(E)-4-Aryl-4-oxo-2-butenoic acid amides, chalcone-aroylacrylic acid chimeras: Design, antiproliferative activity and inhibition of tubulin polymerization

Article abstract of DOI:10.1016/j.ejmech.2013.01.006

Antiproliferative activity of twenty-nine (E)-4-aryl-4-oxo-2-butenoic acid amides against three human tumor cell lines (HeLa, FemX, and K562) is reported. Compounds showed antiproliferative activity in one-digit micromolar to submicromolar concentrations.

Full text of DOI:10.1016/j.ejmech.2013.01.006

European Journal of Medicinal Chemistry 62 (2013) 40e50
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
(E)-4-Aryl-4-oxo-2-butenoic acid amides, chalconeearoylacrylic acid
chimeras: Design, antiproliferative activity and inhibition of tubulin
polymerization
,
b
ꢀ a
ꢀ
ꢀ b
ꢁ
*
ꢀ
Maja D. Vitorovic-Todorovic , Aleksandra Eric-Nikolic , Branka Kolundzija ,
ꢀ d
ꢀ e
ꢀ e
c
Ernest Hamel , Slavica Ristic , Ivan O. Juranic , Branko J. Drakulic
a
ꢀ
Military-Technical Institute, Ratka Resanovica 1, 11000 Belgrade, Serbia
b Institute for Oncology and Radiology, Pasterova 14, 11000 Belgrade, Serbia
c Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute,
Frederick National Laboratory for Cancer Research, National Institutes of Health, Frederick, MD 21702, USA
d
ꢁ
Biomedical Research, R&D Institute, Galenika a.d. Batajnicki Drum b.b., 11000 Belgrade, Serbia
e
ꢁ
Department of Chemistry, IChTM, University of Belgrade, Njegoseva 12, 11000 Belgrade, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
Antiproliferative activity of twenty-nine (E)-4-aryl-4-oxo-2-butenoic acid amides against three human
tumor cell lines (HeLa, FemX, and K562) is reported. Compounds showed antiproliferative activity in
one-digit micromolar to submicromolar concentrations. The most active derivatives toward all the cell
lines tested bear alkyl substituents on the aroyl moiety of the molecules. Fourteen compounds showed
Received 28 September 2012
Received in revised form
29 December 2012
Accepted 4 January 2013
Available online 11 January 2013
tubulin assembly inhibition at concentrations <20
mM. The most potent inhibitor of tubulin assembly
was unsubstituted compound 1, with IC50 ¼ 2.9 M. Compound 23 had an oral LD50 in vivo of 45 mg/kg in
m
mice. Cell cycle analysis on K562 cells showed that compounds 1, 2 and 23 caused accumulation of cells
in the G2/M phase, but inhibition of microtubule polymerization is not the principal mode of action of
the compounds. Nevertheless, they may be useful leads for the design of a new class of antitubulin
agents.
Keywords:
Antitumor agents
Structureeactivity relationships
4-Aryl-4-oxo-2-butenoic acid amides
Inhibition of tubulin polymerization
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
antiproliferative activity bear mainly eOH, MeOe, and halogen
substituents [12]. Alkyl substitution, especially on ring A, occurs
Chalcones, or 1,3-diaryl-2-propen-1-ones (I, Fig. 1), are consid-
ered as the intermediate precursors to all flavonoid compounds (II,
Fig. 1). Diverse biological activities have been attributed to these
compounds, in particular antioxidative [1], antimalarial [2], anti-
leishmanial [3], antiinflamatory [4], and anticancer [5] properties.
Numerous SAR studies of their anticancer activities have been
described [6e9]. Chalcones that originate in plants have eOH and
MeOe substitution on rings A and B, due to biosynthetic pathways
[10]. Health benefits of natural chalcones can be ascribed to their
redox properties and their ability to scavenge radical oxygen spe-
cies [11]. Along with scavenging of free radicals, biological activity
of chalcones can be ascribed to their ability to act as Michael ac-
ceptors with nucleophilic moieties, especially with the thiol groups
of biological targets. So far, synthetic chalcones that exert
relatively infrequently.
Chalcones and structurally similar compounds exert their anti-
proliferative activity through various mechanisms. For example,
recently described chalcone-based compounds that are proteo-
some inhibitors probably act as covalent binders, involving their
ketovinyl moiety [13]. It has been postulated that the presence of
a 4-amino function on ring A of the chalcone structure, protonated
at physiological pH, increases their reactivity and thus their anti-
proliferative activity [14,15]. It has also been confirmed that N-
alkylmaleimides exert antiproliferative activity by inhibition of
topoisomerase
I [16,17]. This led to the design of the 4-
aminochalcone maleamic acids (III) and imides (IV) [18]. Most of
these derivatives exert antiproliferative activity in the low micro-
molar range. Moreover, it was shown that phenylcinnamides (V)
bind to tubulin, causing inhibition of its polymerization and
thereby altering the tubulinemicrotubule equilibrium [19]. Struc-
turally similar cinnamoyl anthranilates (VI) also act as antitubulin
agents [20]. In addition, another type of molecule containing
the ketovinyl moiety, aroylacrylic acids (C, Scheme 1), exert
* Corresponding author.
ꢀ
E-mail addresses: majavitod@gmail.com, mvitod@chem.bg.ac.rs (M.D. Vitorovic-
ꢀ
Todorovic).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.006

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M.D. Vitorovic-Todorovic et al. / European Journal of Medicinal Chemistry 62 (2013) 40e50
41
Fig. 1. Structures of some compounds with the ketovinyl moiety that have antiproliferative activity: (I) chalcones, (II) flavones, (III) 4-aminochalcone maleamic acid, (IV) 4-
aminochalcone maleimides, (V) phenylcinnamides, (VI) cinnamoyl anthranilates.
antiproliferative activity toward HeLa cells [21a] and other human
dedifferentiated cell lines [21b].
which also proved E geometry of the double bond. The same
compound having Z geometry, as obtained by optimization on the
semiempirical level of theory, showed significantly shorter distance
of eCH]CHe protons, as expected (Figure S2 in Supplementary
material). Molecular geometry, as obtained by single crystal X-ray
analysis (unpublished results), of derivative 12 also proved the E
geometry of the double bond (Figure S3 in Supplementary
material). NMR spectra of representative compounds 7, 8, 9 and
23 are shown in Supplementary material, Figure S4. During eval-
uation of the purity of compounds by HPLC analysis, we observed
a trend between the retention times (RT) and virtual log P values
(r ¼ 0.900, n ¼ 29). This finding will be examined in detail
separately.
This rationale led us to design chalconeearoylacrylic acid chi-
meras, by incorporation of the amidic moiety between the
a,b-
unsaturated carbonyl moiety and the B ring of chalcones. We have
synthesized twenty-nine aroylacrylic acid amides, tested their
inhibitory potency on three tumor cell lines, and examined their
effects on tubulin polymerization.
2. Results and discussion
2.1. Chemistry
The synthetic pathway to 1e29 is shown in Scheme 1. Friedele
Crafts acylation of the commercially available substituted benzenes
(A) with maleic acid anhydride (B) yielded aroylacrylic acids (C)
[22]. Subsequently, the acids were converted to acid chlorides in
dry THF by phosphorous oxychloride and then in situ reacted with
equimolar amounts of aromatic or alicyclic amines to give the
corresponding aroylacrylic acid amides (D). The coupling constants
of vinylic protons (3JHH) of c.a. 15 Hz proved the E configuration of
the double bond in all compounds (see characterization data within
Section 5.1 Experimental/Chemistry). Along with this, the NOESY
spectrum of compound 11 also proved the E configuration of the
double bond. We chose this derivative due to the presence of para-
substituted aromatic rings, having a simple AA0BB0 1H NMR pattern.
Distance between ortho- and meta-protons on the aroyl ring was
used as reference. The eCH]CHe NOESY integral appears less than
1% of the chosen reference (Figure S1 in Supplementary material),
2.2. Antiproliferative activities of 1e29
Previously, we showed that aroylacrylic acids exert anti-
proliferative activity toward human tumor cell lines [21] at
micromolar concentrations. The aim of this study was to investigate
the effects of replacement of the carboxyl group with a cyclo-
alkylamide or arylamide moiety. The presence of two aromatic
rings bonded directly to each other or separated by one to four
carbon atoms, so that they are spatially close but not coplanar, is
a significant structural attribute of a number of anticancer com-
pounds, especially antitubulin agents [23].
We tested the antiproliferative activity of 1e29 on HeLa (human
cervix carcinoma), FemX (human melanoma) and K562 (human
chronic myelogenous leukemia) cells. The results are shown in
Table 1. Most of the compounds showed antiproliferative activity at
Scheme 1. Synthetic pathway to compounds 1e29 (Table 1). Reagents: (a) AlCl3 in CH2Cl2; (b) POCl3, dry THF. For compounds 8 and 17, cyclohexylamine was used to obtain the
corresponding aroylacrylic acid amides (D). Benzylamine was used to obtain derivatives 9, 10, 15 and 17. For the rest of the compounds, aniline or substituted anilines were used.

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M.D. Vitorovic-Todorovic et al. / European Journal of Medicinal Chemistry 62 (2013) 40e50
42
Table 1
Antiproliferative activity of the synthesized compounds (1e29) toward cancer cell lines.
Comp. no.
IC50 (mM)
R1
R2
HeLa
FemX
K562
1
2
3
4
5
6
7
8
H
Ph-
Ph-
Ph-
4.91 ꢀ 0.99
0.74 ꢀ 0.13
2.95 ꢀ 0.27
2.89 ꢀ 0.24
2.84 ꢀ 0.15
3.09 ꢀ 1.78
1.98 ꢀ 0.24
1.38 ꢀ 0.65
2.24 ꢀ 0.79
1.37 ꢀ 0.11
2.92 ꢀ 0.04
0.68 ꢀ 0.13
0.83 ꢀ 0.61
0.75 ꢀ 0.29
1.52 ꢀ 0.39
2.67 ꢀ 0.82
2.14 ꢀ 0.30
1.80 ꢀ 0.17
1.63 ꢀ 0.44
2.11 ꢀ 0.09
1.06 ꢀ 0.30
0.86 ꢀ 0.53
2.99 ꢀ 0.65
2.91 ꢀ 0.29
2.80 ꢀ 0.07
2.29 ꢀ 0.98
3.50 ꢀ 0.54
2.55 ꢀ 0.35
1.36 ꢀ 0.38
4.4 ꢀ 0.30
2.20 ꢀ 0.50
0.62 ꢀ 0.13
1.84 ꢀ 0.65
1.17 ꢀ 0.22
1.79 ꢀ 0.01
0.64 ꢀ 0.13
0.75 ꢀ 0.21
0.81 ꢀ 0.03
1.48 ꢀ 0.54
0.49 ꢀ 0.21
1.06 ꢀ 0.07
0.59 ꢀ 0.15
0.95 ꢀ 0.06
0.78 ꢀ 0.06
1.01 ꢀ 0.09
1.26 ꢀ 0.29
0.99 ꢀ 0.05
1.02 ꢀ 0.09
0.80 ꢀ 0.15
0.73 ꢀ 0.16
0.62 ꢀ 0.06
0.65 ꢀ 0.15
0.63 ꢀ 0.32
2.84 ꢀ 0.37
2.30 ꢀ 0.28
2.30 ꢀ 0.33
2.34 ꢀ 0.48
0.52 ꢀ 0.09
0.76 ꢀ 0.33
4.7 ꢀ 0.30
5.30 ꢀ 0.08
0.34 ꢀ 0.08
0.72 ꢀ 0.19
0.45 ꢀ 0.01
1.12 ꢀ 0.15
0.48 ꢀ 0.02
0.45 ꢀ 0.08
0.55 ꢀ 0.08
1.19 ꢀ 0.42
0.34 ꢀ 0.01
0.68 ꢀ 0.16
0.38 ꢀ 0.10
0.71 ꢀ 0.18
0.39 ꢀ 0.06
0.73 ꢀ 0.07
0.70 ꢀ 0.11
0.52 ꢀ 0.13
0.41 ꢀ 0.04
0.36 ꢀ 0.01
0.52 ꢀ 0.06
0.45 ꢀ 0.02
0.55 ꢀ 0.05
0.55 ꢀ 0.07
0.61 ꢀ 0.07
0.51 ꢀ 0.10
0.40 ꢀ 0.12
1.31 ꢀ 0.25
0.38 ꢀ 0.03
0.82 ꢀ 0.35
5.7 ꢀ 0.30
2,5-di-Me
3,4-di-Me
3,4-di-Me
3,4-di-Me
4-i-Pr
4-i-Pr
4-i-Pr
2,5-di-Me
4-i-Pr
4-i-Pr
3,5-di-OMe-Ph-
4-i-Pr-Ph-
Ph-
3,5-di-OMe-Ph-
Ch-
Bn-
Bn-
4-OMe-Ph-
Ph-
3,5-di-OMe-Ph-
4-i-Pr-Ph
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Cisplatina
2,4-di-i-Pr
2,4-di-i-Pr
2,4-di-i-Pr
2,4-di-i-Pr
Bn-
b
b
b
b
b
-tetralinyl
-tetralinyl
-tetralinyl
-tetralinyl
-tetralinyl
3,5-di-OMe-Ph-
Ch-
4-i-Pr-Ph-
Bn-
Ph-
Ph-
3,5-di-OMe-Ph-
Ph-
Ph-
Ph-
Ph-
Ph-
4-n-Bu
2,3,5,6-tetra-Me
4-F
4-Cl
3,4-di-Cl
4-Br
4-OMe
4-OMe
3,5-di-OMe-Ph-
3,5-di-OMe-Ph-
/
3,5-di-Me-4-OMe
/
a
Adopted from Reference [25].
lower concentrations as compared with similarly substituted
aroylacrylic acids [21].
K562 cells were the most sensitive toward the newly synthe-
sized compounds. Most compounds had IC50 values in the narrow
HeLa cells were the least sensitive toward the newly synthesized
compounds, with IC50 values in the one-digit micromolar range for
the majority of compounds. Compounds having alkyl substituents
at positions 2 and 5 or at 2 and 4 on the aroyl phenyl ring (2, 12, 13,
14, and 22) exerted potency at submicromolar concentrations on
the HeLa cells. A similar relationship between aroyl substitution
and antiproliferative activity was observed for aroylacrylic acids
[21]. The influence of structural variations of the amidic moiety on
antiproliferative activity could not be interpreted in a straightfor-
ward manner, but we observed that more ovoid molecules, having
larger apolar surface areas (ASA), displayed higher potency. Trends
between ASA and an oval shape of the molecules and their potency
toward HeLa cells are shown in Supplementary material, Figure S5a
and b. We did not observe such a trend with the estimated virtual
log P values [24].
range of 0.34e0.82 mM. The exceptions were the unsubstituted 1
and compounds 5 and 27.
The majority of the compounds contain phenylamide or 3,5-di-
MeO-phenylamide moieties. It should be noted that among the
phenylamide derivatives (1e3, 6, 12, 20, 21, 23, 24e27), compounds
that have a more lipophilic aroyl moiety exerted a more pro-
nounced antiproliferative activity toward all cell lines tested. We
observed the same trend for 3,5-di-MeO-phenylamide derivatives
(4, 7, 13, 16, 22, 28, 29) toward HeLa cells. Among the 3,5-di-MeO-
phenylamide derivatives, the less lipophilic derivatives exerted
a more pronounced antiproliferative activity toward FemX and
K562 cell lines (see for example 28 vs. 13). Due to the narrow range
of IC50’s toward FemX and K562 cells, this conclusion should be
taken with caution.
A number of the compounds had submicromolar activity against
all three cell lines examined. These were compounds 2, 12, 13, 14
and 22.
The majority of alkyl-substituted derivatives with unsubstituted
phenyl groups at the amidic portion of the molecule exerted sub-
micromolar activities toward the FemX cell line. The least active
were unsubstituted 1 and derivatives with halogen or methoxy
substituents (23e27) at the aroyl moiety. These compounds had
2.3. Inhibition of tubulin polymerization by 1e29 and cell cycle
analysis
IC50 values above 2 mM. Generally, the presence of a 4-i-Pr sub-
stituent at the phenylamidic moiety of the molecules (5, 14, 18)
slightly decreased antiproliferative activity in comparison with
their unsubstituted counterparts. In most instances, 3,5-dimethoxy
substitution on the amidic portion of the molecule had an unfa-
vorable influence on antiproliferative activity. We did not observe
other specific structural features that affected the antiproliferative
potency of 1e29 toward FemX cells.
Turning to molecular mechanism of action, we first considered
tubulin as a potential target, based on the structural similarity of 1e
29 with the known tubulin inhibitory activity of phenylcinnamides
(V) [19] and cinnamoyl anthranilates (VI) [20] (Fig. 1). Compounds
1e29 were compared with combretastatin A-4 as a reference
compound for inhibition of tubulin assembly (Table 2).

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43
Table 2
Inhibition of tubulin polymerization and colchicine binding by 1e29.
Table 3
Cell cycle analysis.
Comp. No.
Tubulin
polymerization
inhibition
Inhibition of
colchicine
binding (% ꢀ SD)
Compound no.
Concentration
G0/G1 (%)
S (%)
G2/M (%)
Control
1
39.55
35.93
40.24
40.56
42.92
40.94
37.48
44.38
41.80
37.70
40.76
21.12
24.03
23.71
26.17
39.18
35.26
36.54
27.44
32.37
37.32
24.74
29.01
37.56
24.52
16.86
17.78
18.02
12.95
19.84
18.32
17.30
12.57
17.72
19.04
14.67
16.41
19.13
12.30
21.75
24.14
23.58
2 ꢁ IC50
4 ꢁ IC50
8 ꢁ IC50
2 ꢁ IC50
4 ꢁ IC50
8 ꢁ IC50
2 ꢁ IC50
4 ꢁ IC50
8 ꢁ IC50
8 ꢁ IC50
IC50
(m
M ꢀ SD)
50
m
M
5 mM
1
2
3
4
5
6
7
8
2.9 ꢀ 0.3
5.2 ꢀ 0.2
6.5 ꢀ 0.08
>20
39 ꢀ 5
24 ꢀ 0.8
2
38 ꢀ 1
9.3 ꢀ 3
14 ꢀ 5
7.0 ꢀ 4
/
/
/
/
23
>20
9.7 ꢀ 1
25 ꢀ 2
3.1 ꢀ 3
>20
/
/
14
11 ꢀ 2
8 ꢀ 0.08
>20
14 ꢀ 0.8
9.6 ꢀ 5
Colchicine
2
4
8
m
m
m
M
M
M
9
10
11
19 ꢀ 4
12 ꢀ 0.01
/
/
/
/
>20
12
>20
/
/
13
>20
/
/
14
15
16
>20
>20
>20
/
/
/
/
/
/
compounds 1, 2 and 23, which inhibited tubulin polymerization
with IC50 values below 5 M, were examined, as well as the effect of
m
17
18
19
20
21
22
23
24
25
26
27
28
11 ꢀ 0.07
13 ꢀ 2
8.9 ꢀ 2
compound 14. The latter compound exerted significant anti-
proliferative potency toward all cells tested and bears bulky alkyl
substituents both on the aroyl phenyl and the phenylamido moi-
eties. Colchicine was used as the reference compound. Compound 1
caused an increase in the proportion of cells in the G2/M phase,
without dose dependence. In contrast, compounds 2 and 23 caused
an increase in the proportion of G2/M cells in a dose-dependent
manner, as compared with the control. Compound 14 did not
cause an increase in the proportion of G2/M cells, even at 8 times
the IC50 value. The increase in the proportion of cells in the G2/M
phase caused by compounds 1, 2 and 23 was less than the effect of
colchicine or of structurally similar compounds with an antitubulin
mechanism of action (see, for example, reference [20]). Thus, it
seems likely that there is another, more dominant molecular
mechanism of action for our compounds through which they exert
their antiproliferative activity. Our compounds are powerful
Michael acceptors, acting as unsaturated ketones (kinetic study of
the addition of the model thiol and amino compounds on repre-
sentative congeners will be described elsewhere), so they probably
also act as alkylating agents.
>20
/
/
>20
/
/
/
/
18 ꢀ 0.9
11 ꢀ 0.1
>20
3.5 ꢀ 0.3
11 ꢀ 0.6
10 ꢀ 2
8.6 ꢀ 0.5
7.8 ꢀ 1
>20
20 ꢀ 5
2.7 ꢀ 4
/
/
31 ꢀ 4
11 ꢀ 3
2.8 ꢀ 1
0.3 ꢀ 2
6.2 ꢀ 3
5.9 ꢀ 5
/
19 ꢀ 0.8
20 ꢀ 5
28 ꢀ 4
24 ꢀ 2
/
/
/
29
>20
1.2 ꢀ 0.007
/
Combretastatin A-4
99 ꢀ 0.7
Combretastatin A-4 was generously provided by Dr. George R.
Pettit, Arizona State University.
Fourteen compounds inhibited tubulin polymerization with IC50
values below 20
mM. The unsubstituted derivative 1 had the lowest
IC50 value (2.9 M), while the other effective inhibitors all had small
m
substituents on the aroyl moiety and no substituent on the phe-
nylamide portion of the molecule. Compounds substituted on the
phenylamide moiety (4-i-Pr, 3,5-di-MeO-) showed only weak
inhibitory activity in the polymerization assay or were inactive. The
halogen and methoxy substituted derivatives (23e27) that exerted
inferior antiproliferative activities, as compared with other com-
pounds, were relatively active as inhibitors of tubulin polymer-
ization. Four (compounds 12, 13, 14 and 22) of the five best
antiproliferative agents were inactive as antitubulin agents.
Therefore, the SAR of 1e29 for the inhibition of tubulin polymer-
ization was quite different from the SAR observed in the cancer cell
line screen.
2.4. In vivo acute toxicity
Compound 23 was chosen for an in vivo acute toxicity test. This
compound exerted both significant antiproliferative potency to-
ward all cell lines tested and showed significant inhibition of
tubulin polymerization. Compound 23 is also the only fluoro-
substituted derivative in the set. The fluoro substituent at posi-
tion 4 of the aroyl phenyl ring makes it, probably, more stable to-
ward metabolic degradation by cytochromes (see, for example,
[27]). Due to insufficient water solubility and the absence of ion-
izable moieties amenable to prepare the salt, the compound was
dissolved in DMSO and given orally to mice. The LD50 value was
45 mg/kg. Further structural optimization is required to obtain less
toxic compounds.
Thirteen compounds that showed activity as inhibitors of
tubulin polymerization were tested as inhibitors of colchicine
binding, at 5 and at 50 mM (Table 2). All tested compounds showed
some, albeit modest, inhibition of colchicine binding. The greatest
inhibition of colchicine binding was observed with compounds 1, 2
and 23, which were also the three best inhibitors of tubulin poly-
merization. It is possible that these derivatives exert antitubulin
activity by binding at the same site as colchicine, which was also
observed with structurally similar antitubulin chalcones [26].
Molecules that alter tubulin assembly in cells invariably cause
alteration of cell cycle distribution, with preferential G2/M block-
ade. We therefore examined the mechanism of action of four of the
3. Molecular modeling
All compounds exert significant antiproliferative potency in low
micromolar to submicromolar concentrations, spanning a narrow
range of IC50 values. A narrow range of activity values usually does
not produce statistically significant correlations, so we did not
perform a QSAR study for data on the antiproliferative potency of
1e29. Among 1e29, roughly, compounds having a solvent-assess-
able area <620 Å2 and a volume <305 Å3 inhibited tubulin
newly synthesized derivatives (Table
Supplementary material) by cytofluorimetric analysis, using pro-
pidium iodide to label the DNA of K562 cells. The effects of
3 and Figure S6 in

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M.D. Vitorovic-Todorovic et al. / European Journal of Medicinal Chemistry 62 (2013) 40e50
44
polymerization with IC50 values <20
m
M
(Table S7 in
were determined in open capillary tubes on an SMP-10 Stuart
apparatus and are uncorrected. ESI-MS spectra were recorded on an
Agilent Technologies 6210-1210 TOF-LC-ESI-MS instrument in
positive mode. For HPLC analysis the Agilent HPLC system 1200
(equipped with vacuum degasser, binary pump, autosampler,
thermostatted column compartment and DAD UV detector) with
Supplementary material). The majority of tubulin assembly in-
hibitors that target the colchicine site, reported thus far, resemble
colchicine or combretastatin A-4, bearing two or three MeO/OH
substituents on the aromatic rings, which are connected by a two-
to four-carbon spacer [23,28]. Within 1e29, the unsubstituted
compound or compounds bearing a small substituent at position 4
of the aryl moiety are the most potent inhibitors of tubulin poly-
merization and of colchicine binding. Also, an unsubstituted phenyl
of the amidic moiety is preferred in our compound series. There-
fore, in our modeling study, among compounds cocrystallized with
tubulin, we chose the minimally substituted compounds E70, from
the PDB entry 3HKC, and N16, from the PDB entry 3HKD [29]
(structures in Figure S8, Supplementary material) in their
tubulin-bound, cocrystallized conformations, for comparison with
compounds 1e3, 6, 8, 9, 17, 21, 20, and 23e27. The shape and the
pharmacophoric pattern of the compounds were compared using
the ROCS program [30]. We found a trend between pharmacophoric
similarity with E70 of the set studied (OpenEye Color force field/
Explicit Meals-Dean [31], Tanimoto scoring) and the percentage of
Zorbax Extend C18 column (RRHT, 50 ꢁ 4.6 mm, 1.8
Compounds were dissolved in MeOH (0.5 mg/mL), sample volume
was 0.2
L, column temperature was 40 ꢂC. The UV detector was set
mm) was used.
m
on 280 and 310 nm. Mobile phase A was 0.2% HCOOH in H2O,
mobile phase B was MeCN. Gradient mode: 0e2 min 40% A: 60% B;
2e10 min 60% B e 100% B. All compounds had >99.5% purity. 1H
and 13C NMR spectra were recorded in DMSO-d6 or CDCl3 on
a Bruker AVANCE 500/125 MHz or a Varian Gemini2000 200/
50 MHz instrument. NOESY spectrum of compound 11 was recor-
ded on a Bruker AVANCE 500 instrument. Chemical shifts are
reported in parts per million (ppm) relative to tetramethylsilane
(TMS), and spin multiplicities are given as follows: s (singlet),
d (doublet), t (triplet), h (heptet), m (multiplet) or br (broad).
General procedure for synthesis of compounds is given under
2.1 Chemistry.
inhibition of colchicine binding at 50
mM (r ¼ 0.89), with two
outliers (17 and 24) (Figure S9 in Supplementary material). It
should be mentioned that pharmacophoric similarity between
templates and compounds studied did not exceed 0.23 for E70 or
0.35 N16, as obtained with the Tanimoto function. Other statisti-
cally significant correlations or trends were not observed. Despite
this, compounds 1, 2, and 23 were always highly ranked using
a combined scoring function that includes both the shape and the
pharmacophoric similarity (Tanimoto combo), having a score >0.80
for E70, and >1.00 for N16 (Figure S10 in Supplementary material).
The Tanimoto combo score for all compounds studied exceed 0.62
for E70, and 0.95 for N16.
5.2. Characterization of compounds 1e29
5.2.1. (E)-4-oxo-4-phenyl-2-butenoic acid phenylamide (1)
C16H13NO2, starting from (E)-4-phenyl-4-oxo-2-butenoic acid
(0.017 mol) and a corresponding amount of aniline, 1.70 g of 1 was
obtained, 39.81% yield, light yellow solid, m.p. ¼ 143e145 ꢂC
decomposition, (AcOEt). 1H NMR (500 MHz, DMSO-d6)
d
¼ 7.15 (t,
J ¼ 7.63 Hz, 1H, amido p-phenyl), 7.36e7.32 (multiplet, 3H,
including w7.33 (d, J ¼ 14.35 Hz, 1H, C(O)eCH]CHe) and 7.35 (t.
J ¼ 7.32 Hz, 2H, aroyl m-phenyl)), 7.50 (t, J ¼ 7.93 Hz, 2H, amido m-
phenyl), 7.62 (t, J ¼ 7.32 Hz, 1H, aroyl p-phenyl), 7.67 (d, J ¼ 7.94 Hz,
2H, amido o-phenyl), 8.04 (d, J ¼ 7.02 Hz, 2H, aroyl o-phenyl), 8.11
(d, 14.95 Hz, 1H, C(O)eCH]CHe), 8.45 (s,b, 1H, NHeC(O)). 13C NMR
4. Conclusion
(E)-4-aryl-4-oxo-2-butenoic acid amides exert antiproliferative
activity toward three human tumor cell lines (HeLa, FemX, and
K562) with one-digit micromolar to submicromolar IC50 values. All
derivatives had antiproliferative activity that was significantly
greater than that of their precursors, aroylacrylic acids. The
replacement of the carboxyl group of aroylacrylic acids with an
arylamide or cycloalkylamide moiety proved beneficial for anti-
proliferative activity. The most active derivatives toward all three
cell lines tested were compounds with alkyl substituents on the
aroyl moiety. Although fourteen compounds inhibited tubulin as-
(125 MHz, DMSO-d6)
134.01, 136.21, 136.79, 137.65, 162.10, 190.05. HR MS (ESI): 252.1003
(M þ 1), Calc. 252.1025. HPLC purity >99.5%; RT 3.447 min.
d
¼ 120.19, 125.03, 128.92, 129.10, 133.79,
5.2.2. (E)-4-(2,5-dimethylphenyl)-4-oxo-2-butenoic acid
phenylamide (2)
C18H17NO2, starting from (E)-4-(2,5-dimethylphenyl)-4-oxo-2-
butenoic acid (0.017 mol) and a corresponding amount of aniline,
as described above, 2.56 g of 6 was obtained, 70.52% yield, yellow
solid, m.p. ¼ 159e160 ꢂC, (AcOEt). 1H NMR (500 MHz, DMSO-d6)
d:
sembly with IC50 values <20
mM, cell cycle analysis of compound-
2.34 (s, 3H, m-CH3), 2.50 (s, 3H, o-CH3), 6.97 (d, J ¼ 15.11 Hz, 1H,
C(O)eCH]CHe), 7.11 (t, J1,2 ¼ 7.16 Hz, 1H, amido p-phenyl), 7.24 (d,
J ¼ 7.95, 1H, aroyl p-phenyl), 7.30 (doublet like peaks, 1H, aroyl m-
phenyl), 7.35 (t, J1,2 ¼ 8.71 Hz, 2H, amido m-phenyl), 7.43 (d,
J ¼ 15.11 Hz, 1H, C(O)eCH]CHe), 7.46 (s, 1H, aroyl o-phenyl), 7.69
(d, J ¼ 8.75 Hz, 2H, amido o-phenyl), 10.54 (1H, s, NHeC(O)). 13C
treated K562 cells showed that only a few compounds caused
accumulation of cells in the G2/M phase. The mode of action
competitive to tubulin polymerization inhibition obviously exists.
Nevertheless, the newly synthesized compounds may represent
good leads for the design of a new class of antitubulin agents with
the SAR data we obtained. The most potent inhibitor of tubulin
polymerization was the compound unsubstituted on both the aroyl
NMR (125 MHz, DMSO-d6)
d
¼ 19.65, 20.36, 119.38, 124.07, 128.88,
129.23, 131.40, 132.10, 133.91, 135.10, 136.39, 137.22, 138.65, 161.80,
193.75. HR MS (ESI): 280.1338 (M þ 1), Calc. 280.1338. HPLC purity
>99.5%; RT 4.696 min.
and the arylamido moieties, having an IC50 ¼ 2.9
mM. Our data
further indicate that bulkier substituents on both the aroyl and
amidic moieties of the molecules impose steric hindrance for the
binding of this class of compounds to tubulin. Structural mod-
ification to further test this hypothesis is underway.
5.2.3. (E)-4-(3,4-dimethylphenyl)-4-oxo-2-butenoic acid
phenylamide (3)
C18H17NO2, starting from (E)-4-(3,4-dimethylphenyl)-4-oxo-2-
butenoic acid (0.013 mol) and a corresponding amount of aniline,
2.00 g of 3 was obtained, 55.10% yield, yellow solid, m.p. ¼ 163e
5. Experimental
5.1. Chemistry
165 ꢂC, (AcOEt). 1H NMR (500 MHz, DMSO-d6)
d
¼ 2.32 (s, 6H,
m,p-CH3), 7.12 (t, J1,2 ¼ 7.30 Hz, 1H, amido p-phenyl), 7.22 (d,
J ¼ 15.11 Hz, 1H, C(O)eCH]CHe), 7.37 (m, 3H, amido m-phenyl and
aroyl m-phenyl), 7.73 (d, J ¼ 7.30 Hz, 2H, amido o-phenyl), 7.80 (d,
All chemicals were purchased from Fluka, Aldrich or Merck,
having >98% purity, and were used as received. Melting points

ꢀ
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M.D. Vitorovic-Todorovic et al. / European Journal of Medicinal Chemistry 62 (2013) 40e50
45
J ¼ 8.32 Hz, 1H, aroyl o-phenyl), 7.85 (s, 1H, aroyl o-phenyl), 7.92 (d,
1H, J ¼ 15.11 Hz, C(O)eCH]CHe), 10.60 (s, 1H, NHeC(O)). 13C NMR
3.76 (s, 6H, eOCH3), 6.97 (d, J ¼ 2.14 Hz, 2H, amido o-phenyl), 7.35
(d, J ¼ 8.12 Hz, 2H, aroyl m-phenyl), 7.39 (d, J ¼ 14.96 Hz, 1H, C(O)e
CH]CHe), 8.00 (d, J ¼ 8.33 Hz, 2H, aroyl o-phenyl), 8.13 (d,
J ¼ 14.75 Hz, 2H, C(O)eCH]CHe), 8.74 (sb, 1H, NHeC(O)). 13C NMR
(125 MHz, DMSO-d6)
d
¼ 19.28,19.66,119.44,124.04,128.49,128.90,
129.59, 130.08, 132.08, 133.00, 134.48, 136.27, 137.22, 138.75, 143.37,
162.00, 189.05. HR MS (ESI): 280.1335 (M þ 1), Calc. 280.1338. HPLC
purity >99.5%; RT 4.618 min.
(125 MHz, CDCl3)
d
¼ 23.55, 34.34, 55.36, 97.60, 98.39, 127.07,
129.26, 133.83, 134.69, 136.03, 139.56, 155.88, 161.03, 162.36, 189.61.
ESI-MS HR: 354.1709 (M þ H), Calc. 354.1705. HPLC purity >99.5%;
RT 5.616 min.
5.2.4. (E)-4-(3,4-dimethylphenyl)-4-oxo-2-butenoic acid (3,5-
dimethoxyphenyl)amide (4)
C20H21NO4, starting from (E)-4-(3,4-dimethylphenyl)-4-oxo-2-
butenoic acid (0.0114 mol) and a corresponding amount of 3,5-
dimethoxyaniline, 2.32 g of 4 was obtained, 59.95% yield, yellow
solid, m.p. ¼ 162e164 ꢂC, decomposition (AcOEt). 1H NMR
5.2.8. (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid
cyclohexylamide (8)
C19H25NO2, starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-
butenoic acid (0.0092 mol) and a corresponding amount of cyclo-
hexylamine, 0.65 g of 8 was obtained, 23.78% yield, yellow solid,
(500 MHz, CDCl3)
d
¼ 2.31 (s, 3H, p-CH3), 2.34 (s, 3H, m-CH3), 3.77
(s, 6H, eOCH3), 6.26 (t, J ¼ 2.35 Hz, 1H, amido p-phenyl), 7.01
(doublet like peak, 2H, aroyl o-phenyl), 7.25 (d, J ¼ 7.70 Hz, 1H, aroyl
m-phenyl), 7.39 (d, J ¼ 14.96 Hz, 1H, C(O)eCH]CHe), 7.81 (d,
J ¼ 7.89 Hz, 1H, aroyl o-phenyl), 7.84 (s, 1H, aroyl o-phenyl), 8.12 (d,
J ¼ 14.75 Hz, 1H, C(O)eCH]CHe), 8.85 (sb, 1H, NHeC(O)). 13C NMR
m.p. ¼ 136e138 ꢂC, (AcOEt); 1H NMR (500 MHz, CDCl3)
d
¼ 1.16e
1.25 (m, 3H, cyclohexyl-CH2e), 1.27 (d, J ¼ 7.0 Hz, 6H, cyclohexyl-
CH2e), 1.36-1.44 (m, 2H, cyclohexyl-CH2e), 1.62e1.67 (m, 1H,
cyclohexyl-CH2e), 1.72e1.76 (m, 2H, cyclohexyl-CH2e), 1.97e2.00
(m, 2H, cyclohexyl-CH2), 2.96 (h, J ¼ 6.5 Hz, 1H, i-PrCH3), 3.93 (m,
1H, cyclohexyl-CH), 6.56 (d, J ¼ 8 Hz, 1H, NHeC(O)), 7.10 (d,
J ¼ 15.00 Hz, 1H, C(O)eCH]CHe), 7.34 (d, J ¼ 8.5 Hz, 2H, aroyl m-
phenyl), 7.97 (d, J ¼ 8.5 Hz, 2H, aroyl o-phenyl), 7.98 (d, J ¼ 15.50 Hz,
(125 MHz, CDCl3)
d
¼ 19.68, 20.18, 55.39, 97.51, 98.48, 126.79,
130.09,130.20, 133.94, 134.79, 134.79, 135.95, 137.53, 139.70,144.05,
161.06,162.48,189.79. ESI-MS HR: 340.1544 (M þ 1), Calc. 340.1549.
HPLC purity >99.5%; RT 4.802 min.
1H, C(O)eCH]CHe). 13C NMR (125 MHz, CDCl3)
25.46, 32.88, 34.26, 48.68, 126.87, 129.11, 132.78, 134.76, 135.88,
155.43, 163.17, 189.54. ESI-MS HR: 300.1958 (M þ 1), Calc. 300.1964.
HPLC purity >99.5%; RT 5.440 min.
d
¼ 23.53, 24.76,
5.2.5. (E)-4-(3,4-dimethylphenyl)-4-oxo-2-butenoic acid (4-
isopropylphenyl)amide (5)
C21H23NO2, starting from (E)-4-(3,4-dimethylphenyl)-4-oxo-2-
butenoic acid (0.0076 mol) and a corresponding amount of 4-
isopropylaniline, 1.65 g of 5 was obtained, 67.62% yield, yellow
solid, m.p. ¼ 129e130 ꢂC, decomposition (AcOEt). 1H NMR
5.2.9. (E)-4-(2,5-dimethylphenyl)-4-oxo-2-butenoic acid
benzylamide (9)
C19H19NO2, starting from (E)-4-(2,5-dimethylphenyl)-4-oxo-2-
butenoic acid (0.0078 mol) and a corresponding amount of ben-
zylamine, as described above,1.04 g of 9 was obtained, 45.41% yield,
yellow solid, m.p. ¼142e144 ꢂC, (AcOEt). 1H NMR (500 MHz, CDCl3)
(500 MHz, CDCl3)
d
¼ 1.24 (d, J ¼ 6.83 Hz, 6H, i-PrCH3), 2.29 (s, 3H,
p-CH3), 2.33 (s, 3H, m-CH3), 2.89 (h, J ¼ 6.82 Hz, 1H, i-PrCH), 7.20 (d,
J ¼ 8.58 Hz, 2H, amido m-phenyl), 7.24 (d, J ¼ 7.88 Hz, 1H, aroyl m-
phenyl), 7.45 (d, J ¼ 14.87 Hz, 1H, C(O)eCH]CHe), 7.64 (d,
J ¼ 8.58 Hz, 2H, amido o-phenyl), 7.88 (d, J ¼ 7.88 Hz, 1H, aroyl o-
phenyl), 7.83 (s, 1H, aroyl o-phenyl), 8.13 (d, J ¼ 14.87 Hz, 1H, C(O)e
CH]CHe), 8.91 (sb, 1H, NHeC(O)). 13C NMR (125 MHz, CDCl3)
d
¼ 2.33 (s, 3H, m-CH3), 2.38 (s, 3H, o-CH3), 4.52 (d, J ¼ 5.92 Hz, 2H,
benzyl-CH2e), 6.80 (sb, 1H, benzylamide-NH), 6.87 (d, J ¼ 15.36 Hz,
1H, C(O)eCH]CHe), 7.12 (d, J ¼ 7.84 Hz, 1H, aroyl p-phenyl), 7.20
(dd, J ¼ 7.68 Hz, 1H, aroyl m-phenyl), 7.24e7.30 (m, 5H, benzyl-CH),
7.40 (d, J ¼ 1.12 Hz, 1H, aroyl o-phenyl), 7.65 (d, J ¼ 15.20 Hz, 1H,
d
¼ 19.95, 20.12, 23.95, 33.63, 120.36, 126.79, 126.90, 130.05, 130.13,
133.47, 133.47, 134.77, 135.61, 136.60, 137.39, 143.91 145.61, 162.27,
189.88. ESI-MS HR: 322.1814 (M þ 1), Calc. 321.1729. HPLC purity
>99.5%; RT 6.703 min.
C(O)eCH]CHe). 13C NMR (125 MHz, CDCl3)
d
¼ 20.31, 20.74, 43.94,
127.60, 127.79, 128.68, 129.74, 131.68, 132.58, 134.97, 135.25, 135.51,
136.71, 137.01, 137.48, 164.07, 194.09. ESI-MS HR: 294.1490 (M þ 1)
100%, Calc. 294.1494. HPLC purity >99.5%; RT 4.123 min.
5.2.6. (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid
phenylamide (6)
C19H19NO2, starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-
butenoic acid (0.017 mol) and a corresponding amount of aniline,
2.15 g of 6 was obtained, 42.99% yield, yellow solid, m.p. ¼ 132e
136 ꢂC, decomposition (AcOEt). 1H NMR (500 MHz, CDCl3)
5.2.10. (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid
benzylamide (10)
C20H21NO2, starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-
butenoic acid (0.0092 mol) and a corresponding amount of ben-
zylamine, 0.94 g of 10 was obtained, 33.29% yield, yellow solid,
m.p. ¼ 125e127 ꢂC, decomposition (AcOEt). 1H NMR (200 MHz,
d
¼ 1.29 (d, J ¼ 6.94 Hz, 6H, i-PrCH3), 2.99 (m, 1H, i-PrCH), 7.16 (t,
J1,2 ¼ 7.38 Hz, 1H, amido p-phenyl), 7.33e7.37 (overlapping multi-
plets, 5H, amido m-phenyl, aroyl m-phenyl and C(O)eCH]CHe),
7.69 (d, J ¼ 7.73 Hz, 2H, amido o-phenyl); 8.00 (d, J ¼ 8.23 Hz, 2H,
aroyl o-phenyl), 8.13 (d, J ¼ 14.97 Hz, 1H, C(O)eCH]CHe), 8.50 (s,
DMSO-d6)
d
¼ 1.27 (d, J ¼ 7.30 Hz, 6H, i-PrCH3), 2.96 (h, J ¼ 6.74 Hz,
1H, i-PrCH), 4.57 (d, J ¼ 5.62 Hz, 2H, benzyl-CH2e), 7.23e7.53
(overlapped m, 9H, benzyl-CHe, aroyl m-phenyl, C(O)eCH]CHe,
and benzylamide-NH), 7.83 (d, J ¼ 6.74 Hz, 2H, aroyl o-phenyl),
8.03 (d, J ¼ 15.16 Hz, 1H, C(O)eCH]CHe). 13C NMR (50 MHz,
1H, NHeC(O)). 13C NMR (125 MHz, DMSO-d6)
d
¼ 23.57, 34.36,
120.20, 124.97, 127.07, 129.09, 129.28, 133.94, 134.67, 135.90, 137.74,
155.89, 162.25, 189.56. HR MS (ESI): 294.1493 (M þ H), Calc.
294.1494. HPLC purity >99.5%; RT 5.464 min.
DMSO-d6)
d
¼ 23.47, 34.20, 43.94, 95.68, 126.89, 127.51, 127.83,
128.63, 129.11, 132.97, 134.52, 135.46, 137.57, 155.53, 164.28, 189.48.
ESI-MS HR: 308.1647 (M þ 1), Calc. 308.1651. HPLC purity >99.5%;
RT 4.849 min.
5.2.7. (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid (3,5-
dimethoxyphenyl)amide (7)
5.2.11. (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid (4-
methoxyphenyl)amide (11)
C20H21NO3, starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-
butenoic acid (0.0078 mol) and a corresponding amount of 3,5-
dimethoxyaniline, 0.71 g of 11 was obtained, 28.09% yield, yellow
solid, m.p. ¼ 142e144 ꢂC, decomposition (AcOEt). 1H NMR
C21H23NO4, starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-
butenoic acid (0.0114 mol) and a corresponding amount of 3,5-
dimethoxyaniline, 1.70 g of 7 was obtained, 41.44% yield, yellow
solid, m.p. ¼ 132e134 ꢂC, (AcOEt). 1H NMR (500 MHz, CDCl3)
d
¼ 1.27 (d, J ¼ 6.84 Hz, 6H, i-PrCH3), 2.98 (h, J ¼ 2.98 Hz,1H, i-PrCH),

ꢀ ꢀ
M.D. Vitorovic-Todorovic et al. / European Journal of Medicinal Chemistry 62 (2013) 40e50
46
(500 MHz, CDCl3)
d
¼ 1.27 (d, J ¼ 6.71 Hz, 6H, i-PrCH3), 2.97 (h,
5.2.15. (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoic acid
benzylamide (15)
J ¼ 6.72 Hz, 1H, i-PrCH), 3.78 (s, 3H, eOCH3), 6.86 (d, J ¼ 9.06 Hz, 2H,
amido m-phenyl), 7.33 (d, J ¼ 8.39 Hz, 2H, aroyl m-phenyl), 7.41 (d,
J ¼ 15.10 Hz, 1H, C(O)eCH]CHe), 7.62 (d, J ¼ 8.06 Hz, 2H, amido o-
phenyl), 7.97 (d, J ¼ 8.39 Hz, 2H, aroyl o-phenyl), 8.11 (d,
J ¼ 14.77 Hz, 1H, C(O)eCH]CHe), 8.90 (sb, 1H, NHeC(O)). 13C NMR
C23H27NO2, starting from (E)-4-(2,4-diisopropylphenyl)-4-oxo-
2-butenoic acid (0.0077 mol) and a corresponding amount of
benzylamine, 0.92 g of 15 was obtained, 35.11% yield, yellow solid,
m.p. ¼ 125e127 ꢂC, (AcOEt). 1H NMR (500 MHz, CDCl3)
d
¼ 1.22 (d,
(125 MHz, CDCl3)
d
¼ 23.54, 34.31, 55.40, 114.16, 121.93, 127.00,
J ¼ 6.85 Hz, 6H, p-i-PrCH3), 1.26 (d, J ¼ 6.85 Hz, 6H, o-i-PrCH3), 2.93
(h, J ¼ 7.00 Hz, 1H, p-i-PrCH), 3.37 (h, J ¼ 6.85 Hz, 1H, o-i-PrCH), 4.52
(d, J ¼ 5.79 Hz, 2H, benzyl-CH2e), 6.48 (m,1H, benzyl-p-CH), 6.75 (d,
J ¼ 15.37 Hz, 1H, C(O)eCH]CHe), 7.09 (d, J ¼ 8.06 Hz, 1H, aroyl m-
phenyl), 7.26e7.30 (m, 6H, benzyl-CHe, aroyl m-phenyl and benzyl-
NHe), 7.38 (d, J ¼ 8.06 Hz, 1H, aroyl o-phenyl), 7.53 (d, J ¼ 15.37 Hz,
129.23, 131.02, 133.28, 134.69, 136.30, 155.76, 155.77, 162.15, 189.66.
ESI-MS HR: 324.1599 (M þ 1), Calc. 324.1600. HPLC purity >99.5%;
RT 5.129 min.
5.2.12. (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoic acid
phenylamide (12)
1H, C(O)eCH]CHe). 13C NMR (125 MHz, CDCl3)
29.49, 34.33, 44.01, 123.32, 124.72, 127.69, 127.87, 128.74, 134.90,
135.04, 137.45, 137.75, 148.79, 152.80, 164.02, 195.06. ESI-MS HR:
d
¼ 23.70, 24.10,
C22H25NO2, starting from (E)-4-(2,4-diisopropylphenyl)-4-oxo-
2-butenoic acid (0.013 mol) and corresponding amount of aniline,
2.06 g of 12 was obtained, 45.87% yield, yellow solid, m.p. ¼ 122e
350.2114 (M
6.590 min.
þ
1), Calc. 350.2120. HPLC purity >99.5%; RT
124 ꢂC, (AcOEt). 1H NMR (500 MHz, DMSO-d6)
d
¼ 1.20 (d,
J ¼ 6.86 Hz, 6H, p-i-PrCH3), 1.24 (d, J ¼ 6.75 Hz, 6H, o-i-PrCH3); 2.96
(m, 1H, p-i-PrCH), 3.19 (m, 1H, o-i-PrCH); 6.91 (d, J ¼ 14.93 Hz, 1H,
C(O)eCH]CHe), 7.10 (t, J1,2 ¼ 6.97 Hz, 1H, amido p-phenyl), 7.21,
7.22 (doublet like peaks, 1H, aroyl m-phenyl), 7.30 (d, J ¼ 14.93 Hz,
1H, C(O)eCH]CHe), 7.34 (t, J1,2 ¼ 8.30 Hz, 2H, amido m-phenyl),
7.37 (br, 1H, aroyl m-phenyl), 7.42 (d, J ¼ 7.94 Hz, 1H, aroyl o-phe-
nyl), 7.67 (d, J ¼ 8.63 Hz, 2H, amido o-phenyl), 10.54 (s, 1H, NHe
5.2.16. (E)-4-oxo-4-(5,6,7,8-tetrahydronaphthalenyl)-2-butenoic
acid (3,5-dimethoxyphenyl)-amide (16)
C22H23NO4, starting from (E)-4-(5,6,7,8-tetrahydronaphtalenyl)-
4-oxo-2-butenoic acid (0.0065 mol) and a corresponding amount of
3,5-dimethoxyaniline, 1.48 g of 16 was obtained, 62.40% yield,
yellow solid, m.p. ¼ 172e174 ꢂC, decomposition (AcOEt). 1H NMR
C(O)). 13C NMR (125 MHz, DMSO-d6)
d
¼ 23.63, 23.90, 29.23,
(500 MHz, CDCl3)
d
¼ 1.83 (m, 4H, tetralinoyl-CH2e), 2.82 (m, 4H,
33.57, 119.28, 123.39, 124.08, 128.88, 134.95, 137.40, 137.57, 136.63,
147.58, 151.82, 161.72, 196.11. HR MS (ESI): 336.1975 (M þ 1), Calc.
336.1964. HPLC purity >99.5%; RT 7.254 min.
tetralinoyl-CH2e), 3.78 (m, 6H, eOCH3), 6.29 (s, 1H, amido p-phe-
nyl), 6.95 (m, 2H, amido o-phenyl), 7.17 (d, J ¼ 8.50 Hz, 1H, aroyl m-
phenyl), 7.30 (d, J ¼ 14.72 Hz, 1H, C(O)eCH]CHe), 7.79 (m, 2H,
aroyl o-phenyl), 8.12 (d, J ¼ 15.05 Hz, 1H, C(O)eCH]CHe), 8.37 (sb,
5.2.13. (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoic acid (3,5-
dimethoxyphenyl)amide (13)
C24H29NO4, starting from (E)-4-(2,4-diisopropylphenyl)-4-oxo-
2-butenoic acid (0.0096 mol) and a corresponding amount of 3,5-
dimethoxyaniline, 1.90 g of 13 was obtained, 50.00% yield, yellow
solid, m.p. ¼ 120e122 ꢂC, decomposition (AcOEt). 1H NMR
1H, NHeC(O)). 13C NMR (125 MHz, CDCl3)
d
¼ 22.71, 22.86, 29.26,
29.78, 55.39, 97.51, 98.37, 125.98, 129.74, 129.99, 134.17, 134.32,
135.57, 136.02, 139.47, 144.64, 161.08, 162.33, 189.63. ESI-MS HR:
366.1710 (M
5.906 min.
þ
1), Calc. 366.1705. HPLC purity >99.5%; RT
(500 MHz, CDCl3)
d
¼ 1.23 (d, J ¼ 6.72 Hz, 6H, p-i-PrCH3), 1.27 (d,
5.2.17. (E)-4-oxo-4-(5,6,7,8-tetrahydronaphthalenyl)-2-butenoic
acid cyclohexylamide (17)
C20H25NO2, starting from (E)-4-(5,6,7,8-tetrahydronaph-
thalenyl)-4-oxo-2-butenoic acid (0.0078 mol) and a correspond-
ing amount of cyclohexylamine, 1.06 g of 17 was obtained, 43.46%
yield, yellow solid, m.p. ¼ 166e168 ꢂC, decomposition (AcOEt). 1H
J ¼ 6.88 Hz, 6H, o-i-PrCH3), 2.94 (h, J ¼ 6.72 Hz, 1H, p-i-PrCH), 3.42
(h, J ¼ 6.72 Hz, 1H, o-i-PrCH), 3.73 (s, 6H, eOCH3), 6.26 (s, 1H, amido
p-phenyl), 6.86 (s, 2H, amido o-phenyl), 7.98 (d, J ¼ 15.03 Hz, 1H,
C(O)eCH]CHe), 7.11 (d, J ¼ 7.67 Hz, 1H, aroyl m-phenyl), 7.28 (s,
1H, aroyl m-phenyl), 7.43 (d, J ¼ 7.84 Hz,1H, aroyl o-phenyl), 7.65 (d,
J ¼ 15.20 Hz, 1H, C(O)eCH]CHe), 8.11 (sb, 1H, NHeC(O)). 13C NMR
NMR (500 MHz, CDCl3)
d
¼ 1.16e1.29 (m, 3H, cyclohexyl-CH2e),
(125 MHz, CDCl3)
d
¼ 23.71, 24.14, 29.58, 34.36, 55.53, 97.55, 98.26,
1.39e1.43 (m, 2H, cyclohexyl-CH2e), 1.64 (m, 1H, cyclohexyl-CH2e
), 1.75 (m, 2H, cyclohexyl-CH2e), 1.82 (sb, 4H, tetralinoyl-CH2e),
2.00 (m, 2H, cyclohexyl-CH2-), 2.81 (sb, 4H, tetralinoyl-CH2e),
3.93 (m, 1H, cyclohexyl-CHe), 6.66 (d, J ¼ 8.10 Hz, 1H, amide-
123.45, 124.82, 128.87, 135.00,135.64, 136.12,139.28, 148.93, 153.09,
161.02, 162.18, 194.99. ESI-MS HR: 396.2169 (M þ 1), Calc. 396.2175.
HPLC purity >99.5%; RT 7.327 min.
NHe), 7.13 (d,
J
¼
15.15 Hz, 1H, C(O)eCH]CH-), 7.17 (d,
5.2.14. (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoic acid (4-
isopropylphenyl)amide (14)
J ¼ 8.56 Hz, 1H, aroyl m-phenyl), 7.75 (m, 2H, aroyl o-phenyl), 7.97
(d, J ¼ 14.95 Hz, 1H, C(O)eCH]CHe). 13C NMR (125 MHz, CDCl3)
C25H31NO2, starting from (E)-4-(2,4-diisopropylphenyl)-4-oxo-
2-butenoic acid (0.0096 mol) and a corresponding amount of 4-
isopropylaniline, 2.30 g of 14 was obtained, 63.53% yield, yellow
solid, m.p. ¼ 119e121 ꢂC, decomposition (AcOEt). 1H NMR
d
¼ 22.66, 22.81, 24.77, 25.46, 29.44, 29.66, 32.89, 48.67, 125.86,
129.52, 129.78, 132.79, 134.38, 135.79, 137.71, 144.16, 163.25, 189.74.
ESI-MS HR: 312.1956 (M þ 1), Calc. 312.1964. HPLC purity >99.5%;
RT 5.692 min.
(500 MHz, DMSO-d6)
d
¼ 1.23 (d, J ¼ 6.50 Hz, 6H, aroyl o-i-PrCH3),
1.17e1.20 (m, 12 H, aroyl p-i-PrCH3, amido p-i-PrCH3), 2.96 (h,
J ¼ 6.31 Hz, 1H, aroyl p-i-PrCH3), 2.84 (h, J ¼ 6.31 Hz, 1H, amido p-i-
PrCH3), 3.18 (h, J ¼ 6.31 Hz, 1H, aroyl o-i-PreCH), 6.90 (d,
J ¼ 15.49 Hz,1H, C(O)eCH]CHe), 7.19e7.21 (m, 3H, aroyl m-phenyl,
amido m-phenyl), 7.29 (d, J ¼ 15.49 Hz, 1H, C(O)eCH]CHe), 7.37 (s,
1H, aroyl m-phenyl), 7.41 (d, J ¼ 7.65 Hz, 1H, aroyl o-phenyl), 7.59 (d,
J ¼ 7.27 Hz, 2H, amido o-phenyl), 10.5 (sb, 1H, NHeC(O)). 13C NMR
5.2.18. (E)-4-oxo-4-(5,6,7,8-tetrahydronaphthalenyl)-2-butenoic
acid (4-isopropylphenyl)amide (18)
C23H25NO2, starting from (E)-4-(5,6,7,8-tetrahydronaph-
thalenyl)-4-oxo-2-butenoic acid (0.0096 mol) and a correspond-
ing amount of 4-isopropylaniline, 1.76 g of 18 was obtained, 52.54%
yield, yellow solid, m.p. ¼ 146e148 ꢂC, decomposition (AcOEt). 1H
NMR (500 MHz, CDCl3)
d
¼ 1.23 (d, J ¼ 6.99 Hz, 6H, i-PrCH3),1.81 (m,
(125 MHz, CDCl3)
d
¼ 23.62, 23.85, 23.89, 29.23, 32.89, 33.57,119.43,
4H, tetralinoyl-CH2-), 2.79 (db, 4H, tetralinoyl-CH2-), 2.88 (h,
J ¼ 6.99 Hz, 1H, i-PrCH), 7.15 (d, J ¼ 8.43 Hz, 2H, amido m-phenyl),
7.19 (d, J ¼ 8.43 Hz, 1H, aroyl m-phenyl), 7.47 (d, J ¼ 14.81 Hz, 1H,
C(O)eCH]CHe), 7.65 (d, J ¼ 8.43 Hz, 2H, amido o-phenyl), 7.76 (m,
123.36, 124.44, 126.57, 128.34, 134.99, 136.42, 137.19, 137.72, 144.20,
147.51, 151.76, 161.46, 196.12. ESI-MS HR: 378.2421 (M þ 1), Calc.
378.2433. HPLC purity >99.5%; RT 7.511 min.

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M.D. Vitorovic-Todorovic et al. / European Journal of Medicinal Chemistry 62 (2013) 40e50
47
2H, aroyl o-phenyl), 8.13 (d, J ¼ 14.81 Hz, 1H, C(O)eCH]CHe), 9.06
(sb, 1H, NHeC(O)). 13C NMR (125 MHz, CDCl3)
23.94, 29.93, 29.71, 33.60, 120.35, 125.98, 126.85, 129.64, 129.92,
133.43, 134.35,135.65, 136.93, 137.86,144.49, 145.53, 162.23, 189.92.
ESI-MS HR: 348.1957 (M þ 1), Calc. 348.1964. HPLC purity >99.5%;
RT 7.832 min.
d
¼ 2.03 (s, 6H, o- and m-CH3), 2.19 (s, 6H, m- and p-CH3), 3.67 (s, 6H,
d
¼ 22.60, 22.80,
eOCH3), 6.23 (t, J1,2 ¼ 2.16 Hz, 1H, amido p-phenyl), 6.80 (d,
J ¼ 15.41 Hz,1H, C(O)eCH]CHe), 6.79 (s,1H, amido o-phenyl), 6.80
(s, 1H, amido o-phenyl), 6.99 (s, 1H, aroyl o-phenyl), 7.31 (d,
J ¼ 15.41 Hz, 1H, C(O)eCH]CHe), 8.38 (s, 1H, NHeC(O)). 13C NMR
(50 MHz, CDCl3)
d
¼ 16.19, 19.36, 55.17, 97.45, 97.99, 129.35, 132.31,
134.52,137.32,138.52,139.20,139.52,160.92,162.03, 201.92. ESI-MS
HR: 368.1856 (M þ 1), Calc. 368.1862. HPLC purity >99.5%; RT
5.551 min.
5.2.19. (E)-4-oxo-4-(5,6,7,8-tetrahydronaphthalen-2-yl)-2-
butenoic acid benzylamide (19)
C21H21NO2, starting from (E)-4-(5,6,7,8-tetrahydronaphtalenyl)-
4-oxo-2-butenoic acid (0.0078 mol) and a corresponding amount of
benzylamine, 1.03 g of 19 was obtained, 41.20% yield, yellow solid,
m.p. ¼ 148e150 ꢂC, decomposition (AcOEt). 1H NMR (500 MHz,
5.2.23. (E)-4-(4-fluorophenyl)-4-oxo-2-butenoic acid phenylamide
(23)
C16H12FNO2, starting from (E)-4-(4-fluorophenyl)-4-oxo-2-
butenoic acid (0.013 mol) and a corresponding amount of aniline,
1.19 g of 23 was obtained, 34.00% yield, yellow solid, m.p. ¼ 163e
CDCl3)
d
¼ 1.81 (sb, 4H, tetralinoyl-CH2e), 2.80 (mb, 4H, tetralinoyl-
CH2e), 4.57 (d, J ¼ 5.94 Hz, 2H, benzylamine-CH2e), 6.87 (m, 1H,
benzylamineeCHe), 7.09 (d, J ¼ 15.07 Hz, 1H, C(O)eCH]CHe), 7.13
(d, J ¼ 8.06 Hz, 1H, aroyl m-phenyl), 7.27 (m, 1H, benzyleNHe), 7.31
(m, 4H, benzylamine-CHe), 7.65 (d, J ¼ 7.91 Hz, 1H, aroyl o-phenyl),
7.70 (sb, 1H, aroyl o-phenyl), 8.00 (d, J ¼ 14.92 Hz, 1H, C(O)eCH]
173 ꢂC, (AcOEt). 1H NMR (500 MHz, DMSO-d6)
d
¼ 7.13 (t,
J1,2 ¼ 7.54 Hz, 1H, amido p-phenyl), 7.23 (d, J ¼ 15.38 Hz, 1H, C(O)e
CH]CHe), 7.37 (t, J1,2 ¼ 7.57 Hz, 2H, amido m-phenyl), 7.42 (t,
J1,2 ¼ 7.42 Hz, 2H, aroyl m-phenyl), 7.73 (d, J ¼ 7.66 Hz, 2H, amido o-
phenyl), 7.92 (d, J ¼ 15.32 Hz, 1H, C(O)eCH]CHe), 8.17 (m,
J1,2 ¼ 5.55 Hz, J1,3 ¼ 9.07 Hz; 2H, aroyl o-phenyl), 10.60 (s, 1H, NHe
CHe). 13C NMR (125 MHz, CDCl3)
d
¼ 22.69, 22.83, 29.26, 29.71,
44.02, 125.85, 127.62, 127.83, 128.73, 129.62, 129.86, 133.55, 134.25,
134.79, 137.59, 137.77, 144.29, 164.19, 189.53. ESI-MS HR: 320.1649
(M þ 1), Calc. 320.1651. HPLC purity >99.5%; RT 5.082 min.
C(O)). 13C NMR (125 MHz, DMSO-d6)
124.08, 128,90, 131.82, 131.89, 132.73, 133.28, 133.30, 136.75, 138.66,
161.89, 166.37, 164.36, 188.22. HR MS (ESI): 270.0935 (M þ 1), Calc.
270.0930. HPLC purity >99.5%; RT 3.610 min.
d
¼ 115.52, 116.23, 119.41,
5.2.20. (E)-4-(5,6,7,8-tetrahydronaphtalenyl)-4-oxo-2-butenoic
acid phenylamide (20)
C20H19NO2, starting from (E)-4-(5,6,7,8-tetrahydronaphthal-
enyl)-4-oxo-2-butenoic acid (0.017 mol) and a corresponding
amount of aniline, 3.10 g of 20 was obtained, 78.08% yield, yellow
solid, m.p. ¼ 135e167 ꢂC, decomposition (AcOEt). 1H NMR
5.2.24. (E)-4-(4-chlorophenyl)-4-oxo-2-butenoic acid phenylamide
(24)
C16H12ClNO2, starting from (E)-4-(4-chlorophenyl)-4-oxo-2-
butenoic acid (0.017 mol) and a corresponding amount of aniline,
2.30 g of 24 was obtained, 47.42% yield, yellow solid, m.p. ¼ 181e
183 ꢂC, decomposition (AcOEt). 1H NMR (500 MHz, DMSO-d6)
(500 MHz, CDCl3)
d
¼ 1.82 (m, 4H, tetralinoyl-CH2e), 2.82 (m, 4H,
tetralinoyl-CH2e), 7.14e7.19 (overlapping peaks, 2H, amido p-phe-
nyl and aroyl m-phenyl), 7.36 (t, J1,2 ¼ 8.32 Hz, 2H, amido m-phe-
nyl), 7.41 (d, J ¼ 14.94 Hz, 1H, C(O)eCH]CHe), 7.71 (d, J ¼ 8.63 Hz,
2H, amido o-phenyl), 7.77 (s, 1H, aroyl m-phenyl), 7.78 (s, 1H, aroyl
o-phenyl), 8.14 (d, J ¼ 14.94 Hz, 1H, C(O)eCH]CHe), 8.68 (s, 1H,
d
¼ 7.12 (t, J1,2 ¼ 7.26 Hz, 1H, amido p-phenyl), 7.22 (d, J ¼ 15.29 Hz,
1H, C(O)eCH]CHe), 7.37 (t, J1,2 ¼ 7.58 Hz, 2H, amido m-phenyl),
7.72 (d, J ¼ 7.44 Hz, 2H, amido o-phenyl), 7.80 (d, J ¼ 8.42 Hz, 2H,
aroyl m-phenyl), 7.87 (d, J ¼ 15.29 Hz, 1H, C(O)eCH]CHe), 8.00 (d,
J ¼ 8.42 Hz, 2H, aroyl o-phenyl), 10.60 (s, 1H, NHeC(O)). 13C NMR
NHeC(O)). 13C NMR (125 MHz, CDCl3)
d
¼ 22.69, 22.84, 29.30, 29.77,
120.20, 124.91, 126.02, 129.06, 129.73, 129.97, 133.90, 134.34, 135.94,
137.85, 137.97, 144.66, 162.32, 189.85. HR MS (ESI): 306.1491
(M þ 1), Calc. 306.1494. HPLC purity >99.5%; RT 5.756 min.
(125 MHz, DMSO-d6)
d
¼ 119.42, 124.09, 128.00, 128.01, 130.73,
132.08, 132.61, 135.55, 137.00, 138.66, 161.76, 188.91. HR MS (ESI):
286.0634 (M þ 1), Calc. 286.0635. HPLC purity >99.5%; RT
4.387 min.
5.2.21. (E)-4-(4-n-butylphenyl)-4-oxo-2-butenoic acid
phenylamide (21)
5.2.25. (E)-4-(3,4-dichlorophenyl)-4-oxo-2-butenoic acid
phenylamide (25)
C20H21NO2, starting from (E)-4-(4-n-buthylphenyl)-4-oxo-2-
butenoic acid (0.013 mol) and a corresponding amount of aniline,
1.29 g of 21 was obtained, 32.25% yield, yellow solid, m.p. ¼ 133e
C16H11Cl2NO2, starting from (E)-4-(3,4-dichlorophenyl)-4-oxo-
2-butenoic acid (0.013 mol) and a corresponding amount of aniline,
2.80 g of 25 was obtained, 67.31% yield, yellow solid, m.p. ¼ 187e
135 ꢂC, (AcOEt). 1H NMR (500 MHz, DMSO-d6)
d
¼ 0.91 (t,
J1,2 ¼ 7.07 Hz, 3H, CH3CH2),1.32 (sp, J1,2 ¼ 7.48 Hz, J1,3 ¼ 14.95 Hz, 2H,
CH3CH2CH2), 1.59 (m, J1,2 ¼ 7.47 Hz, J1,3 ¼ 14.95 Hz, 2H, CH3CH2CH2),
2.68 (t, J1,2 ¼ 7.47 Hz, 2H, CH3CH2CH2CH2), 7.12 (t, J1,2 ¼ 7.08 Hz, 1H,
amido p-phenyl), 7.21 (d, J ¼ 15.33 Hz, 1H, C(O)eCH]CHe), 7.37 (t,
J1,2 ¼ 7.47 Hz, 2H, amido m-phenyl), 7.42 (d, J ¼ 7.87 Hz, 2H, aroyl m-
phenyl), 7.73 (d, J ¼ 7.47 Hz, 2H, amido o-phenyl), 7.89 (d,
J ¼ 15.33 Hz, 1H, C(O)eCH]CHe), 7.99 (d, J ¼ 8.26 Hz, 2H, aroyl o-
phenyl), 10.59 (s, 1H, NHeC(O)). 13C NMR (125 MHz, DMSO-d6)
194 ꢂC, decomposition (AcOEt). 1H NMR (500 MHz, DMSO-d6)
d:
7.12 (t, J1,2 ¼ 7.35 Hz, 1H, amido p-phenyl), 7.24 (d, J ¼ 15.30 Hz, 1H,
C(O)eCH]CHe), 7.37 (t, J1,2 ¼ 7.65 Hz, 2H, amido m-phenyl), 7.72
(d, J ¼ 7.65 Hz, 2H, amido o-phenyl), 7.84 (d, J ¼ 8.46 Hz,1H, aroyl m-
phenyl), 7.87 (d, J ¼ 15.32 Hz, 1H, C(O)eCH]CHe), 8.03 (dd, 1H,
J1,2 ¼ 2.12 Hz, J1,3 ¼ 8.48 Hz, aroyl o-phenyl), 8.24 (s, 1H, aroyl o-
phenyl), 10.61 (s, 1H, NHeC(O)). 13C NMR (500 MHz, DMSO-d6)
d
¼ 119.43, 124.11, 128.77, 128.90, 130.49, 131.29, 132.08, 132.35,
d
¼ 13.70, 21.71, 32.65, 34.81, 119.41, 124.02, 128.88, 149.11, 161.92,
136.61, 136.69, 137.45, 136.64, 161.67, 187.85. HR MS (ESI): 320.0241
189.06. HR MS (ESI): 308.1656 (M þ 1), Calc. 308.1651. HPLC purity
>99.5%; RT 6.572 min.
(M þ 1), Calc. 320.0245. HPLC purity >99.5%; RT 5.37 min.
5.2.26. (E)-4-(4-bromophenyl)-4-oxo-2-butenoic acid phenylamide
(26)
5.2.22. (E)-4-(2,3,5,6-tetramethylphenyl)-4-oxo-2-butenoic acid
(3,5-dimethoxyphenyl)amide (22)
C22H25NO4, starting from (E)-4-(2,3,5,6-tetramethylphenyl)-4-
oxo-2-butenoic acid (0.0094 mol) and a corresponding amount of
3,5-dimethoxyaniline, 2.50 g of 22 was obtained, 72.46% yield,
yellow solid, m.p. ¼198e200 ꢂC, (AcOEt). 1H NMR (200 MHz, CDCl3)
C16H12BrNO2, starting from (E)-4-(4-bromophenyl)-4-oxo-2-
butenoic acid (0.013 mol) and a corresponding amount of aniline,
as described above, 2.60 g of 26 was obtained, 60.60% yield, yellow
solid, m.p. ¼ 187e194 ꢂC, decomposition (AcOEt). 1H NMR
(500 MHz, DMSO-d6)
d
¼ 7.12 (t, J1,2 ¼ 7.55 Hz, 1H, amido p-phenyl);

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M.D. Vitorovic-Todorovic et al. / European Journal of Medicinal Chemistry 62 (2013) 40e50
48
7.23 (d, J ¼ 14.37, 1H, C(O)eCH]CHe); 7.37 (t, J1,2 ¼ 7.85 Hz, 2H,
amido m-phenyl); 7.73 (d, J ¼ 7.60, 2H, amido o-phenyl); 7.82 (d,
J ¼ 8.74, 2H, aroyl m-phenyl); 7.88 (d, J ¼ 15.26, 1H, C(O)eCH]CHe
); 8.00 (d, J ¼ 8.77 Hz, 2H, aroyl o-phenyl); 10.60 (s, 1H, NHeC(O)).
culture medium (Sigma Chemical Co. St Louis, MO) supplemented
with 3 mmol/L -glutamine, 100 g/mL streptomycin, 100 IU/mL
penicillin, 10% heat inactivated fetal bovine serum (FBS e Sigma
Chemical Co.), and 25 mM Hepes, adjusted to pH 7.2 with a bicar-
bonate solution.
L
m
13C NMR (125 MHz, DMSO-d6)
d
¼ 119.41, 124.08, 127.99, 128.90,
130.72, 132.07, 132.60, 135.54, 136.99, 138.65, 161.75, 188.90. HR MS
(ESI): 330.0120/332.0102 (M þ 1), Calc. 330.0124/332.0105. HPLC
purity >99.5%; RT 4.626 min.
5.3.1.2. Treatment of cells. Cells were seeded into 96-well microti-
ter plates, 2000 cells in 0.1 mL of culture medium per well. After
20 h, to wells with the cells, five different concentrations of com-
pounds 1e29 were applied, except to the control wells, in which the
cells were grown in medium only. All concentrations were set up in
triplicate. Culture medium with corresponding concentrations of
investigated agent, but without cells, was used as a blank, also in
triplicate.
5.2.27. (E)-4-(4-methoxyphenyl)-4-oxo-2-butenoic acid
phenylamide (27)
C17H15NO3, starting from (E)-4-(4-methoxyphenyl)-4-oxo-2-
butenoic acid (0.017 mol) and a corresponding amount of aniline,
2.50 g of 27 was obtained, 52.30% yield, yellow solid, m.p. ¼ 157e
161 ꢂC, decomposition (AcOEt). 1H NMR (500 MHz, DMSO-d6)
d
¼ 3.88 (s, 3H, p-OCH3), 6.95 (d, J ¼ 9.03, 2H, aroyl m-phenyl), 7.15
5.3.1.3. Determination of FemX, HeLa, and K562 cell survival.
Cell survival was determined by the MTT test, according to the
method of Mosmann [32] as modified by Ohno and Abe [33], 72 h
(t, J1,2 ¼ 7.35 Hz, 1H, amido p-phenyl), 7.35 (t, J1,2 ¼ 8.16 Hz, 2H,
amido m-phenyl), 7.42 (d, J ¼ 14.81 Hz,1H, C(O)eCH]CHe), 7.70 (d,
J ¼ 8.18, 2H, amido o-phenyl), 8.04 (d, J ¼ 9.03 Hz, 2H, aroyl o-
phenyl), 8.13 (d, J ¼ 14.81 Hz, 1H, C(O)eCH]CHe), 8.87 (1H, s, NHe
after compound addition. Briefly, 20 mL of MTT solution (5 mg/mL in
phosphate buffered saline) was added to each well. Cells were
C(O)). 13C NMR (125 MHz, DMSO-d6)
d: 55.57, 114.18, 120.25, 124.87,
incubated for 4 h at 37 ꢂC in a humidified atmosphere with 5% CO2.
129.04, 128.85, 131.46, 133.69, 135.84, 137.88, 162.43, 164.40, 188.36.
HR MS (ESI): 282.1133 (M þ 1), Calc. 282.1134. HPLC purity >99.5%;
RT 3.360 min.
Then, 100 mL of 10% SDS was added to each well. Absorbance was
measured at 570 nm. Cell survival was measured/quantified using
absorbance at 570 nm of a sample with cells grown in the presence
of various concentrations of agent divided by the absorbance of the
control sample (the absorbance of cells grown only in nutrient
medium). Absorbance of blank was always subtracted from
absorbance of a corresponding sample with cells. All exper-
imentally obtained IC50 data, reported in Table 1, are means of at
least three measurements done in triplicate.
5.2.28. (E)-4-(4-methoxyphenyl)-4-oxo-2-butenoic acid (3,5-
dimethoxyphenyl)amide (28)
C19H19NO5, starting from (E)-4-(4-methoxyphenyl)-4-oxo-2-
butenoic acid (0.0076 mol) and a corresponding amount of 3,5-
dimethoxyaniline, 0.81 g of 28 was obtained, 31.40% yield, yellow
solid, m.p. ¼ 141e143 ꢂC, decomposition (AcOEt). 1H NMR 500 MHz
(CDCl3)
d
¼ 3.76 (s, 6H, eOCH3), 3.90 (s, 3H, eOCH3), 6.29 (sb, 1H,
5.3.1.4. Cell cycle determination. Aliquots of 2.5 ꢁ 105 control cells
or cells treated with investigated compounds for 24 h (concentra-
tions corresponded to 2 ꢁ IC50, 4 ꢁ IC50, and 8 ꢁ IC50 values) were
fixed in 70% ethanol for 1 h on ice, then at ꢃ20 ꢂC for at least 1
week. The cells were then collected by centrifugation. The pellets
amido p-phenyl), 6.94 (m, 2H, amido o-phenyl), 6.98 (d, J ¼ 8.96 Hz,
2H, aroyl m-phenyl), 7.29 (d, J ¼ 14.72 Hz, 1H, C(O)eCH]CHe), 8.07
(d, J ¼ 8.64 Hz, 2H, aroyl o-phenyl), 8.11 (d, J ¼ 15.04 Hz, 1H, C(O)e
CH]CHe), 8.33 (sb, 1H, NHeC(O)). 13C NMR (125 MHz, CDCl3)
d
¼ 55.41, 55.60, 97.53, 98.37, 114.21, 129.86, 131.44, 134.05, 135.35,
were treated with RNase (100
incubated with propidium iodide (40
m
g/mL) at 37 ꢂC for 30 min and then
g/mL) for at least 30 min.
139.44, 161.09, 162.36, 164.41, 188.05. ESI-MS HR: 342.1338 (M þ 1),
Calc. 342.1341. HPLC purity >99.5%; RT 3.500 min.
m
DNA content and cell cycle distribution were analyzed using
a Becton Dickinson FAC-Scan flow cytometer. Flow cytometric
analysis was performed using a CellQuestR (Becton Dickinson, San
Jose, CA, USA) on a minimum of 10,000 cells per sample [34].
5.2.29. (E)-4-(4-methoxy-3,5-dimethylphenyl)-4-oxo-2-butenoic
acid (3,5-dimethoxyphenyl)-amide (29)
C21H23NO5, starting from (E)-4-(4-methoxy-3,5-dimethyl-
phenyl)-4-oxo-2-butenoic acid (0.0076 mol) and a corresponding
amount of 3,5-dimethoxyaniline, as described above, 0.93 g of 29
was obtained, 33.02% yield, yellow solid, m.p. ¼ 160e162 ꢂC,
5.3.2. Inhibition of tubulin polymerization and colchicine binding
The tubulin assembly and [3H]colchicine binding assays were
performed with electrophoretically homogenous bovine brain
decomposition (AcOEt). 1H NMR 500 MHz (CDCl3)
d
¼ 2.19 (s, 3H,
tubulin [35]. The assembly assay [36] was performed with 10 mM
eCH3), 2.54 (s, 3H, eCH3), 5.70 (s, 6H, eOCH3), 3.88 (s, 3H, eOCH3),
6.25 (s, 1H, amido p-phenyl), 6.67 (s, 1H, aroyl o-phenyl), 6.91 (s, 2H,
amido o-phenyl), 7.12 (d, J ¼ 14.88 Hz, 1H, C(O)eCH]CHe), 7.58 (s,
1H, aroyl o-phenyl), 7.89 (d, J ¼ 14.88 Hz, 1H, C(O)eCH]CHe), 8.55
(1.0 mg/mL) tubulin, 0.8 M monosodium glutamate (pH 6.6 with
HCl in a 2.0 M stock solution), 0.4 mM GTP, and 4% (v/v) dimethyl
sulfoxide (as the compound solvent). Tubulin and varying com-
pound concentrations were preincubated without GTP for 15 min at
30 ꢂC, samples were placed on ice, and GTP was added. The samples
were transferred to 0 ꢂC cuvettes in Beckman DU7400 and DU7500
recording spectrophotometers equipped with electronic tempera-
ture controllers. After baselines were established at 350 nm, the
temperature was jumped to 30 ꢂC (less than 1 min), and sample
turbidity was followed for 20 min. The IC50 was the compound
concentration that reduced the turbidity reading at 20 min by 50%,
relative to a control reaction mixture without compound.
(s, 1H, NHeC(O)). 13C NMR (125 MHz, CDCl3)
d
¼ 15.77, 21.94, 55.30,
55.42, 97.46, 98.28, 113.33, 123.97, 128.92, 132.95, 134.61, 137.32,
139.53, 140.25, 160.77, 160.95, 162.62, 191.61. ESI-MS HR (solvent
MeOH/CH2Cl2, positive mode): 370.1649 (M þ 1), Calc. Mass
369.1576. HPLC purity >99.5%; RT 4.863 min.
5.3. Biological assays
5.3.1. Cytotoxicity assays
The colchicine binding assay was described in detail by Verdier-
Pinard et al. [37]. The reaction mixtures contained a tubulin stabi-
5.3.1.1. Preparation of compound solutions. Stock solutions of the
investigated compounds were made in dimethyl sulfoxide (Fluka
Chemie AG Buchs, Switzerland) at a concentration of 20 mM, fil-
tered through Millipore filters (0.22
diluted to various working concentrations with RPMI-1640 cell
lizing buffer system [37] and 1.0
[3H]colchicine (from PerkineElmer, Boston, MA), and compounds
at 5.0 or 60 M in a final dimethyl sulfoxide concentration of 5% (v/
v). The 0.1 mL reaction mixtures were incubated for 10 min at 37 ꢂC,
mM (0.1 mg/mL) tubulin, 5.0 mM
m
m) before use and afterwards
m

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ꢀ
M.D. Vitorovic-Todorovic et al. / European Journal of Medicinal Chemistry 62 (2013) 40e50
49
at which time the reaction is about 40e60% complete in the
absence of the compounds. The reactions were stopped with 3 mL
of ice water, and each diluted reaction mixture was filtered through
a stack of two DEAE-cellulose filters from Whatman. The amount of
radiolabeled colchicine bound to the tubulin adsorbed to the filters
was determined in a scintillation counter.
activity are shown in Table S1 in Supplementary material. Struc-
tures of the E and the Z isomer of compound 11 were optimized in
MOPAC2009 [41], using PM6 semiempirical MO method [42], with
eigenvector-following optimizer to root-mean-square gradient
>0.01 kcal/mol/Å.
Acknowledgements
5.3.3. Acute toxicity
5.3.3.1. Animals. Male albino mice (NMRI Hann) weighing 18e22 g,
obtained from VivariumeGalenika a.d. Belgrade, Serbia, selected by
random sampling, were used in the study. They were housed under
standard animal conditions, at 23 ꢀ 2 ꢂC with 50% relative air hu-
midity. The animals had free access to food (standard pallet diet)
and water. This study was performed following the National (‘The
Law on the Experimental Animal Treatment’, Republic of Serbia),
and European (Directive 2010/63/EU; European convention for the
protection of vertebrate animals used for experimental and other
scientific purposes) regulations and standards. The acute toxicity
tests were performed in accordance with OECD-423 guidelines.
Experiments were approved by the Animal Ethics Committee-
Galenika a.d. (Permission No 3/12).
This work was supported by the Ministry of Education and
Science of the Republic of Serbia Grant 172035. Authors gratefully
ꢀ
acknowledge Mr Milka Jadranin and Dr Nenad Milosavic for help
with HPLC analysis.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.01.006.
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