similarities to
the synthesized compounds, inhibit specifically rat lens ALR2 [68].
Our analogues were tested for their ability to inhibit purified rat
cardiovascular diseases and cancer. Inhibitors of LOX have attracted
attention initially as potential agents for the treatment of inflam-
matory and allergic diseases and certain types of cancer
[12,50e54]. Recently in our laboratory, we have reported a series of
aryl-acetic and aryl-hydroxamic acids acting as multi-target agents,
possessing anti-LOX, anti-inflammatory and anticancer activities
[55]. Curcumin has also been reported to inhibit 5-LOX activity both
in in vitro and in vivo models [56e58].
LOXs contain a “non-heme” iron per molecule in the enzyme
active site, as high-spin Fe2þ in the native state and the high-spin
Fe3þ in the activated state. The four classes of direct 5-lipoxygenase
inhibitors encompass: a) redox-active compounds keeping the
active site iron in the ferrous state, thereby, uncoupling the catalytic
cycle of the enzyme, b) iron ligand inhibitors, c) non-redox type
inhibitors that compete arachidonic acid or lipid hydroperoxides
for binding to 5-LOX and d) a novel class of inhibitors that may act
in an allosteric manner [59].
Thus, we evaluated the ability of our analogues to inhibit
soybean LOX by the UV absorbance based enzyme assay (Table 2)
[60]. It is known that soybean lipoxygenase, which converts linoleic
to 13-hydroperoxylinoleic acid, is inhibited by non-steroidal anti-
inflammatory drugs (NSAIDs) in a qualitatively similar way to
that of the rat mast cell lipoxygenase and may be used as a reliable
screen for such activity [60].
lens ALR2 at 100 mM concentration. The human and rat sequences
of ALR2 are characterized by 81% identity and 89% homology, while
the proposed active sites of both enzymes are identical [69]. The
assay, performed, was based on the spectrophotometric monitoring
of NADPH oxidation from D, L-glyceraldehyde and is proven to be
quite a reliable method [70]. Sorbinil was used as a positive control.
Results are presented in Table 2 and are compared to curcumin
(BML-EI135).
Under our experimental conditions compounds 1a, 1b, 1c, 1d,
1h, 2b were more potent inhibitors of ALR2 enzyme than curcumin.
Among all the analogues 1b seems to be the most potent followed
by 1h, 1a and 1c. The presence of the acryloyl group in 2b reduces
inhibitory activity. The 3-indolyl analogue 1d inhibits ALR2,
whereas the 5-indolyl 1e had no effect under our experimental
conditions. The substitution position of the methyl group on the
thienyl rings influenced the biological response. Thus, analogue 1i
with a 5-CH3 group did not present any activity, whereas the 3-CH3
substituted analogue (1h) exhibited enhanced inhibition (56%)
compared to the unsubstituted thienyl representative 1c. The imi-
dazolyl analogue 1g also did not elicit any response. Perusal of %
inhibition of ALR2 activity delineates the role of lipophilicity since
the most potent analogue 1b also exhibits the highest lipophilicity
value (clog P ¼ 6.06) compared to 1a, 1b, 1c, 1d, 1g and 1h.
Curcumin (BML-EI135) was used as a reference compound
(inhibition 38% at 100
(IC50 37 M) was the most potent analogue followed by 1d, 1c and
1e. Compounds 1a and 1f exhibited lower inhibition. Analogue 1g
at 100 M seemed to be equipotent to curcumin whereas 1h and 1i
mM). Among the free bases, compound 1b
m
m
do not present any inhibitory activity. Between the two indolyl-
derivatives, the 3-substituted analogue (1d) was by far more
potent compared to the 5-substituted analogue (1e). The acryloyl
derivative 2b presented high inhibitory activity (IC50 47
mM)
however, lower than that induced by the corresponding free base
1b. Comparing the activities within the thienyl subgroup, only 1c
inhibited soybean LOX. It is also interesting that the imidazolyl-
derivative 1g was more potent than the phenyl analogue 1a.Both
compounds share the 4-piperidinone moiety with the two attached
olefinic bonds. However, the replacement of phenyl by the imida-
zolyl ring increased the inhibitory activity. In previous studies, we
2.2.3. In vitro anti-proliferative activity
The in vitro anti-proliferative activity studies were performed on
three human cancer cell lines originating from solid tumours:
NCI-H460 (Non small cell lung cancer), MCF7 (Breast Cancer) and
SF268 (Central Nervous System, glioma) using the Sulforhodamine
B assay [71]. The cells were preincubated for 24 h and subsequently

2726
A.-M. Katsori et al. / European Journal of Medicinal Chemistry 46 (2011) 2722e2735
Table 3
In vitro anti-proliferative activity of all analogues against NCI-H460 (Non small cell lung cancer), MCF7 (Breast Cancer) and SF268 (Central Nervous System, glioma);
Concentrations are in M and represent the mean of three independent experiments each one run in triplicates. CV<15%.
m
Cell lines
NCI-H460
Parameters
1a
2.0
6.1
10.3
5.2
0.8
4.1
9.0
11.6
0.9
1b
1c
1d
1e
1f
1g
1h
1i
2b
1.2
Curcumin
GI50
TGI
LC50
TI
GI50
TGI
LC50
TI
GI50
TGI
LC50
TI
6.0
24.5
98.3
16.4
6.8
29.8
97.5
14.3
6.2
>100
>100
>100
ncb
>100
>100
>100
ncb
3.5
6.1
8.7
2.4
3.7
6.0
8.2
2.2
3.3
5.7
8.2
2.5
>100
>100
>100
ncb
>100
>100
>100
ncb
49.9
96.8
>100
>100
>100
ncb
>100
>100
>100
ncb
>100
>100
>100
ncb
73.2
99.3
16.7
50.1
83.4
5.0
4.7
8.3
67.2
14.2
5.1
9.2
61.0
11.9
>100
>100
>100
>100
5.5
9.8
8.1
1.4
5.3
9.2
6.6
0.6
1.1
8.3
13.8
1.4a
1.0a
MCF7
SF268
69.5
63.0
>100
>100
1.6a
53.8
>100
>100
1.9a
80.7
>100
>100
1.2a
65.2
>100
>100
1.5a
>100
>100
1.4a
>100
>100
>100
ncb
>100
2.0a
98.8
>100
>100
>100
ncb
4.7
9.1
10.0
25.2
>100
>100
1.0a
>100
16.0a
GI50: Growth Inhibitory Activity 50%; TGI: Total Growth Inhibitory activity; LC50: Lethal Concentration 50%; TI: Therapeutic index (¼LC50/GI50).
a
The LC50 could not be determined as it was higher than 100
nc: not calculated.
m
M, the highest concentration of the compounds tested. Thus the TI was calculated by assuming LC50 ¼ 100
mM.
b
incubated for 48 h with various concentrations of the analogues at
37 ꢂC, 5% CO2. The dose response parameters such as growth
inhibition 50% (GI50), total growth inhibition (TGI) and lethal
concentration 50% (LC50) were calculated. GI50 is the concentration
of drug required to decrease the cell growth to 50%, compared with
that of the untreated cell number. TGI is the concentration of drug
required to totally inhibit cell growth. LC50 is the concentration of
drug required to decrease the cell population by 50% of the initial
cell number (Table 3, Figs. 2 and 3). Analogues 1g and 1c found to be
aryl substituents, expressed as molar refractivity MR-Ar values
(molar refractivity depends on volume and polarisability) (Table 1).
The results suggest that steric hindrance plays an important role
[MR-Ar-1a 2.586 < MR-Ar-1d 3.703 < MR-Ar-1b 4.274] [72]. The fact
that at least one of the analogues synthesized in this work (1a) has
already been reported to possess significant anti-leukaemic activity
[47] strengthens further the notion that these compounds may be
promising anticancer agents and need to be more thoroughly
investigated. The presence of a naphthyl or of a 3-indolyl group
appears to be critical in terms of structure activity relations.
inactive up to 100 mM, the highest concentration tested against all
three cell lines used and the 1e and 1f derivatives demonstrate
a low growth inhibitory activity only against the MCF7 cell line.
Among the thienyl subgroup, the methyl substituted derivatives 1h
and 1i seemed to be more potent compared to 1c, with 1h having
the lower GI50 value. It seems that increase of lipophilicity
enhances the biological response. Among analogues 1aei,
compounds 1a, 1b, 1d exhibited significant growth inhibitory
2.3. In vivo anti-inflammatory activity
In in vivo acute toxicity experiments, the examined compounds
did not cause toxic effects in doses up to 0.2 mmol/kg body weight.
Ulcerogenicity was not observed. Compounds were tested for their
anti-inflammatory activity in vivo (dose intraperitoneally 0.01 mmol/
kg body weight). The in vivo anti-inflammatory effects of the tested
compounds were assessed by using the functional model of
carrageenin-induced rat paw oedema (CPE) [73] and are presented in
Table 2, aspercentinhibitionofcarrageenin-induced ratpawoedema.
After 3.5 h, compound 1g provided very low protection (11.5%)
against carrageenin-induced paw oedema while the reference drug
indomethacin (IMA) induced 47% protection at an equivalent dose.
Compounds 1b and 1c did not inhibit carrageenin-induced rat paw
oedema, whereas compound 1i caused the highest inhibition
among the tested compounds (62%) followed by the 1f (49%). No
difference was observed between the two indolyl analogues which
showed to be equipotent. The side ring’s attachment site did not
seem to influence the biological response (1d and 1e, 33% and 36%).
Concerning the thienyl derivatives it is interesting that the potency
increased when a methyl group was attached to the 5-position of
the ring (1i). Reduced anti-inflammatory activity was observed for
the 1h (3-substituted) and the activity was diminished for the
unsubstituted derivative 1c. Herein the role of lipophilicity is
marginal (1i > 1c, 1h > 1c). The “parent” derivative 1a produces
satisfactory inhibition of the carrageenin induced rat paw oedema
(43%). As shown in Table 2, analogue 1b appeared inactive, whereas
the acryloyl 2b possessed anti-inflammatory properties. This
behaviour might be attributed to the conformation imposed by the
addition of the acryloyl group in structure 1b.
activity (below 10
experimental conditions used. The most active analogue, within
series 1, was found to be 1a, exhibiting a GI50 close to 1 M, against
mM) against all three cell lines, under the
m
all cell lines. Compound 1d exhibited lower inhibitory activity
(higher GI50 value) than curcumin in all three cell lines. No differ-
ences in cytotoxic activity were detected when 1d was compared to
analogue 1a, as this is reflected by the LC50 values. Therefore the
3-indolyl group seems to play a key role in the growth inhibitory
activity of the analogues. This assumption is supported by the
observation that the 3-indolyl-substitution (1d) resulted in
complete inactivation. On the contrary the 5-indolyl analogue (1e)
had no activity, revealing the detrimental role of the indolyl moiety
ring 3/5 attachment. The analogue 1b exhibited a significant
growth inhibitory activity higher than that of curcumin in
NCI-H460 cell line. However, this compound demonstrated as well,
a much lower cytotoxic activity (LC50 close to 100
mM). Thus,
considering the ratio of LC50/GI50 as a Therapeutic Index (TI),
compound 1b presented the best TI among the most active
analogues, including curcumin (Table 3). When an acryloyl group
was further added to the 1b, the resulting analogue 2b showed
increased both inhibitory and cytotoxic activities (Table 3). These
data are in accordance to previous reports [47]. A detrimental
contribution of lipophilicity is also shown in this case, since
analogue 2b possesses higher clog P than 1b (Table 1).
Altogether the results from the preliminary screen suggest that
1a, 1b, 1d and 2b analogues, exhibit significant growth inhibitory
activity against solid tumour derived cell lines. The observed GI50
values for these four derivatives upon the NCI-H460, MCF7 and
SF268 cancer cell lines, reveal that the anti-proliferative activity
within analogues 1aei is inversely correlated with the size of the
2.4. Physicochemical studies
The variations in the in vivo anti-inflammatory activity of the
tested compounds could partially be attributed to differences in
their physicochemical properties, which determine their

A.-M. Katsori et al. / European Journal of Medicinal Chemistry 46 (2011) 2722e2735
2727
Fig. 2. Percent growth rates of the three cell lines H460, MCF7 and SF268 after treatment with various concentrations of the four analogues 1a, 1b, 1d and 2b for 48 h. The curves
were generated using the SRB assay. Each point represents the mean of three independent experiments, each one run in triplicates ꢁ SD.
distribution in the body and their ability to cross membranes and
enter cells [74,75]. Lipophilicity is a significant physicochemical
property, determining distribution, bioavailability, metabolic
activity and elimination. Thus, this parameter was experimentally
determined for each compound as RM value. The Reverse Phase
Thin-Layer Chromatography (RPTLC) method was used [61] and RM
values were compared to the corresponding theoretically calculated
clog P values in n-octanol [72]. Even though, this is considered to be

2728
A.-M. Katsori et al. / European Journal of Medicinal Chemistry 46 (2011) 2722e2735
Fig. 3. Morphology of untreated cells from the three cell lines used for the in vitro screening (Ut) and after treatment with a concentration of 10 mM of the analogues 1a, 1b, 1d and
2b for 48 h. At the end of the 48 h incubation time cells were fixed with TCA and stained with SRB. DMSO which used as a vehicle solvent did not exhibit any activity (not shown).
Pictures are from one representative out of three independent experiments. Magnification 10ꢃ at a Zeiss Axiovert 200M inverted microscope.
a reliable, fast and convenient method for expressing lipophilicity,
our results (Table 1) indicate that RM values could not be used as
a successful relative measure of the overall lipophilic/hydrophilic
balance of these molecules. This can be attributed to the different
nature of the hydrophilic and lipophilic phases in the two systems
and to the presence of basic nitrogen atoms in the examined
compounds, which could disturb the absorption/desorption
process.
with the inhibitor IDD 594 (PDB entry 1US0), was chosen [76], since
it was determined at the highest resolution (0.66 Å) among all the
available structures. Docking was carried out using the automated
docking program Autodock Vina [77].
The structure of ALR2 active site has been extensively analyzed
by both crystallographic and modelling studies. It has proved to be
highly hydrophobic in nature and is formed by aromatic residues
(Trp20, Tyr48, Trp79, Trp111, Phe121, Phe122 and Trp219), apolar
residues (Val47, Pro218, Leu300 and Leu301), and polar residues
(Gln49, Cys298 and His110). The nicotinamide ring of NADPþ is
centered in the cavity and the three possible proton donors
responsible for catalysis are Tyr48, His110 and Trp111 all composing
the anionic binding pocket [64,78].
2.5. Molecular docking studies
2.5.1. Docking studies on aldose reductase
In terms of analyzing the interactions of the synthesized
analogues in the active site of ALR2, docking simulations were
performed. The human aldose reductase holoenzyme, complexed
In Fig. 4A the alignment of our analogue 1b with both the
inhibitor of ALR2 and curcumin is clearly shown, in the active site of

A.-M. Katsori et al. / European Journal of Medicinal Chemistry 46 (2011) 2722e2735
2729
Fig. 4. Docked poses of (A) alignment of ligand IDD 594 (cyano), curcumin (white) and 1b (purple), (B) inhibitor IDD 594 in the active center of ALR2, (cyano) (C) curcumin (grey)
and (D) analogue 1b (purple) in the ALR2 binding site. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
ALR2. In the catalytic site of ALR2 the interactions of the inhibitor
IDD 594 are mostly polar (Fig. 4B). IDD 594 binds to the active site of
ALR2 with the acidic function forming hydrogen bonds with Tyr48,
His110 and Trp111, which are the three key residues in the binding
site [79,80]. Additional strong electrostatic interactions with
NADPþ occur. The obtained docking results are in accordance with
docking results already reported [76,81].
The best binding modes of the most active analogue 1b and
curcumin are presented in Fig. 4C and D. Compound 1b exhibits
binding energy slightly lower (ꢀ13.2 kcal/mol) than the selective
inhibitor IDD 594 (ꢀ9.7 kcal/mol). Curcumin presents even higher
binding energy (ꢀ8.4 kcal/com). These results seem to be in
accordance with our biological data (Table 2).
Previous molecular docking studies with both keto and enol
forms of curcumin revealed similar results with both tautomers
[67]. Thus, for simplicity, we present our molecular docking data
with the keto form of curcumin. The carbonyl group strongly
interacts via hydrogen bonds with Trp20. Hydrogen bonds are also
observed between one of the methoxy groups and Leu300 in the
active site of the protein.
Analogue 1b is well fitted in the active site of ALR2. Compared to
the other two compounds, it creates different type of interactions
with amino acids of the active site. No hydrogen bonding is
observed in the case of analogue 1b. Lipophilicity seems to play
crucial role in 1b inhibitory activity. The naphthyl groups are
properly oriented to the more lipophilic areas of the ALR2 binding
site whereas hydrophobic bonds are created with Trp111, Leu300
and Ala299.
2.5.2. Docking studies on soybean lipoxygenase
The lack of structural data for human LOX, lead us to model
human LOX using soybean enzyme because of its availability and
highly characterized structure [82]. The possible mechanism of
action, as well as the differences in activity of our analogues toward
soybean lipoxygenase could be explained by docking calculations.
In our lab, we have a long experience in LOX inhibitors. We have
recently presented a study of new LOX inhibitors, performing
docking simulations on the most active compound to get some
clues on the nature and type of interactions governing the inhibitor
binding [83].
For the docking studies of lipoxygenase we have used the 1IK3
and 1RRH (soybean lipoxygenase) available from the Protein Data
Bank (PDB) with a resolution of 2 Å [84]. We used the 1RRH from
PDB with Feþ3 running blind docking. Two soybean lipoxygenase
models were derived from 1RRH. One with a ligand taken from
1IK3 (ligand ID: 9OH) for the docking studies into the catalytic
cavity and another one without any ligand in the catalytic cavity for
the blind docking simulation. The metal center in both models has
been considered as a cation, with charge q ¼ þ3 and no bond
restraint was applied between the iron and the ligands. Len-
nardeJones parameters for Fe(III) force field can be resumed as:
svdw ¼ 2.138157 ee01 nm, 3vdw ¼ 2.092 ee1 kJ/mol. The validation

2730
A.-M. Katsori et al. / European Journal of Medicinal Chemistry 46 (2011) 2722e2735
complex is stabilized by hydrophobic interactions between the
aromatic moieties. From the above, the possibility of an allosteric
effect might be considered. Furthermore, blockage of the cavity
entrance might disturb the access to the catalytic site. The latter
merits further investigation, since analogue 1b could be a lead for
the design of allosteric lipoxygenase inhibitors.
3. Conclusion
This study has demonstrated that curcumin analogues bearing
a variety of heterocyclic substituents in the aromatic part combine
anti-inflammatory and anti-proliferative activity. Some of our
analogues proved to be more potent in vitro inhibitors of soybean
lipoxygenase and aldose reductase (ALR2) enzymes than curcumin.
The highest activity was exhibited by the naphthyl analogue 1b in
both assays. Compounds 1a, 1b, 1d exhibited significant growth
inhibitory activity (below 10 mM) against all three cancer cell lines.
When an acryloyl group was further inserted to 1b, the resulting
analogue 2b showed higher inhibitory activity whereas the anti-
inflammatory activity was not enhanced. Compound 1i presented
a promising anti-inflammatory profile with high anti-oedematous
activity, causing the highest inhibition of carrageenin induced paw
oedema, among the tested compounds. The influence of lip-
ophilicity is well documented. Lipophilicity correlated with the
biological activity of the compounds. Furthermore, the presence of
a naphthyl, or of a 3-indolyl group was critical for their effectiveness.
Molecular docking studies were carried out on the active
compound 1b. Modelling studies were found to be in accordance
with our experimental biological results. The lipophilic analogue 1b
was well fitted into the active site of ALR2 enzyme, developing
hydrophobic interactions. In the case of LOX enzyme the inhibitory
activity of our analogue 1b might be attributed to its ability to act in
an allosteric way.
However, further investigation is needed in order to gain insight
into the mechanism of action of the examined compounds. Some of
the synthesized compounds might constitute initial leads for the
design of new more potent multi-target therapeutic agents.
Fig. 5. Docked poses of (A) curcumin (dark grey) and (B) compound 1b (purple) in the
soybean lipoxygenase binding site. Side-chains of relevant binding site residues are
rendered as ball and stick models. The iron ion is rendered as an orange sphere. (for
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
4. Experimental
of our docking study was made with the ligands crystallized in the
protein complexed to 1IK3, where our docking results were in good
agreement with the structure of the molecules in the active site of
the protein.
The dockingorientationsofthecompoundsarepresentedinFig. 5.
It is important to mention that analogue 1b exhibits lower binding
energy (ꢀ9.2 kcal/mol) to the protein than the reference compound
curcumin. However, it was impossible for 1b to get into the cavity,
where curcuminwas freely inserted (binding energy: ꢀ8.7 kcal/mol).
The docking study suggests that the biological response of the
examined compounds could be explained by the proper geometry of
the predicted conformations at the active site.
In this docking approach it seems that curcumin creates
significant interaction with many amino acids in the inner part of
the cavity, near to the iron ion, forming hydrogen bonds with the
amino acids of the cavity. The carboxylic group forms hydrogen
bonding with His513 whereas one of the hydroxyl groups interacts
with Arg726 in the lipoxygenase cavity. Significant bonds are
observed between curcumin and the following amino acids:
Ser510, Asn713, Asp766, Ile770, Leu773 and Ile857 (Fig. 5A).
As it is shown in Fig. 5B, analogue 1b is well fitted in another
area of the protein and not to the central cavity of LOX. Probable
4.1. General
All starting materials and solvents were obtained from
commercial sources and used without further purification. Melting
points were determined in open glass capillaries using a Mel-
TempII apparatus (Lab. Devices, Holliston, MA, USA). UVevis
spectra were obtained on a Hitachi U-2001 spectrophotometer and
on a Shimadzu UV-1700 PharmSpec (UVprobe Ver. 2.21). Infrared
spectra (film as Nujol mulls or KBr) were recorded with a Shimadzu
FT IR-8101 M and with a PerkineElmer FT-IR System BX. The 1H
NMR spectra were recorded at 300 MHz on a Bruker AM 300
spectrometer (Bruker Analytische Messtechnik GmbH, Rheinstet-
ten, Germany) in CDCl3 or DMSO using tetramethylsilane (TMS) as
an internal standard, unless otherwise stated. 13C NMR spectra
were obtained at 75.5 MHz on a Bruker AM 300 spectrometer in
CDCl3 or DMSO solutions with tetramethylsilane as internal refer-
ence unless otherwise stated. MS (ESI) on a Shimadzu LC-MS 2010
EV. Elemental analyses were obtained in an acceptable range
(ꢁ0.4%) in a PerkineElmer 240 B CHN analyzer (The PerkineElmer
Corporation Ltd). Reactions were monitored by thin-layer chro-
matography (TLC) by Fluka, on aluminium cards precoated with
0.2 mm of silica gel and fluorescent indicator. For RPTLC silica gel
Merck F254 plates were used.
reason for this could be the bulk of naphthyl groups. p-interactions
are developed among analogue 1b and amino acids: Glu197,
For the biological assays soybean lipoxygenase, linoleic acid
Trp257, Leu258, Gly265 and Phe272. The enzyme-inhibitor
sodium salt NADPH, D-L-glyceraldehyde and indomethacin were

A.-M. Katsori et al. / European Journal of Medicinal Chemistry 46 (2011) 2722e2735
2731
obtained from SigmaeAldrich Corporation and carrageenin, type K,
was commercially available. Curcumin (BML-EI135) was obtained
from Biomol (Enzo Life Science) and Sorbinil from Pfizer, Inc. The
cell lines NCI-H460 (Non small cell lung cancer), MCF7 (Breast
Cancer) and SF268 (Central Nervous System, glioma) were obtained
from the National Cancer Institute, NIH (Bethesda, MD, USA). Tri-
chloro acetic acid (TCA), sulphorhodamine B (SRB) acetic acid,
DMSO and trizma base were purchased from SigmaeAldrich
(St. Louis, MO, USA). All cell culture reagents were purchased from
Euroclone Life Sciences Division (Milano, Italy). Microscopy analysis
was performed with Axiovert 200M, Carl Zeiss, Gottingen, Germany
equipped with a Sony digital camera. For the in vivo experiments,
male and female Fischer-344 rats were used.
absorption of piperidone were not observed in the 1H NMR spectra
of the compounds as it has already been refined by other
researchers [47a]. 13C NMR as hydrochloride salt (DMSO-d6)
d: 45,
45.12,110.8,110.9,111.2,112.9,117.7, 118,119,120.5, 120.8, 122, 122.8,
127.5, 128, 128.2, 136, 144, 146, 146.9, 153, 154.7, 158.2, 182.2 ppm.
LC-MS m/z: 354 [M þ H] þ, 352 [M ꢀ H] þ, 376 [M þ Na] þ, 392
[M þ K] þ. Anal. calcd for C23H19N3O: C 78.16, H 5.42 N 11.89. Found:
C 78.04, H 5.36 N 11.49.
4.2.1.5. 3,5-Bis((1H-indol-5-yl)methylene)piperidin-4-one
(1e). Method A: 82% yield, red solid, m.p. 120 ꢂC decomposes. Rf
(EtOH) 0.9. UV (ethanol absolute) lmax: 254, 298 3max: 5380, 3520.
IR (Nujol, cmꢀ1): 1670, 3200e3450. 1H NMR (DMSO-d6)
d: 4.36 (br,
4H), 7.95e7.96 (m, 6H), 8.03e8.20 (br, 6H), 10.26 (s, 2H) ppm. The
NH absorption of piperidone were not observed in the 1H NMR
spectra of the compounds as it has already been refined by other
researchers [47a]. LC-MS m/z: 354 [M þ H] þ. Anal. calcd for
C23H19N3O: C 78.16, H 5.42 N 11.89. Found: C 78.18, H 5.18 N 11.49.
4.2. Synthesis
4.2.1. Synthesis of analogues 1aei
Two different methods were used for the synthesis of the
compounds.
General Method A [44,47a]
4.2.1.6. 3,5-Bis(4-(4-bromobenzyloxy)benzylidene)piperidin-4-one
(1f). Method A: 28% yield, yellow solid, m.p. 210e213 ꢂC. Rf (8.5
benz:1.5 MeOH) 0.8. UV (ethanol absolute) lmax: 351 3max: 7510. IR
A ClaiseneSchmidt condensation was performed between 4-
piperidine hydrochloride monohydrate and the appropriate aryl
aldehyde at a molar ratio 1:2 in acetic acid. Dry hydrogen chloride
was passed through the mixture for at least 1 h. After stirring at room
temperature, the end of the reaction was monitored by TLC. The
precipitate was then collected and added to a mixture of saturated
aqueous potassium carbonate solution (25% w/v, 25 mL) and acetone
(25 ml). The resultant mixture was stirred for 0.5 h. The free base is
collected, washed with water (50 mL) and recrystallized from 95%
aqueous ethanol. In the case where no precipitate was formed after
the water dropping, extraction with 3 ꢃ 50 ml chloroform was
performed and the organic base was collected and dried over K2CO3.
General Method B [48]
An aldol condensation between 4-piperidine hydrochloride
monohydrate and the appropriate aryl aldehyde was performed at
a molar ratio 1:2 respectively, in alcoholic NaOH (10% w/v). The
solution was stirred at room temperature. The progress of the
reaction was monitored using TLC. The separated solid was filtered,
washedwith waterandrecrystallized fromthe 95%aqueous ethanol.
(Nujol, cmꢀ1): 1670, 3300. 1H NMR (DMSO-d6)
d: 4.14 (s, 4H), 5.06
(s, 4H), 6.97e7.00 (m, 4H), 7.17e7.54 (m, 12H), 7.76e7.80 (br, 2H)
ppm. The NH absorption of piperidone were not observed in the 1H
NMR spectra of the compounds as it has already been refined by
other researchers [47a]. 13C NMR (DMSO-d6)
d: 39.6, 39.9, 76.5, 77,
110.9, 111, 114, 115, 116, 119, 121, 123, 124, 126, 127, 129, 131, 133, 135,
136, 137, 138, 139.8, 141, 143, 146, 147, 149,þ151, 155, 159, 161,
183.6 ppm. LC-MS m/z: 646 [M
C33H27Br2NO3: C 61.41, H 4.22 N 2.17. Found: C 61.3, H 4.08 N 1.88.
þ
H]
. Anal. calcd for
4.2.1.7. 3,5-Bis((1H-imidazol-2-yl)methylene)piperidin-4-one hydro-
chloride (1g). Method A: 92% yield, white solid, m.p. 215 ꢂC. Rf (8.5
benz:1.5 MeOH) 0.4. UV (ethanol absolute) lmax: 283 3max: 3940. IR
(Nujol, cmꢀ1): 1660, 3180, 3300. 1H NMR (DMSO-d6)
d: 4.19e4.26 (s,
4H), 6.09 (br, 2H), 7.42e7.49 (m, 2H), 9.70e9.73 (m, 4H) ppm. The NH
absorption of piperidone were not observed in the 1H NMR spectra
of the compounds as it has already been refined byother researchers
[47a]. The same problemwas faced for the [Hþ] of the hydrochloride.
LC-MS m/z: 254 [M þ H] þ. Anal. calcd for C13H14ClN5O: C 53.52, H
4.84 N 24.01. Found: C 53.86, H 4.41 N 23.64.
4.2.1.1. 3,5-Dibenzylidenepiperidin-4-one (1a) [44,47a]
4.2.1.2. 3,5-Bis(naphthalen-1-ylmethylene)piperidin-4-one
(1b).
Method A: 70% yield, yellow solid, m.p. 182e185 ꢂC. Rf (8.5 benz:1.5
MeOH) 0.7. UV (ethanol absolute) lmax: 270, 354 3max: 16580,
4.2.1.8. 3,5-Bis((3-methylthiophen-2-yl)methylene)piperidin-4-one
(1h). Method B: 83.4% yield, yellow solid, m.p. 159e162 ꢂC. Rf (8.5
12390. IR (Nujol, cmꢀ1): 1660, 3400. 1H NMR (DMSO-d6)
d:
benz:1.5 MeOH) 0.6. UV (ethanol absolute) lmax: 261, 400 3max
8540, 23700. IR (Nujol, cmꢀ1): 1645, 3300. 1H NMR (DMSO-d6)
: 1.15e1.35 (br, 6H), 4.05 (s, 4H), 7.08e7.35 (m, 4H), 7.66e7.72 (m,
2H) ppm. The NH absorption of piperidone were not observed in
the 1H NMR spectra of the compounds as it has already been
refined by other researchers [47a] LC-MS m/z: 316 [M þ H] þ, 314
[M ꢀ H] þ. Anal. calcd for C17H17NOS2: C 64.73, H 5.43 N 4.44.
Found: C 64.84, H 5.32 N 4.16.
:
4.36e4.43 (s, 4H), 7.33 (d,1H, J 6 Hz), 7.48 (d,1H, J 8.1 Hz), 7.50e7.59
(m, 6H), 7.61e7.79 (m, 2H), 7.83e7.99 (m, 6H) ppm. The NH
absorption of piperidone were not observed in the 1H NMR spectra
of the compounds as it has already been refined by other
d
researchers [47a]. 13C NMR (DMSO-d6)
d: 40, 42.3, 103.8, 117.7, 122,
123, 124.3, 126, 129, 129.4, 130.2, 132, 134, 136, 138, 141.5, 142, 143,
144, 146, 147.6, 148, 152.3, 155, 157, 160, 179 ppm. LC-MS m/z: 376
[M þ H] þ, 398 [M þ Na] þ. Anal. calcd for C27H21NO: C 86.37, H 5.64,
N 3.73. Found: C 86.20, H 5.70, N 3.76.
4.2.1.9. 3,5-Bis((5-methylthiophen-2-yl)methylene)piperidin-4-one
(1i). Method B: 44% yield, orange solid, m.p. 181e182 ꢂC. Rf (8.5
4.2.1.3. 3,5-Bis(thiophen-2-ylmethylene)piperidin-4-one (1c) [85]
benz:1.5 MeOH) 0.5. UV (ethanol absolute) lmax: 265, 402 3max
:
:
10440, 25520. IR (Nujol, cmꢀ1): 1650, 3300. 1H NMR (DMSO-d6)
d
4.2.1.4. 3,5-Bis((1H-indol-3-yl)methylene)piperidin-4-one
(1d).
1.35e1.60 (br, 6H), 4.10 (s, 4H), 6.70e6.81 (br, 2H), 7.15e7.24 (m, 2H),
7.85 (s, 2H) ppm. The NH absorption of piperidone were not observed
in the 1H NMR spectra of the compounds as it has already been
Method A: 10% yield, orange solid, m.p. 255 ꢂC decomposes. Rf as
hydrochloride salt (8.5 benz:1.5 MeOH) 0.2. UV as hydrochloride
salt (ethanol absolute) lmax: 260, 280, 453 3max: 14170, 13530,
29980. IR as hydrochloride salt (KBr, cmꢀ1): 1650, 3400. 1H NMR as
refined by other researchers [47a]. 13C NMR (DMSO-d6)
d: 22, 21.4,
45.1, 46, 108.7, 111.7, 126.6, 133.8, 136, 137, 138.1, 147.1, 147.8, 148, 152,
155.3,183.5 ppm. LC-MSm/z: 316 [M þ H] þ, 314 [Mꢀ H]þ. Anal. calcd
for C17H17NOS2: C 64.73, H 5.43 N 4.44. Found: C 64.38, H 5.07 N 4.12.
hydrochloride salt (DMSO-d6)
d: 4.46 (s, 4H), 7.20e7.29 (m, 4H),
7.49e7.54 (m, 4H), 7.82e7.84 (m, 2H), 11.87 (m, 2H) ppm. The NH

2732
A.-M. Katsori et al. / European Journal of Medicinal Chemistry 46 (2011) 2722e2735
4.2.2. Synthesis of the acryloyl derivative 2b [47]
experiments conform to the law for the protection of experimental
animals (Republic of Greece) and are registered at the Veterinary
Administration of the Republic of Greece. ALR2 from rat lens was
partially purified according to the reported procedure [78] as
follows: lenses were quickly removed from rats following eutha-
nasia and stored at ꢀ20 ꢂC until used. The lenses were homoge-
nized in 5 vol of cold distilled water. The homogenate was
centrifuged at 10000 g at 0e4 ꢂC for 15 min. The supernatant was
precipitated with saturated ammonium sulphate at 40% salt satu-
ration and this solution was centrifuged at 10000 g at 0e4 ꢂC for
15 min. The latter supernatant was either used directly or stored for
maximum 24 h at ꢀ80 ꢂC.
Acryloylchloride (1.11 mmol) was added to a suspension of 3,5-
bis(naphthalen-1-ylmethylene)piperidin-4-one (0.72 mmol) in
acetone (4 mL) and a solution of potassium carbonate (3.70 mmol)
in water (1.3 mL) which was cooled externally with an ice bath.
After stirring at room temperature, the reaction was monitored by
TLC. The mixture was collected and diluted with water-ice (50e50).
The precipitate was collected, washed with water and dried.
4.2.2.1. 1-Acryloyl-3,5-bis(naphthalen-1-ylmethylene)piperidin-4-
one (2b)
91% yield, yellowish solid, m.p. 163e168 ꢂC. Rf (8.5 benz:1.5
MeOH) 0.7. UV (ethanol absolute) lmax: 264, 534 3max: 18250,
ALR2 activity was assayed spectrophotometrically by deter-
mining NADPH consumption at 340 nm. In order to determine ALR2
14500. IR (Nujol, cmꢀ1): 1640, 1653. 1H NMR (CDCl3)
d: 4.08 (s, 2H),
inhibitory activity D, L-glyceraldehyde was used as a substrate. The
4.65e4.82 (br, 2H), 5.30e5.35 (dd, 1H, J 15 Hz), 6.02e6.05 (m, 2H),
target compounds were tested at the concentration of 100 mM.
7.40e7.58 (m, 10H), 7.91e7.93 (m, 4H), 8.00e8.03 (m, 2H) ppm. 13C
NMR (CDCl3)
d: 50.6, 52, 103.4, 104, 112.1, 122, 123, 125.2, 126, 127.1,
6.1.3. Cell cultures
128.7, 129.4, 130, 132, 134, 136, 138, 141.5, 142, 143, 144, 146, 147.6,
148, 149, 152.3, 155, 156.6, 162.7, 187 ppm. LC-MS m/z: 452 [M þ Na]
þ. Anal. calcd for C30H23NO2: C 83.50, H 5.84 N 3.25. Found: C 83.50,
H 5.45 N 3.05.
The cell lines were adapted to propagate in RPMI 1640 medium
supplemented with 5% heat-inactivated foetal calf serum, 2 mM
L-glutamine and antibiotics. The cultures were grown in a humidi-
fied 37 ꢂC-incubator with 5% CO2 atmosphere.
5. Physicochemical studies
6.1.3.1. In vitro anti-proliferative activity [71]. Cell viability was
assessed at the beginning of each experiment by the trypan blue
dye exclusion method, and was always greater than 95%. Cells were
5.1. Determination of lipophilicity as RM values [61]
seeded into 96-well microtiter plates in 100
mL of medium at
Reversed phase TLC (RPTLC) was performed on silica gel plates
impregnated with 55% (v/v) liquid paraffin in light petroleum ether.
The mobile phase was a methanol/water mixture (77:23 v/v). The
plates were developed in closed chromatography tanks saturated
with the mobile phase at 24 ꢂC. Spots were detected under UV light.
RM values were determined from the corresponding Rf values (from
10 individual measurements) using the equation RM ¼ log [(1/
Rf) ꢀ 1].
a density of 5.000e7.500 cells/well and subsequently, the plates
were incubated at standard conditions for 24 h to allow the cells to
resume exponential growth prior to addition of the compounds.
Then, in order to measure the cell population, cells in one plate
were fixed in situ with TCA followed by SRB staining, as described
elsewhere [71]. To determine the compounds activity, each
compound dissolved in DMSO was added at 10-fold dilutions (from
100 to 0.01 mM) and incubation continued for an additional period
of 48 h. The assay was terminated by addition of cold TCA followed
by SRB staining and absorbance measurement at 530 nm, in an EL-
311 BIOTEK micro elisa reader (BioTek, Winooski, VT, USA). The
dose response parameters such as growth inhibitory activity 50%
5.2. Determination of physicochemical parameters (lipophilicity as
clog P and molar refractivity of substituents as MR-Ar) [72]
Lipophilicity was theoretically calculated as clog P values in
n-octanol-buffer by CLOGP Programe of Biobyte Corp. Molar
refractivity (MR-Ar) of selected substituents was also theoretically
calculated by CLOGP [72].
(GI50) [71], total growth inhibitory activity (TGI) and lethal
concentration 50% (LC50) were calculated.
6.2. In vivo assay
6. Biological experiments
6.2.1. Inhibition of the carrageenin-induced oedema [61]
6.1. In vitro assays
Oedema was induced in the right hind paw of Fisher-344 rats
(150e200g) by the intradermal injection of 0.1 mL of 2% carra-
geenin in water. Both sexes were used. Pregnant females were
excluded. Each group consisted of 6e15 animals. The animals, bred
in our laboratory, were housed under standard conditions and
received a diet of commercial food pellets and water ad libitum
during the maintenance but they were entirely fasted during the
experiment period. Our studies were in accordance with recog-
nized guidelines on animal experimentation.
The tested compounds, 0.01 mmol/kg body weight, were diluted
(HCl salt) in water or suspended in water (bases), with few drops of
Tween 80 and ground in a mortar before use and were given
intraperitoneally simultaneously with the carrageenin injection.
The rats were euthanized 3.5 h after carrageenin injection. The
difference between the weight of the injected and uninjected paws
was calculated for each animal. The change in paw weight was
compared with that in control animals (treated with water) and
expressed as percent inhibition of the oedema; CPE% values
(Table 1). Indomethacin, at 0.01 mmol/kg (47%), was used as
In the in vitro assays each experiment was performed at least in
triplicate and the standard deviation of absorbance was less than
10% of the mean.
6.1.1. Soybean lipoxygenase (LOX) inhibition study in vitro [61]
In vitro study was evaluated as reported previously [61]. The
tested compounds, dissolved in DMSO, were incubated at room
temperature with sodium linoleate (0.1 mM) and 0.2 mL of enzyme
solution (1/9 ꢃ 10ꢀ4, w/v in saline). The conversion of sodium
linoleate to 13-hydroperoxylinoleic acid at 234 nm was recorded
and compared with the appropriate standard inhibitor. NDGA was
used as an appropriate standard.
6.1.2. Aldose reductase (ALR2) enzyme assay
The target compounds as well as curcumin were dissolved in
10% aqueous solution of DMSO. Lenses were quickly removed from
Fischer-344 rats of both sexes following euthanasia. The

A.-M. Katsori et al. / European Journal of Medicinal Chemistry 46 (2011) 2722e2735
2733
[6] R. Morphy, Z. Rankovic, Designing multiple ligands - medicinal chemistry
strategies and challenges, Curr. Pharm. Des. 15 (2009) 587e600.
[7] F. Colotta, P. Allavena, A. Sica, C. Garlanda, A. Mantovani, Cancer-related
inflammation, the seventh hallmark of cancer: links to genetic instability,
Carcinogenesis 30 (2009) 1073e1081.
[8] A. Mantovani, P. Allavena, A. Sica, F. Balkwill, Cancer-related inflammation,
Nature 454 (2008) 436e444.
[9] B.B. Aggarwal, Inflammation, a silent killer in cancer is not so silent!, Curr.
Opin. Pharmacol. 9 (2009) 347e350.
[10] M. Cuendet, J.M. Pezzuto, The role of cyclooxygenase and lipoxygenase in
cancer chemoprevention, Drug Metabol. Drug Interact. 17 (2000) 109e157.
[11] D.M. Klurfeld, A.W. Bull, Fatty acids and colon cancer in experimental models,
Am. J. Clin. Nutr. 66 (1997) 1530Se1538S.
[12] K. Kashfi, S.J. Enna, W. Michael, Anti-inflammatory agents as cancer thera-
peutics, Advances in Pharmacology. Academic Press, 2009, 31e89.
[13] A.T. Chan, S. Ogino, C.S. Fuchs, Aspirin and the risk of colorectal cancer
in relation to the expression of COX-2, N. Engl. J. Med. 356 (2007)
2131e2142.
[14] G.A. Piazza, A.B. Keeton, H.N. Tinsley, J.D. Whitt, B.D. Gary, B. Mathew,
R. Singh, W.E. Grizzle, R.C. Reynolds, NSAIDs: old drugs reveal new anticancer
targets, Pharmaceuticals 3 (2010) 1652e1667.
[15] W.T. Gunning, P.M. Kramer, V.E. Steele, M.A. Pereira, Chemoprevention by
lipoxygenase and leukotriene pathway inhibitors of vinyl carbamate-induced
lung tumors in mice, Cancer Res. 62 (2002) 4199e4201.
[16] J. Epstein, I.R. Sanderson, T.T. MacDonald, Curcumin as a therapeutic agent:
the evidence from in vitro, animal and human studies, Br. J. Nutr. 103 (2010)
1545e1557.
[17] P. Anand, C. Sundaram, S. Jhurani, A.B. Kunnumakkara, B.B. Aggarwal, Cur-
cumin and cancer: an “old-age” disease with an “age-old” solution, Cancer
Lett. 267 (2008) 133e164.
[18] E. Sikora, G. Scapagnini, M. Barbagallo, Curcumin, inflammation, ageing and
age-related diseases, Immun. Ageing 7 (2010) 1.
[19] M.-H. Teiten, S. Eifes, M. Dicato, M. Diederich, CurcumindThe paradigm of
a multi-target natural compound with applications in cancer prevention and
treatment, Toxins 2 (2010) 128e162.
[20] B.B. Aggarwal, Y.-J. Surh, S. Shishodia, The molecular targets and therapeutic
uses of curcumin in health and disease, Advances in Experimental Medicine
and Biology. Springer, US, New York, 2007.
[21] A. Shehzad, F. Wahid, Y.S. Lee, Curcumin in cancer chemoprevention:
molecular targets, pharmacokinetics, bioavailability, and clinical trials, Arch.
Pharm. (Weinheim) 343 (2010) 489e499.
[22] A. Shehzad, Y.S. Lee, Curcumin: multiple molecular targets mediate multiple
pharmacological actions - a review, Drugs Future 35 (2010) 113e119.
[23] B.B. Aggarwal, B. Sung, Pharmacological basis for the role of curcumin in
chronic diseases: an age-old spice with modern targets, Trends Pharmacol.
Sci. 30 (2009) 85e94.
[24] W.P. Steward, A.J. Gescher, Curcumin in cancer management: recent results of
analogue design and clinical studies and desirable future research, Mol. Nutr.
Food Res. 52 (2008) 1005e1009.
a reference compound. CPE% values are the mean from two different
experiments with a standard error of the mean less than 10%.
7. Computational methods, docking simulations
All the molecules were constructed with ChemDraw program
[86] and converted in 3D-Structures with the OpenBabel program
[87], by using MMFF94 force field.
The X-ray structure of human ALR2 complexed with the inhib-
itor IDD 594 (PDB code 1US0) [76] was used in our docking calcu-
lations, after deletion of the inhibitor IDD 594 from the PDB file,
obtained from the Brookhaven Protein Data Bank (PDB) [88].
Although the in vitro inhibition assays of our compounds were
conducted on rat ALR2, the use of the human ALR2 crystal structure
for docking is justified by the facts, that the crystal structure of rat
ALR2 is unknown, while the human and rat sequences of this
enzyme are characterized by 81% identity and 89% homology [69].
Protein setup was performed using the UCSF Chimera software
[89,90]. AnteChamber PYthon Parser interfacE (ACPYPE) tool [91]
was employed to generate the topologies of the ligands. ACPYPE
tool is written in python to use Antechamber [91,92] to generate
topologies for chemical compounds was used for the parametri-
zation of the ligands. Energy minimizations where carried out with
the molecular simulation toolkit GROMACS [93] using the
AMBER99SB-ILDN force field [94].
Docking calculations were performed with the software Auto-
dock Vina [77,95]. PyRx program [96] was employed to generate the
docking input files and to analyze the docking results. The proteins
were considered rigid. Performing a blind docking to the ligand IDD
594 and to all developed inhibitors it was found that all the
molecules were properly aligned in the active site of 1US0. For all
the ligands, the single bonds were considered as active torsional
bonds. Docking was carried out with an exhaustiveness value of 64
and a maximum output of 100 binding modes. The conformation
obtained for the ligand IDD 594 was almost identical to the crys-
tallized (rmsd 0.090 Å) using the same protocol as above. Simul-
taneously the results of ligands from protein 1IK3 presented
identical conformation in the binding site of 1RRH as they were
crystallized in 1IK3.
[25] H. Hatcher, R. Planalp, J. Cho, F. Torti, S. Torti, Curcumin: from ancient
medicine to current clinical trials, Cell. Mol. Life Sci. 65 (2008) 1631e1652.
[26] G. Bar-Sela, R. Epelbaum, M. Schaffer, Curcumin as an anti-cancer agent:
review of the gap between basic and clinical applications, Curr. Med. Chem.
17 (2010) 190e197.
The final output of the docking procedure is a set of solutions
ranked according to the corresponding scoring function values,
each defined by the 3D coordinates of its atoms and expressed as
a PDB file.
[27] A. Goel, A.B. Kunnumakkara, B.B. Aggarwal, Curcumin as “Curecumin”: from
kitchen to clinic, Biochem. Pharmacol. 75 (2008) 787e809.
[28] P. Anand, A.B. Kunnumakkara, R.A. Newman, B.B. Aggarwal, Bioavailability of
curcumin: problems and promises, Mol. Pharm. 4 (2007) 807e818.
[29] E. Burgos-Morón, J.M. Calderón-Montaño, J. Salvador, A. Robles, M. López-
Lázaro, The dark side of curcumin, Int. J. Cancer 126 (2010) 1771e1775.
[30] P. Anand, S.G. Thomas, A.B. Kunnumakkara, C. Sundaram, K.B. Harikumar,
B. Sung, S.T. Tharakan, K. Misra, I.K. Priyadarsini, K.N. Rajasekharan,
B.B. Aggarwal, Biological activities of curcumin and its analogues (congeners)
made by man and Mother Nature, Biochem. Pharmacol. 76 (2008)
1590e1611.
[31] D.K. Agrawal, P.K. Mishra, Curcumin and its analogues: potential anticancer
agents, Med. Res. Rev. 30 (2010) 818e860.
[32] M.J.C. Rosemond, L. St. John-Williams, T. Yamaguchi, T. Fujishita, J.S. Walsh,
Enzymology of a carbonyl reduction clearance pathway for the HIV integrase
inhibitor, S-1360: role of human liver cytosolic aldo-keto reductases, Chem.
Biol. Interact. 147 (2004) 129e139.
Acknowledgements
The authors would like to thank Drs. C. Hansch, A. Leo and
Biobyte Corp. 201 West 4th Street, Suite 204, Claremont, CA 91711,
USA for free access to the C-QSAR program. Katsori A.-M. is thankful
to “Bodossakis Foundation” and Chatzopoulou M. to the Greek State
Scholarship Foundation (IKY) and the Research Committee of
Aristotle University of Thessaloniki for financial support.
References
[33] B. Yadav, S. Taurin, R.J. Rosengren, M. Schumacher, M. Diederich, T.J. Somers-
Edgar, L. Larsen, Synthesis and cytotoxic potential of heterocyclic cyclohexa-
none analogues of curcumin, Bioorg. Med. Chem. 18 (2010) 6701e6707.
[34] U. Das, R.K. Sharma, J.R. Dimmock, 1,5-diaryl-3-oxo-1,4-pentadienes: a case
for antineoplastics with multiple targets, Curr. Med. Chem. 16 (2009)
2001e2020.
[1] L.M. Espinoza-Fonseca, The benefits of the multi-target approach in drug
design and discovery, Bioorg. Med. Chem. 14 (2006) 896e897.
[2] A. Petrelli, S. Giordano, From single- to multi-target drugs in cancer therapy:
when aspecificity becomes an advantage, Curr. Med. Chem. 15 (2008)
422e432.
[35] B.K. Adams, E.M. Ferstl, M.C. Davis, M. Herold, S. Kurtkaya, R.F. Camalier,
M.G. Hollingshead, G. Kaur, E.A. Sausville, F.R. Rickles, J.P. Snyder, D.C. Liotta,
M. Shoji, Synthesis and biological evaluation of novel curcumin analogues as
anti-cancer and anti-angiogenesis agents, Bioorg. Med. Chem. 12 (2004)
3871e3883.
[3] O. Keskin, A. Gursoy, B. Ma, R. Nussinov, Towards drugs targeting multiple
proteins in a systems biology approach, Curr. Top. Med. Chem. 7 (2007)
943e951.
[4] F. Sams-Dodd, Target-based drug discovery: is something wrong? Drug Dis-
cov. Today 10 (2005) 139e147.
[36] P. Lagisetty, D.R. Powell, V. Awasthi, Synthesis and structural determination of
3,5-bis(2-fluorobenzylidene)-4-piperidone analogues of curcumin, J. Mol.
Struct. 936 (2009) 23e28.
[5] G.R. Zimmermann, J. Lehár, C.T. Keith, Multi-target therapeutics: when the
whole is greater than the sum of the parts, Drug Discov. Today 12 (2007)
34e42.

2734
A.-M. Katsori et al. / European Journal of Medicinal Chemistry 46 (2011) 2722e2735
[37] P. Lagisetty, P. Vilekar, K. Sahoo, S. Anant, V. Awasthi, CLEFMA-An anti-
proliferative curcuminoid from structure-activity relationship studies on
3,5-bis(benzylidene)-4-piperidones, Bioorg. Med. Chem. 18 (2010)
6109e6120.
[38] A. Jha, K.M. Duffield, 3,5-Bis(arylmethylene)-4-piperidone derivatives as novel
anticancer agents, Indian J. Chem. Sect. B. Org. Chem. Incl. Med. Chem. 45B
(2006) 2313e2320.
[39] M.V. Makarov, E.S. Leonova, E.Y. Rybalkina, P. Tongwa, V.N. Khrustalev,
T.V. Timofeeva, I.L. Odinets, Synthesis, characterization and structure-activity
relationship of novel N-phosphorylated E, E-3,5-bis(thienylidene)piperid-4-
ones, Eur. J. Med. Chem. 45 (2010) 992e1000.
[40] U. Das, J. Alcorn, A. Shrivastav, R. Sharma, E. De Clercq, J. Balzarini, J. Dimmock,
Design, synthesis and cytotoxic properties of novel 1-[4-(2-alkylaminoe-
[59] C. Pergola, O. Werz, 5-Lipoxygenase inhibitors: a review of recent develop-
ments and patents, Expert Opin. Ther. Pat. 20 (2010) 355e375.
[60] I.B. Taraporewala, J.M. Kauffman, Synthesis and structure-activity relation-
ships of anti-inflammatory 9,10-dihydro-9-oxo-2-acridine-alkanoic acids and
4-(2-carboxyphenyl)aminobenzenealkanoic acids, J. Pharm. Sci. 79 (1990)
173e178.
[61] E. Pontiki, D. Hadjipavlou-Litina, Antioxidant and anti-inflammatory activity
of aryl-acetic and hydroxamic acids as novel lipoxygenase inhibitors, Med.
Chem. 2 (2006) 251e264.
[62] E. Pontiki, D. Hadjipavlou-Litina, Lipoxygenase inhibitors:
a comparative
QSAR study review and evaluation of new QSARs, Med. Res. Rev. 28 (2008)
39e117.
[63] K. Muller, 5-Lipoxygenase and 12-lipoxygenase: attractive targets for the
development of novel antipsoriatic drugs, Arch. Pharm. (Weinheim) 327
(1994) 3e19.
thoxy)phenylcarbonyl]-3,5-bis(arylidene)-4-piperidones
and
related
compounds, Eur. J. Med. Chem. 42 (2007) 71e80.
[41] U. Das, M. Kawase, H. Sakagami, A. Ideo, J. Shimada, J. Molnar, Z. Barath,
Z. Bata, J. Dimmock, 3-(3,4,5-Trimethoxyphenyl)-1-oxo-2-propene: a novel
pharmacophore displaying potent multidrug resistance reversal and selective
cytotoxicity, Bioorg. Med. Chem. 15 (2007) 3373e3380.
[42] A.L. Kasinski, Y. Du, S.L. Thomas, J. Zhao, S.Y. Sun, F.R. Khuri, C.Y. Wang,
M. Shoji, A. Sun, J.P. Snyder, D. Liotta, H. Fu, Inhibition of IkappaB kinase-
nuclear factor-kappaB signaling pathway by 3,5-bis(2-flurobenzylidene)
piperidin-4-one (EF24), a novel monoketone analog of curcumin, Mol. Phar-
macol. 74 (2008) 654e661.
[43] U. Das, P. Selvakumar, R.K. Sharma, T.A. Haas, J.R. Dimmock, N-Acyl-3,5-
bis(arylidene)-4-piperidones and related compounds which stimulate fyn
kinase, J. Enzyme Inhib. Med. Chem. 22 (2007) 451e455.
[44] J.R. Dimmock, V.K. Arora, S.L. Wonko, N.W. Hamon, J.W. Quail, Z. Jia,
R.C. Warrington, W.D. Fang, J.S. Lee, 3,5-Bisbenzylidene-4-piperidones and
related compounds with high activity towards P388 leukemia cells, Drug Des.
Deliv. 6 (1990) 183e194.
[64] P. Alexiou, K. Pegklidou, M. Chatzopoulou, I. Nicolaou, V.J. Demopoulos, Aldose
reductase enzyme and its implication to major health problems of the 21(st)
century, Curr. Med. Chem. 16 (2009) 734e752.
[65] K.V. Ramana, S.K. Srivastava, Aldose reductase: a novel therapeutic target for
inflammatory pathologies, Int. J. Biochem. Cell Biol. 42 (2010) 17e20.
[66] N. Halder, S. Joshi, S.K. Gupta, Lens aldose reductase inhibiting potential of
some indigenous plants, J. Ethnopharmacol. 86 (2003) 113e116.
[67] P. Muthenna, P. Suryanarayana, K.G. Shravan, J.M. Petrash, G.B. Reddy, Inhi-
bition of aldose reductase by dietary antioxidant curcumin: mechanism of
inhibition, specificity and significance, FEBS Lett. 583 (2009) 3637e3642.
[68] Z.-Y. Du, Y.-D. Bao, Z. Liu, W. Qiao, L. Ma, Z.-S. Huang, L.-Q. Gu, A.S.C. Chan,
Curcumin analogs as potent aldose reductase inhibitors, Arch. Pharm.
(Weinheim) 339 (2006) 123e128.
[69] T. Gui, T. Tanimoto, Y. Kokai, C. Nishimura, Presence of a closely related
subgroup in the aldo-ketoreductase family of the mouse, Eur. J. Biochem. 227
(1995) 448e453.
[45] G. Liang, L. Shao, Y. Wang, C. Zhao, Y. Chu, J. Xiao, Y. Zhao, X. Li, S. Yang,
Exploration and synthesis of curcumin analogues with improved structural
stability both in vitro and in vivo as cytotoxic agents, Bioorg. Med. Chem.
17 (2009) 2623e2631.
[46] M.W. Amolins, L.B. Peterson, B.S.J. Blagg, Synthesis and evaluation of electron-
rich curcumin analogues, Bioorg. Med. Chem. 17 (2009) 360e367.
[47] a) J.R. Dimmock, M.P. Padmanilayam, R.N. Puthucode, A.J. Nazarali,
N.L. Motaganahalli, G.A. Zello, J.W. Quail, E.O. Oloo, H.-B. Kraatz, J.S. Prisciak,
T.M. Allen, C.L. Santos, J. Balzarini, E. De Clercq, E.K. Manavathu,
A conformational and structure-activity relationship study of cytotoxic 3,5-
bis(arylidene)-4-piperidones and related N-acryloyl analogues, J. Med. Chem.
44 (2001) 586e593;
[70] A. Del Corso, L. Costantino, G. Rastelli, F. Buono, U. Mura, Aldose reductase
does catalyse the reduction of glyceraldehyde through
a stoichiometric
oxidation of NADPH, Exp. Eye Res. 71 (2000) 515e521.
[71] K. Dimas, S. Hatziantoniou, S. Tseleni, H. Khan, A. Georgopoulos,
K. Alevizopoulos, J. Wyche, P. Pantazis, C. Demetzos, Sclareol induces
apoptosis in human HCT116 colon cancer cells in vitro and suppression of
HCT116 tumor growth in immunodeficient mice, Apoptosis 12 (2007)
685e694.
[72] BioByte Corporation, C-QSAR database 201 West 4th Str., Suite 204, Clar-
emont, California 91711, USA.
[73] C. Kontogiorgis, K. Litinas, A. Makri, D. Nicolaides, A. Vronteli, D. Hadjipavlou-
Litina, E. Pontiki, A. Siohou, Synthesis and biological evaluation of novel
angular fused pyrrolocoumarins, J. Enzyme Inhib. Med. Chem. 23 (2008)
43e49.
b) J.R. Dimmock, V.K. Arora, M. Chen, T.M. Allen, G.Y. Kao, Cytotoxic evaluation
of some N-acyl and N-acyloxy analogs of 3,5-bis(arylidene)-4-piperidones,
Drug Des. Discov. 12 (1994) 19e28.
[74] P. Brooks, R. Day, Nonsteroidal antiinflammatory drugsedifferences and
similarities, N. Engl. J. Med. 324 (1991) 1716e1725.
[48] H.I. El-Subbagh, S.M. Abu-Zaid, M.A. Mahran, F.A. Badria, A.M. Al-Obaid,
Synthesis and biological evaluation of certain alpha, beta-unsaturated ketones
and their corresponding fused pyridines as antiviral and cytotoxic agents,
J. Med. Chem. 43 (2000) 2915e2921.
[49] D.V. Jovanovic, J.C. Fernandes, J. Martel-Pelletier, F.C. Jolicoeur, P. Reboul,
S. Laufer, S. Tries, J.P. Pelletier, In vivo dual inhibition of cyclooxygenase and
lipoxygenase by ML-3000 reduces the progression of experimental osteoar-
thritis: suppression of collagenase 1 and interleukin-1beta synthesis, Arthritis
Rheum. 44 (2001) 2320e2330.
[75] V.R. Shanbhag, A.M. Crider, R. Gokhale, A. Harpalani, R.M. Dick, Ester and
amide prodrugs of ibuprofen and naproxen: synthesis, anti-inflammatory
activity, and gastrointestinal toxicity, J. Pharm. Sci. 81 (1992) 149e154.
[76] E.I. Howard, R. Sanishvili, R.E. Cachau, A. Mitschler, B. Chevrier, P. Barth,
V. Lamour, M. Van Zandt, E. Sibley, C. Bon, D. Moras, T.R. Schneider,
A. Joachimiak, A. Podjarny, Ultrahigh resolution drug design I: details of
interactions in human aldose reductaseeinhibitor complex at 0.66 Å, Proteins
55 (2004) 792e804.
[50] C. Menna, F. Olivieri, A. Catalano, A. Procopio, Lipoxygenase inhibitors for
cancer prevention: promises and risks, Curr. Pharm. Des. 16 (2010) 725e733.
[51] L.G. Melstrom, D.J. Bentrem, M.R. Salabat, T.J. Kennedy, X.Z. Ding, M. Strouch,
S.M. Rao, R.C. Witt, C.A. Ternent, M.S. Talamonti, R.H. Bell, T.A. Adrian, Over-
expression of 5-lipoxygenase in colon polyps and cancer and the effect of
5-LOX inhibitors in vitro and in a murine model, Clin. Cancer Res. 14 (2008)
6525e6530.
[52] L.T. Soumaoro, S. Iida, H. Uetake, M. Ishiguro, Y. Takagi, T. Higuchi, M. Yasuno,
M. Enomoto, K. Sugihara, Expression of 5-lipoxygenase in human colorectal
cancer, World J. Gastroenterol. 12 (2006) 6355e6360.
[77] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of
docking with
a new scoring function, efficient optimization, and multi-
threading, J. Comput. Chem. 31 (2010) 455e461.
[78] M. Chatzopoulou, E. Mamadou, M. Juskova, C. Koukoulitsa, I. Nicolaou,
M. Stefek, V.J. Demopoulos, Structure activity relations on [1-(3,5-difluoro-4-
hydroxyphenyl)-1H-pyrrol-3-yl]phenylmethanone. The effect of methoxy
substitution on aldose reductase inhibitory activity and selectivity, Bioorg.
Med. Chem. 19 (2011) 1426e1433.
[79] K.M. Bohren, C.E. Grimshaw, C.J. Lai, D.H. Harrison, D. Ringe, G.A. Petsko,
K.H. Gabbay, Tyrosine-48 is the proton donor and histidine-110 directs
substrate stereochemical selectivity in the reduction reaction of human aldose
reductase: enzyme kinetics and crystal structure of the Y48H mutant enzyme,
Biochemistry 33 (1994) 2021e2032.
[53] J. Clària, M. Romano, Pharmacological intervention of cyclooxygenase-2 and
5-lipoxygenase pathways. Impact on inflammation and cancer, Curr. Pharm.
Des. 11 (2005) 3431e3447.
[54] X.Z. Ding, R. Hennig, T.E. Adrian, Lipoxygenase and cyclooxygenase metabo-
lism: new insights in treatment and chemoprevention of pancreatic cancer,
Mol. Cancer 2 (2003) 10.
[80] C.E. Grimshaw, K.M. Bohren, C.-J. Lai, K.H. Gabbay, Human aldose reductase:
pK of tyrosine 48 reveals the preferred ionization state for catalysis and
inhibition, Biochemistry 34 (1995) 14374e14384.
[55] E. Pontiki, D. Hadjipavlou-Litina, G. Geromichalos, A. Papageorgiou, Anticancer
activity and quantitative-structure activity relationship (QSAR) studies of
a series of antioxidant/anti-inflammatory aryl-acetic and hydroxamic acids,
Chem. Biol. Drug Des. 74 (2009) 266e275.
[56] C.V. Rao, Regulation of COX and LOX by curcumin. in: B.B. Aggarwal, Y.-J. Surh,
S. Shishodia (Eds.), The Molecular Targets and Therapeutic Uses of Curcumin
in Health and Disease. Springer, US, 2007, pp. 213e226.
[57] E. Skrzypczak-Jankun, N.P. McCabe, S.H. Selman, J. Jankun, Curcumin inhibits
lipoxygenase by binding to its central cavity: theoretical and X-ray evidence,
Int. J. Mol. Med. 6 (2000) 521e526.
[81] A. Podjarny, R.E. Cachau, T. Schneider, M. Van Zandt, A. Joachimiak, Subatomic
and atomic crystallographic studies of aldose reductase: implications for
inhibitor binding, Cell. Mol. Life Sci. 61 (2004) 763e773.
[82] W. Minor, J. Steczko, B. Stec, Z. Otwinowski, J.T. Bolin, R. Walter, B. Axelrod,
Crystal structure of soybean lipoxygenase L-1 at 1.4 Å resolution, Biochem-
istry 35 (1996) 10687e10701.
[83] E. Pontiki, D. Hadjipavlou-Litina, K. Litinas, O. Nicolotti, A. Carotti, Design,
synthesis and pharmacobiological evaluation of novel acrylic acid deriva-
tives acting as lipoxygenase and cyclooxygenase-1 inhibitors with antioxi-
dant and anti-inflammatory activities, Eur. J. Med. Chem. 46 (2011)
191e200.
[58] J. Hong, M. Bose, J. Ju, J.-H. Ryu, X. Chen, S. Sang, M.-J. Lee, C.S. Yang, Modu-
lation of arachidonic acid metabolism by curcumin and related
b
-diketone
[84] H. Berman, K. Henrick, H. Nakamura, Announcing the worldwide Protein Data
Bank, Nat. Struct. Mol. Biol. 10 (2003) 980.
derivatives: effects on cytosolic phospholipase A2, cyclooxygenases and
5-lipoxygenase, Carcinogenesis 25 (2004) 1671e1679.
[85] J.R. Dimmock, U. Das, Antineoplastic Compounds, WO/2007/059613 (2007).

A.-M. Katsori et al. / European Journal of Medicinal Chemistry 46 (2011) 2722e2735
2735
[86] CambridgeSoft, USA, ChemDraw UltraÓ v.12 (1986e2009).http://www.
cambridgesoft.com/software/ChemDraw/.
[92] J. Wang, W. Wang, P.A. Kollman, D.A. Case, Automatic atom type and bond
type perception in molecular mechanical calculations, J. Mol. Graph. Model.
25 (2006) 247e260.
[93] B. Hess, C. Kutzner, D. van der Spoel, E. Lindahl, GROMACS 4: algorithms for
highly efficient, load-balanced, and scalable molecular simulation, J. Chem.
Theory Comput. 4 (2008) 435e447.
[87] OpenBabelÓ v.2.2.3 (2006).http://sourceforge.net/projects/openbabel/.
[88] RCSB Protein Data Bank, operated by the Research Collaboratory for Structural
Bioinformatics. www.pdb.org/.
[89] http://www.cgl.ucsf.edu/chimera
[90] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt,
E.C. Meng, T.E. Ferrin, UCSF ChimeraeA visualization system for exploratory
research and analysis, J. Comput. Chem. 25 (2004) 1605e1612.
[91] J. Wang, R.M. Wolf, J.W. Caldwell, P.A. Kollman, D.A. Case, Development
and testing of a general amber force field, J. Comput. Chem. 25 (2004)
1157e1174.
[94] K. Lindorff-Larsen, S. Piana, K. Palmo, P. Maragakis, J.L. Klepeis, R.O. Dror,
D.E. Shaw, Improved side-chain torsion potentials for the Amber ff99SB
protein force field, Proteins 78 (2010) 1950e1958.
[95] Autodock Vina v. 1.1.1. CopyrightÓ 1989e2010, The Scripps Research Institute.
[96] Sargis Pallakyan, PyRx-Python Prescription, v.0.5. The Scripps Research
Institute, 2008e2010.http://pyrx.scripps.edu/.