Synthesis and evaluation of a series of novel imidazolidinone analogues of 6-aminoflavone as anticancer and anti-inflammatory agents

Article abstract of DOI:10.1007/s00044-013-0486-7

The flavone moiety is a potential pharmacophore known for its diverse range of pharmacological activities. Aminoflavones have recently been the subject of considerable attention as lead molecules in several cancer research projects. Imidazolidinone heterocycles represent another biologically active scaffold with known cytotoxic properties. In an attempt to provide synergistic cytotoxic activity, these two moieties have been combined, and the resulting novel analogues evaluated for their anticancer and anti-inflammatory activities. The results revealed that the cytotoxicities of these compounds were fivefold greater than those of aminoflavone. DNA histograms obtained from cell cycle analysis in the presence of these compounds were apoptotic in their nature. Furthermore, the in vivo screening of these compounds using Ehrlich's ascites tumour model showed an increase in life span, whereas an in vivo anti-inflammatory study resulted in the enhancement of the anti-inflammatory potential. The results therefore supported the hypothesis that there is a relationship between inflammation and cancer.

Full text of DOI:10.1007/s00044-013-0486-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:5066–5075
DOI 10.1007/s00044-013-0486-7
ORIGINAL RESEARCH
Synthesis and evaluation of a series of novel imidazolidinone
analogues of 6-aminoflavone as anticancer and anti-inflammatory
agents
•
Sudheer Moorkoth K. K. Srinivasan
•
•
N. Gopalan Kutty Alex Joseph M. Naseer
•
Received: 4 September 2012 / Accepted: 11 January 2013 / Published online: 7 February 2013
Ó Springer Science+Business Media New York 2013
Abstract The flavone moiety is a potential pharmaco-
phore known for its diverse range of pharmacological
activities. Aminoflavones have recently been the subject of
considerable attention as lead molecules in several cancer
research projects. Imidazolidinone heterocycles represent
another biologically active scaffold with known cytotoxic
properties. In an attempt to provide synergistic cytotoxic
activity, these two moieties have been combined, and the
resulting novel analogues evaluated for their anticancer and
anti-inflammatory activities. The results revealed that the
cytotoxicities of these compounds were fivefold greater
than those of aminoflavone. DNA histograms obtained
from cell cycle analysis in the presence of these com-
pounds were apoptotic in their nature. Furthermore, the
in vivo screening of these compounds using Ehrlich’s
ascites tumour model showed an increase in life span,
whereas an in vivo anti-inflammatory study resulted in the
enhancement of the anti-inflammatory potential. The
results therefore supported the hypothesis that there is a
relationship between inflammation and cancer.
Keywords 6-Aminoflavone ꢀ Imidazolidinone ꢀ
Cytotoxicity ꢀ Ehrlich’s ascites ꢀ Anti-inflammatory ꢀ
Carrageenan
Introduction
Flavonoids are a group of natural substances with a broad
range of biological activities that can be found abundantly in
many food items. Research in the field of flavonoids has
increased steadily since the discovery of the French paradox.
The flavonoid heterocycle is an established pharmacophore
containing a chromone ring system. Compounds of this
structural class typically possess a variety of different bio-
logical activities at non-toxic concentrations (Ren et al.,
2003). These molecules can be useful in the prevention of
cancer because of their strong antioxidant potential (Stefani
et al., 1999; Fotsis et al., 1997) and have established them-
selves as promising anticancer agents (Ren et al., 2003).
Recent studies report an interesting relationship between the
chromone structures in flavonoids and their potential anti-
cancer activity (Nijveldt et al., 2001). According to the
reports of Scambia et al. (1994) and Ferte et al. (1999),
flavonoids possess multidrug resistance (MDR) modulatory
activity. Quercetin and some flavonoids containing N-benzyl
piperazine side chains in particular have been proven to
potentiate Doxorubicin cytotoxicity in MDR cells.
S. Moorkoth (&)
Department of Pharmaceutical Quality Assurance,
MCOPS, Manipal University, Manipal, India
e-mail: sudheermoorkoth@gmail.com
Aminoflavones are a class of flavonoids that contains
amino groups attached to the flavone nucleus. They do not
occur naturally and must therefore be synthesized. Although
reports into the syntheses and biological activities of amin-
oflavones are scarce, the available literature suggests that
compounds containing the aminoflavone moiety have good
potential as anticancer agents. Compounds of this particular
structural class are potent inhibitors of protein tyrosine
K. K. Srinivasan ꢀ A. Joseph
Department of Pharmaceutical Chemistry, MCOPS,
Manipal University, Manipal, India
N. Gopalan Kutty ꢀ M. Naseer
Department of Pharmacology, MCOPS,
Manipal University, Manipal, India
123

Med Chem Res (2013) 22:5066–5075
5067
kinase (Cushman et al., 1994). Aminoflavones have also
been reported as a new class of antitumour agents in breast
cancer (Akama et al., 1997). Reports suggest that aminof-
lavone is uniquely able to induce its own metabolic activa-
tion by CYP1A1/1A2 induction to directly form a cytotoxic
DNA damaging species in tumour cells (Kuffel et al., 2002).
The basis for this selective toxicity of aminoflavone has been
reported to be the result of aryl hydrocarbon receptor acti-
vation, leading to a 100-fold induction of CYP1A1 mRNA
and a corresponding increase in ethoxyresorufin-O-deethy-
lase activity (Loaiza-Perez et al., 2004). Aminoflavone has
also been reported to induce the formation of DNA protein
cross links, DNA single strand breaks, and Histone H2AX
phosphorylation (Meng et al., 2005). In a separate report,
non-malignant cells (MCF10A) were shown to be resistant
to the cytotoxic effects of aminoflavone (McLean et al.,
2008).
Results and discussion
The 6-aminoflavone (5) required for the synthesis of the
imidazolidinone analogues was synthesized from paracet-
amol, as shown in Scheme 1. Acetylation of paracetamol
followed by the base-mediated condensation of the product
with benzaldehyde yielded the corresponding chalcone 3.
The formation of the chalcone with the acetamido group
intact was confirmed by the appearance of the corre-
sponding molecular ion peak at m/z 281 in the mass
1
spectrum of the compound. The presence of a H NMR
signal at d 2.26 was also consistent with the three hydrogen
atoms of the COCH3 group in the product, whereas the
singlet at d 10.02 was consistent with the NH proton of the
NHCOCH3 group. Cyclisation of the chalcone in DMSO
with a catalytic amount of iodine yielded 6-acetamidof-
lavone (Nitin and Soni, 2005), which was hydrolysed to
give 6-aminoflavone in good yield (92 %). The formation
of 6-aminoflavone was confirmed by the presence of the
molecular ion at m/z 237 in the mass spectrum of the
4-Imidazolidinones represent another heterocyclic
structural class with established cytotoxic properties
against a variety of different cell lines (Al-Madi et al.,
2001; Jung et al., 1998). The inclusion of some aryl
functionality at the 5-position of 4-imidazolidinones has
been reported to be essential to their anticancer activity,
and accordingly, 5-substituted 2-cyanoimino-4-imidazoli-
dinones have been shown to exhibit cytotoxic properties at
nano-molar concentrations (Chern et al., 2004; Khodair
et al., 1998). The importance of the imidazolidinone motif
to the cytotoxicity of 4-phenyl 1-arylsulphonyl imidazo-
lidinones has also been reported (Kim and Jung, 2002; Jung
et al., 2001).
1
product, and by the presence of a H NMR signal at d 5.5
corresponding to the two amino protons. The 6-aminof-
lavone was refluxed with a series of different azlactones in
pyridine for 6–8 h to give the proposed imidazolidinone
analogues 7a–e in yields in the range of 48–55 %. The
formation of the products was confirmed by the disap-
pearance of the primary amino-proton signals of the am-
inoflavones at d 5.5 in the 1H NMR spectra of the products.
Product formation was also confirmed by mass spectral and
13C NMR spectral analyses. The azlactones used in the
syntheses described above were prepared by the Erlen-
meyer azlactone synthesis (Brian et al., 1989), as shown in
Scheme 2. The five azlactones 6a–e used in the current
study were synthesized from the corresponding benzalde-
hydes, including benzaldehyde, 4-methoxybenzaldehyde,
3,4-dichlorobenzaldehyde, 3,4-dimethoxybenzaldehyde and
4-hydroxybenzaldehyde, respectively.
Based on the results outlined above, we became inter-
ested in the preparation of imidazolidinone analogues of
6-aminoflavone and the assumption that the combination of
these two pharmacophores may result in compounds with
synergistic anticancer activity and reduced levels of tox-
icity. The general structure of the proposed analogues is
shown in Fig. 1.
All of the newly synthesized imidazolidinone analogues
and the original aminoflavone were screened for their
in vitro cytotoxicity against HeLa (human epithelial cer-
vical cancer), and MDA MB 435 (human breast cancer)
cell lines using the methyl tetrazolium (MTT) and sul-
phorhodamine B (SRB) assays, respectively. Two different
assay techniques were used for the cytotoxicity screening,
based on the fact that the aminoflavone is coloured yellow
and could interfere with the colour formed in the assay.
The occurrence of a matching result across the two assays
was expected to confirm the validity of the results because
the principles involved in the two techniques were differ-
ent. The results of the preliminary cytotoxicity screen
revealed that all of the newly synthesized imidazolidinone
analogues registered higher levels of cytotoxicity than
6-aminoflavone in both the cell lines. The most active
3'
4'
5'
2'
B
8
1
O
7
2"
O
6**
3"
4"
2
3
6'
A
C
4**
5**
4
N
3**
5
6"
N
1**
5"
O
2**
6*
5*
2*
3*
4*
Fig. 1 Structure of the proposed imidazolidinone analogue of flavone
123

5068
Med Chem Res (2013) 22:5066–5075
OH
CH3
O
OH
CH3COCl
H3COCHN
H3COCHN
Anhydrous AlCl3,
Nitrobenzene, Reflux
5-acetamino-2-hydroxylacetophenone [2]
[1]
paracetamol
,
l
o
n
CHO
a
h
t
E
,
H
O
K
OH
O
DMSO, I2
CH
H3COCHN
H3COCHN
Reflux, 175°C, 30 min
O
O
6-acetaminoflavone [4]
5'-acetamino-2'-hydroxychalcone [3]
O
s
i
s
R1
y
l
o
O
N
r
d
y
R2
h
O
N
O
O
d
i
R1
R2
c
A
N
O
Azlactones [6(a-e)]
H2N
Pyr
idin
e,
Ref
lux
O
130-140°C, 6 h
6-aminoflavone
[5]
Imidazolidinone analogue [7(a-e)]
Scheme 1 Synthesis of imidazolidinone analogues of 6-aminoflavone
O
R1
R2
O
OH
R1
H
H
R2
H
OCH3
Cl
HN
O
a
b
c
d
e
N
O
R1
R2
CHO
(CH3CO)2O
CH3COONa,
Cl
+
Heat, 2h
OCH3 OCH3
OH
H
Aromatic aldehydes (a-e)
Benzoyl glycine
Azlactones [6(a-e)]
Scheme 2 Synthesis of azlactones 6a–e
compound in the screen (Compound 7d) was about five
times more active than the parent 6-aminoflavone. The
results are presented in Table 1 and Fig. 2. The data
obtained showed that the conversion of the aminoflavone to
its imidazolidinone analogue effectively enhanced the
cytotoxic potential of the original compound. Of the imi-
dazolidinones synthesized and tested for their cytotoxic
activity, compounds containing hydroxy and methoxy
substituents on the imidazolidinone segment were more
cytotoxic than those containing chloro substituents or no
substitution at all. Furthermore, the presence of two
methoxy groups on the benzene ring had a considerable
impact on the cytotoxicity, as can be seen in the case of
compound 7d. The increase in the activity of this particular
123

Med Chem Res (2013) 22:5066–5075
5069
Table 1 Results of the in vitro
cytotoxicity studies
Compound
IC50 valuea (lg/mL)
MTT assay
SRB assay
HeLa cell line
MDA MB cell line
HeLa cell line
MDA MB cell line
5
55.75 2.37
20.12 1.54
14.24 2.75
15.23 0.89
9.87 3.13
16.40 0.56
0.69 0.12
59.48 1.11
24.29 2.62
18.50 1.03
16.89 0.60
10.79 2.23
18.03 0.69
0.77 0.19
63.61 1.38
20.89 1.86
17.62 0.79
15.98 1.39
11.08 2.57
18.90 2.60
0.81 0.12
78.37 0.89
26.37 0.55
21.74 0.86
20.15 0.19
11.95 0.50
21.36 1.16
0.93 0.19
7a
7b
7c
7d
7e
Dox
Dox Doxorubicin standard
a
Values are the average of
three determinations SDEV
70
60
50
40
30
20
10
100
80
60
40
20
0
MTT assay results
SRB assay results
HeLa cells
MDA MB 435 cells
HeLa cells
MDA MB 435 cells
0
5
7a
7b
7c
7d
7e
DOX
5
7a
7b
7c
7d
7e
DOX
Compounds screened
Compounds screened
Fig. 2 Comparison of results of the in vitro cytotoxicity studies performed for compounds 7a–e using MTT and SRB assays
Fig. 3 Microscopic pictures of
HeLa cells taken after 24 h of
treatment during the MTT assay
compound may be attributed to the presence of two elec-
tron-donating methoxy groups. The presence of a single
hydroxyl, methoxy or halogen group did not provide sub-
stantial increases in the level of cytotoxic activity. During
the cytotoxicity assay, microscopic pictures were also
taken 24 h after the initiation of the treatment with the
compounds. These pictures revealed characteristic mor-
phological changes such as cell rounding and detachment
of cells from the surface of the well, which confirmed the
cytotoxicities of the compounds being tested (Fig. 3).
The most active compound (7d) in the in vitro cytotoxicity
screen was selected to further study the possibility of any
apoptotic potential. Apoptotic potential was studied at a
concentration equal to the IC50 value of the compound.
Accordingly, HeLa cells treated with compound 7d were
stained with Hoechst 33342 stain, and the appearance of
chromatin condensation and the fragmentation of the nuclei
were monitored according to the procedure described by Hu
et al. (2009). Morphological changes in the nuclei of the
apoptotic cells were visualised by fluorescence microscopy.
123

5070
Med Chem Res (2013) 22:5066–5075
The results of the Hoechst stain analysis revealed that the
control cells possessed regular and round-shaped nuclei,
whereas the cells treated with compound 7d and Doxorubi-
cin (standard drug) showed condensation and fragmentation
of their nuclei characteristic of apoptotic cells. As can be
seen from the results obtained with the Hoechst staining
technique (Fig. 4), the cells showed DNA fragmentation
characterised by the occurrence of a condensed nucleus
(highly fluorescent), which is characteristic of apoptosis.
DNA fragmentation is considered to be the key biochemical
event in apoptosis. One method for identifying DNA frag-
mentation involves analysis of the DNA content by flow
cytometry (Telford et al., 1991, 1992). Fluorescence-acti-
vated cell sorting (FACS) analyses were performed on
compound 7d to study the effect of the synthesized com-
pound on cell cycle progression. Figure 5 shows the results
of the FACS analysis following 48 h of treatment as DNA
histograms. Compound 7d provided a DNA histogram
characteristic of apoptosis, showing an increased percentage
of DNA in the SubG0 phase. The presence of the SubG0
phase DNA content is a single parameter indicator of
apoptotic cells. Compound 7d also provided an increase in
the percentage DNA content from 18.65 (untreated control)
to 28.73 % in the G2/M phase, which indicated that the
compound 7d had also caused G2/M phase arrest.
Following the confirmation of its cytotoxicity and
apoptotic potential, compound 7d was further studied for
its in vivo anticancer activity. The safe dose was deter-
mined to be 100 mg/kg/day as per the results of the acute
toxicity study (Ghosh, 1984). In the in vivo studies,
parameters such as mean survival time, % increase in life
span (%ILS) and % increase in body weight were measured
and compared to those of the standard drug Cisplatin. The
mean survival time and percentage increase in life span
were studied using Ehrlich’s Ascites Carcinoma (EAC)-
induced mice with two doses of 100 and 50 mg/kg/day.
The results for the mean survival time and the % ILS of
EAC-induced mice are presented in Table 2. The results
were analysed for statistical significance as per Dunnett’s
multiple comparison test for one-way analysis of variance.
All of the results were found to be significant with p \ 0.01
when compared with the control. A regular rapid increase
in ascites tumour volume was also noted in the tumour-
bearing mice. In the case of treated mice, as well as in the
case of the positive control, the tumour volume was found
to be lower. Treatment with compound 7d led to an
Untreated HeLa cells
HeLa cells treated with doxorubicin
HeLa cells treated with compound 7d
Fig. 4 Results of the Hoechst stain analysis (fluorescent microscopic pictures)
Untreated (control) Hela cells
HeLa Control (G2: R1 & R2)
Hela cells treated with Doxorubicin
Doxorubicin (G2: R1 & R2)
Hela cells treated with compound 7d
Compound 7d (G2: R1 & R2)
Total
R3
R4
S
R5
R6
Total
R3
G0/G1
R4
S
R5
G2/M SubG0
1.15
R6
Total
R3
G0/G1
R4
S
R5
G2/M SubG0
2.22
R6
Phase
G0/G1
G2/M SubG0
Phase
Phase
% Hist
100
36.8 14.1 28.37 21.63
% Hist 100 74.27 6.34 18.65
% Hist 100 16.62 4.01 76.76
Fig. 5 Results of the cell cycle analysis (DNA content histograms)
123

Med Chem Res (2013) 22:5066–5075
5071
Table 2 Mean survival time and percentage increase in life span of EAC-induced mice during the study
Treatment code
Dose (mg/kg/day)
Mean life span
Median life span
Mean survival time (days)
Mean (%ILS)
Median survival time (days)
Median (%ILS)
Negative control
Cisplatin
7d
–
17.33 0.21
34 0.63*
–
17
–
3.5
100
50
96.19
32.72
28.87
34
100
35.29
32.35
23 0.36*
23
22.33 0.33*
22.5
N = 6 animals in each group, Days of treatment = 9, values are expressed as mean SEM
* p \ 0.01 versus control as per Dunnet multiple comparisons test for one-way analysis of variance.
Table 3 Percentage increase in body weight of EAC-induced mice during the study
Treatment code
Average % increase in body weight in grams SEM
Day 3
Day 6
Day 9
Day 12
Day 15
Negative control (untreated)
Cisplatin (3.5 mg/kg/day)
7d (100 mg/kg/day)
4.56 0.52
3.12 1.2
3.58 0.90
16.31 0.48
4.69 1.34*
9.39 1.32***
34.31 1.06
6.97 1.060*
13.63 1.29*
44.42 2.82
9.0 0.99*
17.47 1.2*
58.81 2.67
10.54 0.90*
19.99 1.29*
N = 6 animals in each group, Days of treatment = 9, values are expressed as mean SEM
* p \ 0.001, *** p \ 0.05 versus control as per Tukey multiple comparisons test for one-way analysis of variance
increase in the survival time of the tumour-bearing mice
and also led to a reduction in the ascites fluid volume. The
average % increase in body weight was calculated for the
EAC-induced liquid tumour-bearing mice at a dose level of
100 mg/kg/day. The intra-peritoneal inoculation of EAC
cells in the mice produced a significant increase in their
body weight. The increase in tumour weight was attributed
to the accumulation of peritoneal fluid, which in turn
resulted in an abnormal enlargement of the peritoneal
cavity. The untreated mice showed an average increase of
58.8 % in body weight on the 15th day of the treatment
programme. In the case of the positive control (Cisplatin
treated mice), there was only a 10.54 % increase in body
weight on the 15th day of the treatment programme, and
the amount of ascitic fluid was also much lower. The %
increase in body weight on the 15th day for the mice
treated with compound 7d was 19.99 %, demonstrating a
significant antitumour activity (Table 3).
The in vivo anti-inflammatory activities of compounds
7a–e were studied in Wistar rats according to the method
described by Winter et al. (1962) involving a Carrageenan-
induced rat paw oedema model. A dose of 100 mg/kg/day
was selected based on the acute toxicity study. The results
revealed an increase in paw volume following Carrageenan
administration in the control (0.46 mL) and ibuprofen-
treated groups (0.15 mL), which corresponded well with
the findings of a previous report (Khaksari et al., 2004). All
of the imidazolidinone analogues registered anti-inflam-
matory activity with a reduction in the paw volume from
0.46 mL (untreated control) to as low as 0.23 mL
(Table 4). Compound 7d was found to be the most active
of the compounds tested, suggesting that the presence of
Table 4 Effect of compounds
7a–e on Carrageenan-induced
rat paw oedema
Compound code
Dose (mg/kg, p.o)
Mean rat paw oedema volume
after 3 h (ml SEM*) (N = 6)
Percentage
inhibition SEM
Control
CMC 2 %
100
0.46 0.01
–
5
0.35 0.01*
0.29 0.02*
0.25 0.02*
0.29 0.01*
0.23 0.008*
0.24 0.01*
0.15 0.01*
24.18 2.8
38.25 6.1
45.11 3.6
36.45 3.1
49.81 1.9
47.64 2.5
67.14 2.5
7a
100
7b
100
7c
100
7d
100
One-way ANOVA followed by
post hoc Scheffe’s test
7e
100
Ibuprofen
50
* p \ 0.05 versus control group
123

5072
Med Chem Res (2013) 22:5066–5075
the methoxy group may have an influence on the anti-
inflammatory activity.
precipitated product was collected by filtration, washed
with water to remove pyridine and dried. The dried product
was recrystallised from ethanol yielding imidazolidinone
analogue in pure form. A series of such analogues 7a–e
were prepared using corresponding oxazolones 6a–e. The
newly synthesized 6-imidazolidinone analogues were
characterised by their physical properties and spectral
characteristics. Various experimental and spectral data
Materials and methods
Chemicals required for the synthesis such as paracetamol,
benzaldehyde, 3,4-dichlorobenzaldehyde, 3,4-dimethoxy-
benzaldehyde, 4-methoxybenzaldehyde and 4-hydroxybenz-
aldehyde were purchased from Sigma Aldrich Chemical
Company, USA. The melting points of the synthesized
compounds were determined using digital melting point
apparatus (V Scientific India). Thin layer chromatography
(TLC) studies were performed on silica gel G coated alu-
minium plates supplied by Merck, Germany with a solvent
system composition of Hexane: Acetone (6:4). FTIR spectra
were recorded using 8310 FTIR instrument, Shimadzu, Japan.
NMR spectral study was performed with the help of AMX
400, Bruker Spinwin, Germany. EI MS data were obtained
using GCMS QP5050, Shimadzu, Japan and ESI–MS data
were recorded using Enquire 3000 plus, Bruker Daltonics,
Germany. UV kmax values were determined using UV Visible
Spectrophotometer, Model UV-2400PC, Shimadzu, Japan.
Various chemicals used for biological activity studies such as
MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide], SRB, Hoechst 33342 stain, Propidium iodide dye,
Carrageenan etc. were obtained from Sigma Aldrich Chemi-
cal company, USA. Doxorubicin hydrochloride which was
used as the standard for in vitro anticancer study was procured
from Sigma Aldrich Chemical Company, USA. Cisplatin the
positive control used in the in vivo anticancer study was
obtained as a gift sample from Dabur India ltd. Ibuprofen, the
standard drug for anti-inflammatory activity, was purchased
from Sd fine chemicals, India. The cell lines used such as
HeLa (human epithelial cervix cancer) and MDA MB 435
(human breast cancer) were the ones that were maintained
at the Department of MRDG, Indian Institute of Science,
Bangalore. The anti-inflammatory measurements were car-
ried out on a plethysmometer. Hoechst staining of the cells
was viewed on a fluorescent microscope (Olympus, Japan).
The cell cycle analysis was carried out on a Fluorescence
Assisted Cell Sorting (FACS) machine, MoFlo equipped with
488 nm solid state laser, and data were analysed using
Summit software (Beckman Coulter).
1
such as melting point, Rf value, % yield, UV, IR, MS, H
NMR and 13C NMR etc. were recorded.
4-Benzylidene-1-(4-oxo-2-phenyl-4H-chromen-6-yl)-2-
phenyl-1H-imidazol-5-one (7a)
Yield: 55 %; m.p.: 244 °C; Rf value: 0.388; UV kmax (nm):
294; IR (KBr) cm-1: 1685 (C=O str. imidazolidinone),
1649 (C=O str. flavone), 1600 (C=N str. Imidazolidinone).
EI–MS m/z (% abundance): 468 M? (10), 324 (4), 237 (48),
247 (13), 135 (30), 116 (5). 1H NMR DMSO-d6, 400 MHz,
d 7.22 (s, 1H, Ar–CH=C), 8.48 (s, 1H, H-5), 7.32 (d, 1H,
H-8) J = 12 Hz, 7.8 (d, 1H, H-7) J = 12 Hz, 7.35–7.70 (m,
15H, aromatic protons of the three phenyl rings); 13C NMR
DMSO-d6, 100 MHz, d 164.52 (C-2), 106.48 (C-3), 176.99
(C-4), 118.81 (C-5), 131.78 (C-6), 134.16 (C-7), 114.46
(C-8), 151.78 (C-9), 123.41 (C-10), 131.20 (C-10), 128.55
(C-20, C-60), 127.91 (C-30, C-50), 133.39 (C-40), 162.39
(C-2**), 129.52 (C-4**), 166.07 (C-5**), 106.48 (C-6**),
136.78 (C-100), 126.66 (C-200, C-600), 128.36 (C-500), 128.55
(C-300), 130.76 (C-400), 127.91 (C-1*), 126.32 (C-2*, C-6*),
129.10 (C-5*, C-3*), 130.76 (C-4*).
4-(4-Methoxybenzylidene)-1-(4-oxo-2-phenyl-4H-
chromen-6-yl)-2-phenyl-1H-imidazol-5-one (7b)
Yield: 53 %; m.p.: 267 °C; Rf value: 0.327; UV kmax (nm):
310; IR (KBr) cm-1: 1680 (C=O str. imidazolidinone),
1639 (C=O str. flavone), 1604 (C=N str. imidazolidinone).
EI–MS m/z (% abundance): 498 M? (2), 237 (12), 279 (5),
1
146 (5), 135 (20), 105 (100), 77 (41). H NMR DMSO-d6,
400 MHz, d 3.55 (s, 3H, OCH3) 7.22 (s, 1H, Ar–CH=C),
8.48 (s, 1H, H-5), 7.79 (d, 1H, H-8) J = 12 Hz, 8.18 (d,
1H, H-7) J = 12 Hz, 8.08 (dd, 4H, H-200, 300, 500, 600),
J = 9.4 Hz, J = 9.38 Hz, 7.58 (m, 8H, 2-phenyl of imi-
dazolidinone (5H), 30, 40, 50), 6.97 (d, 2H, H-20, 60)
J = 10.16 Hz; 13C NMR DMSO-d6, 100 MHz, d 162.58
(C-2), 103.58 (C-3), 179.79 (C-4), 120.99 (C-5), 129.45
(C-6), 130.62 (C-7), 117.24 (C-8), 153.12 (C-9), 124.97
(C-10), 130.40 (C-10), 125.59 (C-20, C-60), 128.21 (C-30,
C-50), 129.98 (C-40), 167.39 (C-2**), 131.80 (C-4**),
170.28 (C-5**), 108.58 (C-6**), 127.98 (C-100), 126.16
(C-200, C-600), 128.38 (C-500), 128.91 (C-300), 130.73 (C-400),
127.54 (C-1*), 127.39 (C-2*, C-6*), 116.20 (C-5*, C-3*),
129.96 (C-4*), 56.26 (C–OCH3).
Synthesis of imidazolidinone analogues 7a–e
To an equimolar mixture of 5 and azlactone 6, was added
pyridine (quantity sufficient to dissolve), and the mixture
was refluxed at 130–140 °C for 6 h. The completion of the
reaction was monitored by TLC. The reaction mixture was
then cooled and poured on to 50 ml ice cold water con-
taining 5 N HCl, with stirring until the pH was neutral. The
123

Med Chem Res (2013) 22:5066–5075
5073
4-(3,4-Dichlorobenzylidene)-1-(4-oxo-2-phenyl-4H-
Ar–CH=C), 8.45 (s, 1H, H-5), 7.8 (d, 1H, H-8)
J = 5.48 Hz, 8.19 (d, 1H, H-7) J = 7.4 Hz, 8.08 (dd, 4H,
H-200, 300, 500, 600) J = 7.32 Hz, J = 7.8 Hz, 7.69 (d, 2H,
H-60, 6*) J = 8.64 Hz, 7.5–7.6 (m, 6H, 30, 40, 50, 3*, 4*,
5*), 7.16 (d, 2H, H-20, 2*) J = 8.6 Hz, 9.78 (s, 1H, H–Ar–
OH); 13C NMR DMSO-d6, 100 MHz, d 163.87 (C-2),
104.85 (C-3), 183.31 (C-4), 120.20 (C-5), 127.45 (C-6),
128.27 (C-7), 117.94 (C-8), 152.88 (C-9), 124.14 (C-10),
130.33 (C-10), 126.35 (C-20, C-60), 128.71 (C-30, C-50),
128.00 (C-40), 165.92 (C-2**), 130.83 (C-4**), 170.38 (C-
5**), 109.18 (C-6**), 127.80 (C-100), 125.68 (C-200, C-600),
114.89 (C-500, C-300), 158.37 (C-400), 128.41 (C-1*), 126.94
(C-2*, C-6*), 128.88 (C-5*, C-3*), 130.62 (C-4*).
chromen-6-yl)-2-phenyl-1H-imidazol-5-one (7c)
Yield: 58 %; m.p.: 268 °C; Rf value: 0.367; UV kmax (nm):
292; IR (KBr) cm-1: 1685 (C=O str. imidazolidinone),
1649 (C=O str. flavone), 1600 (C=N str. imidazolidinone);
ESI–MS m/z 555.4 (M ? H2O); 1H NMR DMSO-d6,
400 MHz, d 7.02 (s, 1H, H-3), 7.16 (s, 1H, Ar–CH=C),
8.46 (s, 1H, H-5), 7.8 (d, 1H, H-8) J = 9.04 Hz, 8.18 (d,
1H, H-7) J = 8.84 Hz, 8.01 (d, 2H, H-20, 2*) J = 7.4 Hz,
8.1 (d, 2H, H-60, 6*) J = 6.84 Hz, 7.89 (s, 1H, H-200) 7.67
(d, 1H, H-600) J = 8.4 Hz, 7.58 (m, 7H, 30, 40, 50, 500, 3*,
4*, 5*); 13C NMR DMSO-d6, 100 MHz, d 163.86 (C-2),
105.39 (C-3), 183.02 (C-4), 120.18 (C-5), 129.14 (C-6),
131.37 (C-7), 117.86 (C-8), 152.62 (C-9), 124.16 (C-10),
130.34 (C-10), 126.48 (C-20, C-60), 128.69 (C-30, C-50),
128.01 (C-40), 164.87 (C-2**), 130.57 (C-4**), 171.73
(C-5**), 108.89 (C-6**), 134.81 (C-100), 125.66 (C-200),
127.76 (C-600), 133.69 (C-500), 130.57 (C-300), 132.67
(C-400), 128.01 (C-1*), 126.28 (C-2*, C-6*), 128.85 (C-5*,
C-3*), 130.61 (C-4*).
Biological activity
The synthesized analogues 7a–e were evaluated for their
in vitro cytotoxic potential by the MTT assay (Denizot and
Lang, 1986) and SRB assay (Skehan et al., 1990). The
most active compound from the above cytotoxicity screen
was studied further by Hoechst staining analysis and flow
cytometric analysis (Duvall and Wyllie, 1986; Hu et al.,
2009) to establish the apoptotic potential. After confirming
the apoptotic potential of compound 7d, it was further
studied to evaluate the in vivo anticancer activity. Ehrlich
tumour is a rapidly growing carcinoma with very aggres-
sive behaviour and is able to grow in almost all mice strains
(Chen and Watkins, 1970). The in vivo study was per-
formed using Ehrlich’s ascites tumour model on Albino
mice, and the activity was compared to that of Cisplatin.
The study was carried out as per the procedure reported by
earlier workers (Devi et al., 1998). The results obtained for
in vivo anticancer studies were analysed statistically and
the level of significance determined by one-way ANOVA
followed by Dunnet multiple comparisons test in the case
of mean survival time and by Tukey test in the case of
percentage increase in body weight. An in vivo anti-
inflammatory screen by the method of Winter et al. (1962)
also was performed. Statistical analysis was carried out
using one-way ANOVA followed by Post hoc Scheffe’s
test, and p \ 0.05 was considered significant in the case of
anti-inflammatory evaluation. The animal care and han-
dling was carried out in accordance with the guidelines
issued by the Institutional Animal Ethics Committee,
Kasturba Medical College, Manipal University, Manipal,
and the study was approved by the committee (Clearance
certificate no.—IAEC/KMC/02/2006–2007).
4-(3,4-Dimethoxybenzylidene)-1-(4-oxo-2-phenyl-4H-
chromen-6-yl)-2-phenyl-1H-imidazol-5-one (7d)
Yield: 48 %; m.p.: 240 °C; Rf value: 0.306. UV kmax (nm):
296; IR (KBr) cm-1: 1681 (C=O str. imidazolidinone),
1631 (C=O str. flavone), 1604 (C=N str. imidazolidinone);
ESI–MS m/z 547.1 (M ? H2O); 1H NMR DMSO-d6,
400 MHz, d 3.62 (d, 6H, H–OCH3) J = 12.6 Hz, 6.98 (s,
1H, H-3), 7.01 (s, 1H, Ar–CH=C), 8.448 (s, 1H, H-5), 7.8
(d, 1H, H-8) J = 9.08 Hz, 8.2 (d, 1H, H-7) J = 7.4 Hz, 8.1
(d, 4H, H-20, 2*, 60, 6*) J = 5.92 Hz, 7.23 (d, 1H, H-200)
J = 8.2 Hz, 7.3 (d, 2H, H-500, 600) J = 13.48 Hz, 7.51–7.6
(m, 6H, 30, 40, 50, 3*, 4*, 5*); 13C NMR DMSO-d6,
100 MHz, d 164.62 (C-2), 104.92 (C-3), 182.26 (C-4),
120.89 (C-5), 127.48 (C-6), 128.72 (C-7), 118.18 (C-8),
152.89 (C-9), 124.63 (C-10), 130.43 (C-10), 125.98 (C-20,
C-60), 128.97 (C-30, C-50), 127.89 (C-40), 163.79 (C-2**),
130.38 (C-4**), 170.36 (C-5**), 108.93 (C-6**), 128.18
(C-100), 120.61 (C-200), 110.67 (C-600), 149.94 (C-500),
119.73 (C-300), 151.71 (C-400), 128.58 (C-1*), 126.21
(C-2*, C-6*), 128.93 (C-5*, C-3*), 130.14 (C-4*), 55.89
(C–OCH3 at C-400 and C-500).
4-(4-Hydroxybenzylidene)-1-(4-oxo-2-phenyl-4H-
chromen-6-yl)-2-phenyl-1H-imidazol-5-one (7e)
Yield: 51 %; m.p.: 262 °C; Rf value: 0.347; UV kmax (nm):
296; IR (KBr) cm-1: 3244 (br. Ar–OH str), 1674 (C=O str.
imidazolidinone), 1626 (C=O str. flavone), 1601 (C=N str.
imidazolidinone); ESI–MS m/z 503.1 (M ? H2O); 1H
NMR DMSO-d6, 400 MHz, d 7.01 (s, 1H, H-3), 7.2 (s, 1H,
Conclusion
Five novel imidazolidinone analogues of flavone have been
synthesized in good yields in an attempt to explore the
123

5074
Med Chem Res (2013) 22:5066–5075
Cushman M, Zhu H, Geahlen RL, Kraker AJ (1994) Synthesis and
biochemical evaluation of a series of aminoflavones as potential
inhibitors of protein-tyrosine kinases p56lck, EGFr, and p60v-
src. J Med Chem 37(20):3353–3362
Denizot F, Lang R (1986) Rapid colorimetric assay for cell growth
and survival. Modifications to the tetrazolium dye procedure
giving improved sensitivity and reliability. J Immunol Methods
89(2):271–277
Devi PU, Rao BS, Solomon FE (1998) Effect of plumbagin on the
radiation induced cytogenetic and cell cycle changes in mouse
Ehrlich ascites carcinoma in vivo. Indian J Exp Biol 36(9):
891–895
Duvall E, Wyllie AH (1986) Death and the cell. Immunol Today
7(4):115–119
Ferte J, Kuhnel JM, Chapuis G, Rolland Y, Lewin G, Schwaller MA
(1999) Flavonoid-related modulators of multidrug resistance:
synthesis, pharmacological activity, and structure–activity rela-
tionships. J Med Chem 42(3):478–489
Fotsis T, Pepper MS, Aktas E, Breit S, Rasku S, Adlercreutz H,
Wahala K, Montesano R, Schweigerer L (1997) Flavonoids,
dietary-derived inhibitors of cell proliferation and in vitro
angiogenesis. Cancer Res 57(14):2916–2921
Ghosh MN (1984) Toxicity studies: fundamentals of Experimental
Pharmacology. Scientific Book Agency, Calcutta
Hu YW, Liu CY, Du CM, Zhang J, Wu WQ, Gu ZL (2009) Induction
of apoptosis in human hepatocarcinoma SMMC-7721 cells
in vitro by flavonoids from Astragalus complanatus. J Ethno-
pharmacol 123(2):293–301
possibilities of higher anticancer potential. All of the novel
compounds were completely characterised by ultraviolet
(UV), infrared (IR), NMR and mass spectral methods.
Preliminary in vitro cytotoxicity studies carried out using
MTT and SRB assays on two different cell lines revealed
that the conversion to the imidazolidinone analogues
enhanced the cytotoxic potential of aminoflavone. Of the
analogues tested, compound 7d [4-(3,4-dimethoxybenzy-
lidene)-1-(4-oxo-2-phenyl-4H-chromen-6-yl)-2-phenyl-1H-
imidazol-5-one] showed promising levels of activity with
an IC50 value of 9.87 lg/mL. It is noteworthy that the most
active compound possessed two methoxy groups in its
structure. The study also revealed the apoptotic nature of
the compound 7d. Hoechst staining experiment and cell
cycle analysis carried out using compound 7d showed that
the cell death that occurred was characteristic of apoptosis.
In vivo anticancer studies performed on EAC-induced mice
also resulted in promising levels of activity. The synthe-
sized compounds 7a–e were also evaluated for their anti-
inflammatory activities and registered significant levels of
activity. These results effectively support the observed
anticancer potential of the compounds. Based on these
results, we have concluded that SAR studies and anticancer
screening against a panel of cancer cell lines need to be
performed to confirm and build upon the findings of this
study.
Jung SH, Lee HS, Song JS, Kim HM, Han SB, Lee CW, Lee M, Choi
DR, Lee JA, Chung YH, Yoon SJ, Moon EY, Hwang HS, Seong
SK, Lee DK (1998) Synthesis and antitumor activity of
4-phenyl-1-arylsulfonyl imidazolidinones. Bioorg Med Chem
Lett 8(12):1547–1550
Jung SH, Park KL, Lee HS, Whang JS (2001) Evaluation of the role
of imidazolidinone motif of antineoplastic 4-phenyl-1-arylsulfo-
nylimidazolidinones using 4-phenyl-2-arylsulfonyloxazolines.
Arch Pharm Res 24(6):499–502
Khaksari M, Mahani SE, Mahmoodi M (2004) Calcium chanel
blockers reduce inflammatory edema in the rat. Indian J
Pharmacol 36(6):351–354
Khodair AI, El-subbagh HI, Al-obaid AM (1998) Synthesis, confor-
mational analysis and antitumor testing of 5-(Z)-arylidene-4-
imidazolidinone derivatives. Phosphorus Sulfur Silicon Relat
Elem 140(1):159–181
Acknowledgments The authors would like to acknowledge the All
India Council for Technical Education (AICTE) of New Delhi for
funding this work under Quality Improvement Programme (QIP). The
authors would also like to thank Dr. Annapurni Rangarajan from the
Department of Molecular Reproduction Development and Genetics
(MRDG) at the Indian Institute of Science in Bangalore for providing
the facility to perform the cytotoxicity assays, and the Hoechst
staining and flow cytometric FACS analyses. The authors would also
like to acknowledge the NMR Research centre at the Indian Institute
of Science IISC in Bangalore for the NMR spectral data.
Kim IW, Jung SH (2002) Recognition of the importance of
imidazolidinone motif for cytotoxicity of 4-phenyl-1-arylsulfo-
nylimidazolidinones using thiadiazolidine-1,1-dioxide analogs.
Arch Pharm Res 25(4):421–427
References
Akama T, Ishida H, Shida Y, Kimura U, Gomi K, Saito H, Fuse E,
Kobayashi S, Yoda N, Kasai M (1997) Design and synthesis of
potent antitumor 5,40-diaminoflavone derivatives based on
metabolic considerations. J Med Chem 40(12):1894–1900
Al-Madi SH, Al-Obaid AM, EI-Subbagh HI (2001) The in vitro
antitumor assay of 5-(Z)-arylidene-4-imidazolidinones in screens
of AIDS-related leukemia and lymphomas. Anticancer Drugs
12(10):835–839
Brian SF, Antony JH, Smith Peter WG, Austin RT (1989) Vogel’s
Textbook of practical organic chemistry. Longman Scientific &
Technical, England
Chen L, Watkins JF (1970) Evidence against the presence of H2
histocompatibility antigens in Ehrlich ascites tumour cells.
Nature 225(5234):734–735
Kuffel MJ, Schroeder JC, Pobst LJ, Naylor S, Reid JM, Kaufmann
SH, Ames MM (2002) Activation of the antitumor agent
aminoflavone (NSC 686288) is mediated by induction of tumor
cell cytochrome P450 1A1/1A2. Mol Pharmacol 62(1):143–153
Loaiza-Perez AI, Kenney S, Boswell J, Hollingshead M, Alley MC,
Hose C, Ciolino HP, Yeh GC, Trepel JB, Vistica DT, Sausville
EA (2004) Aryl hydrocarbon receptor activation of an antitumor
aminoflavone: basis of selective toxicity for MCF-7 breast tumor
cells. Mol Cancer Ther 3(6):715–725
McLean L, Soto U, Agama K, Francis J, Jimenez R, Pommier Y,
Sowers L, Brantley E (2008) Aminoflavone induces oxidative
DNA damage and reactive oxidative species-mediated apoptosis
in breast cancer cells. Int J Cancer 122(7):1665–1674
Meng LH, Kohlhagen G, Liao ZY, Antony S, Sausville E (2005)
DNA-protein cross-links and replication-dependent histone
H2AX phosphorylation induced by aminoflavone (NSC
686288), a novel anticancer agent active against human breast
cancer cells. Cancer Res 65(12):5337–5343
Chern JH, Shia KS, Chang CM, Lee CC, Lee YC, Tai CL, Lin YT,
Chang CS, Tseng HY (2004) Synthesis and in vitro cytotoxicity of
5-substituted 2-cyanoimino-4-imidazodinone and 2-cyanoimino-4-
pyrimidinone derivatives. Bioorg Med Chem Lett 14(5):1169–1172
123

Med Chem Res (2013) 22:5066–5075
5075
Nijveldt RJ, Van Nood E, Van Hoorn DE, Boelens PG, Van Norren
K, Van Leeuwen PA (2001) Flavonoids: a review of probable
mechanisms of action and potential applications. Am J Clin Nutr
74(4):418–425
colorimetric cytotoxicity assay for anticancer-drug screening.
J Natl Cancer Inst 82(13):1107–1112
Stefani ED, Boffetta P, Deneo-Pellegrini H, Mendilaharsu M,
Carzoglio JC, Ronco A, Olivera L (1999) Dietary antioxidants
and lung cancer risk: a case-control study in Uruguay. Nutr
Cancer 34(1):100–110
Telford WG, King LE, Fraker PJ (1991) Evaluation of glucocorticoid-
induced DNA fragmentation in mouse thymocytes by flow
cytometry. Cell Prolif 24(5):447–459
Telford WG, King LE, Fraker PJ (1992) Comparative evaluation of
several DNA binding dyes in the detection of apoptosis-
associated chromatin degradation by flow cytometry. Cytometry
13(2):137–143
Nitin NA, Soni PA (2005) Reaction of 20-hydroxy-50-acetamidoch-
alcones with dimethyl sulfoxide-iodine, pyridine-mercuric(II)
acetate and triethanolamine. Indian J Chem 44B:2601–2603
Ren W, Qiao Z, Wang H, Zhu L, Zhang L (2003) Flavonoids:
promising anticancer agents. Med Res Rev 23(4):519–534
Scambia G, Ranelletti FO, Benedetti P, Panici R, Vincenzo G, Bonanno
G, Ferrandina, Piantelli MS, Bussa CR, Cianfriglia M (1994)
Quercetin potentiates the effect of adriamycin in a multidrug-
resistant MCF-7 human breast-cancer cell line: p-glycoprotein as a
possible target. Cancer Chemother Pharmacol 34(6):459–464
Skehan P, Storeng R, Scudiero D, Monks A, Mc Mohan J, Vistica D,
Warren JT, Bokesch H, Kenny S, Boyd MR (1990) New
Winter CA, Risley EA, Nuss GW (1962) Carrageenan induced edema
in hind paw of rat as an assay for anti-inflammatory drugs. Proc
Soc Exp Biol Med 111:544–547
123