Anti-tumor and anti-angiogenic activity of novel hydantoin derivatives: Inhibition of VEGF secretion in liver metastatic osteosarcoma cells

Article abstract of DOI:10.1016/j.bmc.2009.06.004

A series of new azaspiro bicyclic hydantoin derivatives has been designed and synthesized. Initially, the anti-proliferative effect of the hydantoin derivatives was evaluated against human ovarian cancer cells (SKOV-3 and OVSAHO) and murine osteosarcoma c

Full text of DOI:10.1016/j.bmc.2009.06.004

Bioorganic & Medicinal Chemistry 17 (2009) 4928–4934
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Anti-tumor and anti-angiogenic activity of novel hydantoin derivatives:
Inhibition of VEGF secretion in liver metastatic osteosarcoma cells
Basappa a,c, , C. S. Ananda Kumar b, , S. Nanjunda Swamy b, Kazuyuki Sugahara a,
Kanchugarakoppal S. Rangappa b,
*
a Graduate School of Life Science, Hokkaido University, Sapporo 001-0021, Japan
b Department of Studies in Chemistry, University of Mysore, Mysore 570 006, India
c Department of Studies in Chemistry, Central College Campus, Bangalore University, Bangalore 560 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new azaspiro bicyclic hydantoin derivatives has been designed and synthesized. Initially, the
anti-proliferative effect of the hydantoin derivatives was evaluated against human ovarian cancer cells
(SKOV-3 and OVSAHO) and murine osteosarcoma cells (LM8 and LM8G7). Among the tested compounds,
8-(3-fluorobenzyl)-10-(4-(methylsulfonyl)benzyl)-8-azaspiro[bicyclo[3.2.1]octane-3,40-imidazolidine]-20,50-
dione (7h) and 8-(3,4-difluorobenzyl)-10-(4-(methylsulfonyl)benzyl)-8-azaspiro[bicyclo[3.2.1]octane-
3,40-imidazolidine]-20,50-dione (7i) showed a significant anti-proliferative activity against the OVSAHO
and LM8G7 cells. The real-time monitoring of theeffect of the compounds 7h and 7iagainst theproliferation
Received 11 April 2009
Revised 31 May 2009
Accepted 2 June 2009
Available online 9 June 2009
Keywords:
Anti-tumor
Hydantoin
Anti-angiogenesis
VEGF
of LM8G7 was revealed that resulting IC50 values were 102 lM and 13 lM, respectively. We reasoned that
the presence of fluorine atom at the 3rd position of the phenyl ring of the hydantoin side chain may deter-
mine the potency of the molecule. Furthermore, the compound 7i inhibited the tube formation of the mouse
endothelial cells. Finally, the treatment of the compound 7i against the proliferation of LM8G7 cells dem-
onstrated the down regulation of the secretion of VEGF, indicate the potential angioinhibitory effects. In
conclusion, our findings demonstrate the suppression of the secretion of VEGF by LM8G7 cells by the com-
pound 7i might contribute at least in part to the antitumor action.
Metastasis
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
development of novel antitumor agents. The present invention is
to explore the effect of novel hydantoin derivatives on the prolifer-
With a limited improvement in diagnosis, surgical techniques,
patient care, and adjuvant therapies, most of the death from cancer
is due to metastases.1 When cancer has metastasized, it may be
treated with radiosurgery, chemotherapy, radiation therapy, bio-
logical therapy, hormone therapy, or a combination of these.
Mainly, the macroscopic growth of tumor masses requires not only
tumor cell proliferation but also concomitant angiogenesis.2 Angi-
ogenesis has been described as one of the hallmarks of cancer.3 The
stimuli that promote tumor angiogenesis may be provided directly
by the tumor cells themselves or indirectly by host inflammatory
cells that are attracted to the tumor site. Endothelial cell activation
and proliferation is regulated by multiple proangiogenic factors, of
which the most widely studied is vascular endothelial growth fac-
tor (VEGF). VEGF, a potent endothelial cell mitogen in vivo whose
function is critical for angiogenesis in both normal development
and in tumour cell expansion and invasion.4,5 Thus, the inhibition
of tumor angiogenesis affords attractive targets for the
ation of human ovarian tumor cells such as SKOV-3, OVSAHO and
as well as murine osteosarcoma cells like LM8 and LM8G7, which
have metastatic potential to lung and liver, respectively.6 Hydan-
toin lesions have been demonstrated to be highly mutagenic both
in vitro and in vivo.7 The derivatives of hydantoin have attracted
much interest in drug discovery because of their wide range of bio-
logical activities and therapeutic applications depending on the
nature of substitution on the hydantoin ring.8 In continuation of
our ongoing research,9 herein we report the antitumor and anti-
angiogenic activity for the newly synthesized compounds 7 (a–i).
The possible mechanism for the anti-tumor activity was examined
under in vitro condition.
2. Results
2.1. Chemistry
The synthetic pathway utilized in the preparation of the azaspi-
ro bicyclic hydantoin derivatives is outlined as shown in Scheme 1.
A variety of combinatorial approaches have been described by
* Corresponding author. Tel.: +91 821 2419661; fax: +91 821 2412191.
E-mail address: rangappaks@gmail.com (K.S. Rangappa).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.004

Basappa et al. / Bioorg. Med. Chem. 17 (2009) 4928–4934
4929
O
O
O
HN
NH
3(a-c), K2CO3
DMF
(Boc)2, TEA
THF
KCN, (NH4)2CO3
O
ethanol, water, reflux
N
H
N
N
Boc
1
2
3
Boc
O
O
O
R1
R1
R1
N
N
N
NH
NH
NH
MDC
6(a-c), K2CO3
DMF
O
O
O
ether in HCl
N
N
H
N
Boc
R2
4(a-c)
7(a-i)
5(a-c)
3a: 1-(bromomethyl)benzene
3b: 3-bromoprop-1-ene
3c: 1-(bromomethyl)-4-(methylsulfonyl)benzene
6a: 1-(bromomethyl)-4-fluorobenzene
6b: 1-(bromomethyl)-3-fluorobenzene
6c: 4-(bromomethyl)-1,2-difluorobenzene
Scheme 1.
Table 1
Chemical structures, physical data and purity of compounds 7(a–i)
which pharmacophoric groups were attached to such a relatively
rigid scaffold. The synthesis begins with the protection of 8-azabi-
cyclo[3.2.1]octan-3-one 1 with Boc anhydride using dry THF as a
solvent. Under Bucherer-Bergs condition,10 construction of azaspi-
ro bicyclic hydantoin ring 3 was made. The reaction was carried
out in aqueous ethanolic media by using potassium cyanide and
ammonium carbonate at reflux temperature. The introduction of
the substituted alkyl/aryl groups at N-3 position of hydantoin ring
was achieved via selective N-alkylation reaction by reacting alkyl/
aryl halides in presence of K2CO3 and DMF solvent. Target key
intermediates 5 (a–c) was accomplished by deprotection of Boc
group from the compound 4 (a–c) with ether in HCl and followed
by basification with NaHCO3 solution. The aim of the fifth step was
to introduce respective fluoro substituted aryl halides at the 8th
position of azaspiro bicyclic moiety to lead the desired compounds
7 (a–i) for SAR study (Table 1). This was furnished by normal con-
densation reaction with good yield. The formation of the hydantoin
ring was confirmed by 1H NMR and IR spectra.
Compound R1
R2
Mp
Yield Purity
(°C)
(%)
(%)
7a
168–170 82
142–144 77
181–183 75
101–103 78
139–141 80
156–158 65
117–119 80
119–121 79
132–134 75
97.9
F
7b
7c
7d
7e
7f
98.7
F
F
99.1
F
F
F
98.40
97.39
97.9
F
F
F
2.2. Biology
2.2.1. Effects of hydantoin derivatives against the proliferation
of normal and tumor cells
The anti-proliferative activity of hydantoin derivatives 7 (a–i)
against four adherent tumor cells was studied. The inhibitory
effects of the compounds 7 (a–i) against the human ovarian tumor
cells like SKOV-03 and OVSAHO are summarized (Figs. 1 and 2).
Similarly, the inhibitory effects of the compounds 7 (a–i) against
the murine osteosarcoma cells such as LM8 and LM8G7 are sum-
marized (Figs. 3 and 4). Among the tested compounds, 7h and 7i
were significantly inhibited the proliferation of tumor cells.
Further, we dynamically monitored the effects of the compound
7h or 7i on the proliferation of LM8G7 cells using RT-CESTM
O
S
O
7g
7h
7i
98.6
H3C
H3C
H3C
F
O
S
O
99.2
F
F
O
S
O
97.87

4930
Basappa et al. / Bioorg. Med. Chem. 17 (2009) 4928–4934
(Real-Time Cell Electronic Sensing) system.11 The kinetic values
(IC50) were calculated from concentration–response curves by a
non-linear regression analysis using Prism software (GraphPad
software, Sandiego, CA). Compounds 7h and 7i inhibited the prolif-
eration of VEGF-expressing LM8G7 cells dose-dependently with an
IC50 value of 102 and 13 lM, respectively (Figs. 6 and 7). Therefore,
the above results indicated that the compound 7i markedly inhib-
its the growth of tumor cells. The cytotoxicity of the compounds 7
(a–i) were tested using mouse endothelial cells (Fig. 5).
2.2.2. The compound 7i inhibits angiogenesis in vitro
Since the compound 7i showed anti-tumor activity, we tested
the anti-angiogenic effects of compound 7i using an in vitro angi-
ogenesis model. The cultured mouse endothelial cells (40,000 cells/
well) on MatrigelTM in the presence of DMSO (0.1%) and VEGF
(2 ng/mL) rapidly align and form a hollow tube like structures
(Fig. 8A). Compound 7i selectively inhibited the tube formation
Figure 1. The inhibitory effects of hydantoin derivatives on the proliferation of
SKOV-3 cells (MF, mipefristone).
Figure 2. The inhibitory effects of hydantoin derivatives on the proliferation of
OVSAHO cells.
Figure 5. The inhibitory effects of hydantoin derivatives on the proliferation of
UV$2 cells.
Figure 3. The inhibitory effects of hydantoin derivatives on the proliferation of LM8
cells (CP, cisplatin).
Figure 6. Real-time monitoring of the effects of compound 7h on the proliferation
of LM8G7 cells (the arrow indicates the time of the addition of compound 7h).
Figure 4. The inhibitory effects of hydantoin derivatives on the proliferation of
LM8G7 cells.
Figure 7. Real-time monitoring of the effects of compound 7i on the proliferation of
LM8G7 cells (the arrow indicates the time of the addition of compound 7i).

Basappa et al. / Bioorg. Med. Chem. 17 (2009) 4928–4934
4931
on MatrigelTM (Fig. 8B) indicates the anti-angiogenic property of
the molecule.
2.2.3. Effects of the compound 7i on the secretion of VEGF by
LM8G7 cells
Since the molecule, 7i showed the potent inhibition against the
proliferation of LM8G7 cells, which express high amount of VEGF
mRNA.6c Using the Quantikine mouse VEGF immunoassay kit, we
measured the amount of VEGF secreted by the LM8G7 cells.12
LM8G7 (1 ꢀ 106) cells were cultured in 6-well plates for 24 h. Then
the cells were treated with or without the compound 7i at various
concentrations. After 48 h, the cells supernatant was taken, centri-
fuged, and estimated the protein content. Using the known amount
of protein, the VEGF content in the cells supernatant was mea-
sured. Compound 7i inhibited the secretion of VEGF in a dose-
Figure 9. Determination of the amount of VEGF secreted by LM8G7 cells after the
treatment with the compound 7i at various concentrations.
dependent manner (Fig. 9). Compound 7i at 56, 102 and 204 lM
effectively inhibited the secretion of VEGF by 23.8%, 46.7% and
63.9%, respectively.
are induced to undergo tube formation and form cell–cell contacts
that lead to branched networks, which are similar to capillary-like
3. Discussion
blood vessels. However, when UV$2 cells were cultured on poly-
TM
merized Matrigel in the presence of 7i (50 lM), the cells failed
The inhibitory effects of hydantoin derivative on the prolifera-
tion of tumor cells were compared with cisplatin and mifepristone.
The anti-proliferative activity of 7i is considerably higher than the
activity of all other hydantoin derivatives tested. The experiment
clearly demonstrates the presence of 4-methylsulfonyl benzyl
group and 3,4-difluoro benzene moiety, which has an influence
on the anti-proliferative activity of hydantoin derivatives. The
real-time monitoring of the effect of the compound 7i against the
proliferation of LM8G7 cells confirms the potency of the molecule.
Further, VEGF is considered to be one of the major stimulators
of tumor angiogenesis.13 The compound 7i was evaluated for its
ability to inhibit angiogenesis in vitro. Endothelial cells (UV$2)
to organize into capillary-like structures. At these concentrations
no other compounds tested inhibited tube formation.
In order to understand the mechanism of action, the compound
7i, was treated with LM8G7 cells at different concentration to
check its effects on the secretion of VEGF. The compound 7i effec-
tively inhibited the secretion of VEGF in a dose-dependent manner.
These results indicate that the compound 7i might be inhibited the
growth factor-induced proliferation of LM8G7 cells.
4. Conclusion
In conclusion, our current findings are consistent with the data
concerning the inhibitory effects of the compound 7i on the growth
of human/mouse tumor cells. Further, the compound 7i inhibited
the tube formation of UV$2 cells. Treatment with compound 7i
demonstrated the down regulation of the secretion of VEGF in
LM8G7 cells indicating the potential angioinhibitory effect of the
compound. In conclusion, our findings demonstrate the suppres-
sion of the secretion of VEGF by LM8G7 cells might contribute at
least in part to the antitumor action of compound 7i. However, fur-
ther experimental study and preclinical trials could be useful to
fully elucidate mechanism of their anti-proliferative action and
possible pharmacological strategy for the future treatment of
patients with osteosarcoma cancer.
5. Experimental
Melting points were determined using SELACO-650 hot stage
melting point apparatus and were uncorrected. Infrared (IR) spec-
tra were recorded using a Jasco FTIR-4100 series. Nuclear magnetic
resonance (1H NMR) spectra were recorded on Shimadzu AMX
400-Bruker, 400 MHz spectrometer using CDCl3 as a solvent and
TMS as internal standard (chemical shift in d ppm). Spin multiplets
are given as s (singlet), d (doublet), t (triplet) and m (multiplet).
Mass and purity were recorded on a LC-MSD-Trap-XCT. Elemental
(CHNS) analyses were obtained on Vario EL III Elementar. Silica gel
column chromatography was performed using Merck 7734 silica
gel (60–120 mesh) and Merck made TLC plates.
5.1. Synthesis of tert-butyl 3-oxo-8-azabicyclo[3.2.1]octane-8-
carboxylate 2
Figure 8. Compound 7i inhibits the tube formation in vitro. The UV$2 cells were
seeded in 6-well plates coated with MatrigelTM and the cells were incubated with or
without compound 7i (50 lM). After 18 h of culture, the reorganization of the sub-
confluent monolayer of UV$2 cells in 3-dimensional MatrigelTM was monitored and
A solution of 8-azabicyclo[3.2.1]octan-3-one 1 (15 g, 119 mmol)
was taken in dry THF, cooled to 0 °C. Triethyl amine (30.0 g,
photographed in a light microscope attached to a 3CCD camera.

4932
Basappa et al. / Bioorg. Med. Chem. 17 (2009) 4928–4934
297 mmol) and Boc anhydride (25.9 g, 119 mmol) were added. The
reaction mixture was allowed to stirred at room temperature for
6 h. The progress of the reaction was monitored by TLC. Upon
completion, the solvent was removed under reduced pressure and
residue was taken in water and extracted with ethyl acetate. Finally
organic layer was washed with water and dried with anhydrous
Na2SO4. The organic layer was evaporated to get tert-butyl 3-oxo-
8-azabicyclo[3.2.1]octane-8-carboxylate in good yield (77%, 20.6 g).
5.6.1. Synthesis of 10-benzyl-8-(4-fluorobenzyl)-8-azaspiro
[bicyclo[3.2.1]octane-3,40-imidazolidine]-20,50-dione 7a
A
solution of 10-benzyl-8-azaspiro[bicyclo[3.2.1]octane-3,40-
imidazolidine]-20,50-dione 5a (0.20 g, 0.70 mmol) in dimethyl form-
amide was taken, anhydrous K2CO3 (0.48 g, 3.5 mmol) was added to
the solution and stirred for 10 min 1-(bromomethyl)-4-fluoroben-
zene 6a (0.13 g, 0.70 mmol) was added. The reaction mixture was
stirred at room temperature for 8 h and progress of the reaction
was monitored by TLC. Upon completion, the solvent was removed
under reduced pressure and residue was taken in water and ex-
tracted with ethyl acetate. Finally water wash was given to the or-
ganic layer and dried with anhydrous Na2SO4.The solvent was
evaporated, crude product was purified by column chromatography
over silica gel (60–120 mesh) using chloroform/methanol (9:1) as an
eluent to get 7a (0.22 g) as pale yellow crystalline solid. 1H NMR
(CDCl3, 400 MHz) d: 8.53 (s, 1H, –NH), 7.24–7.36 (m, 5H, Ar-H),
7.07–7.1 (m, 3H, Ar-H), 4.63 (s, 2H, –CH2–), 3.61 (s, 2H, –CH2–),
2.42–2.46 (m, 2H, –CH–), 2.0–2.15 (m, 4H, –CH2–), 1.77–1.84 (m,
4H, –CH2–). MS (ESI + ion): m/z = 394.1. IR (KBr, cmꢁ1): 3360, 1653.
5.2. Synthesis of tert-butyl 20,50-dioxo-8-azaspiro
[bicyclo[3.2.1]octane-3,40-imidazolidine ]-8-carboxylate 3
A solution tert-butyl 3-oxo-8-azabicyclo[3.2.1]octane-8-carbox-
ylate 2 (19.0 g, 84 mmol), ammonium carbonate (24.2 g, 252 mmol)
were taken in ethanol and water. Then potassium cyanide (16.4 g,
252 mmol) dissolved in water and it was added the above solution.
The reaction mixture was refluxed for 40 h. The progress of the reac-
tion was monitored by TLC. Upon completion, the reaction mixture
was filtered and washed with water to get hydantoin product 3 in
good yield (76%, 18.8 g).
5.6.2. Synthesis of 10-benzyl-8-(3-fluorobenzyl)-8-azaspiro
[bicyclo[3.2.1]octane-3,40-imidazolidine]-20,50-dione (7b)
The general experimental procedure described above afforded
7b, the product obtained (0.21 g) was pale yellow crystalline solid
5.3. Synthesis of tert-butyl 10-benzyl-20,50-dioxo-8-azaspiro
[bicyclo[3.2.1]octane-3,40-imidazolidine]-8-carboxylate 4a
A
solution of tert-butyl 20,50-dioxo-8-azaspiro[bicyclo[3.2.1]
from
10-benzyl-8-azaspiro[bicyclo[3.2.1]octane-3,40-imidazoli-
octane-3,40-imidazolidine]-8-carboxylate 3 (2.0 g, 6.77 mmol) in
dimethyl formamide was taken, anhydrous K2CO3 (2.8 g,
20.30 mmol) and 1-(bromomethyl) benzene 3a (1.15 g, 6.77 mmol)
were added to the solution. The reaction mixture was stirred at room
temperature for 7 h and progress of the reaction was monitored by
TLC. Upon completion, the solvent was removed under reduced
pressure and residue was taken in water and extracted with
ethyl acetate. Finally water wash was given to the organic layer
and dried with anhydrous Na2SO4. The solvent was evaporated,
crude product was purified by column chromatography over silica
gel (60–120 mesh) using chloroform/methanol (9:1) as an eluent
(80%, 2.0 g).
dine]-20,50-dione 5a (0.20 g, 0.70 mmol), 1-(bromomethyl)-3-fluo-
robenzene (0.13 g, 0.70 mmol), and K2CO3 (0.48 g, 3.5 mmol). 1H
NMR (CDCl3, 400 MHz) d: 8.58 (s, 1H, –NH), 7.24–7.36 (m, 5H, Ar-
H), 7.07–7.1 (m, 3H, Ar-H), 4.65 (s, 2H, –CH2–), 3.63 (s, 2H, –CH2–),
2.41–2.46 (m, 2H, –CH–), 2.0–2.17 (m, 4H, –CH2–), 1.75–1.85 (m,
4H, –CH2–). MS (ESI + ion): m/z = 394. IR (KBr, cmꢁ1): 3360, 1653.
5.6.3. Synthesis of 10-benzyl-8-(3,4-difluorobenzyl)-8-azaspiro
[bicyclo[3.2.1]octane-3,40-imidazolidine]-20,50-dione (7c)
Thegeneralexperimentalproceduredescribedaboveafforded7c,
the product obtained (0.21 g) was pale yellow crystalline solid
from
10-benzyl-8-azaspiro[bicyclo[3.2.1]octane-3,40-imidazoli-
dine]-20,50-dione 5a (0.20 g, 0.70 mmol), 4-(bromomethyl)-1,2-
difluorobenzene (0.14 g, 0.70 mmol), and K2CO3 (0.48 g, 3.5 mmol).
1H NMR (CDCl3, 400 MHz) d: 8.51 (s, 1H, –NH), 7.24–7.36 (m, 5H, Ar-
H), 7.07–7.1 (m, 3H, Ar-H), 4.65 (s, 2H, –CH2–), 3.60 (s, 2H, –CH2–),
2.43–2.47 (m, 2H, –CH–), 2.0–2.17 (m, 4H, –CH2–), 1.76–1.82 (m,
4H, –CH2–). MS (ESI + ion): m/z = 412.2. IR (KBr, cmꢁ1): 3354, 1649.
5.4. Synthesis of tert-butyl 10-allyl-20,50-dioxo-8-azaspiro
[bicyclo[3.2.1]octane-3,40-imidazolidine]-8-carboxylate 4b
The general experimental procedure described above afforded 4b
(1.7 g) from tert-butyl 20,50-dioxo-8-azaspiro[bicyclo[3.2.1]octane-
3,40-imidazolidine]-8-carboxylate 3 (2.0 g, 6.77 mmol), 3-bromo-
prop-1-ene (0.98 g, 8.12 mmol) and K2CO3 (2.8 g, 20.3 mmol).
5.6.4. Synthesis of 10-allyl-8-(4-fluorobenzyl)-8-azaspiro
[bicyclo[3.2.1]octane-3,40-imidazolidine]-20,50-dione (7d)
The general experimental procedure described above afforded
7d, the product obtained (0.22 g) was white crystalline solid from
10-allyl-8-azaspiro[bicyclo[3.2.1]octane-3,40-imidazolidine]-20,50-
dione 5b (0.20 g, 0.85 mmol), 1-(bromomethyl)-4-fluorobenzene
(0.16 g, 0.85 mmol), and K2CO3 (0.58 g, 4.25 mmol). 1H NMR
(CDCl3, 400 MHz) d: 8.52 (s, 1H, –NH), 7.35–7.39 (m, 2H, Ar-H),
7.15 (t, 2H, Ar-H), 5.73–5.86 (m, 1H, @CH), 5.0–5.09 (m, 2H,
CH2@), 3.94 (d, 2H, –CH2), 3.53 (s, 2H, –CH2), 2.44–2.50 (m, 2H,
–CH–), 2.1–2.2 (m, 4H, –CH2–), 1.92–1.99 (m, 4H, –CH2–). MS
(ESI + ion): m/z = 344.1. IR (KBr, cmꢁ1): 3355, 1653.
5.5. Synthesis of tert-butyl 10(4-(methylsulfonyl)benzyl-20,50-
dioxo-8-azaspiro [bicyclo[3.2.1]octane-3,40-imidazolidine]-8-
carboxylate 4c
The general experimental procedure described above afforded 4c
(2.26 g) from tert-butyl 20,50-dioxo-8-azaspiro[bicyclo[3.2.1]octane-
3,40-imidazolidine]-8-carboxylate 3 (2.0 g, 6.77 mmol), 1-(bromo-
methyl)-4-(methylsulfonyl)benzene (1.68 g, 6.77 mmol) and K2CO3
(2.8 g, 20.3 mmol).
5.6. Synthesis of 10-benzyl-8-azaspiro[bicyclo[3.2.1]octane-3,40-
imidazolidine]-20,50-dione 5a
5.6.5. Synthesis of 10-allyl-8-(3-fluorobenzyl)-8-azaspiro
[bicyclo[3.2.1]octane-3,40-imidazolidine]-20,50-dione (7e)
The general experimental procedure described above afforded
7e, the product obtained (0.23 g) was white crystalline solid from
10-allyl-8-azaspiro[bicyclo[3.2.1]octane-3,40-imidazolidine]-20,50-
dione 5b (0.20 g, 0.85 mmol), 1-(bromomethyl)-3-fluorobenzene
(0.16 g, 0.85 mmol), and K2CO3 (0.58 g, 4.25 mmol). 1H NMR
(CDCl3, 400 MHz) d: 8.50 (s, 1H, –NH), 7.30 (m, 1H, Ar-H), 7.17
Compound 4a was taken in dry MDC, cooled to 0 °C. Ether in HCl
was added and allowed to stirred at room temperature for 3 h.
Deprotected salt compound was basified with NaHCO3 solution
and extracted with ethylacetate, organic layer was concentrated
to get 5a. Similar experimental procedure was followed for the
compounds 5b and 5c.

Basappa et al. / Bioorg. Med. Chem. 17 (2009) 4928–4934
4933
(d, 2H, Ar-H), 6.97 (m, 1H, Ar-H), 5.77–5.86 (m, 1H, @CH), 5.16–
5.20 (m, 2H, CH2@), 4.1 (d, 2H, –CH2), 3.65 (s, 2H, –CH2), 2.46–
2.51 (m, 2H, –CH–), 2.17–2.20 (m, 4H, –CH2–), 1.77–1.82 (m, 4H,
–CH2–). MS (ESI + ion): m/z = 344.1. IR (KBr, cmꢁ1): 3350, 1660.
containing 66
Incubated the plates for 48 h at 37 °C under 5% CO2. After the indi-
cated time, 5 L of TetraColor One was added to each well and the
mixture was incubated for an additional 2 h. The viability of cells
treated with DMBO was measured using TetraColor One.14 Absor-
bance at 450 nm was monitored and the percentage viability of
the cells was calculated. The % inhibition of the proliferation of nor-
mal or tumor cells by the hydantoin derivatives was presented.
Results are reported as the average four replicates.
lg/mL concentrations of hydantoin derivatives.
l
5.6.6. Synthesis of 10-allyl-8-(3,4-difluorobenzyl)-8-azaspiro
[bicyclo[3.2.1]octane-3,40-imidazolidine]-20,50-dione (7f)
The general experimental procedure described above afforded
7f, the product obtained (0.19 g) was white crystalline solid from
10-allyl-8-azaspiro[bicyclo[3.2.1]octane-3,40-imidazolidine]-20,50-
dione 5b (0.20 g, 0.85 mmol), 4-(bromomethyl)-1,2-difluoroben-
zene (0.17 g, 0.85 mmol), and K2CO3 (0.58 g, 4.25 mmol). 1H NMR
(CDCl3, 400 MHz) d: 8.52 (s, 1H, –NH), 7.32–7.39 (m, 2H, Ar-H),
7.17–7.2 (m, 1H, Ar-H), 5.72–5.79 (m, 1H, @CH), 4.93–5.08 (m,
2H, CH2@), 3.94 (d, 2H, –CH2), 3.52 (s, 2H, –CH2), 2.44–2.49 (m,
2H, –CH–), 2.15–2.2 (m, 4H, –CH2–), 1.92–1.98 (m, 4H, –CH2–).
MS (ESI + ion): m/z = 362.2. IR (KBr, cmꢁ1): 3371, 1658.
5.6.11. Real-time proliferation assay
LM8G7 (10,000 cells/well) were seeded in ACEA0s 96X e-plateTM
in a final volume of 150
when the cells were in the log growth phase, they were incubated
with 150 L of medium containing various concentrations of com-
l
L.11 Approximately, 24 h after seeding,
l
pound 7h or 7i or DMSO as a solvent control. The effect of the com-
pounds on the proliferation of LM8G7 cells was monitored
dynamically for every 10 min. The cell index (quantitative mea-
surement of cells) against the time was plotted. The IC50 values
were calculated from concentration–response curves by a non-lin-
ear regression analysis using Prism software (GraphPad Software,
San Diego, CA).
5.6.7. Synthesis of 8-(4-fluorobenzyl)-10-(4-(methylsulfonyl)
benzyl)-8-azaspiro[bicyclo [3.2.1]octane-3,40-imidazolidine]-
20,50-dione (7g)
The general experimental procedure described above afforded 7g,
the product obtained (0.20 g) was off-white crystalline solid from
10-(4-(methylsulfonyl)benzyl)-8-azaspiro[bicyclo[3.2.1]octane-3,40-
imidazolidine]-20,50-dione 5c (0.20 g, 0.55 mmol), 1-(bromomethyl)-
4-fluorobenzene (0.10 g, 0.55 mmol), and K2CO3 (0.38 g, 2.75 mmol).
1H NMR (CDCl3, 400 MHz) d: 8.60 (s, 1H, –NH), 7.91 (d, 2H, Ar-H), 7.55
(d, 2H, Ar-H), 7.27–7.38 (m, 2H, Ar-H), 7.02 (t, 2H, Ar-H), 4.72 (s, 2H,
–CH2–), 3.60 (s, 2H, –CH2–), 3.04 (s, 3H, –CH3), 2.44–2.47 (m, 2H,
–CH–), 2.17–2.19 (m, 4H, –CH2–), 1.74–1.80 (m, 4H, –CH2–). MS
(ESI + ion): m/z = 472.1. IR (KBr, cmꢁ1): 3357, 1650, 1340, 1285.
5.6.12. In vitro angiogenesis assay
MatrigelTM (ECM 625, Chemicon) was added to a 24-well plate
in a final volume of 100 lL and allowed to solidify at 37 °C for
30 min. Single cell suspensions (100 lL) of UV$2 were seeded at
a density of 4 ꢀ 105 cells/mL, to the MatrigelTM coated well, with
or without compound 7i (50 lM) and VEGF (2 ng/mL). After 18 h
of culture, the reorganization of the sub-confluent monolayer of
UV$2 cells in 3-dimensional MatrigelTM was monitored and photo-
graphed in a light microscope attached to a 3CCD camera (Olym-
pus, FX380-model, JAPAN).
5.6.8. Synthesis of 8-(3-fluorobenzyl)-10-(4-(methylsulfonyl)
benzyl)-8-azaspiro[bicyclo [3.2.1]octane-3,40-imidazolidine]-
20,50-dione (7h)
5.6.13. VEGF quantification assay
The general experimental procedure described above afforded
7h, the product obtained (0.19 g) was off-white crystalline solid
LM8G7 (1 ꢀ 106) cells were seeded into a 6-well plates and
incubated overnight. After 24 h, the media was replaced with fresh
from
10-(4-(methylsulfonyl)benzyl)-8-azaspiro[bicyclo[3.2.1]
serum-free medium containing 56 lM, 102 lM, and 204 lM of
octane-3,40-imidazolidine]-20,50-dione 5c (0.20 g, 0.55 mmol), 1-
(bromomethyl)-3-fluorobenzene (0.10 g, 0.55 mmol), and K2CO3
(0.38 g, 2.75 mmol). 1H NMR (CDCl3, 400 MHz) d: 8.66 (s, 1H,
–NH), 7.89 (d, 2H, Ar-H), 7.45 (d, 2H, Ar-H), 7.32–7.38 (m, 1H, Ar-
H), 7.19 (t, 2H, Ar-H), 7.07 (t, 1H, Ar-H), 4.64 (s, 2H, –CH2–), 3.57 (s,
2H, –CH2–), 3.15 (s, 3H, –CH3), 2.49–2.50 (m, 2H, –CH–), 2.19–2.23
(m, 4H, –CH2–), 1.93–2.0 (m, 4H, –CH2–). MS (ESI + ion): m/z = 472.
IR (KBr, cmꢁ1): 3355, 1648, 1333, 1280.
compound 7i. The cells were further incubated for 48 h at 37 °C.
The cell supernatant was collected, centrifuged at 5000 rpm for
5 min at 4 °C and the protein concentrations were determined by
BCA (bicinchoninic acid) assay kit (Thermo Fisher Scientific Inc.
USA).15 The quantification of the mouse VEGF was done using
the Immuno Assay Kit (Quantikine, R&D systems). Briefly, the
known amount of recombinant VEGF as standard and the samples
to be tested were added to a 96-well microplate, which was pre-
coated with the polyclonal antibody specific for mouse VEGF and
incubated for 2 h. The bound VEGF was detected by adding horse-
radish peroxidase conjugated polyclonal antibody against mouse
VEGF. After 2 h, the substrate chromogen was added and allowed
to react for 30 min with the peroxidase conjugate, the reaction
was stopped and the VEGF content was quantified in a micro-plate
reader at 450 nm (BIORAD, Model 680). The decrease in the
amount of VEGF secretion by the compound 7i was presented.
5.6.9. Synthesis of 8-(3,4-difluorobenzyl)-10-(4-(methylsulfonyl)
benzyl)-8-azaspiro[bicyclo [3.2.1]octane-3,40-imidazolidine]-
20,50-dione (7i)
The general experimental procedure described above afforded 7i,
the product obtained (0.195 g) was off-white crystalline solid from
10-(4-(methylsulfonyl)benzyl)-8-azaspiro[bicyclo[3.2.1]octane-3,40-
imidazolidine]-20,50-dione 5c (0.20 g, 0.55 mmol), 4-(bromomethyl)-
1,2-difluorobenzene (0.11 g, 0.55 mmol), and K2CO3 (0.38 g,
2.75 mmol). 1H NMR (CDCl3, 400 MHz) d: 8.64 (s, 1H, –NH), 7.87 (d,
2H, Ar-H), 7.46 (d, 2H, Ar-H), 7.30–7.36 (m, 1H, Ar-H), 7.19 (t, 1H,
Ar-H), 7.04 (t, 1H, Ar-H), 4.60 (s, 2H, –CH2–), 3.55 (s, 2H, –CH2–),
3.17 (s, 3H, –CH3), 2.45–2.50 (m, 2H, –CH–), 2.19–2.25 (m, 4H,
–CH2–), 1.95–2.0 (m, 4H, –CH2–). MS (ESI + ion): m/z = 490. IR (KBr,
cmꢁ1): 3351, 1653, 1340, 1284.
Acknowledgements
This work was supported in part by the INSA (Indian National
Science Academy)-JSPS (Japan Society for the Promotion of Sci-
ence) program between India (to K.S.R.) and Japan (to K.S.). Profes-
sor K.S.R. grateful to UGC, Govt. of India for financial support under
the project UGC-SAP (Phase I) DRS Programme DV4/375/2004–05
and one of the authors, Basappa thanks JSPS, Japan for the award
of JSPS Postdoctoral fellowship. We thank Prof. M. Miyasaka, Dr.
K. N. Sugahara and Dr. C. M. Lee for providing the LM8 and
LM8G7 cells.
5.6.10. In vitro cell proliferation assay
SKOV-3, OVSAHO, LM8, LM8G7, and UV$2 cells were seeded at a
density of 15,000 cells/well in a 96-well plate and incubated over-
night at 37 °C. The medium was changed to the new medium

4934
Basappa et al. / Bioorg. Med. Chem. 17 (2009) 4928–4934
Manguikian, R. A.; Buckheit, R. W.; Truss, J. J. W.; Shannon, W. M.; Secrist, J. A. J.
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