Cytotoxic Mannich bases of 6-(3-aryl-2-propenoyl)-2(3H)-benzoxazolones

Article abstract of DOI:10.1016/j.ejmech.2007.02.019

A series of 12 new Mannich bases with chalcone core structure were synthesized as potential antineoplastic agents, via N-aminomethylation of two parent 6-(3-aryl-2-propenoyl)-2(3H)-benzoxazolones. The newly synthesized compounds as well as the chalcone prototypes were evaluated for cytotoxicity in the human pre-B-cell leukemia cell line BV-173 using the MTT-dye reduction assay. The tested compounds exhibited concentration-dependent cytotoxic effects at low micromolar concentrations. Ten of the Mannich bases characterized by significant activity in BV-173 were further evaluated against the chronic myeloid leukemia cell line K-562 and were found to suppress the growth of these cells at relatively higher concentrations as compared to the former tumor model. Selected Mannich bases induced programmed cell death in BV-173 at a concentration of 2.5 μM as evidenced by the encountered DNA-laddering. Taken together our data suggest that the presented heterocyclic chalcone derived Mannich bases necessitate detailed pharmacological evaluation in order to define further the structure activity relationships, in a larger spectrum of tumor models and to elucidate the mechanisms implicated in the observed cytotoxic effects.

Full text of DOI:10.1016/j.ejmech.2007.02.019

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European Journal of Medicinal Chemistry 42 (2007) 1382e1387
http://www.elsevier.com/locate/ejmech
Short communication
Cytotoxic Mannich bases of 6-(3-aryl-2-propenoyl)-
2(3H )-benzoxazolones
*
Y. Ivanova, G. Momekov, O. Petrov , M. Karaivanova, V. Kalcheva
University of Sofia St. Kliment Ohridski, Faculty of Chemistry, Department of Applied Organic Chemistry, 1 James Bourchier Avenue,
1164 Sofia, Bulgaria
Received 13 September 2006; received in revised form 15 February 2007; accepted 26 February 2007
Available online 12 March 2007
Abstract
A series of 12 new Mannich bases with chalcone core structure were synthesized as potential antineoplastic agents, via N-aminomethylation
of two parent 6-(3-aryl-2-propenoyl)-2(3H )-benzoxazolones. The newly synthesized compounds as well as the chalcone prototypes were eval-
uated for cytotoxicity in the human pre-B-cell leukemia cell line BV-173 using the MTT-dye reduction assay. The tested compounds exhibited
concentration-dependent cytotoxic effects at low micromolar concentrations. Ten of the Mannich bases characterized by significant activity in
BV-173 were further evaluated against the chronic myeloid leukemia cell line K-562 and were found to suppress the growth of these cells at
relatively higher concentrations as compared to the former tumor model. Selected Mannich bases induced programmed cell death in BV-173
at a concentration of 2.5 mM as evidenced by the encountered DNA-laddering. Taken together our data suggest that the presented heterocyclic
chalcone derived Mannich bases necessitate detailed pharmacological evaluation in order to define further the structure activity relationships, in
a larger spectrum of tumor models and to elucidate the mechanisms implicated in the observed cytotoxic effects.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Chalcone; 2(3H )-Benzoxazolone; Mannich base; Cytotoxicity; MTT assay; Apoptosis
1. Introduction
The tumor inhibiting properties of chalcones pose special
interest and have been found to arise from their effects on
malignant cell proliferation, tumor angiogenesis and/or on
the established neoplastic vasculature. In contrast to the major-
ity of conventional cytotoxic agents the chalcones are charac-
terized by a low propensity to interact with DNA, which to
a great extent eliminates the risk of mutagenicity and carcino-
genicity common in various chemotherapeutics [1]. This
favorable toxicological profile has solicited intensive research
with natural and synthetic chalcones in view of developing
novel, patient-friendly antineoplastic agents [1,3,6]. The
mechanisms underlying the antiproliferative effects of chal-
cones appear to be complex and frequently multimodal and
include binding to tubulin with consequent disruption of the
microtubule assembly [7e9], interference with the p-53-
MDM2 interactions [10], tyrosine kinase inhibition [11],
depletion of cellular thiol depots [1], and/or triggering of
Chalcone (1,3-diaryl-2-propen-1-one) derivatives constitute
an important group of secondary metabolites found ubiqui-
tously in the plant kingdom. These compounds exhibit a constel-
lation of biological activities e.g. analgesic, anti-inflammatory,
antibacterial, antimycotic, antiviral, antiprotozoal, chemopre-
ventive, anticancerous, etc [1e5]. The complex pharmacologi-
cal activities together with the easy synthetic reproduction and
derivatization of the core structure 1 (depicted in Fig. 1) have
solicited considerable interest towards the exploitation of the
unique chalcone template for the discovery of prospective
lead compounds [1,3].
* Corresponding author. Fax: þ359 2 962 54 38.
E-mail address: opetrov@chem.uni-sofia.bg (O. Petrov).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.02.019

Y. Ivanova et al. / European Journal of Medicinal Chemistry 42 (2007) 1382e1387
1383
3
using acid or base as catalysts in some applications of the Man-
nich reaction, this was not necessary in our study as reported
previously [17]. The physical properties were determined and
structures were confirmed by IR, NMR and elemental analyses.
All spectral data are in accordance with the assumed struc-
tures. IR spectra of the compounds showed no absorption
bands at 3100e3400 cmꢀ1, indicating the absence of an NH-
group, which is an evidence for the addition reaction. The lac-
tam and ketone C]O stretching bands were seen at about
3'
A
4
5
2
1
2'
1'
4'
5'
B
6'
6
O
Fig. 1. Structure of the chalcone (1,3-diphenyl-2-propen-1-one), indicating the
ring A and ring B atom numbering.
1760 cmꢀ1 and 1650 cmꢀ1
.
apoptotic cell-signaling pathways [3,12]. Owing to the com-
plex, multimodal pharmacodynamics and the structural diver-
sity of synthetic and natural chalcones, the structural features,
affording optimal cytotoxicity vary with the predominant
mode of action and there are no generally applicable structure
activity rules, valid for the various classes of chalcone ana-
logues [3]. Nevertheless the abundance of the enone system
and the substitution of the aryl moieties appear to be crucial
for the cytotoxic activity of chalcones [1,3,8,9].
1
In H NMR, methylene protons between the two nitrogen
atoms in 2(3H )-benzoxazolone were observed as a singlet at
4.65 ppm. The aromatic ring protons were observed at 7.2e
8.0 ppm. All of the synthesized Mannich bases were initially
E-conformers, which was proved by coupling constant value
J ¼ 15.6 Hz for vinyl protons, observed at 7.4e7.8 ppm. The
details of protons belonging to the secondary amines are given
in Section 5.
In addition to the plethora of chalcone derivatives bearing
hydroxy, methoxy and/or methyl functions in the aryl rings
that have been designed as potential cytotoxic agents [7,13],
attention has been brought to the development of tumor
inhibiting chalcones via introduction of nitrogen-containing
substituents as well. Thus, a series of Mannich bases with
1,3-diaryl-2-propen-1-one core structure [1] and more recently
40-aminochalcones and related Schiff bases and maleamic
acids have been reported to exert considerable cytotoxic activ-
ity at low micromolar concentrations against P388 and L1210
murine leukemia cell lines [14]. In line with these advances we
prepared a series of 12 Mannich bases, derived from a chalcone
template with a condensed oxazole ring and evaluated their
cytotoxic effects in vitro.
3. Pharmacology
The chalcones 3a, b and the derived Mannich 4ael bases
under investigation were tested on the BV-173 pre-B-cell
leukemia-derived cell line (following 72 h exposure) as
a pre-screen test system, using the MTT-dye reduction assay.
Selected compounds were thereafter evaluated on the
chronic myeloid leukemia K-562 cells as well. The clinically
utilized anticancer drugs etoposide and cisplatin were used
as positive controls throughout the cytotoxicity determina-
tion studies.
As evident from the IC50 values, summarized in Table 1 all
tested compounds exerted cytotoxic effects on BV-173 lym-
phoid cells in a concentration-dependent manner, causing
50% inhibition of the malignant cell proliferation at low mi-
cromolar concentrations, ranging between 4.7 and 18.4 mM.
The comparative evaluation of the N non-substituted com-
pounds 3a and 3b (Scheme 1) revealed that the 4-methoxy
function in the B-ring of the latter affords a substantial, 2-
fold increase in the cytotoxic efficacy against BV-173. Such
a trend was not unambiguously encountered with the Mannich
bases and even more, in case of 4g and 4h the presence of the
methoxy substituent caused significant decrease in potency
than their corresponding analogues 4a and 4b.
The juxtaposition of the cytotoxic effects within non-me-
thoxy substituted analogues 4aef and their prototype 3a re-
vealed that all of the Mannich bases bearing bulky basic
substituents at the nitrogen of the oxazole ring were more potent
than the prototype. Within the series of methoxy chalcones,
namely 3b and its N-substituted analogues 4gel the similar or
even equivalent activity encountered thereof does not allow
clear-cut structure activity relationships to be defined.
Compounds 4aef and 4iel, were tested on the chronic my-
eloid leukemia-derived K-562 cell line as well (Table 1). They
induced 50% inhibition of the malignant cell growth at higher
concentrations relative to their effects on BV-173. Most prob-
ably this is due to the higher expression of the non-receptor
tyrosine kinase bcr-abl in K-562 as compared to BV-173,
2. Chemistry
Twelve new 3-(aminomethyl)-6-(3-aryl-2-propenoyl)-2(3H )-
benzoxazolone derivatives (compounds 4ael) were synthesized.
The synthesis of Mannich bases was carried out by the sequence
of reactions summarized in Scheme 1.
2(3H )-Benzoxazolone was acylated in AlCl3eDMF with
acetyl chloride by a modified method of FriedeleCrafts. In
acylation, since both the 3-nitrogen and 1-oxygen atoms are
electron-donating, both 5- and 6-positions are activated and
therefore, the regioselectivity in the C-acylation of 2(3H )-ben-
zoxazolone cannot be predicted. However, it was reported that
the substitution was directed by the nitrogen atom of 2(3H )-
benzoxazolone ring and only the 6-acyl derivative was formed
[15].
The chalcone 3a and its 4-methoxy analogue 3b was
synthesized by a classic ClaiseneSchmidt condensation of 6-
acetyl-2(3H )-benzoxazolone 2 with benzaldehyde and 4-me-
thoxybenzaldehyde [16].
The Mannich reaction was used for the synthesis of com-
pounds 4ael. In this method the initial compounds 3a and 3b
are heated in ethanol with 37% formaldehyde solution and sec-
ondary amines. Even though the classical literature suggests

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Y. Ivanova et al. / European Journal of Medicinal Chemistry 42 (2007) 1382e1387
H
N
H
N
H
N
R1
i
ii
O
O
O
H3C
O
O
O
O
O
3a,b
2
R2
N
R1
iii
O
O
O
4
Reagents and conditions: (i) CH3COCl, AlCl3-DMF; (ii) R1C6H4CHO, aq. KOH, EtOH;
(iii) secondary amine, 37% CH2O, EtOH
Compound
R1
-H
R2
Compound
R1
R2
-OCH3
-H
-H
3a
3b
CH3
CH3
CH3
4a
4g
CH2
N
-OCH3
-H
CH2
N
CH3
CH2
CH2
CH2
CH2
N
N
N
N
-OCH3
-OCH3
-OCH3
-OCH3
4b
4c
4d
4e
CH2
CH2
CH2
CH2
N
N
N
N
4h
4i
-H
-H
-H
O
S
O
4j
S
4k
N
-H
-H
N
H3CO
H3CO
4f
4l
CH2
N
N CH2
-OCH3
CH2
N
N CH2
Scheme 1. Schematic representation of the synthesis and chemical structures of the tested compounds: prototype chalcones 2 and 3 and the respective series of
Mannich bases 4ael.
which renders the former cell line less responsive to pro-apo-
ptotic stimuli including chemotherapeutic agents [18].
of heterocyclic chalcones represents a prospective class of
cytotoxic agents. Notwithstanding the fact that the intimate
molecular targets and the precise mode of action of these novel
synthetic chalcones are yet to be determined, it is clearly evi-
dent from the encountered DNA-laddering that the induction
of apoptosis at least partly mediates their cytotoxic activity.
Currently novel large series of analogues have been synthe-
sized and are subjected to pharmacological testing in view of
defining prospective lead compounds for further evaluations.
In order to elucidate the mechanisms underlying the estab-
lished cytotoxicity the methoxy-compounds 4j and 4k were
tested for their ability to trigger DNA fragmentation. Both
compounds were applied at concentrations lower than their
IC50 and were found to evoke a typical DNA-laddering phe-
nomenon in BV-173 (Fig. 2). These results indicate that the in-
duction of apoptosis at least partly mediates the cytotoxic
activity of the investigated compounds as repeatedly reported
for diverse chalcones and related plant polyphenols [3,12,19].
5. Experimental protocols
4. Conclusions
5.1. Chemistry
The results obtained from the conducted bioassay of the pre-
sented compounds give us reason to conclude that this series
Melting points were determined on a Boetius hot-stage
microscope and were uncorrected. IR spectra (nujol) were

Y. Ivanova et al. / European Journal of Medicinal Chemistry 42 (2007) 1382e1387
1385
Table 1
(8.1 g, 60 mmol) and acetyl chloride (6.4 ml, 90 mmol) were
added. The reaction mixture was heated at 80 ꢂC under stirring
for 2 h. Then mixture was poured on ice and conc. HCl
(30 ml). The crude product was collected by filtration, air-
dried and recrystallized from ethanol. Yield 6.7 g (63%);
m.p. 227e228 ꢂC [Ref. [20], m.p. 228 ꢂC].
Cytotoxic activity of tested heterocyclic chalcone compounds and the refer-
ence cytotoxic agents on BV-173 and K-562 cells after 72 h incubation
(MTT assay)
Compound
IC50 value (mM) ꢁ SD
BV-173
K-562
3a
3b
18.4 ꢁ 2.2
9.2 ꢁ 1.4
6.6 ꢁ 0.9
8.2 ꢁ 1.3
7.4 ꢁ 1.7
7.9 ꢁ 2.1
9.2 ꢁ 2.7
9.4 ꢁ 2.3
17.7 ꢁ 3.1
15.3 ꢁ 1.3
4.7 ꢁ 1.1
6.1 ꢁ 1.6
5.4 ꢁ 1.2
6.2 ꢁ 2.1
0.9 ꢁ 0.5
7.9 ꢁ 1.8
N.d.a
N.d.
5.1.2. General procedure for preparation of 6-(3-aryl-2-
propenoyl)-2(3H )-benzoxazolones 3a, b
4a
4b
27.2 ꢁ 1.7
38.3 ꢁ 3.3
31.7 ꢁ 2.4
42.2 ꢁ 3.1
30.1 ꢁ 1.7
29.5 ꢁ 2.8
N.d.
Compounds 3a, b were prepared following reported proce-
dures [16]. To a solution of 6-acetyl-2(3H )-benzoxazolone
(2.65 g, 10 mmol) in mixture of 10% aq. KOH (10 ml) and
ethanol (5 ml), the appropriate aldehyde (11 mmol) was
added. After stirring for a few hours at room temperature,
the mixture precipitated. The mixture was poured on 200 ml
water, warmed and acidified with 10% HCl. The crystalline
product was filtered, washed to neutrality, dried and recrystal-
lized from ethanol.
4c
4d
4e
4f
4g
4h
4i
N.d.
22.7 ꢁ 3.1
21.2 ꢁ 3.9
12.4 ꢁ 1.6
24.2 ꢁ 2.7
3.1 ꢁ 1.3
29.2 ꢁ 2.9
4j
4k
4l
Etoposide
Cisplatin
a
5.1.2.1. 6-(3-Phenyl-2-propenoyl)-2(3H )-benzoxazolone 3a.
Yield 84%; m.p. 223e225 ꢂC; IR: 3100e3300 (NH), 1765 (lac-
tam C]O), 1655 (ketone C]O) cmꢀ1; 1H NMR: 7.20 (d, 1H,
arom. H, J ¼ 8.2 Hz); 7.41 (m, 3H, arom. H); 7.53 (d, 1H,
]CHCO, J ¼ 15.6 Hz); 7.62 (m, 2H, arom. H); 7.84 (d, 1H,
ArCH], 15.6 Hz); 7.93 (d, 1H, arom. H, J ¼ 1.5 Hz), 7.96
(dd, 1H, arom. H, J ¼ 1.5 Hz, J ¼ 8.2 Hz), 10.93 (br. 1H, NH).
Not determined.
recorded on a Specord 71 spectrometer. 1H NMR spectra were
recorded on a Bruker AM 250 operating in a field strength of
250 MHz. Chemical shifts are given in parts per million (d)
values downfield from TMS as internal standard and coupling
constants (J ) in Hz. The results of elemental analyses for C, H
and N were within ꢁ0.4% of the theoretical values.
5.1.2.2. 6-(3-(4-Methoxyphenyl)-2-propenoyl)-2(3H )-benzoxa-
zolone 3b. Yield 81%; m.p. 209e211 ꢂC; IR: 3100e3350
5.1.1. 6-Acetyl-2(3H )-benzoxazolone 2
1
(NH), 1760 (lactam C]O), 1645 (ketone C]O) cmꢀ1; H
Dimethylformamide (13 ml, 172 mmol) was slowly added
to aluminum chloride (80 g, 600 mmol). The mixture was
stirred and maintained at 45 ꢂC, while 2(3H )-benzoxazolone
NMR: 3.85 (s, 3H, OCH3); 6.94 (d, 2H, arom. H,
J ¼ 8.7 Hz); 7.24 (d, 1H, arom. H, J ¼ 8.2 Hz); 7.40 (d, 1H,
]CHCO, J ¼ 15.5 Hz); 7.60 (d, 2H, arom. H, J ¼ 8.7 Hz);
7.82 (d, 1H, ArCH], J ¼ 15.5 Hz); 7.98 (d, 1H, arom. H,
J ¼ 1.4 Hz); 7.95 (dd, 1H, arom. H, J ¼ 1.5 Hz, J ¼ 8.2 Hz),
11.14 (br. 1H, NH).
5.1.3. General procedure for preparation of Mannich bases
4ael
A mixture of 6-(3-aryl-2-propenoyl)-2(3H )-benzoxazolone
(0.27 g, 1 mmol), 37% formaldehyde solution (0.1 ml, 1.3
mmol) and ethanol (8 ml) was heated and secondary amine
(1 mmol) was added. After standing for 24 h at room tempera-
ture the mixture crystallized, filtered off, washed with ethanol,
dried and recrystallized from ethanol.
5.1.3.1. 3-(Dimethylaminomethyl)-6-(3-phenyl-2-propenoyl)-
2(3H )-benzoxazolone 4a. Yield 53%; m.p. 225e227 ꢂC; IR:
1
1766 (lactam C]O), 1653 (ketone C]O) cmꢀ1; H NMR:
2.44 (s, 6H, CH3); 4.64 (s, 2H, CH2), 7.22 (d, 1H, arom. H,
J ¼ 8.2 Hz); 7.43 (m, 3H, arom. H); 7.52 (d, 1H, ]CHCO,
J ¼ 15.6 Hz); 7.65 (m, 2H, arom. H); 7.85 (d, 1H, ArCH],
J ¼ 15.6 Hz); 7.91 (d, 1H, arom. H, J ¼ 1.5 Hz), 7.96 (dd,
1H, arom. H, J ¼ 1.5 Hz, J ¼ 8.2 Hz).
Fig. 2. Imaging of DNA-laddering, evidencing for induction of apoptosis, fol-
lowing treatment of BV-173 with 4j and 4k at a concentration lower than their
IC50 values (2.5 mM). DNA was extracted from the cytosolic fraction of
1 ꢃ 106 exponentially growing BV-173 cells exposed for 24 h to either
2.5 mM 4j (lane 3), 2.5 mM 4k (lane 4) or to the reference agent etoposide
(at 0.25 mM, lane 5); solvent treated cells served as controls (lane 2); lane 1
represents a DNA-laddering marker. Thereafter the isolated DNA was ana-
lyzed by 0.8% agarose gel electrophoresis, ethidium bromide staining and
UV-transillumination.
5.1.3.2. 6-(3-Phenyl-2-propenoyl)-3-(1-piperidinylmethyl)-2(3H )
-benzoxazolone 4b. Yield 78%; m.p. 159e162 ꢂC; IR: 1766

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Y. Ivanova et al. / European Journal of Medicinal Chemistry 42 (2007) 1382e1387
1
(lactam C]O), 1653 (ketone C]O) cmꢀ1; H NMR: 1.44e
1.59 (m, 6H, piperidine H-3, 4, 5); 2.66 (m, 4H, piperidine
H-2, 6); 4.69 (s, 2H, CH2); 7.22 (d, 1H, arom. H, J ¼ 8.2 Hz);
7.43 (m, 3H, arom. H); 7.52 (d, 1H, ]CHCO, J ¼ 15.6 Hz);
7.65 (m, 2H, arom. H); 7.84 (d, 1H, ArCH], J ¼ 15.6 Hz);
7.89 (d, 1H, arom. H, J ¼ 1.4 Hz); 7.96 (dd, 1H, arom. H,
J ¼ 8.2 Hz, J ¼ 1.5 Hz).
1H, ArCH], J ¼ 15.5 Hz); 7.98 (d, 1H, arom. H,
J ¼ 1.4 Hz); 7.94 (dd, 1H, arom. H, J ¼ 1.5 Hz, J ¼ 8.2 Hz).
5.1.3.8. 6-(3-(4-Methoxyphenyl)-2-propenoyl)-3-(1-piperidi-
nylmethyl)-2(3H )-benzoxazolone 4h. Yield 86%; m.p. 158e
160 ꢂC; IR: 1766 (lactam C]O), 1640 (ketone C]O)
1
cmꢀ1; H NMR: 1.39e1.60 (m, 6H, piperidine H-3, 4, 5);
2.66 (m, 4H, piperidine H-2, 6); 3.86 (s, 3H, OCH3); 4.69
(s, 2H, CH2); 6.95 (d, 2H, arom. H, J ¼ 8.8 Hz); 7.21 (d,
1H, arom. H, J ¼ 8.2 Hz); 7.40 (d, 1H, ]CHCO,
J ¼ 15.6 Hz); 7.61 (d, 2H, arom. H, J ¼ 8.8 Hz); 7.82 (d,
1H, ArCH], J ¼ 15.6 Hz); 7.88 (d, 1H, arom. H,
J ¼ 1.4 Hz); 7.95 (dd, 1H, arom. H, J ¼ 1.5 Hz, J ¼ 8.2 Hz).
5.1.3.3.
3-(4-Morpholinylmethyl)-6-(3-phenyl-2-propenoyl)-
2(3H )-benzoxazolone 4c. Yield 67%; m.p. 177e180 ꢂC; IR:
1766 (lactam C]O), 1653 (ketone C]O) cmꢀ1; H NMR:
1
2.71 (m, 4H, morpholine H-3, 5); 3.71 (m, 4H, morpholine
H-2, 6); 4.68 (s, 2H, CH2); 7.20 (d, 1H, arom. H,
J ¼ 8.2 Hz); 7.43 (m, 3H, arom. H); 7.52 (d, 1H, ]CHCO,
J ¼ 15.6 Hz); 7.65 (m, 2H, arom. H); 7.84 (d, 1H, ArCH],
J ¼ 15.6 Hz); 7.91 (d, 1H, arom. H, J ¼ 1.4 Hz); 7.96 (dd,
1H, arom. H, J ¼ 8.2 Hz, J ¼ 1.5 Hz).
5.1.3.9. 6-(3-(4-Methoxyphenyl)-2-propenoyl)-3-(4-morpholi-
nylmethyl)-2(3H )-benzoxazolone 4i. Yield 66%; m.p. 168e
170 ꢂC; IR: 1766 (lactam C]O), 1640 (ketone C]O)
1
cmꢀ1; H NMR: 2.71 (m, 4H, morpholine H-3, 5); 3.71 (m,
5.1.3.4. 6-(3-Phenyl-2-propenoyl)-3-(4-thiomorpholinylmethyl)-
4H, morpholine H-2, 6); 3.86 (s, 3H, OCH3); 4.68 (s, 2H,
CH2); 6.95 (d, 2H, arom. H, J ¼ 8.7 Hz); 7.19 (d, 1H, arom.
H, J ¼ 8.2 Hz); 7.40 (d, 1H, ]CHCO, J ¼ 15.6 Hz); 7.62 (d,
2H, arom. H, J ¼ 8.7 Hz); 7.82 (d, 1H, ArCH],
J ¼ 15.6 Hz); 7.90 (d, 1H, arom. H, J ¼ 1.4 Hz); 7.95 (dd,
1H, arom. H, J ¼ 1.5 Hz, J ¼ 8.2).
2(3H )-benzoxazolone 4d. Yield 87%; m.p. 180e183 ꢂC; IR:
1
1766 (lactam C]O), 1653 (ketone C]O) cmꢀ1; H NMR:
2.68 (m, 4H, thiomorpholine H-2, 6); 3.00 (m, 4H, thiomorpho-
line H-3, 5); 4.70 (s, 2H, CH2); 7.17 (d, 1H, arom. H,
J ¼ 8.2 Hz); 7.43 (m, 3H, arom. H); 7.52 (d, 1H, ]CHCO,
J ¼ 15.6 Hz); 7.66 (m, 2H, arom. H); 7.85 (d, 1H, ArCH],
J ¼ 15.6 Hz); 7.90 (d, 1H, arom. H, J ¼ 1.4 Hz); 7.96 (dd, 1H,
arom. H, J ¼ 8.2 Hz, J ¼ 1.5 Hz).
5.1.3.10. 6-(3-(4-Methoxyphenyl)-2-propenoyl)-3-(4-thiomor-
pholinylmethyl)-2(3H )-benzoxazolone 4j. Yield 95%; m.p.
181e185 ꢂC; IR: 1766 (lactam C]O), 1640 (ketone C]O)
1
5.1.3.5. 3-((4-(2-Methoxyphenyl)-1-piperazinyl)methyl)-6-(3-
phenyl-2-propenoyl)-2(3H )-benzoxazolone 4e. Yield 98%;
m.p. 149e151 ꢂC; IR: 1766 (lactam C]O), 1653 (ketone
cmꢀ1; H NMR: 2.68 (m, 4H, thiomorpholine H-2, 6); 3.00
(m, 4H, thiomorpholine H-3, 5); 3.86 (s, 3H, OCH3); 4.70
(s, 2H, CH2); 6.95 (d, 2H, arom. H, J ¼ 8.8 Hz); 7.16 (d,
1H, arom. H, J ¼ 8.2 Hz); 7.39 (d, 1H, ]CHCO,
J ¼ 15.6 Hz); 7.62 (d, 2H, arom. H, J ¼ 8.8 Hz); 7.82 (d,
1H, ArCH], J ¼ 15.6 Hz); 7.89 (d, 1H, arom. H,
J ¼ 1.4 Hz); 7.95 (dd, 1H, arom. H, J ¼ 1.5 Hz, J ¼ 8.2).
1
C]O) cmꢀ1; H NMR: 2.95e3.09 (m, 8H, piperazine H);
3.82 (s, 3H, CH3); 4.82 (s, 2H, CH2); 6.84e6.92 (m, 4H,
arom. H); 7.23 (d, 1H, arom. H, J ¼ 8.2 Hz); 7.43 (m, 3H,
arom. H); 7.52 (d, 1H, ]CHCO, J ¼ 15.6 Hz); 7.66 (m, 2H,
arom. H); 7.85 (d, 1H, ArCH], J ¼ 15.6 Hz); 7.90 (d, 1H,
arom. H, J ¼ 1.4 Hz); 7.98 (dd, 1H, arom. H, J ¼ 8.2 Hz,
J ¼ 1.5 Hz).
5.1.3.11. 3-((4-(2-Methoxyphenyl)-1-piperazinyl)methyl)-6-(3-
(4-methoxyphenyl)-2-propenoyl)-2(3H )-benzoxazolone
4k.
Yield 86%; m.p. 142e144 ꢂC; IR: 1766 (lactam C]O),
1
5.1.3.6. 3-((4-Benzyl-1-piperazinyl)methyl)-6-(3-phenyl-2-pro-
penoyl)-2(3H )-benzoxazolone 4f. Yield 80%; m.p. 167e
170 ꢂC; IR: 1766 (lactam C]O), 1653 (ketone C]O)
1640 (ketone C]O) cmꢀ1; H NMR: 2.87e3.01 (m, 8H, pi-
perazine H); 3.75 (s, 3H, CH3); 3.79 (s, 3H, CH3); 4.74 (s,
2H, CH2); 6.75e6.94 (m, 4H, arom. H); 6.87 (d, 2H, arom.
H, J ¼ 8.8 Hz); 7.15 (d, 1H, arom. H, J ¼ 8.2 Hz); 7.33 (d,
1H, ]CHCO, J ¼ 15.5 Hz); 7.54 (d, 2H, arom. H,
J ¼ 8.8 Hz); 7.74 (d, 1H, ArCH], J ¼ 15.5 Hz); 7.82 (d,
1H, arom. H, J ¼ 1.5 Hz), 7.88 (dd, 1H, arom. H,
J ¼ 1.5 Hz, J ¼ 8.2 Hz).
1
cmꢀ1; H NMR: 2.50e2.77 (m, 8H, piperazine H); 3.51 (s,
2H, CH2eAr); 4.72 (s, 2H, CH2); 7.17 (d, 1H, arom. H,
J ¼ 8.2 Hz); 7.28 (m, 5H, arom. H); 7.43 (m, 3H, arom. H);
7.52 (d, 1H, ]CHCO, J ¼ 15.6 Hz); 7.66 (m, 2H, arom.
H); 7.84 (d, 1H, ArCH], J ¼ 15.6 Hz); 7.92 (d, 1H, arom.
H, J ¼ 1.4 Hz); 7.95 (dd, 1H, arom. H, J ¼ 8.2 Hz,
J ¼ 1.5 Hz).
5.1.3.12. 3-((4-Benzyl-1-piperazinyl)methyl)-6-(3-(4-methoxy-
phenyl)-2-propenoyl)-2(3H )-benzoxazolone 4l. Yield 81%;
m.p. 168e171 ꢂC; IR: 1766 (lactam C]O), 1640 (ketone
;
C]O) cmꢀ1 1H NMR: 2.47e2.74 (m, 8H, piperazine H-
2, 6); 3.47 (s, 2H, CH2eAr); 3.85 (s, 3H, OCH3); 4.71 (s,
2H, CH2); 6.93 (d, 2H, arom. H, J ¼ 8.8 Hz); 7.15 (d, 1H,
arom. H, J ¼ 8.2 Hz); 7.27 (m, 5H, arom. H); 7.37 (d, 1H,
]CHCO, J ¼ 15.6 Hz); 7.60 (d, 2H, arom. H, J ¼ 8.8 Hz);
7.80 (d, 1H, ArCH], J ¼ 15.6 Hz); 7.86 (d, 1H, arom.
5.1.3.7. 3-(Dimethylaminomethyl)-6-(3-(4-methoxyphenyl)-2-
propenoyl)-2(3H )-benzoxazolone 4g. Yield 72%; m.p. 139e
142 ꢂC; IR: 1766 (lactam C]O), 1640 (ketone C]O)
1
cmꢀ1; H NMR: 2.44 (s, 6H, NCH3); 3.87 (s, 3H, OCH3);
4.64 (s, 2H, CH2); 6.95 (d, 2H, arom. H, J ¼ 8.7 Hz); 7.20
(d, 1H, arom. H, J ¼ 8.2 Hz); 7.40 (d, 1H, ]CHCO,
J ¼ 15.5 Hz); 7.63 (d, 2H, arom. H, J ¼ 8.7 Hz); 7.82 (d,

Y. Ivanova et al. / European Journal of Medicinal Chemistry 42 (2007) 1382e1387
1387
H, J ¼ 1.4 Hz); 7.91 (dd, 1H, arom. H, J ¼ 1.5 Hz,
J ¼ 8.2).
Zafiroska for the completion of the pharmacological investiga-
tions. They also thank Dr. Ivan Kozekov from Vanderbilt Uni-
versity (Nashville, Tennessee) for NMR spectra.
5.2. Pharmacology
5.2.1. Cell lines, culture conditions and cytotoxicity
determination
References
All of the procedures regarding the cell culture mainte-
nance, drug solution preparation and treatment of cells were
carried out in a ‘‘Heraeus’’ laminar flow cabinet. The stock so-
lutions of the referent antineoplastic agents e etoposide and
cisplatin were prepared using the commercially available ster-
ile dosage forms for clinical application, while the tested chal-
cones were dissolved in DMSO. The stock solutions were
consequently diluted with RPMI-1640 medium to yield the de-
sired final concentrations (in case of DMSO stocks at the final
dilutions obtained, the concentration of the solvent never ex-
ceeded 0.5%). The human tumor cell lines exploited in this
study, namely the pre-B-cell leukemia BV-173 and the chronic
myeloid leukemia K-562 cell were obtained from the German
Collection of Microorganisms and Cell Cultures (DSMZ
GmbH, Braunschweig, Germany). The cells were grown as
suspension cultures in a controlled environment: RPMI-1640
medium supplemented with 10% FBS and 2 mM L-glutamine
at 37 ꢂC in an incubator ‘‘BB 16-Function Line’’ Heraeus
(Kendro, Hanau, Germany) with humidified atmosphere and
5% CO2. Cells were maintained in log phase by supplementa-
tion with fresh medium after removal of cell suspension ali-
quots, two or three times a week.
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5.2.2. DNA fragmentation analysis
The cytosolic fraction DNA isolation and its electrophoretic
analysis was performed as previously described [22].
Acknowledgments
The authors greatly acknowledge the excellent technical
assistance of Mrs. Theodora Atanassova and Ms. Maya