New heterocyclic chalcones. Part 6. Synthesis and cytotoxic activities of 5- or 6-(3-aryl- 2-propenoyl)-2(3H)-benzoxazolones

Article abstract of DOI:10.1515/hc-2012-0081

A number of chalcones bearing an oxazole cycle were synthesized by Claisen-Schmidt condensation of 5-acetyl-2(3 H )-benzoxazolone or 6-acetyl-2(3 H )-benzoxazolone and the appropriate aldehydes. The chalcones were evaluated for cytotoxic activity against several tumor cell lines - BV-173 (human B cell precursor leukemia), MCF-7 and MDA-MB-231 (human breast adenocarcinoma) using the MTT-dye reduction assay. The tested compounds exhibit concentration- dependent cytotoxic effects at micromolar concentrations. Exposure of the BV-173 tumor cell line to compound 3f results in strong monoand oligonucleosomal fragmentation of genomic DNA, as evidenced by a 'cell death detection' ELISA kit, which unambiguously indicates that the induction of apoptosis is implicated in the cytotoxic mode of action of the tested compound.

Full text of DOI:10.1515/hc-2012-0081

ꢁꢁꢁꢂ
DOI 10.1515/hc-2012-0081ꢀ ꢀHeterocycl. Commun. 2013; 19(1): 23–28
Yordanka B. Ivanova, Georgi T. Momekov and Ognyan I. Petrov*
New heterocyclic chalcones. Part 6. Synthesis
and cytotoxic activities of 5- or 6-(3-aryl-
2-propenoyl)-2(3H)-benzoxazolones
Abstract: A number of chalcones bearing an oxazole cycle
Previous studies have indicated that chalcones
were synthesized by Claisen-Schmidt condensation of and their derivatives demonstrate anticancer activity
5-acetyl-2(3H)-benzoxazolone or 6-acetyl-2(3H)-benzox- in various tumor cells – ovarian cancer cells [9], gastric
azolone and the appropriate aldehydes. The chalcones cancer HGC-27 cells [10], HepG2 hepatocellular carcinoma
were evaluated for cytotoxic activity against several tumor cells [12], human melanoma cells A375 [13], KB human
cell lines – BV-173 (human B cell precursor leukemia), buccal carcinoma cells [14], PC-3 prostate cells, MCF-7
MCF-7 and MDA-MB-231 (human breast adenocarcinoma) breast cells and KB nasopharyngeal cancer cells [15].
using the MTT-dye reduction assay. The tested com-
Our research group is interested in the synthesis and
pounds exhibit concentration-dependent cytotoxic effects investigation of biological effects of chalcones. Recently,
at micromolar concentrations. Exposure of the BV-173 we have reported the synthesis of some chalcones sub-
tumor cell line to compound 3f results in strong mono- stituted with a 2(3H)-benzoxazolone moiety that have
and oligonucleosomal fragmentation of genomic DNA, showed good to excellent cytotoxic activity [16–18]. Our
as evidenced by a ‘cell death detection’ ELISA kit, which studies with BV-173 leukemic cells have suggested that the
unambiguously indicates that the induction of apoptosis cytotoxic effect of these agents is at least partly mediated
is implicated in the cytotoxic mode of action of the tested by induction of apoptotic cell death. We have also investi-
compound.
gated the influence of an appended thiazole system in the
chalcone scaffold [19] on the cytotoxic activity against the
Keywords: apoptosis; 2(3H)-benzoxazolone; chalcone; human chronic lymphoid leukemia SKW-3 tumor cells. In
cytotoxicity; MTT.
this report, we further extended our research on oxazole-
bearing chalcone derivatives and evaluated their cytotoxic
activity.
*Corresponding author: Ognyan I. Petrov, Faculty of Chemistry
and Pharmacy, Department of Applied Organic Chemistry,
Sofia University St. Kl. Ohridski, 1164 Sofia, Bulgaria,
e-mail: opetrov@chem.uni-sofia.bg
Yordanka B. Ivanova: Faculty of Ecology and Landscape
Architecture, Department of Plant Pathology and Chemistry,
University of Forestry, 1756 Sofia, Bulgaria
Georgi T. Momekov: Faculty of Pharmacy, Department of
Pharmacology, Pharmacotherapy and Toxicology, Medical
University, Sofia, Bulgaria
Results and discussion
Synthesis
New 5- and 6-(3-aryl-2-propenoyl)-2(3H)-benzoxazolones
3a–l (Equation 1) were prepared by treating 6-acetyl-2(3H)-
benzoxazolone (1) or 5-acetyl-2(3H)-benzoxazolone (2)
with different methoxy-substituted benzaldehydes. The
general synthetic strategy employed to prepare the chal-
cones (3a–l) was based on Claisen-Schmidt condensation.
The reaction was performed under standard condi-
Introduction
Natural and synthetic chalcones have become impor-
tant building blocks in medicinal chemistry and a tions by maintaining the mixture in aqueous ethanolic
number of derivatives endowed with anti-inflammatory, solution containing 10% KOH at room temperature for 24 h.
antimicrobial, antifungal, antioxidant, cytotoxic, antitu- The structures of the new compounds were confirmed by
mor, anticancer and chemopreventive effects have been IR, 1H NMR, 13C NMR and elemental analysis. In particular,
synthesized [1–7]. These compounds are important inter- analysis of 1H NMR spectra revealed that all structures are
mediates for the synthesis of heterocyclic systems and geometrically pure with the E configuration, as derived
play a role in organic syntheses as Michael acceptors [8].
from coupling constant J = 15.6 Hz for vinyl protons.

ꢁꢁꢁꢂ
24ꢀ
ꢀY. B. Ivanova et al.: New heterocyclic chalcones
R2
R2
R3
R4
R1
R3
R4
R1
KOH
X
Y
X
Y
(1)
O
O
Me
EtOH
CHO
O
1, 2
3a-l
O
1: X = NH; Y = O
2: X = O; Y = NH
3f: X = NH; Y = O; R1 = H; R2 = R3 = R4 = OMe
3g: X = O, Y = NH; R1 = OMe; R2 = R3 = R4 = H
1
2
3
4
1
2
3
4
3a
3b
3h
: X = O; Y = NH; R = H; R = OMe; R = R = H
: X = NH; Y = O; R = OCH3; R = R = R = H
1
2
3
4
1
2
3
4
3i
: X = NH; Y = O; R = H; R = OMe; R = R = H
: X = O; Y = NH; R = R = H; R = OMe; R = H
3c: X = NH; Y = O; R1 = R2 = H; R3 = OMe; R4 = H
3j: X = O; Y = NH; R1= OMe; R2 = H; R3 = OMe; R4 = H
3d: X = NH; Y = O; R1 = OMe; R2 = H; R3 = OMe; R4 = H 3k: X = O; Y = NH; R1 = H; R2 = R3 = OMe; R4 = H
3e: X = NH; Y = O; R1 = H; R2 = R3 = OMe; R4 = H 3l: X = O; Y = NH; R1 = H; R2 = R3 = R4 = OMe
The lactam and ketone C=O stretching bands in the IR as shown in method B of Scheme 1. Commercially avail-
spectra are seen at approximately 1760 сm-1 and 1650 сm-1, able salicylamide (8) was acetylated with acetyl chloride
respectively.
by the Friedel-Crafts method to obtain 5-acetylsalicyla-
mide (9) exclusively. The product was isolated with high
yield and purity. In this work, the Hofmann-type rear-
rangement of 9 to 2 was carried out in the presence of
C6H5I(OAc)2. The use of this hypervalent iodine reagent
gave the desired product 2 in an 89% yield. The mecha-
nism of this reaction apparently involves generation of
an isocyanate from the amide function followed by an
intramolecular nucleophilic attack on the isocyanate of
the adjacent hydroxyl group. The advantages of method
B are high total yield (89%), a two-step synthesis and the
use of only 3 equiv of AlCl3. By contrast, method A is less
efficient (40%), involves three steps and requires the use
of 8 equiv of AlCl3.
H
N
H
N
CH3COCl, AlCl3 - DMF
O
O
O
(2)
Me
O
O
4
1
We aimed to synthesize 2(3H)-benzoxazolone deriva-
tives bearing 3-phenyl-2-propenoyl substituents in posi-
tions 5 or 6, in order to study the influence of these func-
tionalities on cytotoxicity. To meet this objective, the
starting ketones were synthesized using different syn-
thetic routes. As indicated in Equation 2, 6-acetyl-2(3H)-
benzoxazolone (1) was synthesized by direct acetylation
of 2(3H)-benzoxazolone (4) with acetyl chloride in the
presence of the AlCl3-DMF complex [16]. The product Cytotoxic activity
was obtained in good yield and purity. Because the
C-acylation of 2(3H)-benzoxazolone is regioselective and The cytotoxic effects of compounds 3a–l were examined
always leads to a 6-acyl derivative, it was necessary to use against a panel of human tumor cell lines, namely BV-173
another synthetic route for preparation of 5-acetyl-2(3H)- (chronic myeloid leukemia), MCF-7 (estrogen receptor-
benzoxazolone (2). Aichaoui et al. [20] suggested a three- positive breast cancer) and MDA-MB-231 (estrogen recep-
step synthesis for converting 2-acetamidophenol (5) to tor-negative breast cancer) following a 72-h exposure. The
5-acetyl-2(3H)-benzoxazolone (2), as shown in method A activity was assessed by the MTT-dye reduction assay as
of Scheme 1. Following this procedure to prepare the start- described by Mosmann, with minor modifications [22]. All
ing ketone we modified the last step. Thus, the cyclization compounds exhibited concentration-dependent cytotox-
of 2-aminophenol to 2(3H)-benzoxazolone was conducted icity which enabled the construction of the concentration-
under mild conditions and with a higher yield than that response curves and the calculation of the corresponding
reported by using 1,1′-carbonyldiimidazole [21].
IC50 values (Table 1). The clinically utilized anticancer
Amide, azide and hydroxamic acid derivatives of sali- drug cisplatin was used as a positive control throughout
cylic acid can be used for the synthesis of 2(3H)-benzoxa- the cytotoxicity determination studies.
zolones by Hofmann, Curtius and Lossen rearrangements.
The data indicate that almost all compounds display
For the preparation of 5-acetyl-2(3H)-benzoxazolone (2), certain cytotoxic activity against the three tumor cells,
we developed an alternative two-step synthetic route whereby invariably BV-173 cells are more sensitive, as
using Hofmann rearrangement of 5-acetylsalicylamide (9) compared with the breast cancer-derived cell lines. Within

ꢂꢁꢁꢁ
Y. B. Ivanova et al.: New heterocyclic chalconesꢀ
ꢀ25
O
O
O
NHCOMe
OH
NHCOMe
OH
NH2
OH
a
b
e
Me
Me
Me
5
8
6
9
7
2
c
O
H
CONH2
OH
CONH2
OH
N
O
O
Me
d
Scheme 1ꢀMethod A: (a) MeCOCl, AlCl3-DMF; (b) conc. HCl; (c) 1,1′-carbonyldiimidazole, THF; Method B: (d) MeCOCl, AlCl3, MeNO2, CH2Cl2;
(e) C6H5I(OCOMe)2, KOH, MeOH.
Table 1ꢁCytotoxic effects of compounds 3a–l against the panel of
human tumor cell lines as assessed by the MTT-dye reduction assay
after 72-h exposure.
In general, the greatest chemosensitivity to the tested
series was established in BV-173 cells followed by the two
breast cancer cell lines MDA-MB-231 > MCF-7.
To elucidate the mechanisms underlying the estab-
lished cytotoxicity, we investigated the level of apoptotic
fragmentation of genomic DNA, using a commercially
available ‘cell death detection’ ELISA kit. This method
allows semiquantitative determination of the histone-
associated mono- and oligonucleosomal DNA fragments
using ‘sandwich’ ELISA. The clinically applied anticancer
drug cisplatin was used as a reference compound.
As indicated in Figure 1, chalcone 3f treatment of
BV-173 cells, even in concentrations lower than its IC50
value, leads to a significant elevation of the enrichment
factor (corresponding to the level of histone-associated
DNA fragments). The established proapoptotic activity is
Compound
IC50 value (μm) SD
MDA-MB-231
BV-173
MCF-7
3a
22.9 1.1
151.9 11.1
9.3 1.4
27.4 1.8
28.7 1.4
8.3 1.5
38.3 2.1
288.9 12.0
144.7 6.5
282.9 11.9
67.7 4.2
131.2 5.1
25.9 1.9
41.2 2.2
400
204.2 9.8
42.7 1.9
34.8 1.9
8.7 2.1
27.9 1.4
192.5 10.2
92.2 2.9
179.3 5.1
45.7 3.1
15.5 1.1
21.7 1.3
33.8 1.8
262.9 7.3
38.9 2.6
142.4 4.4
16.7 0.9
7.9 1.8
3b
3c
3d
3e
3f
3g
3h
3i
10.5 1.3
12.1 2.7
58.8 3.9
71.1 2.4
11.7 1.2
4.9 0.7
3j
3k
3l
Cisplatin
7.6 1.7
Control
Chalcone 3f
Cisplatin
5
4
3
2
1
0
the BV-173 bioassay in both series of compounds, 3,4,5-tri-
methoxy analogs 3f and 3l displayed superior activity,
causing 50% inhibition of cellular viability at low micro-
molar concentrations. Our data demonstrate that chal-
cone 3l exerts the most pronounced cytotoxic activity with
an IC50 value of 4.9 μm, whereas an IC50 value for cisplatin
is 7.6 μm under similar conditions. This compound is also
relatively active against the breast cancer cell lines. Among
the compounds bearing one methoxy group in ring B, the
ortho-position gives promising cytotoxic activity against
MDA-MB-231 and MCF-7 cell lines. Derivatives with the
meta-methoxy group show significantly less activity in the
series of 6-substituted 2(3H)-benzoxazolones. This finding
is in contrast within activity of the 5-substituted 2(3H)-
benzoxazolone derivatives, where chalcone 3i bearing the
methoxy group in para-position shows diminished activ-
ity. Introduction of an additional methoxy group in B-ring
A
B
C
Figure 1ꢀIncrease in the levels of histone-associated DNA frag-
ments (expressed as enrichment factor), following 24 h chalcone 3f
treatment in BV-173 [at IC50 (plot A), ½ IC50 μm (plot B), and ¼ IC50
imparts insignificant modulation of the biological activity. (plot C)] as assessed by the ‘cell death detection’ ELISA.

ꢁꢁꢁꢂ
26ꢀ ꢀY. B. Ivanova et al.: New heterocyclic chalcones
comparable to that of the reference drug cisplatin. These
findings suggest that cytotoxicity of these agents is medi-
ated by induction of cell death through apoptosis, as
already demonstrated for a series of previously described
cytotoxic chalcones [23, 24].
General procedure for synthesis of chalcones
3a–l
To a solution of 5- or 6-acetyl-2(3H)-benzoxazolone [20] (2 mmol)
in a mixture of 10% aq. KOH (2 mL) and ethanol (3 mL), aldehyde
(2.2 mmol) was added. Afer stirring for 24 h at room temperature,
the mixture precipitated, was poured on 30 mL water, warmed and
acidified with 10% HCl. The crystalline product was filtered, washed
to neutrality and dried.
Conclusions
6-[3-(2-Methoxyphenyl)-2-propenoyl]-2(3Н)-benzoxazolone
1
(3a)ꢀYield 98%; mp 212–215°C; IR: 1779, 1643 (C=O) cm-1; Н NMR:
Although the precise mode of action of tested compounds
is yet to be determined, a possible mechanism may involve
inhibition of tubulin polymerization, as firmly established
for diverse structurally related chalcones. The SAR investi-
gations failed to indicate the optimal substitution patterns
governing cytotoxic potencies and it appears that com-
pounds with multiple methoxy groups are characterized
by superior activity.
δ 3.90 (s, 3H, OCH3); 7.04 (m, 1H, arom H); 7.12 (d, 1H, arom H, J = 8.0
Hz); 7.24 (d, 1H, arom H, J = 8.0 Hz); 7.43–7.48 (m, 1H, arom H); 7.91
(d 1H, =CHCO, J = 15.7 Hz); 8.00–8.08 (m, 4H, ArCH=, arom H); 12.09
(br s, 1H, NH); 13C NMR: 55.7, 109.3, 109.5, 111.7, 120.6, 121.4, 122.9, 125.6,
128.3, 131.9, 132.3, 134.9, 138.1, 143.5, 154.5, 158.2, 187.4. Anal. Calcd for
C17H13NO4: C, 69.15; H, 4.44; N, 4.74. Found: C, 69.40; H, 4.54; N, 4.72.
6-[3-(3-Methoxyphenyl)-2-propenoyl]-2(3Н)-benzoxazolone (3b)
Yield 98%; mp 229–232°C; IR: 1743, 1657 (C=O) cm-1; 1Н NMR: δ 3.84 (s,
3H, OCH3); 7.03 (m, 1H, arom H); 7.24 (d, 1H, arom H, J = 8.2 Hz); 7.42
(m, 3H, arom H); 7.72 (d, 1H, =CHCO, J = 15.5 Hz); 7.99 (d, 1H, ArCH=,
J = 15.5 Hz); 8.08 (dd, 1H, arom H, J1 = 8.2 Hz, J2 = 1.6 Hz); 8.13 (d, 1H,
arom H, J = 1.6 Hz); 12.10 (br s, 1H, NH); 13C NMR: 55.2, 109.5, 109.5,
113.3, 116.6, 121.7, 121.9, 125.7, 129.8, 131.7, 134.9, 136.1, 143.4, 143.7, 154.4,
159.6, 187.2. Anal. Calcd for C17H13NO4: C, 69.15; H, 4.44; N, 4.74. Found:
C, 68.76; H, 4.70; N, 4.77.
Experimental section
All research chemicals were purchased from Sigma-Aldrich (St. Louis,
MO, USA). Solvents were dried and purified according to literature
procedures, as necessary. Reactions were monitored by thin layer
chromatography (TLC) on precoated silica gel plates from E. Merck 6-[3-(4-Methoxyphenyl)-2-propenoyl]-2(3Н)-benzoxazolone (3c)
(Darmstadt, Germany). Melting points were determined on a Boetius Yield 89%; mp 209–211°C; IR: 1760, 1645 (C=O) cm-1; 1Н NMR: δ 3.85 (s,
3H,OCH3);6.94(d,2H,aromH,J =8.7Hz);7.24(d,1H,aromH,J =8.2Hz);
7.40 (d, 1H, =CHCO, J = 15.5 Hz); 7.60 (d, 2H, arom H, J = 8.7 Hz); 7.82
(d, 1H, ArCH=, J = 15.5 Hz); 7.98 (d, 1H, arom H, J = 1.5 Hz); 7.95 (dd, 1H,
arom H, J1 = 1.5 Hz, J2 = 8.2 Hz), 11.14 (br s, 1H, NH); 13C NMR: 55.3,
109.4, 114.3, 119.2, 125.5, 127.3, 130.8, 132.0, 134.7, 143.4, 143.7, 154.4,
161.3, 187.1. Anal. Calcd for C17H13NO4: C, 69.15; H, 4.44; N, 4.74. Found:
C, 69.33; H, 4.24; N, 4.62.
hot-stage microscope and were uncorrected. IR spectra (Nujol) were
recorded on a Specord 71 spectrometer. 1H NMR spectra were record-
ed in DMSO-d6 on a Bruker DRX 250 operating at 250 MHz. 13C NMR
spectra were recorded in DMSO-d6 at 62.5 MHz.
Synthesis of 5-acetyl-2(3H)-benzoxazolone (2)
6-[3-(2,4-Dimethoxyphenyl)-2-propenoyl]-2(3Н)-benzoxazolone
(3d)ꢀYield 84%; mp 202–204°C; IR: 1785, 1643 (C=O) cm-1; 1Н NMR: δ
3.85 (s, 3H, OCH3); 3.91 (s, 3H, OCH3); 6.62–6.65 (m, 2H, arom H); 7.22
(d, 1H, arom H, J = 8.1 Hz); 7.79 (d, 1H, =CHCO, J = 15.6 Hz); 7.96 (m,
4H, ArCH=, arom H); 12.04 (br s, 1H, NH); 13C NMR: 55.5, 55.8, 98.2,
106.3, 109.2, 109.3, 115.9, 118.6, 125.3, 129.9, 132.2, 134.6, 138.3, 143.4,
154.4, 159.8, 163.0, 187.2. Anal. Calcd for C18H15NO5: C, 66.46; H, 4.65;
N, 4.31. Found: C, 66.78; H, 4.72; N, 4.49.
A mixture of dichloromethane (25 mL), nitromethane (5 mL) and alu-
minum chloride (9.98 g, 75 mmol) was stirred briefly, and afer cool-
ing to 0°C, was treated with salicylamide (3.43 g, 250 mmol). Acetyl
chloride (3.93 g, 50 mmol) was added dropwise with stirring afer the
suspension became a clear solution. The mixture was stirred at room
temperature for an additional 4–5 h, and then poured on ice (100 g)
with conc. HCl (20 mL). The crude 5-acetylsalicylamide (9) was fil-
tered, washed with water, dried and crystallized from ethanol; yield
4.27 g (95%); mp 216–217°C.
To a solution of KOH (1.32 g, 20 mmol) in methanol (20 mL),
5-acetylsalicylamide (1.8 g, 10 mmol) was added. The resulting
suspension was cooled to 0°C and iodobenzene diacetate (3.22 g,
10 mmol) was added within 5 min. The mixture was stirred for 1 h and
acidified with 10% HCl. Water (20 mL) and petroleum ether (20 mL)
were then added and the mixture stirred for 10 min. The product was
filtered and washed with water and petroleum ether, and crystallized
from ethanol; yield 1.58 g (89%); mp 230–232°C; IR: 3100–3300 (NH),
1780, 1660 (CO) cm-1; 1Н NMR: δ 2.57 (s, 3H, CH3), 7.37 (d, 1H, arom H,
6-[3-(3,4-Dimethoxyphenyl)-2-propenoyl]-2(3Н)-benzoxazolone
1
(3e)ꢀYield 83%; mp 210–212°C; IR: 1785, 1643 (C=O) cm-1; Н NMR:
δ 3.82 (s, 3H, OCH3); 3.86 (s, 3H, OCH3); 7.02 (d, 1H, arom H, J = 8.3 Hz);
7.24 (d, 1H, arom H, J = 8.2 Hz); 7.39 (dd, 1H, arom H, J1 = 8.3 Hz, J2 =
1.5 Hz); 7.55 (d, 1H, arom H, J = 1.5 Hz); 7.70 (d, 1H, =CHCO, J = 15.4
Hz); 7.86 (d, 1H, ArCH=, J = 15.4 Hz); 8.07–8.13 (m, 2H, arom H). Anal.
Calcd for C18H15NO5: C, 66.46; H, 4.65; N, 4.31. Found: C, 66.51; H, 4.46;
N, 4.79.
6-[3-(3,4,5-Trimethoxyphenyl)-2-propenoyl]-2(3Н)-benzo-
J = 8.4Hz),7.57(d,1H,aromH,J = 1.8Hz),7.78(dd,1H,aromH,J = 1.8Hz, xazolone (3f)ꢀYield 87%; mp 218–220°C; IR: 1650, 1780 (C=O) cm-1;
J = 8.4 Hz), 11.8 (br s, 1H, NH).
1Н NMR: δ 3.72 (s, 3H, OCH3); 3.86 (s, 6H, OCH3); 7.24 (m, 2H, arom H);

ꢂꢁꢁꢁ
Y. B. Ivanova et al.: New heterocyclic chalconesꢀ
ꢀ27
7.26 (d, 1H, arom H, J = 8.2 Hz); 7.70 (d 1H, =CHCO, J = 15.5 Hz); 7.92 7.75 (d, 1H, arom H, J = 1.7 Hz); 7.91 (d, 1H, ArCH=, J = 15.5 Hz); 8.05
(d, 1H, ArCH=, J = 15.5 Hz); 8.08 (dd, 1H, arom H, J1 = 8.2 Hz, J2 = 1.5 Hz); (dd, 1H, arom H, J1 = 8.4 Hz, J2 = 1.7 Hz); 13C NMR: 56.0, 60.0, 106.4,
8.12 (d, 1H, arom H, J = 1.5 Hz); 12.09 (br s, 1H, NH); 13C NMR: 56.1, 109.4, 120.9, 123.9, 130.1, 130.9, 133.7, 133.7, 139.7, 144.4, 146.7, 153.0,
60.1, 106.5, 109.5, 120.8, 125.6, 130.2, 131.9, 134.9, 139.6, 143.4, 144.2, 154.2, 187.7. Anal. Calcd for C19H17NO6: C, 64.22; H, 4.82; N, 3.94. Found:
153.0, 154.4, 187.1. Anal. Calcd for C19H17NO6: C, 64.22; H, 4.82; N, 3.94. C, 64.33; H, 4.61; N, 3.94.
Found: C, 64.51; H, 5.05; N, 4.24.
5-[3-(2-Methoxyphenyl)-2-propenoyl]-2(3Н)-benzoxazolone (3g)
Biological activities
Yield 93%; mp 199–201°C; IR: 1790, 1643 (C=O) cm-1; 1Н NMR: δ 3.90 (s,
3H, OCH3); 7.04 (m, 1H, arom H); 7.12 (d, 1H, arom H, J = 7.9); 7.45 (dd,
2H, arom H, J1 = 8.4, J2 = 1.9 Hz) 7.74 (d, 1H, arom H, J = 1.7 Hz); 7.89 (d,
1H, =CHCO, J = 15.7 Hz); 7.99 (dd, 2H, arom H, J1 = 8.4 Hz, J2 = 1.7 Hz);
8.06 (d, 1H, ArCH=, J = 15.7 Hz); 11.94 (br s, 1H, NH); 13C NMR: 55.7,
109.3, 109.4, 111.7, 120.6, 121.6, 123.8, 128.5, 130.9, 132.3, 133.7, 138.4,
146.7, 146.7, 158.2, 158.2, 187.8. Anal. Calcd for C17H13NO4: C, 69.15; H,
4.44; N, 4.74. Found: C, 69.36; H, 4.57; N, 4.79.
Cytotoxic activity (MTT-dye reduction assay)
The cytotoxic activity of the tested compounds was
assessed by the MTT-dye reduction assay as described by
Mosmann, with minor modifications [22]. Briefly, expo-
nentially growing cells were seeded into 96-well plates
(100 μL aliquots/well at a density of 1 × 105 cells/mL).
Following a 24-h adaptation period, they were exposed
to various concentrations of the tested compounds for
72 h. After the treatment period, MTT solution (10 mg/mL
in PBS) was added (10 μL/well). Plates were further
incubated for 4 h at 37°C and the MTT-formazan crys-
5-[3-(3-Methoxyphenyl)-2-propenoyl]-2(3Н)-benzoxazolone (3h)
1
Yield 95%; mp 180–181°C; IR: 1785, 1657 (C=O) cm-1; Н NMR: δ 3.84
(s, 3H, OCH3); 7.03 (m, 1H, arom H); 7.44–7.49 (m, 3H, arom H); 7.72
(d, 1H, =CHCO, J = 15.5 Hz); 7.77 (d, 1H, arom H, J = 1.7 Hz) 7.97 (d,
1H, ArCH=, J = 15.5 Hz); 8.05 (dd, 1H, arom H, J1 = 8.4 Hz, J2 = 1.7 Hz);
11.95 (br s, 1H, NH); 13C NMR: 55.2, 109.3, 113.4, 116.6, 121.6, 122.1, 123.9,
129.8, 130.9, 133.6, 135.9, 143.9, 146.8, 154.2, 159.5, 159.6, 187.8. Anal.
Calcd for C17H13NO4: C, 69.15; H, 4.44; N, 4.74. Found: C, 68.86; H, 4.28; tals formed were dissolved by adding 100 μL/well of 5%
N, 4.88.
formic acid in 2-propanol. Absorption was measured on
an ELISA reader (Uniscan® Titertek, Helsinki, Finland)
5-[3-(4-Methoxyphenyl)-2-propenoyl]-2(3Н)-benzoxazolone (3i)
at 540 nm. For each concentration at least eight wells
Yield 81%; mp 222–225°C; IR: 1771, 1650 (C=O) cm-1; 1Н NMR: δ 3.82 (s,
3H, OCH3); 7.01–7.03 (m, 2H, arom H); 7.44 (d, 1H, arom H, J = 8.4 Hz);
7.71 (d, 1H, =CHCO, J = 15.5 Hz); 7.74–7.79 (m, 2H, arom H, ArCH=); 7.86
(d, 2H, arom H, J = 7.2 Hz); 8.01 (d, 1H, arom H, J = 8.4 Hz); 11.93 (br
s, 1H, NH); 13C NMR: 55.3, 109.2, 109.3, 114.3, 114.3, 119.2, 123.7, 127.2,
130.7, 130.8, 130.9, 133.9, 143.9, 146.6, 154.2, 161.3, 187.6. Anal. Calcd for
C17H13NO4: C, 69.15; H, 4.44; N, 4.74. Found: C, 69.55; H, 4.56; N, 4.62.
were used. A mixture of 100 μL RPMI-1640 medium with
10 μL MTT stock and 100 μL 5% formic acid in 2-propa-
nol served as a blank solution. The cell viability (% of
untreated control) for each treatment group was calcu-
lated using the formula:
AT
% of untreated control= ×100
AC
5-[3-(2,4-Dimethoxyphenyl)-2-propenoyl]-2(3Н)-benzoxazolone
1
(3j)ꢀYield 85%; mp 239–243°C; IR: 1793, 1635 (C=O) cm-1; Н NMR:
δ 3.90 (s, 9H, OCH3); 6.60–6.67 (m, 2H, arom H); 7.43 (d, 1H, arom H,
J = 8.4 Hz); 7.71 (d, 1H, arom H, J = 1.4 Hz); 7.76 (d, 1H, =CHCO, J =
15.6 Hz); 7.91–8.02 (m, 3H, arom H, ArCH=); 11.91 (br s, 1H, NH); 13C
NMR: 55.5, 55.8, 98.2, 98.2, 106.3, 109.1, 109.3, 115.8, 118.7, 123.5, 130.0,
130.8, 134.1, 138.6, 146.5, 159.9, 163.0, 187.8. Anal. Calcd for C18H15NO5:
C, 66.46; H, 4.65; N, 4.31. Found: C, 66.52; H, 4.29; N, 4.47.
where AT denotes MTT-formazan absorption of the test
sample and AC denotes MTT-formazan absorption of the
control (solvent treated) sample.
Concentration response curves were generated and
the corresponding IC50 values were extrapolated using
Origin plot Software for PC.
5-[3-(3,4-Dimethoxyphenyl)-2-propenoyl]-2(3Н)-benzoxazolone
1
(3k)ꢀYield 88%; mp 208–210°C; IR: 1785, 1664 (C=O) cm-1; Н NMR:
δ 3.82 (s, 3H, OCH3); 3.87 (s, 3H, OCH3); 7.03 (d, 1H, arom H, J = 8.4 Hz);
7.40 (dd, 1H, arom H, J1 = 8.4, J2 = 2.0 Hz); 7.46 (d, 1H, arom H, J = 8.4
Hz); 7.55 (d, 1H, arom H, J = 2.0 Hz); 7.70 (d, 1H, =CHCO, J = 15.4 Hz);
7.75 (d, 1H, arom H, J = 1.8 Hz); 7.84 (d, 1H, ArCH=, J = 15.4 Hz); 8.03
(dd, 1H, arom H, J1 = 8.4 Hz, J2 = 1.8 Hz); 13C NMR: 55.6, 109.2, 110.7,
111.4, 119.3, 123.7, 123.9, 127.3, 130.8, 133.8, 142.6, 144.4, 145.7, 146.6,
148.9, 151.2, 154.2, 187.7. Anal. Calcd for C18H15NO5: C, 66.46; H, 4.65; N,
4.31. Found: C, 66.70; H, 4.78; N, 4.07.
Cell-death detection
The characteristic for apoptosis oligonucleosomal DNA
fragmentation was examined using a commercially
available ‘cell death detection’ ELISA kit (Roche Applied
Science). This method allows semiquantitative deter-
mination of the characteristic for the apoptotic process
of histone-associated mono- and oligonucleosomal
DNA fragments using ‘sandwich’ ELISA. Exponentially
5-[3-(3,4,5-Trimethoxyphenyl)-2-propenoyl]-2(3Н)-benzo-
xazolone (3l)ꢀYield 98%; mp 210–212°C; IR: 1771, 1657 (C=O) cm-1;
1Н NMR: δ 3.73 (s, 3H, OCH3); 3.87 (s, 6H, OCH3); 7.23–7.26 (m, 2H, arom
H); 7.48 (d, 1H, arom H, J = 8.4 Hz); 7.70 (d, 1H, =CHCO, J = 15.5 Hz); growing cells were exposed to varying concentrations of

ꢁꢁꢁꢂ
28ꢀ ꢀY. B. Ivanova et al.: New heterocyclic chalcones
the tested compounds and thereafter cytosolic fractions between the absorption in the treated vs. the solvent-
of 1 × 104 cells per group (treated or untreated) served treated control samples).
as an antigen source in a ‘sandwich’ ELISA, utilizing a
primary antihistone antibody-coated microplate and a Acknowledgments: The authors are thankful to the Uni-
secondary peroxidase-conjugated anti-DNA antibody. versity of Forestry, Sofia, Bulgaria (contract number
The photometric immunoassay for histone-associated 133/14.03.2012) and the University of Sofia, Sofia, Bulgaria
DNA fragments was executed according to the manu- (contract number 99/15.04.2011) for their financial support.
facturer’s instructions at 405 nm, using an ELISA reader
(Labexim LMR-1). The results are expressed as the oli-
Received May 20, 2012; accepted October 10, 2012; previously
gonucleosomal enrichment factor (representing a ratio published online January 23, 2013
References
[1] Di Carlo, G.; Mascolo, N.; Izzo, A.; Capasso, F. Flavonoids: old
and new aspects of a class of natural therapeutic drugs. Life
Sci. 1999, 65, 337–353.
[2] Dimmock, J.; Elias, D.; Beazely, M.; Kandepu, N. Bioactivities
of chalcones. Curr. Med. Chem. 1999, 6, 1125–1149.
[3] Go, M.; Wu, X.; Liu, X. Chalcones: an update on cytotoxic
and chemoprotective properties. Curr. Med. Chem. 2005,
12, 483–499.
[4] Katsori, A.-M.; Hadjipavlou-Litina, D. Chalcones in cancer:
understanding their role in terms of QSAR. Curr. Med. Chem.
2009, 16, 1062–1081.
[5] Kontogiorgis, C.; Mantzanidou, M.; Hadjipavlou-Litina, D.
Chalcones and their potential role in inflammation. Mini Rev.
Med. Chem. 2008, 8, 1224–1242.
progression and induce apoptosis in human melanoma cell
A375. Biol. Pharm. Bull. 2009, 32, 1109–1113.
[14] Quintin, J.; Desrivot, J.; Thoret, S.; Le Menez, P.; Cresteil, T.;
Lewin, G. Synthesis and biological evaluation of a series of
tangeretin-derived chalcones. Bioorg. Med. Chem. Lett. 2009,
19, 167–169.
[15] Reddy, M.; Su, C.; Chiou, W.; Liu, Y.; Chen, R.; Bastow, K.;
Lee, K.; Wua, T. Design, synthesis, and biological evaluation of
Mannich bases of heterocyclic chalcone analogs as cytotoxic
agents. Bioorg. Med. Chem. 2008, 16, 7358–7370.
[16] Ivanova, Y.; Petrov, O.; Gerova, M.; Momekov, G. Synthetic
chalcones of 2(3H)-benzothiazolones with potential
cytotoxic activity. Compt. Rend. Acad. Bulg. Sci. 2007, 60,
642–650.
[6] Makita, H.; Tanaka, T.; Fujitsuka, H.; Tatematsu, N.; Satoh, K.;
Hara, A.; Mori, H. Chemoprevention of 4-nitroquinoline 1-oxide-
induced rat oral carcinogenesis by the dietary flavonoids
chalcone, 2-hydroxychalcone, and quercetin. Cancer Res.
1996, 56, 4904–4909.
[7] Wattenberg, L.; Coccia, J.; Galbraith, A. Inhibition of
carcinogen-induced pulmonary and mammary carcinogenesis
by chalcone administered subsequent to carcinogen exposure.
Cancer Lett. 1994, 83, 165–169.
[8] Ceylan, M.; Gezegen, H. Preparation of 1,5-diketones by addition
of cyclohexanone to chalcones under solvent-free phase transfer
catalyst condition. Turk. J. Chem. 2008, 32, 55–61.
[9] De Vincenzo, R.; Scambia, G.; Benedetti, P.; Ranelletti, F.;
Bonanno, G.; Ercoli, A.; Delle, M.; Ferrari, F.; Piantelli, M.;
Mancuso, S. Effect of synthetic and naturally occurring
chalcones on ovarian cancer cell growth: structure-activity
relationships. Anti-Cancer Drug Des. 1995, 10, 481–490.
[10] Shibata, S. Anti-tumorigenic chalcones. Stem Cells 1994, 12,
44–52.
[11] Li, T.; Satomi, Y.; Katoh, D.; Shimada, J.; Baba, M.; Okuyama, T.;
Nishino, H.; Kitamura, N. Induction of cell cycle arrest and
p21CIP1/WAF1 expression in human lung cancer cells by
isoquiritigenin. Cancer Lett. 2004, 207, 27–35.
[17] Ivanova, Y.; Momekov, G.; Petrov, O. Synthesis of novel
substituted 1,3-diarylpropenone derivatives and their in vitro
cytotoxic activity. Lett. Drug Des. Discov. 2009, 6, 353–357.
[18] Petrov, O.; Ivanova, Y.; Momekov, G.; Kalcheva, V.
New synthetic chalcones: cytotoxic Mannich bases of
6-(4-chlorocinnamoyl)-2(3H)-benzoxazolone. Lett. Drug Des.
Discov. 2008, 5, 358–362.
[19] Ivanova, Y.; Momekov, G.; Petrov, O.; Karaivanova, M.;
Kalcheva, V. Cytotoxic Mannich bases of 6-(3-aryl-2-propenoyl)-
2(3H)-benzoxazolones. Eur. J. Med. Chem. 2007, 42,
1382–1387.
[20] Aichaoui, H.; Poupaert, J. H.; Henichart, J. P. Regioselectivity in
the C-acylation of 2(3H)-benzoxazolones. Tetrahedron 1991, 47,
6649–6654.
[21] Ivanova, Y.; Momekov, G.; Kalcheva, V.; Petrov, O. Synthesis
of chalcones condensed with an 1,3-azole ring using a SOCl2/
EtOH catalytic system. Compt. Rend. Acad. Bulg. Sci. 2008, 61,
41–48.
[22] Bakalova, A.; Buyukliev, R.; Tcholakova, I.; Momekov, G.;
Konstantinov, S.; Karaivanova, M. Synthesis, physicochemical
investigation and cytotoxic activity of new Pt(II) complexes
with hydantoin ligands. Eur. J. Med. Chem. 2003, 38, 627–632.
[23] Ducki, S. Antimitotic chalcones and related compounds as
inhibitors of tubulin assembly. Anti-Cancer Agents Med. Chem.
2009, 9, 336–347.
[24] Hadfield, J.; Ducki, S.; Hirst, N.; McGown, A. Tubulin and
microtubules as targets for anticancer drugs. Prog. Cell Cycle
Res. 2003, 5, 309–325.
[12] Yang, E.; Guo, Y.; Zang, K.; Chen, Y.; Mack, P. Inhibition of
epidermal growth factor receptor tyrosine kinase by chalcone
derivatives. Biochim. Biophys. Acta 2001, 1150, 144–152.
[13] Henmi, K.; Hiwatashi, Y.; Hikita, E.; Toyama, N.; Hirano, T.
Methoxy- and fluoro-chalcone derivatives arrest cell cycle

Copyright of Heterocyclic Communications is the property of De Gruyter and its content may not be copied or
emailed to multiple sites or posted to a listserv without the copyright holder's express written permission.
However, users may print, download, or email articles for individual use.