Anticancer activities of some newly synthesized pyrazole and pyrimidine derivatives

Article abstract of DOI:10.1007/s12272-013-0163-x

A series of pyrazolopyridine and pyridopyrimidine derivatives 2-6 were newly synthesized using 3,5-bisarylmethylene-1-methylpiperidone as the starting material. The anticancer activities of the synthesized compounds were evaluated using 59 different human

Full text of DOI:10.1007/s12272-013-0163-x

Arch. Pharm. Res.
DOI 10.1007/s12272-013-0163-x
RESEARCH ARTICLE
Anticancer activities of some newly synthesized pyrazole
and pyrimidine derivatives
•
Ashraf M. Mohamed Weal A. El-Sayed
•
•
•
Musaed A. Alsharari Husam R. M. Al-Qalawi
Mousa O. Germoush
Received: 18 February 2011 / Accepted: 2 June 2011
Ó The Pharmaceutical Society of Korea 2013
Abstract A series of pyrazolopyridine and pyridopyr-
imidine derivatives 2–6 were newly synthesized using
3,5-bisarylmethylene-1-methylpiperidone as the starting
material. The anticancer activities of the synthesized
compounds were evaluated using 59 different human tumor
cell lines, representing cancers of CNS, ovary, renal,
breast, colon, lung, leukemia, and melanoma, prostate as
well as kidney. Some of the tested compounds, especially
those with a fluorine substituent at the para-position in the
phenyl ring and those with a pyridopyrimidine-2-thione
with a free –NH or –SH, exhibited greater in vitro anti-
tumor activities at low concentrations (log 10 [GI50] =
-4.6) against the human tumor cell lines. Additionally,
some of the compounds had moderate inhibitory effects on
the growth of the cancer cell lines. The detailed synthesis,
spectroscopic data and antitumor properties of the synthe-
sized compounds are reported.
Keywords N-methylpiperidone ꢀ Pyrimidine derivatives ꢀ
Human tumor ꢀ Anticancer activities
Introduction
Electron-rich nitrogen heterocycles play important roles in
diverse biological activities. For example, pyrimidines are
an important class of compounds and have widespread
applications from pharmaceuticals to industrial materials
(Selvam et al. 2012) with activities such as Tie-2 kinase
inhibitors (Matloobi and Kappe 2007), HIV-1 inhibitors
(Gadhachanda et al. 2007), antimalarials (Agarwal et al.
2005), adenosine A1 receptor antagonism (Chang et al.
2004), anticancer agents (Capdeville et al. 2002), analge-
sics (Zaki et al. 2006), cardiovascular agents (Atwal 1988)
and antiallergic agents (Ozeki et al. 1989). On the other
hand, the importance of the pyridine ring in the chemistry
of biological systems has attracted much attention because
of their presence in the substructures of many natural
products of therapeutic importance. The potent biological
activity of various vitamins and drugs (Joule et al. 1974;
Henry 2004; Vacher et al. 1999) is primarily a result of the
presence of the pyridine ring in their molecular structure.
The pyridine ring is found in the skeleton of many com-
pounds with potent antibacterial, antifungal and anticancer
properties (Millet et al. 2004; Mallea et al. 2003). Pyrazole
derivatives exhibit pharmacological activities; they can act
as hypotensives, antibacterials, anti-inflammatory agents
and antitumor agents (Hardy 1984; Orth 1968; Elnagdi
et al. 1987; Elnagdi et al. 1990). 2-Pyrazoline derivatives
have antimicrobial (Turan-Zitouni et al. 2005), anti-
Present Address:
A. M. Mohamed (&) ꢀ M. A. Alsharari ꢀ H. R. M. Al-Qalawi
Department of Chemistry, College of Science, Al Jouf
University, Sakaka, Al Jouf, Kingdom of Saudia Arabia
e-mail: ammewas@yahoo.com
A. M. Mohamed
Department of Applied Organic Chemistry, National Research
Centre, Dokki, Cairo, Egypt
W. A. El-Sayed (&)
Department of Photochemistry, National Research Centre,
Dokki, Cairo, Egypt
e-mail: waelshendy@gmail.com
Present Address:
W. A. El-Sayed
Faculty of Science, Northern Borders University, Arar, Kingdom
of Saudia Arabia
M. O. Germoush
Department of Biology, College of Science, Al Jouf University,
Sakaka, Al Jouf, Kingdom of Saudia Arabia
123

A. M. Mohamed et al.
inflammatory (Nasr and Said 2003), and antihypertensive
(Turan-Zitouni et al. 2000) activities. In particular, con-
densed pyrazoles are known for their various biological
activities; e.g., the pyrazolo[3,4-b]pyridine system has
interesting biological and pharmacological properties such
as adrenocorticotropic hormone–releasing factor [cortico-
tropin-releasing factor (CRF)] antagonist activity; CRF
antagonists are believed to be effective in the treatment of a
wide variety of stress-related illnesses, such as depression,
Alzheimer’s disease, gastrointestinal diseases, anorexia
nervosa, hemorrhage stress, drug and alcohol withdrawal
symptoms, drug addiction and infertility (Chen 1995). On
the other hand, many pyridopyrimidine derivatives possess
anticancer and kinase inhibiting activities (McKennon
et al. 2004; Newlander and Parrich 2010). Pyrazolo[3,
4-b]pyridines are generally prepared by reaction of
5-aminopyrazole and substituted a,b-unsaturated nitriles in
organic solvents (Quiroga et al. 1999, 2001) but most of
them suffer from drawbacks such as lower yields, and use
of organic solvents. In view of these facts, and in contin-
uation of our interest in the synthesis of new candidates
with potential activity (Mohamed et al. 2011; Amr et al.
2003, 2006 ; Abd El-Salam et al. 2010; Abdel-Hafez et al.
2009; Rashad et al. 2010; El-Sayed et al. 2010a, b), we
herein report the synthesis and anticancer activity of new
pyrazolopyridine and pyridopyrimidine derivatives. In
addition, the effect of substituents on the aromatic ring on
activity was investigated. The results on activity show the
effects of substitution at C-2 in the synthesized pyrido-
pyrimidine ring system with oxo-, thioxo- and imino
groups.
Germany) and the spots were detected by exposure to a UV
lamp at k245 nm for a few seconds. The starting material,
3,5-bisarylmethylene-1-methyl-4-piperidone (1), was syn-
thesized according to reported procedures (Lyles et al.
1974; Mcelvain and Kurt 1948).
3-Aryl-7-arylmethylene-3,3a,4,5,6,7-hexahydro-5-
methyl-2-phenyl-2H-pyrazolo[4,3-c]pyridine (2)
To a solution of compound 1 (10 mmol) in acetic acid
(10 mL), phenylhydrazine (1.1 mL, 10 mmol) was added.
The mixture was stirred at room temperature for 7 h,
poured into 25 mL of cold water and mixed with dilute
50 % hydrochloric acid (2 mL). The solid formed was
filtered and crystallized from 75 % aqueous ethanol. The
crystals included the following reaction products (2a–e).
7-Benzylidine-3,3a,4,5,6,7-hexahydro-5-methyl-2,3-
diphenyl-2H-pyrazolo[4,3-c]pyridine (2a)
3.4 g (89 % yield), m.p 183–184 °C; IR (KBr, cm-1): 1630
1
(C=N); H NMR (DMSO-d6): d 7.40–6.61 (m, 16H, Ar–
H ? C=CH), 4.1 (d, 1H, J = 6.1 Hz, H-3), 3.11–3.20 (dd,
2H, J = 7.0 Hz, J = 6.5 Hz, H-6), 2.31–2.61 (m, 3H,
H-3a ? H-4,40), 2.31 (s, 3H, N–CH3); 13C NMR:
d = 155.80 (C=N), 143.81, 141.11, 138.10 (Ar–C, 3C),
132.51 (C=CH), 129.70, 128.90,128.70 (Ar–C, 6CH-m),
128.51, 126.11, 122.47, 118.51 (Ar–C, C-7 and 6CH-o),
118.10, 117.12, 116.20 (Ar–C, 3CH-p), 57.51 (C-4), 53.70
(C-3 ? C-6), 48.41 (C-4a), 43.81 (CH3); MS, m/z (%): 379
[M?] (100), 350 (M?–CH3–N) (75.5), 336 (M?–CH3–
N=CH2) (83.5), 302 (M?–C6H5) (81.5); anal. calcd. for
C26H25N3 (379.49); C, 82.28; H, 6.64; N, 11.07. Found: C,
82.30; H, 6.62; N, 11.06.
Materials and methods
Melting points were determined on open glass capillaries
using an Electrothermal IA 9000 digital melting point
apparatus and are uncorrected. Elemental analyses were
done using an Elementar, Vario El, Microanalytical Unit,
National Research Centre, Cairo, Egypt and were found to
be within 0.4 % of theoretical values. Infrared (IR)
spectra were recorded on a Carlzeise Spectrophotometer
model ‘UR 10’ spectrophotometer using the KBr disc
7-(4-Flourobenzylidine)-3-(4-flourophenyl)-3,3a,4,5,6,
7-hexahydro-5-methyl-2-phenyl-2H-pyrazolo[4,
3-c]pyridine (2b)
3.5 g (86 % Yield), m.p 180–183 °C; IR (KBr, cm-1):
1
1632 (C=N); H NMR (acetone-d6): d 7.42–6.66 (m, 14H,
1
technique. H NMR spectra were recorded on a Varian
Ar–H ? C=CH), 4.62, 3.71 (dd, 1H, H-3), 3.40–3.20 (m,
3H, H-3a ? H-4,40), 2.60 (m, 2H, H-6,60), 2.41 (s, 3H, N–
CH3); 13C NMR: d = 155.30 (C=N), 143.61, 138.11,
133.41, 133.20, 131.51, 129.50, 129.12, 128.50, 127.06,
117.41, 113.51 (18Ar–C and C-7), 132.11 (C=CH), 57.52
(C-4), 53.71 (C-3 ? C-6), 48.42 (C-4a), 43.81 (CH3); MS,
m/z (%): 415 [M?] (100), 372 (M?–CH3–N=CH2) (80.5),
320 (M?–F–C6H4) (39.6); anal. calcd. for C26H23N3F2
(415.454); C, 75.15; H, 5.58; N, 10.11. Found: C, 75.18; H,
5.55; N, 10.12.
Gemini 200 MHz spectrometer (DMSO-d6 or CDCl3) and
the chemical shifts are reported as d (parts per million)
downfield from tetramethylsilane as an internal standard.
Splitting patterns were designated as follows; s: singlet; d:
doublet; t: triplet: m: multiplet. Mass spectra (MS) were
measured using a Finnigan SSQ 7000 mass spectrometer.
Follow-up of the reactions and checking the purity of the
compounds was done by TLC on silica gel-precoated alu-
minum sheets (Type 60 F254, Merck, Darmstadt,
123

Anticancer activities of pyrazole and pyrimidine derivatives
368 (M?–CHC6H4–Br) (100); anal. calcd. for C26H23Br2N3
(537.29); C, 58.12; H, 4.31; N, 7.82. Found: C, 58.10; H,
4.33; N, 7.80.
7-(4-Methoxybenzylidine)-3-(4-methoxyphenyl)-
3,3a,4,5,6,7-hexahydro-5-methyl-2-phenyl-2H-
pyrazolo[4,3-c]pyridine (2c)
3.1 g (76 % Yield), m.p 189–191 °C; IR (KBr, cm-1):
1634 (C=N); H NMR (DMSO-d6): d 7.61–6.82 (m, 14H,
4-Aryl-8-arylmethylene-3,4,5,6,7,8-hexahydro-6-
1
methylpyrido[4,3-d] pyrimidine-2(1H)-one (3)
Ar–H ? C=CH), 4.51 (d, 1H, J = 6.0 Hz, H-3), 3.48 (s,
3H, OCH3), 3.41 (s, 3H, OCH3), 3.20–3.11 (dd, 2H, J = 7,
6.5 Hz, H-6,60), 2.80–2.61 (m, 3H, H-3a ? H-4,40), 2.31
(s, 3H, N–CH3); 13C NMR: d = 155.81 (C=N), 143.81,
141.10, 138.10 (Ar–C, 3C), 132.15 (C=CH), 129.17,
128.19, 128.70 (Ar–C, 6CH-m), 128.50, 126.10, 113.50
(Ar–C, 6CH-o and C-7), 128.01, 125.92, 117.21 (Ar–C,
2CH-p), 57.52 (C-4), 55.81 (2OCH3), 53.81 (C-3 ? C-6),
48.62 (C-4a), 43.92 (CH3). MS, m/z (%): 439 [M?] (15),
332 (M?–CH3O–C6H4) (71), 362 (M?–C6H5) (100); anal.
calcd. for C28H29N3O2 (439.52); C, 76.51; H, 6.65; N, 9.56.
Found: C, 76.53; H, 6.63; N, 9.53.
A solution of compound 1 (10 mmol) in 25 ml of sodium
ethoxide (0.2 g Na in 25 mL of absolute ethanol) was
mixed with urea (10 mmol). The mixture was refluxed for
3 h, left to cool, and then poured gradually, with stirring,
into 50 mL cold water. The solid that formed was filtered,
washed with water and crystallized from ethanol. The
following reaction products 3a–e were identified.
8-Benzylidene-3,4,5,6,7,8-hexahydro-6-methyl-4-
phenylpyrido[4,3-d]pyrimidine-2(1H)-one (3a)
2.8 g (85 % Yield), m.p 216–219 °C; IR (KBr, cm-1):
3350, 3294 and 3153 (NH), 1670 (C=O); 1H NMR
(CDCl3): d 9.23–9.18 (bs, 2H, 2NH, exchangeable with
D2O), 6.73–7.51 (m, 11H, Ar–H ? C=CH), 5.02 (d, 1H,
J = 4.6 Hz, H-4), 3.78 (s, 2H, CH2), 3.98 (s, 2H, CH2),
2.28 (s, 3H, CH3); 13C NMR: d = 162.12 (C=O),
123.08–157.06 (12Ar–C, C-4a, 8a and C=CH), 70.05
(C-4), 58.61 (C-7), 53.52 (C-5), 43.50 (CH3); MS, m/z (%):
331 [M?] (24); anal. calcd. for C21H21N3O (331.38); C,
76.11; H, 6.39; N, 12.68. Found: C, 76.50; H, 5.83; N,
12.74.
7-(4-Chlorobenzylidine)-3-(4-chlorophenyl)-3,3a,4,5,6,
7-hexahydro-5-methyl-2-phenyl-2H-pyrazolo[4,
3-c]pyridine (2d)
4.1 g (90 % Yield), m.p 253–255 °C; IR (KBr, cm-1):
1
1635 (C=N); H NMR (acetone-d6): d 7.62–6.75 (m, 14H,
Ar–H ? C=CH), 4.72 (d, 1H, J = 5.9 Hz, H-3), 3.30–3.21
(dd, 2H, J = 7.0, J = 6.6 Hz, H-6,60), 3.11–3.90 (m, 3H,
H-3a ? H-4,40), 2.32 (s, 3H, CH3); 13C NMR: d = 155.42
(C=N), 143.72, 138.21, 133.61, 133.30, 131.51, 129.52,
129.11, 128.60, 127.51, 117.32, 113.50 (18Ar–C and C-7),
132.21 (C=CH), 57.62 (C-4), 53.90 (C-3 ? C-6), 42.51
(C-3a), 48.91 (CH3); MS, m/z (%): 448 [M?] (100), 452
[M??4] (33.6), 450 [M??2] (66.7) due to the presence of
two chlorine atoms, 403 (447–CH3–N=CH2, –H?) (83.2),
336 (447–Cl–C6H4) (30.3); anal. calcd. for C26H23N3Cl2
(448.384); C, 69.64; H, 5.17; N, 9.37. Found: C, 69.66; H,
5.15; N, 9.39.
8-(4-Fluoroenzylidene)-3,4,5,6,7,8-hexahydro-6-methyl-4-
(4-fluoro phenyl)pyrido[4,3-d]pyrimidin-2(1H)-one (3b)
3.3 g (91 % Yield), m.p 209–212 °C; IR (KBr, cm-1):
3332, 3296 and 3172 (NH), 1662 (C=O); 1H NMR
(CDCl3): d 9.44–9.38 (bs, 2H, 2NH, exchangeable with
D2O), 7.69–6.61 (m, 9H, Ar–H ? C=CH), 5.15 (d, 1H,
J = 6.8 Hz, H-4), 3.59 (s, 2H, CH2), 3.92 (s, 2H, CH2),
2.29 (s, 3H, CH3); 13C NMR: d = 162.12 (C=O),
160.19–124.28 (12Ar–C, C-7a, 8a and C=CH), 72.05
(C-4), 58.51 (C-7), 53.60 (C-5), 40.51 (CH3); MS, m/z (%):
367 [M?] (65); anal. calcd. for C21H19N3F2O (367.39); C,
68.65; H, 5.21; N, 11.44. Found: C, 69.01; H, 4.90; N,
11.53.
7-(4-Bromobenzylidine)-3-(4-bromophenyl)-3,3a,4,5,6,7-
hexahydro-5-methyl-2-phenyl-2H-pyrazolo[4,3-c]pyridine
(2e)
4.7 g (88 % Yield), m.p 161–164 °C; IR (KBr, cm-1):
1
1630 (C=N); H NMR (DMSO-d6): d 7.52–6.73 (m, 14H,
Ar–H ? C=CH), 4.92 (d, 1H, J = 6 Hz, H-3), 3.41–3.31
(dd, 2H, J = 7, 6.5 Hz, H-6,60), 3.23–3.12 (m, 3H,
H-3a ? H-4,40), 2.30 (s, 3H, CH3); 13C NMR: d = 155.72
(C=N), 143.80, 139.12, 134.61, 134.31, 132.21, 129.70,
129.21, 128.50, 127.41, 117.52, 113.60 (18Ar–C and C-7),
132.31 (C=CH), 57.71 (C-4), 53.81 (C-3 ? C-6), 42.70
(C-4a), 47.81 (CH3); MS, m/z (%): 537 [M?] (13.61), 539
[M??2] (16.77), 541 [M??4] (8.43) due to the presence of
two bromine atoms, 492 (M?–CH3–N=CH2) (71.2) and
8-(4-Methoxybenzylidene)-3,4,5,6,7,8-hexahydro-6-
methyl-4-(4-methoxyphenyl)pyrido[4,3-d]pyrimidin-2(1H)-
one (3c)
3.5 g (90 % Yield), m.p 135–137 °C; IR (KBr, cm-1):
3338, 3268 and 3175 (NH), 1665 (C=O); 1H NMR
(CDCl3): d 9.60–9.53 (bs, 2H, 2NH, exchangeable with
D2O), 7.80–6.67 (m, 9H, Ar–H ? C=CH), 5.16 (d, 1H,
123

A. M. Mohamed et al.
J = 6.8 Hz, H-4), 3.61 (s, 2H, CH2), 3.57 (s, 2H, CH2),
3.44, 3.49 (2s, 6H, 2 OCH3), 2.32 (s, 3H, CH3); 13C NMR:
d = 163.30 (C=O), 1243.53–161.10 (12Ar–C, C-5a, 8a and
C=CH), 72.51 (C-4), 58.71 (C-7), 56.90 (C-5), 56.90
(2CH3O), 43.52 (CH3); MS, m/z (%): 391 [M?] (50); anal.
calcd. for C23H25N3O3 (391.41); C, 70.57; H, 6.44; N,
10.73. Found: C, 70.90; H, 5.97; N, 10.80.
2NH, exchangeable with D2O), 8.42 (s, 1H, NH, exchange-
able with D2O), 7.61–7.05 (m, 11H, Ar–H ? C=CH), 5.15
(s, 1H, H-4), 4.10 (s, 2H, CH2), 3.91 (s, 2H, CH2), 2.70 (s, 3H,
CH3); 13C NMR: d 163.61 (C=NH), 140.30 (C-8a), 137.26
(C-4a), 136.91 (C-8), 53.61 (C-4), 143.30, 135.41, 128.80,
128.70, 128.61, 128.12, 127.01, 126.62, 126.41 (12Ar–C),
124.62 (C=CH), 52.80 (C-7), 50.91 (C-5), 44.40 (CH3); MS,
m/z (%): 330 [M?] (88), 329 (M?-H?) (100), 314 (329–
CH3) (9.9), 254 (M?–C6H5, –H?) (19.9); anal. calcd. for
C21H22N4 (330.426); C, 76.32; H, 6.71; N, 16.95. Found: C,
76.35; H, 6.70; N, 16.92.
8-(4-Chlorobenzylidene)-3,4,5,6,7,8-hexahydro-6-methyl-
4-(4-chlorophenyl)pyrido[4,3-d]pyrimidine-2(1H)-one (3d)
3.6 g (91 % Yield), m.p 162–164 °C; IR (KBr, cm-1):
3352, 3282 and 3170 (NH), 1665 (C=O); 1H NMR
(CDCl3): d 9.31–9.26 (bs, 2H, 2NH, exchangeable with
D2O), 7.60–6.71 (m, 9H, Ar–H ? C=CH), 5.14 (d, 1H,
J = 6.2 Hz, H-4), 3.60 (s, 2H, CH2), 3.55 (s, 2H, CH2),
2.35 (s, 3H, CH3); 13C NMR: d = 164.12 (C=O),
123.21–160.60 (12Ar–C, C-4a, 8a and C=CH), 72.01
(C-4), 58.62 (C-7), 53.51 (C-5), 43.52 (CH3); MS, m/
z (%):400 [M?] (75), 401 [M??1] (100), 402 [M??2]
(46), 404 [M??4] (22); anal. calcd. for C21H19N3Cl2O
(400.30); C, 63.01; H, 4.78; N, 10.50. Found: C, 63.30; H,
4.39; N, 10.52.
8-(4-Fluorobenzylidene)-3,4,5,6,7,8-hexahydro-6-methyl-
4-(4-fluorophenyl)pyrido[4,3-d]pyrimidine-2(1H)-imine
(4b)
3.5 g (94 % Yield), m.p 130–132 °C; IR (KBr) m cm-1: 3155,
1
3310 and 3452 (NH); H NMR (DMSO-d6): d 9.60 (s, 2H,
2NH, exchangeable with D2O), 8.61 (s, 1H, NH, exchangeable
with D2O), 6.85–7.04 (m, 9H, Ar–H ? C=CH), 4.95 (s, 1H,
H-4), 4.02 (s, 2H, CH2), 3.82 (s, 2H, CH2), 2.72 (s, 3H, CH3);
13C NMR: d 163.27 (C=NH), 140.38 (C-8a), 137.04 (C-4a),
136.29 (C-8), 53.65 (C-4), 143.41, 135.40, 128.76, 128.49,
128.39, 128,127, 126.61, 126.20 (12Ar–C), 124.71 (C=CH),
52.77 (C-7), 50.99 (C-5), 44.45 (CH3); MS, m/z (%): 366 [M?]
(80), 347 (M?–F) (51), 271 (M?–C6H4–F) (100), 243 (M?–F–
C6H4–CH=NH?) (63); anal. calcd. for C21H20N4F2 (366.39);
C, 68.83; H, 5.50; N, 15.29. Found: C, 68.81; H, 5.50; N, 15.30.
8-(4-Bromobenzylidene)-3,4,5,6,7,8-hexahydro-6-methyl-
4-(4-bromophenyl)pyrido[4,3-d]pyrimidin-2(1H)-one (3e)
4.3 g (89 % Yield), m.p 208–211 °C; IR (KBr, cm-1): 3318,
1
3272 and 3190 (NH), 1672 (C=O); H NMR (CDCl3): d
9.28–9.21 (bs, 2H, 2NH, exchangeable with D2O), 7.66–6.96
(m, 9H, Ar–H ? C=CH), 5.12 (d, 1H, J = 6.2 Hz, H-4), 3.59
(s, 2H, CH2), 3.54 (s, 2H, CH2), 2.32 (s, 3H, CH3); 13C NMR:
d = 164.12 (C=O), 123.22–160.52 (12Ar–C, C-4a, 8a and
C=CH), 76.22 (C-4), 58.51 (C-7), 53.51 (C-5), 43.50 (CH3);
MS, m/z (%): 488 [M?-1] (65); anal. calcd. for
C21H19N3OBr2 (489.20); C, 51.56; H, 3.91; N, 8.59. Found:
C, 51.75; H, 3.59; N, 8.64.
8-(2-Chlorobenzylidene)-3,4,5,6,7,8-hexahydro-6-methyl-
4-(2-chlorophenyl)pyrido[4,3-d]pyrimidine-2(1H)-imine
(4c)
3.7 g (92 % Yield), m.p 142–145 °C; IR (KBr) m cm-1: 3158,
1
3310 and 3455 (NH); H NMR (DMSO-d6): d 9.21 (s, 2H,
2NH, exchangeable with D2O), 8.31 (s, 1H, NH, exchangeable
with D2O), 6.82–7.12 (m, 9H, Ar–H ? C=CH), 4.98 (s, 1H,
H-4), 4.04 (s, 2H, CH2), 3.70 (s, 2H, CH2),2.71 (s, 3H, CH3);
13C NMR: d 163.29 (C=NH), 141.08 (C-8a), 138.04 (C-4a),
137.59 (C-8), 54.66 (C-4), 144.40, 137.42, 128.66, 128.53,
128.43, 128,12, 126.7, 126.56 (12Ar–C), 124.91 (C=CH),
52.73 (C-7), 50.90 (C-5), 44.50 (CH3); MS, m/z (%): 399 [M?]
(68), 401 (M??2) (45.5), 403 (M??4) (10.3), due to the
presence of two chlorine atoms, 384 (M?–CH3) (10.2), 288
(M?–Cl–C6H5) (36); anal. calcd. for C21H20N4Cl2 (399.32); C,
63.16; H, 5.04; N, 14.03. Found: C, 63.13; H, 5.07; N, 14.06.
4-Aryl-8-arylmethylene-3,4,5,6,7,8-hexahydro-6-
methylpyrido-[4,3-d]pyrimidine-2(1H)imine (4)
A mixture of compound 1 (10 mmol) in 25 mL absolute
ethanol and sodium hydroxide (0.5 g) was mixed with gua-
nidine hydrochloride (0.96 g, 10 mmol) and refluxed for 3 h.
It was left to cool and then decanted gradually, with stirring,
into cold water. The solid formed was filtered, washed with
water and crystallized from the proper solvent.
8-(4-Chlorobenzylidene)-3,4,5,6,7,8-hexahydro-6-methyl-
4-(4-chlorophenyl)pyrido[4,3-d]pyrimidine-2(1H)-imine
(4d)
8-Benzylidene-3,4,5,6,7,8-hexahydro-6-methyl-
4-phenylpyrido[4,3-d]pyrimidine-2(1H)-imine (4a)
2.8 g (84 % Yield), m.p 155–158 °C; IR (KBr) m cm-1: 3150,
3300 and 3450 (NH); H NMR (DMSO-d6): d 9.15 (s, 2H,
3.5 g (89 % Yield), m.p 213–215 °C; IR (KBr) m cm-1
3150, 3312 and 3458 (NH); H NMR (DMSO-d6): d 8.91
:
1
1
123

Anticancer activities of pyrazole and pyrimidine derivatives
(s, 2H, 2NH, exchangeable with D2O), 8.40 (s, 1H, NH,
exchangeable with D2O), 6.79–7.15 (m, 9H, Ar–
H ? C=CH), 5.01 (s, 1H, H-4), 4.11 (s, 2H, CH2), 3.70 (s,
2H, CH2), 2.70 (s, 3H, CH3); 13C NMR: d 162.77 (C=NH),
141.45 (C-8a), 138.02 (C-4a), 136.19 (C-8), 55.25 (C-4),
144.21, 134.50, 129.61, 128.49, 127.62, 126.11, 126.31,
126.05 (12Ar–C), 124.25 (C=CH), 52.62 (C-7), 50.94 (C-
5), 44.43 (CH3); MS, m/z (%): 399 [M?] (73), 401 (M??2)
(48.8), 403 (M??4) (13), due to the presence of two
chlorine atoms, 356 (M?–CH3–N=CH2) (20.2), 288 (M?
–Cl–C6H5) (46); anal. calcd. for C21H20N4Cl2 (399.32); C,
63.16; H, 5.04; N, 14.03. Found: C, 63.17; H, 5.02; N,
14.06.
143.29, 136.19, 129.49, 128.74, 128.62, 128,13, 126.65
126.92 (12Ar–C), 125.63 (C=CH), 54.39 (C-7), 53.91 (C-
5), 44.48 (CH3); MS, m/z (%): 347 [M?] (80.7), 346 (M?–
H?) (100), 214 (346–C6H5CH=NH) (52.7), 254 (M?–
C6H5, –H?) (19.9); anal. calcd. for C21H21N3S (347.468);
C, 72.58; H, 6.1; N, 12.1; S, 9.22. Found: C, 72.56; H, 6.12;
N, 12.07; S, 9.25.
8-(4-Fluorobenzylidene)-3,4,5,6,7,8-hexahydro-6-methyl-
4-(4-fluorophenyl)pyrido[4,3-d]pyrimidine-2(1H)-thione
(5b)
4.5 g (91 % Yield), m.p 155–157 °C; (KBr) m cm-1: 3344,
1
3204 (NH); H NMR (DMSO-d6): d 9.51, 9.21 (2s, 2H,
8-(3-Bromobenzylidene)-3,4,5,6,7,8-hexahydro-6-methyl-
4-(3-bromophenyl)pyrido[4,3-d]pyrimidine-2(1H)-imine
(4e)
2NH, exchangeable with D2O), 7.50–7.11 (m, 9H, Ar–
H ? C=CH), 4.96 (s, 1H, H-4), 4.10 (s, 2H, CH2), 3.60 (s,
2H, CH2), 2.20 (s, 3H, CH3); 13C NMR: d 180.56 (C=S),
140.41 (C-8a), 137.28 (C-4a), 136.81 (C-8), 53.72 (C-4),
143.33, 135.43, 128.89, 128.75, 128.61, 128.14, 126.29,
126.47 (12Ar–C), 124.19 (C=CH), 52.71 (C-7), 50.93
(C-5), 44.47 (CH3); MS, m/z (%): 383 [M?] (100), 339
(M?–C=S) (11.4), 259 (383–F–C6H4CH=NH) (71.4); anal.
calcd. for C21H19N3SF2 (383.432); C, 65.77; H, 4.99; N,
10.96; S, 8.36. Found: C, 65.75; H, 5.01; N, 10.94; S, 8.38.
4.4 g (90 % Yield), m.p 145–148 °C; IR (KBr) m cm-1
:
1
3156, 3309 and 3452 (NH); H NMR (DMSO-d6): d 9.11
(s, 2H, 2NH, exchangeable with D2O), 8.51 (s, 1H, NH,
exchangeable with D2O), 6.68–7.06 (m, 9H, Ar–
H ? C=CH), 4.85 (s, 1H, H-4), 4.31 (s, 2H, CH2), 3.61(s,
2H, CH2), 2.77 (s, 3H, CH3); 13C NMR: d 163.31 (C=NH),
140.35 (C-8a), 137.10 (C-4a), 135.28 (C-8), 55.85 (C-4),
142.44, 135.45, 129.71, 128.78, 128.51, 128,17, 126.38,
126.07 (12Ar–C), 124.71 (C=CH), 52.80 (C-7), 50.95
(C-5), 43.85 (CH3); MS, m/z (%): 488 [M?] (56), 490
(M??2) (100), 492 (M??4) (54), due to the presence of two
bromine atoms, 408 (M?–Br) (5.6), 332 (M?–Br–C6H5)
(36); anal. calcd. for C21H20N4Br2 (488.23); C, 51.66; H,
4.13; N, 11.47. Found: C, 51.64; H, 4.12; N, 11.49.
8-(2-Chlorobenzylidene)-3,4,5,6,7,8-hexahydro-6-methyl-
4-(2-chlorophenyl)pyrido[4,3-d]pyrimidine-2(1H)-thione
(5c)
3.4 g (82 % Yield), m.p 200–202 °C; IR (KBr) m cm-1
:
3351, 3304 (NH); 1H NMR (DMSO-d6): d 9.71, 9.10
(2s, 2H, 2NH, exchangeable with D2O), 7.55–7.15 (m, 9H,
Ar–H ? C=CH), 5.41 (s, 1H, H-4), 4.21 (s, 2H, CH2),
3.71(s, 2H, CH2), 2.20 (s, 3H, CH3); 13C NMR: d 180.59
(C=S), 140.42 (C-8a), 137.32 (C-4a), 136.28 (C-8), 53.71
(C-4), 143.33, 135.43, 128.89, 128.74, 128.62, 128,19,
126.31, 126.52 (12Ar–C), 124.22 (C=CH), 52.73 (C-7),
50.98 (C-5), 44.44 (CH3); MS, m/z (%): 416 [M?] (50),
418 (M??2) (35), 420 (M??4) (8.5), due to the presence
of two chlorine atoms, 275 (M?–Cl–C6H4CH=NH, –H2)
(100); anal. calcd. for C21H19N3SCl2 (416.36); C, 60.57; H,
4.6; N, 10.1; S, 7.7. Found: C, 60.60; H, 4.57; N, 10.07; S,
7.71.
4-Aryl-8-arylmethylene-3,4,5,6,7,8-hexahydro-6-
methyl-pyrido[4,3-d]pyrimidine-2-(1H)thione (5)
To a solution of compound 1 (10 mmol) in 25 ml absolute
ethanol, 0.5 g potassium hydroxide and thiourea (0.76 g,
10 mmol) were added. The mixture was refluxed for 3 h,
left to cool, and poured gradually into cold water. The solid
formed was filtered, washed with water and crystallized
from the proper solvent. The following compounds were
identified.
8-Benzylidene-3,4,5,6,7,8-hexahydro-6-methyl-4-
phenylpyrido[4,3-d]pyrimidine-2(1H)-thione (5a)
8-(4-Chlorobenzylidene)-3,4,5,6,7,8-hexahydro-6-methyl-
4-(4-chlorophenyl)pyrido[4,3-d]pyrimidine-2(1H)-thione
(5d)
3.1 g (89 % Yield), m.p 190–193 °C; IR (KBr) m cm-1
:
1
3502, 3194 (NH); H NMR (DMSO-d6): d 9.48, 9.18 (2s,
2H, 2NH, exchangeable with D2O), 7.40–7.15 (m, 11H,
Ar–H ? C=CH), 4.95 (s, 1H, H-4), 4.09 (s, 2H, CH2), 3.85
(s, 2H, CH2), 2.20 (s, 3H, CH3); 13C NMR: d 180.61 (C=S),
143.31 (C-8a), 138.26 (C-4a), 137.92 (C-8), 54.35 (C-4),
3.7 g (88 % Yield), m.p 202–205 °C; IR (KBr) m cm-1
:
1
3248, 3176 (NH); H NMR (DMSO-d6): d 9.60, 9.10 (2s,
2H, 2NH, exchangeable with D2O), 7.6–7.1 (m, 9H, Ar–
H ? C=CH), 5.02 (s, 1H, H-4), 4.31 (s, 2H, CH2), 3.63 (s,
2H, CH2), 2.32 (s, 3H, CH3); 13C NMR: d 180.82 (C=S),
123

A. M. Mohamed et al.
8-(4-Bromobenzylidene)-3,4,5,6,7,8,-hexahydro-6-methyl-
2-(methylthio)-4-(4-bromophenyl)pyrido[4,3-
d]pyrimidine (6b)
140.46 (C-8a), 137.36 (C-4a), 136.88 (C-8), 53.75 (C-4),
143.37, 135.43, 128.89, 128.79, 128.61, 128,15, 126.42,
126.50 (12Ar–C), 124.28 (C=CH), 52.74 (C-7), 50.96 (C-
5), 44.58 (CH3); MS, m/z (%): 416 [M?] (87.6), 418
(M??2) (59), 420 (M??4) (13), due to the presence of two
chlorine atoms, 414 (M?–H2) (100), 275 (M?–Cl–
C6H4CH=NH, –H2) (72.2); anal. calcd. for C21H19N3SCl2
(416.36); C, 60.57; H, 4.60; N, 10.09; S, 7.70. Found: C,
60.55; H, 4.62; N, 10.13; S, 7.68.
4.3 g (82 % Yield), m.p 163–165 °C; IR (KBr) m cm-1
:
1
1645 (C=N); H NMR (DMSO-d6): d 9.69 (s, 1H, NH),
6.9-7.65 (m, 9H, Ar–H ? C=CH), 4.99 (s, 1H, H-4), 3.61
(s, 2H, CH2), 3.03 (s, 2H, CH2), 2.47 (s, 3H, N–CH3), 2.26
(s, 3H, SCH3); 13C NMR: d 159.48 (C=NH), 141.72
(C-8a), 138.38 (C-4a), 136.02 (C-8), 56.79 (C-4), 144.13,
135.72, 128.52, 128.31, 128.41, 127.28, 126.21, 126.30
(12Ar–C), 123.70 (C=CH), 52.40 (C-7), 50.45 (C-5), 44.35
(CH3), 26.79 (SCH3); MS: m/z (%): 519 [M?] (21), 521
[M??2] (43), 523 [M??4] (22), due to the presence of two
bromine atoms, 472 (M?–SCH3) (100), 363 (M?–C6H4Br)
(51); anal. calcd. for C22H21N3SBr2 (519.276); C, 50.88; H,
4.08; N, 8.09; S, 6.17. Found: C, 51.10; H, 3.98; N, 8.10; S,
6.21.
8-(4-Bromobenzylidene)-3,4,5,6,7,8-hexahydro-6-methyl-
4-(4-bromophenyl)pyrido[4,3-d]pyrimidine-2(1H)-thione
(5e)
4.3 g (85 % Yield), m.p 180–182 °C; IR (KBr) m cm-1
:
1
3346, 3275 (NH); H NMR (DMSO-d6): d 9.42, 9.01 (2 s,
2H, 2NH, exchangeable with D2O), 7.61–7.30 (m, 9H, Ar–
H ? C=CH), 5.31 (s, 1H, H-4), 4.22 (s, 2H, CH2), 3.53 (s,
2H, CH2), 2.23 (s, 3H, CH3); 13C NMR: d 180.57 (C=S),
140.44 (C-8a), 137.29 (C-4a), 136.84 (C-8), 53.74 (C-4),
143.37, 135.45, 128.87, 128.70, 128.71, 128,21, 126.31,
126.48 (12Ar–C), 124.23 (C=CH), 52.74 (C-7), 50.95 (C-
5), 44.49 (CH3); MS, m/z (%): 505 [M?] (47), 507 (M??2)
(95), 509 (M??4) (45), due to the presence of two bromine
atoms, 349 (M?–Br–C6H4,) (100); anal. calcd. for
C21H19N3SBr2 (505.27); C, 49.91; H, 3.79; N, 8.32; S,
6.34. Found: C, 49.90; H, 3.80; N, 8.31; S, 6.35.
8-(4-Bromobenzylidene)-2-(ethylthio)-3,4,5,6,7,8,-
hexahydro-6-methyl-4-(4-bromophenyl)pyrido[4,3-
d]pyrimidine (6c)
4.3 g (81 % Yield), m.p 129–131 °C; IR (KBr) m cm-1
:
1
1643 (C=N); H NMR (DMSO-d6): d 9.48 (s, 1H, NH),
7.72–6.91(m, 9H, Ar–H ? C=CH), 4.84 (s, 1H, H-4), 3.72
(q, 2H, J = 6.2 Hz, CH2CH3), 3.63 (s, 2H, CH2), 3.13
(s, 2H, CH2), 2.78 (s, 3H, N–CH3), 1.21 (t, 3H, J = 6.2 Hz,
CH2CH3); 13C NMR: d 160.05 (C=NH), 141.84 (C-8a),
138.31 (C-4a), 136.05 (C-8), 57.10 (C-4), 143.16, 135.67,
128.29, 128.14, 128.02, 127.13, 126.02, 126.15 (12Ar–C),
123.70 (C=CH), 52.30 (C-7), 51.18 (C-5), 46.38 (CH2),
45.22 (CH3), 27.59 (SCH3); MS: m/z (%): 533 [M?] (18),
535 [M??2](37), 537[M??4] (19), due to the presence of
two bromine atoms, 375 (M?–C6H4Br) (100); anal. calcd.
for C23H23N3SBr2 (533.32); C, 51.79; H, 4.35; N, 7.88; S,
6.01. Found: C, 51.97; H, 3.99; N, 7.90; S, 6.06.
4-Aryl-8-arylmethylene-2-(alkylthio)-3,4,5,6,7,
8,-hexahydro-6-methylpyrido[4,3-d]pyrimidine-2-
alkylthione (6)
To a mixture of compound 5 (10 mmol) in 50 mL of ethyl
alcohol, sodium hydroxide (0.3 g) and methyl iodide or
ethyl iodide (10 mmol) was added. The mixture was
refluxed for 2 h, excess ethanol was evaporated and the
solid formed was collected and crystallized from ethanol.
8-Benzylidene-3,4,5,6,7,8,-hexahydro-6-methyl-2-
(methylthio)-4-phenylpyrido[4,3-d]pyrimidine (6a)
8-(4-Fluorobenzylidene)-3,4,5,6,7,8,-hexahydro-6-methyl-
2-(methylthio)-4-(4-fluorophenyl)pyrido[4,3-d]pyrimidine
(6d)
3.1 g (85 % Yield), m.p 119–121 °C; IR (KBr) m cm-1
:
1
1642 (C=N); H NMR (DMSO-d6): d 9.24 (s, 1H, NH),
7.62–7.20 (m, 11H, Ar–H ? C=CH), 4.97 (s, 1H, H-4),
4.20 (s, 2H, CH2), 3.70 (s, 2H, CH2), 2.45 (s, 3H, N–CH3),
2.20 (s, 3H, SCH3); 13C NMR: d 159.37 (C=NH), 141.26
(C-8a), 138.25 (C-4a), 135.94 (C-8), 56.77 (C-4), 143.06,
135.58, 128.28, 128.03, 128.1, 127,21, 126.21, 126.15
(12Ar–C), 123.68 (C=CH), 52.26 (C-7), 50.38 (C-5), 44.11
(CH3), 26.16 (SCH3); MS: m/z (%): 361 [M?] (19), 318
(M?–CH3–N=CH2) (51), 284 (M?–C6H5) (100); anal.
calcd. for C22H23N3S (361.484); C, 73.09; H, 6.41; N,
11.62; S, 8.87. Found: C, 73.11; H, 6.43; N, 11.60; S, 8.85.
3.4 g (86 % Yield), m.p 160–162 °C; IR (KBr) m cm-1
:
1
1649 (C=N); H NMR (DMSO-d6): d 9.52 (s, 1H, NH),
6.91–7.45 (m, 9H, Ar–H ? C=CH), 4.96 (s, 1H, H-4), 3.61
(s, 2H, CH2), 3.25 (s, 2H, CH2), 2.47 (s, 3H, N–CH3), 2.27
(s, 3H, SCH3); 13C NMR: d 160.11 (C=NH), 141.93
(C-8a), 138.49 (C-4a), 136.28 (C-8), 58.05 (C-4), 143.81,
135.95, 128.40, 128.22, 128.90, 127.85, 126.53, 126.19
(12Ar–C), 123.99 (C=CH), 52.75 (C-7), 51.62 (C-5), 45.29
(CH3), 26.38 (SCH3); MS: m/z (%): 395 [M?] (20), 300
(M?–C6H4F) (100); anal. calcd. for C22H21N3SF2 (397.48);
123

Anticancer activities of pyrazole and pyrimidine derivatives
C, 66.48; H, 5.33; N, 10.57; S, 8.07. Found: C, 66.83; H,
5.11; N, 10.64; S, 8.12.
as log 10 GI50 which is the drug concentration (M) causing
a 50 % reduction in the net protein increase in control cells
during the drug incubation.
8-(4-Fluorobenzylidene)-2-(ethylthio)-3,4,5,6,7,8,-
hexahydro-6-methyl-4-(fluorophenyl)pyrido[4,3-
d]pyrimidine (6e)
Results and discussion
3.3 g (81 % Yield), m.p 142–144 °C; IR (KBr) m cm-1
:
1
1651 (C=N); H NMR (DMSO-d6): d 9.72 (s, 1H, NH),
6.92–7.44 (m, 9H, Ar–H ? C=CH), 4.97 (s, 1H, H-4), 3.73
(q, 2H, J = 6.2 Hz, CH2CH3), 3.60 (s, 2H, CH2), 3.22
(s, 2H, CH2), 2.51 (s, 3H, N–CH3), 1.21 (t, 3H, J = 6.2 Hz,
CH2CH3); 13C NMR: d 159.27 (C=NH), 141.21 (C-8a),
138.10 (C-4a), 136.20 (C-8), 57.11 (C-4), 143.16, 135.66,
128.29, 128.18, 127.62, 126.84, 126.14, 125.65 (12Ar–C),
123.64 (C=CH), 52.14 (C-7), 51.18 (C-5), 46.29 (CH2),
45.08 (CH3), 27.84 (SCH3); MS: m/z (%): 411 [M?] (100),
316 (M?–C6H4F) (95); anal. calcd. for C23H23N3SF2
(411.491); C, 67.13; H, 5.63; N, 10.21; S, 7.79. Found: C,
67.11; H, 5.62; N, 10.24; S, 7.81.
3,5-Bisarylmethylene-1-methyl-4-piperidon (1) was syn-
thesized according to the reported procedure (Lyles et al,
1974; Mcelvain and Kurt 1948) and used as the starting
material. It was reacted with phenyl hydrazine in dioxan
and/or acetic acid to afford the corresponding pyrazolo[4,3-
c]pyridine derivative 2. Both IR and 1H NMR spectral data
showed an absence of NH groups, indicating that the for-
mation of the cyclic product 2A is favored over the iso-
meric structure 2B. The 13C NMR of 2a–e revealed signals
at 155.25–155.79 ppm that were assigned to C=N. The
presence of a signal corresponding to C=N in the pyraz-
olopyridine system in compounds 2a–e indicated that
products 2A are favored over their isomeric structure 2B.
Compound 1 was condensed with urea in the presence of
ethanolic potassium hydroxide, guanidine hydrochloride in
the presence of sodium ethoxide, and/or thiourea in the
presence of ethanolic potassium hydroxide under reflux to
afford the corresponding pyrimidine derivatives 3, 4 and 5.
The structures of the pyridopyrimidine derivatives pro-
duced were confirmed by their spectral and analytical data.
The IR spectra of compounds 3a–e showed characteristic
absorption bands for NH, C=O and C=N groups. The
presence of signals corresponding to C-4a and C-8a in the
13C NMR spectra of 3a–e proved formation of the pyri-
dopyrimidine ring system in these compounds. The 1H
NMR spectra of compounds 4 and 5 revealed the presence
of NH signals, signals of methyl protons, as well as aro-
matic proton signals (see ‘‘Materials and methods’’ sec-
tion). The 13C NMR spectra of 5a–e showed signals at
180.16–180.22 ppm corresponding to C=S groups.
8-(4-Chlorobenzylidene)-3,4,5,6,7,8,-hexahydro-6-methyl-
2-(methylthio)-4-(4-chlorophenyl)pyrido[4,3-d]pyrimidine
(6f)
3.7 g (87 % Yield), m.p 132–134 °C; IR (KBr) m cm-1
:
1
1648 (C=N); H NMR (DMSO-d6): d 9.47 (s, 1H, NH),
7.60-7.22 (m, 9H, Ar–H ? C=CH), 4.99 (s, 1H, H-4), 3.71
(s, 2H, CH2), 3.45 (s, 2H, CH2), 2.55 (s, 3H, N–CH3), 2.20
(s, 3H, SCH3); 13C NMR: d 159.04 (C=NH), 141.39
(C-8a), 138.18 (C-4a), 135.73 (C-8), 57.02 (C-4), 142.93,
135.11, 128.04, 127.84, 127.42, 127.05, 126.02, 126.03
(12Ar–C), 123.60 (C=CH), 52.31 (C-7), 51.26 (C-5), 45.19
(CH3), 27.18 (SCH3); MS: m/z (%): 430 [M?] (22),432
[M??2] (15), 434 [M??4] (3.8), due to the presence of
two chlorine atoms, 306 (M?–ClC6H4–CH) (100); anal.
calcd. for C22H21N3SCl2 (430.39); C, 61.39; H, 4.92; N,
9.76; S, 7.45. Found: C, 61.64; H, 4.89; N, 9.83; S, 7.46.
When compounds 5a–e were allowed to react with
methyl iodide and/or ethyl iodide in the presence of alco-
holic sodium hydroxide, sodium ethoxide or acetic acid and
sodium acetate, the corresponding alkylated products 6a–
f were afforded in 80–86 % yields. The 1H NMR spectra of
the resulting S-alkyl derivatives showed signals corre-
sponding to either the alkyl groups as singlets or the ethyl
proton signals as triplet and quartets. The spectra also
showed signals at 9.24–9.72 ppm corresponding to NH
groups which were assigned to position N-3. The absence
of a signal corresponding to C=S in the 13C NMR spectra
of compounds 6a–f indicates that alkylation had taken
place at the sulfur atom, not the nitrogen atom. This is also
Antitumor activity
Some of the synthesized compounds were screened for
their anticancer activity. Each test compound was studied
at five different concentrations against 59 different human
tumor cell lines, representing cancers of lung, leukemia,
brain, colon, breast, prostate, ovary, renal, melanoma as
well as kidney, according to a previously reported proce-
dure (Ally et al. 1988; Grever et al. 1992; Boy and Paull
1995). Measurements of in vitro growth in micro culture
wells by cell-mediated reduction of tetrazolium showed a
good correlation with measurements of cellular protein in
adherent cell line cultures as well as viable cell counts in
suspensions of cell line cultures. The results are expressed
1
in agreement with their H NMR spectra which revealed a
relatively low chemical shift for CH3 or CH2CH3
123

A. M. Mohamed et al.
indicating their attachment to the sulfur atom as S–CH3 or
S–CH2CH3 (Scheme 1).
concentrations against 59 different human tumor cell lines, rep-
resenting cancers of lung, leukemia, brain, colon, breast, prostate,
ovary, renal, melanoma as well as kidney. Some compounds
showed little or no activity while other compounds such as 2b,
2d, 3a, 4b, 5b and 5d exhibited moderate to high activities.
The results are expressed as log 10 GI50, which is the
drug concentration (M) causing a 50 % reduction in the net
Antitumor activity
The synthesized compounds were screened for their anticancer
activity. Each compound was tested at five different
N
Hb
N
NH
N
Hd
Hc
X
Ha
N
X
X
X
N
CH3
CH3
2
B
A
X
2
a
b
c
d
e
H
2-F
OCH3
4-Cl
4-Br
H
Dioxan or
Acetic acid
N NH2
O
N
H
X
H
X
N
O
N
NH
NH
X
X
(NH2)2C=O
(NH2)2C=NH.HCl
EtONa
N
NH
N
H3C
H3C
CH3
E
tO
H
/ K
O
H
1
X
X
X
3
(NH2)2C=S
X
4
a
b
c
d
e
H
F
OCH3
4-Cl
a
b
c
d
e
H
F
2-Cl
4-Cl
3-Br
EtOH / KOH
4-Br
RI
H
X
X
N
S
N
SR
NH
N
NH
N
H3C
H3C
X
X
X
H
4-Br
R
6
X
5
a
CH3
CH3
C2H5
CH3
C2H5
CH3
a
b
c
d
e
H
F
2-Cl
4-Cl
4-Br
b
c
d
e
f
4-Br
4-F
4-F
4-Cl
Scheme 1 Synthesis of fused N-methyl pyridine derivatives
123

Anticancer activities of pyrazole and pyrimidine derivatives
Table 1 Concentrations resulting in 50 % inhibition of (log 10 GI50
of the growth of in vitro human tumor cell lines
)
Table 1 continued
Panel/cell line
Compound
2b 2d
Panel/cell line
Compound
2b 2d
3a
4b
5b
5d
3a
4b
5b
5d
Prostate cancer
PC-3
Non-small cell lung cancer
A 549/ATCC
EKVX
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
-5.3 -5.3 -5.3 -5.3 -5.3 -5.3
-5.3 -5.3 -5.3 -5.3 -5.3 -5.3
-5.3 -5.3 -5.3 -5.3 -5.3 -5.3
-5.3 -5.3 -5.3 -5.3 -5.3 -5.3
-5.3 -5.3 -5.3 -5.3 -5.3 -5.3
-5.3 -5.3 -5.3 -5.3 -5.3 -5.3
-5.3 -5.3 -5.3 -5.3 -5.3 -5.3
-5.3 -5.3 -5.3 -5.3 -5.3 -5.3
-5.3 -5.3 -5.3 -5.3 -5.3 -5.3
DU-145
Renal cancer
786-0
HOP-62
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
HOP-92
A 498
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
ACHN
CAKI-1
RXF-393
SN12C
TK-10
UO-31
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
Melanoma
LOXIMVI
MALME-3 M
M 14
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
MoLT-4
RPMI-8226
SR
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
CNS cancer
SF-268
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
SF-295
SNB-19
SNB-75
protein increase in control cells during the drug incubation.
The results are presented in Table 1.
U 251
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
From the in vitro data it was noticed that compounds 2b,
2d, 3a, 4b, 5b and 5d were the most active derivatives
against all tested cell lines. An inspection of these data
shows that the majority of the compounds tested exhibit
activity against lung cancer at low concentration compa-
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
-4.0 -4.3 -4.3 -4.3 -4.3 -4.3
HT29
rable with that of 5-fluorodeoxyuridine (log 10 GI50
=
KM12
-4.7), which was used as the reference compound (Bhatt
et al. 1994) (see Table 1). The results obtained clearly
showed differences in activity between compounds with
different substituents on the phenyl ring, indicating the
effect of substituents on the resulting activity.
SW-620
Breast cancer
MCF 7
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
NCI/ADR-RES
MDA-MB-231/ATCC -4.3 -4.3 -4.3 -4.3 -4.3 -4.3
From the results outlined in Table 1 and structure
activity correlations it was concluded that pyrazole and
pyrimidine moieties fused to nitrogen-methylpiperidone
ring systems result in increased antitumor activities. It is
also suggested that the presence of fluorine and chlorine in
the para-position especially in the pyrazolopyrimidine ring
system results in enhancement of anticancer activity.
Substitution in the para-position with a fluorine atom in the
aromatic ring attached to pyrazolopyrimidine, 2-imino-
pyridopyrimidine and pyridopyrimidine-2-thione systems
is essential for enhanced activity. We also conclude that
substitution on the pyridopyrimidine-2-thione ring system
HS 578T
MDA-MB-435
BT-549
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
-4.3 -4.3 -4.3 -4.3 -4.3 -4.3
T-47D
Ovarian cancer
IGROVI
-4.3 -4.0 -4.0 -4.0 -4.3 -4.3
-4.3 -4.0 -4.0 -4.0 -4.3 -4.3
-4.3 -4.0 -4.0 -4.0 -4.3 -4.3
-4.3 -4.0 -4.0 -4.0 -4.3 -4.3
-4.3 -4.0 -4.0 -4.0 -4.3 -4.3
-4.3 -4.0 -4.0 -4.0 -4.3 -4.3
OVCAR-3
OCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
123

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123