Synthesis and antitumor activity of indolylpyrimidines: Marine natural product meridianin D analogues

Article abstract of DOI:10.1016/j.bmc.2006.11.023

Marine indole alkaloid meridianin D analogues have been synthesized starting from the appropriate 3-cyanoacetyl indole. A facile two-step conversion of 3-cyanoacetyl indole to the corresponding cyano meridianin D analogue by treatment with dimethylformamide-dimethylacetal and further cyclization of the resulting enaminonitrile with aminoguanidine is described. Then, alkaline hydrolysis of cyano meridianin D afforded the carboxylic acid analogue. The treatment of acid with 75% H₂SO₄ afforded the desired 6-debromomeridianin D. Simply treatment of cyano meridianin D analogue with hydrazine hydrate afforded the amidrazone analogue. The biological evaluation indicated that cyano analogue showed good cytotoxic activity with IC₅₀ values of 0.85 and 2.65 μg (against MCF7 and HeLa, respectively), but acid and amidrazone analogues showed high cytotoxicity with IC₅₀ values of 0.75 and 0.25 μg, respectively (against MCF7).

Full text of DOI:10.1016/j.bmc.2006.11.023

Bioorganic & Medicinal Chemistry 15 (2007) 1206–1211
Synthesis and antitumor activity of indolylpyrimidines: Marine
natural product meridianin D analogues
a,
b
*
Mohamed A. A. Radwan and Mahmoud El-Sherbiny
a
Applied Organic Chemistry Department, National Research Centre, Dokki, Giza, Egypt
b
Medicinal Chemistry Department, National Research Centre, Dokki, Giza, Egypt
Received 12 June 2006; revised 9 November 2006; accepted 13 November 2006
Available online 16 November 2006
Abstract—Marine indole alkaloid meridianin D analogues have been synthesized starting from the appropriate 3-cyanoacetyl
indole. A facile two-step conversion of 3-cyanoacetyl indole to the corresponding cyano meridianin D analogue by treatment with
dimethylformamide-dimethylacetal and further cyclization of the resulting enaminonitrile with aminoguanidine is described. Then,
2 4
alkaline hydrolysis of cyano meridianin D afforded the carboxylic acid analogue. The treatment of acid with 75% H SO afforded
the desired 6-debromomeridianin D. Simply treatment of cyano meridianin D analogue with hydrazine hydrate afforded the amid-
razone analogue. The biological evaluation indicated that cyano analogue showed good cytotoxic activity with IC50 values of 0.85
and 2.65 lg (against MCF7 and HeLa, respectively), but acid and amidrazone analogues showed high cytotoxicity with IC50 values
of 0.75 and 0.25 lg, respectively (against MCF7).
ꢀ
2006 Elsevier Ltd. All rights reserved.
1. Introduction
H N
2
N
Marine organisms represent a very promising source of
unique bioactive molecules and antitumor secondary
metabolites. Various biologically active indole alkaloids
have been produced from the marine invertebrates over
the past few years. Along with these, 3-substituted in-
doles represented a promising structural class of marine
alkaloids based upon their high degree of biological
activity. The substitution at 3-position of the indole ring
connecting an extra heterocyclic ring: imidazole (topsen-
N
R1
R4
R2
R3
1
–4
N
H
A R1=OH, R2=R3=R4=H
B R1=OH, R2=R4=H, R3= Br
C R1=R3=R4=H, R2=Br
D R1=R2=R4=H, R3=Br
E R1=OH,R2=R3=H,R4=Br
4
,5
6
Meridianins
tins and nortopsentins ); dihydroimidazole (discoder-
7
mindole ); oxazole (martefrgin, amazol ); oxadiazine
(
(
8
9
1
0
11
alboinon ); maliemide (didemidines ); and piperazine
3
dragmacidon ) takes place. Recently, five novel indole
Figure 1. Structure of meridianins A–E.
alkaloids, meridianins A–E (Fig. 1), have been isolated
1
2
from the tunicate Splidium meridiaum. They show
1
cytotoxicity toward murine tumor cell lines and show
2. Chemistry
3
1
4
potent inhibition against several protein kinases and
are therefore interesting synthetic targets. This forms
the basis for the aim of the synthesis of the marine in-
dole alkaloids.
The natural meridianins have previously been synthesized
by two different strategies: either the construction of the 2-
aminopyrimidine ring from a b-dicarbonylic substituted
or by a cross-coupling reaction be-
tween the indole and pyrimidine moieties. This synthetic
methodology has also been applied for the synthesis of
1
5,16
indole precursor,
1
7
1
8
natural Variolin B. Recently, Stanovnik et al. reported
the synthesis of both condensed indolylpyrimidones as
Keywords: Alkaloids; Indole; Enaminonitrile; Heterocycles; Meridia-
nines; Cyclizations; Antitumor.
1
9
*
Corresponding author. Tel.: +20 2 7201588; fax: +20 2 3370931;
e-mail: m1radwan@yahoo.com
meridianin analogues and polycyclic meridianin ana-
2
logues with uracil structural unit.
0
0
968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.11.023

M. A. A. Radwan, M. El-Sherbiny / Bioorg. Med. Chem. 15 (2007) 1206–1211
1207
CN
3 (Scheme 2). This indolylpyrimidine derivative 4 can be
0
O
considered as 5 -cyano-6-debromomeridianin D. The
structure of compound 4 was established on the basis
of its elemental analysis and spectral data. For example,
its IR spectrum showed the presence of absorption peak
a
N
N
ꢀ
1
1
H
H
at 2222 cm due to a cyano group and its H NMR
spectrum revealed a singlet signal at d 8.53 characteristic
of the pyrimidine proton (H-6 ), which was confirmed
1
0
1
3
Scheme 1. Reagents and condition: (a) cyanoactic acid, Ac
2
O, D.
by C NMR and HSQC experiments at d 163.11 (see
Section 5).
Here a facile synthesis of indolylpyrimidines was
achieved via the cyanoacetyl side chain at the indole
Alkaline hydrolysis of compound 4 with 20% NaOH
solution in ethanol under refluxing temperature for 6 h
afforded the carboxylic acid analogue 5. The structure
of compound 5 was established on the basis of its ele-
mental analysis and spectral data. For example, its IR
spectrum showed the presence of absorption peaks at
3
2
-position using C2 moiety for the construction of the
-aminopyrimidine ring, through the cyclization of
(2Z)-3-dimethylamino-2-(1H-indole-3-carbonyl)acrylo-
nitrile 2 with aminoguanidine as N,N-1,3-dinucleophile.
ꢀ
1
The starting compound 3-cyanoacetyl indole 1 was pre-
2
3125 and 1690 cm
groups, respectively, and its H NMR spectrum revealed
due to the OH and carbonyl
1
1
viously prepared by Kreher and Wagner and recently
2
2
by Bergman et al. via a new facile approach starting
from indole and cyanoacetic acid (Scheme 1). A novel
a singlet signal at d 8.25 characteristic of the pyrimidine
proton (H-6 ). Decarboxylation of acid 5 with 75%
0
(
2Z)-3-dimethylamino-2-(1H-indole-3-carbonyl)acrylo-
nitrile 2 was prepared from 3-cyanoacetyl indole 1 by
treatment with dimethylformamide-dimethylacetal
DMF-DMA) in dry toluene at 120 ꢁC for 15 min, the
H
the previously prepared 6-debromomeridianin D 6, but
2
SO
under refluxing temperature for 6 h afforded
4
1
7
in a higher yield. In addition, simply treatment of cya-
no meridianin D analogue with hydrazine hydrate in
absolute ethanol under refluxing temperature for 3 h
afforded the desired amidrazone analogue 7 (Scheme
3). The structure of compound 7 was established on
the basis of its elemental analysis and spectral data.
For example, its IR spectrum showed the presence of
(
enaminonitrile 2 was obtained in a good yield (84.5%).
The structure of compound 2 was established on the ba-
sis of its elemental analysis and spectral data. For exam-
ple, its H NMR spectrum revealed a singlet signal at d
8
1
.30 characteristic of the olefinic proton and two singlet
ꢀ
1
signal at d 3.25 and 3.30 as a result of magnetically non-
equivalent N(CH ) group.
absorption peaks at 3425, 3345, 3285 cm due to 3
1
NH groups and its H NMR spectrum revealed a sin-
2
3
2
glet signal at d 8.45 characteristic of the pyrimidine pro-
ton (H-6 ).
0
Pyrimido annelation took place by further cyclization of
the enaminonitrile 2 with guanidine hydrochloride in
absolute ethanol in the presence of anhydrous potassi-
um carbonate under refluxing temperature for 12 h to
give 2-amino-4-(1H-indolyl-3-)-pyrimidine-5-carbonitri-
le 4 in 71.4% yield through the nonisolable intermediate
The structures of the novel compounds were determined
1
13
by spectroscopic methods (IR, H and C NMR,
HSQC, NOE and mass spectrometry) and by elemental
analyses for C, H, and N. Compound 2 was character-
CN
CN
O
O
H
N
a
N
N
H
H
2
1
C
H N
2
N
CN
N
O
H
CN
N
NH
N
H
H
2
N
N
H
4
3
Scheme 2. Reagents and conditions: (a) DMF-DMA, toluene, 120 ꢁC, 15 min; (b) H
2 2 2 3
N(C@NH)NH ÆHCl, K CO , ethanol, reflux, 12 h.

1208
M. A. A. Radwan, M. El-Sherbiny / Bioorg. Med. Chem. 15 (2007) 1206–1211
H N
toxic activity in in vitro disease-oriented antitumor
screening using sulforhodamine B (SRB) assay includ-
ing 4 human tumor cell lines representing different
2
N
H N
2
N
N
N
24
a
cancer types. And also tested for its cytotoxicity
2
COOH
3
CN
against Ehrlich Ascites Carcinoma.
The cyano
meridianin D 4 analogue exhibited a good cytotoxic
activity against breast carcinoma cell line (MCF7)
and cervix cell line (HeLa) with the IC50 values of
N
H
N
H
5
0
.85 and 2.65 lg, respectively, but carboxylic acid
4
analogue 5 and amidrazone analogue 7 showed high
cytotoxicity with IC50 values of 0.75 and 0.25 lg,
respectively (against MCF7).
c
b
H N
The natural product, meridianin D, showed weak anti-
tumor activities against a variety of tumor cell lines,
and its analogue 6-debromomeridianin D 6 did not
2
H N
N
2
N
N
N
NH2
13
show any antitumor activity at all.
N
H N
The tested compounds were proven to have no cytotoxic
activities against liver carcinoma and brain tumor cell
lines. And it showed no effect on the viability of the Ehr-
lish Ascites Carcinoma at concentrations 25, 50, and
2
N
N
H
H
7
6
-debromo meridianin D 6
1
00 lg/mL.
Scheme 3. Reagents and conditions: (a) 20% NaOH, ethanol 220ꢁC, 6 h;
(b) 75% H
2
SO
4
, reflux, 6 h; (c) hydrazine hydrate, ethanol, reflux, 3 h.
In summary, we have synthesized cyano 4, carboxylic
acid 5, and amidrazone 7 meridianin D analogues that
showed good cytotoxic effects in the growth of different
human cell lines in vitro, especially, cyano analogue 4
against breast and cervix carcinoma cell lines. And car-
boxylic acid 5 and amidrazone 7 analogues showed high
antitumor activity against breast carcinoma cell line. In
CN
O
H
3.3
N
3.2
H
8.03
13
comparison, these meridianin D analogues were con-
sidered to be more potent than the natural meridianin
N
NOE
H
D which was isolated from the tunicate Splidium
1
meridiaum.
Figure 2. NOE experiment of (Z)-isomer 2.
2
ized as pure (Z)-olefin, of which the configuration
around the olefinic double bond was determined by
NOE experiment (Fig. 2).
5. Experimental
5
5
.1. Chemistry
.1.1. General methods. Melting points were deter-
3. Conclusions
In summary, simple, cost-effective, and convenient
method for the synthesis of four meridianin D ana-
logues starting from easily prepared 3-cyanoacetyl in-
dole was demonstrated. In this approach cyanoacetyl
group at the heteroaromatic 3-position is used as a
precursor to construct the appropriately substituted
pyrimidines. In this method, we do not need to pro-
mined on a Gallenkamp apparatus and are uncorrect-
ed. IR spectra were recorded on Shimadzu FT-IR
8101 PC infrared spectrophotometer. H NMR spec-
1
tra were recorded on a Jeol EX-270 MHz spectrom-
eter using DMSO-d as solvent and TMS as the
6
1
3
internal standard, C NMR and 2D NMR spectra
were recorded on a varian Mercury VX 300 NMR
using DMSO-d as solvent and TMS as an internal
1
6
tect the N-indole compared to the previous methods.
6
Further application of (2Z)-3-dimethylamino-2-(1H-in-
dole-3-carbonyl)acrylonitrile 2 in the synthesis of a
variety of other meridianin analogues and 3-indole
heterocyclic compounds is currently underway. The
cytotoxic activity in vitro and structure–activity rela-
tionship (SAR) of the newly synthesized compounds
were provided.
standard. Mass spectra were recorded on a Finnigan
mat. SSQ-7000 GC-MS spectrometer. Microanalyses
were performed at the Microanalytical Center of Cai-
ro University.
5.1.2. 3-(1H-Indol-3-yl)-3-oxo-propionitrile 1. Prepared
2
0
according to the literature procedure; colorless prisms;
yield (91%); mp 240–241 ꢁC. IR: 3214 (NH), 2251 (CN),
ꢀ
1 1
633 (C@O), 1580 (C@C) cm . H NMR (270 MHz,
1
4
. Biological evaluation
DMSO-d ): d 4.48 (s, 2H), 7.22–7.24 (m, 2H, H-5, H-
6
6
8.37 (br s, 1H, H-2), 12.16 (br s, 1H, NH). C NMR:
d 29.4 (t), 112.4 (d), 114.4 (s), 116.4 (s), 121.0 (d),
), 7.49–7.51 (m, 1H, H-7), 8.13–8.15 (m, 1H, H-4),
1
3
The synthesized meridianin D analogues were evaluat-
ed in National Cancer Institute (Egypt) for its cyto-

M. A. A. Radwan, M. El-Sherbiny / Bioorg. Med. Chem. 15 (2007) 1206–1211
1209
0
1
(
22.3 (d), 123.3 (d), 125.1 (s), 135.4 (d), 136.6 (s), 182.8
s).
(s, 1H, H-6 ), 8.41 (s, 1H, H-2), 8.50–8.55 (dd, 1H, H-
C
1
3
4), 11.91 (br s, 1H, NH), 11.97 (br s, 1H, OH).
NMR (300 MHz, DMSO-d ): d 111.14 (C-7), 112.13
(C-3), 112.44 (C-5 ), 120.85 (C-3a), 122.56 (C-6),
123.56 (C-5), 126.24 (C-4), 129.32 (C-2), 136.17 (C-7a),
161.23 (C-6 ), 163.12 (C-4 ), 163.34 (C-2 ), 167.57
(COOH); EIMS m/z (%) 254 (M+, 100), 237 (91), 209
(54). Anal. Calcd for C H N O : C, 61.41; H, 3.96;
6
0
5
.1.3.
(2Z)-3-Dimethylamino-2-(1H-indole-3-carbon-
yl)acrylonitrile 2. To a solution of 3-(1H-indol-3-yl)-3-
oxo-propionitrile 1 (1.18 g, 6.4 mmol) in dry toluene
(10 mL) was added a solution of dimethylformamide-
dimethylacetal (1.2 g, 10 mmol) in the same solvent
0
0
0
1
3
10
4
2
(5 mL). The resultant solution was heated at 120 ꢁC
for 15 min. After cooling, the mixture was filtered to
N, 22.04, O, 12.59. Found: C, 61.12; H, 3.71; N,
21.89; O, 12.67.
leave enaminonitrile 2 as a yellow solid (1.35 g,
8
4.5%). Recrystallization from EtOH gave analytically
5.1.6. 6-Debromomeridianin D (6). To a 75% aqueous
solution of sulfuric acid (5 mL), compound 5 (0.25 g,
0.98 mmol) was added gradually and the mixture was
heated at 90 ꢁC for 2 h and at 120 ꢁC for further 4 h.
The obtained mixture was cooled, poured into 20%
aqueous solution of sodium hydroxide (5 mL), and
extracted with hexane–ethyl acetate. The extract was
washed with water, concentrated, and purified by silica
gel column chromatography (eluent, hexane/ethyl ace-
tate, 4:6) to give 6-debromomeridianin D. Yield: 0.18 g
1
pure material; mp 160–163 ꢁC (yellow prisms).
H
NMR (270 MHz, DMSO-d ) 3.20 (br s, 3H, NCH ),
6
3
3
7
.30 (br s, 3H, NCH ), 7.15–7.19 (m, 2H, H-5, H-6),
3
.45–7.48 (m, 1H, H-7), 8.03 (br s, 1H, H-2), 8.13–8.15
(
m, 1H, H-4), 8.30 (br s, 1H, H-b), 11.80 (br s, 1H,
+
NH). MS: m/z (%) (EI positive) 238, M ꢀ1, (57), 222
53), 210 (30), 195 (24), 184 (69), 159 (43), 144 (100),
30 (29), 116 (61), 89 (65), 77 (38), 62 (55). Anal. Calcd
for C H N O: C, 70.28; H, 5.48; N, 17.56; O, 6.69.
(
1
1
4
13
3
1
7
Found: C, 70.18; H, 5.41; N, 1767; O, 6.74.
(87%); mp 263–265 ꢁC (lit. 262–264 ꢁC).
0
5.1.4. 5 -Cyano-6-debromomeridianin D 4. A mixture of
enaminonitrile 2 (2.0 g, 8.36 mmol), guanidine hydro-
IR (KBr) mmax 3409 (NH ), 3329 (NH ), 3172 (NH),
1659, 1569, 1454, 1416, 1241, 1129, 808, 741,
2
2
ꢀ
1
1
chloride (1.15 g, 12.0 mmol), anhydrous K CO (2.0 g,
2
5.0 mmol), and absolute ethanol (20 mL) was heated
at reflux temperature for 12 h. After cooling, the mixture
was filtered off to leave 5 -cyano-6-debromomeridianin
D 4 as a yellow solid (1.4 g, 71.4%). Recrystallization
684 cm ; H NMR (270 MHz, DMSO-d ): d 6.4 (br
3
6
0
1
s, 2H, NH ), 7.03 (d, 1H, H-5 ), 7.15 (m, 2H, H-5,
0
2
H-6), 7.44–7.46 (d, 1H, H-7), 8.11 (d, 1H, H-6 ), 8.19
(s, 1H, H-2), 8.58–8.61 (d, 1H, H-4), 11.65 (br s, 1H,
0
1
3
NH). C NMR (300 MHz, DMSO-d ): d 105.2 (C-
6
0
from EtOH gave analytically pure material; mp 258–
2
5 ), 111.71 (C-7), 113.70 (C-3), 120.21 (C-3a), 121.85
(C-6), 122.32 (C-5), 125.30 (C-4), 128.10 (C-2), 136.90
0 0 0
C-7a), 156.91 (C-6 ), 162.62 (C-4 ), 163.40 (C-2 );
EIMS m/z (%) 210 (M+, 100), 209 (36), 169 (49), 155
59 ꢁC (yellow prisms).
(
IR (KBr) mmax 3408 (NH ), 3329 (NH ), 3174 (NH),
2
2
2
8
7
7
222 (CN), 1658, 1569, 1494, 1436, 1335, 1241, 1129,
(4), 140 (10), 114 (8). Anal. Calcd for C H N : C,
1
68.56; H, 4.79; N, 26.65. Found: C, 68.72; H, 4.76;
N, 26.47.
2
10
4
ꢀ1 1
08, 741, 684 cm ; H NMR (270 MHz, DMSO-d ): d
6
.17–7.22 (m, 2H, H-5, H-6), 7.47–7.49 (dd, 1H, H-7),
.55–7.65 (d, 2H, NH ), 8.45 (s, 1H, H-2), 8.53 (s, 1H,
2
0
H-6 ), 8.70–8.75 (dd, 1H, H-4), 11.93 (br s, 1H, NH).
0
5.1.7. 2-Amino-4-(1H-indol-3-yl)pyrimidine-5-carbohy-
drazonamide (7), amidrazone analogue. To a solution of
compound 4 (0.2 g, 0.85 mmol) in 5 mL ethanol, hydra-
zine hydrate (99%) was added. The mixture was refluxed
for 3 h and the solvent was removed under reduced pres-
sure. The precipitate was crystallized from ethanol as a
white solid. Yield: 0.18 g (79%); mp 267–269 ꢁC; IR
1
3
C NMR (300 MHz, DMSO-d ): d 89.31 (C-5 ),
6
1
1
1
2
1
6
11.57 (C-7), 111.99 (C-3), 119.76 (CN), 121.04 (C-3a),
22.75 (C-6), 123.22 (C-5), 125.72 (C-4), 130.01 (C-2),
0
0
36.35 (C-7a), 163.11 (C-6 ), 163.16 (C-4 ), 163.27 (C-
0
); EIMS m/z (%) 235 (M+, 100), 208 (11), 194 (37),
80 (14), 166 (15), 139 (18), 117 (19), 97 (9), 77 (10),
1 (12). Anal. Calcd for C H N : C, 66.37; H, 3.86;
(KBr) m
1559 (C@N groups) cm
DMSO-d ): d 4.85 (br s, 2H, NH ), 5.65 (br s, 2H,
3389 (NH ), 3296 (NH ), 3172 (NH), 1619,
1
3
9
5
max
2
2
ꢀ
1
1
N, 29.77. Found: C, 66.18; H, 3.56; N, 29.83.
;
H NMR (270 MHz,
6
2
5
.1.5. 2-Amino-4-(1H-indol-3-yl)-pyrimidine-5-carboxylic
NH ), 7.22 (m, 2H, H-5, H-6), 7.43 (br s, 2H, NH ),
2 2
7.47 (d, 1H, H-7), 8.31 (s, 1H, H-2), 8.45 (d, 1H, H-
6 ), 8.66 (d, 1H, H-4), 11.76 (br s, 1H, NH); EIMS m/
z (%) 267 (M+, 100), 251 (76), 209 (34). Anal. Calcd
acid (5), carboxylic acid analogue. A mixture of com-
pound 4 (0.3 g, 1.28 mmol), aqueous NaOH solution
0
(
5 mL, 20%), and ethanol (15 mL) was heated at reflux-
ing temperature of 220 ꢁC for 6 h. The reaction mixture
was filtered off while hot and the filtrate left to cool and
acidified with 6 N HCl. The solid precipitate was fil-
tered, washed with water, and air-dried to form 5 as
an orange solid (0.27 g, 83.1%). Recrystallization from
ethanol gave analytically pure material; mp 157–159 ꢁC.
for C H N : C, 58.42; H, 4.90; N, 36.68. Found: C,
1
3
13
7
58.14; H, 4.89; N, 36.43.
5.2. Materials and methods
5.2.1. Human tumor cell lines
IR (KBr) mmax 3401 (NH ), 3331(NH ), 3125 (OH),
1
a– MCF7 (Breast carcinoma cell line)
b– HeLa (Cervix carcinoma cell line)
c– U251 (Brain tumor cell line)
2
2
ꢀ
1 1
690, 1570, 1494, 1446, 1256, 745, 688 cm ; H NMR
(
270 MHz, DMSO-d ): d 7.15–7.20 (m, 2H, H-5, H-6),
6
7
.42–7.45 (dd, 1H, H-7), 7.45–7.50 (d, 2H, NH ), 8.25
d– HEPG2 (Liver carcinoma cell line) (Fig. 3).
2

1210
M. A. A. Radwan, M. El-Sherbiny / Bioorg. Med. Chem. 15 (2007) 1206–1211
Figure 3. Cytotoxic activities of the compound 4 (at concentrations 1, 2.5, 5, and 10 lg/mL) against: (a) breast carcinoma cell line; (b) cervix
carcinoma cell line; (c) brain tumor cell line; (d) liver carcinoma cell line.
5
.2.2. Ehrlich ascites carcinoma (EAC). Maintained in
– Different concentrations of the compounds under test
(0, 1, 2.5, 5, and 10 lg/mL) were added to the cell
monolayer. Triplicate wells were prepared for each
individual dose.
the laboratory by weekly interperitoneal transplantation
in female Swiss albino mice.
5
.2.3. Animals. Female Swiss albino mice from the ani-
– Monolayer cells were incubated with the compound
mal house of National Cancer Institute (Egypt), weigh-
ing 18–22 g, were used. Animals were maintained on
standard pellet diet and water.
for 48 h at 37 ꢁC and in atmosphere of 5% CO
.
2
– After 48 h, cells were fixed, washed, and stained with
sulforhodamine B stain.
–
Excess stain was washed out with acetic acid and
attached stain was recovered with EDTA buffer.
5.2.4. Antitumor activity of the Ehrlich Ascites Carci-
noma (EAC). A set of sterile test tubes were used where
– Color intensity was measured in an ELISA reader.
– The relation between surviving fraction and tested
compound concentrations is plotted to get the sur-
vival curve of each tumor cell line after the cytotox-
icity of the specified compound and IC50 (dose of
the tested compound which reduces survival to
50%) were evaluated.
2
.5 · 105 tumor cells per ml were suspended in phos-
phate-buffered saline. Then 25, 50, and 100 lg/mL from
tested compound were added to the suspension, kept at
3
carried out to calculate the percentage of nonviable
7 ꢁC for 2 h. Trypan blue dye exclusion test was then
2
3
cells.
5.2.5. Measurement of potential cytotoxicity by SRB
assay. Potential cytotoxicity of the compounds was test-
ed using the method of Skehan et al.
Acknowledgments
2
4
The authors are grateful to National Cancer Institute,
Cancer Biology Department, Egypt, for biological eval-
uation. Also, the authors thank the Central Services
Unit (National Research Center, Cairo, Egypt) for run-
ning the spectral data.
–
Cells were plated in 96-multiwell plate (104 cells/well)
for 24 h before treatment with the compounds to
allow attachment of cell to the wall of the plate.

M. A. A. Radwan, M. El-Sherbiny / Bioorg. Med. Chem. 15 (2007) 1206–1211
1211
References and notes
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