Synthesis, anticancer activity and radiosensitizing evaluation of some new 2-pyridone derivatives

Article abstract of DOI:10.1055/s-0031-1299695

Based on the reported anticancer activity of 2-pyridone, a new series of 6-amino-5-cyano-1-(3-ethylphenyl)-2-oxo-4-substituted-1,2-dihydropyridine-3- carbo-nitriles 4a-p were synthesized and tested for in-vitro anticancer activity against Ehrlich Ascites Carcinoma (EAC) cell line and liver human tumor cell line (HEPG2). Radiosensitizing activity was also evaluated. The starting material 2-cyano-N-(3-ethylphenyl)-acetamide 3 was obtained via reaction of 3-ethyl aniline 1 with ethyl cyanoacetate under condition of fusion. Upon treatment of compound 3 with aromatic aldehyde and malononitrile in the presence of catalytic amount of piperidine yielded the corresponding 1,2-dihydropyridine derivative 4a-p. Also chromenes 5 and 6 were obtained in good yield via reaction of compound 3 with salicyladehyde under different condition. The chromene derivatives 5 and 6 were further reacted with malononitrile in NH ₄OAc, afford the corresponding chromenopyridones 7 and 8. The structures of the synthesized compounds 3-8 were confirmed by analytical and spectral data. Compounds 4d, 4e, 5 and 6 showed higher anticancer activity against EAC cell line with IC₅₀ values (75.32, 20.77, 73.1 and 67.05 M) compared to doxorubicin as positive control with IC₅₀ value (68.13 M), moreover, these compounds showed potent activity on HEPG2 cell line with IC₅₀ values (26.5, 19.2, 39.3, 44.9 M), respectively, compared to doxorubicin (CAS 29042-30-6) (38.46 M) and their activity increased synergistically when combined with γ-radiation. Georg Thieme Verlag KG Stuttgart · New York.

Full text of DOI:10.1055/s-0031-1299695

Original Article 149
Synthesis, Anticancer Activity and Radiosensitizing
Evaluation of Some New 2-Pyridone Derivatives
Authors
M. S. El-Said1, M. G. El-Gazzar2, M. S. Al-Dosari3, M. M. Ghorab1
Affiliations
1 Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud University,
Riyadh, Saudi Arabia
2 Department of Drug Radiation Research, National Center for Radiation Research and Technology, Nasr City, Cairo, Egypt
3 Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
Key words
Abstract
▼
obtained in good yield via reaction of compound
3 with salicyladehyde under different condition.
The chromene derivatives 5 and 6 were further
reacted with malononitrile in NH4OAc, afford the
corresponding chromenopyridones 7 and 8. The
structures of the synthesized compounds 3–8
were confirmed by analytical and spectral data.
Compounds 4d, 4e, 5 and 6 showed higher anti-
cancer activity against EAC cell line with IC50 val-
ues (75.32, 20.77, 73.1 and 67.05μM) compared
to doxorubicin as positive control with IC50 value
(68.13μM), moreover, these compounds showed
potent activity on HEPG2 cell line with IC50
values (26.5, 19.2, 39.3, 44.9μM), respectively,
compared to doxorubicin (CAS 29042-30-6)
(38.46μM) and their activity increased synergis-
tically when combined with γ-radiation.
▶
● synthesis
▶
● 2-pyridone
Based on the reported anticancer activity of
2-pyridone, a new series of 6-amino-5-cyano-
1-(3-ethylphenyl)-2-oxo-4-substituted-1,2-
dihydropyridine-3-carbo-nitriles 4a-p were
synthesized and tested for in-vitro anticancer
activity against Ehrlich Ascites Carcinoma (EAC)
cell line and liver human tumor cell line (HEPG2).
Radiosensitizing activity was also evaluated. The
starting material 2-cyano-N-(3-ethylphenyl)-
acetamide 3 was obtained via reaction of 3-ethyl
aniline 1 with ethyl cyanoacetate under condi-
tion of fusion. Upon treatment of compound 3
with aromatic aldehyde and malononitrile in
the presence of catalytic amount of piperidine
yielded the corresponding 1,2-dihydropyridine
derivative 4a-p. Also chromenes 5 and 6 were
▶
● anticancer
▶
● γ-radiation
received 17.11.2011
accepted 07.12.2011
Introduction
▼
vatives 4a-p, also, benzochromenes 5, 6 and
chromenopyridine derivatives 7, 8 were synthe-
sized to evaluate their in-vitro anticancer and
Bibliography
Many naturally occurring and synthetic com-
pounds containing the 2-pyridone scaffold pos-
sess oncogenic properties [1]. A series of novel
4,6-diaryl-2-oxo-1,2-dihydropyridine-3-carbon-
itriles (e.g. compounds I, II) were identified as
inhibitors of the oncogenic serine/threonine
kinase PIM-1, which plays a role in cancer sur-
vival, differentiation and proliferation, PIM-1
kinase has been shown to be overexpressed in a
variety of cancer cell lines [2–5]. Furthermore,
milrinone III and amrinone IV are 5-pyridyl-2-
oxopyridine derivatives used for the treatment of
congestive heart failure, their mechanism of
action involve PDE3 inhibition, leading to high
levels of CAMP and consequent inotropic effect,
recent studies showed that PDE3, PDE4, PDE5 are
overexpressed in cancerous cells compared with
normal cells [6–10]. Based on the above informa-
tions and as a continuation of previous work on
anticancer agents [11–16], we report the syn-
thesis of novel 2-oxo-3,5-dicyanopyridine deri-
DOI http://dx.doi.org/
10.1055/s-0031-1299695
Published online:
January 23, 2012
Arzneimittelforschung 2012;
62: 149–156
© Georg Thieme Verlag KG
Stuttgart · New York
ISSN 0004-4172
▶
radiosensitizing activity (● Fig. 1) .
Experimental
▼
Chemistry
2- Cyano-N-(3-ethylphenyl) acetamide (3)
A mixture of 3-ethylaniline 1 (1.21g, 0.01mol)
and ethyl cyanoacetate (1.13g, 0.01mol) was
fused at 220 °C for 3h. The reaction mixture was
concentrated and cooled. The obtained product
was crystallized from ethanol to give 3.
Yield, 88%, m.p. 86–88°C; IR (KBr, cm–1): 3317
(NH), 3100 (CH. arom.), 2960, 2870 (CH aliph.),
2260 (C≡N), 1670 (C=O). 1HNMR spectrum of 3 in
(DMSO-d6) δ : 1.2[t, 3H, CH3], 2.6[q, 2H, CH2], 3.6[s,
2H, CH2], 4.2[s, 1H, NH, exchangeable with D2O],
7.03–7.7[m, 4H, Ar-H]. Anal. Calcd. For C11H12N2O:
C, 70.21; H, 6.38, 14.89. Found: C, 70.50; H, 6.10; N,
14.60.
Correspondence
M. M. Ghorab
Medicinal, Aromatic and
Poisonous Plants Research
Center (MAPPRC)
College of Pharmacy
King Saud University
P.O.box 2457
Riyadh 11451
Saudi Arabia
Tel.:+966/053/4292 860
Fax: +966/01/4670 560
mmsghorab@yahoo.com
El-Said MS et al. Synthesis, Anticancer Activity and… Arzneimittelforschung 2012; 62: 149–156

150 Original Article
Fig. 1 Synthetic compounds containing the
2-pyridone scaffold (I–IV) and structures of the
target compounds (4 a-p, 7, 8).
N
CN
O
CN
O
CN
OH
Br
N
H
N
H
N
H
O
III
II
Milrinone (WIN 47203)
I
NH2
NC
N
R
NC
CN
N
NH2
O
H2N
N
O
O
X
N
O
H
7, X= O
8, X= NH
IV
Amrinone (WIN 40680)
4 a-p
6-Amino-5-cyano-1-(3-ethylphenyl)-2-oxo-4-
CH2], 8.3[s, 2H, NH2, exchangeable with D2O], 7.2–7.7[m, 8H,
Ar-H]. Anal. Calcd. for : C21H15N5O3: C, 65.54; H, 3.89; N, 18.18.
Found: C, 65.20; H, 3.50; N, 18.00.
substitutedaryl 1,2-dihydropyridine-3-carbonitriles (4a-p)
General procedure: A mixture of compound 3 (1.88g, 0.01mol),
appropriate aldehyde (0.0l mol) and malononitrile (0.66g,
0.01mol) in ethanol (50ml) containing a catalytic amount of
piperidine was refluxed for 4h the obtained solid was recrystal-
lized from dioxane to give 4a-p, respectively.
6-Amino-5-cyano-1-(3-ethylphenyl)-2-oxo-4-(4-
nitrophenyl)-1,2-dihydropyridine-3-carbonitrile (4e)
Yield, 78%; m.p. 140–142°C; IR (KBr, cm−1): 3227, 3209 (NH2),
3082 (CH arom.), 2964, 2931, 2872 (CH aliph.), 2195 (C≡N),
1680 (C=O). 1H-NMR in (DMSO-d6) δ: 1.2[t, 3H, CH3], 2.6[q, 2H,
CH2], 3.6[s, 3H, OCH3], 7.03–7.7[m, 8H, Ar-H], 8.6[s, 3H, NH2,
exchangeable with D2O]. Anal. Calcd. for : C21H15N5O3: C, 65.45;
H, 3.89; N, 18.18 . Found: C, 65.70; H, 4.20; N, 18.50.
6-Amino-5-cyano-1-(3-ethylphenyl)-2-oxo-4-phenyl-1,2-
dihydropyridine-3-carbonitriles (4a)
Yield, 71%; m.p. 248–250 °C; IR (KBr, cm−1): 3309, 3207 (NH2),
3059 (CH arom.), 2966, 2931, 2875 (CH aliph.), 2214 (C≡N),
1676 (C=O). 1H-NMR in (DMSO-d6) δ: 1.2[t, 3H, CH3], 2.6[q, 2H,
CH2], 7.03–7.7[m, 9H, Ar-H], 8.2[s, 2H, NH2, exchangeable with
D2O]. Anal. Calcd. For C21H16N4O: C, 74.11; H, 4.70; N, 16.47.
Found: C, 74.40; H, 4.30; N, 16.10.
6-Amino-5-cyano-1-(3-ethylphenyl)-2-oxo-4-
(benzo[d][1,3]dioxol-5-yl)-1,2-dihydropyridine-3-
carbonitrile (4f)
Yield, 81%; m.p.>300 °C; IR (KBr, cm−1): 3309, 3201 (NH2),
3080 (CH arom.), 2910, 2840 (CH aliph.) 2210 (C≡N), 1662
(C=O). 1H-NMR (DMSO-d6) δ: 1.1[t, 3H, CH3], 2.6[q, 2H, CH2],
6.2[s, 3H, CH2 dioxol], 7.03–7.7[m, 7H, Ar-H], 8.2[s, 2H, NH2,
exchangeable with D2O]. Anal. Calcd. for : C22H16N4O3: C, 68.75;
H, 4.16; N, 14.58. Found: C, 68.50; H, 4.40; N, 14.20.
6-Amino-5-cyano-1-(3-ethylphenyl)-2-oxo4-(2-
methoxyphenyl)-1,2-dihydropyridine-3-carbonitrile (4b)
Yield, 84%; m.p. 278–280°C’ IR (KBr, cm−1); 3259, 3182 (NH2)
3066 (CH arom.), 2968, 2937, 2841 (CH aliph.), 2212 (C≡N),
1672 (C=O). 1H-NMR in (DMSO-d6) δ: 1.4[t, 3H, CH3], 2.6[q, 2H,
CH2], 3.7[s, 3H, OCH3], 7.03–7.7[m, 8H, Ar-H], 8.2[s, 2H, NH2,
exchangeable with D2O]. Anal. Calcd. For C22H18N4O2: C, 71.35;
H, 4.86; N, 15.13. Found: C, 71.50; H, 4.60; N, 15.40.
6-Amino-5-cyano-1-(3-ethylphenyl)-2-oxo-4-(3-ethoxy-4-
methoxyphenyl)-1,2-dihydropyridine-3-carbonitrile (4g)
Yield, 64%; m.p. 244–246 °C, IR (KBr) 3325, 3219 (NH2), 3084
(CH arom.), 2931, 2837 (CH aliph.) 2214 (C≡N), 1670 (C=O). 1H-
NMR (DMSO-d6) δ: 1.3[m, 6H, 2CH3], 2.6[q, 2H, CH2], 3.4[s, 3H,
OCH3], 4.6[q, 2H, CH2], 6.5–7.7[m, 7H, Ar-H], 8.3[s, 2H, NH2,
exchangeable with D2O]. Anal. Calcd. for C24H22N4O3: C, 69.56;
H, 5.31; N, 13.52. Found: C, 69.20; H, 5.60; N, 13.80.
6-Amino-5-cyano-1-(3-ethylphenyl)-2-oxo-4-(4-methoxy-
phenyl)-1,2-dihydro-pyridine-3-carbonitrile (4c)
Yield, 89%; m.p.> 300°C; IR (KBr, cm−1): 3309, 3207 (NH2),
3088 (cH arom.), 2966, 2839 (CH aliph.), 2214 (C≡N), 1676
(C=O). 1H-NMR in (DMSO-d6) δ: 1.2[t, 3H, CH3], 2.6[q, 2H, CH2],
3.6[s, 3H, OCH3], 8.6[s, 3H, NH2, exchangeable with D2O], 7.03–
7.7[m, 8H, Ar-H]. Anal. Calcd. for C22H18N4O2: C, 71.35; H, 4.86;
N, 15.13. Found: C, 71.00; H, 5.10; N, 14.80.
6-Amino-5-cyano-1-(3-ethylphenyl)-2-oxo-4-(2,4-
dichlorophenyl)-1,2-dihydropyridine-3-carbonitrile (4h)
Yield, 84%; m.p.>300; IR (KBr, cm−1): 3373, 3311 (NH2), 3100
(CH arom., 2966, 2935, 2862 (CH aliph.), 2212 (C≡N), 1683
(C=O). 1H-NMR (DMSO-d6) δ: 1.2[t, 3H, CH3], 2.6[q, 2H, CH2],
7.1–7.7[m, 7H, Ar-H], 8.2[s, 2H, NH2, exchangeable with D2O].
Anal. Calcd. for C21H14Cl2NO2O : C, 61.62; H, 2.42; N, 13.69.
Found: C, 61.30; H, 2.10; N, 13.90.
6-Amino-5-cyano-1-(3-ethylphenyl)-2-oxo-4-(3-
nitrophenyl)-1,2,-dihydropyridine-3-carbonitrile (4d)
Yield, 80%; m.p. 145–147°C; IR (KBr, cm−1): 3227, 3209 (NH2),
3082 (CH arom.), 2964, 2931, 2872 (CH aliph.), 2194 (C≡N),
1680 (C=O). 1H-NMR in (DMSO-d6) δ: 1.2[t, 3H, CH3], 2.6[q, 2H,
El-Said MS et al. Synthesis, Anticancer Activity and… Arzneimittelforschung 2012; 62: 149–156

Original Article 151
6-Amino-5-cyano-1-(3-ethylphenyl)(-2-oxo-4-(3-
bromophenyl)-1,2-dihydropyridine-3-carbonitrile (4i)
Yield, 62%; m.p. 96–98°C; IR (KBr, cm−1): 3334, 3246 (NH2),
3086 (CH arom.), 2981, 2829 (CH aliph.), 2212 (C≡N), 1649
(C=O). 1H-NMR (DMSO-d6) δ: 1.2[t, 3H, CH3], 2.6[q, 2H, CH2],
7.1–7.9[m, 8H, Ar-H], 8.6[s, 2H, NH2, exchangeable with D2O].
Anal. Calcd. for C21H15BrN4O : C, 60.14; H, 3.57; N, 13.36. Found:
C, 60.50; H, 3.20; N, 13.70.
CH3 furyl], 2.6[q, 2H, CH2], 6.9–7.8[m, 6H, Ar-H], 8.2[s, 2H, NH2,
exchangeable with D2O]. Anal. Calcd. for C20H16N4O2 : C, 69.75;
H, 4.65; N, 16.27. Found: C, 69.40; 4.90; N, 16.50.
6-Amino-5-cyano-1-(3-ethylphenyl)-2-oxo-4-(2-thienyl)-
1,2-dihydropyridine-3-carbonitrile (4p)
Yield, 71%; m.p. 105–107 °C; IR (KBr, cm−1): 3325, 3209 (NH2),
3100 (CH arom.), 2931, 2872 (CH aliph.), 2210 (C≡N), 1658
(C=O), 1H-NMR (DMSO-d6) δ: 1.2[t, 3H, CH3], 2.6[q, 2H, CH2],
7.2–8.4[m, 7H, Ar-H], 8.7[s, 2H, NH2, exchangeable with D2O].
Anal. Calcd. for C19H14N4OS : C, 65.89; H, 4.04; N, 16.18. Found:
C, 65.50; H, 3.70; N, 16.40.
6-Amino-5-cyano-1-(3-ethylphenyl)-2-oxo-4-(4-amino-
dimethylphenyl)-1,2-dihydropyridine-3-carbonitrile (4j)
Yield, 76%; m.p. 260–262°C; IR (KBr, cm−1): 3350, 3305 (NH2),
3088 (CH arom.), 2962, 2925, 2872 (CH aliph.), 2200 (C≡N),
1672 (C=O). 1H-NMR (DMSO-d6) δ: 1.2[t, 3H, CH3], 2.3[s, 6H,
N(CH3)2], 2.6[q, 2H, CH2], 6.9–7.2[m, 6H, Ar-H], 8.3[s, 2H, NH2,
exchangeable with D2O]. Anal. Calcd. for C23H21N5O : C, 72.06; H,
5.48; N, 18.27. Found: C, 72.30; H, 5.10; N, 18.50.
3-[N-(3-Ethylphenyl)-carbaxamido[-Benzochromene-2-
one(5)
To a solution of compound 3 (1.88g, 0.01mol) in acetic anhy-
dride (20ml), 2-hydroxy-1-naphthaldehyde (1.56g, 0.01mol)
and fused sodium acetate (0.8g, 0.01mo) was added. The reac-
tion mixture was refluxed for 2h, cooled and the solid obtained
was crystallized from ethanol to give 5.
6-Amino-5-cyano-1-(3-ethylphenyl)-2-oxo-4-(styryl-N-
dimethyl-4)-1,2-dihydropyridine-3-carbonitrile (4k)
Yield, 59%; m.p. 190–192°C; IR (KBr, cm−1): 3350, 3246 (NH2),
2900, 2819 (CH aliph.) 2220(C≡N), 1647 (C=O). 1H-NMR
(DMSO-d6) δ: 1.3[t, 3H, CH3], 2.6[q, 2H, CH2], 3.2[s, 6H, 2CH3],
6.9[2d, 2H, CH=CH, J=7.3, 7.2Hz], 7.1–7.7[m, 8H, Ar-H], 8.2[s,
2H, NH2, exchangeable with D2O]. Anal. Calcd. for C25H23N5O : C,
73.34; H, 5.62; N, 17.11. Found: C, 73.60; H, 5.30; N, 17.40.
Yield, 69%; m.p. 117–119 °C; IR (KBr, cm−1): 3417(NH), 1766
1
(2C=O), HNMR (DMSO-d6) δ: 1.3(t, 3H, CH3) 2.6 (q, 2H, CH2),
7.3–8.0 (m, 10H, Ar-H), 8.35 (s, 1H, CH), 8.42 (s, 1H, NH). Anal.
Calcd. for C22H17NO3 : C, 76.96; H, 4.95; N, 4.08. Found: C, 76.60;
H, 4.30; N, 4.30.
3-[N-(3-ethylphenyl)-carboxamido]-2-imino-2H-
benzochromene (6)
A mixture of 3 (1.88g, 0.01mol), 2-hydroxy-1-naphthaldehyde
(1.56g, 0.01mol) and anhydrous ammonium acetate (1.15g,
0.15mol) in ethanol (20ml) was refluxed for 2h. The solid
obtained was recrystallized from ethanol to give 6.
Yield, 59%; m.p. >300°C; IR (KBr, cm−1): 3344, 3166 (2NH),
1720 (C=O), 1570 (C=N) 1H-NMR (DMSO-d6) δ: 1.1 (t, 3H, CH3)
2.7 (q, 2H, CH2), 6.9–8.3 (m, 10H, Ar-H), 8.7 (s, 1H, CH), 9.5 (s, 1H,
NH imino), 12.5 (s, 1H, NH CO). Anal. Calcd. for C22H18N2O2 : C,
77.19; H, 5.26, 8.18. Found: C, 77.40; H, 5.50; N, 8.50.
6-Amino-5-cyano-1-(3-ethylphenyl)-2-oxo-4-(2-hydroxy-1-
naphthalene)-1,2-dihydropyridine-3-carbonitrile (4l)
Yield, 71%; m.p. >300°C; IR (KBr, cm−1): 3446 (OH), 3334, 3236
(NH2), 3057) (CH arom.), 2940, 2836 (CH aliph.) 2210(C≡N),
1656 (C=O). 1H-NMR (DMSO-d6) δ: 1.2[t, 3H, CH3], 2.6[q, 2H,
CH2], 6.2[s, 1H, OH, exchangeable with D2O], 7.1–7.9[m, 10H,
Ar-H], 8.2[s, 2H, NH2, exchangeable with D2O]. Anal. Calcd. for
C25H18N4O2 : C, 73.89; H, 4.43; N, 13.79 Found: C, 73.50; H, 4.10;
N, 13.40.
6-Amino-5-cyano-1-(3-ethylphenyl)-2-oxo-4-(2-methoxy-1-
naphthalene)-1,2-dihydropyridine-3-carbonitrile (4m)
Yield, 67%; m.p. >300°C; IR (KBr, cm−1): 3304, 3199 (NH2),
3055(CH arom.), 2964, 2935, 2845 (CH aliph.), 2210(C≡N),
1662 (C=O). 1H-NMR (DMSO-d6) δ: 1.2[t, 3H, CH3], 2.6[q, 2H,
CH2], 3.7[s, 3H, OCH3], 6.2[s, 1H, OH, exchangeable with D2O],
7.1–7.9[m, 10H, Ar-H], 8.2[s, 2H, NH2, exchangeable with D2O].
Anal. Calcd. for C26H20N4O2 : C, 74.28; H, 4.76; N, 13.33. Found:
C, 74.50; H, 4.40; N, 13.70.
2.1.21. 2-Amino-3-(3-ethylphenyl)-4,5-dioxo-4,5-
dihydro 3H-Chromeno[3,4-c]pyridine-1-carbonitrile (7).
2-Amino-3-(3-ethylphenyl)-5-imino-4-oxo-4,5-dihydro-3H-
benzochromeno (3,4-c) pyridine-1- carbonitrile (8)
Equimolar amounts of compounds 5 or 6, malononitrile (0.66g,
0.01mol) and anhydrous ammonium acetate (1.115g, 0.01mol)
in ethanol (50ml) were refluxed for 4h. The solid obtained by
filtration was recrystallized from dioxane to give 7 and 8, respec-
tively.
6-Amino-5-cyano-1-(3-ethylphenyl)-2-oxo-4-(4-methoxy-1-
naphthalene)-1,2-dihydropyridine-3-carbonitrile (4n)
Yield, 81%; m.p. 240–242°C; IR (KBr, cm−1): 3387, 3317 (NH2),
3070 (CH arom.), 2935, 2839 (CH aliph.), 2208 (C≡N), 1654
(C=O). 1H-NMR (DMSO-d6) δ: 1.4[t, 3H, CH3], 2.6[q, 2H, CH2],
3.8[s, 3H, OCH3], 6.1[s, 1H, OH, exchangeable with D2O], 7.1–
7.9[m, 10H, Ar-H], 8.5[s, 2H, NH2, exchangeable with D2O]. Anal.
Calcd. for C26H20N4O2 : C, 74.28; H, 4.76; N, 13.33. Found: C,
73.90; H, 5.10; N, 13.00.
7: Yield, 61%, m.p.> 300°C; IR (KBr, cm−1): 3336, 3232 (NH2)
1
2200 (C≡N), 1684, 1654 (2C=O). H-NMR (DMSO-d6) δ: 1.1 (t,
3H, CH3) 2.7 (q, 2H, CH2), 6.9–8.3 (m, 10H, Ar-H), 8.5 [s, 2H, NH2,
exchangeable with D2O]. Anal. Calcd. for C25H17N3O3: C, 73.71;
H, 4.17; N, 10.31 Found: C, 73.40; H, 4.50; N, 10.60.
8: Yield, 58%, m.p.>300°C; IR (KBr, cm−1): 3360, 3331, 3216
(NH, NH2) 2925, 2853 (CH aliph.) 2217 (C≡N), 1684, (C=O). 1H-
NMR (DMSO-d6) δ: 1.1 (t, 3H, CH3) 2.7 (q, 2H, CH2), 6.9–8.3 (m,
10H, Ar-H), 8.5 [s, 2H, NH2, exchangeable with D2O], 8.7 (s, 1H,
NH imino). Anal. Calcd. for C25H18N4O2: C, 73.89; H, 4.43; N,
13.79 Found: C, 73.44; H, 4.12; N, 13.40.
6-Amino-5-cyano-1-(3-ethylphenyl)-2-oxo-4(5-methyl-2-
furyl)-1,2-dihydropyridine-3-carbonitrile (4o)
Yield, 69%; m.p. 108–110°C; IR (KBr, cm−1): 3339, 3209 (NH2),
3034 (CH arom.), 2964, 2931, 2872 (CH aliph.), 2208 (C≡N),
1660 (C=O). 1H-NMR (DMSO-d6) δ: 1.2[t, 3H, CH3], 2.3[s, 3H,
El-Said MS et al. Synthesis, Anticancer Activity and… Arzneimittelforschung 2012; 62: 149–156

152 Original Article
Table 1 In-vitro anticancer screening of the newly synthesized compounds against EAC cells.
Cpd. No.
Non-viable cells (%)
IC50a (μg/ml)
IC50a (μM)
Concentration (μg/ml)
100
50
25
10
3
100
100
100
100
100
100
90
50
55
60
60
100
100
50
40
70
50
30
30
50
0
25
35
20
20
60
80
25
20
45
25
0
10
5
50
48
265.95
141.17
132.43
121.62
75.32
20.77
129.53
132.85
100.24
119.33
156.65
163.81
140.44
238.1
238.1
290.6
–
4a
4b
4c
4d
4e
4f
0
49
10
30
60
10
5
45
29
8
50
4g
4h
4i
100
100
100
100
85
55
20
10
0
41
50
4j
60
4k
4l
0
0
67
100
50
25
0
10
0
50
4m
4n
4o
4p
5
100
100
100
>100*
25
50
0
0
0
50
20
10
70
85
20
10
68
0
0
40
0
0
95
50
60
10
0
10
25
5
73.1
6
100
50
23
67.05
245.7
237.74
68.13
7
100
97
8
60
0
Dox.
100
30
24
37
a IC50 value: corresponds to the compound concentration causing 50% mortality in net cells
*Compounds with IC50 >100 lg/mL are considered to be inactive
In-vitro anticancer screening
Total cells=cells/ml×the original volume of fluid from which the
Animals, chemicals and facilities
cell sample was removed
Ehrlich Ascites Carcinoma (EAC) cells were maintained in female
Swiss albino mice weighing 25–30g (the holding company for
biological products and vaccines, VACSERA, Cairo, Egypt) were
housed at a constant temperature (24 °C) with alternating 12-h
light and dark cycles and fed standard laboratory food (Milad
%
cell non-viability=total non-viable cells (stained)/total
cells×100
The results of in-vitro anticancer activity experiments are pre-
sented in● Table 1.
▶
CO., Cairo, Egypt) and water ad libitum. All chemicals and rea- In-vitro anticancer activity using liver human tumor cell
gents were of the highest grade commercially available. Facili- lines (HEPG2)
ties including animal house, biochemical equipments have been
made available by the National Center for Radiation Research
and Technology (NCRRT), Atomic Energy Authority (AEA), Cairo,
Egypt. Animal care and handling was done according to the
guidelines set by the world health organization, Geneva, Swit-
zerland and approved from the committee for animals care at
NCRRT.
The human tumor cell line (HEPG2) was available at the National
Cancer Institute, Cairo, Egypt. Irradiation was performed in the
National Cancer Institute, Cairo, Egypt using Gamma cell-40
(
60CO) source. The anticancer activity of the newly synthesized
compounds was measured using the Sulfo-Rhodamine-B stain
(SRB) assay by the method of Skehan et al. [19] (1990). Cells
were plated in 96-multiwell plate (104 cells/well) for 24h before
treatment with the compounds to allow attachment of cell to
the wall of the plate. Tested compounds were dissolved in DMSO
and diluted with saline to the appropriate volume. Different
concentrations of the compounds under test (5, 12.5, 25 and
50μM) were added to the cell monolayer. Triplicate wells were
prepared for each individual dose. Monolayer cells were incu-
bated with the compounds for 48h at 37°C and in atmosphere of
5% CO2. After 48h, cells were fixed, washed and stained for
30min with 0.4% (wt/vol) with SRB dissolved in 1% acetic acid.
Unbounded dye was removed by 4 washes with 1% acetic acid,
and attached stain was recovered with Tris-EDTA buffer. Color
intensity was measured in an ELISA reader. The relation between
surviving fraction and drug concentration was plotted to get the
survival curve of each tumor cell line after the specified time.
The concentration required for 50% inhibition of cell viability
(IC50) was calculated and compared with the reference drug dox-
In-vitro anticancer activity using EACs
EAC cells were obtained by needle aspiration of ascetic fluid
from preinoculated mice; under aseptic conditions. Tumor cells
suspension (2.5 _106 per ml) was prepared in RPMI-1640 media.
Tested compounds were prepared with various dilutions by dis-
solving: 100, 50, 25 & 10mg of the tested compounds in DMSO
(1ml). In a set of sterile test tubes 0.8ml RPMI-1640 media con-
taining (glutamine, fetal calf serum as nutrient, streptomycin
and penicillin), 0.1ml of each of the tested compounds (corre-
sponding to 100, 50, 25, 10mg) were mixed then 0.1ml of tumor
cell suspension (2×106) was added. The test tubes were incu-
bated at 37 °C for 2h. Trypan blue exclusion test was carried out
to calculate the percentage of non-viable cells after 2h of incu-
bation [17–18]. The total number of cells/ml will be determined
using the following calculations:
Cells/ml=average cells count per 5 squares×dilution factor×104
orubicin and the results are given in● Table 2.
▶
El-Said MS et al. Synthesis, Anticancer Activity and… Arzneimittelforschung 2012; 62: 149–156

Original Article 153
Table 2 In-vitro anticancer evalu-
ation of compounds 4d, 4e, 5 and
6 against HEPG2.
Cpd. No.
Compound concentration (μM)
IC 50 (μM)
5 (μM)
12.5 (μM)
25 (μM)
50 (μM)
Surviving fraction (mean±SE) a
Dox.
4d
4e
5
0.921±0.020
0.756±0.002
0.426±0.018
0.921±0.012
0.922±0.064
0.846±0.020
0.761±0.010
0.412±0.028
0.362±0.022
0.621 ±0.047
0.630±0.016
0.494±0.030
0.213±0.047
0.271±0.012
0.373±0.022
0.446±0.012
38.46
26.5
19.2
39.3
44.9
0.634±0.009
0.394±0.031
0.792±0.041
0.746±0.014
6
a Each value is the mean of 3 experiments±standard error
Table 3 In-vitro anticancer evalu-
ation of compounds 4d, 4e, 5 and
6 against HEPG2 after radiation.
Cpd. No.
Compound concentration (μM) + irradiation (8Gy)
IC 50 (μM)
Surviving fraction (mean±SE) a
5
12.5
25
50
Dox.
4d
4e
5
0.87±0.0012
0.512±0.01
0.340±0.07
0.471±0.01
0.564±0.04
0.65±0.047
0.478±0.02
0.290±0.01
0.371±0.01
0.321±0.01
0.38±0.085
0.39±0.05
0.270±0.01
0.245±0.01
0.218±0.01
0.063±0.111
0.10±0.06
20.2
18.4
12
0.240±0.08
0.140±0.07
0.100±0.06
14.2
15.14
6
a Each value is the mean of 3 experiments±standard error
2.3 Radiosensitizing evaluation
7 and 8 was elucidated from elemental analyses and spectral
data.
The most potent compounds resulted from the in vitro antican-
cer screening; compounds 4d, 4e, 5 and 6, were selected to be
evaluated again for their in-vitro anticancer activity in combina-
tion with γ-radiation and compared to the reference drug doxo-
rubicin. This study was conducted to evaluate the ability of these
compounds to enhance the cell killing effect of γ-radiation. Cells
were subjected to a single dose of γ-radiation at a dose level of 8
Gy with a dose rate of 2 Gy/min. Irradiation was performed in
the National Cancer Institute, Cairo University, using Gamma
cell-40 (60CO) source. The surviving fractions were expressed as
In-vitro anticancer activity
Doxorubicin, the reference drug used in this study is one of the
most effective antitumor agents used to produce regressions in
acute leukemias, Hodgkin’s disease, and other lymphomas. The
relationship between survival fraction and drug concentration
was plotted to obtain the survival curve of EAC cells and
HEPG2. The response parameter calculated was the IC50 value
▶
(● Tables 1, 2), which corresponds to the compound concentra-
▶
means ± standard error. The results are given in● Table 3.
tion causing 50% mortality in net cells.
In-vitro anticancer activity using EAC cell line
Results and Discussion
▼
The cytotoxicity of 21 compounds was examined on EAC cells. It
▶
is clear from the results in ● Table 1, that the most potent com-
Chemistry
pound in this study was compound 4e (IC50 =20.77μM) which
was found to be more potent than the reference drug (doxoru-
bicin) (IC50 =68.13μM) this may be attributed to the presence of
4-nitrophenyl substitution on 2-pyridone ring which may give
an idea about the possible importance of nitro group to enhance
activity, especially, because the 3-nitrophenyl derivative 4d
showed also significant activity (IC50 =75.32μM). Moreover, the
chromene derivatives 5, 6 showed significant activity (IC50 =73.1
and 67.05μM) which is nearly as potent as the reference drug
and also they found to be more active than the starting material
3 (IC50 =265.95μM), while, their cyclization to the correspond-
ing chromenopyridine 7, 8 resulted in a drop in their activity
(IC50 =245.7 and 237.74μM).
▶
● Figs. 2, 3 outline, the synthestic pathway used to obtain
1,2-dihydropyridines, 4a-p, chromenes 5,6, and chromenopyrid-
ines 7, 8. The starting material, 3-cyano-N-(3-ethylphenyl)
acetamide 3 was prepared via reaction of 3-ethylaniline 1 with
ethyl cyanoacetate 2. Compound 3 was confirmed by elemental
analysis, IR, 1H-NMR, and mass spectral data. Upon treatment of
compound 3 with required aldehyde and malononitrile in the
presence of catalytic amount of piperidine furnished 6-amino-
5-cyano-1-(3-ethylphenyl)-2-oxo-4-substituted-aryl-
▶
1,2-dihydropyridine-3-carbonitriles 4a-p (● Fig. 2). The
structure of compounds 4a-p was deduced from elemental anal-
yses and spectral data.
Furthermore, Perkin reaction was carried out by reaction of
2-hydroxy-1-naphthaldehyde in the presence of sodium acetate
to give the corresponding chromene-2-one derivative 5, while
conducting the same reaction in the presence of ammonium
acetate in ethanol furnished 2-iminochromene 6. The structure
of compounds 5 and 6 was supported in the basis of elemental
analyses and spectral data. The chromene derivatives 5 and 6
were further reacted with malononitrile in the presence of
ammonium acetate to give the corresponding chromenopyrid-
In-vitro anticancer activity using HEPG2
This study was performed to evaluate the anticancer activity of
the most potent 4 compounds resulted from EAC assay (com-
pounds 4d, 4e, 5, 6). We can conclude from the results obtained
▶
from (● Table 2) that indeed these compounds showed potent
activity on HEPG2 cell line. The most potent was compound
4e (IC50 =19.2μM) which is more potent than doxorubicin
(IC50 =38.46μM), while compounds 4d, 5, 6 showed lesser activ-
ity (IC50 =26.5, 39.3, 44.9μM) which is nearly as active as doxo-
rubicin as positive control.
▶
ines 7 and 8, respectively (● Fig. 3). The structure of compounds
El-Said MS et al. Synthesis, Anticancer Activity and… Arzneimittelforschung 2012; 62: 149–156

154 Original Article
Fig. 2 Synthetic pathways for compounds 4a–p.
R
O
NC
H 2
CN
O
CN
NH 2
HN
3
O
Fusion
N
N
RCHO
NC
+
O
2
CN
CN
1
4 a-p
4a, R= C6H5
4i, R= C6H4 Br-3
4b. R= C6H 4 OCH3 -2
4c, R= C6H4 OCH3-4
4d, R= C6H4 NO2-3
4e, R= C6H 4 NO2-4
4j, R= C6H11 N(CH3 )-4
4k, R= CH=CHC 6H 4 N(CH 3
4l, R= C10 H6 OH-2
)
2 -4
4m, R= C10 H6 OCH3 -2
4n, R= C10 H6 OCH3 -4
4o, R= 5-methyl-2-furyl
4p, R= 2-thienyl
4f, R= (benzo[d][1,3]dioxol-5-yl
4g, R= C6H 3 OC 2H5 -3, OCH3-4
4h, R= C6H 3 Cl 2 -2, 4
Fig. 3 Synthetic pathways for compounds 5–8.
CHO
OH
CHO
O
OH
O
O
N
H
NH
N
H
3
Ac2O / NaOAc
EtOH / NH4OAc
O
6
O
5
EtOH
EtOH
NH4OAc
NH4OAc
NH2
N
CN
CN
CN
CN
NC
O
O
X
7, 8
7, X= O
8, X= NH
effective if chemotherapy is sufficiently active to eradicate sub-
clinical metastases and if the primary local tumor is effectively
treated by radiotherapy. In this regard, no interaction between
radiotherapy and chemotherapy is required. The other idea is
the enhancement of radiation effects. Cytotoxic agents can
enhance radiation effects by direct enhancement of the initial
radiation damage by incorporating drugs into DNA, inhibiting
cellular repair, accumulating cells in a radiosensitive phase or
eliminating radioresistant phase cells, eliminating hypoxic cells
or inhibiting the accelerated repopulation of tumor cells [20].
Consequently, the ability of the 4 most active compounds,
compounds 4d, 4e, 5 and 6, to enhance the cell killing effect of
γ-irradiation was studied. From the results obtained in
1.2
1
y = –0.0145x + 0.8712
y = –0.0142x + 0.7581
0.8
0.6
0.4
0.2
0
▶
● Table 2, compound 4d showed an in-vitro cytotoxic activity
with IC50 value of 26.4μM, when the cells were subjected to dif-
ferent concentrations of the compound alone. While, when the
cells were subjected to the same concentrations of compound
4d, and irradiated with a single dose of γ-radiation at a dose
0
20
40
60
Concentration (uM)
▶
level of 8Gy, as shown in● Table 3, the IC50 value was synergis-
▶
tically decreased to 18.4μM (● Fig. 4). Similarly, compounds 4e,
Cpd.4d
Linear (Cpd.4d)
Linear (Cpd.4d+8Gy)
5, 6 showed IC50 values of 19.2, 39.3 and 44.9μM, respectively,
Cpd.4d+8Gy
▶
when used alone, as shown in ● Table 2. The IC50 value was
decreased to 12, 14.2 and 15.14μM, respectively, when the cells
were treated with compounds 4e, 5, 6 in combination with
Fig. 4 Survival curve for HEPG2 cell line for compound 4d alone and in
combination with γ-irradiation (8Gy), the dotted lines represent the best
fitting lines for each curve, and their equations are shown.
▶
γ-radiation (● Fig. 5, 6, 7). From these results, we can conclude
that the combination of compounds 4d, 4e, 5 or 6 and ionizing
radiation synergistically enhanced growth inhibition on liver
cancer cells, compared with each agent alone and these com-
pound showed better radiosensitizing activity then doxorubicin
(20.2μM). For better comparison, the change in IC50 before and
Radiosensitizing evaluation
The rationale for combining chemotherapy and radiotherapy is
based mainly on 2 ideas, one being spatial cooperation, which is
El-Said MS et al. Synthesis, Anticancer Activity and… Arzneimittelforschung 2012; 62: 149–156

Original Article 155
1.2
1.2
1
y = –0.0099x + 0.673
y = –0.0097x + 0.608
y = –0.0108x + 0.9492
y = –0.0147x + 0.7129
1
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
0
20
40
60
0
20
40
60
Concentration (uM)
–0.2
Concentration (uM)
Linear (Cpd.6)
Linear (Cpd.6+8Gy)
Linear (Cpd.4e)
Linear (Cpd.4e+8Gy)
Cpd.4e
Cpd.4e+8Gy
Cpd.6
Cpd.6+8Gy
Fig. 5 Survival curve for HEPG2 cell line for compound 4e alone and in
combination with γ-irradiation (8Gy), the dotted lines represent the best
fitting lines for each curve, and their equations are shown.
Fig. 6 Survival curve for HEPG2 cell line for compound 6 alone and in
combination with γ-irradiation (8Gy), the dotted lines represent the best
fitting lines for each curve, and their equations are shown.
1.2
y = –0.0125x + 0.9722
y = –0.0132x + 0.6899
1
44.9
0.8
0.6
0.4
0.2
0
45
39.3
40
35
26.5
30
25
20
15
10
5
19.2
18.4
15.14
14.2
12
0
20
Concentration (uM)
Linear (Cpd.5)
Linear (CPd.5+8GY)
40
60
0
4d
4e
5
6
Compound no.
Cpd.5
CPd.5+8GY
before rad. after rad
Fig. 8 Change in IC50 before and after irradiation on HEPG2 cell line for
compounds 4d, 4e, 5 and 6.
Fig. 7 Survival curve for HEPG2 cell line for compound 5 alone and in
combination with γ-irradiation (8Gy), the dotted lines represent the best
fitting lines for each curve, and their equations are shown.
anticancer activity on EAC, the most potent compounds were
compounds 4d, 4e, 5, 6 which also showed promising activity
HEPG2. While, combining these compounds with radiation at
the same concentrations enhanced their activity which demon-
strates the importance of the combination therapy for the
patients with cancer to decrease the side effects of both drug
and radiation.
after irradiation on HEPG2 cell line for compounds 4d, 4e, 5 or
▶
6 is plotted in a histogram (● Fig. 8) .
Conclusion
▼
We report here the synthesis of new 2-pyridone derivatives. It
was clearly observed from the results of in-vitro anticancer
screening that the synthesized compounds exhibited significant
El-Said MS et al. Synthesis, Anticancer Activity and… Arzneimittelforschung 2012; 62: 149–156

156 Original Article
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Acknowledgement
▼
The authors are grateful to the sponsorship of the Research
Center, College of Pharmacy and the Deanship of Scientific
Research, King Saud University, Riyadh, Saudi Arabia.
Conflict of Interest
▼
The authors declare that they have no conflict of interest with
respect to this paper.
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