Regioselective one-pot synthesis and anti-proliferative and apoptotic effects of some novel tetrazolo[1,5-a]pyrimidine derivatives

Article abstract of DOI:10.1016/j.bmc.2010.02.028

An easy and efficient route for the synthesis of some tetrazolo[1,5-a]-pyrimidine derivatives was described through the reaction of sodium salts of formyl cycloalkanones with 5-aminotetrazole monohydrate. The derivative 6,7,8,9-tetrahydrotetrazolo[1,5-a]quinazoline (6b) has profound anti-tumor cytotoxic effects against Ehrlich ascites carcinoma (EAC) both in vivo and in vitro and against hepatocellular carcinoma (HepG2) cell line in vitro. These anti-tumor effects may be mediated via stimulation of cell cycle arrest and apoptosis through down-regulation of Bcl-2 and up-regulation of p53 transcription factors.

Full text of DOI:10.1016/j.bmc.2010.02.028

Bioorganic & Medicinal Chemistry 18 (2010) 2639–2644
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Regioselective one-pot synthesis and anti-proliferative and apoptotic effects
of some novel tetrazolo[1,5-a]pyrimidine derivatives
a
b,*
Ahmed M. Hussein , Osama M. Ahmed
a
Chemistry Department (Organic Chemistry Division), Faculty of Science, Beni-Suef University, Egypt
Zoology Department (Physiology Division), Faculty of Science, Beni-Suef University, Egypt
b
a r t i c l e i n f o
a b s t r a c t
Article history:
An easy and efficient route for the synthesis of some tetrazolo[1,5-a]-pyrimidine derivatives was
described through the reaction of sodium salts of formyl cycloalkanones with 5-aminotetrazole monohy-
drate. The derivative 6,7,8,9-tetrahydrotetrazolo[1,5-a]quinazoline (6b) has profound anti-tumor cyto-
toxic effects against Ehrlich ascites carcinoma (EAC) both in vivo and in vitro and against
hepatocellular carcinoma (HepG2) cell line in vitro. These anti-tumor effects may be mediated via stim-
ulation of cell cycle arrest and apoptosis through down-regulation of Bcl-2 and up-regulation of p53 tran-
scription factors.
Received 11 December 2009
Revised 15 February 2010
Accepted 17 February 2010
Available online 20 February 2010
Key words:
5
-Aminotetrazole
Ó 2010 Elsevier Ltd. All rights reserved.
Tetrazolopyrimidine
Formyl salt
Anti-tumor cytotoxicity
EAC
HepG2
Apoptosis
1
. Introduction
murine cell line B16.18 Although many investigators are interested
in the synthesis of new tetrazolopyrimidine derivatives,1
9,20
there
Tetrazolopyrimidines have been reported to be used in the
are scarce publications concerning the anti-tumor effects of these
compounds with tetrazole and pyrimidine nuclei together.
Thus, this study was designed to synthesize new tetrazolo[1,5-
a]pyrimidine derivatives. One of these derivatives, 6,7,8,9-tetrahy-
drotetrazolo[1,5-a]quinazoline (6b), was tested for its anti-tumor
cytotoxicity effects against Ehrlich ascites carcinoma (EAC) and
hepatocellular carcinoma (HepG2) cell line. In addition, the effect
on the anti-apoptotic protein Bcl-2 and tumor suppressor protein
p53 was also investigated.
treatment of obesity, diabetes, atherosclerosis, hypertension, coro-
nary heart disease, hypercholesterolemia, hyperlipidemia, thyroid
cancer, hypothyroidism, depression, glaucoma, cardiac arrhyth-
mias, and congestive heart failure.1
–6
Despite significant advances in medical technology for protec-
tion and treatment, cancer is still widely producing threat mortal-
ity. Thereby, the search for new anti-cancer chemopreventive and
chemotherapeutic agents continues to be an active area of research
at many companies and research centers.7
–10
Apoptosis, programmed cell death, has been recognized as a
tightly controlled mechanism invoked to prevent unrestricted
2
2
. Results and discussion
1
1
growth. The recognition of tumor development involves an
imbalance between proteins promoting cell viability like Bcl-2,
.1. Chemistry (synthesis)
12,13
and those mediate cell death or cell cycle arrest, p53,
which
Since the direct introduction of some specific substances for
is the current dogma in tumor biology.14
construction of tetrazolopyrimidines nucleus is not always easy
and the most promising methods for synthesis of these compounds
are multi-step reactions, our method has succeeded to prepare
some novel tetrazolo[1,5-a]pyrimidines through the reaction of
readily available starting materials and in a one-step fast reaction.
Thus, treatment of 5-amino-1H-tetrazole hydrate (4) with the for-
Pyrimidine is found as a core structure in large variety of com-
15,16
pounds that exhibit biological activity.
Some novel substituted
pyrimidine derivatives showed anti-tumor cytotoxicity effects
against different human carcinoma cell lines.1
0,16,17
Also, some
substituted tetrazolo compounds had anti-tumor effects against
myl salts of cycloalkanones, namely sodium (2-oxocycloalkyli-
*
Corresponding author. Tel.: +20 101084893; fax: +20 822334551.
E-mail addresses: osamamoha@yahoo.com, dr_osamamahmed@yahoo.com
21–24
dine)methenoates (2)
in the presence of aqueous piperidine
acetate and acetic acid, afforded in a good yield (69–73%) of the
(
O.M. Ahmed).
0
968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.028

2
640
A. M. Hussein, O. M. Ahmed / Bioorg. Med. Chem. 18 (2010) 2639–2644
solid reaction products. The reaction products were suggested to
be cycloalkane ring-fused tetrazolo[1,5-a]pyrimidines (6) and not
the isomeric tetrazolopyrimidines (8) as outlined in Chart 1.
The reaction mode for the formation of the products is sug-
gested to proceed through the initial nucleophilic attack by the
exocyclic amino group at the formyl group of compound (3), that
formed in situ due to the reaction of the formyl salts (2) with
water, followed by cyclization through the elimination of two
water molecules leading to the formation of the reaction products
sence of the bands related to NH or NH
2
of the start and revealed
ꢀ1
ꢀ1
bands at
m
= 2948 cm , (paraffinic CH), 1655 cm (C@N), and at
ꢀ
1
1
1619 cm (C@C). The H NMR spectrum revealed the presence
of signals at d = 1.96–2.11 ppm (m, 4H, 2CH ); 2.91–3.33 (m, 4H,
2CH ), 8.70 (s, 1H, pyrimidine proton). Its mass spectrum showed
2
2
a molecular ion peak at m/z = 175 (32.8%) coincident with the
molecular weigh of the compound (175.19) and revealed the base
peak at 107 (100%). The fragmentation pattern of compound (6b)
was illustrated in Scheme 1.
(
6).
The suggestion of the formation of the alternative isomeric pla-
2.2. Biological studies
ner products (8) is based on the probability of the initial attack of
the indocyclic NH of the tetrazole ring, which is expected to be
more nucleophilic at the formyl group in (3) followed by cycliza-
tion and elimination of water. This suggestion is principally ex-
cluded due to the more steric hindrance around the endocyclic
nitrogen atom than the exocyclic one that can easily attack the
unhindered and the electronically favoured formyl group of com-
For determination of lethal dose (LD)50 of 6,7,8,9-tetrahydrotet-
razolo[1,5-a]quinazoline (6b), single gradual increasing doses were
orally administered to various groups of normal albino mice. After
48 h, the number of dead animals in each group was determined
and LD50 was calculated. LD50 of 6b was found to be 500 mg/kg
b.w. (Table 1). Based on this toxicity study, the therapeutic dose
for subsequent in vivo study was chosen to be 10 mg/kg b.w. (1/
2
3,25
pound (3).
The characterization of the reaction products was confirmed by
using the elemental analysis (C, H, N) and spectral data (IR, MS, H
NMR). Thus, the IR spectrum of compound (6b) showed the ab-
50 of LD50) which is so far from LD50.
The Ehrlich ascites carcinoma (EAC), in this study, was induced
in female mice by transplantation of EAC-cells into peritoneal cav-
1
_
+
O Na
CH ONa
3
+
HCOOC H
2 5
(
)
(
n
(
)
O
n
O
1) a, n = 1
(2)
b, n = 2
c, n = 4
d, n = 8
H O
2
OH
O
(
)
(
)
n
n
O
(3)
OH
NH2
NH
N
pip. acetate/
AcOH
N
N
(
4)
H
OH
OH
H
N
N
H
(
)
N
N
N
n
(
)
N
n
OH HN
N
HO
H
N
N
(7)
(5)
-
2H O
- 2H O
2
2
N
(
)
N
N
n
N
N
N
N
(
)
N
n
N
N
(
6)
a, n = 1
b, n = 2
c, n = 4
d, n = 8
(
8)
Chart 1.

A. M. Hussein, O. M. Ahmed / Bioorg. Med. Chem. 18 (2010) 2639–2644
Table 3
2641
.
. +
+
Effect of 6,7,8,9-tetrahydrotetrazolo[1,5-a]quinazoline on percent inhibition of EAC-
N
N3
N
cell viability in vitro after 2 h of incubation
N
N
N
N
Compound
% inhibition of cell viability
50 g/ml 100
25.00 ± 2.24 45.00 ± 6.71
N
2
5
l
g/ml
l
lg/ml
*
*
**
Tetrazolo[1,5-a]pyrimidine
10.00 ± 1.29
m/z = 175 (32.8 %)
CH =CH (28)
m/z = 175
*
*
p <0.01: Values are highly significantly different as compared with value at
lowest concentration.
-
2
2
.
+
.
+
N
N
The inhibitory effect of tetrazolo[1,5-a]pyrimidine derivative
(6b) on EAC-cells in vivo was examined in terms of EAC-aliquot
volume and number of total, alive and dead EAC-cell number and
dead EAC-cells percent of EAC-bearing mice treated with vehicle
or compound. The obtained data (Table 2) revealed that adminis-
tration of 6b for 2 weeks induced a profound decrease (p <0.01)
of EAC-aliquot volume, total number of EAC-cells and number of
alive cells, on one hand, and marked increase of count (p <0.05)
and percent (p <0.01) of dead EAC-cells, on the other. Similarly,
to in vivo, the tested derivative (6b) has potent anti-tumor cyto-
toxic effects on EAC-cells and HepG2 cell line in vitro. The treat-
N
N
N
-
N2 (28)
N
N
N
m/z = 119 (16.2%)
CH =CH (28)
m/z = 147 (7.10%)
-
2
2
.
.
+
+
N
-
N (28)
2
N
N
N
ment of EAC-cells in vitro with 6b at doses 25, 50 and 100 lg/ml
m/z = 91 (11.6%)
m/z = 63 (11.8%)
produced 10.00 ± 1.29, 25.00 ± 2.24 and 45.00 ± 6.71 percent inhi-
bition of cell viability (Table 3). Furthermore, the incubation of
HepG2 cell line with gradual increased concentrations of the tested
compound (6b) for 48 h produced moderate dose-dependent inhi-
bition of cell growth and anti-tumor activity (Fig. 1).
Scheme 1.
Table 1
In trial to explain the cytotoxic and anti-proliferative effects, the
effect of tetrazolo[1,5-a]pyrimidine derivative (6b) on anti-apopto-
tic mediator Bcl-2 and tumor suppressor protein p53 was checked
by immunohistochemical techniques. Bcl-2 has been reported to
function primarily by blocking the apoptosis pathway.28 On the
other hand, p53 activates the transcription of downstream genes
such as p21 and Bax to induce apoptotic process, inhibiting the
Determination of LD50 of 6,7,8,9-tetrahydrotetrazolo[1,5-a] quinazoline in albino
mice
P(zꢁd)
Dose (mg/kg
b.w.)
Total number of
animals
Number of dead
animals
z
d
0
5
5
5
5
5
5
0
1
2
3
4
5
—
—
—
2
4
6
8
1
00
00
00
00
000
0.5 200 100
1.5 200 300
2.5 200 500
3.5 200 700
4.5 200 900
29,30
growth of cells with damaged DNA or cancer cells.
It is re-
3
1,32
quired for cells to initiate apoptotis.
Thus, whether a cell be-
comes committed to apoptosis partly depends upon the balance
between proteins that mediate death such as p53 and proteins that
z: Mean number of dead animals in two successive doses.
d: Constant factor between two successive doses.
3
3–35
promote cell viability, such as Bcl-2.
The photomicrographs of
LD50 = the biggest dose which kill all animals ꢀ
P(zꢁd)/n = 1000–2500/5 = 500 mg/
immunohistochemically stained EAC sections revealed that the
concentration of Bcl-2 in the cytoplasm of EAC-cells was pro-
foundly decreased in EAC-bearing mice treated with tetrazol-
o[1,5-a]pyrimidine derivative (6b) (Fig. 2b and 2d) as compared
with EAC-bearing mice control (Fig. 2a and 2c). On the other hand,
the amount of p53 was potentially increased in EAC-bearing mice
treated with the tested derivative (6b) (Fig. 2f) as compared with
EAC-bearing mice control (Fig. 2e). Based on these results, it could
be suggested that the decreased number of total and alive EAC-
cells and increased number and percent of dead cells, may be
attributed, at least in part, to the apoptotic effects of the tested tet-
razolo[1,5-a]pyrimidine derivative (6b). In addition, the stimulat-
kg b.w.
ity. The EAC-cell is a spontaneous murine mammory adenocarci-
noma adopted to ascites form and carried in outbred mice by serial
2
6,27
passage.
In the present study, the survival percentage of EAC-bearing
mice treated with tetrazolo[1,5-a]pyrimidine derivative (6b) for
2
weeks was profoundly increased to reach 83.33% that was higher
as compared with that of control (66.66%). Thus, this tetrazolo[1,5-
a]pyrimidine derivative decreased the mortality and improved the
survival rate of EAC-bearing mice.
Table 2
Effect of 6,7,8,9-tetrahydrotetrazolo[1,5-a]quinazoline administration for 2 weeks on EAC-aliquot volume, EAC-cells number and percent of dead cells in EAC-bearing mice
Group
Parameter
EAC-aliquot
volume (ml)
Total EAC-cells
numberꢁ10
Alive EAC-cells
numberꢁ10
Dead EAC-cells
numberꢁ10
Percent of dead
EAC-cells
7
7
7
EAC-bearing mice control (n = 8)
EAC-bearing mice treated with tetrazolo[1,5-a]
pyrimidine (n = 10)
5.77 ± 0.39
2.52 ± 0.25
125.76 ± 8.35
71.01 ± 2.99
122.83 ± 8.24
66.39 ± 2.74
2.64 ± 0.29
4.62 ± 0.61
2.28 ± 0.22
8.24 ± 1.28
*
*
**
**
*
**
LSD at the 5% level
LSD at the 1% level
0.94
1.29
17.26
23.79
16.83
23.18
1.56
2.16
3.09
4.26
Data are expressed as mean ± standard error (SE).
*
p <0.05: Difference is significant.
*
p <0.01: Difference is highly significant.
*

2
642
A. M. Hussein, O. M. Ahmed / Bioorg. Med. Chem. 18 (2010) 2639–2644
X
1
0
0
.25
1
LD50 ¼ the biggest dose which kill all animals ꢀ
ðz ꢁ dÞ=n
where n: number of animals in groups = five animals in each group.
3
.2.1.2. Induction of EAC-bearing and animal group-
ing. Normal female albino mice were obtained from animal
house, Institute of Ophthalmology, Giza, Egypt. EAC-bearing stock
female mice were obtained from Cancer Biology Department, Na-
tional Cancer Institute, Cairo University, Egypt. To induce EAC in
mice for the experimental study, 0.2 ml EAC-aliquot aspirated from
stock mice was added to 9.8 ml saline (dilution is 1:50) and 0.2 ml
of this diluted EAC was intraperitoneally administered by syringe
into each mice. The EAC-injected mice were divided into two
groups each of 12 animals. Mice of group 1 (control group) was
administered with 10% DMSO as a vehicle in a volume equivalent
to that given to treated animals. Group 2 was treated with tetraz-
olo[1,5-a]pyrimidine derivative (6b), dissolved in 10% DMSO, at
dose of 10 mg/kg b.w. (1/50 of LD50) The compound and vehicle
were orally applied by gastric intubation between 10 and 12 AM
daily for 2 successive weeks beginning from the 1st day of EAC-
injection.
.75
0
.5
.25
0
0
25
50
75
100
125
Concentration (µg/ml)
Figure 1. Anti-proliferative effect of 6,7,8,9-tetrahydrotetrazolo[1,5-a] quinazoline
on HepG2 cell lines in vitro after 48 h of incubation.
The number of animals survived in each group was detected at
the end of the experiment and the survival percent in each group
was calculated as follow:
ing effect of this tetrazolo[1,5-a]pyrimidine derivative on apoptosis
is not restricted to one mediator but may be mediated via affecting
many apoptotic and cell cycle arrest signal like p53 and anti-apop-
totic factors like Bcl-2.
Survival percent ¼ ðnumber of survived animals=
total number of animalsÞ ꢂ 100
3
3
. Experimental protocols
3
.2.1.3. Sampling and detection of EAC-volume and cell num-
.1. Chemistry
ber. At the end of the experimental period, animals were sac-
rificed under diethyl ether anesthesia and 0.2 ml saline was
intraperitoneally injected. One minute later, EAC-aliquot was aspi-
rated by a sterile syringe into test tube. The volume of EAC-aliquot
for each mouse was measured. The number of alive and dead EAC-
cells was determined using trypan blue exclusion assay. Alive and
dead EAC-cells were counted by Neubauer haemocytometer.
All melting points were determined on an electrothermal appa-
ratus and are uncorrected. IR spectra were recorded (KBr discs) on
a BRUKER IFS-25 FT-IR spectrophotometer at the region 400–
ꢀ1 1
4
3 3 2
000 cm . H NMR spectra were recorded in CDCl and (CD ) SO
solutions on a Varian Gemini 300 MHz spectrometer and chemical
shifts are expressed in d units using TMS as an internal reference.
Mass spectra were recorded on a GC–MS QP 1000 EX Shimadzu.
Elemental analyses were carried out at the Microanalytical Center
of the Cairo University, Giza, Egypt. Piperidine acetate was pre-
pared by addition of 5 ml piperidine to a mixture of 4 ml acetic acid
Briefly, 40 ll of EAC-aliquot was added to 4 ml 2% trypan blue (dis-
solved in 0.9% saline) and the mixture was left for 5 min. One drop
from mixture was taken on Neubauer haemocytometer and the
number of stained cells (dead cells) and non-stained cells (viable
or alive cells) were counted. Total number of EAC-cells and percent
of dead EAC-cells were calculated for each EAC-bearing mouse.
Part of EAC-aliquot from each tumor-bearing mouse was centri-
fuged at 3000 r.p.m. for 15 min and the precipitated EAC-cells were
fixed in neutral buffered formalin for immunohistochemical
studies.
2
1
and 10 ml water.
3
.1.1. Synthesis of tetrazolo[1,5-a]pyrimidine derivatives 6a–d
Respective mixtures of 5-aminotetrazole monohydrate (4)
0.01 mol) were refluxed in a solution of sodium salts (2)
0.012 mol) and piperidine acetate (1.5 ml) for 3–5 min. Acetic acid
1.5 ml) was added to the reaction mixture while boiling, then the
mixture was cooled and solid product was collected by filtration
and recrystallized from the a suitable solvent (Tables 4 and 5).
(
(
(
3.2.1.4. Immunohistochemical investigations.
The fixed
samples were transferred to the Department of Pathology, National
Cancer Institute for processing, blocking, sectioning and mounting
onto positive-charged slides (Fisher Scientific, Pittsburgh, PA) to de-
tect the Bcl-2 and p53 reactivity or apoptotic cells using the TUNEL
3
.2. Biology
3
7
3
3
.2.1. In vivo studies
.2.1.1. Acute toxicity study and determination of LD50 in
assay. Bcl-2 and p53 reactivity were determined following Hua
3
8
and Ya-wei method. Briefly, before the incubation with antibodies,
endogenous peroxidase activity was quenched, slides washed and
then incubated in a blocking solution of hydrogen peroxide 1% in
methanol, in darkness for 15 min. Antigen retrieval occurred with
citrate buffer 10 mM, pH 6. After cooling, sections were rinsed in
tap water and then phosphate buffer saline 1 M. Primary antibodies
for either Bcl-2 or p53, diluted 1:150 and 1:100, respectively in PBS,
were applied for 1 h at 37 °C. Secondary biotinylated antibody di-
luted 1:100 and 1:200 in PBS was applied for a period of 30 min at
37 °C. Streptavidin–biotin or avidin–biotin peroxidase (ABC/ HRP)
was applied for 10 min at room temperature. Bound antibody com-
normal mice.
LD50 of the studied compound (6b) was deter-
36
mined as described by Afifi et al. In this experiment, six groups of
albino mice weighing 20–25 g were used. One group serves as con-
trol and other groups of mice were orally administered the tested
compound by gastric intubation in gradual increasing doses (200,
4
00, 600, 800 and 1000 mg/kg b.w.). After 48 h of administration,
the number of dead animals in each group, the mean of dead ani-
mals in two successive doses (z) and the constant factor between
two successive doses (d) were recorded and LD50 was calculated
as follow:

A. M. Hussein, O. M. Ahmed / Bioorg. Med. Chem. 18 (2010) 2639–2644
2643
0
39
plex was visualized by the reaction of 3,3 -diaminobenzidine sub-
strate and counter stained with haematoxylin.
the method Mclimans et al. Briefly, EAC-cells at concentration
5
of 2.5 ꢂ 10 cells/ml suspended in phosphate buffer saline were
2
incubated at 37 °C and 5% CO for 2 h in the presence of three dif-
3
3
.2.2. In vitro studies
.2.2.1. Evaluation of anti-tumor cytotoxicity against EAC-
ferent concentrations of the compound. At the end of incubation
period, equal volume of trypan blue solution was added to sample
cells, then the stained cells (dead cells) and unstained cells (alive
cells) were counted using Neubauer haemocytometer. The percent
of dead cells for each test was calculated.
cells.
tions (25, 50 and 100
tive (6b) was tested by trypan blue exclusion assay according to
The viability of cells as a result of 3 different concentra-
l
g/ml) of tetrazolo[1,5-a]pyrimidine deriva-
Figure 2. Photomicrographs of EAC-immunohistochemically-stained sections showing the effect of 6,7,8,9-tetrahydrotetrazolo[1,5-a]quinazoline on anti-apoptotic marker
Bcl-2 (2b; ꢂ100 and 2d; ꢂ400) and apoptotic marker p53 (2f; ꢂ100) expression as compared with the corresponding control (2a; ꢂ100 and 2c; ꢂ400 for Bcl-2 and 2e; ꢂ100
for p53).

2
644
A. M. Hussein, O. M. Ahmed / Bioorg. Med. Chem. 18 (2010) 2639–2644
Table 4
Acknowledgements
The melting point, color, yield, molecular formula and elemental analysis of the newly
synthesized compounds
The authors are grateful to Pharmacology Unit, Cancer Biology
Department (National Cancer Institute, Cairo, Egypt) for supplying
EAC-bearing mice and human carcinoma cell lines. The study was
partially funded by the Faculty of Science, Beni-Suef University,
Egypt.
Compd MP °C
Color
yield%
Mol. formula
(M.Wt.)
Elemental analysis
calcd/found
No.
solvent
C
H
N
6
6
6
6
a
b
c
182–183
EtOH
119–121
EtOH
Pale brown
69.1
Orange
73.3
C
C
C
C
7
H
H
7
N
5
5
(161)
(175)
52.17 4.35 43.47
52.22 4.19 43.66
54.85 5.14 40.00
54.88 5.00 39.79
8
9
N
References and notes
103–105 EtOH White 64
10
H
13
N
5
5
(203) 59.11 6.40 34.48
8.89 6.65 34.45
(259) 64.86 8.11 27.03
4.49 8.21 27.00
1
2
.
.
Takayama, Y.; Yoshida, Y.; Uehata, M. US Patent No. 7109208, 2006.
Fujii, A.; Tanaka, H.; Otsuki, M.; Kawaguchi, T.; Oshita, K. US Patent No.
5
d
110–112 EtOH White 73
14
H
21
N
6930115, 2005.
6
3
4
.
.
Takaya, T.; Murata, M.; Ito, K. US Patent No. 4725600, 1988.
Utsunomiya, T.; Niki, T.; Kikuchi, T.; Watanabe, J.; Yamagishi, K.; Nishioka, M.;
Suzuki, H.; Furusato, T.; Miyake, T. WO/1999/006380, PCT/JP1998/003397,
1999.
Table 5
The spectral data of the newly synthesized compounds
5. Aspnes, G.E.; Chiang, Y.P. US Patent No. 6441015, 2002.
6. Uehata, M.; Ono, T.; Satoh, H.; Yamagami, K.; Kawahara, T. US Patent No.
6218410, 2001.
Compd No. Spectra
(cmꢀ1): 2920–2823 (CH paraffinic); 1665 (C@N)
IR
7. Bridges, A. J. Chem. Rev. 2001, 101, 2541.
8.
Wang, J. D.; Miller, K.; Boschelli, D. H.; Ye, F.; Wu, B.; Floyd, M. B.; Powell, D. W.;
Wissner, A.; Weber, J. M.; Boschelli, F. Bioorg. Med. Chem. Lett. 2000, 10, 2477.
Shenone, S.; Bruno, O.; Bondavalli, F.; Ranise, A.; Mosti, L.; Menozzi, G.; Fossa,
P.; Donnini, S.; Santoro, A.; Ziche, M.; Manetti, F.; Botta, M. Eur. J. Med. Chem.
6
a
m
and 1594 (C@C)
9.
1
H NMR d
m, 4H, 2CH
Mass (m/z): 161 (M , 10.0%); 151 (65.5%); 122 (73.0%);
5 (81.5%) and 67 (100.0%)
IR
(cmꢀ1): 2948 (paraffinic CH), 1655 (C@N), and
619 (C@C)
H
(ppm): 1.90–2.22 (m, 2H, CH
2
); 2.87–3.16
(
2
) and 8.72 (s, 1H, pyrimidine H)
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Å+
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9
2009, 44, 3519.
6
b
m
11. Ahmed, R. R. J. Egypt Ger. Soc. Zool. 2008, 57C, 257.
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7–28.
1
1
H NMR d
H
(ppm): 1.96–2.11 ppm (m, 4H, 2CH
) and 8.70 (s, 1H, pyrimidine H)
(ppm): 17.9 (2CH ); 34.6 (2CH ); 140.1
2
);
2
.91–3.33 (m, 4H, 2CH
2
13
14. Kanzler, S.; Galle, P. Cancer Biol. 2000, 10, 173.
C NMR d
C
2
2
1
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(
(
C@C–C@N); 156.2 (C@C–N); 160.6 (–C@N–C@N); 170.6
tetrazole C)
Mass (m/z): 175 (MÅ+, 32.8%); 146 (10.4%); 107 (MÅ+, 100%)
and 79 (39.6%)
1
1
6. Xie, F.; Zhao, H.; Zhao, L.; Lou, L.; Hu, Y. Bioorg. Med. Chem. Lett. 2009, 19, 275.
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c
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589 (C@C)
m
(cmꢀ1): 2943–2845 (CH paraffinic); 1658 (C@N) and
1
342, 299.
1
H NMR d
m, 4H, 2CH
s, 1H, pyrimidine H)
H
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1
1
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2
232.
+
Mass (m/z): 204 (M +1, 31.6%); 203 (48.7%); 174 (13.50%);
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IR
46 (31.3%); 107 (48.0%) and 81 (48.7%)
ꢀ
1
6
d
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+
Å
1606 (C@C). Mass (m/z): 259 (M , 7.3%); 233 (56.1%);
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2
2
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0
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1
00
Cytotoxicity and anti-proliferative activity against HepG2 cell line
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2
lg/ml streptomycin at 37 °C in atmosphere of 5% CO .
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1
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9
1
6-multiwell plate were incubated with different concentrations (0,
0, 25, 50 and 100 g/ml) of each compound for 48 hours at 37 °C in
. At the end of the incubation period, cells were
l
36. Afifi, N. A.; Ramadan, A.; El-Kashoury, E. A.; El-Banna, H. A. Vet. Med. J. Giza
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atmosphere of 5% CO
2
3
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acetic acid and attached stain was recovered with tris EDTA buffer
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tween survival fraction and compound concentration was plotted.
3
39. Mclimans, W.; Davis, E.; Glover, F.; Rake, G. J. Immunol. 1957, 79, 428.
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4
0. Zhu, B.; Yao, Z.; Luo, S.; Jiang, L.; Xiao, J.; Liu, S.; Sun, J.; Pei, Z. Hepatobiliary
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.3. Statistical analysis
1107.
4
2. Roa, M.; Blane, K.; Zonneberg, M. One-way Analysis of Variance, PC-STAT,
Data were expressed as mean ± standard error (SE) and were ana-
lyzedbyone-wayANOVAfollowedbyLSDtestusingPC-STAT program.
University of Georgia, USA, 1985.
42