Synthesis and anti-tumor activities of some new pyridines and pyrazolo[1,5-a]pyrimidines

Article abstract of DOI:10.1016/j.ejmech.2009.03.042

Cyclocondensation of cyanoacetamide, cyanothioacetamide and 3-aminopyrazols with sodium salt of 3-hydroxy-1-(2-naphthyl)prop-2-en-1-one gives pyridin-2-one, pyridin-2(1H)-thione, and pyrazolo[1,5-a]pyrimidine derivatives. These derivatives showed potent a

Full text of DOI:10.1016/j.ejmech.2009.03.042

European Journal of Medicinal Chemistry 44 (2009) 3519–3523
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anti-tumor activities of some new pyridines and pyrazolo
[1,5-a]pyrimidines
,
c
b
d
Osama M. Ahmed a , Mahmoud A. Mohamed , Rasha R. Ahmed , Sayed A. Ahmed
*
a Zoology Department, Physiology Division, Faculty of Science, Beni-Suef University, Salah Salim Street, P.O. 62514, Beni-Suef 62514, Egypt
b Zoology Department, Cytology and Histology Division, Faculty of Science, Beni-Suef University, Beni-Suef, Egypt
c Textile Department, Faculty of Industrial Education, Beni-Suef University, Beni-Suef, Egypt
d Chemistry Department (Organic Chemistry Division), Faculty of Science, Beni-Suef University, Beni-Suef, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclocondensation of cyanoacetamide, cyanothioacetamide and 3-aminopyrazols with sodium salt of
3-hydroxy-1-(2-naphthyl)prop-2-en-1-one gives pyridin-2-one, pyridin-2(1H)-thione, and pyrazolo[1,
5-a]pyrimidine derivatives. These derivatives showed potent anti-tumor cytotoxic activity in vitro using
different human cancer cell lines.
Received 20 February 2009
Received in revised form
19 March 2009
Accepted 24 March 2009
Available online 8 April 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
2-Acetylnaphthalene
Cyanoacetamide
Cyanothioacetamide
3-Aminopyrazols
Anti-tumor cytotoxicity
1. Introduction
2-thione and pyrazolo[1,5-a]pyrimidine for evaluation of anti-
cancer properties.
The search for new anticancer chemotherapeutic agents
continues to be an active area of research at many companies and
research centers [1–3]. In the last several decades, pyridin-2-thione
and pyrazolopyrimidine derivatives have received considerable
attention due to their wide-range of applications such as anti-
inflammatory, anti-tumor, antimycobacterial, antifungal and anti-
viral activities [4–6]. Recently, it was reported that many pyridine
derivatives showed strong cytotoxicity against several human
cancer cell lines [6–9]. Pyrazolo[4,3-d]pyrimidine analogue and
pyrazolo[1,5-a]pyrimidine derivatives were reported to be inhibi-
tors of tyrosine kinase and cyclin-dependent kinases (CDK) which
are involved in mediating the transmission of mitogenic signals and
numerous other cellular events [3,10–15], including cell prolifera-
tion, migration, differentiation, metabolism and immune response.
It was also found that many of these derivatives may block prolif-
eration of various cancer cell lines [16].
2. Results and discussion
2.1. Synthesis
Pyridin-2-thione derivatives are provided to be useful synthetic
intermediates for synthesis of biologically active deazafolates’ ring
system and pyrimidine nucleosides, which reported to be signifi-
cantly active both in vitro and vivo [17,18], inhibitor of dihydrofolate
reductase [19], cytotoxicity against various experimental tumors as
potentially as methotrexate [20,21] and one of the most effective
antimetabolites currently used in treatment of various solid tumors
[22,23]. Owing to our plane to develop an efficient and simple
procedure for the synthesis of new antimetabolites [24,25],
recently we have approaches for the synthesis of this class of
compounds. We report in this part new synthesis of 2-pyridone,
pyridin-2-thione and pyrazolo[1,5-a]pyrimidine derivatives. Thus,
it has been found that sodium salt of 3-hydroxy-1-(2-naph-
In view of these reports and in continuation with the previous
work, we have herein synthesized new derivatives of pyridin-
thyl)prop-2-en-1-one
1 reacted with cyanamides 2 to give
a trisubstituted 2-pyridone (pyridin-2-thione). Two modes of
cyclization are feasible, giving a 2,3,6- or 2,3,4-trisubstituted
products, as outlined in Scheme 1. In the first one, initial attack by
* Corresponding author. Tel.: þ20 101084893; fax: þ20 822334551.
E-mail address: osamamoha@yahoo.com (O.M. Ahmed).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.03.042

3520
O.M. Ahmed et al. / European Journal of Medicinal Chemistry 44 (2009) 3519–3523
-
+
O Na
H
2 O
NH2
H2
O
O
NC
+
i) Pip. Acetate/
ii) H+
X
i) Pip. Acetate/
ii) H+
2
a, X = O
b, X = S
1
H
OH
CN
OH
CN
X
OHH2N
X
OH H2N
4
3
-2 H2O
-2 H2O
CN
X
CN
X
N
H
N
H
5
a, X = O
b, X = S
6
Scheme 1.
a carbanion takes place at the formyl group of salt 1 and subsequent
Michael cyclization followed by elimination of 2 mol of water leads
to the 2,3,6-trisubstituted product 5, whereas in the second, initial
nucleophilic attack by the methylene carbon takes place at the
ketonic group, followed by cyclization and elimination of water
leading to 2,3,4-trisubstituted isomer 6. In fact only one isomer was
obtained. The structure of 5 was expected due to the fact that initial
attack of the active methylene carbon at the unhindered formyl
group leading to 5 being much more probable than attack at the
hindered and electronically disfavored ketonic group. Spectral
studies did not allow us to distinguish between structures 5 and 6.
1H NMR for the product revealed the presence of an NH proton at
2.2. Cytotoxicity against different human cancer cell lines in vitro
For evaluation of anti-tumor cytotoxicity of the synthesized
compounds (5b and 9f), four different human cancer cell lines were
used: HepG2 (liver carcinoma cell line), MCF7 (breast carcinoma
cell line), HCT116 (colon carcinoma cell line), and HeLa (cervix
carcinoma cell line). Cytotoxicity of pyridin-2(1H)-thione (PT) (5b)
and pyrazolo[1,5-a]pyrimidine (9f) against above human cancer
cell lines is shown in Fig. 1. The IC50 values are represented in
Table 1. Both tested substances showed potent anti-tumor toxicity
against HepG2. The survival fraction was gradually decreased as the
concentration of 5b and 9f was increased. IC50 of 9f (1.88
mg/ml)
d
¼ 14.30 ppm in solutions. No significant amounts of the alterna-
was higher than that of 5b (1.01 g/ml); thus 5b seemed to be more
m
tive regioisomers could be detected. In order to establish unam-
biguously the structure of the product, the crystal structure of
a similar previous work has been reported [24–26]. The X-ray
analysis of this work confirms the exclusive presence of the
regioisomer 5 in the solid state; all hydrogen atoms could be
located unambiguously [25].
Finally, treatment of sodium 3-hydroxy-1-(2-naphthyl)prop-2-
en-1-one 1 with the appropriate 3-aminopyrazoles 7 in the pres-
ence of piperidine acetate afforded pyrazolo[1,5-a]pyrimidines 9
(Scheme 2).
potent in inducing cytotoxicity against hepatocellular carcinoma
cell line. With regards to the breast carcinoma cell line (MCF7), both
5b and 9f caused vigorous and rapid decrease in the survival frac-
tion at concentration of 1
less steady as the concentration of 5b and 9f was beyond 1
and 2.5 g/ml, respectively. The IC50 of 5b (0.34 g/ml) was lower
than that of 9f (0.74 g/ml). In comparison with the anti-tumor
mg/ml. Thereafter, the effect was more or
mg/ml
m
m
m
cytotoxicity effect on HepG2, both 5b and 9f produce a much more
potent effect against MCF7. Also, HCT116 and HeLa exhibited
marked decrease in the surviving fraction as a result of 5b and 9f.
Moreover, 9f showed rapid decrease in the surviving activity at
Structure of the product 9f was confirmed by elemental analysis
and spectral data. Thus, 1H NMR for the product revealed the pres-
1 mg/ml and has a stronger anti-tumor cytotoxic potential than 5b
ence of an NH proton at
d
¼ 10.09 ppm in solutions. The reaction
on both HCT116 and HeLa. Overall, 5b was more effective in causing
anti-tumor cytotoxicity against HepG2 and MCF7 cell lines while 9f
was more effective against HCT116 and HeLa. These results are in
agreement with several publications dealing with other pyridine
and pyrazolopyrimidine derivatives. It was reported that some
pyrazolo[3,4-d] pyrimidine derivatives demonstrated significant
anti-tumor cytotoxic activity and anti-proliferative effects against
different human cancer cell lines [3,11,30–32]. Son et al. [6]
revealed that most of the prepared 2,6-diaryl-substituted pyridine
derivatives showed anti-tumor cytotoxicity effects on human cell
seemed to be via the initial nucleophilic attack by the exocyclic
amino group at the ketonic group, which formed in situ from 1 with
water, followed by cyclization and elimination of 2 mol of water
leading to the formation of the product 9f (Scheme 2). The sugges-
tion of the formation of the alternative isomeric product 11f is based
on the initial attack of endocyclic amino group at the ketonic group.
The latter suggestion is excluded due to the higher nucleophilicity of
the exocyclic primary amino group than the endocyclic amino group
and it has been previously reported [26–29].

O.M. Ahmed et al. / European Journal of Medicinal Chemistry 44 (2009) 3519–3523
3521
R1
H2N
H2O
H2O
+
1
H N
R2
i) Pip. Acetate/
+
i) Pip. Acetate/
+
N
H
ii)
H
ii)
7
OH
OH
H2N
N
R1
N
HN
R2
R2
N
N
H
OH
OH
R1
10
8
-2H2O
-2H2O
R1
N
N
R2
N
R2
N
N
N
R1
11
9
R2
R1
7, 9
a
H5
H5
SCH3
NHC6
SCH3
CONH-C6
H
b
c
CONH-C6
CONH-C6
CONH-C6
5
H4 -4-CH
H4 -4-CH
3
3
NHC6H5
d
e
f
SCH3
H4 -4-Cl
CONH-C6
CONH-C6
NHC6H5
H4 -4-Cl
Scheme 2.
colon adenocarcinoma (HCT15), ovary carcinoma (SK-OV-3), lung
carcinoma (A549) and malignant melanoma (SK-MEL-2) cell lines.
Furthermore, it was elucidated that pyridine, pyrane and pyrimi-
dine derivatives, that were newly synthesized using nitro-
cancer cell lines may be attributed, at least, to free radicals (pyridyl
thiyl radicals) which might be involved in cell damage [35].
3. Experimental protocols
benzosuberone as
a starting material, exhibited anti-tumor
activities against different human tumor cell lines representing
leukemia, melanoma and cancer of lung colon, brain, ovary, breast,
prostate as well as kidney [33]. A new pyrazolo[4,3-d]pyrimidine of
analogue of myoseverin, described by Krystof et al. [16], blocked
cells’ proliferation of the breast carcinoma line (BT474). The
explanation of the anti-tumor activities of the pyridine thione and
pyrazolopyrimidine was suggested by various publications.
Shenone et al. [11] and Angelucci et al. [13] reported that
pyrazolopyrimidine derivatives are inhibitors of protein tyrosine
kinases (PTKs) and Scr which are critical component of signaling
transduction cascades and participate in a wide variety of cellular
activities including proliferation, secretion, adhesion and responses
to mitogen and stress. Krystof et al. [16] suggested that pyr-
azolopyrimidine derivatives may inhibit tubulin polymerization
and that the microtubular cytoskeleton is their primary target in
the cells. Jorda et al. [34] found that pyrazolopyrimidine derivatives
increase the cellular level of tumor suppressor protein p53, stabilize
its nuclear utilization and subsequently, activates transcription of
some p53-regulated genes; this effect probably results from inhi-
bition of cyclin-dependent kinases (CDK). On the other hand, the
anti-tumor cytotoxicity of pyridine thione against the examined
3.1. Chemistry
All melting points are uncorrected. IR spectra were obtained
(KBr disk) on a Perkin Elmer 11650 FT-IR instrument. The 1H NMR
spectra were measured on a Varian 400 MHz spectrometer for
solutions in (CD3)2SO using Si(CH3)4 as an internal standard. Mass
spectra were recorded on a Varian MAT 112 spectrometer. Analyt-
ical data were obtained from the Microanalytical Data Center at
Cairo University.
3.1.1. Sodium salt of 3-hydroxy-1-(2-naphthyl)prop-2-ene-1-one (1)
In a three necked flask containing 0.01 mol of sodium meth-
oxide and 20 ml ether, 0.01 mol of 2-acetyl naphthalene 16 with
0.01 mol of ethyl formate was poured over it through separating
funnel with efficient stirring. The solid product was collected at the
pump and used directly in the reactions.
3.1.2. Pyridin-2-thione (2-pyridone) derivatives (5a, b)
A solution of (0.01 mol) sodium salt of 1-naphthyl-3-hydroxy-2-
propene-1-one
1 (0.01 mol), cyanamides 2 (0.01 mol) and

3522
O.M. Ahmed et al. / European Journal of Medicinal Chemistry 44 (2009) 3519–3523
HepG2 (5b)
HepG2 (9f)
1.25
1
1.25
1
0.75
0.5
0.75
0.5
0.25
0
0.25
0
2.5
5
7.5
Concentration
10
12.5
0
2.5
5
Concentration
7.5
10
12.5
MCF7 (5b)
MCF7 (9f)
1.25
1.25
1
1
0.75
0.5
0.75
0.5
0.25
0
0.25
0
0
2.5
5
7.5
Concentration
10
0
2.5
5
Concentration
7.5
10
12.5
HCT116 (5b)
HCT116 (9f)
1.25
1
1.25
1
0.75
0.5
0.25
0
0.75
0.5
0.25
0
0
2.5
5
7.5
Concentration
10
12.5
0
2.5
5
Concentration
7.5
10
12.5
HELA (5b)
HELA (9f)
1.25
1
1.25
1
0.75
0.5
0.25
0
0.75
0.5
0.25
0
0
2.5
5
7.5
10
12.5
0
2.5
5
7.5
Concentration
10
12.5
Concentration
Fig. 1. Anti-tumor cytotoxicity of different concentrations (mg/ml) of pyridin-2-thione derivative (5b) and pyrazolo[1,5-a]pyrimidine analogue (9f) against different human cancer
cell lines in vitro.
piperidine acetate (1 ml) in H2O (3 ml) was refluxed for 10 min.
Acetic acid (1 ml) was added to the hot solution. The solid product
was filtered off and recrystallized from ethanol.
5a: yellow (yield 85%), m.p. >300 ꢀC, nmax/cmꢁ1 (KBr) 3392 (NH),
Table 1
2224 (CN) and 1656 (CO); 1H NMR (DMSO-d6)
d
¼ 7.12–8.43 (m, 9H,
IC50 of pyridin-2-thione analogue (5b) and pyrazolo[1,5-a]pyrimidine derivative (9f)
against different human cancer cell lines in vitro.
aromatic), 14.29 (s, br, 1H, NH); m/z 246 (Calcd for C16H10N2O: C,
78.04; H, 4.09; N, 11.38%. Found C, 78.23; H, 4.31; N, 11.14%). 5b:
yellow (yield 75%), m.p. >300 ꢀC, nmax/cmꢁ1 (KBr) 3432 (NH) and
Compound
IC50 (mg/ml) using different cell lines
HepG2
MCF7
HCT116
HeLa
2210 (CN); 1H NMR (DMSO-d6)
10.41 (s, br,1H, NH); m/z 262 (Calcd for C16H10N2S: C, 73.26; H, 3.84;
d
¼ 7.23–8.46 (m, 9H, aromatic),
5b
9f
1.01
1.88
0.34
0.47
0.49
0.54
0.60
0.40
N, 10.68; S, 12.22%. Found C, 73.52; H, 3.63; N, 10.71; S, 12.35%).

O.M. Ahmed et al. / European Journal of Medicinal Chemistry 44 (2009) 3519–3523
3523
3.1.3. Compounds 7a–f
Appendix. Supplementary material
These were prepared as previously reported [36,37].
Supplementary material associated with this article can be
found in the online version at doi:10.1016/j.ejmech.2009.03.042.
3.1.4. 7-(2-Naphthyl)pyrazolo[1,5-a]pyrimidine derivatives (9a–f)
A solution of (0.01 mol) sodium salt of 1-naphthyl-3-hydroxy-2-
propene-1-one 1 (0.01 mol), 3-aminopyrazoles 7 (0.01 mol) and
piperidine acetate (1 ml) in H2O (3 ml) was refluxed for 10 min.
Acetic acid (1 ml) was added to the hot solution. The solid product
was filtered off and recrystallized from ethanol.
References
[1] A.J. Bridges, Chem. Rev. 101 (2001) 2541–2572.
[2] J.D. Wang, K. Miller, D.H. Boschelli, F. Ye, B. Wu, M.B. Floyd, D.W. Powell, A. Wissner,
J.M. Weber, F. Boschelli, Bioorg. Med. Chem. Lett. 10 (2000) 2477–2480.
[3] S. Shenone, O. Bruno, F. Bondavalli, A. Ranise, L. Mosti, G. Menozzi, P. Fossa,
S. Donnini, A. Santoro, M. Ziche, F. Manetti, M. Botta, Eur. J. Med. Chem. 39
(2004a) 939–945.
[4] A.E. Rashad, M.I. Hegab, R.E. Abdel-Megeid, J.A. Micky, F.M.E. Abdel-Megeid,
Bioorg. Med. Chem. 16 (2008) 7102–7106.
[5] F. Shaheen, A. Badashah, M. Gielen, C. Gieck, M. Jamil, D. de Vos, J. Organomet.
Chem. 693 (2008) 1117–1126.
[6] J. Son, L. Zhao, A. Basnet, P. Thapa, R. Karki, Y. Na, Y. Jahng, T.Ch. Jeong, B. Jeong,
C. Lee, E. Lee, Eur. J. Med. Chem. 43 (2008) 675–682.
[7] L.X. Zhao, J. Sherchan, J.K. Park, Y. Jhang, B.S. Jeong, T.C. Jeong, C.S. Lee, E.S. Lee,
Arch. Pharm. Res. 29 (12) (2006) 1091–1095.
[8] A. Basnet, P. Thapa, R. Karki, Y. Na, Y. Jahng, B.S. Jeong, T.C. Jeong, C.S. Lee,
E.S. Lee, Bioorg. Med. Chem. 15 (2007) 4351–4359.
[9] S. Cesarini, A. Spallarossa, A. Ranise, S. Schenone, G. Rosano, P. La Colla,
G. Sanna, B. Bernardetta, R. Loddo, Eur. J. Med. Chem. (2008) 1–13.
[10] D.C. Kim, Y.R. Lee, B. Yang, K.J. Shin, D.J. Kim, B.Y. Chung, K.H. Yoo, Eur. J. Med.
Chem. 38 (2008) 525–532.
[11] S. Shenone, O. Bruno, A. Ranise, F. Bondavalli, P. Fossa, L. Mosti, G. Menozzi,
F. Carraro, A. Naldini, C. Bernini, F. Manetti, M. Botta, Bioorg. Med. Chem. Lett.
14 (2004b) 2511–2517.
[12] R. Morisi, M. Celano, E. Tosi, S. Schenone, M. Navarra, E. Ferretti, G. Costante,
C. Durante, G. Botta, M. D’Agostino, C. Brullo, S. Filetti, M. Botta, D. Russo, J.
Endocrinol. Invest. 30 (10) (2007) RC31–RC34.
[13] A. Angelucci, S. Schenone, G.L. Gravina, P. Muzi, C. Festuccia, C. Vicentini,
M. Botta, M. Bologna, Eur. J. Cancer 42 (2006) 2838–2845.
[14] M.E. Farley, W.F. Hoffman, R.S. Rubino, R.W. Hungate, A.J. Tebben,
R.Z.R.Z. Rutledge, R.C. McFall, W.R. Huckle, R.L. Kendall, K.E. Coll, K.A. Thomas,
Bioorg. Med. Chem. Lett. 12 (2002) 2767–2770.
[15] M.E. Farley, R.S. Rubino, W.F. Hoffman, S.R. Hambaugh, K.L. Arrington,
R.W. Hungate, M.T. Bilodeau, A.J. Tebben, R.Z. Rutledge, R.L. Kendall, R.C. McFall,
W.R.Huckle, K.E. Coll, K.A. Thomas, Bioorg.Med.Chem. Lett.12(2002)3537–3541.
[16] V. Krystof, D. Moravcova, M. Paprskarova, P. Barbier, V. Peyrot, A. Hlobikova,
L. Havlicek, M. Stramd, Eur. J. Med. Chem. 41 (2006) 1405–1411.
[17] C. Temple, R.D. Elliot, J.A. Montgomery, J. Org. Chem. 47 (1982) 761–764.
[18] E.C. Taylor, D.C. Palmer, T.J. George, S.R. Fletcher, C.P. Tseng, P.J. Horrion,
G.P. Beardsley, J. Org. Chem. 48 (1983) 4852–4860.
9a: Colorless (yield 72%), m.p. 188 ꢀC, nmax/cmꢁ1 (KBr) 3560,
3433 (NH) and 1670 (CO); 1H NMR (DMSO-d6)
d
¼ 2.52 (s, 3H,
SCH3) 7.13–8.54 (m, 14H, aromatic) and 11.53 (s, 1H, NH); m/z
410 (Calcd for C24H18N4OS: C, 70.22; H, 4.42; N, 13.65; S, 7.81%.
Found: C, 70.36; H, 4.22; N, 13.25; S, 7.63%). 9b: Colorless (yield
65%), m.p. 235 ꢀC, nmax/cmꢁ1 (KBr) 3540, 3450 (NH) and 1655
(CO); 1H NMR (DMSO-d6)
d
¼ 7.15–8.45 (m, 19H, aromatic), 10.31
(s, 1H, NH) and 11.62 (s, 1H, NH); m/z 455 (Calcd for C29H21N5O:
C, 76.47; H, 4.65; N, 15.37%. Found: C, 76.56; H, 4.45; N, 15.03%).
9c: Colorless (yield 75%), m.p. 200 ꢀC, nmax/cmꢁ1 (KBr) 3500,
3410 (NH) and 1640 (CO); 1H NMR (DMSO-d6)
d
¼ 2.5 (s, 3H,
SCH3), 3.2 (s, 3H, CH3), 7.22–8.62 (m, 13H, aromatic) and 10.5
(s, 1H, NH), m/z 424 (Calcd for C25H20N4OS: C, 70.73; H, 4.75; N,
13.20; S, 7.55%. Found: C, 70.53; H, 4.85; N, 13.51; S 7.75%). 9d:
Colorless (yield 70%), m.p. 240 ꢀC, nmax/cmꢁ1 (KBr) 3550, 3470
(NH), 1660 (CO); 1H NMR (DMSO-d6)
d
¼ 3.52 (s, 3H, CH3), 7.12–
846 (m, 18H, aromatic), 9.6 (s, 1H, NH) and 10.20 (s, 1H, NH); m/z
469 (Calcd for C30H23N5O: C, 76.74; H, 4.94; N, 14.92%. Found: C,
76.45; H, 4.73; N, 14.76%). 9e: Colorless (yield 65%), m.p. 180 ꢀC,
nmax/cmꢁ1 (KBr) 3565, 3414 (NH) and 1655 (CO); 1H NMR
(DMSO)
d
¼ 2.44 (s, 3H, SCH3), 7.0–8.5 (m, 13H, aromatic) and
10.33 (s, 1H, NH); m/z 444 (Calcd for C24H17ClN4OS: C, 64.79; H,
3.85; N, 12.59; S, 7.21%. Found: C, 64.59; H, 3.65; N, 12.46; S,
7.32%). 9f: Colorless (yield 75%), m.p. 250 ꢀC, 3523, 3430, 3410
(NH) and 1630 (CO); 1H NMR (DMSO-d6)
d
¼ 6.94–8.99 (m, 18H,
aromatic), 9.38 (s, 1H, NH), 10.09 (s, 1H, NH); m/z 489 (Calcd for
C29H20ClN5O: C, 71.09; H, 4.11; N, 14.29%. Found: C, 70.94; H,
4.32; N, 14.55%).
[19] S.R.Stone,J.A.Montgomery, J.F.Morrision, Biochem. Pharmacol. 33(1974)175–179.
[20] E.M. Grivsky, S. Lee, S.W. Sigel, D.S. Durch, C.A. Nichol, J. Med. Chem. 23 (1980)
327–329.
[21] W.D. Ensminger, G.B. Grindey, J.A. Hoglind, in: A. Rosowsky (Ed.), Advances in
cancer chemotherapy, vol. 1, Marcel Dekker, New York, 1976, pp. 61–109.
[22] J.I. DeGraw, P.H. Christine, R.L. Kisliuk, Y. Gaumont, F.M. Sirotnak, J. Med. Chem.
33 (1990) 673–677.
[23] E.C. Taylor, P.J. Harrington, S.R. Fletcher, G.P. Beardsley, R.G. Moran, J. Med.
Chem. 28 (1985) 914–921.
[24] G.H. Elgemeie, A.H. Elghandour, A.M. Elzanate, M.A. Mohamed, Mansora Sci.
Bull. 31 (2) (2004).
[25] G.H. Elgemeie, M.A. Mohamed, P.G. Jones, Acta Crystallogr. E58 (2002) 1293–1295.
[26] S.A. Ahmed, A.M. Hussein, W.G.M. Hozayen, A.H.H. El-Ghandour,
A.O. Abdelhamid, J. Heterocycl. Chem. 44 (2007) 803–808.
3.2. Anti-tumor cytotoxicity bioassay in vitro
HepG2 (hepatocellular carcinoma cell line), MCF7 (breast
carcinoma cell line), HCT116 (colon carcinoma cell line), HeLa
(cervix carcinoma cell line) were obtained from the Pharmacology
Unit, Cancer Biology Department, National Cancer Institute, Cairo
University, Egypt. Cells were maintained in DMEM medium with
10% foetal calf serum, sodium pyruvate, 100 U/ml penicillin and
100 m
g/ml streptomycin at 37 ꢀC and 5% CO2. Potential cytotoxicity
[27] S.A. Ahmed, A.H.H. El-Ghandour, A.O. Abdelhamid, M.A. Mohamed,
B.M. Mohamed, J. Chem. Res. 1 (2008) 26–31.
[28] C.R. Lefert, H. Siripumchidchidbouree, S. Hamspons, S. Workman, D. Sigee,
H.A.S. Epton, A. Harbour, J. Appl. Bacteriol. 78 (1955) 97.
of 5b and 9f was tested using the method of Skeha et al. [38].
Briefly, 104 cells/well were plated onto 96-well dishes overnight
before the treatment with the tested compounds to allow the
attachment of cells to the wall of the plate. Different concentrations
[29] S.A. Ahmed, Mansoura J. Chem. 35 (2008) 1–14.
[30] S. Shenone, O. Bruno, F. Bondavalli, A. Ranise, L. Mosti, G. Menozzi, P. Fossa,
F. Manetti, L. Morbidelli, L. Trincavelli, C. Martini, A. Lucacchini, Eur. J. Med.
Chem. 39 (2004c) 153–160.
of each tested compound (0.1, 2.5, 5, 10 mg/ml) were added to the
cell monolayer; triplicate wells were used for each individual dose.
Monolayer cells were incubated with the tested agent(s) for 48 h at
37 ꢀC and 5% CO2. At the end of the incubation period, the cells
were fixed and stained with sulforhodamine B dissolved in acetic
acid. Unbound stain was removed by washing four times with 1%
acetic acid and the protein bound dye was extracted with tris–
EDTA buffer. Absorbance was measured in an ELISA reader. The
relation between surviving fraction and compound concentration
was plotted to get the survival curve of each tumor cell line and
IC50, the concentration of an agent that causes a 50% growth
inhibition for each tested agent using each cell line was obtained
from the survival curve.
[31] B.S. Holla, M.S. Mahalinga, M.S. Karthekyan, P.M. Akberali, N.S. Shetti, Bioorg.
Med. Chem. 14 (2006) 2040–2047.
[32] S. Shenone, C. Brullo, O. Bruno, F. Bondavalli, L. Mosti, G. Maga, E. Crespan,
F. Carraro, F. Manetti, C. Tintori, M. Botta, Eur. J.Med.Chem. 43(2008)2665–2676.
[33] A.E. Amr, A.M. Mohamed, S.F. Mohamed, N.A. Abdel-Hafez, A.G. Hammam,
Bioorg. Med. Chem. 14 (2006) 5481–5488.
[34] R. Jorda, V. Krystof, L. Havlicek, M. Strnad, Poster Session, 07 July 2008, p. 84.
[35] M. Mo¨ller, W. Adam, S. Marquardt, C.R. Saha-Mo¨ller, H. Stopper, Free Radic.
Biol. Med. 39 (2005) 473–482.
[36] G.H. Elgemeie, A.H. Elghandour, A.M. Elzanate, S.A. Ahmed, J. Chem. Soc.
Perkin Trans. 1 (1997) 3285–3290.
[37] G.H. Elgemeie, A.H. Elghandour, A.M. Elzanate, S.A. Ahmed, J. Chem. Res. (S)
162 (1998) 162–163.
[38] P. Skeha, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J.T. Warren,
H. Bokesch, S. Kenney, M.R. Boyd, J. Natl. Cancer Inst. 82 (1990) 1107–1112.