Substituted 2-hydroxy-N-(arylalkyl)benzamides induce apoptosis in cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2013.08.009

Variously substituted 2-hydroxy-N-(arylalkyl)benzamides were prepared and screened for antiproliferative and cytotoxic activity in cancer cell lines in vitro. Five compounds, out of 33 showed single-digit micromolar IC₅₀ values against several human cancer cell lines. One of the most potent compounds N-((R)-1-(4-chlorophenylcarbamoyl)-2-phenylethyl)-5-chloro-2-hydroxybenzamide (6k) reduced proliferation and induced apoptosis in the melanoma cell line G361 in a dose-dependent manner, as shown by decrease in 5-bromo-2′- deoxyuridine incorporation and increase in several apoptotic markers, including subdiploid population increase, activation of caspases and site-specific poly-(ADP-ribose)polymerase (PARP) cleavage.

Full text of DOI:10.1016/j.ejmech.2013.08.009

European Journal of Medicinal Chemistry 68 (2013) 253e259
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Substituted 2-hydroxy-N-(arylalkyl)benzamides induce apoptosis
in cancer cell lines
a
,
b
,
c
a
b
a
ꢀ
*
ꢀ
ꢀ
ꢀ
Ales Imramovský , Radek Jorda , Karel Pauk , Eva Reznícková , Jan Dusek ,
a
b
ꢀ
ꢀ
Jirí Hanusek , Vladimír Krystof
a Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, Studentská 573, 53210 Pardubice, Czech Republic
Laboratory of Growth Regulators, Faculty of Science, Palacký University & Institute of Experimental Botany, Slechtitelu 11, 78371 Olomouc, Czech Republic
Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Zlutý kopec 7, Brno 656 53, Czech Republic
b
ꢀ
ꢁ
c
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
Variously substituted 2-hydroxy-N-(arylalkyl)benzamides were prepared and screened for anti-
proliferative and cytotoxic activity in cancer cell lines in vitro. Five compounds, out of 33 showed single-
digit micromolar IC50 values against several human cancer cell lines. One of the most potent compounds
N-((R)-1-(4-chlorophenylcarbamoyl)-2-phenylethyl)-5-chloro-2-hydroxybenzamide (6k) reduced pro-
liferation and induced apoptosis in the melanoma cell line G361 in a dose-dependent manner, as shown
by decrease in 5-bromo-20-deoxyuridine incorporation and increase in several apoptotic markers,
including subdiploid population increase, activation of caspases and site-specific poly-(ADP-ribose)po-
lymerase (PARP) cleavage.
Received 15 April 2013
Received in revised form
31 July 2013
Accepted 1 August 2013
Available online 14 August 2013
Keywords:
Diamides
Apoptosis
Cytotoxicity
Cancer
Ó 2013 Elsevier Masson SAS. All rights reserved.
Autophagy
p53
1. Introduction
cancers, treatment effective in one cancer is not effective in another
at all [2]. Moreover, standard treatment options such as radio-
According to 2008 annual report of the World Health Organi-
zation, cancer remains one of the largest causes of death in the
developed world. Today, it is estimated that one of every five death
is caused by cancer, mainly due to increased longevity of the pop-
ulation and to environmental factors. Moreover, it is estimated that
the annual number of deaths due to cancers will increase from 7.6
million in 2008 to 13 million in 2030 [1]. Therapy is complicated by
the fact that cancer is not a single disease; due to the variability of
therapy and DNA-damaging chemotherapy sometimes induce a
different cancer in surviving patients, but the risk of secondary
cancer is outweighed by the benefit of curing the original one.
Another serious problem in cancer therapy relates to developing
resistance to used drugs and in this sense, there is an urgent need
for new drugs and new drug combinations, based on classical
cytotoxic chemotherapy, molecularly targeted drugs or new
chemical entities, that can be collectively more active, specific and
with less harmful side effects [2].
In our search for new chemical entities with potential anticancer
activity, weexploredthebiologicalpropertiesofvariouslysubstituted
2-hydroxy-N-(arylalkyl)benzamides that were described earlier as
unexpected major products isolated during preparation of amino
acids esters with salicylanilides [3,4]. First examples of this rear-
rangement described interesting synthetic pathway for preparation
of these compounds [3]. Later on, our study confirms generality of
rearrangement for wide range of substituents including various
amino acids [4]. These compounds share interesting structural
motive with some known proteasomal inhibitors (e.g. bortezomib or
carfilzomib) [5] or other agents displaying promising anticancer ac-
tivity [6]. First, our library was evaluated for antifungal as well as
Abbreviations: PET, photosynthetic electron transport; CDK, cyclin-dependent
kinase; BrdU, 5-bromo-20-deoxyuridine; SAR, structureeactivity relationship; GSK,
glycogen synthase kinase; Rb, retinoblastoma protein; IC50, growth inhibition 50%;
Cbz, benzyloxycarbonyl group; DMF, N,N0-dimethylformamide; DCC, N,N0-dicyclo-
hexylcarbodiimide; Et3N, triethyl amine; DCM, dichloromethane; Et2O, dieth-
ylether; EtOAc, ethyl acetate; EDC$HCl, N-(3-dimethylaminopropyl)-N0-
ethylcarbodiimide hydrochloride; HOBt$H2O, 1-hydroxybenzotriazole hydrate;
ERK1/2, extracellular-signal-regulated kinases; PARP-1, poly-(ADP-ribose)polymer-
ase-1; Bcl-2, B-cell lymphoma 2; Bcl-xL, B-cell lymphoma-extra large; Mdm2,
mouse double minute 2; HMGB1, high-mobility group protein B1; RT, room tem-
perature (approx. 20 ꢀC); LC3, microtubule-associated protein light chain 3.
* Corresponding author.
E-mail address: Ales.Imramovsky@upce.cz (A. Imramovský).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.08.009

254
A. Imramovský et al. / European Journal of Medicinal Chemistry 68 (2013) 253e259
antimycobacterial inhibitory activities of its members [7]. This paper
describes their antiproliferative and proapoptotic activity in human
cancer cell lines. We were also interested in the targeted synthesis of
these compounds, where synthesized intermediates were the same
for a chosen group of compounds, and the last reaction step with
different anilines produced the desired series of substituted 2-
hydroxy-N-(arylalkyl)benzamides.
methyl ester hydrochloride 2a in the DCM as solvent and in the
presence of triethyl amine, EDC$HCl and HOBt$H2O. Thus prepared
ester 3a was isolated using column chromatography. During the
isolation, decomposition of isolated esters was observed. The
product of decomposition was identified as deacetylated ester 4a.
Deprotection of carboxylic group as well as the acetyl group was
carried out in a mixture of 1,4-dioxaneewater in the presence of
lithium hydroxide. Intermediate 5a was isolated by simple extrac-
tion. These compounds formed the final diamides 6b and 6s with
chosen anilines in the presence of EDC$HCl (Scheme 2).
2. Results and discussion
As mentioned above, during isolation of 3a, a mixture with 4a
was obtained due to low stability of the acetyl ester group. Hence,
we switched to benzyl ether as a new protection of the phenolic
hydroxyl group of substituted salicylic acid. These derivatives were
prepared using a two-step procedure. The first step was prepara-
tion of substituted benzyl 2-(benzyloxy)benzoate followed by the
second step, where benzyl ester was hydrolytically cleaved in a
methanolic solution of sodium hydroxide [6]. The following syn-
thetic pathway was similar to the synthesis with an acetyl group
(Scheme 3). Substituted 2-(benzyloxy)chlorobenzoic acid 1b or 1c
2.1. Chemistry
Our original method for the synthesis of 2-hydroxy-N-(arylalkyl)
benzamides [3] (including the rearrangement depicted in Scheme 1)
is somewhat general. It was demonstrated that the described rear-
rangement is independent of the substitution of salicylic or anilide
moiety of the molecule as well as of the amino acid type [4]. The
crucial step in the synthesis was not the rearrangement or even the
formation of hydrobromide salt but the first step e esterification of
N-protected (the Cbz was mostly used) amino acid with substituted
salicylanilides. The reaction was carried out in DMF in the presence
of DCC at low temperature. Due to low stability of prepared esters,
during column chromatography, we abandoned this purification
method as unsuitable for the isolation these esters. Several crys-
tallizations were necessary for complete removal of the impurities,
including dicyclohexylurea. In some cases, there was only one
possibility for isolation of esters, although the methodology
involved caused significant loss in total yields.
The synthetic pathway developed by the group of Shibasaki as
well [8], did not meet our needs. For each final 2-hydroxy-N-(ary-
lalkyl)benzamide, it is necessary to synthesize intermediates,
which serves only for the synthesis of one targeted molecule. Our
intention was to develop a synthetic strategy, where intermediates
would be stable and their isolation would be possible with simple
crystallization or column chromatography and one intermediate
would be the starting material for several other target molecules.
For this reason, we developed a new synthetic method for
preparation of substituted 2-hydroxy-N-(arylalkyl)benzamides
(Scheme 2). The principle of the synthesis involves gradual building
of the desired molecules. All intermediates were found to be stable
and could be isolated using column chromatography with in high
yields. Both facts were the main advantage over the original
method. In general, the synthetic pathway starts from the O-pro-
tected salicylic acid [9] which forms an amide bond with chosen
carboxyl-protected amino acid. The carboxyl group of such inter-
mediate is liberated and this intermediate can react with several
anilines to form 2-hydroxy-N-(arylalkyl)benzamides containing a
protected phenolic hydroxyl group, terminated by its deprotection.
All intermediates and final products were fully characterized by
melting points, infrared spectroscopy, nuclear magnetic resonance,
mass spectrometry (for suitable low molecular weight compounds
GCeMS was used) and elemental analyses.
reacts with commercially available
drochloride 2b, -tert-leucine methyl ester hydrochloride 2e or
prepared hydrochloride salts of methyl esters of -phenylalanine 2c,
-phenylalanine 2d -cyclohexylalanine 2f, eventually -alanine 2g.
L-alanine tert-butyl ester hy-
L
L
D
L
L
The reactions were performed in DCM and in the presence of
EDC$HCl. The prepared esters 3beh were isolated using column
chromatography on silica. Hydrolysis of 3beh in lithium hydroxide
solution (1,4-dioxaneewater) gave appropriate 2-(2-(benzyloxy)
benzamido) acids 4beh. These acids were the key intermediates for
the formation of O-benzyl-2-hydroxy-N-(arylalkyl)benzamides
5bei. Hydrogen release of the phenolic hydroxyl group (H2/Pd) was
the final step of the developed synthetic pathway. The obtained
compounds 6c, 6k, 6r, 6t, 6u, 6ee, 6ff and 6gg were the same as the
compounds isolated in the procedure shown in Scheme 1.
2.2. Cytotoxicity
All prepared 2-hydroxy-N-(arylalkyl)benzamides were evalu-
ated for their cytotoxicity against K562 and MCF-7 cell lines derived
from chronic myelogenous leukemia and breast adenocarcinoma.
The data are presented in Table 1 and clearly show that most of the
new diamides displayed no cytotoxicity except for compounds 6k,
6reu, whose IC50 values reached the single-digit micromolar range
and are comparable or even lower than those for well-known
imatinib inhibitor Bcr-Abl or roscovitine (CDK inhibitor), respec-
tively. Hence we tested these five compounds against additional
tumor-derived cell lines (Table S2) and, due to the observed
apoptotic-like cell death, in a one-step cellular caspase-3/7 activity
assay, too (Fig. 2). Of these, only 6k, 6t and 6u were able to strongly
activate cellular caspases 3 and 7. Compounds 6r and 6s were found
to be nearly inactive in the caspase assay and we speculate that
they induce other type of cell death.
Initially, we chose 2-acetoxy-5-chlorobenzoic acid 1a as a
All diamides 6 share chlorine at position 4 or 5 of the salicylic
starting material. This compound directly reacts with
L-valine
moiety (R1 substituent), but differ in substitution in positions R2
O
O
R3
O
O
O
R2
b
R2
R2
H
N
N
H
N
H
N
H
N
H
R1
R1
R1
R1
R2
c
a
O
O
OH
OH
H
N
+
R3
NH2*HCl
6a-dd
O
Cbz
O
Cbz
R3
R3
HOOC
N
H
Scheme 1. Original synthetic pathway including unexpected rearrangement. a ¼ DMF, DCC. ꢁ15 ꢀC 4h, 4 ꢀC 16 h, b ¼ 33% HBr in acetic acid, 30 min, Et2O, RT, c ¼ CHCl3, Et3N,
30 min, RT (rearrangement) [4].

A. Imramovský et al. / European Journal of Medicinal Chemistry 68 (2013) 253e259
255
O
O
O
O
O
Cl
O
Cl
O
Cl
O
O
N
H
N
H
OH
+
b
a
O
O
+
NH2*HCl
OH
O
O
2a
1a
3a
4a
O
NH2
R2
O
H
N
Cl
OH
+
Cl
N
H
N
c
R2
b
H
O
O
OH
OH
R2 = 4-CF3 6s
R2 = 4-Cl
6b
5a
Scheme 2. Synthetic pathway led to substituted 2-hydroxy-N-(arylalkyl)benzamides using acetyl group for protection OH group. a ¼ EDC$HCl, Et3N, DCM, RT, 16 h; b ¼ LiOH$H2O,
dioxaneewater 1:1, 50 ꢀC, 45 min; c ¼ aniline, EDC$HCl, Et3N, DCM, RT, 16 h.
and R3. Exploration of the influence of R2 substitution on the
cytotoxicity showed preferences for halogenated mono-substituted
anilines (6k, 6reu) over disubstituted (6j, 6len, 6cc, 6dd) or non-
halogenated (6oeq, 6v) anilines (Table 1). Interestingly, diamides
bearing trifluoromethyl moiety at position R2 (6ret) were the most
potent compounds in this series. Diversity in substituents at posi-
tion R3 allowed us to compare the activities of diverse compounds
having alkyl, cycloalkyl or aromatic chains. A pronounced cytotoxic
effect was observed in cells treated by diamides 6t and 6u bearing
cyclohexyl groups and by derivatives 6r and 6s with tert-butyl and
isopropyl substitution at position R3. Additionally, these three de-
rivatives (6ret) shared the same substituent at position R2. A
negative effect on the cell viability was also determined for the
most potent diamide 6k with the R-phenyl ring, while others
sharing this type of substituent (6e, 6n, 6aa) showed no toxicity in
tested cells.
G361 cells. Compound 6k was dosed to asynchronously growing
G361 cells that were subsequently analyzed by flow cytometry. As
shown in Fig. 1A, treatment with 6k led to decrease in the S-phase
population in treated cells, more specifically in cells actively
replicating DNA (i.e. BrdU-positive cells). Besides clear cell cycle
suppression, significant increase in the sub-G1 population on
treatment was observed. This indirect marker of apoptotic cell
death was already obvious at concentrations above 1
of treatment.
mM after 24 h
Changes in some proteins known to be principal regulators of
proliferation and the cell cycle were monitored in G361 cells treated
with compound 6k by immunoblotting (Fig. 1B). A significant
reduction was observed in protein levels of CDK4 and cyclins A and
B. In contrast, changes in expressions of CDK1, CDK2 (not shown)
and cyclin E were only marginal. An interesting trend was observed
in the expression of CDK inhibitor p27KIP1 that was dramatically up-
regulated by 670 nM 6k, while higher doses had no effect on the
protein level. This effect is an indication of multiple molecular tar-
gets for 6k, affected separately depending on concentration.
2.3. Antiproliferative activity of 6k
Due to the anticancer activity of the five tested diamides, we
sought the underlying mechanism using the most potent com-
pound 6k. First, we analyzed the effect of 6k on the cell cycle of
In addition, we analyzed the influence of 6k on functional status
of ERK1/2 and AKT, two pivotal protein kinases involved in mito-
genic signal transduction to the cell cycle control system. Both
R3 R4
O
O
O
O
R3
OH
N
H
O
O
R1
R1
+
O
a
b
O
R4
NH2*HCl
1b, 1c
2a - 2h
3b - 3h
O
R3
NH2
R4
O
R3
OH
+
H
N
N
H
R1
N
H
O
b
c
R1
R2
O
O
O
R5
6c, 6k, 6r,
6t, 6u,
6ee, 6ff, 6gg
5b - 5h
4b - 4h
Scheme 3. Synthetic pathway led to substituted 2-hydroxy-N-(arylalkyl)benzamides using benzyl group for protection OH group. a) DCM, EDC$HCl, Et3N, 16 h, RT; b) dioxanee
water 1:1, LiOH$H2O, 45 min, 50 ꢀC; c) DCM, aniline, EDC$HCl, Et3N, 16 h, RT.

256
A. Imramovský et al. / European Journal of Medicinal Chemistry 68 (2013) 253e259
Table 1
Cytotoxicity of compounds 6 in human cancer cell lines.
O
R3
H
N
N
H
R1
R2
O
OH
Compound
R1
R2
R3
IC50
K562
(m
M)a
MCF-7
Roscovitine
Imatinib
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
6w
6x
6y
e
e
e
e
e
45.5
0.5
>20
12.3
>10
>20
>10
7.9
>10
18.4
>10
>10
>10
>10
>20
3.5
>10
>10
>10
>10
>10
>10
8.8
e
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
5-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
(R)-CH3
(S)-CH(CH3)2
(S)-CH2Ph
(S)-CH2Ph
(R)-CH2Ph
H
>20
>10
>10
>20
>10
>10
>10
>10
>20
(S)-CH3
(S)-CH(CH3)2
(R)-CH(CH3)2
H
(R)-CH2Ph
(S)-CH(CH3)2
(S)-CH2Ph
(R)-CH2Ph
(S)-CH(CH3)2
(S)-CH2eindole
(S)-CH3
(S)-C(CH3)3
(S)-CH(CH3)2
(S)-CH2ecyclohexyl
(S)-CH2ecyclohexyl
(S)-CH(CH3)2
(S)-CH3
4,3-diCl
4-Cl
2.5
4,3-diCl
4,3-diCl
4,3-diCl
4-OCH3
4-CH3
4-CH3
3-CF3
4-CF3
3-CF3
4-Br
H
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Br
>10
>10
>10
>10
>10
>10
5.9
4.5
3.4
3.0
>10
>10
>10
>10
>10
>10
>10
>10
>10
>12.5
>12.5
>12.5
7.3
2.3
1.9
>10
>10
>10
>10
9.1
>10
>10
>10
7.3
(R)-CH3
(S)-CH(CH3)2
(S)-CH2Ph
(R)-CH2Ph
(S)-CH(CH3)2
(S)-CH(CH3)2
(S)-CH2Ph
(S)-CH3
6z
6aa
6bb
6cc
6dd
6ee
6ff
6gg
3,4-diCl
3,4-diCl
4-CF3
3,4-diCl
4-CF3
>12.5
>12.5
>12.5
(S)-CH3
(S)-CH3
a
Cytotoxicity of prepared compounds is an average from at least two de-
terminations (inactive compounds were tested twice, more active compounds were
tested four times).
pathways showed deactivation in cells treated by 6k through de-
phosphorylations of ERK1/2 and AKT (Fig. 1C). The observed
changes probably contribute to the antiproliferative activity of 6k
as these kinases regulate transcription of cyclin D1, deactivation of
Fig. 1. Antiproliferative effect of 6k in asynchronous G361 cells exposed for 24 h. (A)
The influence of different doses of compound 6k in G361 cells on the level of sub-G1
population and BrdU incorporation analyzed by flow cytometry. Immunoblot analysis
of (B) selected cell cycle regulators or (C) ERK1/2 and Akt phosphorylation. Actin level
is included as a control for equal protein loading.
GSK-3b
and stability of p27KIP1 [10,11].
2.4. Induction of cell death by 6k
7 and 9 are effectors involved in the mitochondrial apoptotic
pathway and for this reason we also monitored the levels of some
mitochondrial proteins involved in apoptosis. While expression and
phosphorylation of Bcl-2 increased on treatment of cells with 6k,
the level of anti-apoptotic protein Bcl-xL showed a large dose-
dependent decrease. These changes are consistent with the roles
of Bcl-2 and Bcl-xL in depolarization of mitochondria and subse-
quent caspase activation [13,14].
Mitochondrial cell death is often mediated by the tumor sup-
pressor p53, activated for example by DNA damage, blocked tran-
scription or hypoxic condition [15]. As the diamide 6k is not
expected to induce any of these stress situations, it was not a sur-
prise that it increased neither the p53 level (Fig. 3) nor its
Flow cytometric analysis revealed that 6k increases the sub-G1
population which is a typical marker of apoptosis. We also analyzed
the ability of five potently cytotoxic diamides to activate apoptosis
in the melanoma cell line G361, using a one-step cellular caspase-3/
7 activity assay [12]. All tested compounds proved able to activate
caspases-3/7 in cells treated for 24 h at a single dose of 2 mM (Fig. 2).
Compounds 6k, 6u and 6t increased caspase activity more than
ten-times over untreated control cells. The most active compound
6k was then selected for immunoblotting experiments that clearly
confirmed apoptosis as a mechanism of cell death; we observed
cleavage of caspase-9 and a decrease in zymogenes of caspases-3
and 7 (Fig. 3). Monitoring of the cleavage of PARP-1, a nuclear
target of caspase-3, further confirmed the above results. Caspases 3,

A. Imramovský et al. / European Journal of Medicinal Chemistry 68 (2013) 253e259
257
promising diamides bearing trifluoromethyl moiety showed sig-
nificant antiproliferative effect against cancer cell lines with IC50
values reaching the single-digit micromolar range. Our candidate
6k was then studied in a more detail. Diamide 6k showed an
inhibitory effect on DNA replication in treated cells without any
effect on protein expression of known cell cycle regulators except of
p27KIP1 whose expression was dramatically up-regulated by
670 nM 6k. We also analyzed an induction of cell death by 6k with
interesting results indicating activation of apoptosis as well as
autophagy that was previously shown only in a few studies. We also
investigated protein expression of tumor suppressor p53 that was
surprisingly down regulated via 6k despite of its rapid activation by
treatment by the most anticancer agents.
4. Experimental section
4.1. Chemistry
Fig. 2. Relative caspase-3/7 activity in G361 melanoma cells after treatment with the
most potent diamides.
All reagents and solvents were purchased from commercial
sources (SigmaeAldrich, Merck, Acros Organics). Commercial grade
reagents were used without further purification. Reactions were
monitored by thin layer chromatography plates coated with
0.2 mm silica gel 60 F254 (Merck). TLC plates were visualized by the
UV irradiation (254 nm).
All melting points were determined on a Melting Point B-545
apparatus (Buchi, Switzerland) and are uncorrected. Infrared
spectra (KBr pellets) were recorded on FT-IR spectrometer Nicolet
6700 FT-IR in the range of 400e4000 cmꢁ1. The NMR spectra were
measured in DMSO-d6 solutions at ambient temperature on a
Bruker Avance III 400 spectrometer (400 MHz for 1H, 100 MHz for
13C). The coupling constants are quoted in Hz. Elemental analyses
(C, H, N) were performed on an automatic microanalyser Flash
2000 Organic elemental analyzer.
transcriptional activity in a cell based assay (data not shown).
Rather unexpectedly, 6k triggered down regulation of p53 as well
as its target product Mdm2. We therefore sought alternative
mechanisms that could explain mitochondria- and caspase-
involved cell death. Interestingly, we observed the fragmentation
of microtubule-associated protein 1-light chain 3 (LC3) (Fig. 3) that
is considered a marker of autophagic activity in cells. For some
cellular settings, apoptosis and autophagy can interplay as partners
in the execution of cell death in a cooperative fashion [16,17]. On
the other hand, autophagy is often viewed as maintenance of
cellular viability under various stress conditions [18]. It is known
that p53 regulates the balance between tumor cell death and sur-
vival too through autophagy, as a negative regulator of HMGB1/
Beclin 1 complex [19]. Decrease in p53 level caused by 6k thus
could promote autophagy that can eventually lead to cell death
executed by caspases.
4.1.1. General experimental procedure for synthesis of amino acids
esters hydrochlorides 2
For the synthesis of compounds 2a, 2ce2g, we used slightly
modificated known procedure [1]. To a stirred solution of amino
acid (32.2 mmol) in dry methanol (100 mL) was added drop-wise
thionyl chloride (64.4 mmol). The temperature was kept
between ꢁ10 and ꢁ5 ꢀC. After complete addition, the reaction was
stirred at RT overnight. After 16 h, the solution was evaporated to
dryness. The product was diluted with EtOAc and collected by
filtration. The residue was dried under reduced pressure to give an
amino acid methyl ester hydrochloride as white crystalline powder.
The yields were higher in all experiments than 80%. Melting point
and 1H as well as 13C NMR spectra in D2O were used for charac-
terization of the prepared compounds. The data are in good
agreement with literature data [20].
3. Conclusion
The library of substituted 2-hydroxy-N-(arylalkyl)benzamides
were prepared using an original synthetic pathway. The most
4.1.2. General procedure for the synthesis of amino acid salicylate
esters 3
To a stirred solution of substituted acetyl or benzyloxy salicylic
acid 1 (4 mmol) in dichloromethane (25 mL), amino acid ester
hydrochloride 2 (4 mmol), was added in one portion. The solution
was cooled to 5 ꢀC and EDC$HCl (4.4 mmol) as well as HOBt$H2O
(4.0 mmol) were added. Triethyl amine (4 mmol) was added
dropwise. The clear mixture was stirred at the same temperature
for 30 min. After additional stirring at RT overnight, the reaction
mixture was quenched with water and the resulting mixture was
treated with dichloromethane (3 ꢂ 20 mL). The organic phase was
treated with saturated solution of sodium hydrogen carbonate, 5%
water solution of citric acid, and brine, then dried over anhydrous
Na2SO4. The organic solvent was removed under reduced pressure
and residue was purified by silica gel column chromatography
Fig. 3. Effect of 6k on the induction of cell death in G361 cell line upon 24 h treatment.
Immunoblot analysis of selected markers of cell death. Actin level was detected to
verify equal protein loading.

258
A. Imramovský et al. / European Journal of Medicinal Chemistry 68 (2013) 253e259
(EtOAc/hexane 1:1) to give desired esters. The purification of the
reaction mixture of 1a and 2a gave the desired compound 3a with a
mixture of deacetylated product. The reactions of O-benzyl salicylic
acids 1b or 1c with amino acid esters 2be2g was done using the
same procedure as mentioned above. Appropriate esters 3be3h
were isolated as colorless oils.
4.1.6.2. 4-Chloro-2-hydroxy-N-[(2S)-1-[(3,4-dichlorophenyl)amino]-
1-oxopropan-2-yl]benzamide 6ff. White solid; Yield 37%; mp 235e
237 ꢀC. IR (KBr pellet): 3333, 3248, 3066, 1691(C]O amide),
1667(C]O amide), 1634, 1576, 1536, 1493, 1474, 1401, 1371, 1341,
1295, 1232,m 1169, 1122, 1111, 1069, 1044, 954, 914, 900, 816, 797,
669, 607 cmꢁ1 1H NMR (400 MHz, DMSO-d6):
. d 12.44 (1H, brs,
OH), 10.45 (1H, s, NH), 9.04 (1H, d, J ¼ 6.8 Hz, NH), 7.96e8.05 (2H,
m, AreH), 7.49e7.59 (2H, m, AreH), 6.97e7.05 (2H, m, Ar-H),
4.58e4.67 (1H, m, NHCH), 1.25 (3H, d, J ¼ 7.2 Hz, CHCH3), 13C NMR
(100 MHz, DMSO-d6): 172.0, 167.5, 160.4, 139.6, 138.3, 131.7, 131.3,
125.5, 121.1, 120.0, 119.7, 117.5, 115.7, 50.4, 18.5. Anal Calc. for
C16H13Cl3N2O3 (387.65): C, 49.57; H, 3.38; N, 7.23. Found: C, 49.29;
H, 3.27; N 7.02.
4.1.3. General procedure for synthesis of amino acid salicylate acids
4a and amino acid O-benzyl-salicylate acids 4be4g
To a stirred solution of esters 3a (a mixture with deacetylated
product) or other compounds 3 (2 mmol) in mixture of 1,4-
dioxaneeH2O (50 mL) (2:1) was added LiOH$H2O (20 mmol) at
50 ꢀC. The reaction was monitored by TLC. When compounds 3
disappeared from the mixture, the reactions were quenched with
3 N HCl and the residues were treated with EtOAc (3 ꢂ 50 mL).
Organic phases were collected and dried over anhydrous Na2SO4
followed by removing of the solvent under reduced pressure.
Acid 4a and other O-benzyl salicyloic acids 4be4h were
obtained.
4.1.6.3. 4-Chloro-2-hydroxy-N-{(2S)-1-[(4-(trifluoromethyl)phenyl)
amino]-1-oxopropan-2-yl}benzamide 6gg. White solid; Yield 63%;
mp 232e236 ꢀC. IR (KBr pellet): 3337, 3552, 3132, 1691(C]O
amide), 1666(C]O amide), 1634, 1607, 1577, 1550, 1534, 1494,
1462, 1413, 1335, 1262, 1232, 1162, 1121, 1068, 1044, 1018, 953,
913, 874, 839, 769, 751, 729, 708, 667, 643 cmꢁ1
.
1H NMR
d 12.45 (1H, brs, OH), 10.54 (1H, s, NH),
4.1.4. General procedure for synthesis of 2-O-benzyloxy-N-
(arylalkyl)benzamides 5be5h or N-(arylalkyl)benzamides 6s and
6b
(400 MHz, DMSO-d6):
8.90e9.01 (1H, m, NH), 7.95e8.05 (1H, m, AreH), 7.83 (2H, d,
J ¼ 8.4 Hz, AreH), 7.68 (2H, d, J ¼ 8.8 Hz, AreH), 6.89e7.05 (2H,
m, AreH), 4.61e4.73 (1H, m, NHCH), 1.47 (3H, d, J ¼ 7.2 Hz,
CHCH3). 13C NMR (100 MHz, DMSO-d6): 172.2, 167.4, 159.8, 143.1,
138.3, 131.4, 126.7 (q, 3J(19F, 13C) ¼ 3.7 Hz), 125.0 (q, 1J(19F,
13C) ¼ 270.7 Hz), 124.0 (q, 2J(19F, 13C) ¼ 32.2 Hz), 119.7, 119.6,
117.5, 116.4, 115.7, 50.4, 18.6. Anal Calc. for C17H14ClF3N2O3
(386.75): C, 52.79; H, 3.65; N, 7.24. Found: C, 53.18; H, 3.91;
N 7.08.
To a stirred solution of amino acid 4 (4 mmol) in dichloro-
methane (25 mL) chosen aniline (4 mmol) was added in one
portion. The solution was cooled to 5 ꢀC and EDC$HCl (4.4 mmol) as
well as HOBt$H2O (4.0 mmol) were added. Triethyl amine (4 mmol)
was added dropwise. The clear mixture was stirred at the same
temperature for 30 min. After additional stirring at RT overnight,
the reaction mixture was quenched with water and the resulting
mixture was treated with dichloromethane (3 ꢂ 20 mL). Separated
organic phase was treated with saturated solution of sodium
hydrogen carbonate, 5% water solution of citric acid, and brine, and
then dried over Na2SO4. Organic solvent was removed under
reduced pressure and the residue was purified by silica gel column
chromatography (EtOAc/hexane 1:1) to give 6s or 6b eventually
5be5h if the starting acids were 4be4g.
Characterization of all new intermediates can be found in the
Supplementary material of this article.
4.2. Cell lines
G361, MCF-7, K562, HOS, HCT-116 and HeLa cell lines were
maintained in DMEM supplemented with 10% fetal bovine serum,
penicillin (100 U/ml) and streptomycin (100
mg/ml). All cell lines
4.1.5. General procedure for synthesis of 2-hydroxy-N-(arylalkyl)
benzamides 6
were cultivated at 37 ꢀC in 5% CO2.
A
mixture of 2-benzyloxy-N-(arylalkyl)benzamide 5be5h
4.3. Cytotoxicity assays
(2 mmol) in EtOAc (80 mL) with 10% Pd on carbon (0.2 mmol) was
hydrogenated at 1 atm for 12 h. The catalyst was filtered off, and the
filtrate was evaporated under reduced pressure. The residue was
purified by silica gel column chromatography (EtOAc/hexane 1:3.5)
to give the desired 2-hydroxy-N-(arylalkyl)benzamides 6.
The cytotoxicity of the studied compounds was determined
using cell lines of different histological origin as described earlier
[21,22]. The cells were assayed with compounds using three-fold
dilutions in triplicate. Treatment lasted for 72 h, followed by
addition of Calcein AM solution, and measurement of the fluores-
cence of live cells at 485 nm/538 nm (ex/em) with a Fluoroskan
Ascent microplate reader (Labsystems). The IC50 value, the drug
concentration lethal to 50% of the tumor cells, was calculated from
the obtained dose response curves. The IC50 for compounds 6ee, 6ff
4.1.6. Spectral characterization of new compounds 6
4.1.6.1. 5-Chloro-2-hydroxy-N-[(2S)-1-oxo-1-[[4-(trifluoromethyl)
phenyl]amino]propan-2-yl]benzamide 6ee. White solid; Yield 66%;
mp 230.1e232.8 ꢀC. IR (KBr pellet): 3335, 3257, 1692(C]O
amide), 1667(C]O amide), 1633, 1580, 1538, 1499, 1469, 1400,
1371, 1341, 1295, 1230, 1168, 1130, 1112, 1060, 1048, 952, 916, 899,
and 6gg were >12.5 mM (Table 1).
820, 797, 669, 607 cmꢁ1 1H NMR (400 MHz, DMSO-d6):
. d 12.20
(1H, s, AreOH), 10.50 (1H, s, AreNH), 9.11 (1H, d, J ¼ 6.2 Hz, NH),
8.11 (1H, s, AreH), 8.05 (1H, d, J ¼ 4.9 Hz, AreH), 7.80 (1H, d,
J ¼ 8.5 Hz, AreH), 7.56 (1H, t, J ¼ 8.0 Hz, AreH), 7.45 (2H, m, Are
H), 6.97 (1H, d, J ¼ 8.78 Hz, AreH), 4.64 (1H, m, NHeCH), 1.46 (3H,
4.4. Immunoblotting
Immunoblotting analysis was performed as described earlier
[21,22]. Briefly, cellular lysates were prepared by harvesting cells in
Laemmli sample buffer. Proteins were separated on SDS-
polyacrylamide gels and electroblotted onto nitrocellulose mem-
branes. After blocking, the membranes were incubated with spe-
cific primary antibodies overnight, washed and then incubated
with peroxidase-conjugated secondary antibodies. Finally, peroxi-
dase activity was detected with ECL þ reagents (AP Biotech) using a
CCD camera LAS-4000 (Fujifilm).
d, J ¼ 7.03 Hz, CHeCH3). 13C NMR (150 MHz, DMSO-d6):
d 172.0,
167.0, 158.4, 140.2, 134.0, 130.7, 130.0 (q, 2J(19F, 13C) ¼ 31.1 Hz),
128.9, 124.7 (q, 1J(19F, 13C) ¼ 272.4 Hz), 123.5, 123.2, 120.5 (q,
3J(19F, 13C) ¼ 3.6 Hz), 119.8, 117.9, 116.0 (q, 3J(19F, 13C) ¼ 3.6 Hz),
50.5, 18.5. 19F NMR (376.46 MHz, DMSO-d6):
for C17H14ClF3N2O3 (386.75): C, 52.79; H, 3.65; N, 7.24. Found: C,
52.99; H, 3.72; N, 7.62.
d
ꢁ61.3. Anal. Calc.

A. Imramovský et al. / European Journal of Medicinal Chemistry 68 (2013) 253e259
259
4.5. Antibodies
Appendix A. Supplementary material
Specific antibodies were purchased from Calbiochem (anti-Bcl-2,
clone Ab-2), SigmaeAldrich (peroxidase-labeled secondary anti-
bodies, anti-LC3B), Santa Cruz Biotechnology (anti-p27, clone F-8;
anti-cyclin B, clone GNS1; anti-Mdm-2, clone SMP14; anti-PARP,
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.08.009.
References
clone F-2; anti-b-actin, clone C4; anti-CDK1, clone B-6), Cell
Signaling (anti-pMAPK (Erk1/2) phosphorylated at Thr202/Tyr204,
clone D13.14.4E; anti-MAPK (Erk1/2); anti-pAkt (Ser473), clone
D9E; anti-Rb, clone 4H1; anti-cyclin A, clone BF683; anti-cyclin E,
clone HE12; anti-Bcl-xl, clone 54H6; anti-cleaved caspase-9; anti-
caspase-7; anti-caspase-3, clone 3G2) or were a generous gift from
[1] www.who.int.
[2] B. Al-Lazikani, U. Banerji, P. Workman, Combinatorial drug therapy for cancer
in the post-genomic era, Nat. Biotechnol. 30 (2012) 679e692.
[3] A. Imramovský, J. Vinsová, J.M. Férriz, J. Kunes, M. Pour, M. Dolezal, Salicyla-
nilide esterification: unexpected formation of novel seven-membered rings,
Tetrahedron Lett. 47 (2006) 5007e5011.
ꢀ
ꢀ
ꢀ
ꢀ
[4] A. Imramovský, J.M. Férriz, K. Pauk, M. Krátky, J. Vinsová, Synthetic route for
ꢀꢀ
Dr. B. Vojtesek from Masaryk Memorial Cancer Institute, Brno, Czech
the preparation of 2-hydroxy-N-[1-(2-hydroxyphenylamino)-1-oxoalkan-2-
yl]benzamides, J. Comb. Chem. 12 (2010) 414e416.
Republic (anti-p53, clone DO-1; anti-CDK4; anti-p21waf1, clone 118).
[5] S. Tsukamoto, H. Yokosawa, Targeting the proteasome pathway, Expert Opin.
Ther. Targets 13 (2009) 605e621.
[6] I. Kuriyama, A. Miyazaki, Y. Tsuda, H. Yoshida, Mizushina, Inhibitory effect of
novel somatostatin peptide analogues on human cancer cell growth based on
4.6. One-step cellular caspase-3/7 activity assay
the selective inhibition of DNA polymerase b, Bioorg. Med. Chem. 15 (2013)
The activity of cellular caspases-3/7 was measured according to
published procedures [12]. Briefly, G361 cells were incubated in a
density of 20,000 cells/well in a 96-well plate overnight. Next day,
403e411.
ꢀ
ꢀ
ꢀ
ꢀ
[7] A. Imramovský, M. Pesko, K. Králová, M. Vejsová, J. Stolariková, J. Vinsová,
J. Jampílek, Investigating spectrum of biological activity of 4- and 5-chloro-2-
hydroxy-N-[2-(arylamino)-1-alkyl-2-oxoethyl]benzamides, Molecules 16
(2011) 2414e2430.
the compounds were added (the final concentration reached 2 mM)
and the cells were incubated for the next 24 h. After incubation, 3x
caspase-3/7 assay buffer (150 mM HEPES pH 7.4, 450 mM NaCl,
150 mM KCl, 30 mM MgCl2, 1.2 mM EGTA, 1.5% Nonidet P40, 0.3%
[8] T. Mahsiko, K. Hara, D. Tanaka, Y. Fujiwara, N. Kumagai, M. Shibasaki, En route
to an efficient catalytic asymmetric synthesis of AS-3201, J. Am. Chem. Soc.
129 (2007) 11342e11343.
[9] L.A. Gaudioso, M.A. Weglarz, Novel Process for the Synthesis of Exochelins.
WO 2000/09547.
[10] C. Marshall, How do small GTPase signal transduction pathways regulate cell
cycle entry? Curr. Opin. Cell Biol. 11 (1999) 732e736.
[11] M. Qiao, S. Sheng, A.B. Pardee, Metastasis and AKT activation, Cell Cycle 7
(2008) 2991e2996.
CHAPS, 30% sucrose, 30 mM DTT, 3 mM PMSF) with 37.5 mM Ac-
DEVD-AMC as a substrate (SigmaeAldrich) was added to the
wells and plates were incubated at 37 ꢀC. The caspase-3/7 activity
was measured after 4 h using Fluoroskan Ascent microplate reader
(Labsystems) at 346 nm/442 nm (excitation/emission).
[12] R.A. Carrasco, N.B. Stamm, B.K.R. Patel, One-step cellular caspase-3/7 assay,
Biotechniques 34 (2003) 1064e1067.
[13] T. Kuwana, D.D. Newmeyer, Bcl-2-family proteins and the role of mitochon-
dria in apoptosis, Curr. Opin. Cell Biol. 15 (2003) 691e699.
[14] A. Gross, J.M. McDonnell, S.J. Korsmeyer, BCL-2 family members and the
mitochondria in apoptosis, Genes Dev. 13 (1999) 1899e1911.
[15] S. Haupt, M. Berger, Z. Goldberg, Y. Haupt, Apoptosis e the p53 network, J. Cell
Sci. 116 (2003) 4077e4085.
[16] A. Eisenberg-Lerner, S. Bialik, H.U. Simon, A. Kimchi, Life and death partners:
apoptosis, autophagy and the cross-talk between them, Cell Death Differ. 16
(2009) 966e975.
[17] H. Rikiishi, Novel insights into the interplay between apoptosis and auto-
phagy, Int. J. Cell Biol. (2012) 317645.
[18] C. He, D.J. Klionsky, Regulation mechanisms and signaling pathways of
autophagy, Annu. Rev. Genet. 43 (2009) 67e93.
4.7. Cell cycle analysis
Sub-confluent cells were treated with test compounds at
different concentrations for 24 h. The cultures were pulse-labeled
with 10
m
M BrdU for 30 min at 37 ꢀC prior to harvesting. The
cells were then washed in PBS, fixed with 70% ethanol, and dena-
tured in 2 M HCl. Following neutralization, the cells were stained
with anti-BrdU fluorescein-labeled antibodies, washed, stained
with propidium iodide and analyzed by flow cytometry using a
488 nm laser (Cell Lab Quanta SC, Beckman Coulter) as described
previously [21,22].
[19] K.M. Livesey, R. Kang, P. Vernon, W. Buchser, P. Loughran, S.C. Watkins,
L. Zhang, J.J. Manfredi, H.J. Zeh III, L. Li, M.T. Lotze, D. Tang, p53/HMGB1
complexes regulate autophagy and apoptosis, Cancer Res. 72 (2012) 1996e
2005.
Acknowledgments
[20] S. Anantharaj, M. Jayakannan, Polymers from amino acids: development of
dual ester-urethane melt condensation approach and mechanistic aspects,
Biomacromolecules 13 (2012) 2446e2455.
This research was supported by the Czech Science Foundation
(GACR 305/12/0783, GACR 301/08/1649) and by the Internal Grant
Agency of Palacký University (project PrF_2013_023). The authors
also wish to acknowledge the institutional financial support of the
Faculty of Chemistry and Chemical Technology, University of Par-
dubice and Czech Ministry of Education, Youth and Sports. The
work was supported by the European Regional Development Fund
and the State Budget of the Czech Republic (RECAMO, CZ.1.05/
2.1.00/03.0101).
ꢀ
[21] R. Jorda, L. Havlícek, I.W. McNae, M.D. Walkinshaw, J. Voller, A. Sturc,
ꢀ
J. Navratilová, M. Kuzma, M. Mistrik, J. Bartek, M. Strnad, V. Krystof, Pyrazolo
[4,3-d]pyrimidine bioisostere of roscovitine: evaluation of a novel selective
inhibitor of cyclin-dependent kinases with antiproliferative activity, J. Med.
Chem. 54 (2011) 2980e2993.
ꢀ
[22] V. Krystof, P. Cankar, I. Frýsová, J. Slouka, G. Kontopidis, P. Dzubak,
M. Hajduch, J. Srovnal, W.F.J. de Azevedo, M. Orsag, M. Paprskarova, J. Rolcik,
A. Latr, P.M. Fischer, M. Strnad, 4-Arylazo-3,5-diamino-1H-pyrazole CDK in-
hibitors: SAR study, crystal structure in complex with CDK2, selectivity, and
cellular effects, J. Med. Chem. 49 (2006) 6500e6509.