Synthesis, antiproliferative activity and possible mechanism of action of novel 2-acetamidobenzamides bearing the 2-phenoxy functionality

Article abstract of DOI:10.1016/j.bmc.2015.08.027

Several new 2-(2-phenoxyacetamido)benzamides 17a-v, 21 and 22 were synthesized by stirring in pyridine the acid chlorides 16a-e and the appropriate5-R-4-R₁-2-aminobenzamide 15a-e and initially evaluated in vitro for antiproliferative activity against the K562 (human chronic myelogenous leukemia) cell line. Some of 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). The most active compounds caused an arrest of K562 cells in the G0-G1 phase of cell cycle and induction of apoptosis, which was mediated by caspase activation.

Full text of DOI:10.1016/j.bmc.2015.08.027

Bioorganic & Medicinal Chemistry 23 (2015) 6305–6316
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, antiproliferative activity and possible mechanism of
action of novel 2-acetamidobenzamides bearing the 2-phenoxy
functionality
a,
a
a
a
Demetrio Raffa a, , Benedetta Maggio , Fabiana Plescia , Stella Cascioferro , Maria Valeria Raimondi ,
Gabriella Cancemi a, Antonella D’Anneo b, Marianna Lauricella c, Maria Grazia Cusimano a, Ruoli Bai d,
Ernest Hamel d, Giuseppe Daidone a
⇑
⇑
a Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Via Archirafi, 32, 90123 Palermo, Italy
b Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, Laboratory of Biochemistry, University of Palermo, 90123 Palermo, Italy
c Department of Experimental Biomedicine and Clinical Neurosciences, Laboratory of Biochemistry, University of Palermo, 90123 Palermo, Italy
d Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Frederick National Laboratory
for Cancer Research, 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:
Received 1 July 2015
Revised 3 August 2015
Accepted 25 August 2015
Available online 28 August 2015
Several new 2-(2-phenoxyacetamido)benzamides 17a–v, 21 and 22 were synthesized by stirring in
pyridine the acid chlorides 16a–e and the appropriate5-R-4-R1-2-aminobenzamide 15a–e and initially
evaluated in vitro for antiproliferative activity against the K562 (human chronic myelogenous leukemia)
cell line. Some of 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). The most active
compounds caused an arrest of K562 cells in the G0–G1 phase of cell cycle and induction of apoptosis,
which was mediated by caspase activation.
Keywords:
2-(2-Phenoxyacetamido)benzamides
Antiproliferative activity
G0/G1 arrest
Ó 2015 Elsevier Ltd. All rights reserved.
Apoptosis
Pro-caspase 3
1. Introduction
Thus, following our studies in the new anticancer agents,14–17
and on the basis of our previous work on acetamidobenzamides,7,8
Acetamidobenzamides represent a class of biologically active
substances of great importance in medicinal chemistry. Despite
their wide range of biological activities,1–6 a review of the litera-
ture revealed that no anticancer activity had been described for
these compounds. This omission led us to explore the potential
of this class of compounds as anticancer agents by synthesizing and
screening for antileukemic activity a series of novel 2-cinnamamido,
2-(3-phenylpropiolamido) and 2-(3-phenylpropanamido)benza-
mides.7,8Among the synthesized compounds, 1–5 showed the best
antiproliferative activity (Fig. 1).
as well as of data reported for some active compounds containing
the phenoxyacetamido scaffold,9–13 we decided to investigate the
influence of the cinnamamido and 3-phenylpropiolamido scaffolds
when substituted for the phenoxyacetamido scaffold. Moreover,
the displacement of the 2-phenoxyacetamido moiety from the
ortho to the para and meta positions was investigated.
A series of novel 2-(2-phenoxyacetamido)benzamide derivatives,
including the 3-(2-phenoxyacetamido) and the 4-(2-phenoxyac-
etamido)benzamide derivatives, were therefore synthesized and
screened for their antileukemic activity.
Several examples of active compounds containing the
phenoxyacetamido scaffold in their structure include the kinase
2. Results and discussion
2.1. Chemistry
inhibitor 6,9 the carboxypeptidase R39 inhibitor 7,10 the HIF-
a
prolyl hydroxylase inhibitor 8,11 the FLT3 inhibitor 912 and the
HIV-protease inhibitor 1013 (Fig. 2).
The synthesis of 2-(2-phenoxyacetamido)benzamides 17a,18
17b, 17c,19 17e–f, 17g,19 and 17h–v was achieved as described in
Scheme 1. A mixture of the appropriate acid chlorides 16a–e and
the appropriate 5-R-4-R1-2-aminobenzamides 15a–f in pyridine
⇑
Corresponding authors. Tel.: +39 91 23891917 (D.R.), +39 91 23891949 (B.M.).
E-mail addresses: demetrio.raffa@unipa.it (D. Raffa), benedetta.maggio@unipa.it
(B. Maggio).
http://dx.doi.org/10.1016/j.bmc.2015.08.027
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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D. Raffa et al. / Bioorg. Med. Chem. 23 (2015) 6305–6316
Figure 1. The most active compounds among the previously synthesized benzamides.7,8
In particular, the 5-iodo-2-aminobenzamide (15c) was
obtained, as reported in the literature,21 by treatment of anthranil-
amide (15a) with iodine in an aqueous solution of sodium
bicarbonate.
The 5-methylanthranilamide (15d), the 5-methoxyanthranil-
amide (15e) and the 4,5-dimethoxyanthranilamide (15f) were
obtained by reduction of 2-nitro-5-methyl benzamides 13a–c as
shown in Scheme 1. Compounds 13a–c were obtained by reaction
of the acids 11a–c with thionyl chloride to afford 12a–c, followed
by treatment of 12a–c with aqueous ammonia.
The anthranilamide derivative 15b was obtained by stirring the
6-chloro-1H-benzo[d][1,3]oxazine-2,4-dione 14a in a 25% aqueous
ammonia solution (Scheme 1).22
The 4-(2-phenoxyacetamido)benzamide (21) and the 3-(2-
phenoxyacetamido)benzamide (22) were obtained, following the
same synthetic route reported for compounds 17a–v, first by trans-
formation of the commercially available nitrobenzoyl chlorides
18a,b to nitrobenzamides 19a,b,7,8 then by reduction with palla-
dium on activated charcoal in a Parr apparatus (Scheme 2).
The structures of the new compounds were determined by ana-
lytical and spectroscopic measurements. In particular, 17a–v, 21
and 22 showed 1H NMR signals attributable to aromatic protons
in the range 6.24–8.67 d and a singlet in the range 11.99–12.92
d, exchangeable with D2O, for the amidic NH. The signals of the
NH2 benzamido protons, exchangeable with D2O, showed a differ-
ent behavior depending on the solvent. As shown in Figure 3a for
17b, taken as an example, the spectrum recorded in chloroform
showed
a broad signal attributable to the NH2 moiety at
6.07 ppm. In DMSO-d6 as solvent, the presence of an intramolecu-
lar hydrogen bond rendered the benzamido NH2 protons diastereo-
topic. Ha appeared as a singlet at 7.39 d whereas Hb was found at a
lower field as a singlet at 8.25 d (Fig. 3b). The presence of an
intramolecular hydrogen bond was confirmed by performing the
1H NMR spectrum of 17b at 90 °C. Figure 3c showed that, at
90 °C, the two singlets collapsed into a single broad signal at 7.64
d. Furthermore, the 1H NMR spectrum of 17b in a diluted solution
of DMSO-d6 was similar to that in a concentrated solution, proving
that no intermolecular interaction occurred.
Figure 2. Some examples of active compounds from the literature bearing the
phenoxyacetamido scaffold.
was stirred magnetically in an ice bath. Crude benzoylchlorides
16a–e, not commercially available, were obtained starting from
the corresponding acid by treatment with thionyl chloride.20
The anthranilamide 15a is commercially available, while the
5-R-4-R1-2-aminobenzamides 15b–f were obtained according to
Scheme 1.
Moreover, the 1H NMR spectra of methyl and methoxy-substi-
tuted derivatives showed signals in the range 2.24–2.39 d and
2.24–3.90 d, respectively, arising from the methyl and methoxyl

D. Raffa et al. / Bioorg. Med. Chem. 23 (2015) 6305–6316
6307
Scheme 1. Synthetic pathway for the formation of 2-[(phenoxyacetyl)amino]benzamides 17a–v. Reagents and conditions: (i) SOCl2, (ii) aqueous NH3, (iii) I2, aqueous
NaHCO3, (iv) SnCl2, HCl.
Scheme 2. Synthetic pathway for the formation of benzamides 20a,b, 4-(2-phenoxyacetamido)benzamide (21) and 3-(2-phenoxyacetamido)benzamide (22).
groups. Finally, a signal in the range 4.61–4.91 d was found, and
this was due to the methylene group of the phenoxy moiety.
antileukemic activity against the K562 (human chronic myeloge-
nous leukemia) cell line; colchicine, whose antileukemic activity
is well known, was used as a reference compound.
2.2. Biological activity
The percent growth inhibition at a screening concentration of
10 lM and the IC50 values for compounds that exhibited at least
All synthesized 2-(2-phenoxyacetamido)benzamido derivatives
50% growth inhibition in the 10
lM screening assay are shown in
17a–v, 21 and 22 were initially tested in vitro for their
Table 1.

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D. Raffa et al. / Bioorg. Med. Chem. 23 (2015) 6305–6316
Figure 3. 1H NMR spectrum of 17b in (a) chloroform, (b) DMSO-d6 and (c) DMSO-d6 performed at 90 °C.
Table 1
seems that the simultaneous introduction of a substituent into
both the benzamido and phenoxyacetamido moieties is unfavour-
able for inhibition of K562 cell growth relative to the unsubstituted
2-cinnamamidobenzamide 4 (Fig. 1), considering that antiprolifer-
ative activity was only maintained in the ortho-phenoxyacetamido
derivatives (compounds 17g, 17l, 17n, 17s and 17v, Table 1).
To obtain more insights into the cytotoxic potential of the test
compounds for normal human cells, compounds 17a,17f,17g,
17j,17k,17l,17m,17r and 17u were also tested in vitro for their
cytotoxicity against HuDe cells, a primary cell culture from human
epidermis (Table 2).
The data in Table 2 show that the K562-active compounds were
significantly less cytotoxic against the HuDe cell line (HuDe/K562
index ranging from 21 to 1987), indicating the possibility that
these compounds may have limited toxicity in vivo at doses that
would limit tumor growth. This contrasts with a very low HuDe/
K562 index (0.85) obtained for colchicine.
Compounds 17f, 17j, 17l, 17r and 17u were selected by the NCI
to evaluate in their in vitro antiproliferative assay at a single high
dose (10À5 M) against 60 human cell lines derived from nine
clinically isolated human cancer types (leukemia, lung, colon, mel-
anoma, renal, ovarian, brain, breast and prostate) according to the
NCI standard protocol.23
The antitumor activity is given by three parameters for each cell
line; GI50 (compound’s molar concentration inhibiting 50% net cell
growth), TGI (compound’s molar concentration for total inhibition
of net cell growth), and LC50 (compound’s molar concentration
inducing 50% net cell death).
Percent growth inhibition of compounds 17a–v at 10
compounds that exhibited at least 50% growth inhibition in the 10
assay against the K562 cell line
l
M and IC50 values (
l
M) for
lM screening
Compd
10
l
M
IC50 (lM)
17a
17b
17c
17d
17e
17f
17g
17h
17i
17j
17k
17l
17m
17n
17o
17p
17q
17r
57.4 3.4
19.7 2.0
ns
ns
ns
8.9 0.9
87.0 4.3
75.9 1.9
24.1 0.2
18.8 1.0
73.0 0.7
49.8 3.5
74.7 1.2
72.0 1.1
45.6 0.4
30.3 1.8
35.4 0.9
30.8 1.9
85.3 3.2
40.1 08
48.1 0.7
72.8 3.1
17.7 0.1
23.7 0.8
ns
1.3 0.1
1.61 0.7
0.16 0.02
6.9 0.3
4.7 0.6
1.1 0.17
17s
17t
17u
17v
21
0.58 0.03
22
Colchicine
63.6 1.7
0.02 0.003
Values are the means of at least three independent determinations. ns = not
significant (% inhibition <10).
Among these, 17f, 17j, 17r and 17u, showing the best mean
graph midpoints (MG_MID) (Table 3), and therefore they were
progressed to the 5-dose assay against the NCI full panel of 60 cell
lines.23
These data showed that the best antiproliferative agent was
compound 17j, with an IC50 value of 0.16 lM.
As far as structure–activity relationships are concerned, it
seems that on average the substitution of the cinnamamido moiety
with the phenoxyacetamido moiety had little effect on inhibition
of K562 cell growth (Table 1). In particular, the average inhibitory
Table 2
Comparison of IC50 values (lM) in HuDe and K562 cells
activity at 10 lM on K562 cell growth, showed by all the previ-
Compd
HuDe
K562
HuDe/K562
ously tested compounds,7,8 was about 46%7 and 44%,8 respectively,
while with the phenoxyacetamido moiety, average inhibition was
49%. Moreover, shifting the phenoxyacetamido moiety from the
ortho (compound 17a) to the para or meta position (compounds
21 and 22, Table 1) resulted in reduced activity.
17a
17f
17g
17j
17k
17l
17m
17r
17u
523.0 7.8
807.8 11.7
27.3 3.9
318.0 15.7
355.7 12.1
33.8 2.3
8.9 .09
1.3 0.1
1.61 0.7
0.16 0.02
10 1.5
6.9 0.3
4.7 0.6
1.1 0.17
0.58 0.03
0.02 0.003
58.76
621.38
16.96
1987.5
35.57
4.9
21.13
856.28
280.52
0.85
As for the previously reported benzamide analogs,7,8 our data
(Table 1) showed positive effects following substitution at the 5
position of the benzamido moiety (17f, 17j, 17m, 17r and 17u,
Table 1), especially when the substituent(s) was iodine or
4,5-dimethoxy (compounds 17j and 17u, Table 1). Moreover, it
99.3 1.4
941.9 4.7
162.7 1.9
0.017 0.001
Colchicine
Values are the means of at least three independent determinations.

D. Raffa et al. / Bioorg. Med. Chem. 23 (2015) 6305–6316
6309
Table 3
Table 4
Overview of the results of the in vitro single high dose (10À5 M) antitumor screening
Results of multi-dose growth inhibition assay (GI50
and 17u
,
lM) for compounds 17f, 17j, 17r
for compounds 17f, 17j, 17r and 17ua
Compd
Growth percentb
MG_MIDc
Cell line
17f
17j
17r
17u
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
3.07
3.29
0.382 0.487 0.562
0.339 0.340 0.293
17f
17j
17l
17r
17u
46.40
24.31
87.28
25.81
27.38
0.854 0.161 0.245 0.310
8.14
4.33
1.10
0.553 1.17
2.00
2.91
2.27
0.777 0.221 0.392 0.515
a
Non-small cell lung cancer A549/ATCC
7.06
5.88
5.70
>100
5.90
7.09
3.00
3.78
0.732 0.908 0.917
0.824 1.01 1.75
0.414 17.7
Data obtained from the NCI’s in vitro disease-oriented human tumor cells
screen.
HOP-62
HOP-92
b
1.50
4.94
Growth percent is the percent of growth inhibition at the single high dose of
10À5 M.
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
21.5
0.657 1.38
0.705 2.10
42.8
1.32
5.77
c
MG_MID = mean graph midpoint = arithmetical mean value for all tested cancer
cell lines.
0.425 0.484 0.529
0.630 1.19 0.410
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
2.51
12.1
2.72
1.57
1.74
2.13
2.58
0.353 0.487 0.453
2.95 2.34 3.33
The GI50 values for the test compounds were reported in Table 4.
0.517 0.518 0.620
0.253 0.432 0.804
0.349 0.388 0.437
0.308 0.388 0.443
0.454 0.448 0.646
All the compounds 17f, 17j, 17r and 17u tested by NCI were found
to possess low cytotoxicity. On the other hand, they turn out to be
inhibitor of cell growth with GI50 values ranging from 0.101 to
KM12
SW-620
42.8 lM. Among these, compounds 17j, 17r and 17u were particu-
larly active, against the majority of type of tumor cell lines inves-
tigates, in the sub micromolar concentration. The best activity
was showed by compound 17j that caused 50% growth inhibition
at submicromolar concentrations against 44 of 56 cellular lines.
Compounds 17r and 17u resulted less active with respect to 17j
causing 50% growth inhibition at submicromolar concentrations
against 30 of 56 and 26 of 58 cellular lines, respectively. On the
contrary, compound 17f resulted less active with respect to 17j,
17r and 17u being a micro molar inhibitor with GI50 values at sub-
micromolar concentrations only against 3 of 57 cellular lines.
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 5), the most active compound of the series was derivative
17j, at the GI50, TGI and LC50 levels, followed by 17r.
In our previous studies on the cinnamamido and 3-phenylpro-
piolamido benzamides (compounds 1–5, Fig. 1),7,8 the major
evidence that suggested these agents interacted with tubulin was
derived from cellular studies, such as an increased proportion of
cells in the G2/M phase of the cell cycle, increased proportion
of apoptotic cells, disappearance of cellular microtubules and
COMPARE analysis of NCI cytotoxicity data.
Therefore we first wondered whether the same mechanism of
action occurred with the phenoxyacetamido scaffold and we exam-
ined the most active compounds in the series (17f, 17j, 17r and
17u) for potential effects on the assembly of purified tubulin and
on the inhibition of colchicine binding to tubulin, but negligible
effects were observed (data not presented). Our initial studies with
the K562 cells did yield evidence for mitotic arrest following treat-
ment with the four compounds. However further studies using
compounds 17j and 17u, which were the most effective antiprolif-
erative agents, failed to validate the initial observations.
CNS cancer
Melanoma
SF-268
SF-295
SF539
SNB-19
SNB75
U251
9.89
1.96
1.80
11.7
1.81
2.34
0.934 2.42
0.223 0.650 0.930
0.249 0.441 0.563
0.913 3.20
0.215 0.245 0.547
0.396 0.664 0.777
6.77
3.31
LOX IMVI
M14
8.33
2.21
0.822 0.869 1.81
0.389 0.514 4.00
MDA-MB-435 0.356 0.101 0.121 0.614
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
5.08
6.25
7.45
>100
5.42
0.463 0.611 0.203
1.79 0.502 24.2
0.533 1.68
41.7 >100
0.701 1.05
0.766
>100
2.52
Ovarian cancer
IGROV1
5.60
2.30
>100
3.86
7.06
1.43
8.15
1.37 4.75
0.321 0.375 0.472
4.07
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
nt
1.68
>100
5.40
>100
15.0
3.01
0.904 3.13
0.344 0.418 0.487
3.17
2.99
3.58
Renal cancer
786-0
A498
9.54
1.41
10.7
4.18
3.93
17.0
63.8
17.5
1.07
1.02
1.32
2.10
0.304 2.77
2.25 8.12
7.32
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
0.519 0.721 3.80
0.413 1.40
0.945 3.09
1.69
3.12
14.4
13.5
8.11
2.87
23.8
8.72
Prostate cancer
Breast cancer
PC-3
DU-145
3.22
6.01
0.428 0.464 2.23
0.444 1.87 0.914
MCF7
MDA-MB-231 9.01
HS 578T
BT-549
T-47D
1.81
0.230 0.497 0.661
1.28 2.03 2.39
0.630 0.592 0.771
4.65
nt
6.87
nt
nt
0.770
3.46
0.556 nt
MDA-MB-468 1.742 0.351 0.345 0.848
nt = not tested.
In order to elucidate the antiproliferative effect of 17j or 17u
compounds, K562 cells were treated with different doses of the
agents for 48 h. Clear morphological changes were observed in
cells exposed to both the compounds.
(Fig. 4e), while a consistent cytotoxic effect was already reached
at the lowest concentration examined of 17j (Fig. 4b).
In fact, under light microscopy, control cells appeared with a reg-
ular rounded shape (Fig. 4a), whereas after incubation with 17j or
To gain additional insight into the mechanism of action of the
compounds, a 10
experiments.
lM dose of 17j and 17u was used in subsequent
17u cells detached themselves from poly-
and showed a remarkable reduction in cell number and cell size.
The effect, which appeared with 10 dose of each
compound (Fig. 4b and d), was further enhanced with 25 M 17u
D lysine coated plates
After propidium iodide (PI) staining of DNA, flow cytometry
analyses were performed to investigate the distribution of cells
in the different phases of the cell cycle. These studies showed that
a
lM
l

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D. Raffa et al. / Bioorg. Med. Chem. 23 (2015) 6305–6316
Table 5
Overview of the results of the in vitro antitumor screening for compounds 17f,j,r,ua
b
d
Compd
No. Studiede
pGI50
pTGIc
pLC50
No. giving
Range
MG_MIDf
No. giving
Range
MG_MIDf
No. giving
Range
MG_MIDf
positive resultse
positive resultse
positive resultse
17f
17j
17r
17u
57
57
56
58
54
57
54
56
6.45–4.00
7.00–4.38
6.92–4.00
6.69–4.00
5.31
6.16
6.05
5.71
10
25
25
23
5.65–4.00
6.46–4.00
6.20–4.00
6.06–4.00
4.14
4.45
4.31
4.31
1
8
2
5
4.13–4.00
4.84–4.00
4.50–4.00
4.70–4.00
4.00
4.05
4.01
4.03
a
b
c
d
e
f
Data obtained from the NCI’s in vitro disease-oriented human tumor cell screen.
pGI50 is the Àlog of the molar concentration that inhibits 50% net cell growth.
pTGI is the Àlog of the molar concentration giving total growth inhibition.
pLC50 is the Àlog of the molar concentration leading to 50% net cell death.
Refers to the number of cell lines for which GI50, TGI or LC50, respectively, are 6100
l
M.
MG_MID = mean graph midpoint = 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.
Figure 4. Effects of 17j and 17u on cell morphology. Cells (8 Â 103/well) were treated with 10
l
M compound for 48 h, then morphology was observed under light microscopy
at 200Â magnification: (a) control, (b) cells treated with 10
l
M 17j, (c) cells treated with 25
l
M 17j, (d) cells treated with 10 M 17u, (e) cells treated with 25 M 17u.
l
l
both compounds caused an arrest of cells in the G0/G1 phase at
24 h. As shown in Figure 5, the proportion of K562 cells in the
G0/G1 phase increased from 54.9% in control cells (Fig. 5a) to
of typical morphological features, such as cell shrinkage and
detachment from the surrounding tissue, nuclear condensation,
fragmentation into membrane-bound apoptotic bodies which are
rapidly engulfed via phagocytosis by neighboring cells. Biochemi-
cal hallmarks of apoptosis include activation of proteases termed
caspases, cleavage of proteins and DNA and exposure of phos-
phatidylserine on the cell surface.24
78.5% in 10
lM 17j-treated cells (Fig. 5b, upper panel) and to
80% in 10 M 17u-treated cells (Fig. 5c, upper panel).
l
Prolonging the time of incubation with the compounds to 48 h a
decrease in the proportion of cells in the G0/G1 phase occurred,
while the percentage of cells in sub-G0/G1 region, characteristic
of apoptotic cells with fragmented DNA, increased, reaching values
Therefore K562 cells were treated with 10 lM of 17j or 17u for
48 h and examined for the characteristic morphological features of
apoptotic cell death under a fluorescence microscope. Using
Hoechst 33342 to stain the nuclei, it was observed (Fig. 6) that,
equal to 32.9% and 20.6% with 10
(Fig. 5b and c, lower panels).
lM 17j and 17u, respectively
In order to establish whether the reduction in cell number and
cell size as well as the observed DNA fragmentation were related to
the apoptotic cell death, additional analyses were performed.
Apoptosis is a programmed cell death that occurs in physiolog-
ical and pathological conditions.24 Defects in apoptotic pathways
are now to contribute to malignant transformation of cells, cancer
progression and resistance to anticancer drugs.24 On the other
hand, many compounds exerts their cytotoxic activity in tumor
cells by inducing apoptosis. Apoptosis is characterized by a series
in comparison with normal cells, treatment with 10 lM 17j or
17u induced in a high percentage of cells chromatin condensation
and nuclear fragmentation, which are morphological changes typ-
ical of apoptosis.25
One of the earlier events of apoptosis includes translocation of
phosphatidylserine (PS), a phospholipid preferentially present in
the inner side of membrane, to the surface of membrane. This
allows early recognition of dead cells by macrophages, resulting
in phagocytosis without the release of pro-inflammatory cellular

D. Raffa et al. / Bioorg. Med. Chem. 23 (2015) 6305–6316
6311
Figure 5. Cell cycle distribution of K562 cells after exposure to 17j or 17u. Cells (1.5 Â 105) were incubated with compounds for 24 or 48 h (upper and lower panels,
respectively). Then, cells were harvested, stained with PI and subjected to flow cytometry. Percentages of cells in the sub G0/G1 region was considered as an index of
apoptosis. At least 10,000 events per sample were measured: (a) control, (b) cells treated with 10
(upper and lower panels, respectively).
lM of 17j for 24 or 48 h, (c) cells treated with 10 lM of 17u for 24 or 48 h
Figure 6. Fluorescence micrographs showing the effects of 17j and 17u on K562 cells after Hoechst 33342 staining. K562 cells (8 Â 103/well) were stained with Hoechst
33342 (2.5 lg/mL medium; blue fluorescence) and then treated with 10 lM of 17j or 17u for 48 h. Cell morphology was visualized with a Leica DC 300F microscope with a
fluorescence filter for DAPI. The images show details of cells with clear signs of apoptosis, such as nuclear shrinkage and condensed and fragmented chromatin: (a) control, (b)
cells treated with 10 M 17j for 48 h, (c) cells treated with 10 M 17u for 48 h.
l
l
components.24 In our study the effects of 17j and 17u treatment on
early and late apoptosis were examined using combined annexin
V/PI staining.26 Annexin V, a Ca2+-dependent phospholipid-binding
protein, has high affinity for PS, and fluorochrome-labeled annexin
V can be used to specifically target and identify cells in the early
stages of apoptosis.26 Propidium iodide (PI) is a DNA-intercalating
dye which enters in the cells only when plasma membrane
integrity is lost, a typical event of late apoptotic or necrotic cells.
Annexin V-FITC and PI positive populations can easily be
distinguished using a flow cytometer. In the scatter plot of a double
variant flow cytometry (Fig. 7), the lower left quadrant of the his-
tograms shows the viable cells, which exclude PI and are negative
for annexin V-FITC staining. The lower right quadrant represents
the early apoptotic cells, which are PI negative and annexin V pos-
itive indicating that cell population endowed with cytoplasmic
membrane integrity and PS externalization. The upper right quad-
rant represents the non-viable necrotic and late-stage apoptotic
cells, which are positive for annexin V binding and PI uptake. This
population has loss of membrane integrity and presents external-
ization of PS. Finally, the upper left quadrant provides information
about necrotic cell population, which has loss of cell membrane
integrity (PI positive) with no PS externalization (Annexin-V nega-
tive). In our experiments, to distinguish early from late apoptotic
cells, the analysis was performed after 24 h incubation with 17j
or 17u. As shown in Figure 7, after exposure to 10 lM 17j, 24.5%
of the K562 cells were found to be in early apoptosis and 12.5%
in late apoptosis, while no necrotic cells were detected. Similar
results were obtained in cells treated with 17u.
In light of these results, we focused on the possible activation of
caspases, known molecular players in the process of apoptosis.
Caspases are cysteine aspartyl proteases involved in the activation
of apoptotic cell death, being critical for certain processes

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D. Raffa et al. / Bioorg. Med. Chem. 23 (2015) 6305–6316
Figure 7. Flow cytometric analysis of apoptotic effects of 17j and 17u in K562 cells. Cells were treated with compounds, then 105 cells/sample were analyzed by flow
cytometry for PS externalization after double staining with annexin V and PI. To distinguish early apoptotic from late apoptotic cells, the analysis was performed after a 24 h
incubation with the compounds. The different cell populations are reported in the C1–C4 areas. The C1 area represents necrotic cells, the C2 area represents late apoptotic
cells, the C3 area represents viable cells and the C4 area early apoptotic cells. Analysis of fluorescence intensity of gated cells in FL-1 (annexin V-FITC) and FL-3 (PI) channels
was from 10,000 events: (a) control, (b) cells treated with 10
experiments.
lM 17j, (c) cells treated with 10 lM 17u. The results shown are representative of three independent
3. Conclusion
The data reported here show that the 2-(2-phenoxyacetamido)
benzamides 17 caused growth inhibition against many tumor cell
lines. The best agents inhibited proliferation at low micromolar
and submicromolar concentrations in every investigated tumor
cell lines. It seems that, on average, the substitution of the
cinnamamido moiety with the phenoxyacetamido moiety had lit-
tle positive effect on inhibition of K562 cell growth, but moving
the phenoxyacetamido moiety from the ortho to the meta or para
position had negative effects on activity. As in our earlier studies
with this class of compounds,7,8 we initially suspected tubulin as
a target, but the most cytotoxic compounds had little antitubulin
activity. In the experiments presented here, we found different
behavior in compounds 17a–v, bearing a phenoxyacetamido scaf-
fold (G0/G1 arrest of cell cycle), in comparison with previously
studied cinnamamido and 3-phenylpropiolamido benzamides
(G2/M arrest of cell cycle).7,8
Figure 8. The effect of 17j and 17u on pro-caspase 3 and pro-caspase 6 expression.
lM 17j or 17u. Cell
extracts were prepared and, following gel electrophoresis, subjected to Western
K562 cells (1.5 Â 105/well) were treated for 48 h with 10
blotting analysis to determine the levels of pro-caspases 3 and 6. Equal loading of
Compounds 17j and 17u caused a G0/G1 arrest of the cell cycle.
Prolonging the treatment to 48 h demonstrated that they caused an
apoptotic cell death through the activation of pro-caspase 3 and 6.
the gel was verified by immunoblotting for b-actin. (a) Cells treated with 10
lM 17j.
(b) Cells treated with 10
independent experiments.
lM 17u. The results are representative of three
4. Experimental section
associated with the dismantling of the cell and the formation of
apoptotic bodies.27 Caspases are normally present as pro-caspases,
inactive proenzymes which must be cleaved and transformed in
functional caspases.27 There are two classes of apoptotic caspases:
initiator caspases, which are associated with the initiation of
apoptosis (caspases 2, 8, 9, 10), and effector caspases, which cleave
cellular substrates needed for the cell’s survival (caspases 3, 6, 7).27
To demonstrate the involvement of caspases in apoptosis induced
by 17j and 17u in K562 cells, activation of the effector caspases 3
and 6 was evaluated by Western blotting analysis. As shown in
Figure 8, with respect to the basal level, the cellular content of
pro-caspase 3 protein decreased by 40% and 50% after 48 h treat-
4.1. Chemistry: general procedures
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 deter-
mined on a Büchi 530 capillary melting point apparatus and are
uncorrected. IR spectra were recorded with a Perkin Elmer Spec-
trum RXI FT-IR System spectrophotometer, with compound as a
solid in a KBr disk. 1H and 13C NMR spectra (250 and 62.90 MHz,
respectively) were obtained using a Bruker AC-E 300 MHz spec-
trometer (tetramethylsilane as internal standard): chemical shifts
are expressed in d 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).
ments with 10 lM 17j or 17u, respectively, thus indicating activa-
tion of caspase 3.
Moreover, a modest reduction in the level of pro-caspase 6 was
observed in 17j-treated cells (À20%) and a greater decrease in
17u-treated cells (À50%). These results demonstrate that the cyto-
toxic effect exerted in K562 by both 17j and 17u was due to the
induction of apoptosis mediated by caspase activation.

D. Raffa et al. / Bioorg. Med. Chem. 23 (2015) 6305–6316
6313
4.1.1. General procedure for preparation of 5-R-4-R1-2-nitro-
benzoyl chlorides 12a–c and 2-phenoxyacetyl chlorides 16a–e20
Substituted benzoyl and phenoxyacetyl chlorides 12a–c and
16a–e were obtained by refluxing for 5 h the appropriate acid
derivatives (0.01 mol) with thionyl chloride (7.25 mL). After evap-
oration under reduced pressure, the crude liquid residue was used
for subsequent reactions without purification.
removed by filtration, washed with water, and crystallized from
the indicated solvent.
4.1.8.1. 2-(2-(o-Tolyloxy)acetamido)benzamide (17b). White
solid (1.27 g, 28% yield); mp 170–173 °C (ethanol); I.R. (KBr)
cmÀ1 3347, 3145 (NH, NH2), 1659, 1620 (2 Â CO); 1H NMR (DMSO)
d 2.39 (s, 3H, CH3); 4.70 (s, 2H, OCH2); 6.87–8.62 (a set of signals,
10H, aromatic protons and exchangeable NH2); 12.28 (s, 1H, NH,
exchangeable). 13C NMR (d) (DMSO) 16.68 (CH3), 67.75 (OCH2),
111.73 (CHAr), 120.85 (CAr), 120.88 (CHAr), 121.76 (CHAr), 123.61
(CHAr), 126.90 (CAr), 127.41 (CHAr), 129.01 (CHAr), 131.21 (CHAr),
132.67 (CHAr), 138.85 (CAr), 155.65 (CAr), 167.71 (CO), 170.91
(CO). Anal. Calcd for C16H16N2O3: C, 67.59; H, 5.67; N, 9.85. Found:
C, 67.52; H, 5.60; N, 9.50.
4.1.2. Preparation of 5-R-4-R1-2-nitrobenzamides 13a–c
To 0.01 mol of 5-R-4-R1-2-nitrobenzoyl chlorides 12a–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 evapo-
rated to give pure 13a–c.
4.1.3. Preparation of 5-R-4-R1-2-aminobenzamides 15d,e,f
To a magnetically stirred suspension of stannous chloride
(0.038 mol) in concentrated HCl (37%), 0.013 mol of 13a–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 NaOH (40%) was added
until the tin salt dissolved. The solution was extracted with ethyl
acetate (3 Â 150 mL), and the extracts dried and evaporated to
obtain pure 15d,e,f.
4.1.8.2. 2-(2-(p-Tolyloxy)acetamido)benzamide (17c).
White
solid (1.54 g, 34% yield); mp 205–208 °C (ethanol); I.R. (KBr) cmÀ1
3378, 3318, 3195 (NH, NH2), 1694, 1651 (2 Â CO); 1H NMR (DMSO)
d 2.24 (s, 3H, CH3); 4.63 (s, 2H, OCH2); 6.96–8.63 (a set of signals,
10H, aromatic protons and exchangeable NH2); 12.53 (s, 1H, NH,
exchangeable). 13C NMR (d) (DMSO) 20.52 (CH3), 67.88 (OCH2),
115.18 (2 Â CHAr), 120.38 (CAr), 120.43 (CHAr), 123.50 (CHAr),
129.07 (CHAr), 130.40 (2 Â CHAr), 130.94 (CAr), 132.81 (CHAr),
139.05 (CAr), 155.56 (CAr), 167.73 (CO), 171.06 (CO). Anal. Calcd for
C
16H16N2O3: C, 67.59; H, 5.67; N, 9.85. Found: C, 67.46; H, 5.54; N,
4.1.4. Preparation of 2-amino-5-chlorobenzamide 15b18
A mixture of 0.01 mol of 6-R-7-R1-1H-benzo[d][1,3]oxazine-
2,4-diones 14a 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.
10.10.
4.1.8.3.
(17d).
2-(2-(2,4-Dichlorophenoxy)acetamido)benzamide
Sand-colored solid (1.08 g, 20% yield); mp 185–187 °C
(ethanol); I.R. (KBr) cmÀ1 3362, 3314, 3222 (NH, NH2), 1695,
1628 (2 Â CO); 1H NMR (DMSO) d 4.85 (s, 2H, OCH2); 7.15–8.56
(a set of signals, 9H, aromatic protons and exchangeable NH2);
12.23 (s, 1H, NH, exchangeable). 13C NMR (d) (DMSO) 68.85
(OCH2), 116.00 (CHAr), 120.99 (CAr), 121.05 (CHAr), 123.41 (CAr),
123.74 (CHAr), 126.09 (CAr), 128.52 (CHAr), 128.97 (CHAr), 129.95
(CHAr), 132.63 (CHAr), 138.72 (CAr), 152.45 (CAr), 166.76 (CO),
170.82 (CO). Anal. Calcd for C15H12Cl2N2O3: C, 53.12; H, 3.57; N,
8.26. Found: C, 52.95; H, 3.50; N, 7.93.
4.1.5. Preparation of 2-amino-5-iodobenzamide (15c)21
Powdered iodine (11.7 g, 46.2 mmol) was added portion-wise
over 1 h to a stirred solution of 2-aminobenzamide (15a) (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 Â 800 mL). After being dried with Na2SO4, the
organic phase was evaporated. The crude product was recrystal-
lized with water/methanol 10:1 v/v (600 mL) to yield pure 15c;
yield 95%, mp 197–198 °C.
4.1.8.4.
(17e).
2-(2-(2,3-Dichlorophenoxy)acetamido)benzamide
White solid (1.52 g, 28% yield); mp 202–205 °C (etha-
nol); I.R. (KBr) cmÀ1 3225–2915 (NH, NH2), 1673 (broad, 2 Â CO);
1H NMR (DMSO) d 4.88 (s, 2H, OCH2); 7.16–8.58 (a set of signals,
9H, aromatic protons and exchangeable NH2); 12.24 (s, 1H, NH,
exchangeable). Anal. Calcd for C15H12Cl2N2O3: C, 53.12; H, 3.57;
N, 8.26. Found: C, 52.75; H, 3.86; N, 8.63.
4.1.6. General procedure for the preparation of compounds 19a,
b
To 0.01 mol of 5-R-4-R1-benzoyl chlorides 18a,b 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 to
give pure 19a,b.28
4.1.8.5.
5-Chloro-2-(2-phenoxyacetamido)benzamide
(17f). White solid (0.97 g, 20% yield); mp 197–200 °C (ethanol);
I.R. (KBr) cmÀ1 3377, 3296, 3178 (NH, NH2), 1698, 1655 (2 Â CO);
1H NMR (DMSO) d 4.70 (s, 2H, OCH2); 6.70–8.67 (a set of signals,
10H, aromatic protons and exchangeable NH2); 12.51 (s, 1H, NH,
exchangeable). 13C NMR (d) (DMSO) 67.67 (OCH2), 115.33
(2 Â CHAr), 122.01 (CAr), 122.19 (2 Â CHAr), 127.27 (CAr), 128.72
(CHAr), 130.10 (2 Â CHAr), 132.45 (CHAr), 137.94 (CAr), 157.55
(CAr), 167.70 (CO), 169.73 (CO). Anal. Calcd for C15H13ClN2O3:
C, 59.12; H, 4.30; N, 9.19. Found: C, 58.90; H, 4.11; N, 9.29.
4.1.7. General procedure for the preparation of compounds 20a,b
To28 a solution of 0.8 mmol of 19a,b in 20 mL of hot ethanol,
15 mg of 10% palladium on activated charcoal as a catalyst was
added. The mixture was left under hydrogenation in a Parr appara-
tus at 40 psi for 24 h to obtain 20a,b after crystallization.29,30
4.1.8. General procedure for preparation of phenyl 2-(2-pheno-
xyacetamido)benzamides 17a,18 17b–c,19 17d 17e–f, 17g,19 17h–
v, 4-(2-phenoxyacetamido)benzamide (21) and 3-(2-phenoxy-
acetamido)benzamide (22)
To a cold (0–5 °C) stirred suspension of aminobenzamides
15a–f and 20a,b (0.016 mol) in pyridine (13 mL), 0.016 mol of
the appropriate phenoxyacetyl chloride 16a–e 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
4.1.8.6.
5-Chloro-2-(2-(o-tolyloxy)acetamido)benzamide
(17g). White solid (0.51 g, 10% yield); mp 218–220 °C (ethanol);
I.R. (KBr) cmÀ1 3411, 3182 (NH, NH2), 1665 (broad, 2 Â CO); 1H
NMR (DMSO) d 2.38 (s, 3H, CH3); 4.71 (s, 2H, OCH2); 6.90–8.64 (a
set of signals, 9H, aromatic protons and exchangeable NH2);
12.21 (s, 1H, NH, exchangeable). 13C NMR (d) (DMSO) 16.72
(CH3), 67.73 (OCH2), 111.80 (CHAr), 121.65 (CHAr), 122.26 (CHAr),

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D. Raffa et al. / Bioorg. Med. Chem. 23 (2015) 6305–6316
126.85 (CAr), 127.17 (CAr), 127.36 (CHAr), 128.71 (CHAr), 130.61
(CAr), 131.20 (CHAr), 132.34 (CHAr), 137.98 (CAr), 157.63 (CAr),
167.22 (CO), 169.48 (CO). Anal. Calcd for C16H15ClN2O3: C, 60.29;
H, 4.74; N, 8.79. Found: C, 60.34; H, 5.03; N, 8.95.
4.1.8.12.
(17m).
5-Methyl-2-(2-phenoxyacetamido)benzamide
White solid (1.91 g, 42% yield); mp 199–202 °C (etha-
nol); I.R. (KBr) cmÀ1 3381, 3302, 3182 (NH, NH2), 1693, 1652
(2 Â CO); 1H NMR (DMSO) d 2.31 (s, 3H, CH3); 4.67 (s, 2H, OCH2);
6.98–8.52 (a set of signals, 10H, aromatic protons and exchange-
able NH2); 12.43 (s, 1H, NH, exchangeable). 13C NMR (d) (DMSO)
20.84 (CH3), 67.73 (OCH2), 115.38 (CHAr), 120.23 (CAr), 120.31
(CHAr), 122.02 (CHAr), 129.44 (CHAr), 130.05 (CHAr), 132.33 (CAr),
133.16 (CHAr), 136.92 (CAr), 157.00 (CAr), 167.14 (CO), 171.08
(CO). Anal. Calcd for C16H16N2O3: C, 67.59; H, 5.67; N, 9.85. Found:
C, 67.45; H, 5.76; N, 9.90.
4.1.8.7.
(17h).
5-Chloro-2-(2-(p-tolyloxy)acetamido)benzamide
White solid (1.12 g, 22% yield); mp 225–227 °C (etha-
nol); I.R. (KBr) cmÀ1 3390, 3253, 3248 (NH, NH2), 1660 (broad,
2 Â CO); 1H NMR (DMSO) d 2.24 (s, 3H, CH3); 4.65 (s, 2H, OCH2);
6.95–8.66 (a set of signals, 9H, aromatic protons and exchangeable
NH2); 12.48 (s, 1H, NH, exchangeable). 13C NMR (d) (DMSO) 20.51
(CH3), 67.86 (OCH2), 115.19 (2 Â CHAr), 122.01 (CAr), 122.17 (CHAr),
127.25 (CAr), 128.71 (CHAr), 130.40 (2 Â CHAr), 131.00 (CAr), 132.45
(CHAr), 137.94 (CAr), 155.52 (CAr), 167.85 (CO), 169.70 (CO). Anal.
Calcd for C16H15ClN2O3: C, 60.29; H, 4.74; N, 8.79. Found: C,
60.39; H, 4.50; N, 8.53.
4.1.8.13. 5-Methyl-2-(2-(o-tolyloxy)acetamido)benzamide
(17n).
White solid (0.72 g, 15% yield); mp 160–162 °C (etha-
nol); I.R. (KBr) cmÀ1 3385, 3344, 3186 (NH, NH2), 1693, 1652
(2 Â CO); 1H NMR (DMSO) d 2.03 (s, 3H, CH3); 2.38 (s, 3H, CH3);
4.67 (s, 2H, OCH2); 6.87–8.50 (a set of signals, 9H, aromatic protons
and exchangeable NH2); 12.14 (s, 1H, NH, exchangeable). 13C NMR
(d) (DMSO) 16.78 (CH3), 20.82 (CH3), 67.78 (OCH2), 111.79 (CHAr),
120.70 (CHAr), 120.78 (CAr), 121.63 (CHAr), 126.91 (CHAr), 127.36
(CAr), 129.37 (CHAr), 131.15 (CHAr), 132.44 (CAr), 133.00 (CHAr),
136.72 (CAr), 155.74 (CAr), 167.28 (CO), 170.92 (CO). Anal. Calcd
for C17H18N2O3: C, 68.44; H, 6.08; N, 9.39. Found: C, 68.65; H,
6.34; N, 9.78.
4.1.8.8. 5-Chloro-2-(2-(2,4-dichlorophenoxy)acetamido)benza-
mide (17i).
White solid (0.65 g, 11% yield); mp 215–218 °C
(ethanol); I.R. (KBr) cmÀ1 3378, 3254, 3246 (NH, NH2), 1684,
1644 (2 Â CO); 1H NMR (DMSO) d 4.91 (s, 2H, OCH2); 6.24–8.64
(a set of signals, 8H, aromatic protons and exchangeable NH2);
12.19 (s, 1H, NH, exchangeable). 13C NMR (d) (DMSO) 63.83
(OCH2), 116.10 (CHAr), 122.72 (CAr), 122.82 (CHAr), 123.43 (CAr),
126.17 (CAr), 127.54 (CAr), 128.53 (CHAr), 128.60 (CHAr), 129.95
(CHAr), 132.96 (CHAr), 137.57 (CAr), 152.40 (CAr), 166.88 (CO),
169.45 (CO). Anal. Calcd for C15H11Cl3N2O3: C, 48.22; H, 2.97; N,
7.50. Found: C, 48.38; H, 2.65; N, 7.66.
4.1.8.14. 5-Methyl-2-(2-(p-tolyloxy)acetamido)benzamide
(17o).
White solid (1.72 g, 36% yield); mp 216–218 °C (etha-
nol); I.R. (KBr) cmÀ1 3390, 3262, 3222 (NH, NH2), 1665, 1660
(2 Â CO); 1H NMR (DMSO) d 2.24 (s, 3H, CH3); 2.30 (s, 3H, CH3);
4.61 (s, 2H, OCH2); 6.95–8.51 (a set of signals, 9H, aromatic protons
and exchangeable NH2); 12.39 (s, 1H, NH, exchangeable). 13C NMR
(d) (DMSO) 20.65 (CH3), 20.81 (CH3), 67.90 (OCH2), 115.22
(2 Â CHAr), 120.88 (CAr), 120.23 (CHAr), 129.42 (CHAr), 130.35
(2 Â CHAr), 130.76 (CAr), 132.30 (CAr), 133.14 (CHAr), 136.91 (CAr),
155.66 (CAr), 167.28 (CO), 171.05 (CO). Anal. Calcd for
4.1.8.9.
(17j).
5-Iodo-2-(2-phenoxyacetamido)benzamide
Sand-colored solid (2.41 g, 38% yield); mp 206-209 °C
(ethanol); I.R. (KBr) cmÀ1 3377, 3295, 3178 (NH, NH2), 1694,
1652 (2 Â CO); 1H NMR (DMSO) d 4.69 (s, 2H, OCH2); 6.99–8.46
(a set of signals, 10H, aromatic protons and exchangeable NH2);
12.50 (s, 1H, NH, exchangeable). 13C NMR (d) (DMSO) 67.73
(OCH2), 87.03 (CAr), 115.13 (2 Â CHAr), 122.20 (CHAr), 122.50
(CAr), 122.57 (CHAr), 130.11 (2 Â CHAr), 137.17 (CHAr), 138.74
(CAr), 141.19 (CHAr), 157.55 (CAr), 167.70 (CO), 169.62 (CO). Anal.
Calcd for C15H13IN2O3: C, 45.47; H, 3.31; N, 7.07. Found: C, 45.40;
H, 3.59; N, 7.25.
C17H18N2O3: C, 68.44; H, 6.08; N, 9.39. Found: C, 68.46; H, 6.19;
N, 9.76.
4.1.8.15. 2-(2-(2,4-Dichlorophenoxy)acetamido)-5-methylben-
zamide (17p).
White solid (1.02 g, 18% yield); mp 214–
216 °C (ethanol); I.R. (KBr) cmÀ1 3391, 3320, 3201 (NH, NH2),
1665, 1660 (2 Â CO); 1H NMR (DMSO) d 2.30 (s, 3H, CH3); 4.82
(s, 2H, OCH2); 7.16–8.44 (a set of signals, 8H, aromatic protons
and exchangeable NH2); 12.07 (s, 1H, NH, exchangeable). 13C
NMR (d) (DMSO) 20.83 (CH3), 68.92 (OCH2), 116.08 (CHAr),
120.84 (CAr), 120.90 (CHAr), 123.41 (CAr), 125.99 (CAr), 128.51
(CHAr), 129.32 (CHAr), 129.95 (CHAr), 132.59 (CAr), 132.96 (CHAr),
136.57 (CAr), 152.57 (CAr), 166.42 (CO), 170.81 (CO). Anal. Calcd
for C16H14Cl2N2O3: C, 54.41; H, 4.00; N, 7.93. Found: C, 54.09; H,
4.20; N, 7.58.
4.1.8.10.
(17k).
5-Iodo-2-(2-(p-tolyloxy)acetamido)benzamide
White solid (1.57 g, 24% yield); mp 220–222 °C (etha-
nol); I.R. (KBr) cmÀ1 3386, 3297, 3195 (NH, NH2), 1693, 1651
(2 Â CO); 1H NMR (DMSO) d 2.24 (s, 3H, CH3); 4.63 (s, 2H, OCH2);
6.94–8.44 (a set of signals, 9H, aromatic protons and exchangeable
NH2); 12.44 (s, 1H, NH, exchangeable). 13C NMR (d) (DMSO) 20.53
(CH3), 67.85 (OCH2), 87.05 (CAr), 115.16 (2 Â CHAr), 122.42 (CAr),
122.53 (CHAr), 130.41 (2 Â CHAr), 130.98 (CAr), 137.16 (CHAr),
138.74 (CAr), 141.18 (CHAr), 155.47 (CAr), 167.96 (CO), 169.86
(CO). Anal. Calcd for C16H15IN2O3: C, 46.85; H, 3.69; N, 6.83. Found:
C, 47.00; H, 3.98; N, 6.79.
4.1.8.16. 2-(2-(2,3-Dichlorophenoxy)acetamido)-5-methylben-
zamide (17q).
White solid (0.56 g, 10% yield); mp 223–
225 °C (ethanol); I.R. (KBr) cmÀ1 3310–2910 (NH, NH2), 1670
(broad, 2 Â CO); 1H NMR (DMSO) d 2.30 (s, 3H, CH3); 4.85 (s, 2H,
OCH2); 7.14–8.44 (a set of signals, 8H, aromatic protons and
exchangeable NH2); 12.07 (s, 1H, NH, exchangeable). Anal. Calcd
for C16H14Cl2N2O3: C, 54.41; H, 4.00; N, 7.93. Found: C, 54.68; H,
3.74; N, 7.47.
4.1.8.11. 2-(2-(2,4-Dichlorophenoxy)acetamido)-5-iodobenza-
mide (17l).
White solid (1.04 g, 14% yield); mp 213–215 °C
(ethanol); I.R. (KBr) cmÀ1 3376, 3261, 3144 (NH, NH2), 1670,
1665 (2 Â CO); 1H NMR (DMSO) d 4.84 (s, 2H, OCH2); 7.17–8.38
(a set of signals, 8H, aromatic protons and exchangeable NH2);
12.11 (s, 1H, NH, exchangeable). 13C NMR (d) (DMSO) 68.93
(OCH2), 87.38 (CAr), 116.10 (CHAr), 122.92 (CAr), 123.02 (CHAr),
123.41 (CAr), 126.07 (CAr), 128.50 (CHAr), 129.96 (CHAr), 137.10
(CAr), 138.66 (CHAr), 140.99 (CHAr), 152.47 (CAr), 166.84 (CO),
169.35 (CO). Anal. Calcd for C15H11Cl2IN2O3: C, 38.74; H, 2.38; N,
6.02. Found: C, 38.37; H, 2.59; N, 5.88.
4.1.8.17.
(17r).
5-Methoxy-2-(2-phenoxyacetamido)benzamide
White solid (0.96 g, 20% yield); mp 178–181 °C
(ethanol); I.R. (KBr) cmÀ1 3391, 3313, 3164 (NH, NH2), 1698,
1654 (2 Â CO); 1H NMR (DMSO) d 3.80 (s, 3H, OCH3); 4.66 (s, 2H,
OCH2); 7.00–8.56 (a set of signals, 10H, aromatic protons and

D. Raffa et al. / Bioorg. Med. Chem. 23 (2015) 6305–6316
6315
exchangeable NH2); 12.27 (s, 1H, NH, exchangeable). 13C NMR (d)
(DMSO) 55.93 (OCH3), 67.69 (OCH2), 113.99 (CHAr), 115.38
(2 Â CHAr), 118.18 (CHAr), 121.64 (CAr), 121.86 (CHAr), 122.01
(CHAr), 130.05 (2 Â CHAr), 132.58 (CAr), 154.82 (CAr), 157.71 (CAr),
166.79 (CO), 170.69 (CO). Anal. Calcd for C16H16N2O4: C, 63.99; H,
5.37; N, 9.33. Found: C, 64.05; H, 5.72; N, 9.64.
(CO), 167.89 (CO). Anal. Calcd for C15H14N2O3: C, 66.66; H, 5.22;
N, 10.36. Found: C, 66.34; H, 4.93; N, 10.32.
4.1.8.23. 3-(2-Phenoxyacetamido)benzamide (22).
Sand-
colored solid (1.12 g, 26% yield); mp 168–170 °C (ethanol); I.R.
(KBr) cmÀ1 3400–3212 (NH, NH2), 1678, 1652 (2 Â CO); 1H NMR
(DMSO) d 4.72 (s, 2H, OCH2); 6.96–8.13 (a set of signals, 11H, aro-
matic protons and exchangeable NH2); 10.24 (s, 1H, NH, exchange-
able). Anal. Calcd for C15H14N2O3: C, 66.66; H, 5.22; N, 10.36.
Found: C, 66.91; H, 5.26; N, 10.24.
4.1.8.18. 5-Methoxy-2-(2-(o-tolyloxy)acetamido)benzamide
(17s).
Brown solid (0.65 g, 13% yield); mp 165–168 °C (etha-
nol); I.R. (KBr) cmÀ1 3347, 3289, 3186 (NH, NH2), 1672 (broad,
2 Â CO); 1H NMR (DMSO) d 2.39 (s, 3H, OCH3); 3.90 (s, 3H,
OCH3); 4.67 (s, 2H, OCH2); 6.87–8.53 (a set of signals, 9H, aromatic
protons and exchangeable NH2); 11.99 (s, 1H, NH, exchangeable).
13C NMR (d) (DMSO) 16.42 (CH3), 55.38 (OCH3), 67.39 (OCH2),
111.62 (CHAr), 113.52 (CHAr), 118.07 (CHAr), 121.98 (CHAr),
122.96 (CAr), 123.11 (CHAr), 126.85 (CAr), 127.46 (CHAr), 131.03
(CAr), 131.30 (CHAr), 155.26 (CAr), 155.40 (CAr), 167.71 (CO),
170.81 (CO). Anal. Calcd for C17H18N2O4: C, 64.96; H, 5.77; N,
8.91. Found: C, 65.16; H, 6.16; N, 9.19.
4.2. Biology
4.2.1. Percent growth inhibition assay
Human chronic myelogenous leukemia cell line K562 was
maintained in suspension culture in RPMI1640 medium
(SIGMA–Aldrich, cat. #R8758) supplemented with 5% fetal bovine
serum (FBS: SIGMA–Aldrich, cat. #F6178), 100 U/mL penicillin,
100 lg/mL streptomycin and 0.25 lg/mL amphotericin B (com-
plete medium) at 37 °C in a 5% CO2 humidified atmosphere.
The trypan blue exclusion assay was performed to assess the
effect of all test compounds on the viability of K562 cells, as
described previously.31 Briefly, about 2 Â 105 cells/mL were cul-
4.1.8.19. 5-Methoxy-2-(2-(p-tolyloxy)acetamido)benzamide
(17t).
Yellow solid (0.85 g, 17% yield); mp 238–240 °C (etha-
nol); I.R. (KBr) cmÀ1 3353, 3245, 3191 (NH, NH2), 1669, 1667
(2 Â CO); 1H NMR (DMSO) d 2.24 (s, 3H, CH3); 3.79 (s, 3H, OCH3);
4.61 (s, 2H, OCH2); 6.95–8.55 (a set of signals, 9H, aromatic protons
and exchangeable NH2); 12.24 (s, 1H, NH, exchangeable). 13C NMR
(d) (DMSO) 20.55 (CH3), 55.91 (OCH3), 67.85 (OCH2), 113.93 (CHAr),
115.20 (CHAr), 118.18 (CHAr), 121.70 (CAr), 121.92 (CHAr), 130.38
(CHAr), 130.82 (CAr), 132.47 (CAr), 154.84 (CAr), 155.64 (CAr),
167.98 (CO), 170.69 (CO). Anal. Calcd for C17H18N2O4: C, 64.96; H,
5.77; N, 8.91. Found: C, 65.21; H, 5.69; N, 9.17.
tured for 2 4 h, and compounds were added at 10 lM (or at differ-
ent 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.
4.2.2. MTT assay
The MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay was performed as described earlier32 and was used
to determine the viability of HuDe cells.
4.1.8.20. 4,5-Dimethoxy-2-(2-phenoxyacetamido)benzamide
(17u).
White solid (2.38 g, 45% yield); mp 219–222 °C (etha-
nol); I.R. (KBr) cmÀ1 3367, 3289, 3173 (NH, NH2), 1693, 1652
(2 Â CO); 1H NMR (DMSO) d 3.81 (s, 6H, 2 Â OCH3); 4.67 (s, 2H,
OCH2); 6.98–8.44 (a set of signals, 9H, aromatic protons and
exchangeable NH2); 12.92 (s, 1H, NH, exchangeable). 13C NMR (d)
(DMSO) 55.58 (OCH3), 56.37 (OCH3), 67.76 (OCH2), 103.33 (CHAr),
111.21 (CAr), 112.13 (CHAr), 115.38 (2 Â CHAr), 121.98 (CHAr),
130.03 (2 Â CHAr), 135.01 (CAr), 144.08 (CAr), 151.88 (CAr), 157.75
(CAr), 167.15 (CO), 170.84 (CO). Anal. Calcd for C17H18N2O5: C,
61.81; H, 5.49; N, 8.48. Found: C, 62.06; H, 5.18; N, 8.83.
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 spectropho-
tometer (ELx800-BioTek instruments). The absorbance correlates
directly with cell number.
Briefly, about 2 Â 105 cells/mL (2 Â 104 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
4.1.8.21. 2-(2-(2,3-Dichlorophenoxy)acetamido)-4,5-dimetho-
xybenzamide (17v).
Yellow solid (0.96 g, 15% yield); mp
10
crystals were solubilized by the addition of 4.5 mL isopropyl alco-
hol, 0.5 mL triton X-100 and 150 L of 37% HCl. Cells grown in cul-
ture media alone or with appropriate concentrations of DMSO as
controls.
ll of MTT (5 mg/mL in PBS) at 37 °C for 3 h. The tetrazolium
248–250 °C (ethanol); I.R. (KBr) cmÀ1 3363, 3259, 3178 (NH,
NH2), 1664, 1616 (2 Â CO); 1H NMR (DMSO) d 3.80 (s, 6H,
2 Â OCH3); 4.85 (s, 2H, OCH2); 7.13–8.35 (a set of signals, 7H, aro-
matic protons and exchangeable NH2); 12.60 s, 1H, NH, exchange-
able). 13C NMR (d) (DMSO) 55.87 (OCH3), 56.28 (OCH3), 68.96
(OCH2), 104.33 (CHAr), 111.70 (CAr), 111.79 (CHAr), 113.11 (CHAr),
121.66 (CAr), 123.48 (CHAr), 128.92 (CHAr), 132.90 (CAr), 134.43
(CAr), 144.25 (CAr), 151.67 (CAr), 154.87 (CAr), 166.44 (CO), 170.63
(CO). Anal. Calcd for C17H16Cl2N2O5: C, 51.14; H, 4.04; N, 7.02.
Found: C, 51.21; H, 4.05; N, 6.77.
l
4.2.3. Hoechst staining and cell cycle distribution
For these experiments, after plating on poly-(D-lysine) (Sigma–
Aldrich, Milan, Italy) coated 96 or 6 well plates, K562 cells were
allowed to adhere overnight and then treated with chemicals or
vehicle only.
In order to determine either changes in nuclear morphology or
plasma membrane damage, the cells were stained with Hoechst
33342 (Sigma–Aldrich, Milan, Italy), a fluorescent cell permeant
dye. Specifically, cells (8 Â 103/well) were stained with Hoechst
4.1.8.22. 4-(2-Phenoxyacetamido)benzamide (21).
Sand-
colored solid (2.81 g, 65% yield); mp 216–218 °C (ethanol); I.R.
(KBr) cmÀ1 3379, 3323-3175 (NH, NH2), 1681, 1645 (2 Â CO); 1H
NMR (DMSO) d 4.74 (s, 2H, OCH2); 6.96–7.88 (a set of signals,
11H, aromatic protons and exchangeable NH2); 10.24 (s, 1H, NH,
exchangeable). 13C NMR (d) (DMSO) 67.48 (OCH2), 115.08
(2 Â CHAr), 119.22 (2 Â CHAr), 121.68 (CHAr), 128.84 (2 Â CHAr),
129.64 (CAr), 129.99 (2 Â CHAr), 141.48 (CAr), 158.19 (CAr), 167.46
33342 (2.5 lg/mL medium) for 30 min, washed with PBS, resus-
pended in culture medium and treated for various times with com-
pounds. Cell morphology was visualized using a Leica DC 300F
microscope (Leica Microsystems, Wetzlar, Germany), and all
images were acquired by Leica Q Fluoro Software using appropriate

6316
D. Raffa et al. / Bioorg. Med. Chem. 23 (2015) 6305–6316
filters to examine Hoechst 33342 (DAPI filter with excitation wave-
length of 372 nm and emission wavelength of 456 nm).
Cell cycle distribution was analyzed by flow cytometry using PI
staining. Cells were seeded in 6 well plates (1.5 Â 105/well), trea-
ted with compounds and harvested for flow cytometric analysis.
Fluorescence of the cells was analyzed by a FACscan (Beckman
Coulter Inc., Brea, CA). The proportion of cells with fragmented
DNA was estimated by evaluating the percentage of events
accumulated in the hypodiploid sub G0/G1 area of each scan. Data
were analyzed using Expo32 software (Beckman Coulter Inc., Brea,
CA).
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Financial support from ‘Fondo di Finanziamento della Ricerca di
Ateneo (ex 60%)’ 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.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.08.027.
33. Carlisi, D.; Lauricella, M.; D’Anneo, A.; Buttitta, G.; Emanuele, S.; Di Fiore, R.;
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