Synthesis and anticancer activity of some novel fused pyridine ring system

Article abstract of DOI:10.1007/s12272-012-1107-6

New series of pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidines (7a,b) and thieno[2,3-b:4,5-b′] dipyridine (11a-c) were synthesized from 4-aryl-6-(4-chlorophenyl)-2-thioxo-1,2-dihydro pyridine-3-carbonitriles 4a,b via application of Thorpe-Zielger reaction. The n

Full text of DOI:10.1007/s12272-012-1107-6

Arch Pharm Res Vol 35, No 11, 1909-1917, 2012
DOI 10.1007/s12272-012-1107-6
Synthesis and Anticancer Activity of Some Novel Fused Pyridine Ring
System
Afaf K. Elansary, Ashraf A. Moneer, Hanan H. Kadry, and Ehab M. Gedawy
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
(Received June 22, 2012/Revised August 7, 2012/Accepted August 31, 2012)
New series of pyrido[3',2':4,5]thieno[3,2-
11a ) were synthesized from 4-aryl-6-(4-chlorophenyl)-2-thioxo-1,2-dihydro pyridine-3-carboni-
triles 4a via application of Thorpe-Zielger reaction. The novel target compounds were evalu-
d]pyrimidines (7a,b) and thieno[2,3-b:4,5-b'] dipyridine
(
-c
,b
ated in vitro for their anticancer activity against human breast adenocarcinoma MCF-7 and
colon carcinoma cell line (HCT 116). Most of the tested compounds exploited potent to moderate
growth inhibitory activity, in particular compound 11d, which exhibited superior potency to the
reference drug Doxorubicin (IC50 = 5.95, 6.09 and 8.48, 8.15 µM, respectively). The structures of
the compounds obtained were determined by spectroscopic data.
Key words: Synthesis, Pyridothienopyrimidine, Thienodipyridine, Anticancer activity
biological activities. Among these derivatives, fused
analogues are often of greater biological interest than
Cancer treatment has been a major endeavor of re- the corresponding monocyclic compounds (Litvinov et
search and development in academia and pharmaceu- al , 2007). Furthermore, it should be emphasized that
tical industry for the last many years as it is one of the combination of pyridine with other hetero cycles is
the leading causes of death (Choo et al , 2002; Abdel- a well-known approach for drug-like molecules build-
Aziz, 2007; Hassan et al , 2011; Taher et al , 2012). up. This may allow achieving new pharmacological
The great incidence of cancer worldwide encourages profiles, action-strengthening or toxicity-lowering.
the search for newer, safer and more efficient antican- On the other hand, compounds containing a fused
INTRODUCTION
.
.
.
.
cer agents, aiming at preventing or curing this illness. pyrimidine ring have attracted attention in the past
Although chemotherapy is the mainstay for cancer few years. This is due by their wide range of biological
treatment, the use of available chemotherapeutics is activities, particularly in cancer and virus research
often limited due to undesirable side effects. In addi- (Baba et al., 1987; Gangjee et al., 1995). Pyrido[2,3-d]
tion, there is a growing incidence of drug resistance to pyrimidines were reported to exhibit antitumor activity
cancer chemotherapeutic agents, which represents a which may be attributed to inhibition of cyclin depend-
serious medical problem (Li et al
2008). As a result of these challenges, significant interest check point kinase (Palmer et al
has developed in the search for new anticancer agents. lian target of rapamycin (Malagu et al
The pyridine nucleus is prevalent in numerous nat- tion, thienopyrimidine derivatives showed promising
ural products and is extremely important in the chem- biological (Devani et al , 1976) and anticancer activity
istry of biological systems (Bringmann et al , 2004). (Dai et al , 2005; Rheault et al , 2009). Several reports
.
, 1995; Rojo et al
.
,
ent kinase (Toogood et al
.
, 2005; VanderWel et al
, 2005) or mamma-
, 2009). In addi-
., 2005),
.
.
.
.
.
.
Derivatives of pyridine were found to have various showed that a single molecule containing more than
one pharmacophore, each with different mechanism of
action, could be beneficial for the treatment of cancer.
Correspondence to: Hanan Hassan Kadry, Pharmaceutical
Accordingly, the combination of these two classes to
Organic Chemistry Department, Faculty of Pharmacy, Cairo
give the tricyclic pyridothienopyrimidines and the
University, Cairo 11562, Egypt
subsequent influence on the biological activities have
Tel: 2-01-0196-0964, Fax: 2-02-2363-5140
E- mail: hanankadry2005@yahoo.com
become a subject of interest (Dave et al., 1989; Quintela
1909

1910
et al
A. K. Elansary et al.
.
, 1998; Amr et al
.
, 2003). However, none of these tive chalcone analogue 2a
:4,5- acetamide
was heated under reflux for 7 h, left to cool to room
Motivated by the above-mentioned findings, the pre- temperature. The separated solid was filtered, dried
,b
(0.01 mol) and cyanothio-
publication discussed the effect of thieno[2,3-
b
3 (1.1 g, 0.011 mol). The reaction mixture
b']dipyridine as antitumor agents.
sent study involves the synthesis and pharmacological and crystallized from suitable solvent.
evaluation of some substituted pyridothienopyrimi-
dines and isosteric analogues thienodipyridines as 6-(4-Chlorophenyl)-4-(2-nitrophenyl)-2-thioxo-1,2-
anticancer agents.
dihydropyridine-3-carbonitrile (4a)
Crystallized from DMF/H2O. Yield: method A (72%),
method B (60%); mp: 208-210oC; IR max/cm−1: 3400
ν
MATERIALS AND METHODS
Chemistry
(NH), 2200 (CN), 1520, 1350 (NO2), 1090 (C=S);1H-NMR
(DMSO-d6 ppm: 6.94 (s, 1H, NH, exch. D2O), 7.50-
)
δ
General remarks: Melting points are uncorrected 8.75 (m, 9H, Ar-H and H-5); MS: m/z 367 [M+, 14.44%];
and determined in one end open capillary tubes using Anal. Calcd for C18H10ClN3O2S (367.81): C, 58.78; H,
Gallen Kamp melting point apparatus MFB-595- 2.74; N, 11.42. Found: C, 58.50; H, 3.00; N, 11.42.
010M (Gallen Kamp). Microanalysis was carried out at
Micro-analytical Unit, Faculty of Science, Cairo Uni- 6-(4-Chlorophenyl)-4-(3-nitrophenyl)-2-thioxo-1,2-
versity, Analyses indicated were within 0.4% of the dihydropyridine-3-carbonitrile (4b)
theoretical values. Infrared Spectra were recorded on Crystallized from ethanol. Yield: method A (75%),
Schimadzu FT-IR 8400S spectrophotometer (Shimadzu), method B (55%); mp: >300oC; IR
ν
max/cm−1: 3400 (NH),
1
and expressed in wave number (cm−1), using potas- 2200 (CN), 1520, 1340 (NO2), 1090 (C=S); H-NMR
sium bromide discs. The NMR spectra were recorded (DMSO-d6) δ ppm: 6.96 (s, 1H, NH, exch. D2O), 6.94-8.47
on a Varian Gemini 200 MHz and Varian Mercury (m, 9H, Ar-H and H-5); Anal. Calcd for C18H10ClN3O2S
1
VX-300 NMR spectrometer. H spectra were run at (367.81): C, 58.78; H, 2.74; N, 11.42. Found: C, 58.99;
300 MHz and 13C spectra were run at 75.46 MHz in H,2.79; N, 11.40.
dimethylsulfoxide (DMSO-d6). Chemical shifts were
quoted in
δ and were related to that of the solvents.
3-Amino-2-aminocyaniminomethyl-4-aryl-6-(4-
chlorophenyl)thieno[2,3- ]pyridines 6a,b
A solution of the appropriate pyridinethione 4a
Mass spectra were recorded using Hewlett Packard
Varian (Varian) and Shimadzu Gas Chromatograph
b
,b
Mass spectrometer-QP 1000 EX (Shimadzu). TLC were (0.003 mol) and 10% aqueous KOH (0.16 g, 0.003 mol)
carried out using Art.DC-Plastikfolien, Kieselgel 60 in DMF (20 mL) was stirred at room temperature for
F254 sheets (Merck), the developing solvents were 5 min. N-cyanochloroacetamidine
5 (0.36 g, 0.0031 mol)
benzene/acetone (4:1) and the spots were visualized at was added and the mixture was stirred for additional
366, 254 nm by UV Vilber Lourmat 77202 (Vilber). 5 min. Finally, additional 10% aqueous KOH (0.33 g,
Compounds 2a
(Huffman and Schaefer, 1963) and
Gewald, 1965) were obtained according to the reported onto cold water, the formed precipitate was filtered,
,
b
(Bickel, 1946),
3
(Howard, 1956),
5
0.006 mol) was added to the reaction mixture with con-
9
(Mowry, 1945; tinuous stirring for 2 h. The reaction mixture was poured
procedures.
washed with water, dried and crystallized from ethanol.
3-Amino-2-aminocyaniminomethyl-6-(4-chloro-
4-Aryl-6-(4-chlorophenyl)-2-thioxo-1,2-dihydro-
pyridine-3-carbonitriles 4a,b
phenyl)-4-(2-nitrophenyl)thieno[2,3-
b]
pyridine
Two methods were adopted for the synthesis of the (6a)
title compounds.
Yield: 63% ; mp: 190-191oC; IR max/cm−1: 3420, 3300
(NH2), 3080 (CH aromatic), 2200 (CN) 1530, 1390
ppm: 7.60-7.62 (br s, 2H,
3 (1 g, 0.01 mol) and NH2, exch. D2O), 7.11-8.62 (m, 9H, Ar-H and H-5), 7.97
ν
Method A: A mixture of
p
-chloroacetophenone
1
(1.55 g, 0.01 mol), the appropriate aromatic aldehydes (NO2); 1H-NMR (DMSO-d6
) δ
(0.01 mol), cyanothioacetamide
ammonium acetate (6.16 g, 0.08 mol) in n-butanol (30 (s, 2H, NH2, exch. D2O); MS m/z: 448 [M+, 2.55%], 446
mL) was heated under reflux for 9 h. The reaction [M-2, 3.06%], 69 [NH2CH=NCN, 22.96%]; Anal. Calcd
mixture was cooled to room temperature. The separated for C21H13ClN6O2S: C, 56.19; H, 2.92; N, 18.72. Found:
solid was filtered, dried and crystallized from suitable C, 56.46; H, 2.70; N, 19.00.
solvent.
Method B: Sodium ethoxide (0.07 g sodium in abso- 3-Amino-2-aminocyaniminomethyl-6-(4-chloro-
lute ethanol 20 mL) was added to a mixture of respec- phenyl)-4-(3-nitrophenyl)thieno[2,3-b] pyridine

Synthesis of Novel Fused Pyridine
1911
(6b)
was added to a stirred solution of sodium (0.5 g, 0.021
Yield: 59%; mp: 225-227oC; IR
ν
max/cm−1: 3300, 3190 mol) in absolute ethanol (50 mL). A solution of the
(NH2), 3050 (CH aromatic), 2200 (CN), 1520, 1320 (NO2); substituted phenacyl bromide (0.002 mol) in dioxane
1H-NMR (DMSO-d6
)
δ
ppm: 7.63 (s, 2H, NH2, exch. (20 mL) was added portion-wise to the reaction mixture
D2O), 7-8.6 (m, 9H, Ar-H and H-5), 7.99 (s, 2H, NH2, and the mixture was stirred at room temperature for
exch. D2O); Anal. Calcd for C21H13ClN6O2S: C, 56.19; 3 h. Finally, the reaction mixture was diluted with
H, 2.92; N, 18.72. Found: C, 55.98; H, 3.19; N, 18.50.
water (50 mL), the formed precipitate was filtered,
dried and crystallized from ethanol.
2,4-Diamino-9-aryl-7-(4-chlorophenyl)pyrido
[3',2':4,5]thieno[3,2-d]pyrimidines 7a,b
3-Amino-2-benzoyl-6-(4-chlorophenyl)-4-(2-nitro-
Two methods were adopted for the synthesis of the phenyl)thieno[2,3-
title compounds.
Yield: 72%; mp: 145-148oC; IR
Method A: A mixture of thienopyridine 6a (0.001 (NH2), 3050 (CH aromatic), 1600 (C=O), 1520, 1340
mol) and sodium (0.07 g, 0.003 mol) in absolute ethanol (NO2); 1H-NMR (DMSO-d6
ppm: 7.28-8.89 (m, 14H,
b
]pyridine (8a)
ν
max/cm−1: 3400-3100
,b
)
δ
(20 mL) was heated under reflux for 6 h. The precipitat- Ar-H and H-5), 10.46 (s, 2H, NH2, exch. D2O); Anal.
ed solid was filtered, washed with cold ethanol, dried Calcd for C26H16ClN3O3S: C, 64.26; H, 3.31; N, 8.64.
and crystallized from ethanol.
Found: C, 64.46; H 3.46; N, 9.05
Method B: A mixture of the appropriate pyridine-
thione 4a
g, 0.001mol) in absolute ethanol (20 mL) was stirred (3-nitrobenzoyl)thieno[2,3-
for 10 min and kept at room temperature. N-Cyano- Yield: 65%; mp: 229-231oC; IR
chloroacetamidine
(0.13 g, 0.0011 mol) was added to 3050 (CH aromatic), 1600 (C=O), 1520, 1340 (NO2); 1H-
the mixture and stirred for 20 min. Sodium (0.07 g, NMR (DMSO-d6 ppm: 7.36-8.78 (m, 13 H, Ar-H and
,b
(0.001 mol) and 10% aqueous KOH (0.056 3-Amino-6-(4-chlorophenyl)-4-(2-nitrophenyl)-2-
]pyridine (8b)
max/cm−1: 3320 (NH2),
b
ν
5
)
δ
0.003 mol) in absolute ethanol (20 mL) was added and H-5), 9.49 (s, 2H, NH2, exch. D2O); MS m/z: 532 [M+2,
the reaction mixture was heated under reflux for 4 h. 5.06], 530 [M+, 11.28]; Anal. Calcd for C26H15ClN4O5S:
The formed precipitate was filtered, washed with etha- C, 58.81; H, 2.84; N, 10.55. Found:C, 58.62; H 3.14; N,
nol, dried and crystallized from ethanol.
10.16.
2,4-Diamino-7-(4-chlorophenyl)-9-(2-nitrophenyl) 3-Amino-6-(4-chlorophenyl)-2-(3-nitrobenzoyl)-4-
pyrido[3',2':4,5]thieno[3,2- ]pyrimidine (7a) (3-nitrophenyl)thieno[2,3- ]pyridine (8c)
Yield: method A (65%), method B (52%); mp: >300oC; Yield: 63%; mp: >300oC; IR
IR
max/cm−1: 3320, 3200 (NH2), 3050 (CH aromatic), 3080 (CH aromatic), 1600 (C=O), 1530, 1340 (NO2);
1520, 1340 (NO2); H-NMR (DMSO-d6 δ ) δ ppm: 7.60-8.80 (m, 13 H, Ar-H
ppm: 7.6 (s, 1H-NMR (DMSO-d6
d
b
1
ν
max/cm− : 3300 (NH2),
ν
1
)
2H, 4-NH2, exch. D2O), 7.6-8.62 (m, 9H, Ar-H and H- and H-5), 8.47 (s, 2H, NH2, exch. D2O); Anal. Calcd for
8), 8.8 (s, 2H, 2-NH2, exch. D2O); MS m/z: 367 [M+, C26H15ClN4O5S: C, 58.81; H, 2.84; N, 10.55. Found: C,
14.44%]; Anal. Calcd for C21H13ClN6O2S: C, 56.19; H, 58.61; H 3.12; N, 10.13.
2.92; N, 18.72. Found: C, 56.43; H, 3.15; N, 18.43.
2-[(6-(4-Chlorophenyl)-3-cyano-4-(2-nitrophen-
yl)pyridin-2-yl)sulfanyl]-1-phenylethylidene
malononitrile (10)
2,4-Diamino-7-(4-chlorophenyl)-9-(3-nitrophenyl)
pyrido[3',2':4,5]thieno[3,2- ]pyrimidine (7b)
d
Yield: method A (74%), method B (58%); mp: >300oC;
A solution of pyridinethione 4a (0.73 g, 0.002 mol) in
IR
ν
max/cm−1: 3400, 3200 (NH2), 3040 (CH aromatic), absolute ethanol (15 mL) containing piperidine (0.16
1
1543, 1330 (NO2); H-NMR (DMSO-d6
)
δ
ppm: 7.15- g, 0.0019 mol) was heated to 50oC with stirring. 3-
8.48 (m, 9H, Ar-H and H-8), 8.33 (s, 2H, 4-NH2, exch. Bromo-2-phenyl-1,1-dicyanopropene
D2O), 8.48 (s, 2H, 2-NH2, exch. D2O); 13C-NMR (DMSO- was added to the reaction mixture with continuous
6) ppm: 165.1, 160.3, 156.1, 153.7, 147.4, 144.2, 137.9, stirring for 12 h. The reaction mixture was diluted with
9 (0.5 g, 0.002 mol)
d
δ
135.3, 132.2, 129.3, 129.1, 124.5, 122.3; Anal. Calcd for cold water (50 mL). The formed solid was filtered,
C21H13ClN6O2S: C, 56.19; H, 2.92; N, 18.72. Found: C, washed with ethanol, dried and crystallized from
56.38; H 3.20; N, 18.66.
ethanol.
Yield: 65%; mp: 145-148oC; IR max/cm−1: 3050 (CH
ν
aromatic), 2920 (CH aliphatic), 2200, 2180 (CN), 1520,
1360 (NO2); H-NMR (DMSO-d6
b (0.002 mol) CH2), 7.32-8.73 (m, 14H, Ar-H and H-5); MS m/z: 533
3-Amino-4-aryl-2-arylcarbonyl-6-(4-chlorophen-
1
) δ ppm: 3.7 (s, 2H,
yl)thieno[2,3-b]pyridines 8a-c
The appropriate pyridinethione 4a
,

1912
A. K. Elansary et al.
[M+, 64.29%]; Anal. Calcd for C29H16ClN5O2S: C, 65.22; 3350-3150 (NH2), 3050 (CH aromatic), 2200 (CN),
1
H, 3.02; N, 13.11. Found: C, 64.90; H, 3.10; N, 13.18.
1520, 1340 (NO2); H-NMR (DMSO-d6
)
δ
ppm: 7.41-
8.36 (m, 13H, Ar-H and H-8), 8.85 (s, 2H, NH2, exch.
D2O); 13C NMR (DMSO-
6) ppm: 168.2, 164.3, 156.1,
149.3, 146.8, 146.2, 144.6, 143.9, 141.8, 139.6, 139.1,
Three methods were adopted for the synthesis of the 138.5, 135.8, 129.5, 129.2, 128.6, 125.3, 124.7, 122.0,
title compounds. 101.9, 88.9; Anal. Calcd for C29H15ClN6O4S: C, 60.15;
Method A: A mixture of the suitable thienopyridine H, 2.61; N, 14.51. Found: C, 60.08; H, 2.92; N, 14.80.
8a (0.001 mol), and malononitrile (0.2 g, 0.003 mol)
in pyridine (10 mL) was heated under reflux for 12 h. 2-Amino-7-(4-chlorophenyl)-4-(3-nitrophenyl)-9-
d
δ
2-Amino-4,9-diaryl-7-(4-chlorophenyl)thieno[2,
3-b:4,5-b']dipyridine-3-carbonitriles 11a-d
-c
After cooling, the reaction mixture was diluted with (3-nitrophenyl)thieno[2,3-
b
:4,5-
b']dipyridine-3-car-
water (20 mL). The formed precipitate was filtered, bonitrile (11d)
dried and crystallized from ethanol.
Method B: A solution of the S-alkylated pyridine- 3450-3320 (NH2), 3060 (CH aromatic), 2200 (CN),
thione 10 (1.06 g, 0.002 mol) in boiling ethanol (30 mL) 1520, 1340 (NO2); 1H-NMR (DMSO-d6
ppm 7.38-8.91
Yield: method A (85%); mp: 180-182oC; IR max/cm−1
ν
)
δ
containing 2-3 drops piperidine was stirred with heat- (m, 13H, Ar-H and H-8), 9.57 (s, 2H, NH2, exch. D2O);
ing at 50oC for 5 h. The formed solid was filtered, Anal. Calcd for C29H15ClN6O4S: C, 60.15; H, 2.61; N,
washed with ethanol, dried and crystallized from 14.51. Found: C, 60.23; H, 2.61; N, 14.53.
ethanol.
Method C: 3-Bromo-2-phenyl-1,1-dicyanopropene
(12.35 g, 0.05 mol) was added to a solution of the
appropriate pyridinethione 4a (0.05 mol) and sodium
9
Biological testing
Materials and methods
The human breast adenocarcinoma cell line (MCF-
,b
(2.3 g, 0.1 mol) in absolute ethanol (50 mL). The reac- 7) was obtained and colon carcinoma cell line HCT-
tion mixture was heated under reflux for 2 h. The pre- 116 were obtained as a gift from NCI, MD,USA.
cipitated solid was filtered, dried and crystallized from
ethanol.
All solvents and chemicals were purchased from
Sigma-Aldrich.
2-Amino-7-(4-chlorophenyl)-4-(2-nitrophenyl)-9-
Measurement of potential cytotoxicity
phenylthieno[2,3-
(11a)
b
:4,5-
b']dipyridine-3-carbonitrile
The cytotoxic activity of the newly synthesized com-
pounds was measured in vitro on human breast adeno-
Yield: method A (83%), method B (71%), method C carcinoma cell line (MCF-7) and Colon carcinoma cell
(90%); mp: >300oC; IR max/cm−1: 3400 (NH2), 3050 line (HCT 116) using Sulforhodamine-B (SRB) stain
(CH aromatic), 2200 (CN), 1520, 1385 (NO2); 1H-NMR assay, applying the method of Skehan et al. (1990) as
(DMSO-d6 ppm: 7.46-8.31 (m, 14H, Ar-H and H-8), follows:
8.76 (s, 2H, NH2, exch. D2O); MS m/z: 533 [M+,
Cells were plated in 96-multiwell plate (104 cells/
57.38%]; Anal. Calcd for C29H16ClN5O2S: C, 65.22; H, well) for 24 h before treatment with the test com-
ν
)
δ
3.02; N, 13.11 Found: C, 65.23; H, 3.02; N, 13.11.
pounds to allow attachment of the cells to the wall of
the plate. Test compounds were dissolved in DMSO
and diluted with saline to the appropriate volume.
2-Amino-7-(4-chlorophenyl)-4-(3-nitrophenyl)-9-
phenylthieno[2,3-
b
:4,5-
b']dipyridine-3-carbonitrile Different concentrations of the test compounds (0, 1,
(11b)
2.5, 5 and 10 mg/mL) were added to the cell
Yield: method B (68%), method C (73%) ; mp: >300oC; monolayer. Triplicate wells were prepared for each
IR
ν
max/cm−1: 3350, 3150 (NH2), 3020 (CH aromatic), individual dose. Monolayer cells were incubated with
1
2200 (CN), 1520, 1380 (NO2); H-NMR (DMSO-d6
)
δ
the test compound for 48 h at 37oC in an atmosphere
ppm: 7.44-8.26 (m, 14H, Ar-H and H-8), 8.67 (s, 2H, of 5% CO2. After 48 h, cells were fixed with trichloro-
NH2, exch. D2O); Anal. Calcd for C29H16ClN5O2S: C, acetic acid, washed with water and stained for 30 min
65.22; H, 3.02; N, 13.11. Found: C, 65.09; H, 3.32; N, with 0.4% (wt/vol) SRB stain dissolved with 1% acetic
13.51.
acid. Excess stain was removed by four washes with
1% acetic acid and attached stain was recovered with
Tris EDTA buffer. Color intensity was measured in
ELISA reader. The relation between surviving fraction
and compound concentration was plotted and IC50 [the
2-Amino-7-(4-chlorophenyl)-4-(2-nitrophenyl)-9-(3-
nitrophenyl)thieno[2,3-
b:4,5-b']dipyridine-3-car-
bonitrile (11c)
Yield: method A (79%); mp: 180-182oC; IR max/cm−1 : concentration required for 50% inhibition of cell via-
ν

Synthesis of Novel Fused Pyridine
1913
Table I. IC50 values in µ ,b and 11a-d
Ma of compounds 7a
thienodipyridine derivatives 11a
tive than 7a
tives 7a . Moreover, regarding the pyridothieno-
pyrimidine derivatives 7a , the presence of 3-nitro-
phenyl substituent at position 4 had a better activity
against MCF7 than the 2-nitrophenyl analogue (IC50
= 11.25, 19.87 µM, respectively). On the other hand,
regarding the influence of substitution at position 4
and position 8 on the activity of thienodipyridine de-
-
d
were more effec-
against MCF7 and HCT 116
,
b than pyridothienopyrimidine deriva-
,b
Cell line
Compound no.
,b
MCF-7
HCT 116
Doxorubicinb
8.48±0.015
19.87±0.032
11.25±0.008
24.34±0.047
15.90±0.023
33.68±0.018
5.95±0.030
8.15±0.015
9.53±0.015
7a
7b
11a
11b
11c
11d
23.61±0.035
8.29±0.021
6.04±0.011
22.10±0.056
6.09±0.025
rivatives 11a-d, the presence of phenyl substituent at
position 8 and 3-nitrophenyl at position 4, compound
11b, demonstrated a higher activity to both cell lines
compared to that bearing 2-nitrophenyl substituent
11a. While regarding the replacement of phenyl group
at position 8 with 3-nitrophenyl in the presence of 2-
nitrophenyl substitution at position 4, 11c led to signifi-
aEach experiment was independently performed three
times and expressed as means±S.D. bDoxorubicin was used
as reference drug.
bility] was calculated for each compound and results cant decrease in activity. The best result was obtained
are given in Table I.
by compound 11d which bears 3-nitrophenyl group in
both positions. On the other hand, in the presence of
phenyl substituent at position 8 and 2-nitrophenyl at
position 4, compound 11a was successful inducing
selectivity against HCT116 cell line. Further studies
RESULTS
In vitro anticancer screening
The cytotoxicity of the newly synthesized compounds are recommended to explore the mechanism of action
was evaluated in vitro against two cell lines, human as well as the effect of substitution on the anticancer
breast adenocarcinoma cell line (MCF7) and colon car- effect of these compounds. We hope that the synthe-
cinoma cell line (HCT 116). For comparison purposes, sized compounds serve as lead chemical entities for
the cytotoxicity of doxorubicin, which is one of the further modification to render them clinically useful
most effective anticancer agents, was evaluated under drug agents.
the same conditions. The relationship between the
surviving fraction and drug concentration was plotted
to obtain the survival curve of breast cancer cell line
(MCF-7) and colon carcinoma cell line (HCT 116). The
response parameter calculated was the IC50 value,
DISCUSSION
Chemistry
The synthetic approaches adopted to obtain the
which corresponds to the concentration required for target compounds are outlined in Schemes 1 and 2.
50% inhibition of cell viability. The IC50 of the synthe- The starting compounds, 4-aryl-6-(4-chlorophenyl)-2-
sized compounds, compared to the reference drug, are thioxo-1, 2-dihydropyridine-3-carbonitriles 4a
shown in Table I. From the results in Table I, it is evi- prepared in a good yield using two pathways. The first
dent that all the tested compounds displayed moderate pathway involved one pot reaction of -chloroaceto-
to potent cancer cell growth inhibition. Generally, all phenone with the appropriate aromatic aldehydes
the tested compounds tended to be more active against and cyanothioacetamide (Howard, 1956) in the pre-
,b were
p
1
3
HCT-116 than against MCF-7 cell lines. Compounds sence of ammonium acetate, following the reaction con-
11b and 11c showed the highest potency against ditions reported for the preparation of related com-
HCT116 while compound 11d was the most active pounds (Fathalla et al
against MCF7 (IC50 = 5.95 M). In particular the thi- way was adopted via refluxing a mixture of respec-
enodipyridine derivative 11b and 11d were found to tive chalcone analogues 2a and cyanothioacetamide
have improved activity against HCT116 (IC50 = 6.04, using sodium ethoxide in absolute ethanol, accord-
6.09 M, respectively). Although the number of tested ing to the previously described procedure for the pre-
compounds in this study is limited, some structural paration of analogous compounds (Vieweg et al , 1989).
features that are important for explanation of their The derivatives 4a were characterized by spectral
cytotoxic effects can be referred. In general, pyrido- studies using IR, 1H-NMR and MS. IR spectra of com-
thienopyrimidine derivatives 7a were more potent pounds 4a showed the characteristic C N stretching
against MCF-7 than thienodipyridine derivatives 11a absorption at
2200 cm−1, in addition to NH absorp-
(with exception of 11d) while in case of HCT-116 tion at
3400 cm−1. The H-NMR spectra of 4a
., 2002). Whereas the second path-
µ
,b
3
µ
.
,b
,b
,b
≡
-
υ
1
d
υ
,b

1914
A. K. Elansary et al.
Scheme 1. Reagent and condition : (i) NaOC2H5, reflux 7 h; (ii) amm acetate,
n-butanol, reflux 9 h; (iii) 10% KOH, DMF,
stir 2 h; (iv) a-10% KOH, ethanol, stir 10 min; b-NaOC2H5 reflux 4 h; (v) NaOC2H5 rreflux 6 h
Scheme 2. Reagent and condition: (i) Dioxane, stir 3, H2O; (ii) CH2(CN)2, pyridine, reflux 12 h; (iii) piperidine/ethanol, stir
12 h, 50oC; (iv) piperidine/ ethanol, stir 5 h; (v) C2H5ONa, reflux 2 h.
revealed an exchangeable proton (NH) at
ppm. Further structural evidence stemmed from the molecular ion peak with m/z 367.
mass spectrum of compound 4a, which corroborates The regioselective reaction of 4a
δ 6.94-6.96 the spectral data and the proposed structure, giving a
,b with N-cyano-

Synthesis of Novel Fused Pyridine
1915
Chart 1. Formation of compounds 6a,b according to Thorpe-Zielger reaction.
chloroacetamidine 5 (Huffman and Schaefer, 1963) in condensed pyridines was synthesized by applying
DMF and excess KOH afforded the corresponding 3- Litvinov's synthetic route (Artyomov et al , 1997)
amino-2-aminocyaniminomethyl-4-aryl-6-(4-chlorophen- which was found to proceed as a result of a sequence
yl)thieno[2,3- ]pyridines 6a . The regioselectivity of of consecutive reactions: nucleophilic substitution at
.
b
,b
the reaction is a consequence of two consecutive reac- the sulfur atom, Thorpe-Ziegler closure of the thio-
tions, namely nucleophilic substitution and Thorpe- phene ring to afford the intermediate thienopyridine
Zielger reaction (Ivanov et al
.
, 1996) (Chart 1).
and finally Thorpe-Guareschi closure of the pyridine
, 1996). This was achieved through
1H-NMR spectrum of 6a
,b
revealed the presence of ring (Ivanov et al.
two exchangeable singlet signals corresponding to three different synthetic routes previously described
protons of the amino group at position-3 and amino for the preparation of analogous compounds (Ivanov et
group of the amidine moiety, respectively. Refluxing al
the substituted 3-amino-2-aminocyaniminomethyl- involved the synthesis of intermediate 3-amino-4-aryl-
thieno[2,3-b] pyridines 6a with sodium ethoxide in 2-arylcarbonyl-6-(4-chlorophenyl)thieno[2,3- ]pyridines
ethanol produced pyridothienopyrimidinederivatives 8a-c via stirring 4,6-disubstituted pyridinethiones
7a (method A) (Artemov et al , 1996). The latter com- 4a with appropriate phenacyl bromide in the pre-
pounds were also obtained by condensation of 4a
sence of sodium ethoxide. The 1H-NMR spectra of 8a
with N-cyanochloroacetamidine in presence of showed a D2O exchangeable singlet signal at 10.46,
., 1996; Artyomov et al., 1997). The first technique
,b
b
,b
.
,b
,b
-
5
c
δ
potassium hydroxide in quantitative amount followed 9.49 and 8.47 attributed to NH2, respectively. Besides,
by addition of sodium ethoxide (method B) (Artemov the mass spectrum of 8b displayed molecular ion
et al
confirmed by IR and 1H-NMR spectra. The IR spectra (M+2) in ratio 3:1. Condensation of 2-substituted ben-
proved useful in tracing the disappearance of the CN zoylthieno [2,3- ] pyridines 8a with malononitrile in
stretching absorption of the parent compounds 4a pyridine afforded the desired substituted thieno [2,3-
:4,5- '] dipyridines 11a 11c and 11d
The second route involved Alkylation of 4,6-disub-
with 3-bromo -2-phenyl-
(Mowry, 1945; Gewald, 1965) in
One of the principle objectives of the present work the presence of piperidine to produce the intermediate
was the synthesis of substituted thieno [2,3-
:4,5- compound 10. 1H-NMR spectrum of compound 10 dis-
']dipyridines 11a (Scheme 2). The formation of these played the appearance of a singlet signal at 3.7 cor-
., 1994). The structures of compounds 7a,b
were peaks at m/z 530 and 532 corresponding to (M+) and
b
-c
,
b
1
and 6a
displayed two singlet signals D2O exchangeable at
7.60-8.33 and
NH2 groups at position 4 and 2.
,b
. The H-NMR spectra of compounds 7a
,b
b
b
,
.
δ
δ
8.48-8.80 corresponding to the two stituted pyridinethiones 4a
,
b
1,1-dicyanopropene
9
b
b
-d
δ

1916
A. K. Elansary et al.
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sorption of the precursor 4a. Cyclization of the latter
compound in the presence of piperidine furnished
.,
thieno[2,3-
involved cyclocondensation of 2-thioxopyridine-3-car-
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pene in the presence of sodium ethoxide yielding the
corresponding thieno [2,3- :4,5- '] dipyridines 11a
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the disappearance of CH2 absorption of the precursor
reagent and the presence of the expected D2O
exchangeable singlet signal at 8.67-9.57, which was
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ACKNOWLEDGEMENTS
We are grateful to all members of the department of
Cancer Biology, pharmacology unit, National Cancer
Institute, Cairo, Egypt, for carrying out the cytotoxicity
testing.
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