2-Cinnamamido, 2-(3-phenylpropiolamido), and 2-(3-phenylpropanamido) benzamides: Synthesis, antiproliferativeactivity, and mechanism of action

Article abstract of DOI:10.1016/j.ejmech.2013.04.068

Several new benzamides 4a-q were synthesized by stirring in pyridine the acid chlorides 3a-q with the appropriate anthranilamide derivatives 2a-g. Some of the synthesized compounds were evaluated for their in vitro antiproliferative activity against a panel of 5 human cell lines (K562 human chronic myelogenous leukemia cells, MCF-7 breast cancer cells, HTC-116 and HT26 colon cancer cells and NCI H460 non-small cell lung cancer cells).

Full text of DOI:10.1016/j.ejmech.2013.04.068

Accepted Manuscript
2-Cinnamamido, 2-(3-phenylpropiolamido), and 2-(3-
phenylpropanamido)benzamides: synthesis, antiproliferative activity, and mechanism
of action
Demetrio Raffa, Benedetta Maggio, Maria Valeria Raimondi, Maria Grazia Cusimano,
Amico Giandomenico, Anna Carollo, Pier Giulio Conaldi, Ruoli Bai, Ernest Hamel,
Giuseppe Daidone
PII:
S0223-5234(13)00301-2
10.1016/j.ejmech.2013.04.068
EJMECH 6179
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 December 2012
Revised Date: 30 April 2013
Accepted Date: 30 April 2013
Please cite this article as: D. Raffa, B. Maggio, M.V. Raimondi, M.G. Cusimano, A. Giandomenico, A.
Carollo, P.G. Conaldi, R. Bai, E. Hamel, G. Daidone, 2-Cinnamamido, 2-(3-phenylpropiolamido), and
2-(3-phenylpropanamido)benzamides: synthesis, antiproliferative activity, and mechanism of action,
European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.04.068.
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ACCEPTED MANUSCRIPT
►A series of 2-cinnamamido, 2-(3-phenylpropiolamido), and 2-(3-phenylpropanamido)benzamides
was synthesized ►Synthesized compounds were evaluated for the antiproliferative activity against
a panel of 5 human cell lines.►They caused 50% growth inhibition at micromolar and
submicromolar concentrations ► The effects on tubulin polymerization on cell cycle and on
apoptosis were studied.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
2-Cinnamamido, 2-(3-phenylpropiolamido), and 2-(3-
phenylpropanamido)benzamides: synthesis, antiproliferative activity, and
mechanism of action
Demetrio Raffaa, Benedetta Maggio*a, Maria Valeria Raimondia, Maria Grazia Cusimanoa,
Amico Giandomenicob,c, Anna Carollod, Pier Giulio Conaldib,c, Ruoli Baie, Ernest Hamele and
Giuseppe Daidonea.
a
Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Via
Archirafi, 32, 90123-Palermo, Italy.
b
Department of Laboratory Medicine and Advanced Biotechnologies - ISMETT
Mediterranean Institute for Transplantation and Advanced Specialized Therapies, Palermo
c
Unit of Regenerative Medicine and Biomedical Technologies - Ri.MED. Foundation,
Palermo
d
Servizio Farmacia Clinica – ISMETT Mediterranean Institute for Transplantation and
Advanced Specialized Therapies, Palermo
e Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer
Treatment and Diagnosis, National Cancer Institute, Frederick National Laboratory for
Cancer Research, Frederick, Maryland 21702, USA.
CORRESPONDING AUTHOR: Phone +39 91 23891949, e-mail benedetta.maggio@unipa.it
ABSTRACT. Several new benzamides 4a-q were synthesized by stirring in pyridine the
acid chlorides 3a-q with the appropriate anthranilamide derivatives 2a-g. Some of the synthesized
compounds were evaluated for their in vitro antiproliferative activity against a panel of 5 human
cell lines (K562 human chronic myelogenous leukemia cells, MCF-7 breast cancer cells, HTC-116
and HT26 colon cancer cells and NCI H460 non-small cell lung cancer cells).
KEYWORDS
2-cinnamamidobenzamides,
2-(3-phenylpropiolamido)benzamides,
2-(3-
phenylpropanamido)benzamides, antiproliferative activity, apoptosis.

ACCEPTED MANUSCRIPT
1. Introduction
Cinnamoyl anthranilates represent a class of biologically active substances of great importance in
medicinal chemistry. Some examples of biological activities of this class of compounds include
activity against human coronary artery smooth muscle and endothelial cells [1], antifibrotic and
antinflammatory properties [2,3], inhibition of cornea pterygium progression and blood vessel
development [4,5] and prevention and treatment of renal glomerular diseases [6]. In a previous
paper from our laboratories [7], we reported the synthesis, antiproliferative activity and mechanism
of action of a series of 2-{[2E]-3-phenylpropenamido}benzamides 1a-v (figure 1).
Synthesized benzamides 1a-v were initially tested in vitro for their antileukemic activity against the
K562 human chronic myelogenous leukemia cell line. In addition, compounds 1a-c,t,u were
evaluated by the NCI against a panel of approximately 60 human cell lines derived from seven
cancer types (lung, colon, melanoma, renal, ovarian, brain, and leukemia). The data reported [7]
show that the 2-{[2E]-3-phenylpropenamido}benzamides 1a-c,t,u caused growth inhibition against
many tumor cell lines. These five compounds inhibited proliferation at low micromolar (1a, 1b, 1c)
or submicromolar concentrations (1t, 1u) against every tumor cell line investigated. The best
activity was obtained when the 5 position of the benzamido moiety was substituted with an iodine
moiety. COMPARE analysis, effects on tubulin polymerization in cells and with purified tubulin,
and effects on cell cycle distribution, including induction of apoptosis, indicate that these new
antiproliferative compounds act as antitubulin agents.
On the basis of these results, we synthesized new derivatives that, starting from the most active
previously synthesized compounds, allowed us to determine the best substituents to insert in the
molecules to improve their activity and expand our understanding of the SAR in this class of
compounds.

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In particular, we carried out the following structural modifications on the cinnamamidobenzamide
skeleton: introduction of new substituents on the benzamido and cinnamamido groups and
substitution of the vinylene group with ethylene and ethynylene moieties, with the goal of
identifying new antitubulin agents (figure 2).
2. Results and Discussion
2.1. Chemistry
The synthesis of benzamido derivatives 4a-e, 4f [4,5], 4g-k, 4l [8], 4m-q, 4r [9], 4s-w was achieved
as described in scheme 1. A mixture of the appropriate acid chloride 3a-h and the appropriate 5-R-
4-R1-2-aminobenzamide 2a-g was stirred magnetically in an ice bath. The attempt to obtain (Z)-
cinnamamidobenzamides 4f-k, starting from the appropriate cis-2-methoxycinnamic acid failed due
to stereochemical conversions during the synthetic procedures, so that only (E)-derivatives were
isolated.
Crude benzoyl chlorides 3a-q, not commercially available, were obtained starting from the
corresponding acid by treatment with thionyl chloride [10].

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The anthranilamide 2a is commercially available, while the 5-R-4-R1-2-aminobenzamides 2b-g
were obtained according to Scheme 2

ACCEPTED MANUSCRIPT
R
CONH2
NO2
R
COCl
NO2
R
COOH
NO2
NH3
R = CH3
SOCl2
R1
R1
R1
7a-c
6a-c
5a-c
SnCl2 / HCl
O
R
R
CONH2
NH2
O
NH3
R1
N
H
O
R1
2b-g
8a,b
I2
2a R=H, R1 =H
2b R=Cl, R1 =H
2c R=Br, R1 =H
2d R= CH3, R1 =H
2e R=I, R1 =H
5c R=OCH3, R1 = OCH3
6a R= CH3, R1 =H
6b R=OCH3, R1 =H
6c R=OCH3, R1 = OCH3
7a R= CH3, R1 =H
R
CONH2
NH2
R1
R=OCH3 , R1 =H
R=OCH3, R1 =H
2f
7b
R=OCH3 , R1 = OCH3
5a R= CH3, R1 =H
5b R=OCH3 , R1 =H
R=OCH3, R1 = OCH3
8a R=Cl, R1 =H
8b R=Br, R1 =H
2g
7c
2a
Scheme 2
In particular, the 5-iodo-2-aminobenzamide 2e was obtained, as reported in the literature [11], by
treatment of anthranilamide 2a with iodine in an aqueous solution of sodium bicarbonate.
The 5-methylanthranilamide 2d, the 5-methoxyanthranilamide 2f and the 4,5-
dimethoxyanthranilamide 2g were obtained by reduction of 2-nitro-5-methyl benzamides 7a-c as
shown in Scheme 2. Compounds 7a-c were obtained by reaction of the acids 5a-c with thionyl
chloride to afford 6a-c, followed by treatment of 6a-c with aqueous ammonia.
The anthranilamide derivatives 2b,c were obtained by stirring the appropriate 2H-3,1-benzoxazine-
2,4(1H)-dione 8a,b in a 25% aqueous ammonia solution (Scheme 2) [12].
The structures of the new compounds were determined by analytical and spectroscopic
measurements. In particular, all derivatives 4a-w showed signals attributable to aromatic protons in
the range 6.18-8.77 δ and a singlet in the range 11.00-13.43 δ, exchangeable with D2O, for the
amidic NH. Different chemical shift values were detected for the signals of the NH2 benzamido
protons according to the different registration solvent. In fact, for compounds 4a-c,f-k,m-q, whose
1H-NMR spectra were recorded in DMSO, this signal falls together with the aromatic signals,
while, in the case of compounds 4d,e,s-w, recorded in CDCl3, a splitting of the signals for the two
1
protons of the NH2 group at 5.84 and 6.29 δ was observed. Moreover, the H-NMR spectra of 5-
methyl, 5-methoxy and 4,5-dimethoxy-substituted derivatives showed signals at about 2.30 δ and

ACCEPTED MANUSCRIPT
1
3.84-3.99 δ, respectively, arising from the methyl and methoxyl groups. H NMR spectra of
compounds 4a-k were consistent with an E-olefinic structure. They showed signals attributable to
the β-olefinic protons at 6.86-6.96 δ, with coupling constants of 15.6-15.9 Hz, as required for E-
structures [13], while the α-olefinic hydrogens were found along with aromatic multiplets. As
regards the 2-(3-phenylpropiolamido)benzamido derivatives 4l-q, the IR spectra showed the
1
characteristic band around 2210 cm-1 attributable to acetylenic bond stretching. Finally, H NMR
spectra of 2-(3-phenylpropanamido)benzamides 4s-w showed two triplets at 2.70 and 3.00 δ,
respectively, due to the ethylene chain.
2.2. Biological activity
Synthesized benzamido derivatives 4a-w were initially tested in vitro for their antiproliferative
activity against a panel of 5 human cell lines: K562 (human chronic myelogenous leukemia), MCF-
7 (breast cancer), HTC-116 (colon cancer), HT26 (colon cancer) and NCI H460 (non-small cell
lung cancer); colchicine was used as a reference compound.
The percent growth inhibition at a screening concentration of 10 µM and the IC50 values for
compounds that exhibited at least 50% growth inhibition in the 10 µM screening assay are shown in
tables 1 and 2, respectively.
Table 1. Percent growth inhibition of compounds 4a-w at 10 µM.
Comp.
K562
NCI H460
MCF-7
HT29
HCT116
ns
ns
ns
ns
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
11.1±1.2
26.6±3.5
19.4±1.9
79.0±2.5
40.7±4.3
40.0±3.9
80.0±0.9
68.0±1.4
56.0±0.6
68.0±2.3
ns
80.0±0.2
27.5±1.2
15.0±1.9
36.2±2.2
65.0±0.7
61.1±2.1
ns
17.1±1.1
15.3±1.6
ns
15.0±0.9
11.0±1.0
ns
36.6±0.6
20.6±0.3
40.6±1.2
ns
21.3±1.9
29.8±0.8
12.6±1.4
19.3±2.0
31.4±3.1
ns
39.7±4.3
53.6±3.4
23.6±3.0
11.9±0.5
43.0±0.4
ns
42.9±3.7
14.6±1.9
45.7±2.9
17.7±2.0
14.9±2.0
26.3±1.2
17.5±1.7
13.5±1.9
35.1±0.4
20.2±1.8
85.0±0.3
30.7±1.0
24.2±1.1
ns±
30.3±0.6
16.5±0.8
16.0±1.2
29.5±3.7
24.5±1.1
36.2±1.7
34.4±0.8
46.8±1.0
72.3±2.2
24.0±1.7
ns
ns
ns
ns
17.2±0.7
ns
13.7±1.0
ns
ns
ns
18.1±2.1
34.5±2.3
13.9±1.8
49.9±0.6
49.9±2.4
nt
45.2±4.1
21.1±1.9
nt
nt
ns
ns
ns
ns
nt
39.0±1.6
21.6±1.1
13.2±0.6
8.6±0.4
ns
ns
4s
4t
4u
4v
66.7±2.2
25.7±1.9
37.8±1.6
59.7±5.4
40.6±5.6
63.6±0.7
15.6±1.0
ns
13.2±0.5
ns
24.1±1.9
19.0±0.2
ns
ns
ns
ns
18.9±2.1
ns
4w
Colchicine
60.1±2.8
58.7±1.3
Values are the mean of at least three independent determinations. ns = not significant (% inhibition <
10%). nt = not tested

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Table 2. IC50 values (µM) for compounds that exhibited at least 50% of growth
inhibition in the 10 µM screening assay.
Comp.
4d
4g
4h
4i
4j
4l
4m
K562
HEP G2
>10
>10
>10
>10
>10
5.9±0.2
>10
NCI H460
>10
HT29
>10
>10
>10
>10
>10
4.4±0.01
3.7±0.003
5.7±0.04
7.9±0.02
7.9±0.009
0.4±0.001
>10
>10
>10
>10
>10
>10
9.7
1.4±0.03
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
4p
4q
4s
0.43±0.009
3.2±0.006
5.6±0.1
>10
>10
>10
0.71±0.02
>10
0.07±0.001
4v
8.0±0.07
0.02±0.003
Colchicine
Values are the mean of at least three indipendent determination.
These data showed that the best antiproliferative agent was compound 4l, which showed
antiproliferative activity against all cell lines investigated, and it yielded the lowest IC50 value in the
K562 cells, in particular. Overall, however, this series of compounds was most active in the K562
cell line.
To obtain more insights into the cytotoxic potential of test compounds for normal human cells,
compounds 4d,g-l,p,q,s,v were also tested in vitro for their cytotoxicity against HuDe (IZSL) cells,
a primary cell culture from human epidermis (table 3).
Table 3. Comparison of IC50 values (µM) in HuDe and K562 cells.
Comp.
4d
4g
4h
4i
4j
4l
4p
HuDe
145.1±0.01
191.3±0.9
260.5±2.1
57.5±1.7
51.5±0.4
191.2±0.09
54.6±0.4
K562
4.4±0.01
3.7±0.003
5.7±0.04
7.9±0.02
7.9±0.009
0.4±0.001
0.43±0.0089
3.2±0.006
5.6±0.1
HuDe/K562
32.9
51.7
45,7
7.3
6.52
478
127
22.4
32.9
4q
4s
4v
71.7±2.5
184.4±3.1
52.4±0.01
0.017±0.001
6.55
0.85
8.0±0.07
0.02±0.003
Colchicine
Values are the mean of at least three independent determination.
The data in table 3 showed that the K562-active compounds were significantly less cytotoxic
against the HuDe cell line (HuDe/K562 index ranging from 7.3 to 478), indicating the possibility
that these compounds may have limited toxicity in vivo at doses that would limit tumor growth;
data from table 3 also showed a very low HuDe/K562 index (0.85) for colchicine.

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As far as structure-activity relationships are concerned, it seems that on average the substitution of
the double bond with either a triple (compounds 4l-q) or single (compounds 4r-w) bond had little
effect on inhibition of K562 cell growth (Table 1). With a double bond (4a-k), average inhibition by
10 µM compound of K562 cell growth was 44.4%, while with a triple (4l-q) or single (4r-w) bond,
average inhibition was 47.5 and 38.4%, respectively. In the cinnamamidobenzamido series
(compounds 4a-k), as previously reported [7], the activity was only maintained in the ortho-styryl
derivatives (compounds 4g-j). Moreover, substitutions in the 4 position of the benzamido moiety
reduced K562 cell growth irrespective of the substituent (compounds 4e and 4k). In the 2-(3-
phenylpropiolamido)benzamido series (compounds 4l-q), antiproliferative activity against the K562
cell line was greatest in the unsubstituted compound 4l. Finally, with regard to the 2-(3-
phenylpropanamido)benzamides (compounds 4r-w), the unsubstituted 4r was the least active of the
series against the K562 cells, while the choro (4s) and methoxy (4v) derivatives were the most
active among the compounds with substituents.
The effects (table 2) of one the most active compounds of each series (4d for 2-
cinnamamidobenzamides, 4l for 2-(3-phenylpropiolamido)benzamides and 4s for 2-(3-
phenylpropanamido)benzamides, respectively) on cell cycle distribution were analyzed in K562
cells. Cells were cultured for 24 h in the presence of each compound at 10 µM. Flow cytometric
analyses of cell cycle distribution are summarized in table 4.
Table 4. Effects of compounds 4d, 4l, 4s on DNA content/cell following
the treatment of K562 cells for 24 h.
SUB G1
G0/G1
S
G2M
± SE
± SE
± SE
± SE
no drug
7.1 ± 1,1
3.0 ± 1,7
40 ± 1,2
29.2 ± 2,2
24 ± 0,8
27.2 ± 1,6 40.6 ± 2,5
28.9 ± 1,3
4d
4l
4s
12.0 ± 4,2 28.8 ± 1,6 23.2 ± 1,7
36.0 ± 3,7
40.9 ± 1,5
63.3 ± 0,5
4.0 ± 1,1
5.6 ± 0,4
27.3 ± 2,1 27.7 ± 0,7
14.3 ± 0,8 16.8 ± 0,1
colchicine
The three compounds increased the proportion of cells in the G2/M phase, although the effect of
colchicine was greater. The increase in G2/M cells occurred at the expense of cells in G0-G1, which
decreased following treatment with all compounds.
Selected compounds (4d, 4l and 4s) were also evaluated in human Burkitt lymphoma CA46 cells by
flow cytometry, because this cell line generally produces a very high proportion of cells arrested in
G2/M when treated with antitubulin agents. The IC50 values obtained in the Burkitt cells were 5.0 ±
1 µM for 4d, 20 ± 2 µM for 4s, and > 100 µM for 4l. For flow cytometry, the cells were treated
with 100 µM of the three agents. The proportion of cells in G2/M was over 90% following
treatment with either 4d or 4s, but the cells treated with 4l showed a control pattern of cell cycle

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distribution. A morphological analysis was also performed with Burkitt cells treated with 4d and 4s
at 100 µM. A mitotic index of 32% was obtained following 4d treatment and of 23% following 4s
treatment (control value, 3%; value following treatment with the potent antitubulin agent
combretastatin A-4, 48%).
Since G2/M arrest and, especially, an increase in the mitotic index is associated with antitubulin
agents, we evaluated a number of the active compounds for potential effects on the assembly of
purified tubulin. None of the examined compounds (4d, 4e, 4l, 4m, 4r, 4s, 4t, 4w) had any effect on
tubulin assembly at 20 µM, in contrast to combretastatin A-4, which yielded an IC50 of 1.1 ± 0.1
µM in these experiments.
Nevertheless, since antitubulin agents invariably cause apoptosis in cells treated with these
compounds, we evaluated compounds 4a-e, i, l-w for apoptotic effects on the cell line HepG2. We
used the annexin V assay. In the early stages of apoptosis, changes occur at the cell surface. One of
these is the translocation of phosphatidylserine (PS), which is normally located exclusively at the
inner side of the plasma membrane, to the external surface of the cell. The Ca2+-dependent
phospholipid-binding protein annexin V has a high affinity for PS. It can be used as a sensitive
probe for PS that is exposed on the outer leaflet of the cell membrane. However, due to the loss of
membrane integrity in necrotic cells, annexin V can enter the cells and bind to the PS exposed on
the inner leaflet of the cell membrane. Simultaneous staining with propidium iodide (PI), a DNA
stain that is not cell membrane permeable, distinguishes apoptotic cells from necrotic cells.
Apoptotic cells will stain annexin V positive, but be PI negative because the PI cannot cross the
intact cell membrane. Necrotic cells will stain positive for both annexin V and PI because the cell
membrane is no longer intact.
A comparison of the percentage of apoptotic cells in the absence of drug (13.9%) with the
percentage following a 72 h treatment with the compounds at 10 µM is shown in Table 5. The
greatest apoptotic activity occurred with compounds 4i (50.9%), 4l (28%), 4p (41.7%), 4q (67.9%),
4s (26.0%), 4t (34.9%) and 4v (40.8%). With the reference compound colchicine there were 40.5%
apoptotic cells. Among these, compounds 4i and 4q showed a marked apoptotic effect compared
with cell death (apoptosis/dead index 1.80 and 2.18, respectively), significantly greater than the
ratio obtained with colchicine.

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. Apoptotic activity on HepG2 cell line of compounds
Table 5
ANNEXIN V (%
4a-e, i, l-w.
APOPTOTIC
PI (% DEAD
CELLS ± SE)
CELLS ± SE)
APOPTOSIS/DEAD
no drug
13.9 ± 1.2
15.9 ± 2.8
11.6 ± 2.7
12.2 ± 3.0
18.1 ± 1.7
16.8 ± 0.9
50.9 ± 2.2
28.0 ± 2.9
19.1 ± 0.8
20.5 ± 0.2
11.1 ± 0.3
41.7 ± 0.8
67.9 ± 0.1
14.1 ± 1.3
26.0 ± 1.2
34.9 ± 1.1
15.9 ± 0.5
40.8 ± 3.3
17.8 ± 1.7
40.5 ± 2.8
0.7 ± 0.2
31.5 ± 6.3
25.0 ± 6.3
27.9 ± 7.4
47.3 ± 3.3
38.7 ± 2
28.2 ± 4.1
62.8 ± 7.3
56.6 ± 2.0
45.2 ± 1.8
34.3 ± 1.0
57.0 ± 2.4
31.2 ± 0.4
39.7 ± 11.6
32.2 ± 3.2
49.4 ± 3.0
27.1 ± 1.3
46.2 ± 2.7
39.5 ± 2.5
41.8 ± 1.9
4a
4b
4c
4d
4e
4i
4l
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
0.50
0.46
0.44
0.38
0.43
1.80
0.45
0.34
0.45
0.32
0.73
2.18
0.36
0.81
0.71
0.59
0.88
0.45
0.97
4w
colchicine
3. Conclusion
To summarize, we have two suggestive pieces of evidence that the most active compounds in this
series may act through an antitubulin mechanism of action. The compounds cause G2/M and
mitotic arrest in treated cells, and they cause an apoptotic cell death. In contrast, however, we could
detect no activity with purified tubulin. There are several potential explanations for such findings.
Possibly the target is another intracellular protein. Possibly our derivatives are metabolized
intracellularly and converted to more active compound(s). On the other hand, the most potent
antitubulin agents have IC50 values in the low nanomolar range and even much lower (the most
active spongistatins and cryptophycins arrest cell growth in the low picomolar range). Our reference
agents here are good examples, with colchicine having a 20 nM IC50 value in the K562 cells and
combretastatin A-4 a 20 nM IC50 in the Burkitt cells. In other words, cells are substantially more
sensitive to these agents than is the purified protein (the most active agents in our hands yield
assembly IC50 values no lower than 0.5 µM), possibly because the intracellular concentration of
agents is much higher than the nominal concentration in the culture medium.

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4. Experimental protocols
4.1. Chemistry
4.1.1. General
Reaction progress was monitored by TLC on silica gel plates (Merck 60, F254, 0.2 mm). Organic
solutions were dried over Na2SO4. Evaporation refers to the removal of solvent on a rotary
evaporator under reduced pressure. All melting points were determined on a Büchi 530 capillary
melting point apparatus and are uncorrected. IR spectra were recorded with a Perkin Elmer
Spectrum RXI FT-IR System spectrophotometer, with compound as a solid in a KBr disc. 1H NMR
spectra were obtained using a Bruker AC-E 300 MHz spectrometer (tetramethylsilane as internal
standard): chemical shifts are expressed in
δ values (ppm). Merck silica gel (Kiesegel 60/230-400
mesh) was used for flash chromatography columns. Microanalyses data (C, H, N) were obtained by
an Elemental Vario EL III apparatus and were within ±0.4% of the theoretical values. Yields refer
to purified products and are not optimized. The names of the products were obtained using the
ACD/I-Lab Web service (ACD/IUPAC Name Free 8.05).
4.1.2. General procedure for preparation of cinnamoyl chlorides 3a-f, 3-phenylpropioloyl chloride
3g, 3-phenylpropanoyl chloride 3h and 5-R-4-R1-2-nitrobenzoyl chloride 6a-c.
Substituted benzoyl and acryloyl chlorides 3a-h, and 6a-c were obtained by refluxing for 5 h the
appropriate acid derivatives (0.01 mole) with thionyl chloride (7.25 ml) [10]. After evaporation
under reduced pressure, the crude liquid residue was used for subsequent reactions without
purification.
4.1.3. Preparation of 5-R-4-R1-2-nitrobenzamides 7a-c
To 0.01 mole of 5-R-4-R1-2-nitrobenzoyl chlorides 6a-c 10 ml of an aqueous ammonia solution
(25%) and 33 ml of acetonitrile were added. The solution was first refluxed for 8 h, then evaporated
under reduced pressure to give pure 7a-c.
4.1.4. Preparation of 5-R-4-R1-2-aminobenzamides 2d,f,g.
To a magnetically stirred suspension of stannous chloride (0.038 mole) in concentrated HCl (37%),
0.013 mole of 7a-c was added (15 ml) at a rate so that the temperature of the slurry was maintained
below 5°C (about 1 h). After addition was complete, the mixture was stirred for 24 h. The white
slurry thus obtained was diluted with cold water (150 ml), and sodium hydroxide (40%) was added
until the tin salt dissolved. The solution was extracted with ethyl acetate (3X150 ml), and the
extracts dried and evaporated in vacuo to obtain pure 2d,f,g.

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4.1.5. General procedure for preparation of 2-amino-5-chlorobenzamide 2b and 2-amino-5-
bromobenzamide 2c [12].
A mixture of 0.01 mole of 6-R-7-R1-1H-benzo[d][1,3]oxazine-2,4-diones 8b,c and 25 ml of
aqueous ammonia solution (25%) was stirred for 1 h. The solid precipitate was removed by
filtration, washed with an aqueous ammonia solution (5%) and crystallized from ethanol.
4.1.6. General procedure for preparation of 2-amino-5-iodobenzamide 2e [11].
Powdered iodine (11.7 g, 46.2 mmol) was added portion-wise over 1 h to a stirred solution of 2-
aminobenzamide 2a (5.72, 42.0 mmol) and NaHCO3 (3.52 g, 42.0 mmol) in water (1.3 l). The
solution was stirred overnight at room temperature. Afterwards NaHSO3 (0.87 g, 8.40 mmol) was
added. The solution was extracted with ethyl acetate (3 X 800 ml). After being dried with Na2SO4,
the organic phase was removed under reduced pressure. The crude product was recrystallized with
water/methanol 10:1 v/v, 600 ml) to yield pure 2e; yield 95%, mp 197-198°C.
4.1.7. General procedure for preparation of 5-R-4-R1-2-cinnamamidobenzamides 4a-e, 4f [4,5], 4g-
k, 5-R-4-R1-2-(3-phenylpropiolamido)benzamide 4l [8], 4m-q and 5-R-4-R1-2-(3-
phenylpropanamido)benzamide 4r [9] 4s-w.
To a cold (0-5°C) stirred suspension of aminobenzamides 2a-g (0.016 mole) in pyridine (13 ml),
0.016 mole of the appropriate cinnamoyl chloride 3a-e, 3-phenylpropioloyl chloride 3f-k and 3-
phenylpropanoyl chloride 3l-o was added over 30 min. After addition was complete, the solution
was stirred for 24 h and then poured onto crushed ice. The precipitate was removed by filtration,
washed with water, and crystallized from the appropriate solvent.
(E)-2-(3-(3-Bromophenyl)acrylamido)-5-iodobenzamide (4a): yield 38.0 %; mp 252-254°C
1
-1
(dioxane); I.R. (KBr) cm 3347, 3145 (NH, NH2), 1659, 1620 (2XCO); H-NMR (DMSO) δ 6.96
(d, 1H, J=15.9 Hz, olefinic CH); 7.36-8.41 (a set of signals, 9H, aromatic protons, olefinic CH and
exchangeable NH2); 11.76 (s, 1H, NH; exchangeable). Anal. (C16H12BrIN2O2) C,H,N.
(E)-2-(3-(3-Chlorophenyl)acrylamido)-5-iodobenzamide (4b): yield 26.4 %; mp 254-256°C
1
-1
(dioxane); I.R. (KBr) cm 3354, 3150 (NH, NH2), 1657, 1618 (2XCO); H-NMR (DMSO) δ 6.96
(d, 1H, J=15.6 Hz, olefinic CH); 7.46-8.44 (a set of signals, 10H, aromatic protons, olefinic CH and
exchangeable NH2); 11.82 (s, 1H, NH; exchangeable). Anal. (C16H12ClIN2O2) C,H,N.

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(E)-2-(3-(4-Chlorophenyl)acrylamido)-5-iodobenzamide (4c): yield 31.6 %; mp 250-252°C
1
-1
(dioxane); I.R. (KBr) cm 3377, 3218 (NH, NH2), 1660, 1616 (2XCO); H-NMR (DMSO) δ 6.86
(d, 1H, J=15.9 Hz, olefinic CH); 7.42-8.40 (a set of signals, 10H, aromatic protons, olefinic CH and
exchangeable NH2); 11.78 (s, 1H, NH; exchangeable). Anal. (C16H12ClIN2O2) C,H,N.
2-Cinnamamido-5-methoxybenzamide (4d): yield 38.3 %; mp 196-198°C (ethanol); I.R. (KBr)
1
-1
cm 3371, 3188 (NH, NH2), 1660, 1616 (2XCO); H-NMR (CDCl3) δ 3.85 (s, 3H, OCH3); 5.68
(broad s, 1H, NH-H, exchangeable); 6.27 (broad s, 1H, NH-H, exchangeable); 6.58 (d, 1H, J=15.6
Hz, olefinic CH); 7.42-8.40 (a set of signals, 9H, aromatic protons, olefinic CH); 11.03 (s, 1H, NH;
exchangeable). 13C-NMR(δ) (CDCl3) 55.74 (OCH3), 113.17 (CHAr), 117.92 (CHAr), 119.90 (CAr),
122.06 (CAr), 123.43 (CHAr), 128.01 (2XCHAr), 128.83 (2XCHAr), 129.82 (CHAr), 133.42 (CAr),
134.80 (CHAr), 141.63 (CAr), 154.78 (CHAr), 164.07 (CO), 170.98 (CO). Anal. (C17H16N2O3)
C,H,N.
2-Cinnamamido-4,5-dimethoxybenzamide (4e): yield 41.6 %; mp 214-216°C (ethanol); I.R.
1
-1
(KBr) cm 3327-3221 (NH, NH2), 1657, 1610 (2XCO); H-NMR (CDCl3) δ 3.89 (s, 3H, OCH3);
3.99 (s, 3H, OCH3); 5.92 (broad s, 2H, NH2, exchangeable); 6.59 (d, 1H, J=15.6 Hz, olefinic CH);
6.97-8.61 (a set of signals, 8H, aromatic protons, olefinic CH); 11.73 (s, 1H, NH; exchangeable).
13C-NMR(δ) (CDCl3) 56.15 (OCH3), 56.52 (OCH3), 104.79 (CHAr), 109.52 (CAr), 110.00 (CHAr),
122.12 (CHAr), 128.07 (2XCHAr), , 128.86 (2XCHAr), 129.92 (CHAr), 134.71 (CAr), 136.77 (CAr),
141.88 (CHAr), 144.09 (CAr), 153.16 (CAr), 164.54 (CO), 171.15 (CO). Anal. (C18H18N2O4) C,H,N.
(E)-5-Chloro-2-(3-(2-methoxyphenyl)acrylamido)benzamide (4g): yield 36 %; mp 235-240°C
1
-1
(ethanol); I.R. (KBr) cm 3365, 3160 (NH, NH2), 1689, 1654 (2XCO); H-NMR (DMSO) δ 3.89
(s, 3H, OCH3); 6.80 (d, 1H, J=15.6 Hz, olefinic CH); 6.98-8.64 (a set of signals, 10H, aromatic
protons, olefinic CH and exchangeable NH2); 11.85 (s, 1H, NH; exchangeable). Anal.
(C17H15ClN2O3) C,H,N.
(E)-2-(3-(2-Methoxyphenyl)acrylamido)-5-methylbenzamide (4h): yield 36 %; mp 216-220°C
1
-1
(ethanol); I.R. (KBr) cm 3384, 3166 (NH, NH2), 1652, 1621 (2XCO); H-NMR (DMSO) δ 2.32
(s, 3H, CH3); 3.89 (s, 3H, OCH3); 6.74 (d, 1H, J=16.0 Hz, olefinic CH); 6.79-8.50 (a set of signals,
10H, aromatic protons, olefinic CH and exchangeable NH2); 11.81 (s, 1H, NH; exchangeable).
Anal. (C18H18N2O3) C,H,N.

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(E)-5-Iodo-2-(3-(2-methoxyphenyl)acrylamido)benzamide (4i): yield 32 %; mp 240-242°C
1
-1
(ethanol); I.R. (KBr) cm 3390, 3214 (NH, NH2), 1662, 1613 (2XCO); H-NMR (DMSO) δ 3.88
(s, 3H, OCH3); 6.77 (d, 1H, J=15.6 Hz, olefinic CH); 6.97-8.41 (a set of signals, 10H, aromatic
protons, olefinic CH and exchangeable NH2); 11.79 (s, 1H, NH; exchangeable). 13C-NMR(δ)
(DMSO) 55.64 (OCH3), 86.14 (CAr), 111.71 (CHAr), 120.70 (CHAr), 110.00 (CHAr), 122.10 (CAr),
122.40 (CHAr), , 122.58 (CHAr), 122.65 (CAr), 128.40 (CHAr), 131.53 (CHAr), 136.04 (CHAr), 136.63
(CHAr), 139.28 (CAr), 140.49 (CHAr), 157.72 (CAr), 163.91 (CO), 169.28 (CO). Anal. (C17H15IN2O3)
C,H,N.
(E)-5-Methoxy-2-(3-(2-methoxyphenyl)acrylamido)benzamide (4j): yield 36 %; mp 158-161°C
1
-1
(ethanol); I.R. (KBr) cm 3382, 3234 (NH, NH2), 1662, 1619 (2XCO); H-NMR (DMSO) δ 3.81
(s, 3H, OCH3); 3.89 (s, 3H, OCH3); 6.82 (d, 1H, J=15.9 Hz, olefinic CH); 6.98-8.50 (a set of
signals, 10H, aromatic protons, olefinic CH and exchangeable NH2); 11.57 (s, 1H, NH;
exchangeable). Anal. (C18H18N2O4) C,H,N.
(E)-4,5-Dimethoxy-2-(3-(2-methoxyphenyl)acrylamido)benzamide (4k): yield 36 %; mp 240-
1
-1
243°C (ethanol); I.R. (KBr) cm 3333, 3202 (NH, NH2), 1654, 1607 (2XCO); H-NMR (DMSO) δ
3.83 (s, 3H, OCH3); 3.85 (s, 3H, OCH3); 3.90 (s, 3H, OCH3); 6.75 (d, 1H, J=15.6 Hz, olefinic CH);
6.98-8.47 (a set of signals, 9H, aromatic protons, olefinic CH and exchangeable NH2); 12.40 (s, 1H,
NH; exchangeable). Anal. (C19H20N2O5) C,H,N
5-Chloro-2-(3-phenylpropiolamido)benzamide (4m): yield 95%; mp 239-240°C (ethanol-water);
1
-1
I.R. (KBr) cm
3390, 3215 (NH, NH2), 2208 (C≡C), 1666, 1649 (2XCO); H-NMR (DMSO) δ
7.47-8.52 (a set of signals, 8H, aromatic protons); 8.55 (s, 2H, NH2, exchangeable); 12.29 (s, 1H,
NH; exchangeable). Anal. (C16H11ClN2O2) C,H,N.
5-Bromo-2-(3-phenylpropiolamido)benzamide (4n): yield 95%; mp 239-240°C (ethanol-
1
-1
dioxane); I.R. (KBr) cm
3392, 3217 (NH, NH2), 2209 (C≡C), 1664, 1651 (2XCO); H-NMR
(DMSO) δ 7.45-8.43 (a set of signals, 8H, aromatic protons); 8.43 (s, 2H, NH2, exchangeable);
12.26 (s, 1H, NH; exchangeable). Anal. (C16H11BrN2O2) C,H,N.
5-Methyl-2-(3-phenylpropiolamido)benzamide (4o): yield 95%; mp 239-240°C (ethanol-
1
-1
dioxane); I.R. (KBr) cm 3393, 3213 (NH, NH2), 2207 (C≡C), 1666, 1647 (2XCO); H-NMR

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(DMSO) δ 2.32 (s 3H, CH3); 7.35-8.27 (a set of signals, 8H, aromatic protons); 8.30 (s, 2H, NH2,
exchangeable); 12.26 (s, 1H, NH; exchangeable). Anal. (C17H14N2O2) C,H,N.
5-Iodo-2-(3-phenylpropiolamido)benzamide (4p): yield 84.0%; mp 235-237°C (ethanol); I.R.
1
-1
(KBr) cm 3402, 3211 (NH, NH2), 2207 (C≡C), 1666, 1647 (2XCO); H-NMR (DMSO) δ 7.50-
8.16 (a set of signals, 8H, aromatic protons); 8.43 (s, 2H, NH2, exchangeable); 12.26 (s, 1H, NH;
exchangeable). 13C-NMR(δ) (DMSO) 83.99 (CHAr), 84.98 (CAr), 87.79 (CHAr), 119.15 (CAr),
122.12 (CAr), 123.00 (CHAr), 129.23 (2XCHAr), 131.01 (CHAr), 132.58 (2XCHAr), 136.97 (CHAr),
138.45 (CAr), 140.99 (CHAr), 150.12 (CO), 169.27 (CO). Anal. (C16H11IN2O2) C,H,N.
4,5-Dimethoxy-2-(3-phenylpropiolamido)benzamide (4q): yield 87.4%; mp 190-193°C
1
-1
(ethanol); I.R. (KBr) cm 3359, 3164 (NH, NH2), 2208 (C≡C), 1677, 1618 (2XCO); H-NMR
(DMSO) δ 3.81 (s 6H, 2XOCH3); 7.40-8.36 (a set of signals, 9H, aromatic protons, exchangeable
NH2); 12.86 (s, 1H, NH; exchangeable). Anal. (C18H16N2O4) C,H,N.
5-Chloro-2-(3-phenylpropanamido)benzamide (4s): yield 76.6 %; mp 165-166°C (ethanol); I.R.
1
-1
(KBr) cm 3347, 3173 (NH, NH2), 1683, 1618 (2XCO); H-NMR (CDCl3) δ 2.72 (t, 2H, CH2);
3.05 (t, 2H, CH2); 5.69 (broad s, 1H, NH-H, exchangeable); 6.15 (broad s, 1H, NH-H,
exchangeable); 6.19-8.64 (a set of signals, 8H, aromatic protons); 11.00 (s, 1H, NH; exchangeable).
13C-NMR(δ) (CDCl3) 31.29 (CH2), 39.92 (CH2), 119.81 (CAr), 123.04 (CHAr), 126.25 (CHAr),
126.99 (CHAr), 127.56 (CAr), 128.37 (2XCHAr), 128.53 (2XCHAr), 133.15 (CHAr), 138.67 (CAr),
140.52 (CAr), 169.81 (CO), 171.09 (CO). Anal. (C16H15ClN2O2) C,H,N.
5-Methyl-2-(3-phenylpropanamido)benzamide (4t): yield 58.8 %; mp 155-158°C (ethanol); I.R.
1
-1
(KBr) cm 3294, 3162 (NH, NH2), 1667, 1630 (2XCO); H-NMR (CDCl3) δ 2.33 (s, 3H, CH3);
2.71 (t, 2H, CH2); 3.06 (t, 2H, CH2); 5.59 (broad s, 1H, NH-H, exchangeable); 6.13 (broad s, 1H,
NH-H, exchangeable); 7.20-8.54 (a set of signals, 8H, aromatic protons); 11.01 (s, 1H, NH;
exchangeable). 13C-NMR(δ) (CDCl3) 20.78 (CH3), 31.41 (CH2), 39.95 (CH2), 118.48 (CAr), 121.61
(CHAr), 126.15 (CHAr), 127.45 (CHAr), 128.38 (2XCHAr), 128.48 (2XCHAr), 132.17 (CAr), 134.03
(CHAr), 137.66 (CAr), 140.73 (CAr), 170.91 (CO), 171.21 (CO). Anal. (C17H18N2O2) C,H,N.
5-Iodo-2-(3-phenylpropanamido)benzamide (4u): yield 67.4 %; mp 183-185°C (ethanol); I.R.
1
-1
(KBr) cm 3388, 3267, 3188 (NH, NH2), 1654, 1610 (2XCO); H-NMR (CDCl3) δ 2.71 (t, 2H,
CH2); 3.04 (t, 2H, CH2); 5.83 (broad s, 1H, NH-H, exchangeable); 6.31 (broad s, 1H, NH-H,

ACCEPTED MANUSCRIPT
exchangeable); 7.19-8.43 (a set of signals, 8H, aromatic protons); 11.03 (s, 1H, NH; exchangeable).
13C-NMR(δ) (CDCl3) 31.24 (CH2), 39.97 (CH2), 85.00 (CAr), 120.54 (CAr), 123.43 (CHAr), 126.24
(CHAr), 128.36 (2XCHAr), 128.53 (2XCHAr), 135.76 (CHAr), 139.70 (CAr), 140.48 (CAr), 141.89
(CHAr), 169.62 (CO), 171.13 (CO). Anal. (C16H15IN2O2) C,H,N.
5-Methoxy-2-(3-phenylpropanamido)benzamide (4v): yield 72.1 %; mp 156-158°C (ethanol);
1
-1
I.R. (KBr) cm 3382, 3280, 3187 (NH, NH2), 1652, 1608 (2XCO); H-NMR (CDCl3) δ 2.75 (t,
2H, CH2); 3.11 (t, 2H, CH2); 3.85(s, 3H, OCH3); 5.98 (broad s, 1H, NH-H, exchangeable); 6.54
(broad s, 1H, NH-H, exchangeable); 7.06-8.52 (a set of signals, 8H, aromatic protons); 10.76 (s, 1H,
NH; exchangeable). 13C-NMR(δ) (CDCl3) 31.43 (CH2), 39.72 (CH2), 55.64 (OCH3), 113.15 (CHAr),
117.58 (CHAr), 120.72 (CAr), 123.31 (CHAr), 126.19 (CHAr), 128.35 (2XCHAr), 128.50 (2XCHAr),
132.75 (CAr), 140.66 (CAr), 154.69 (CAr), 170.86 (CO), 171.02 (CO). Anal. (C17H18N2O3) C,H,N.
4,5-Dimethoxy-2-(3-phenylpropanamido)benzamide (4w): yield 44.3 %; mp 170-172°C
1
-1
(ethanol); I.R. (KBr) cm 3381, 3280, 3230 (NH, NH2), 1660, 1621 (2XCO); H-NMR (CDCl3) δ
2.73 (t, 2H, CH2); 3.06 (t, 2H, CH2); 3.87 (s, 3H, OCH3); 3.96 (s, 3H, OCH3); 5.84 (broad s, 2H,
NH2, exchangeable); 6.92-8.44 (a set of signals, 7H, aromatic protons); 11.43 (s, 1H, NH;
exchangeable). 13C-NMR(δ) (CDCl3) 31.34 (CH2), 40.02 (CH2), 56.13 (OCH3), 56.50 (OCH3),
104.16 (CHAr), 109.50 (CAr), 109.98 (CHAr), 126.21 (CHAr), 128.34 (2XCHAr), 128.53 (2XCHAr),
136.38 (CAr), 140.65 (CHAr), 143.93 (CAr), 153.09 (CAr), 171.06 (CO), 171.17 (CO). Anal.
(C18H20N2O4) C,H,N.
4.2. Biology
4.2.1 Cell lines and cell cultures
Human chronic myelogenous leukemia cell line K562 was maintained in suspension culture in
RPMI1640 medium (SIGMA-Aldrich, cat.#R8758) supplemented with 10% fetal bovine serum
(FBS: SIGMA-Aldrich, cat.#F6178), 100 U/ml penicillin, 100 ꢀg/ml streptomycin and 0.25 ꢀg/ml
amphothericin B (complete medium) at 37°C in a 5% CO2 humidified atmosphere.
The hepatocarcinoma HEP G2 cell line was incubated for 72 h with 105 cells/ well in 24-well
flasks with medium VITO 143, with the addition of ITG (insulin, transferrin, and glucagon), phenol
red, and penicillin and streptomycin, as above. After 72 h, the cells were harvested with EDTA,
washed 2 times with PBS for 5 min with centrifugation at 200 x g and resuspended in 100 ꢀl of PBS
for flow cytometric analysis.

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4.2.2. Trypan blue exclusion assay
The trypan blue exclusion assay was performed to assess the effect of all test compounds on the
viability of K562 cells, as described earlier [14]. Briefly, about 2x105 cells/ml were cultured for 24
h, and compounds were added at 10 µM (or at different concentrations to determine the IC50). The
effects of test compounds on cell viability were assayed on a portion of the cell suspension after 24
h. The cell number was determined with a hemocytometer, and viability was estimated by trypan
blue dye exclusion.
4.2.3. MTT assay
MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed as
described earlier [15,16] and was used to determine viability of NCI-H460, HT29, HCT116, MCF-7
and HuDe cells.
MTT is a yellow-colored tetrazolium salt that is taken up and cleaved only by metabolically active
cells, which reduce it to a colored, water-insoluble formazan salt. The solubilized formazan product
can be quantified via absorbance at 570 nm (690 nm for blank), which is measured using a 96-well-
format spectrophotometer (ELx800-BioTek instruments). The absorbance correlates directly with
cell number.
Briefly, about 2x105 cells/ml (2x104 cells/ml for HuDe) were cultured ; different concentrations of
test drugs or 0.1% DMSO were added to the wells 24 h after seeding. Cells were collected after 72
h and evaluated by the MTT assay. Cells were incubated with 10 ꢀl of MTT (5 mg/ml in PBS) at
37°C for 3 h. The tetrazolium crystals were solubilized by the addition of 4.5 ml isopropyl alcohol
0.5ml tritonX-100 in 150 ꢀl of 37% HCl. Cells grown in culture media alone or with appropriate
concentrations of DMSO were used as controls.
4.2.4. Flow cytometry (FACS).
To analyze the proportion of cells in different phases of the cell cycle and the percentage of
apoptotic cells, K562 and HepG2 cells were analyzed, respectively, by flow cytometry. The cells
were analyzed after growth for 72 h in the compound being examined. In the apoptotic study, one
million cells were treated with fluorescein isothiocyanate (FITC)-conjugated antibody for annexin
V and propidium iodide (PI) for staining of necrotic cells. Side scatter and forward scatter were set
to include all viable cells. The debris was excluded from the population of cells by applying a gate
in the FSC (forward scatter) versus SSC (side scatter). The data were acquired on a Becton-
Dickinson FACS Air II instrument and analyzed by FACSDiva software 6.1.2 (BD Biosciences,

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Franklin Lakes, NJ). We used manual compensation with the instrument to exclude the overlap of
the two emission spectra.
4.2.5. Determination of mitotic index.
Human Burkitt lymphoma CA46 cells were purchased from the ATCC (Manassas, VA, USA) and
grown in RPMI 1640 medium with glutamine (Gibco, Grand Island, NY, USA) supplemented with
20% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, USA). For mitotic index
determinations (that is, percent of cells with condensed chromosomes), 106cells/ml were grown for
16 h at 37°C in a humidified 5% CO2 atmosphere. Cells were harvested by centrifugation for 5 min
at 1,000 rpm in a Beckman Allegra 6KR centrifuge equipped with a GH-3.8A swinging bucket
rotor. The supernatant was decanted, and the cells were washed once in 5 ml of phosphate-buffered
saline (pH 7.4). After centrifugation again, the cells were resuspended in 0.5 ml of 0.034 M sodium-
potassium phosphate (pH 7.2) and left for 10 min at room temperature. After this hypotonic
swelling, the cells were recentrifuged and suspended and fixed in 1.5% ethanol-0.5 % acetic acid.
After 30 min, the cells were again harvested by centrifugation, and the final cell pellet was
suspended in 75% ethanol-25% acetic acid. The cell suspension was smeared onto a microscope
slide, which was air dried. The cells were stained with Giemsa, and the percentage of cells with
condensed chromosomes was determined by visual examination of the slide with a Nikon TMS
light microscope.
4.2.6. Evaluation of tubulin assembly.
Purified bovine brain tubulin [17] was used in turbidimetric assembly studies. Tubulin and the
desired concentration of compound were preincubated at 30°C for 15 min in a 0.24 ml reaction
volume. Reaction mixtures were then chilled on ice, and 10 µL of 10 mM GTP was added. All
concentrations refer to the final reaction volume of 0.25 ml: tubulin at 1.0 mg/ml (10 µM), 0.8 M
monosodium glutamate (pH 6.6 in a 2 M stock solution with HCl), 4% (v/v) dimethyl sulfoxide,
and 0.4 mM GTP. The chilled reaction mixtures were transferred to cuvettes held to 0°C in
recording spectrophotometers (Beckman models DU7400 and DU7500) equipped with electronic
temperature controllers. After baselines were established at 350 nm, the temperature was jumped to
30°C in about 30 s, and the IC50 is defined as the concentration of compound that inhibits turbidity
development by 50% at 30 min. Detailed method description has been published previously [18].
The method is generally most reliable at compound concentrations up to 20 µM. At higher
concentrations, compound precipitation and/or absorbance often leads to interference with the
simple turbidity readings.

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Acknowledgement
Financial support from MIUR (40% funding) is gratefully acknowledged.
REFERENCES AND NOTES
[1].
H. Ogita, Y. Isobe, H.Takaku, R. Sekine, Y. Goto, S. Misawa, H. Hayashi, Synthesis
and structure–activity relationship of diarylamide derivatives as selective inhibitors of the
proliferation of human coronary artery smooth muscle cells. Bioorg. Med. Chem. Lett., 11
(2001) 549-551.
[2].
S.J. Williams, D. Stapleton, S. Zammit, D.J. Kelly, R.E. Gilbert, H., Krum.,
Preparation of cinnamoyl anthranilates and analogs thereof with antifibrotic activity. Patent
WO2008003141 (2008); Chem. Abst. 148:144415 (2008).
[3].
Y. Bo, P.P. Chakrabarti, N. Chen, E.M. Doherty, C.H. Fotsch, N. Han, M.G. Kelly,
Q. Liu, M.H. Norman, X. Wang, J. Zhu, V. Ognynanov, Preparation of vanilloid receptor
ligands and their use in treatments. Patent WO03049702 (2003); Chem. Abst. 139:53025
(2003).
[4].
H. Harada, M. Isaji, H. Miyata, H. Kusama, Y. Nonaka, K. Kamata, T. Yazaki, Y.
Hotei, Acylaminobenzamide derivatives for inhibiting cornea pterygium progress and
preventing its relapse. Patent JP10330254 (1998); Chem. Abst. 130:105331 (1998).
[5].
H. Harada, M. Isaji, H. Kusama, Y. Taketana, Y. Nonaka, T. Kamata, Y. Futai,
Inhibitors of blood vessel development. Patent JP10259129 (1998); Chem. Abst. 129:321163
(1998).
[6].
H. Harada, H. Kusama, Y. Nonaka, K. Kamata, Y. Fotei, Acylaminobenzamide
derivatives for prevention and treatment of glomerular diseases. Patent JP10306024 (1998);
Chem. Abst. 130:47477 (1998)
[7].
D. Raffa, B. Maggio, F. Plescia, S. Cascioferro, S. Plescia, M.V. Raimondi, G.
Daidone, M. Tolomeo, S. Grimaudo, A. Di Cristina, R.M. Pipitone, R. Bai, E. Hamel,
Synthesis, antiproliferative activity, and mechanism of action of a series of 2-{[2E]-3-
phenylprop-2-enoylamino}benzamides. Eur. J. Med. Chem., 46 (2011) 2786-2796.
[8].
P.A. Brownsort, R.M. Paton, A.G. Sutherland, J. Chem. Perkin Trans 1 Org. Bioor.
Chem. (1989) 9, 1679-1686.
[9].
A.V. Purandare, A. Gao, H. Wan, J.Somerville, K. Burke, C. Seachord, W. Vaccaro,
J. Witjak, M.A. Poss, Identification of chemokine receptor CCR4 antagonist. Bioorg. Med.
Chem. Lett., 15 (2005) 2669-2672.

ACCEPTED MANUSCRIPT
[10].
M.H. Palmer, G.J. McVie, Alkylation and acylation reactions. Part II. The interaction
of aryloxyacetyl chlorides with aluminium chloride. J. Chem. Soc. B (1968) 754-751.
[11]. T. Sardon, T. Cotting, J. Xu, A. Giannis, I. Vernos, Development and Biological
Evaluation of a Novel Aurora A Kinase Inhibitor. ChemBioChem (2009), 10, 464-478.
[12]. A.V. Dolzhenco-Podchezertseva, L.M.Korkodinova, M.V. Vaslyuc, V.P. Koteyov,
Synthesis and antiinflammatory activity of n-acyl-5-bromanthranilic acid amides. Pharm.
Chem. J. (2002), 36, 647-648.
[13].
preparation of coumaric acids via styrylisoxazoles. Heterocycles, 20 (1983) 263-267.
[14]. K.K. Chiruvella, V. Kari, B. Choudhary, M. Nambiar, R.G. Ghanta, S.C. Raghavan,
S. Chimichi, F. De Sio, D. Donati, G. Fina, R. Pepino, P. Sarti-Fantoni, The
Methyl angolensate, a natural tetranortriterpenoid induces intrinsic apoptotic pathway in
leukemic cells. FEBS Lett. (2008), 582, 4066–4076.
[15].
M.S. Shahabuddin, M. Nambiar, B. Choudhary, G.M. Advirao, S.C. Raghavan, A
novel DNA intercalator, butylamino-pyrimido[4',5':4,5]selenolo(2,3-b)quinoline, induces cell
cycle arrest and apoptosis in leukemic cells. Invest. New Drugs (2010), 28, 35-48.
[16].
F.M. Freimoser, C.A. Jakob, M. Aebi, U. Tuor, The MTT [3-(4,5-Dimethylthiazol-2-
yl)-2,5-Diphenyltetrazolium Bromide] Assay Is a Fast and Reliable Method for Colorimetric
Determination of Fungal Cell Densities. Appl. Environ. Microbiol. (1999), 65, 3727-3729.
[17].
E. Hamel, C. M. Lin, Separation of active tubulin and microtubule-associated
proteins by ultracentrifugation and isolation of a component causing the formation of
microtubule bundles. Biochemistry (1984), 23, 4173-4184.
[18].
E. Hamel, Evaluation of antimitotic agents by quantitative comparisons of their
effects on the polymerization of purified tubulin. Cell Biochem. Biophys. (2003), 38, 1-22.