Synthesis, antiproliferative activity, and mechanism of action of a series of 2-{[(2E)-3-phenylprop-2-enoyl]amino}benzamides

Article abstract of DOI:10.1016/j.ejmech.2011.03.067

Several new 2-{[(2E)-3-phenylprop-2-enoyl]amino}benzamides 12a-s and 17t-v were synthesized by stirring in pyridine the (E)-3-(2-R1-3-R2-4-R3-phenyl) acrylic acid chlorides 11c-k and 11t-v with the appropriate anthranilamide derivatives 10a-c or the 5-iodoanthranilic acid 13. Some of the synthesized compounds were evaluated for their in vitro antiproliferative activity against the full NCI tumor cell line panel derived from nine clinically isolated cancer types (leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast). COMPARE analysis, effects on tubulin polymerization in cells and with purified tubulin, and effects on cell cycle distribution for 17t, the most active of the series, indicate that these new antiproliferative compounds act as antitubulin agents.

Full text of DOI:10.1016/j.ejmech.2011.03.067

European Journal of Medicinal Chemistry 46 (2011) 2786e2796
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antiproliferative activity, and mechanism of action of a series of
2-{[(2E)-3-phenylprop-2-enoyl]amino}benzamides
,
a
a
a
a
Demetrio Raffa a , Benedetta Maggio , Fabiana Plescia , Stella Cascioferro , Salvatore Plescia ,
Maria Valeria Raimondi a, Giuseppe Daidone a, Manlio Tolomeo b, Stefania Grimaudo b,
Antonietta Di Cristina b, Rosaria Maria Pipitone b, Ruoli Bai c, Ernest Hamel c
*
a Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, Via Archirafi, 32, 90123 Palermo, Italy
b Centro Interdipartimentale di Ricerca in Oncologia Clinica e Dipartimento Biomedico di Medicina Interna e Specialistica, Università di Palermo, Palermo, Italy
c Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick,
National Institutes of Health, Frederick, MD 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Several new 2-{[(2E)-3-phenylprop-2-enoyl]amino}benzamides 12aes and 17tev were synthesized by
stirring in pyridine the (E)-3-(2-R1-3-R2-4-R3-phenyl)acrylic acid chlorides 11cek and 11tev with the
appropriate anthranilamide derivatives 10aec or the 5-iodoanthranilic acid 13. Some of the synthesized
compounds were evaluated for their in vitro antiproliferative activity against the full NCI tumor cell line
panel derived from nine clinically isolated cancer types (leukemia, non-small cell lung, colon, CNS,
melanoma, ovarian, renal, prostate and breast). COMPARE analysis, effects on tubulin polymerization in
cells and with purified tubulin, and effects on cell cycle distribution for 17t, the most active of the series,
indicate that these new antiproliferative compounds act as antitubulin agents.
Received 29 September 2010
Received in revised form
23 March 2011
Accepted 31 March 2011
Available online 6 April 2011
Keywords:
2-{[2E]-3-phenylprop-2-enoylamino}
benzamides
Ó 2011 Elsevier Masson SAS. All rights reserved.
Antimitotic agents
Cytotoxic activity
1. Introduction
treatment of glomerular diseases [6] and diseases caused by the
excessive proliferation of vascular intimal cells [7].
During a screening program to find antiproliferative compounds
in our laboratory’s collection of small organic molecules, the
2-cinnamamido-5-iodobenzamide 1 was found at 10 mM to inhibit
However, despite their wide range of biological activities, a review
of the literature revealed that no anticancer activity had been
describedforcinnamoylanthranilates.Thus, theactivityofcompound
1 asaninhibitorofK562 proliferation led usto explore the potential of
this class of compounds as anticancer agents, and we therefore
synthesized a series of novel cinnamoyl anthranilates and screened
the compounds for antileukemic activity. Our work enabled us to
perform aninitial studyof theirstructure-activityrelationshipsand to
determine their mechanism of action. Compounds 12aes and 17tev
were initially tested in vitro for their antileukemic activity against the
K562 (human chronic myelogenous leukemia) cell line (Table 1).
Among these, 12aec and 17t,u, which showed the best anti-
proliferative activity, were selected by the National Cancer Institute
(NCI) for evaluation in the 60 human tumor cell line screen of the NCI.
proliferation of the leukemic cell line K562 by 74%.
Compound 1 belongs to cinnamoyl anthranilates, which repre-
sent a class of biologically active substances of great importance in
medicinal chemistry. Tranilast (RizabenÒ) 2 is an antiallergic drug
approved in 1982 for use in Japan and South Korea for bronchial
asthma and was also investigated for use as an antiproliferative
agent on drug-eluting stents. Its derivative (E)-2-(3-(3,4-dime-
thoxyphenyl)acrylamido)benzamide 3 [1] is another member of
this class of compounds and is more potent than the lead
compound, Tranilast (Fig. 1).
Other biological activities possessed by this class of compounds
are antifibrotic and antinflammatory properties [2,3] and inhibition
of cornea pterygium progression and blood vessel development
[4,5]. Finally, cinnamoyl anthranilates are useful for prevention and
2. Results and discussion
2.1. Chemistry
A series of 2-{[(2E)-3-phenylprop-2-enoyl]amino}benzamides
12aes and 17tev was synthesized by stirring the (E)-3-(2-R1-3-R2-
* Corresponding author. Tel.: þ39 91 23891917.
E-mail address: demraffa@unipa.it (D. Raffa).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.067

D. Raffa et al. / European Journal of Medicinal Chemistry 46 (2011) 2786e2796
2787
starting materials, 2-cinnamamido-5-iodobenzoic acids 15tev,
were obtained by stirring the 5-iodoanthranilic acid 13 and the
cinnamoyl chlorides 11tev. The reaction gave a mixture of (E)-6-
iodo-2-styryl-4H-benzo[d] [1,3]oxazin-4-ones 14teu and 2-cinna-
mamido-5-iodobenzoic acids 15teu, and the mixtures were treated
with aqueous Na3PO4 [9] to give the corresponding acids 15t [10],
15u,v as the only products. By treating the acids 15tev with ethyl
chloroformate, the 2H-3,1-benzoxazine-2,4-diones 16tev were
obtained [11], which, in turn, converted to the 2-{[2E]-3-phenyl-
prop-2-enoylamino}benzamides 17tev by refluxing them in an
aqueous ammonia solution [8].
The structures of the new compounds were determined by
analytical and spectroscopic measurements. In particular, 1H NMR
spectra of compounds 12aes and 17vet were consistent with
an E-olefinic structure. They showed signals attributable to the
b
-olefinic protons at 6.66e6.97
d
with coupling constants of
-olefinic
16.6e14.7 Hz, as required for E-structures [12], while the
a
hydrogens were found along with aromatic multiplets. Moreover,
their 1H NMR spectra showed both the NH and NH2 amidic signals;
the cinnamamido NH proton appeared as a singlet at 11.77e11.92 d,
while, according to the literature [13], the presence of an intra-
molecular hydrogen bond renders the benzamido NH2 protons
diastereotopic. Ha is easily exchangeable with D2O and appeared as
Fig. 1. Structure of cinnamoyl anthranilates.
a singlet at 8.24e8.44 d. Hb was found at a lower field along with
the aromatic multiplets.
4-R3-phenyl)acrylic acid chlorides 11cek, 11tev and the appro-
priate anthranilamide derivatives 10aec or the 5-iodoanthranilic
acid 13 as described in Schemes 1 and 2.
2.2. Biology
Crude (E)-3-(2-R1-3-R2-4-R3-phenyl)acrylic acids 11cek, 11tev
were obtained by refluxing the appropriately substituted acrylic
acid 8cek, 8tev with thionyl chloride. The 5-methylan-
thranilamide 10a was obtained by reduction of 2-nitro-5-methyl-
benzamideamide 7a as shown in Scheme 1. Compound 7 was
obtained by reaction of the acid 5 with thionyl chloride to afford 6,
followed by treatment of 6 with aqueous ammonia.
The anthranilamide derivatives 10b,c were obtained by stirring
the appropriate 2H-3,1-benzoxazine-2,4(1H)-dione 9b,c in aqueous
ammonia solution (Scheme 1) [8].
Synthesized
2-{[(2E)-3-phenylprop-2-enoyl]amino}benza-
mides 12aes and 17tev were initially tested in vitro for their
antileukemic activity against the K562 (human chronic myeloge-
nous leukemia) cell line. Colchicine 18, whose antileukemic activity
is well known, and the 2-cinnamamidobenzamide 4 [13] (Fig. 1),
were used as reference compounds. The percent growth inhibition
at a screening concentration of 10
compounds that exhibited at least 50% of growth inhibition at
10 M are shown in Table 1. Compounds 12aes and 17tev had
inhibitory activity against the K562 cells ranging from 22.0 to 74.5%
at 10 M, with 12aed, 12k,l, and 17t,u (IC50 0.57e8.1 M) being the
mM and the IC50 values for
m
A different synthetic method was used to obtain the 2-{[(2E)-3-
phenylprop-2-enoyl]amino}benzamides 17tev (Scheme 2). The
m
m
most active compounds. Our data (Table 1) showed positive effects
following substitution with halogens at the 5 position of the ben-
zamido moiety, especially when the substituent was iodine
(compounds 17t and 17u). As far as structure-activity relationships
are concerned, it seems that the introduction of a substituent in
both the benzamido and styryl moieties are favorable for inhibition
of K562 cell growth relative to the unsubstituted 2-cinnamamido-
benzamide 4 (Table 1). However, the best activity was obtained
when the substitutions were present only on the benzamido
moiety (compounds 12aed, 17t). Compounds substituted in both
the benzamido and styryl moieties (compounds 12ees, 17v) were
less active, even if antiproliferative activity was maintained in the
ortho-styryl derivatives (12d,i,l,u).
Compounds 12a,12b,12c,17t and 17u were evaluated by the NCI
for testing against a panel of approximately 60 human cell lines
derived from seven clinically isolated cancer types (lung, colon,
melanoma, renal, ovarian, brain, and leukemia) according to the NCI
standard protocol [14] (Table 2). The data summarized in Table 2
showed that 12a, 12b, 12c, 17t and 17u caused 50% growth inhibi-
tion at micromolar (12a, 12b, 12c) and submicromolar concentra-
tions (17t, 17u) against every type of tumor cell line investigated.
Moreover, a mean graph midpoint (MG_MID) is calculated for
the GI50, TGI and LC50 parameters, giving an average activity
parameter for all the cell lines. For the calculation of the MG_MID,
insensitive cell lines are included and assigned as their values the
highest concentration tested. Considering the MG_MID values
Table 1
Percent growth inhibition obtained with the K562 cell line with compounds at
10
mM and IC50 values (
m
M) with the same cells.
Com.
% inhibition
IC50 (mM)
4
14.2
65.4
64.0
62.4
58.0
28.2
31.0
49.0
27.0
52.2
37.8
64.0
59.0
25.5
22.0
46.2
45.9
43.2
43.8
45.9
74.5
74.1
26.3
63.6
>10
5.5
2.5
5.0
7.4
>10
>10
10
>10
9.5
>10
6.3
8.1
>10
>10
>10
>10
>10
>10
>10
0.57
1.2
>10
0.02
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
12q
12r
12s
17t
17u
17v
18

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D. Raffa et al. / European Journal of Medicinal Chemistry 46 (2011) 2786e2796
Scheme 1. Reagents and conditions: (a) SOCl2, reflux, 5 h; (b) acetonitrile, aqueous ammonia (25%), reflux, 8 h; (c) aqueous ammonia (25%), stirring, rt, 1 h; (d) SnCl2, HCl (35%),
stirring, 0e5 ꢁC, (24 h); (e) pyridine, stirring, rt, 24 h.
(Table 3), the most active compound of the series was derivative
17t, at both the GI50 and TGI levels, followed by 17u and 12c.
Compound 17t was selected by the NCI for evaluation in its vivo
toxicity assay, with the finding that the compound was nontoxic at
a dose of 400 mg/kg in nontumored mice. Compound 17t was also
selected by the NCI for testing in the hollow fiber assay, a prelimi-
nary in vivo screening tool, but the compound was not sufficiently
active for evaluation in xenograft models.
To predict the probable mechanism of action, the NCI’s bio-
informatic tool COMPARE analysis [15] was performed for the most
active compound 17t. When tested as seed against the NCI “standard
agents” Database, the compound showed Pearson Correlation Coef-
ficients (PCC) of 0.492 and 0.476 at the GI50 level and higher values,
0.677, 0.600, 0.597, at the TGI level. In all cases the highest PCC’s were
with compounds NSC 332598 (rhizoxin), NSC 125973 (paclitaxel),
NSC 49842 (vinblastine sulfate) and NSC 153858 (maytansine), which
are all antimitotic agents directed against tubulin.
To confirm the ability of 17t to act on microtubules, the percentage
of cells blocked in mitosis (M) was morphologically determined after
staining cells with ethidium bromide and acridine orange. The
percentage of K562cells blocked inthe M phase after 24 h of exposure
to 1
m
M17t was 35 ꢀ 6 (Fig. 4A, untreatedK562cellascontrol<1%). Of
interest, concentrationsof17t higher than 2
m
M induced morphologic
alterations in almost all treated cells, suggesting an interaction
between 17t and the cytoskeleton of K562 cells (Fig. 4C and D).
To examine the effects of 17t on microtubules (tubulin) and
microfilaments (actin), PtK2 cells were examined by direct immu-
nofluorescence. These cells were selected for this examination
since their flattened morphology yields high quality images of
cytoskeletal elements, particularly the microtubule and microfila-
ment networks. As shown in Fig. 5, the microtubules disappeared
while the microfilaments persisted with micromolar 17t. In addi-
tion, we found that 17t partially inhibited the polymerization of
purified tubulin, but this effect was relatively weak, as shown in
Fig. 6, with a comparison to the much stronger inhibition observed
with colchicine 18. With 17t, we observed a concentration depen-
dent inhibition of the rate of microtubule assembly, beginning at
To verify the prediction of the COMPARE algorithm, the effects of
17t on cell cycle distribution as determined by flow cytometry and
on tubulin polymerization were evaluated.
As shown in Fig. 2, 17t caused a dose dependant increase of
K562 cells in the G2-M phase of the cell cycle and a decrease of
cells in G0-G1 after a 24 h treatment. This is the typical flow
cytometric cell cycle distribution observed with drugs targeting
tubulin (see panel b in Fig. 2), which is the main component of the
mitotic spindle.
about 10
able to examine (up to 40
assembly. It should be pointed out that 17t appeared to partially
precipitate at 40 M. With the classic antitubulin agent colchicine
18, the rate of assembly was 50% inhibited at about 2 M, and the
extent of assembly at about 5 M. At 7 and 10 M colchicine 18,
m
M. However, within the concentration range we were
mM), there was no effect on the extent of
m
m
m
m
After 48 h 17t caused extensive apoptosis in K562 cells, with an
tubulin polymerization was essentially completely inhibited, an
effect that we were unable to achieve with 17t.
AC50 (concentration inducing 50% apoptosis) of 3.7
mM (Fig. 3).
Apoptosis in this experiment was measured as described in the
Experimental Section.
Taken together, our data suggest that 17t interacts with tubulin,
preventing formation of the mitotic spindle and thereby caused

D. Raffa et al. / European Journal of Medicinal Chemistry 46 (2011) 2786e2796
2789
Scheme 2. Reagents and conditions: (a) SOCl2, reflux, 5 h; (b) pyridine, stirring, 0e5 ꢁC, 24 h; (c) aqueous NaPO3 (0.01 M), reflux, 20 h; (d) ethyl chloroformate, acetyl chloride
reflux, (15 min þ 30 min); (e) dioxane, acqueous ammonia (25%), reflux, 3 h.
the block in M phase. Inhibition of tubulin assembly would also
disrupt the integrity of the microtubule cytoskeleton in interphase
cells, causing extensive alterations in cell morphology. Although
its effect on in vitro tubulin assembly is relatively weak, the
cellular effects obtained with 17t are most consistent with those
observed with more potent antitubulin agents. Furthermore,
disruption of cellular microtubules invariably results in activation
of cellular apoptosis.
4. Experimental
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
3. Conclusions
The data reported here show that the 2-{[2E]-3-phenylprop-
2-enoylamino}benzamides 12aes and 17tev caused growth inhi-
bition against many tumor cell lines. The best agents inhibited
proliferation at low micromolar (12a, 12b, 12c) and submicromolar
concentrations (17t, 17u) against every tumor cell line investigated.
The best activity was obtained when the 5 position of the benza-
mido moiety was substituted with an iodine atom. COMPARE
analysis, effects on tubulin polymerization in cells and with puri-
fied tubulin, and effects on cell cycle distribution, including
induction of apoptosis, indicate that these new antiproliferative
compounds act as antitubulin agents.
internal standard): chemical shifts are expressed in
d values
(ppm). Merck silica gel (Kiesegel 60/230e400 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).

2790
D. Raffa et al. / European Journal of Medicinal Chemistry 46 (2011) 2786e2796
Table 2
4.1.3. Preparation of 5-methyl-2-nitrobenzamide 7a
Results of multi-dose growth inhibition assay (GI50,
m
M).
12c
To 0.01 mol of 5-methyl-2-nitrobenzoyl chloride 6a 10 ml of
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.
Cell line
12a
12b
2.96
1.42
17t
17u
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF539
SNB-19
SNB75
U251
2.36
1.77
1.11
3.56
nt
0.429
0.346
0.415
1.86
1.03
1.02
2.41
1.51
1.96
2.61
1.09
0.662
nt
0.341
0.0542 0.165
0.484
0.845
3.95
2.79
3.77
2.83
3.15
2.87
3.00
3.37
1.76
2.08
1.68
0.487
1.98
2.13
1.94
1.69
1.99
2.90
1.40
2.05
3.18
1.84
2.14
4.33
2.68
1.71
0.492
2.75
2.88
0.373
0.920
0.202
0.347
2.65
0.734
0.800
21.2
0.370
1.42
0.772
1.87
1.00
2.30
13.2
1.14
1.39
0.479
0.459
0.797
0.984
0.509
0.987
0.376
0.533
0.605
0.778
0.415
0.444
1.41
4.1.4. Preparation of 5-methyl-2-aminobenzamide 10a
To a magnetically stirred suspension of stannous chloride
(0.038 mol) in concentrated HCl (37%) (15 ml), 0.013 mole of 7a was
added at a rate so that the temperature of the slurry was main-
tained 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 aqueous sodium hydroxide
(40%) was added until the tin salt dissolved. The solution was
extracted with ethyl acetate (3 ꢂ 150 ml), and the extracts dried
and evaporated in vacuo to obtain pure 10a.
Non-small cell
lung cancer
5.81
5.18
7.74
11.4
7.28
3.38
3.59
2.83
1.84
6.45
3.20
3.77
2.31
2.94
3.30
10.9
3.14
2.05
5.69
3.18
4.13
7.65
7.59
2.35
0.660
4.75
5.24
1.01
17.0
3.77
4.37
2.18
11.4
8.20
7.49
2.79
2.79
4.00
5.56
13.5
8.92
nt
1.09
0.840
0.453
0.303
0.343
1.25
0.430
0.586
0.367
0.499
0.541
2.31
0.350
0.445
0.586
0.245
0.501
3.75
1.60
0.429
nt
1.40
2.51
0.383
14.3
0.632
0.917
0.353
1.84
Colon cancer
0.917
nt
0.663
1.04
0.527
0.739
0.705
1.83
0.625
0.819
2.00
2.07
0.887
3.27
1.21
0.482
0.231
nt
0.839
0.435
nt
0.782
1023
0.349
nt
4.1.5. General procedure for preparation of aminobenzamides 10b,c
A mixture of 0.01 mol of 2H-3,1-benzoxazine-2,4(1H)-diones
9b,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.
CNS cancer
Melanoma
1.22
0.663
4.21
4.1.6. General procedure for preparation of 2-{[(2E)-3-phenylprop-
2-enoyl]amino}benzamides 12aes
LOX IMVI
MALME-3M
M14
nt
To a cold (0e5 ꢁC) stirred suspension of aminobenzamides
10aec (0.016 mol) in pyridine (13 ml), 0.016 mol of the appropriate
3-phenylacryloyl chloride 11cek 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 ethanol.
0.698
0.234
0.678
nt
0.367
38.7
0.388
0.352
0.294
2.37
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786e0
15.1
2.52
3.29
1.30
2.77
2.74
3.89
2.24
2.30
2.32
3.61
4.75
0.263
1.71
3.96
5.51
3.57
3.15
2.16
1.74
2.48
2.04
3.19
3.36
1.17
Ovarian cancer
Renal cancer
5-Methyl-2-{[(2E)-3-phenylprop-2-enoyl]amino}benzamide
(12a): yield 85%; mp 228e230 ꢁC (dioxane); I.R. (KBr) cmꢃ1 3400-
3258 (NH, NH2), 1681, 1651 (2XCO); 1H NMR (DMSO)
d 2.31 (s, 3H,
2.92
nt
3.52
1.06
nt
0.744
1.43
2.70
3.70
0.648
3.02
0.635
3.15
4.02
56.0
CH3); 6.89 (d, 1H, J ¼ 15.6 Hz, olefinic CH); 7.32e8.49 (a set of
signals, 9H, aromatic protons and NH-H); 7.58 (d, 1H, J ¼ 15.6 Hz,
olefinic CH); 8.24 (s, 1H, NHeH, exchangeable); 11.80 (s, 1H, NH,
exchangeable). Anal. (C17H16N2O2) C,H,N
0.400
1.41
1.76
1.70
3.36
1.34
0.444
2.96
2.67
1.85
0.684
0.935
0.569
2.19
0.624
1.41
1.57
0.279
0.719
0.539
0.937
0.880
9.86
0.637
0.425
1.55
26.7
1.63
0.832
1.28
0.401
1.41
A498
ACHN
CAKI-1
RXF 393
SN12C
5-Chloro-2-{[(2E)-3-phenylprop-2-enoyl]amino}benzamides
(12b): yield 67%; mp 218e220 ꢁC (dioxane); I.R. (KBr) cmꢃ1 3384,
3218 (NH, NH2), 1682, 1659 (2XCO); 1H NMR (DMSO)
d 6.83 (d, 1H,
9.63
25.8
9.62
3.97
3.66
3.48
7.26
1.94
4.88
4.20
3.12
J ¼ 14.9 Hz, olefinic CH); 7.43e8.63 (a set of signals, 10H, aromatic
protons, olefinic CH and NHeH); 8.42 (s, 1H, NHeH, exchangeable);
11.82 (s, 1H, NH, exchangeable). Anal. (C16H13ClN2O2) C,H,N
5-Bromo-2-{[(2E)-3-phenylprop-2-enoyl]amino}benzamides
(12c): yield 98%; mp 229e231 ꢁC (dioxane); I.R. (KBr) cmꢃ1 3388,
TK-10
UO-31
3.53
Prostate cancer PC-3
DU-145
MCF7
0.628
0.477
0.450
0.596
0.375
9.47
Breast cancer
MDA-MB-231
HS 578T
BT-549
T-47D
MDA-MB-468
3217 (NH, NH2), 1682, 1659 (2XCO); 1H NMR (DMSO)
d 6.82 (d, 1H,
0.362
0.816
19.9
J ¼ 14.9 Hz, olefinic CH); 7.43e8.58 (a set of signals, 10H, aromatic
protons, olefinic CH and NHeH); 8.44 (s, 1H, NHeH, exchangeable);
11.84 (s, 1H, NH, exchangeable). Anal. (C16H13BrN2O2) C,H,N
5-Chloro-2-{[(2E)-3-(2-chlorophenyl)prop-2-enoyl]amino}benza-
mides (12d): yield 36%; mp 268e270 ꢁC (dioxane); I.R. (KBr) cmꢃ1
0.857
nt
0.377
nt ¼ not tested.
GI50
¼
Growth inhibition of 50%; [(TiꢃTz)/(CꢃTz)]
ꢂ
100
¼
50 where
3365, 3155 (NH, NH2), 1686, 1661 (2XCO); 1H NMR (DMSO)
d 6.90
Tz ¼ absorbance at t ¼ 0, Ti ¼ absorbance at t ¼ 48 h, C ¼ absorbance of control at
t ¼ 48 h.
(d, 1H, J ¼ 15.2 Hz olefinic CH); 7.38e8.63 (a set of signals,
9H, aromatic protons, olefinic CH and NHeH); 8.43 (s, 1H,
NHeH, exchangeable); 11.92 (s, 1H, NH, exchangeable). Anal.
(C16H12Cl2N2O2) C,H,N
4.1.2. General procedure for preparation of 5-methyl-2-
nitrobenzoyl chloride 6a and 3-phenylacryloyl chlorides 11cek,
11tev
Substituted benzoyl and acryloyl chlorides 6a, 11cek and 11tev
were obtained by refluxing for 5 h the appropriate acid derivatives
5a, 8cek and 8tev (0.01 mol) with thionyl chloride (7.25 ml) [16].
After evaporation under reduced pressure, the crude liquid residue
was used for subsequent reactions without purification.
5-Chloro-2-{[(2E)-3-(3-chlorophenyl)prop-2-enoyl]amino}benza-
mides (12e): yield 88%; mp 260e261 ꢁC (dioxane); I.R. (KBr) cmꢃ1
3351, 3157 (NH, NH2), 1681, 1662 (2XCO); 1H NMR (DMSO)
d 6.97 (d,
1H, J ¼ 16.6 Hz, olefinic CH); 7.45e8.63 (a set of signals, 9H,
aromatic protons, olefinic CH and NHeH); 8.42 (s, 1H, NHeH,
exchangeable); 11.79 (s, 1H, NH, exchangeable). Anal.
(C16H12Cl2N2O2) C,H,N

D. Raffa et al. / European Journal of Medicinal Chemistry 46 (2011) 2786e2796
2791
Table 3
Overview of the results of the in vitro antitumor screening for compounds 12aec and 17t,u.a
no. Studiede
pGI50b
pTGIc
pLC50d
Comp
no. giving positive
resultse
range
MG_MIDf
no. giving positive
resultse
range
MG_MIDf
no. giving positive
resultse
range
MG_MIDf
12a
12b
12c
17t
17u
58
58
52
59
57
58
58
52
59
57
6.18e4.59
6.58e4.82
6.64e5.47
7.27e4.67
6.78e4.41
5.36
5.65
5.99
6.05
5.97
30
29
23
44
32
5.53e4.00
6.06e4.00
6.18e4.00
6.41e4.00
6.17e4.00
4.31
4.53
4.58
4.71
4.62
2
7
10
17
8
4.44e4.00
5.19e4.00
5.02e4.00
4.94e4.00
4.90e4.00
4.01
4.06
4.07
4.10
4.05
a
Data obtained from the NCI’s in vitro disease-oriented human tumor cells screen.
pGI50 is the elog of the molar concentration that inhibits 50% net cell growth.
pTGI is the eLog of the molar concentration giving total growth inhibition.
pLC50 is the eLog of the molar concentration leading to 50% net cell death.
Refers to the number of cell lines.
b
c
d
e
f
MG_MID ¼ mean graph midpoint ¼ arithmetical mean value for all tested cancer cell lines. If the indicated effect was not attainable within the used concentration interval,
the highest tested concentration was used for the calculation.
5-Chloro-2-{[(2E)-3-(4-chlorophenyl)prop-2-enoyl]amino}benza-
mides (12f): yield 58%; mp 243e244 ꢁC (dioxane); I.R. (KBr) cmꢃ1
3356, 3283, 3177 (NH, NH2), 1675, 1661 (2XCO); 1H NMR (DMSO)
9H, aromatic protons, olefinic CH and NHeH). 8.43 (s, 1H, NHeH,
exchangeable); 11.82 (s, 1H, NH, exchangeable). Anal.
(C16H12Cl2N2O2) C,H,N
d
6.87 (d, 1H, J ¼ 14.9 Hz, olefinic CH); 7.47e8.62 (a set of signals,
2-{[(2E)-3-(2-bromophenyl)prop-2-enoyl]amino}benzamides
(12g): yield 66%; mp 261e262 ꢁC (dioxane); I.R. (KBr) cmꢃ1 3366,
3157 (NH, NH2), 1686, 1662 (2XCO); 1H NMR (DMSO)
d 6.87 (d, 1H,
J ¼ 14.8 Hz, olefinic CH); 7.35e8.63 (a set of signals, 9H, aromatic
protons, olefinic CH and NHeH); 8.43 (s, 1H, NHeH, exchangeable);
11.90 (s, 1H, NH, exchangeable). Anal. (C16H12BrClN2O2) C,H,N
2-{[(2E)-3-(3-bromophenyl)prop-2-enoyl]amino}benzamides
(12h): yield 91%; mp 251e252 ꢁC (dioxane); I.R. (KBr) cmꢃ1 3349,
3167 (NH, NH2), 1678, 1662 (2XCO); 1H NMR (DMSO)
d 6.96 (d, 1H,
J ¼ 15.1 Hz, olefinic CH); 7.34e8.64 (a set of signals, 9H, aromatic
protons, olefinic CH and NHeH); 8.43 (s, 1H, NHeH, exchangeable);
11.80 (s, 1H, NH, exchangeable). Anal. (C16H12BrClN2O2) C,H,N
5-Chloro-2-{[(2E)-3-(2-methylphenyl)prop-2-enoyl]amino}ben-ꢃ1
zamides (12i): yield 42%; mp 231e232 ꢁC (dioxane); I.R. (KBr) cm
3383, 3165 (NH, NH2), 1687, 1660 (2XCO); 1H NMR (DMSO)
d 2.34 (s,
3H, CH3); 6.66 (d, 1H, J ¼ 15.6 Hz, olefinic CH); 7.28e8.42 (a set of
signals, 9H, aromatic protons, olefinic CH and NHeH); 8.42 (s, 1H,
NHeH, exchangeable); 11.89 (s, 1H, NH, exchangeable). Anal.
(C17H15ClN2O2) C,H,N
5-Chloro-2-{[(2E)-3-(3-methylphenyl)prop-2-enoyl]amino}ben-ꢃ1
zamides (12j): yield 82%; mp 238e239 ꢁC (dioxane); I.R. (KBr) cm
3337, 3163 (NH, NH2), 1681, 1660 (2XCO); 1H NMR (DMSO)
d 2.34 (s,
3H, CH3); 6.80 (d, 1H, J ¼ 15.4 Hz, olefinic CH); 7.21e8.65 (a set of
signals, 9H, aromatic protons, olefinic CH and NHeH); 8.43 (s, 1H,
NHeH, exchangeable); 11.83 (s, 1H, NH, exchangeable). Anal.
(C17H15ClN2O2) C,H,N
Fig. 2. Effects of compound 17t on DNA content/cell following treatment of K562 cells
for 24 h. The cells were cultured without compound (control, a), with an antimitotic
drug used as internal standard (60 nM taxol (b)) and with 1 mM (c), 2 mM (d), or 4 mM
(e) 17t. Cell cycle distribution was analyzed by the standard propidium iodide proce-
dure as described in Materials and Methods. Sub-G0eG1 (A), G0eG1, S, and G2-M cells
are indicated in (a).
Fig. 3. Percentage of apoptosis induced by compound 17t in K562 cells. Cells were
cultured with different concentrations of 17t. Apoptosis was evaluated after 48 h of
treatment as described in the experimental section. Bars: ꢀSE.

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D. Raffa et al. / European Journal of Medicinal Chemistry 46 (2011) 2786e2796
Fig. 4. Morphology of K562 cells after 24 h exposure to 2
mM (B), 5 mM (C) and 10 mM (D) compound 17t. (A) Untreated K562 cells (control). Living cells in culture medium were
observed by using a phase contrast invertoscope (40ꢂ magnification).
5-Chloro-2-{[(2E)-3-(4-methylphenyl)prop-2-enoyl]amino}ben-ꢃ1
3379, 3212 (NH, NH2),1682,1660 (2XCO); 1H NMR (DMSO)
d 2.34 (s,
3H, CH3); 6.78 (d, 1H, J ¼ 15.2 Hz, olefinic CH); 7.23e8.62 (a set of
signals, 9H, aromatic protons, olefinic CH and NHeH); 8.43 (s, 1H,
NHeH, exchangeable); 11.77 (s, 1H, NH, exchangeable). Anal.
(C17H15ClN2O2) C,H,N
3366, 3153 (NH, NH2), 1685, 1661 (2XCO); 1H NMR (DMSO)
d 6.87
zamides (12k): yield 82%; mp 238e239 ꢁC (dioxane); I.R. (KBr) cm
(d, 1H, J ¼ 16.5 Hz, olefinic CH); 7.71e8.52 (a set of signals, 9H,
aromatic protons, olefinic CH and NHeH); 8.42 (s, 1H, NHeH,
exchangeable); 11.88 (s, 1H, NH, exchangeable). Anal.
(C16H12Br2N2O2) C,H,N
5-Bromo-2-{[(2E)-3-(3-bromophenyl)prop-2-enoyl]amino}benzꢃa-1
mides (12p): yield 81%; mp 258e259 ꢁC (dioxane); I.R. (KBr) cm
5-Bromo-2-{[(2E)-3-(2-chlorophenyl)prop-2-enoyl]amino}benza-
3348, 3156 (NH, NH2), 1678, 1661 (2XCO); 1H NMR (DMSO)
d 6.97
mides (12l): yield 73%; mp 250e251 ꢁC (dioxane); I.R. (KBr) cmꢃ1
(d, 1H, J ¼ 15.6 Hz, olefinic CH); 7.35e8.56 (a set of signals, 9H,
aromatic protons, olefinic CH and NHeH); 8.43 (s, 1H, NHeH,
exchangeable); 11.78 (s, 1H, NH, exchangeable). Anal.
(C16H12Br2N2O2) C,H,N
3366, 3153 (NH, NH2), 1685, 1661 (2XCO); 1H NMR (DMSO)
d 6.91
(d, 1H, J ¼ 15.1 Hz, olefinic CH); 7.42e8.02 (a set of signals, 9H,
aromatic protons, olefinic CH and NHeH); 8.42 (s, 1H, NHeH,
exchangeable); 11.90 (s, 1H, NH, exchangeable). Anal.
(C16H12BrClN2O2) C,H,N
5-Bromo-2-{[(2E)-3-(2-methylphenyl)prop-2-enoyl]amino}ben-ꢃ1
zamides (12q): yield 98%; mp 238e240 ꢁC (dioxane); I.R. (KBr) cm
5-Bromo-2-{[(2E)-3-(3-chlorophenyl)prop-2-enoyl]amino}benza-
3384, 3160 (NH, NH2), 1686, 1659 (2XCO); 1H NMR (DMSO)
d 2.32 (s,
mides (12m): yield 56%; mp 260e261 ꢁC (dioxane); I.R. (KBr) cmꢃ1
3H, CH3); 6.74 (d, 1H, J ¼ 15.4 Hz, olefinic CH); 7.25e8.56 (a set of
signals, 9H, aromatic protons, olefinic CH and NHeH); 8.42 (s, 1H,
NHeH, exchangeable); 11.89 (s, 1H, NH, exchangeable). Anal.
(C17H15BrN2O2) C,H,N
3352, 3154 (NH, NH2), 1680, 1662 (2XCO); 1H NMR (DMSO)
d 6.97
(d, 1H, J ¼ 16.0 Hz, olefinic CH); 7.45e8.58 (a set of signals,
9H, aromatic protons, olefinic CH and NHeH); 8.44 (s, 1H,
NHeH, exchangeable); 11.81 (s, 1H, NH, exchangeable). Anal.
(C16H12BrClN2O2) C,H,N
5-Bromo-2-{[(2E)-3-(4-chlorophenyl)prop-2-enoyl]amino}benza-
mides (12n): yield 57%; mp 255e256 ꢁC (dioxane); I.R. (KBr) cmꢃ1
3357, 3285, 3178 (NH, NH2), 1673, 1660 (2XCO); 1H NMR (DMSO)
5-Bromo-2-{[(2E)-3-(3-methylphenyl)prop-2-enoyl]amino}ben-ꢃ1
zamides (12r): yield 71%; mp 268e270 ꢁC (dioxane); I.R. (KBr) cm
3338, 3163 (NH, NH2), 1681, 1661 (2XCO); 1H NMR (DMSO)
d 2.34 (s,
3H, CH3); 6.80 (d, 1H, J ¼ 16.5 Hz, olefinic CH); 7.24e8.63 (a set of
signals, 9H, aromatic protons, olefinic CH and NHeH); 8.42 (s, 1H,
NHeH, exchangeable); 11.80 (s, 1H, NH, exchangeable). Anal.
(C17H15BrN2O2) C,H,N
d
6.87 (d, 1H, J ¼ 14.7 Hz, olefinic CH); 7.47e8.55 (a set of sig-
nals, 9H, aromatic protons, olefinic CH and NHeH); 8.42 (s, 1H,
NHeH, exchangeable); 11.79 (s, 1H, NH, exchangeable). Anal.
(C16H12BrClN2O2) C,H,N
5-Bromo-2-{[(2E)-3-(4-methylphenyl)prop-2-enoyl]amino}ben-ꢃ1
zamides (12s): yield 88%; mp 236e237 ꢁC (dioxane); I.R. (KBr) cm
5-Bromo-2-{[(2E)-3-(2-bromophenyl)prop-2-enoyl]amino}benzꢃa-1
3353, 3163 (NH, NH2), 1678,1660 (2XCO); 1H NMR (DMSO)
d
2.33 (s,
mides (12o): yield 81%; mp 258e259 ꢁC (dioxane); I.R. (KBr) cm
3H, CH3); 6.75 (d, 1H, J ¼ 14.8 Hz, olefinic CH); 7.22e8.56 (a set of

D. Raffa et al. / European Journal of Medicinal Chemistry 46 (2011) 2786e2796
2793
Fig. 5. Panels A, C, E, and G, PtK2 cells stained for tubulin with an antibody to
-actin conjugated to FTIC. A and B, untreated cells. C and D, cells treated with 10
cells treated with 1 M latrunculin A (a potent inhibitor of actin assembly).
b
-tubulin conjugated to Cy3. Panels B, D, F, and H, PtK2 cells stained for actin with an antibody to
b
m
M 17t. E and F, cells treated with 1 M combretastatin A-4 (a potent antitubulin drug). G and H,
m
m
signals, 9H, aromatic protons, olefinic CH and NHeH); 8.42 (s, 1H,
NHeH, exchangeable); 11.78 (s, 1H, NH, exchangeable). Anal.
(C17H15BrN2O2) C,H,N
15tev. The mixture was refluxed in 0.01 M aqueous Na3PO4 for
20 h. The solution was allowed to cool to room temperature and,
after filtration, was acidified with HCl (0.1 M) to pH 2 to give the
corresponding acids 15t [10] and 15u as the only products. Finally,
the precipitate was removed by filtration and washed with cold
chloroform to obtain pure 15t,u. In the case of cinnamoyl chloride
11v, treatment with 2-amino-5-iodobenzoic acid 13 (3 mmol) in
anhydrous pyridine (20 ml) directly gave pure (E)-5-iodo-2-(3-o-
tolylacrylamido)benzoic acid 15v.
4.1.7. General procedure for preparation of 2-cinnamamido-5-
iodobenzoic acids 15tev
To an ice cooled (0e5 ꢁC) stirred solution of 2-amino-5-iodo-
benzoic acid 13 (3 mmol) in anhydrous pyridine (20 ml) 2 mmol of
cinnamoyl chlorides 11tev was added. The solution was left stirring
overnight, then poured into cold water. The precipitate was
collected as a mixture of (E)-6-iodo-2-styryl-4H-benzo[d] [1,3]
oxazin-4-ones 14teu and 2-cinnamamido-5-iodobenzoic acids
(E)-2-(3-(2-chlorophenyl)acrylamido)-5-iodobenzoiꢃc1 acid (15u):
yield 80%; mp 250e252 ꢁC (ethanol); I.R. (KBr) cm 3448, 3117
(NH, OH), 1703, 1688 (2XCO); 1H NMR (DMSO)
d 6.94 (d, 1H,

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D. Raffa et al. / European Journal of Medicinal Chemistry 46 (2011) 2786e2796
5-iodo-2-{[(2E)-3-phenylprop-2-enoyl]amino}benzamide (17t):
yield 88%; mp 260e261 ꢁC (dioxane); I.R. (KBr) cmꢃ1 3393, 3215
(NH, NH2), 1680, 1656 (2XCO); 1H NMR (DMSO)
6.82 (d, 1H,
d
J ¼ 15.6 Hz, olefinic CH); 7.43e8.42 (a set of signals, 10H, aromatic
protons, olefinic CH and NHeH); 8.15 (s, 1H, NHeH, exchangeable);
11.80 (s, 1H, NH, exchangeable). Anal. (C16H13IN2O2) C,H,N
5-Iodo-2-{[(2E)-3-(2-chlorophenyl)prop-2-enoyl]amino}benzamides
(17u): yield 88%; mp 260e261 ꢁC (dioxane); I.R. (KBr) cmꢃ13381, 3155
(NH, NH2), 1681, 1660 (2XCO); 1H NMR (DMSO)
d 6.97 (d, 1H,
J ¼ 16.6 Hz, olefinic CH); 7.45e8.63 (a set of signals, 9H, aromatic
protons, olefinic CH and NHeH); 8.42 (s, 1H, NHeH, exchangeable);
11.79 (s, 1H, NH, exchangeable). Anal. (C16H12ClIN2O2) C,H,N
5-iodo-2-{[(2E)-3-(2-methylphenyl)prop-2-enoyl]amino}benza-ꢃ1
mide (17v): yield 88%; mp 260e261 ꢁC (dioxane); I.R. (KBr) cm
3368, 3294, 3192 (NH, NH2), 1673, 1658 (2XCO); 1H NMR (DMSO)
d
2.34 (s, 3H, CH3); 6.97 (d,1H, J ¼ 16.6 Hz, olefinic CH); 7.45e8.63 (a
set of signals, 9H, aromatic protons, olefinic CH and NHeH); 8.14 (s,
1H, NHeH, exchangeable); 11.77 (s, 1H, NH, exchangeable). Anal.
(C17H15IN2O2) C,H,N
Fig. 6. Comparison of the effects of 17t with those of colchicine as an inhibitor of the
polymerization of purified tubulin. Assembly was followed in Gilford model 250
recording spectrophotometers equipped with electronic temperature controllers.
Reaction mixtures were preincubated at 30 ꢁC without GTP and chilled on ice. GTP
(0.4 mM) was added to the samples, which were transferred to cuvettes held at 0 ꢁC.
Baselines were established, and the temperature was jumped to 30 ꢁC over about 60 s
and held there for 20 min. Maximal reaction rates and extent of assembly were
determined for each sample and compared to a control reaction mixture in the same
experiment. Each data point was obtained in triplicate, and standard deviations are
4.2. Biology
4.2.1. Antiproliferative activity
Compounds 12aes and 17tev were initially tested in vitro for
antiproliferative activity against the K562 (human chronic
myelogenous leukemia) cell line. These cell lines were grown at
37 ꢁC in a humidified atmosphere containing 5% CO2, in RPMI-1640
medium supplemented with 10% fetal bovine serum and antibiotics.
K562 cells were suspended at a density of 1 ꢂ 105 cells/ml in
growth medium, transferred to a 24-well plate (1 ml/well), cultured
with or without (in the case of control wells) a screening concen-
shown. Symbols as follows: o, maximum rates obtained with 17t; D, extents of reaction
obtained with 17t; V, maximum rates obtained with colchicine; >, extents of reaction
obtained with colchicine.
J ¼ 15.3 Hz, olefinic CH); 7.44e8.43 (a set of signals, 8H, aromatic
protons, olefinic CH); 11.37 (s, 1H, NHeH, exchangeable); 13.98 (s,
1H, OH, exchangeable). Anal. (C16H11ClINO3) C,H,N
(E)-5-iodo-2-(3-o-tolylacrylamido)benzoic acid (15v): yield 83%;
tration of 10 m
M compounds and incubated at 37 ꢁC for 48 h.
mp 239e240 ꢁC (ethanol); I.R. (KBr) cmꢃ1 3353, 3163 (NH, NH2),
Numbers of viable cells were determined by counting in a hema-
tocytometer after dye exclusion with trypan blue [17]. The anti-
proliferative effects of the compounds were estimated in terms of %
growth inhibition, comparing cell viability of treated and untreated
cells. We determined IC50 values (test agent concentration at
which the cell proliferation was inhibited by 50% as compared with
the untreated control) for compounds that exhibited the best
activity at the screening concentration.
1678, 1660 (2XCO); 1H NMR (DMSO)
d 2.37 (s, 3H, CH3); 6.82 (d, 1H,
J ¼ 15.3 Hz, olefinic CH); 7.27e8.48 (a set of signals, 8H, aromatic
protons, olefinic CH); 11.28 (s, 1H, NHeH, exchangeable); 13.90 (s,
1H, OH, exchangeable). Anal. (C17H14INO3) C,H,N
4.1.8. General procedure for preparation of 6-iodo-1H-benzo[d]
[1,3]oxazine-2,4-diones 16tev
A
mixture of 2-cinnamamido-5-iodobenzoic acids 15tev
(4.6 mmol) and ethylchloroformate (25 ml) was refluxed for 15 min.
After this time, acetyl chloride (1.5 ml) was added, and reflux
continued for an additional 30 min. On cooling, the product
precipitated, was removed by filtration, and was washed with
chloroform to give pure 16tev. Compound 16v easily decomposed
and was used as is without purification.
4.2.2. Tumor cell line screening
The in vitro antiproliferative activity values were obtained by
the Developmental Therapeutics Program, National Cancer Insti-
tute (USA) [18]
The human tumor cell lines of the cancer screening panel are
grown in RPMI 1640 medium containing 5% fetal bovine serum and
1-Cinnamoyl-6-iodo-1H-benzo[d] [1,3] oxazine-2,4-dione (16t):
2 mM
L
-glutamine. For a typical screening experiment, cells are
yield 83%; mp 201e204 ꢁC (CHCl3); I.R. (KBr) cmꢃ1 1760, 1670
inoculated into 96 well microtiter plates in 100
ml at plating
(3XCO); 1H NMR (DMSO)
7.39e8.42 (a set of signals, 9H, aromatic protons and olefinic C).
Anal. (C17H10INO4) C,H,N
d
6.96 (d, 1H, J ¼ 16 Hz, olefinic CH);
densities ranging from 5000 to 40,000 cells/well, depending on the
doubling time of the individual cell lines. After cell inoculation, the
microtiter plates are incubated at 37 ꢁC, 5% CO2, 95% air and 100%
relative humidity for 24 h prior to addition of experimental drugs.
After 24 h, two plates of each cell line are fixed in situ with TCA, to
represent a measurement of the cell population for each cell line at
the time of drug addition (Tz). Experimental drugs are solubilized
in dimethyl sulfoxide at 400-fold the desired final maximum test
concentration and stored frozen prior to use. At the time of drug
addition, an aliquot of frozen concentrate is thawed and diluted to
twice the desired final maximum test concentration with complete
(E)-1-(3-(2-chlorophenyl)acryloyl)-6-iodo-1H-benzo[d] [1,3]oxa-
zine-2,4-dione (16u): yield 83%; mp 201e204 ꢁC (CHCl3); I.R. (KBr)
cmꢃ1 1760, 1660 (3XCO); 1H NMR (DMSO)
olefinic CH); 7.38e8.33 (a set of signals, 8H, aromatic protons and
olefinic C). Anal. (C17H9IClNO4) C,H,N
d
7.01 (d, 1H, J ¼ 16 Hz,
4.1.9. General procedure for preparation of 2-cinnamamido-5-iodo-
benzamides 17t-v
A
solution of 6-iodo-1H-benzo[d] [1,3]oxazine-2,4-diones
medium containing 50
or ½ log serial dilutions are made to provide a total of five drug
concentrations plus control. Aliquots of 100 l of these different
drug dilutions are added to the appropriate microtiter wells already
containing 100 l of medium, resulting in the required final drug
mg/ml gentamicin. Additional four, 10-fold
16tev (2.3 mmol), 25% aqueous ammonia solution (28 ml) and
dioxane (5 ml) was left under reflux for 3 h. After this time, the
solution was kept in a freezer to allow the product to precipitate. It
was removed by filtration and crystallized from dioxane.
m
m

D. Raffa et al. / European Journal of Medicinal Chemistry 46 (2011) 2786e2796
2795
concentrations. Following drug addition, the plates are incubated
for an additional 48 h at 37 ꢁC, 5% CO2, 95% air, and 100% relative
humidity. For adherent cells, the assay is terminated by the addition
percentage of apoptotic cells was determined after counting at least
300 cells.
of cold TCA. Cells are fixed in situ by the gentle addition of 50
m
l of
4.2.5. Determination of apoptosis by annexin-V
cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for
60 min at 4 ꢁC. The supernatant is discarded, and the plates are
washed five times with tap water and air dried. Sulforhodamine B
Cells (1 ꢂ 106) were washed with phosphate-buffered saline
(PBS) and centrifuged at 200 ꢂ g for 5 min. Cell pellets were sus-
pended in 100 ml of staining solution containing FITC-conjugated
(SRB) solution (100
m
l) at 0.4% (w/v) in 1% acetic acid is added to
annexin-V and propidium iodide (Annexine-V-Fluos Staining Kit,
Roche Molecular Biochemicals, Mannheim, Germany) and incu-
bated for 15 min at 20 ꢁC. Annexin-V positive cells were evaluated
by flow cytometry (BectoneDickinson.
each well, and plates are incubated for 10 min at room temperature.
After staining, unbound dye is removed by washing five times with
1% acetic acid, and the plates are air dried. Bound stain is subse-
quently solubilized with 10 mM trizma base, and the absorbance is
read on an automated plate reader at a wavelength of 515 nm. For
suspension cells, the methodology is the same except that the assay
is terminated by fixing settled cells at the bottom of the wells by
4.2.6. Flow cytometric analysis of cell cycle and apoptosis
Cells were washed once in ice-cold PBS and resuspended at
1 ꢂ 106 ml in a hypotonic fluorochrome solution containing pro-
gently adding 50
ml of 80% TCA (final concentration, 16% TCA). Using
pidium iodide (Sigma) 50 mg/ml in 0.1% sodium citrate plus 0.03%
the seven absorbance measurements [time zero (Tz), control
growth (C), and test growth in the presence of drug at the five
concentration levels (Ti)], the percentage growth is calculated at
each of the drug concentration levels. Percentage growth inhibition
is calculated as:
(v/v) nonidet P-40 (Sigma). After 30 min of incubation, the fluo-
rescence of each sample was analyzed as a single-parameter
frequency histogram using
a FACScan flow cytometer (Bec-
toneDickinson, San Jose, CA). Distribution of cells in the cell cycle
was determined using the ModFit LT program (Verity Software
House, Inc.). Apoptosis was determined by evaluating the
percentage of hypodiploid nuclei accumulated in the sub-G0eG1
peak after labeling with propidium iodide.
½ðTi ꢃ TzÞ=ðC ꢃ TzÞꢄ ꢂ 100 for concentrations for which Ti ꢅ Tz
½ðTi ꢃ TzÞ=Tzꢄ ꢂ 100 for concentrations for which Ti < Tz
4.2.7. Immunofluorescence
Three dose response parameters are calculated for each
experimental agent. Growth inhibition of 50% (GI50) is calcu-
PtK2 cells (Potorus tridactylis kidney epithelial cells) were
obtained from the American Type Tissue Collection and grown as
recommended by the supplier. The technique was described in
detail previously [19,20]. Cells were treated with compounds for
24 h at 37 ꢁC prior to fixation and staining with antibodies (Cy3
lated from [(TiꢃTz)/(CꢃTz)]
ꢂ
100
¼
50, which is the
drug concentration resulting in a 50% reduction in the net
protein increase (as measured by SRB staining) in control cells
during the drug incubation. The drug concentration resulting in
total growth inhibition (TGI) is calculated from Ti ¼ Tz. The
LC50 (concentration of drug resulting in a 50% reduction in the
measured protein at the end of the drug treatment as compared
to that at the beginning) indicating a net loss of cells follo-
wing treatment is calculated from [(TiꢃTz)/Tz] ꢂ 100 ¼ ꢃ50.
Values are calculated for each of these three parameters if the
level of activity is reached; however, if the effect is not reached
or is exceeded, the value for that parameter is expressed as
greater or less than the maximum or minimum concentration
tested.
conjugate of anti-b-tubulin clone TUB2.1 and FTIC conjugate of
anti- -actin clone Ac-15 monoclonal antibodies from Sigma). Cells
b
were examined with a Nikon Eclipse E800 microscope equipped
with epifluorescence and appropriate filters, and images were
collected with a Spot digital camera.
4.2.8. Tubulin assembly
The procedure with purified bovine brain tubulin was described
in detail previously [21].
Acknowledgment
4.2.3. Cytotoxicity assays
Financial support from MIUR (40% funding) is gratefully
acknowledged. The authors wish to thank the Developmental
Therapeutics Program of the National Cancer Institute of the United
States of America for performing the antiproliferative screening of
compounds.
To evaluate the number of live and dead neoplastic cells, the
cells were stained with trypan blue and counted on a hemocy-
tometer. To determine the growth inhibitory activity of the drugs
tested, 2 ꢂ 105 cells were plated into 25 mm wells (Costar, Cam-
bridge, UK) in 1 ml of complete medium and treated with different
concentrations of each drug. After 48 h of incubation, the number of
viable cells was determined and expressed as percent of control
proliferation.
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