Synthesis and anticancer activity of some new pyridine derivatives

Article abstract of DOI:10.1007/s11164-011-0413-9

Different substituents were introduced in positions 2 and 6 of 2,6 diaminopyridine in order to obtain new heterocyclic compounds. A new series of aza pyridine, imidazopyridine, benzodiazepine, indole, pyrimidine, and benzimidazole heterocyclic derivatives were synthesized in good yields. The anticancer activities of some of the new compounds were evaluated against liver cancer cell line HEPG2. Compounds 3, 4, 10, 11, 12, and 17 showed the highest activity when compared to 5-flurouracil (5-FU) and doxorubicin (DOX) chemotherapy. Springer Science+Business Media B.V. 2011.

Full text of DOI:10.1007/s11164-011-0413-9

Res Chem Intermed (2012) 38:1291–1310
DOI 10.1007/s11164-011-0413-9
Synthesis and anticancer activity of some new pyridine
derivatives
•
Fatma A. Bassyouni Hanaa A. Tawfik
•
•
Abdel Mohsen Soliman Mohamed Abdel Rehim
Received: 16 August 2011 / Accepted: 22 September 2011 / Published online: 26 October 2011
Ó Springer Science+Business Media B.V. 2011
Abstract Different substituents were introduced in positions 2 and 6 of 2,6
diaminopyridine in order to obtain new heterocyclic compounds. A new series of
aza pyridine, imidazopyridine, benzodiazepine, indole, pyrimidine, and benzimid-
azole heterocyclic derivatives were synthesized in good yields. The anticancer
activities of some of the new compounds were evaluated against liver cancer cell
line HEPG2. Compounds 3, 4, 10, 11, 12, and 17 showed the highest activity when
compared to 5-flurouracil (5-FU) and doxorubicin (DOX) chemotherapy.
Keywords Pyridine derivatives ꢀ Aza pyridine derivatives ꢀ
Pyrimidine derivatives ꢀ Benzodiazepine derivatives ꢀ Anticancer activity
Introduction
The pyridine is among the most common heterocyclic compounds found in
various therapeutic agents. The structure of 2,6-diaminopyridine (DAP) has the
F. A. Bassyouni (&)
Departments of Chemistry of Natural and Microbial Products and Pharmaceutical Research,
Center of Excellence for Advanced Sciences, National Research Centre, Dokki, Cairo 12622, Egypt
e-mail: fatma.nrc@hotmail.com
H. A. Tawfik
Department of Chemistry of Natural and Microbial Products, National Research Centre, Dokki,
Cairo 12622, Egypt
A. M. Soliman
Department of Therapeutic Chemistry, National Research Centre, Dokki, Cairo 12622, Egypt
M. A. Rehim
Department of Clinical Pharmacology & DMPK, AstraZeneca, R&D, Sodertalje, 15185 Sodertalje,
Sweden
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symmetrical feature that possesses three nitrogen lone pairs. N-acyl pyridine
derivatives have also attracted considerable attention due to their hydrogen bonding
motifs [1–3]. Poly functional pyridines are highly reactive reagents that have been
used extensively in heterocyclic synthesis [4–7] and that possess biological as well
as pharmacological activity [8–10]. In addition, triazolopyridines are also interest-
ing compounds due to their pronounced biological activity, as they can be used as
antidepressants [11–13]. Various pyridine derivatives are well known to possess an
array of physiological activities, such as anticancer, muscle relaxant, hypnotic, anti-
inflammatory, diuretic, and antihypertensive activities [14–17]. Imidazole deriva-
tives are part of anticancer drugs like mercaptopurine. Imidazole is also a part of the
theophylline molecule, found in tea leaves and coffee beans, which stimulate the
central nervous system [18]. On the other hand, benzimidazole is a very important
pharmacophore in drug discovery, and its derivatives are an important class of
bioactive molecules in the field of drugs and pharmaceuticals [19]. Pyrimidines
have been used as therapeutic agents possessing analgesics and anti-inflammatory
activity [20–22]. Moreover, indoles and their derivatives are known to possess a
wide variety of biological and pharmacological properties, including antibacterial
and antifungal, cytotoxic, antioxidant and insecticidal activity [23, 24].
In this article, synthesis and characterization of some new heterocyclic
compounds such as pyrido pyrimidine, diazepine, indole and imidazole derivatives
and were performed to be evaluated against liver carcinoma cell line (HEPG2).
Experimental
Chemistry
All melting points are uncorrected and were recorded on an open glass capillary
tube using an Electrothermal IA 9100 digital melting point apparatus. Elemental
microanalyses were carried out at Micro analytical Unit, using Vario Elementar and
were found within 0.5% of the theoretical values. Infrared spectra were recorded
on a Jasco FT/IR 300 E Fourier transform infrared spectrophotometer using the KBr
1
disc technique. The spectra of H-NMR and C13-NMR were determined using a
JEOL EX-270 and/or 500NMR spectrophotometers and were run in deuterated
chloroform (CDCl3) or dimethyl sulphoxide (DMSO-d6). Chemical shifts were
related to that of the solvent. The mass spectra were measured with a JEOL mass
spectrometer and LC/MS-Finnigan at Central Services Laboratory, National
Research Center, Cairo, Egypt. Follow-up of the reactions and checking purity of
the compounds were made by thin-layer chromatograph technique on silica gel pre-
coated aluminum sheets (Type 60, F 254, Merck, Darmstadt, Germany) and the
spots were detected by exposure to UV lamp at 254 nm for a few seconds.
Synthesis of 6-amino-3,4-dihydro-2H-pyrido[1,2-a]pyrimidine-2-one (1)
A suspension of 2,6 diaminopyridine (0.1 mol), chloroacetic acid (10 g, 0.1 mol),
anhydrous sodium acetate (1.5 g) in a mixture of acetic anhydride and acetic acid
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Anticancer activity of some new pyridine derivatives
1293
(1:3) was heated under reflux with stirring at 120 °C for 10 h. The reaction mixture
was then poured onto ice-water (50 mL) and set aside in refrigerator at 0 °C. The
solid formed was collected by filtration and recrystallization from benzene–
petroleum ether (60–80 °C).
1
Yield 72%, mp. 140–142 °C. H-NMR (270 MHz, chloroform) d: 3.60 (2H, t,
CH2), 3.90 (2H, t, CH2), 6.50 (2H, br, J = 11.0 Hz, NH2 D2O-exchangeable),
7.20–7.60 (3H, m, Ar’H pyridine), 13C-NMR (270 MHz, chloroform) d: 39.80,
40.20 (CH2), 120.50, 122.70, 131.90 (pyridine carbon), 158.90 (C=N), 160.90
(C=N), 170.90 (C=O). IR (KBr) cm-1: 3350 (NH2), 1720(C=O), 1640–1650 (C=N).
Anal. Calcd. for C8H9N3O: C, 58.88; H, 5.56; N, 25.75. Found: C, 58.80; H, 5.50;
N; 25.70.
Synthesis of 6-amino-2H-pyrido[1,2-a]pyrimidine-2,4(3H)-dione (2)
and 1-aminobenzo[e]pyrido[1,2-a][1,3]diazepine-6,11-dione (2-3): general
procedure
A mixture of 2,6 diaminopyridine (0.02 mol) and (0.02 mol) malonic acid and/or
phthalic acid anhydride in ethanol (25 mL) in presence of few drops of TEA was
refluxed for 8–10 h. Cooling the mixture followed by filtration and recrystallization
from ethanol afforded the target molecules 2 and 3.
6-amino-2H-pyrido[1,2-a]pyrimidine-2,4(3H)-dione (2)
1
Yield 77%, mp. 130–133 °C. H-NMR (270 MHz, chloroform) d: 3.95 (2H, s,
J = 7.0 Hz, CH2), 6.50 (2H, br, J = 11.0 Hz, NH2 D2O-exchangeable), 7.20–7.60
(3H, m, Ar’H pyridine). 13C-NMR (270 MHz, chloroform) d: 40.20 (CH2), 120.50,
122.70, 131.90 (pyridine carbon), 158.90 (C=N), 160.90 (C=N), 168.90 (C=O),
169.90 (C=O). IR (KBr) cm-1: 3350 (NH2), 1720–1740 (C=O), 1640–1650 (C=N).
Anal. Calcd. for C8H8N3O2: C, 53.93; H, 4.53; N, 23.58. Found: C, 53.90; H, 4.55;
N, 23.68, MS: m/z = 178 [M?].
1-aminobenzo[e]pyrido[1,2-a][1,3]diazepine-6,11-dione (3)
Yield 74%, mp. 154–156 °C. 1H-NMR (270 MHz, chloroform) d: 6.50 (2H, br, NH2
D2O-exchangeable), 7.20–7.50 (3H, m, J = 9.5 Hz, Ar’H pyridine), 7.60–7.80 (4H,
m, Ar’H).13C-NMR (270 MHz, chloroform) d: 120.50, 122.70, 131.90 (pyridine
carbon), 124.90, 125.40, 129.90, 130.70, 132.90, 135.60 (aromatic carbon), 158.90
(C=N), 160.50 (C=N), 166.30 (C=O), 168.90 (C=O). IR (KBr) cm-1: 3350 (NH2),
1710–1720 (C=O), 1640–1650 (C=N). Anal. Calcd. for C13H9N3O2: C, 65.27; H,
3.79; N, 17.56. Found: C, 65.20; H, 3.75; N; 17.50.
General procedure for the synthesis of compounds (4–6)
A mixture of compound 1–3 (0.001 mol) and the appropriate aldehyde, namely,
5-methylfuran-2-carbaldehyde (0.0015 mol) in ethanol (20 mL) and glacial acetic
acid (1 mL) was refluxed for 6–8 h. The reaction mixture was poured into ice-cold
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water and the solid product was filtered off, washed with petroleum ether and
recrystallization from methanol to form compounds 4–6, respectively.
-6-((4-methylfuran-2-yl)methyleneamino)-3,4-dihydro-2H-pyrido
[1,2-a]pyrimidin-2-one (4)
1
Yield 75%, mp. 162–164 °C. H-NMR (270 MHz, DMSO) d: 1.90 (3H, s, CH3),
3.60 (2H, t, CH2), 3.90 (2H, t, CH2), 6.65 (1H, d, furan), 6.75 (1H, d, furan),
7.20–7.50 (3H, m, J = 9.5 Hz, Ar’H pyridine), 7.65 (1H, s, J = 12.0 Hz, N=CH).
IR (KBr) cm-1: 1710 (C=O), 1640–1650 (C=N). Anal. Calcd. for C14H13N3O2:
C, 65.87; H, 5.13; N, 16.46. Found: C, 65.80; H, 5.10; N, 16.40, MS:
m/z = 253[M?].
6-((4-methylfuran-2-yl)methyleneamino)-2H-pyrido
[1,2-a]pyrimidine-2,4(3H)-dione (5)
1
Yield 72%, mp. 170–172 °C. H-NMR (270 MHz, DMSO) d: 1.90 (3H, s, CH3),
3.95 (2H, s, CH2), 6.65 (1H, d, furan), 6.75 (1H, d, furan), 7.20–7.50 (3H, m, Ar’H
pyridine), 7.75 (1H, s, J = 12.0 Hz, N=CH), IR (KBr) cm-1: 1710–1720 (C=O),
1640–1650 (C=N). Anal. Calcd. for C14H11N3O3: C, 62.45; H, 4.12; N, 15.61.
Found: C, 62.50; H, 4.18; N, 15.66.
(4a)-1-((4-methylfuran-2-yl)methyleneamino)benzo[e]pyrido[1,2-a]
[1,3]diazepine-6,11-dione (6)
Yield 75%, mp. 180–183 °C. 1H-NMR (270 MHz, DMSO) d: 1.90(3H, s,
J = 5.0 Hz, CH3), 6.65 (1H, d, furan), 6.75 (1H, d, furan), 7.20–7.50 (3H, m,
Ar’H pyridine), 7.60–7.80 (4H, m, Ar’H), 7.90 (1H, s, J = 12.0 Hz, N=CH). IR
(KBr) cm-1: 1710–1720 (C=O), 1640–1650 (C=N). Anal. Calcd. for C19H13N3O3:
C, 68.88; H, 3.95; N, 12.68. Found: C, 68.85; H, 3.89; N, 12.60, MS: m/z =
331[M?].
Synthesis of N2,N6-bis ((1H-indol-3-yl)methylene)pyridine-2,6-diamine (7)
A mixture of starting compound 2,6 diaminopyridine (0.001 mol) and indole
3-carboxaldehyde (0.001 mol) in ethanol and few drops of glacial acetic acid was
refluxed for 8 h. The reaction mixture was poured into ice-cold water and the solid
product was filtered off, washed with petroleum ether, and recrystallization from
ethanol to give compound 7.
Yield 68%, mp. 182–184 °C. 1H-NMR (270 MHz, DMSO) d: 7.20–7.50 (3H, m,
Ar’H pyridine), 7.60–7.80(5H, m, J = 13.00 Hz, Ar’H), 7.90 (1H, s, J = 11.0 Hz,
N=CH), 10.75 (1H, br, NH benzimidazole). IR (KBr) cm-1: 3290 (NH), 1640
(C=N). Anal. Calcd. for C23H17N5: C, 67.01; H, 4.71; N, 19.27. Found: C, 67.08; H,
4.76; N, 19.20. MS: m/z = 253 [M?].
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Anticancer activity of some new pyridine derivatives
1295
Reaction of compound (7) with oxalyl chloride: preparation of -2-(3-((6-((1-(2-
chloro-2-oxoacetyl)-1H-indol-3-yl)methyleneamino)pyridin-2-yl)methyleneamino)-
1H-indol-1-yl)-2-oxoacetyl chloride (8)
A mixture of compound 7 (0.01 m mol) and oxalyl chloride (0.01 mmol) in diethyl
ether (30 mL) with stirring for 12 h. The reaction mixture was poured into ice water
and the solid product was filtered off, washed with petroleum ether, and
recrystallization from ethanol to obtain compound 8.
Yield 65%, mp. 192–195 °C. 1H-NMR (270 MHz, DMSO) d: 7.20–7.50 (3H, m,
Ar’H pyridine), 7.60–7.80 (5H, m, J = 13.00 Hz, Ar’H), 7.90 (1H, s, J = 11.0 Hz,
N=CH). IR (KBr) cm-1: 1640–1660 (C=N), 1680–1695 (C=O), 1250–1260 (C–Cl).
Anal. Calcd. for C29H21N5O4: C, 60.64; H, 3.68; Cl, 12.34; N, 12.19. Found: C,
60.68; H, 3.62; Cl, 12.39; N, 12.15, MS: m/z = 545 [M? ?1].
Synthesis of 2,20-(3,30-(pyridine-2,6-diylbis(aza-1-yl-1-ylidene))bis(methan-1-yl-1-
ylidene)bis(1H-indole-3,1-diyl))bis(1-(piperazin-1-yl)ethane-1,2-dione) (9)
A mixture of the appropriate compound 8 (0.005 mol) and piperazine (0.005 mol)
was dissolved in hot absolute ethanol (20 mL) and anhydrous potassium
carbonate (0.5 g) was added with stirring. The mixture was heated under reflux
with stirring for 8 h. Then, an excess of solvent was evaporated under vacuum,
poured onto ice and then neutralized with diluted HCl. The residue formed was
treated with petroleum ether, collected and recrystallization from ethanol to form
compound 9.
Yield 62%, mp. 206–208 °C. 1H-NMR (270 MHz, DMSO) d: 2.80–3.30 (8H, m,
4CH2 piprazine), 7.20–7.50 (3H, m, Ar’H pyridine), 7.60–7.80 (5H, m, Ar’H), 7.90
(1H, s, J = 11.0 Hz, N=CH), 9.95 (1H, s, NH piprazine). IR (KBr) cm-1: 3225
(NH), 1640–1660 (C=N), 1680–1695 (C=O). Anal. Calcd. for C36H35N9O4: C,
65.74; H, 5.36; N, 19.17. Found: C, 65.70; H, 5.31; N, 19.10.
Synthesis of 3,30-(pyridine-2,6-diyl)bis (aza-1-yl-1-ylidene)) diindolin-2-one (10)
2,6 diaminopyridine (0.02 mol) was added to 2,3 indolinedione (0.02 mol, 2.94 g)
in (50 mL) absolute ethanol containing few drops of glacial acetic acid. The
reaction mixture was heated under reflux for 6 h and then the reaction mixture was
filtered off. Recrystallization of the product from ethanol then drying afforded
product 10.
Yield 78%, mp. 225–227 °C. 1H-NMR (500 MHz, DMSO) d: 7.20–7.50 (3H, m,
Ar’H pyridine), 7.60–7.90 (4H, m, J = 13.0 Hz, Ar’H), 10.19 (1H, s, NH). 13C-
NMR (500 MHz, DMSO) d: 122.3, 127.98, 132.70 (pyridine carbon), 111.80,
118.90, 124.80, 125.70, 133.60, 135.20 (aromatic carbon), 165.77 (C=N), 167.70
(C=N), 170.90 (C=O). IR (KBr) cm-1: 3340 (NH), 1645–1660 (C=N), 1690 (C=O)
Anal. Calcd. for C21H13N5O2: C, 68.66; H, 3.57; N, 19.06. Found: C, 68.62; H, 3.52;
N, 19.10. MS: m/z = 367 [M?].
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Synthesis of -1-(2-hydroperoxyethyl)-2-oxoindolin-3-ylideneamino) pyridin-2-
ylimino)-2-oxoindolin-1-yl) acetic acid derivative (11)
A mixture of potassium hydroxide (0.01 mol) and compound 10 (0.01 mol) was
dissolved in (25 mL) absolute ethanol. Then chloroacetic acid (0.01 mol, 1.22 g)
was added drop wisely with stirring and then the reaction mixture was refluxed for
8 h. After cooling, the solvent was evaporated under vacuum and the formed solid
was recrystallization from ethanol.
Yield 74%, mp. 285–287 °C. 1H-NMR (500 MHz, DMSO) d: 4.20 (2H, s,
J = 7.5 Hz, CH2), 7.20–7.50 (3H, m, Ar’H pyridine), 7.60–7.95 (4H, m,
J = 9.0 Hz, Ar’H), 11.20 (1H, s, OH). 13C-NMR (500 MHz, DMSO) d: 40.50
(CH2), 122.3, 127.98, 132.70 (pyridine carbon), 115.90, 117.30, 124.50, 125.98,
129.47, 131.77 (aromatic carbon), 155.30 (C=N), 163.22 (C=N), 170.90 (C=O),
173.90 (COOH). IR (KBr) cm-1: 3370 (OH), 1645–1660 (C=N), 1690 (C=O), 1750
(COOH). Anal. Calcd. for C25H17N5O6: C, 62.11; H, 3.54; N, 14.49. Found: C,
62.18; H, 3.54; N, 14.55. MS: m/z = 483 [M?].
Synthesis of 3,30-(pyridine-2,6-diyl)bis(aza-1-yl-1-ylidene))bis(2-oxoindoline-1-yl-
3-ylidene)bis(methylene)bis(1H-benzo[d]imidazole-6-carboxylic acid) (12)
and 3,30-(pyridine-2,6-diylbis(aza-1-yl-1-ylidene))bis(1-((3H-imidazo[4,5-
b]pyridin-2-yl)methyl)indolin-2-one) (13) derivatives: general procedure
Compound 11 (0.01 mol) was dissolved in ethanol (10 mL) and was added to 2,3
diaminopyridine and/or o-phenylendiamine-5-carboxylic acid (0.01 mol) in ethanol
(10 mL) in presence of few drops of TEA. The reaction mixture was heated under
reflux with stirring for 10–12 h. The solvent was evaporated under vacuum and then
crystallization from methanol to afford the products 12–13.
3,30-(pyridine-2,6-diyl)bis(aza-1-yl-1-ylidene))bis(2-oxoindoline-1-yl-3-
ylidene)bis(methylene)bis(1H-benzo[d]imidazole-6-carboxylic acid) derivative (12)
Yield 75%, mp. 196–198 °C. 1H-NMR (500 MHz, DMSO) d: 4.20 (2H, s,
J = 7.0 Hz, CH2), 6.90–7.10 (4H, m, J = 12.0 Hz, Ar’H), 7.20–7.50 (4H, m, Ar’H
pyridine), 7.60–7.90 (4H, m, Ar’H), 10.90 (1H, s, NH), 11.20 (1H, s, OH). 13C-
NMR (500 MHz, DMSO) d: 39.96 (CH2), 122.3, 127.98, 132.70 (pyridine carbon),
114.20, 115.90, 116.50, 117.80, 118.50, 119.20, 121.90, 124.50, 125.50, 126.80,
128.60, 131.70 (aromatic carbon), 163.20 (C=N), 165.90 (C=N), 167.22 (C=N),
170.90 (C=O), 174.90 (COOH), IR (KBr) cm-1: 3340 (OH), 1640–1665 (C=N),
1690 (C=O), 1750 (COOH). Anal. Calcd. for C39H25N9O6: C, 65.45; H, 3.52; N,
17.61. Found: C, 65.52; H, 3.46; N, 17.55.
3,30-(pyridine-2,6-diyl)bis(aza-1-yl-1-ylidene))bis(1-((3H-imidazo[4,5-b]pyridin-2-
yl)methyl)indolin-2-one) derivative (13)
1
Yield 78%, mp. 175–177 °C. H-NMR (500 MHz, DMSO) d: 4.23 (2H, s, CH2),
6.90–7.10 (3H, m, Ar’H pyridine), 7.20–7.50 (3H, m,, Ar’H pyridine), 7.60–7.90
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Anticancer activity of some new pyridine derivatives
1297
(3H, m, J = 11.0 Hz, Ar’H), 10.50 (1H, s, NH). 13C-NMR (500 MHz, DMSO) d:
40.90 (CH2), 122.89, 127.80, 132.50 (pyridine carbon), 115.70, 117.90, 123.80,
126.90, 130.90 (pyridine carbon), 115.90, 117.80, 119.90, 124.50, 126.98, 131.91
(aromatic carbon), 158.40 (C=N), 161.90 (C=N), 163.22 (C=N), 165.24 (C=N),
170.90 (C=O). IR (KBr) cm-1: 3350 (NH),1640–1660 (C=N), 1695 (C=O) Anal.
Calcd. for C35H23N11O2: C, 66.77; H, 3.68; N, 24.47. Found: C, 66.70; H, 3.61; N,
24.40.
Synthesis of 1,10-(pyridine-2,6-diyl) bis (3-isopropylthiourea) (14), 1,10-(pyridine-
2,6-diyl) bis (3-butylurea) (15) and N,N0-(pyridine-2,6-diyl)bis (aza-diyl)) bis
(thioxomethylene) dibenzamid (16) derivatives: general procedure
To a solution of 2,6 diaminopyridine (0.01 mol) in ethanol (20 mL) containing
isocyanate or isothiocyanate derivatives; namely, isopropyl isothiocyanate, butylis-
ocyanate, and benzoyl isothiocyanate (0.02 mol) added dropwisely with stirring.
The reaction mixture was heated under reflux for 6–8 h. After cooling, the reaction
mixture washed with water. The formed solids were collected by filtration, dried,
and crystallization from methanol to produce compounds 14–16.
1,10-(pyridine-2,6-diyl) bis (3-isopropylthiourea) derivative (14)
1
Yield 70%, mp. 103–105 °C. H-NMR (500 MHz, DMSO) d:1.20–1.34 (6H, m,
2CH3), 4.55–4.70 (1H, m, CH), 7.60–7.90 (3H, m, Ar’H pyridine), 8.90 (1H, s,
NH),11.50 (1H, s, NH). IR (KBr) cm-1: 3280–3290 (NH), 1640 (C=N), 1260
(C=S). Anal. Calcd. for C13H21N5S2: C, 50.13; H, 6.80; N, 22.48; S, 20.59 Found:
C, 50.20; H, 6.70; N, 20.60; S, 20.55. MS: m/z = 311 [M?].
1,10-(pyridine-2,6-diyl) bis (3-butylurea) derivative (15)
1
Yield 67%, mp. 130–132 °C. H-NMR (500 MHz, DMSO) d: 1.18–1.30 (3H, m,
CH3), 4.25–4.50 (6H, m, 3CH2), 7.20–7.50 (3H, m, Ar’H pyridine), 8.90 (1H, s,
NH), 9.60 (1H, s, NH). IR (KBr) cm-1: 3250–3270 (NH), 1690 (C=O), 1640 (C=N).
Anal. Calcd. for C15H25N5O2: C, 58.61; H, 8.20; N, 22.78. Found: C, 58.70; H, 4.28;
N, 22.86. MS: m/z = 307 [M?].
N,N0-(pyridine-2,6-diyl)bis(aza-diyl))bis(thioxomethylene)dibenzamide
derivative (16)
1
Yield 70%, mp. 120–122 °C. H-NMR (500 MHz, DMSO) d: 6.90–7.50 (5H, m,
Ar’H), 7.60–7.90 (3H, m, Ar’H pyridine), 10.50 (1H, s, NH), 11.40 (1H, s, NH). IR
(KBr) cm-1: 3260–3280 (NH), 1700 (C=O), 1640 (C=N), 1265 (C=S). Anal. Calcd.
for C21H17N5O2S2: C, 57.91; H, 3.93; N, 16.08; S, 14.72. Found: C, 57.82; H, 4.10;
N, 16.02; S, 14.67.
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Synthesis of 3,30-(pyridine-2,6-diyl)bis(dihydropyrimidine-4,6(1H,5H)-dione)
derivatives (17–19): general procedure
A mixture of compounds 14–16 (0.001 mol) and (0.002 mol) of malonic acid in
presence of sodium methoxide (20 mL) added dropwisely and heated under reflux
for 5–6 h. The mixture was cooled, followed by filtration and recrystallization from
methanol afforded the target molecules 17–19.
3,30-(pyridine-2,6-diyl)bis(1-isopropyl-2-thioxodihydropyrimidine-4,6(1H,5H)-
dione) derivative (17)
1
Yield 70%, mp. 156–158 °C. H-NMR (500 MHz, DMSO) d: 1.20–1.40 (6H, m,
2CH3), 4.10 (2H, s, CH2), 4.50–4.70 (1H, m, CH), 7.60–7.90 (3H, m, Ar’H
pyridine). 13C-NMR (500 MHz, DMSO) d: 19.90 (CH3), 29.70 (CH3), 39.20 (CH3),
50.90 (CH), 124.77, 129.66, 135.32 (pyridine carbon), 166.33 (C=N), 168.92
(C=O), 170.40 (C=O), 179.50 (C=S). IR (KBr) cm-1: 1680–1695 (C=O), 1645
(C=N), 1265 (C=S). Anal. Calcd. for C19H21N5O4S2: C, 51.06; H, 4.69; N, 15.65, S,
14.33 Found: C, 51.02; H, 4.74; N, 15.70; S, 14.30. MS: m/z = 447 [M?].
3,30-(pyridine-2,6-diyl)bis(1-butyl-2-dihydropyrimidine-4,6(1H,5H)-dione)
derivative (18)
1
Yield 67%, mp. 178–180 °C. H-NMR (500 MHz, DMSO) d: 1.20–1.30 (3H, m,
CH3), 4.10 (2H, s, CH2), 4.25–4.50 (6H, m, 3CH2), 7.60–7.90 (3H, m, Ar’H
pyridine). 13C-NMR (500 MHz, DMSO) d: 20.90(CH3), 27.90 (CH2), 29.20 (CH2),
30.99 (CH2), 39.70 (CH2), 124.50, 135.74, 137.70 (pyridine carbon), 149.80 (C=O),
165.70 (C=O), 169.70 (C=O). IR (KBr) cm-1: 1680–1698 (C=O), 1645 (C=N).
Anal. Calcd. for C21H25N5O6: C, 56.88; H, 5.64; N, 15.80. Found: C, 56.80; H, 5.60;
N, 15.86. MS: m/z = 443 [M?].
3,30-(pyridine-2,6-diyl)bis(1-benzoyl-2-thioxodihydropyrimidine-4,6(1H,5H)-dione)
derivative (19)
1
Yield 72%, mp. 146–148 °C. H-NMR (500 MHz, DMSO) d: 4.20 (2H, s, J = 5.0
CH2), 6.90–7.50 (5H, m, J = 10.2, Ar’H), 7.60–7.90 (3H, m, Ar’H pyridine). IR (KBr)
cm-1: 1680–1690 (C=O), 1645 (C=N), 1265 (C=S). Anal. Calcd. for C27H17N5O6S2:
C, 56.74; H, 3.00; N, 12.25; S, 11.22. Found: C, 56.70; H, 3.10; N, 12.28; S, 11.26.
Results and discussion
In the present work, we report on the synthesis and preliminary biological activity
screening of several heterocycles compounds based on 2,6 diaminopyridine (DAP)
could be considered as a starting material was reacted with chloroacetic acid as well
as malonic acid and phthalic acid in refluxing ethanol containing TEA afforded
a single product identified as 6-amino-3,4-dihydro-2H-pyrido[1,2-a]pyrimidin-2-
123

Anticancer activity of some new pyridine derivatives
1299
one (1), 6-amino-2H-pyrido[1,2-a]pyrimidine-2,4(3H)-dione (2) and 1-aminobenzo
[e]pyrido[1,2-a][1,3]diazepine-6,11-dione (3), respectively (Scheme 1). The struc-
tures of compounds 1–3 were identified by elemental analyses and spectroscopic
1
data. The structure of compounds 1, 2, and 3 were confirmed by their IR, H-NMR
and 13C-NMR spectra. The IR spectra of compounds 1–3 appearance of bands at
3,350 cm-1 attributed to NH2 stretching frequency are good evidence for the
structure given to those compounds, 1710–1740 cm-1 due to the presence of two
C=O groups for compound 2 and 3 in addition to C=N groups at 1640–1650 cm-1
All the synthesized compounds 1–3 were confirmed by thin-layer chromatograph.
.
On the other hand, compounds 1–3 were reacted similarly with 5-methylfuran-2-
carbaldehyde in refluxing ethanol containing a few drops of acetic acid to yield
the corresponding 6-((4-methylfuran-2-yl)methyleneamino)-3,4-dihydro-2H-pyrido
[1,2-a]pyrimidin-2-one (4), 6-((4-methylfuran-2-yl)methyleneamino)-2H-pyrido
[1,2-a]pyrimidine-2,4(3H)-dione (5) and -1-((4-methylfuran-2-yl)methyleneami-
no)benzo[e]pyrido[1,2-a][1,3]diazepine-6,11-dione (6), respectively (Scheme 1).
The identities of the isolated products were assigned by their elemental analysis
and spectral data. The 1H-NMR analysis of compounds (4–6) showed characteristic
N
H2N
NH2
i
ii
iii
O
O
O
N
H2
N
1
H2N
N
N
N
3
O
O
H2N
N
N
2
O
N
N
N
H
3
C
CH=N
O
4
O
O
iv
N
H3C
CH=N
1,2,3
O
5
O
O
H3C
CH=N
N
O
N
6
Scheme 1 Synthesis of 2H-pyrido[1,2-a]pyrimidine derivatives 4–5 and pyrido[1,2-a][1,3]diazepine
derivative 6 from 2,6 diaminopyridine (DAP). Reagents and conditions: (i) chloro acetyl chloride/
anhydrous sodium acetate/reflux, (ii) malonic acid/ethanol/TEA/reflux, (iii) phthalic acid/ethanol/TEA/
reflux, (iv) 5-methylfuran-2-carboxaldehyde/ethanol/acetic acid/reflux
123

1300
F. A. Bassyouni et al.
resonance at d 7.20–7.50 ppm due to the presence of (Ar’H pyridine) function
and 7.65–7.90 ppm due to the presence of (N=CH) group of compounds 4–6 and
1.90 ppm for (CH3) group of compounds 4–6, respectively. All synthesized
compounds were confirmed by thin-layer chromatography.
In addition, condensation reaction of 2,6 diaminopyridine with indole 3-carbox-
aldehyde afforded the corresponding of N2,N6-bis ((1H-indol-3-yl)methylene)
pyridine-2,6-diamine (7), which was reacted with oxalyl chloride to give -2-(3-
((6-((1-(2-chloro-2-oxoacetyl)-1H-indol-3-yl)methyleneamino)pyridin-2-yl)methy-
leneamino)-1H-indol-1-yl)-2-oxoacetyl chloride (8). The condensation reaction of
compound 8 with piperazine led to the formation of 2,20-(3,30-(pyridine-2,6-
diylbis(aza-1-yl-1-ylidene))bis(methan-1-yl-1-ylidene)bis(1H-indole-3,1-diyl))bis
(1-(piperazin-1-yl)ethane-1,2-dione) (9) (Scheme 2). All the synthesized com-
pounds 7–9 were characterized by their physical, chemical, and spectral data. Thus,
the IR spectra of compound 7 shows the presence of characteristic absorption peaks
1
around 3,290 cm-1 (NH stretching), C=N at 1,640 cm-1 and H-NMR spectra of
compound 7 showed broad band’s at 10.75 ppm that revealed the presence of NH
absorption peak in addition to MS at m/z = 253 [M?].
In a similar manner, treatment of compound 2,6 diaminopyridine with 2,3
indolinedione upon refluxing in ethanol and few drops of acetic acid gave the
corresponding Schiff’s base 3,30-(pyridine-2,6-diylbis(aza-1-yl-1-ylidene))diindolin-
2-one (10), which reacted with chloroacetic acid yielded the corresponding
H
N
H
N
i
N
N=CH
N
7
H2N
NH2
HC=N
ii
Cl
N
Cl
NH
N
NH
N
N
O
O
O
O
O
O
O
O
N
iii
N
N=CH
N
N=CH
HC=N
HC=N
N
9
8
Scheme 2 Synthesis of 2,20-(3,30-(pyridine-2,6-diylbis(aza-1-yl-1-ylidene))bis(methan-1-yl-1-ylidene)
bis(1H-indole-3,1-diyl))bis(1-(piperazin-1-yl)ethane-1,2-dione) 9 from 2,6 DAP. Reagents and con-
ditions: (i) indole 3-carboxaldehyde/ethanol/glacial acetic acid/reflux, (ii) oxally chloride/diethylether/rt,
(iii) piprazine/ethanol/anhydrous potassium carbonate/reflux
123

Anticancer activity of some new pyridine derivatives
1301
1-(-2-hydroperoxyethyl)-2-oxoindolin-3ylideneamino)pyridin-2-ylimino)-2-oxo-
indolin-1-yl) acetic acid derivative (11), which continue to react with diamines,
namely, o-phenylendiamine-5-carboxylic acid and 2,3 diaminopyridine yielded the
corresponding 3,30-(pyridine-2,6-diyl) bis (aza-1-yl-1-ylidene))bis(2-oxoindoline-1-
yl-3-ylidene)bis(methylene)bis(1H-benzo[d] imidazole-6-carboxylic acid) derivative
(12) and 3,30-(pyridine-2,6-diylbis(aza-1-yl-1-ylidene)) bis (1-((3H-imidazo [4,5-b]
pyridin-2-yl) methyl) indolin-2-one) derivative (13), respectively (Scheme 3). The 1H
-NMR spectra of compounds 10 showed broad band at 10.19 ppm revealed the
presence of the NH peak. The structure of compound 10 was further supported by its
MS spectra, indicating the molecular ion peak was at m/z 367. The structures of
compounds 10, 11, 12, and 13 were confirmed on the basis of elemental analyses as
well as spectral data. The synthesized compounds were confirmed by thin-layer
chromatography.
Moreover, 2,6-diaaminopyridine, which upon treatment with appropriate isocy-
anate and/or isothiocyanate derivatives, namely, isopropyl isothiocyanate, butyl
H
H
N
N
O
i
O
O
+
N
N
H
O
H2N
NH2
N
N
N
10
ii
CH2COOH
CH2COOH
N
N
O
O
N
N
N
11
iii
H
N
H
N
N
HOOC
H
N
COOH
N
H
N
N
N
N
H2C
N
N
CH2
N
H2C
CH2
N
N
O
O
O
O
N
N
N
N
N
N
13
12
Scheme 3 Synthesis of 3,30-(pyridine-2,6-diyl)bis(aza-1-yl-1-ylidene))bis(2-oxoindoline-1-yl-3-ylidene)
bis(methylene)bis(1H-benzo[d]imidazole-6-carboxylic acid) 12 and 3,30-(pyridine-2,6-diylbis(aza-1-yl-1-
ylidene))bis(1-((3H-imidazo[4,5-b]pyridin-2-yl)methyl)indolin-2-one) 13 from 2,6 diaminopyridine
(DAP). Reagents and conditions: (i) 2,3 indolinedione/ethanol/acetic acid/reflux, (ii) chloroacetic acid/
ethanol/reflux, (iii) 2,3 diaminopyridine and/or o-phenylendiamine-5-carboxylic acid/ethanol/TEA/reflux
123

1302
F. A. Bassyouni et al.
isocyanate and benzoyl isothiocyanate, afforded the corresponding urea and
thiourea derivatives 14–16, respectively, which was followed by cyclization when
treated with malonic acid in presence of sodium methoxide to gave the
corresponding pyrimidine derivatives; 3,30-(pyridine-2,6-diyl)bis(1-isopropyl-2-
thioxodihydropyrimidine-4,6(1H,5H)-dione) (17), 3,30-(pyridine-2,6-diyl)bis
(1-butyl-2-dihydropyrimidine-4,6(1H,5H)-dione) (18) and 3,30-(pyridine-2,6-diyl)-
bis(1-benzoyl-2-thioxodihydropyrimidine-4,6(1H,5H)-dione) (19) derivatives. The
spectral data of 14–19 are in agreement with the assigned structures (Scheme 4). All
the synthesized compounds 14–19 were confirmed by thin-layer chromatography.
N
NH2
H2N
i
iii
ii
R3HNSCHN
NHCONHR2
NHCSNHR3
R1HNSCHN
NHCSNHR1
N
N
16
14
R2HNOCHN
N
2
iv
iv
15
O
O
O
O
O
O
O
NR3
O
R1N
N
N
R3N
S
NR1
N
N
N
N
iv
S
S
S
17
19
O
O
O
O
R2N
N
N
NR2
N
O
O
R1= CH (CH3)2
18
R2= (CH2)3 CH3
R3= CO C6H5
Scheme 4 Synthesis of 3,30-(pyridine-2,6-diyl)bis(1-isopropyl-2-thioxodihydropyrimidine-4,6(1H,5H)-
dione) derivative 17, 3,30-(pyridine-2,6-diyl)bis(1-butyl-2-dihydropyrimidine-4,6(1H,5H)-dione) deriv-
ative 18 and 3,30-(pyridine-2,6-diyl)bis(1-benzoyl-2-thioxodihydropyrimidine-4,6(1H,5H)-dione) derivative
19 from 2,6 diaminopyridine. Reagents and conditions: (i) (CH3)2CHNCS/ethanol/reflux, (ii) CH3
(CH2)3NCO/ethanol/reflux, (iii) C6H5CONCS/ethanol/reflux, (iv) HCOOCH2COOH/sodium methoxide/
reflux
123

Anticancer activity of some new pyridine derivatives
1303
Pharmacology
Antitumor activity
Cytotoxic and biological effects of tested compounds against liver cancer cell line
Compounds 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 17, and 18 were soluble in DMSO at
concentrations high enough to allow cell experiments; the in vitro biological activity
of these compounds was evaluated by their growth-inhibitory potency in liver
HEPG2 cancer cell lines. The cytotoxic potency of compounds 3, 4, 5, 6, 7, 8, 10,
11, 12, 13, 17, and 18 were studied in comparison to the known anticancer drugs
5-flurouracil (5-FU) and doxorubicin (DOX). Moreover, the biochemical effects of
the tested compounds on some enzymes such as aspartate and alanine aminotrans-
ferases (AST and ALT) and alkaline phosphates (ALP), in addition to albumin,
globulins, creatinine, total lipids, cholesterol, triglycerides, and bilirubin in serum of
mice were investigated.
Measurement of potential cytotoxicity by SRB assay
Compounds (3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 17, and 18) were evaluated for their
antitumor activity against liver HEPG2 cancer cell lines in comparison to the known
anticancer drugs: 5-FU and DOX. Potential cytotoxicity of the tested derivatives
was tested according the method of Skehan et al. [25] as follows:
Cells were plated in 96-multiwell plates (104 cells/well) for 24 h before treatment
with the tested compounds to allow attachment of cells to the wall of the plate.
Different concentrations of the compound under test (0, 1, 2.5, 5, 10 lg/mL) were
added to the cell monolayer. Triplicate wells were prepared for each individual
dose. Monolayer cells were incubated with the compounds for 48 h at 37 °C and in
an atmosphere of 5% CO2. Cultures were then fixed with trichloroacetic acid and
stained for 30 min with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1%
acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and
protein-bound dye was extracted with 10 mM un-buffered Tris base [tris
(hydroxymethyl) amino methane] for determination of optical density in a
computer-interfaced, 96-well micro titer plate reader. The SRB assay results were
linear with the number of cells and with values for cellular protein measured by
both the Lowry and Bradford assays modified by Zor and Selinger [26] at den-
sities ranging from sparse sub-confluence to multilayered supra-confluence. The
signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well.
The relation between surviving fraction and drug concentration is plotted to get the
survival curve of both cancer cell lines after the specified compounds.
Biochemical analysis
Animals Male albino mice weighing 18–20 g were used in the present study. Mice
were divided into three main groups as follows:
123

1304
F. A. Bassyouni et al.
1- Group (1): untreated or control group (five mice).
2- Group (2): divided into two subgroups (five mice for each subgroup) and
treated with 5-FU or DOX as reference anticancer drugs.
3- Group (3): divided into eight subgroups (five mice for each subgroup) and
treated with the tested compounds.
4- Treatment.
Group (1): each mouse was given a single intraperitoneal injection of 0.1 mL
DMSO.
Group (2): each mouse was given a single intraperitoneal injection of 0.1 mL
containing 12 mg/kg body weight 5-FU or DOX dissolved in sterile water.
Group (3): each mouse was given a single intraperitoneal injection of 0.1 mL
containing 12 mg/kg body weight of the tested compounds (3, 4, 5, 6, 7, 8, 10, 11,
12, 13, 17 and 18, respectively) dissolved in DMSO. Blood was collected after
7 days from all mice groups.
The biochemical effects of the tested compounds, on some liver enzymes such as
aspartate and alanine, aminotransferases (AST and ALT) [27] and alkaline
phosphatase (ALP) [28], were done using blood auto analyzer (Olympus AV 400,
Japan). Moreover, albumin [29], globulins [30] and creatinine [31], total lipids [32],
cholesterol [33], triglycerides [34] and bilirubin [35] in serum of mice were
evaluated in comparison to 5-FU and DOX.
Statistical analysis of the results was performed using Chi-square values (SPSS
computer program).
Preliminary screening of the tested compounds showed that all compounds
exhibited a moderate to strong growth inhibition activity on the tested cell line
between 1 and 10 lg/mL concentrations in comparison to the known anticancer
drugs: 5-flurouracil and doxorubicin. Table 1 indicated the cytotoxic activity of the
tested compounds (3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 17, and 18) against liver HEPG2
cancer cell lines in comparison to the traditional anticancer drugs: 5-FU and DOX.
It can be deduced from the results that compounds (3, 4, 10, 11, 12 and 17) were the
most active and induced a reasonable growth inhibition, in a dose-dependent
manner against HEPG2 when compared to 5-FU and DOX (IC50 equals 2.08, 2.89,
3.02, 3.09, 3.15, and 3.36 lg/mL, while 5-FU and DOX were 5 and 3.56 lg/mL).
Effect of antitumor compounds on the biochemical parameters
Data obtained in Table 2 represented the liver enzymatic activities (ALT, AST,
and ALP) in serum of control and treated groups of mice. The results showed that
the values recorded for AST and ALT were significantly higher (p \ 0.001) with
5-Fu and DOX treated groups of mice than the control. On the other hand,
treatment of compounds, caused inverse effects, where some values recorded for
AST and ALT were non-significant (n.s.) or slightly higher (p \ 0.01) in
comparison to control. Moreover, the recorded data showed that ALP activities
were significantly increased (p \ 0.001) with the treatment of 5-Fu and DOX,
while there were no significant changes in ALP activities upon treatment with
some of the new compounds.
123

Anticancer activity of some new pyridine derivatives
1305
Data listed in Table 3 demonstrated the comparison between the levels of total
lipids, cholesterol, triglycerides, and bilirubin in serum of treated mice and the
control group. It can be deduced from the present data that 5-FU and DOX caused a
significant increase in the level of these parameters while treatment with the
selected compounds showed moderate or no significant changes.
Table 4 represented a comparison between the levels of albumin, globulins, and
creatinine in serum of control and treated groups of mice. It is clear from the results
in the table that there was a slight increase in the level of albumin and creatinine and
globulins in the 5-FU and DOX treated groups of mice while there were moderate or
non-significant changes in the other treated groups.
Cytotoxic drugs are being administered with novel ways of therapy such as
inhibitors of signals [36]. It is therefore important to discover novel cytotoxic agents
with spectra of activity and toxicity that differ from those current agents. The
antitumor activities of compounds were assessed against HEPG2 cancer cell line in
comparison to the traditional anticancer drugs: 5-Fu and DOX. Regarding the
antitumor activity study, some of the compounds showed reasonable antitumor
activity in comparison to 5-FU and DOX. Moreover, study of the induced
biochemical parameters of the tested compounds in mice showed insignificant
differences relative to the control group, which indicates a moderate margin of
safety for the tested compounds. Comparable to 5-FU and DOX, a dose
augmentation of compounds 11 and 4 may be higher potency. Furthermore, the
tested compounds have important potential advantages over 5-FU and DOX because
of their lower toxicity and their ability to induce lower biochemical parameters.
These results are in agreement with Espinosa et al. [37] and Kamalakannan and
Venkappayya [38].
On the basis of monitoring the inhibition of the growth of human cancer cells, a
series of synthesized compounds possessing a broader spectrum of antitumor
activity and fewer toxic side-effects than traditional anticancer drugs have been
Table 1 Effect of some tested
Compound
IC50 (lg/mL)
newly synthesized compounds
on liver carcinoma cell line
(HEPG2)
5-Flurouracil
5
Doxorubicin
3.56
3.36
2.89
4.1
3
4
5
6
7.11
3.92
3.62
3.02
2.08
3.15
3.56
3.09
5.23
7
8
10
11
12
13
17
18
IC50 dose of the compounds
which reduces survival to 50%
123

1306
F. A. Bassyouni et al.
Table 2 Biochemical effects of treatment with 5-FU, DOX, and the benzofuran derivatives on serum
ALT, AST, and ALP in mice
Mice groups
Biochemical parameters
Alanine amino transferase
mean SD ALT (IU/mL)
Aspartate amino transferase
mean SD AST (IU/mL)
Alkaline phosphatase
mean SD ALP
(k.k./dL)
Control
43.5 2.03
51.47 9.02
0.001
108.32 4.19
130.431 8.92
0.001
18.70 1.10
25.485 ? 6.03
0.001
5-FU
p\
Doxorubicin
59.26 12.03
0.001
147.226 16.34
0.001
30.317 5.14
0.001
p\
3
80.7 19.09
0.001
162.17 34.5
0.001
38.58 12.61
0.001
p\
4
38.9 8.9
n.s.
123.9 11.4
0.01
18.83 6.29
n.s.
p\
5
39.56 6.7
n.s.
112.54 12.7
0.01
19.94 4.35
n.s.
p\
6
46.21 4.17
n.s.
107.81 4.25
n.s.
21.94 3.4
0.01
p\
7
53.7 10.08
0.001
142.3 29.7
0.001
45.42 10.41
0.001
p\
8
81.34 27.3
0.001
151.52 45.6
0.001
43.7 8.36
0.001
p\
10
p\
11
p\
12
p\
13
p\
15
p\
17
p\
33.42 7.05
n.s.
111.2 11.05
n.s.
18.32 3.07
n.s.
60.5 9.7
0.001
156.22 20.1
0.001
44.26 7.01
0.001
68.34 11.9
0.001
146.4 28.1
0.001
36.9 9.8
0.001
50.81 12.01
0.01
119 9.56
0.01
22.07 3.42
0.01
73.09 14.2
0.001
140.09 31.01
0.001
30.41 9.22
0.001
46.09 6.13
n.s.
110.06 8.91
n.s.
18.76 3.02
n.s.
Data are expressed as mean ? SD
n.s. non significant
p [ 0.05 insignificant, p \ 0.01: significant, p \ 0.001: highly significant
studied. Twenty tested compounds (3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 17, and 18) were
subjected to a screening system for investigation of their antitumor potency against
liver (HEPG2) cell line. Moreover, the biochemical effects of the tested compounds
on some enzymes such as aspartate and alanine aminotransferases (AST and ALT)
123

Anticancer activity of some new pyridine derivatives
1307
Table 3 Biochemical effects of treatment with 5-FU, DOX and the benzofuran derivatives on serum
total lipids, cholesterol, triglycerides, and bilirubin in mice
Mice groups Biochemical parameters
Total lipids (mg/dL) Cholesterol (mg/dL) Triglycerides (mg/dL) Bilirubin (mg/dL)
Control
5-FU
p\
323.41 27.1
378.2 31.4
0.001
94.32 13.5
105.9 11.7
0.001
108.7 16.8
126.5 19.4
0.001
0.63 0.04
0.75 0.10
0.001
Doxorubicin 366.7 6.10
109.3 14.2
0.001
137.8 17.10
0.001
0.81 0.19
0.001
p\
3
0.001
363.6 29.3
0.001
116.4 8.3
0.01
98.4 10.6
n.s.
0.96 0.8
0.001
p<
4
329.34 19.7
n.s.
95.3 9.4
n.s.
119.7 18.8
0.01
0.65 0.9
n.s.
p<
5
331.63 17.5
n.s.
96.4 10.5
n.s.
118.6 19.70
0.01
0.68 0.11
n.s.
p<
6
317.4 30.7
n.s.
93.24 19.53
n.s.
116.23 20.5
n.s.
0.51 0.08
0.01
p<
7
372.8 37.6
0.001
110.3 17.8
0.01
93.6 9.5
n.s.
1.08 0.7
0.001
p<
8
382.09 23.6
0.001
111.76 34.3
0.01
172.9 36.3
0.001
0.84 0.4
0.01
p<
10
p<
11
p<
12
p\
13
p<
15
p<
17
p<
328.3 13.7
n.s.
96.8 17.2
n.s.
112.3 10.6
n.s.
0.67 0.01
n.s.
374.9 36.6
0.001
123.3 26.09
0.001
132.8 26.03
0.001
0.99 0.07
0.001
326.3 18.7
n.s.
95.6 14.9
n.s.
113.7 8.6
n.s.
0.61 0.04
n.s.
364.19 23.8
0.001
105.6 17.4
0.01
119.8 19.3
0.01
0.76 0.15
0.01
371.23 26.7
0.001
110.9 31.2
0.01
152.6 34.5
0.001
0.77 0.2
0.01
329.71 21.5
n.s.
97.48 16.7
n.s.
112.54 17.8
n.s.
0.53 0.04
0.01
Data are expressed as mean ? SD
n.s. non significant
p [ 0.05 insignificant, p \ 0.01: significant, p \ 0.001: highly significant
and alkaline phosphatase (ALP), in addition to albumin, globulins, creatinine, total
lipids, cholesterol, triglycerides and bilirubin in serum of mice were studied in
comparison to 5-flurouracil and doxorubicin.
The antitumor activity results indicated that most of the compounds showed good
growth inhibition activity against the tested cell line but with varying intensities
123

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F. A. Bassyouni et al.
Table 4 Biochemical effects of treatment with 5-FU, DOX, and the benzofuran derivatives on serum
albumin, globulin, and creatinine in mice
Mice groups
Biochemical parameters
Albumin (mg/dL)
Globulin (mg/dL)
A/G ratio
Creatinine (mg/dL)
Control
5.63 0.51
6.49 0.92
0.01
4.32 0.9
5.75 0.8
0.01
1.3
0.69 0.03
0.81 0.06
0.01
5-FU
1.13
0.01
1.078
0.01
1.003
0.001
1.006
0.001
1.46
n.s.
p\
Doxorubicin
6.37 0.85
0.01
5.91 0.63
0.01
0.78 0.04
0.01
p\
3
7.1 0.31
0.01
7.62 0.76
0.01
0.78 0.03
0.01
p\
4
7.2 0.61
0.01
6.62 0.86
0.01
0.8 0.1
0.01
p\
5
5.97 0.34
n.s.
4.09 0.63
n.s.
0.65 0.09
n.s.
p\
6
5.92 0.82
n.s.
5.12 0.9
n.s.
1.15
n.s.
0.73 0.04
n.s.
p\
7
6.87 0.49
0.01
6.86 0.8
0.01
1.02
0.001
1.13
0.01
1.12
n.s.
1.7 0.43
0.001
p\
8
11.43 1.48
0.001
8.97 0.9
0.001
0.76 0.25
n.s.
p\
10
p\
11
p\
12
p\
13
p\
15
p\
17
p\
5.62 0.68
n.s.
4.68 1.06
n.s.
0.68 0.08
n.s.
6.47 0.46
0.01
6.42 0.7
0.01
1.001
0.001
1.15
n.s.
0.77 0.05
0.01
5.92 0.81
n.s.
4.72 0.91
n.s.
0.84 0.6
0.01
7.73 0.52
0.01
6.25 0.82
0.01
1.23
0.01
1.14
0.01
1.13
n.s.
0.84 0.06
0.01
10.22 1.35
0.001
8.96 0.91
0.001
0.72 0.21
n.s.
5.53 0.71
n.s.
4.88 1.01
n.s.
0.67 0.1
n.s.
n.s. non significant
Data are expressed as mean ? SD
p [ 0.05 insignificant, p \ 0.01: significant, p \ 0.001: highly significant
extents in comparison to the known anticancer drugs: 5-flurouracil and doxorubicin.
Moreover, compounds (11, 4, 10, 17, 12 and 3) showed cytotoxic activity (IC50
equals 2.08, 2.89, 3.02, 3.09, 3.15, and 3.36 lg/mL, respectively). Results of the
biochemical investigations indicated that 5-flurouracil and doxorubicin caused
significant changes in the level of all parameters tested while treatment with the
tested compounds showed slight, moderate, or no significant changes.
123

Anticancer activity of some new pyridine derivatives
1309
Conclusions
In the present investigation, we report the design and synthesis of a new series of 2,
6 disubstituted pyridine, which include in their structures various heterocyclic
moieties directly attached to the pyridine nucleus at positions 2 and 6 aiming to
potentiate the biological activity as anticancer. In addition, our study was extended
to the synthesis of additional derivatives containing a substituted pyridine moiety
directly attached to diazepine, indole, benzimidazole, pyrimidine, and imidazole
nucleus at positions 2 and 6. The anticancer activity data indicate that compounds
11, 4, 10, 17, 12, and 3 were the most active compounds against HEPG2.
Acknowledgments The authors are grateful to the National Research Center for financial support of
this work (Project No: 8040204).
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