Synthesis and antimicrobial activity of some new pyrimidinone and oxazinone derivatives fused with thiophene rings using 2-chloro-6-ethoxy-4-acetylpyridine as starting material

Article abstract of DOI:10.3390/molecules171113642

A series of pyridines, pyrimidinones, oxazinones and their derivatives were synthesized as antimicrobial agents using citrazinic acid (2,6- dihydroxyisonicotinic acid) as a starting material. α,β-Unsaturated ketones 3a-c were condensed with cyanothio-acetamide in the presence of ammonium acetate to give 2-cyanopyridinethiones 4a-c, which were reacted with ethyl chloroacetate to yield the corresponding cyano esters 5a-c. The esters 5a-c were cyclized by action of sodium methoxide to aminoesters 6a-c, which were aminolyzed with ammonia to corresponding aminoamide derivatives 7a-c. Also, the esters 6a-c were hydrolyzed with NaOH to the corresponding sodium salt 8a-c, which were treated with acetic anhydride to afford 2-methyloxazinones 9a-c. The latter compounds were treated with ammonium acetate to afford 2-methylpyrimidinones 10a-c, followed by methylation with methyl iodide to yield 2,3-dimethyl-pyrimidinones 11a-c. The antimicrobial screening showed that many of these compounds have good antibacterial and antifungal activities comparable to streptomycin and fusidic acid used as reference drugs.

Full text of DOI:10.3390/molecules171113642

Molecules 2012, 17, 13642-13655; doi:10.3390/molecules171113642
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Antimicrobial Activity of Some New Pyrimidinone
and Oxazinone Derivatives Fused with Thiophene Rings Using
2-Chloro-6-ethoxy-4-acetylpyridine as Starting Material
Aisha S. M. Hossan 1,*, Hanaa M. A. Abu-Melha 1, Mohamed A. Al-Omar 2,3 and
Abd El-Galil E. Amr 3,4
1
Chemistry Department, Girls College of Science, King Khalid University, Abha 9004, Saudi Arabia
2
Pharmaceutical Chemistry Department, College of Pharmacy, King Saud University, Riyadh 11451,
Saudi Arabia
3
Pharmaceutical Chemistry Department, Drug Exploration & Development Chair,
College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
4
Applied Organic Chemistry Department, National Research Center, Cairo, Dokki 12622, Egypt
* Author to whom correspondence should be addressed; E-Mail: hossanaisha@yahoo.com.
Received: 19 September 2012; in revised form: 9 November 2012 / Accepted: 12 November 2012 /
Published: 19 November 2012
Abstract: A series of pyridines, pyrimidinones, oxazinones and their derivatives were
synthesized as antimicrobial agents using citrazinic acid (2,6-dihydroxyisonicotinic acid)
as a starting material. α,β-Unsaturated ketones 3a–c were condensed with cyanothio-
acetamide in the presence of ammonium acetate to give 2-cyanopyridinethiones 4a–c,
which were reacted with ethyl chloroacetate to yield the corresponding cyano esters 5a–c.
The esters 5a–c were cyclized by action of sodium methoxide to aminoesters 6a–c, which
were aminolyzed with ammonia to corresponding aminoamide derivatives 7a-c. Also, the
esters 6a–c were hydrolyzed with NaOH to the corresponding sodium salt 8a–c, which
were treated with acetic anhydride to afford 2-methyloxazinones 9a–c. The latter
compounds were treated with ammonium acetate to afford 2-methylpyrimidinones 10a–c,
followed by methylation with methyl iodide to yield 2,3-dimethyl-pyrimidinones 11a–c.
The antimicrobial screening showed that many of these compounds have good antibacterial
and antifungal activities comparable to streptomycin and fusidic acid used as reference drugs.
Keywords: citrazinic acid; acryloyl candidates; oxazinone; pyrimidinone; antimicrobial agents

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1. Introduction
In previous work, we have found that certain substituted pyridines and their derivatives showed
antimicrobial, analgesic, anticonvulsant, antiparkinsonian [1–4] and antitumor activities [5–7].
In addition, the biological and analgesic activities of many heterocyclic compounds containing a sulfur
atom have been reviewed [8–10]. On the other hand, thienopyrimidine and thioxopyrimidine
derivatives have promising biological [11,12] and anticancer activity [13]. Recently, some new
oxazinones, thienopyrimidinones and their derivatives have been synthesized as anti-inflammatory,
antimicrobial and anti-HIV agents [14–18]. In view of these observations and in continuation of our
previous work in pyridine chemistry, we have now synthesized some novel heterocyclic compounds
containing the thieno[2,3-b]pyridine moiety fused with a pyridine, oxazinone, or pyrimidinone,
nucleus and tested their antimicrobial activities.
2. Results and Discussion
2.1. Synthesis
The starting materials 3a–c (Table 1) were prepared from 2,6-dihydroxyisonicotinic acid (1) via the
corresponding 2-chloro-6-ethoxy-4-acetylpyridine 2 according to literature methods [1,19]. Acryloyl
derivatives 3a–c were condensed with 2-cyanothioacetamide in the presence of ammonium acetate to
give the corresponding cyanopyridine thione derivatives 4a–c (Table 1). Treatment of 4a–c with ethyl
chloroacetate in the presence of anhydrous K2CO3 gave the corresponding ethyl ester derivative 5a–c
(Table 1), which were cyclized by sodium methoxide in methanol to give the amino ester derivatives
6a–c (Table 1). Aminolysis of compounds 6a–c by action of ammonia gas afforded the corresponding
aminoamide derivatives 7a–c (Scheme 1, Table 1). The IR spectra of 6a–c showed the absence of
ν (CN) for 5a–c and the presence of broad band corresponding to ν (NH2). Also, the IR spectra of 7a–c
showed the absence of ν(C=O, ester) for 6a–c and the presence of a broad band corresponding to ν(NH2).
Table 1. Melting points, crystallization solvents, yields, molecular formulae and molecular
weights of compounds 3–7.
Comp. No.
X
F
Cl
Cl Cl
F
Cl
Cl Cl
F
Cl
Cl Cl
F
Cl
Cl Cl
F
Cl
Cl Cl
Y
H
H
Yield (%)
86
M.p. (°C)
185–187
155–157
203–205
192–194
206–208
225–227
198–200
189–191
245–257
176–178
214–216
235–237
200–202
228–230
256–258
Cryst. Solv.
EtOH
Molecular Formula (Mol. Wt.)
C16H13ClFNO2 (505.73)
C16H13Cl2NO2 (322.19)
C16H12Cl3NO2 (356.63)
C19H13ClFN3OS (385.84)
C19H13Cl2N3OS (402.30)
C19H12Cl3N3OS (436.74)
C23H19ClFN3O3S (471.93)
C23H19Cl2N3O3S (488.39)
C23H18Cl3N3O3S (522.83)
C23H19ClFN3O3S (471.93)
C23H19Cl2N3O3S (488.39)
C23H18Cl3N3O3S (522.83)
C21H16ClFN4O2S (442.89)
C21H16Cl2N4O2S (459.35)
C21H15Cl3N4O2S (493.79)
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
82
85
65
58
70
78
76
69
65
70
58
86
85
84
EtOH
EtOH
H
H
DMF/H2O (2:1)
AcOH/H2O (2:1)
DMF/H2O (2:1)
EtOH/Ether (2:1)
EtOH/Ether (2:1)
EtOH/Ether (2:1)
Dioxane
EtOH
DMF/EtOH (2:1)
MeOH
H
H
H
H
H
H
AcOH
AcOH/H2O (2:1)

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Scheme 1. Synthetic Pathway for Compound 3–7.
CH3
O
i) Halogenation
ii)Esterfication
iii) NaOEt/AcOEt
Cl
HO
O
OH
N
N
1
2
X
X
OEt
OH
ArCHO
TEA/DEA
EtOH
Y
CN
Y
NCCH2CSNH2
CH3COONH4
EtOH
Cl
Cl
N
H
S
O
4a-c
N
N
3a-c
ClCH2COOEt
K2CO3/DMF
OEt
OEt
X
X
Y
CN
Y
S
CH3ONa/
CH3OH
NH2
Cl
N
5a-c
S
CO2Et
Cl
N
N
CO2Et
X
N
6a-c
OEt
OEt
Y
S
NH2
NH3
0oC
Cl
N
CONH2
N
7a-c
OEt
Compounds 6a–c were hydrolyzed by refluxing with ethanolic sodium hydroxide (NaOH) to the
corresponding sodium salts 8a–c, which was treated in situ with refluxing acetic anhydride to give the
corresponding oxazinone derivatives 9a–c (Table 2). Reaction of 9a–c with ammonium acetate in
refluxing acetic acid afforded the corresponding pyrimidinone derivatives 10a–c (Table 2), which were
treated with methyl iodide in N,N-dimethylformamide in the presence of anhydrous K2CO3 to yield the
corresponding 3-methyl-pyrimidinone derivatives 11a–c (Scheme 2, Table 2).

Molecules 2012, 17
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Table 2. Melting points, crystallization solvents, yields, molecular formulae and molecular
weights of compounds 9–11.
Comp. No.
9a
X
F
Cl
Cl Cl
F
Cl
Cl Cl
F
Cl
Y
H
H
Yield (%) M.p. (°C)
Cryst. Solv.
EtOH
AcOH
DMF/H2O (2:1)
Molecular Formula (Mol. Wt.)
C23H15ClFN3O3S (467.90)
C23H15Cl2N3O3S (484.35)
C23H14Cl3N3O3S (518.80)
C23H16ClFN4O2S (466.92)
C23H16Cl2N4O2S (483.37)
C23H15Cl3N4O2S (517.81)
C24H18ClFN4O2S (480.94)
C24H18Cl2N4O2S (497.40)
C24H17Cl3N4O2S (531.84)
75
68
60
80
72
65
78
66
72
195–197
214–216
282–284
178–180 AcOH/H2O (2:1)
188–190 AcOH/H2O (2:1)
256–258
186–188 AcOH/H2O (2:1)
200–202
264–266
9b
9c
10a
10b
10c
11a
11b
11c
H
H
DMF/H2O (2:1)
H
H
AcOH
DMF/H2O (2:1)
Cl Cl
Scheme 2. Synthetic Pathway for Compound 9–11.
X
X
Y
S
Y
NH2
NH2
NaOH/EtOH
Cl
Cl
N
COONa
N
S
CO2Et
N
N
6a-c
8a-c
OEt
OEt
Ac2O
X
X
Y
N
CH3
CH3COONH4
CH3COOH
Y
S
N
O
O
CH3
Cl
N
N
S
H
X
Cl
N
O
10a-c
N
N
9a-c
OEt
OEt
N
CH3I
K2CO3/DMF
Y
S
CH3
CH3
Cl
N
N
N
O
11a-c
OEt

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2.2. Antimicrobial Activity
The antimicrobial activities of some of the synthesized compounds were determined by the agar
diffusion method as recommended by the National Committee for Clinical Laboratory Standards
(NCCLS) [20]. The compounds were evaluated for antimicrobial activity against bacteria, viz.
Streptomyces sp., Bacillus subtilis, Streptococcus lactis, Escherichia coli, and Pseudomonas sp. and
antifungal activity against various fungi, viz. Aspergillus niger, Penicillium sp and yeast Candida albican
and Rhodotorula ingeniosa.
The concentrations of the tested compounds (10 µg/mL) were used according to a modified
Kirby-Bauer’s disk diffusion method. The sterile discs were impregnated with 10 µg/disc of the tested
compound. Each tested compound was performed in triplicate. The solvent DMSO was used as a
negative control and streptomycin/fusidic acid were used as standard calculated average diameters (for
triplicates) of the zone of inhibition (in mm) for tested samples with that produced by the standard
drugs. Four of the synthesized compounds 5a, 7b, 9b and 10b exhibited potent antibacterial and
antifungal bioactivity compared with the standard drug used. The other tested compounds were found
to exhibit a moderate to low antibacterial activity (Table 3).
Table 3. Antimicrobial activities of the newly synthesized compounds 3–11.
Comp.
No.
Fungi
Pen. sp
Yeast
C. a
Bacteria
Gram − ve Gram + ve
Str. sp
A.n
R.i
B.s
14
8
12
11
13
12
23
8
13
20
12
11
13
12
8
20
13
11
11
23
10
13
11
10
S.l
14
7
12
10
11
10
24
7
12
21
13
13
12
13
8
21
12
10
12
22
12
12
10
12
E.c
14
9
11
12
12
12
23
9
10
23
13
12
10
13
6
19
10
12
12
24
11
10
12
11
P. sp
13
11
14
11
12
11
21
8
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
9a
9b
9c
10a
10b
10c
11a
11b
11c
12
12
8
10
11
10
17
4
13
10
8
12
13
7
12
12
19
10
15
23
11
13
10
13
12
12
10
12
12
12
16
5
12
13
8
13
12
5
13
10
20
11
16
22
10
12
12
11
12
10
9
11
13
12
16
4
12
10
6
13
12
8
11
11
19
11
13
22
12
12
11
10
11
11
11
11
11
13
17
3
13
11
7
12
13
9
13
11
19
12
14
20
11
13
11
12
13
9
12
13
14
13
22
7
11
21
11
13
11
6
12
20
11
10
11
11
11
11
13
11
9
23
12
13
9
12
7
20
9
11
13
23
11
9
11
11
Streptomycin
-
-
-
-
21
22
21
22
21
Fusidic acid
17
17
18
18
-
-
-
-
-
A.n: Aspergillus niger; Pen. sp: Penicillium sp; C. a: Candida albican; Str. sp: Streptomyces sp;
R.i: Rhodotorula ingeniosa; B.s: Bacillus subtilis; S.l: Streptococcus lactis; E.c: Escherichia coli;
P. sp: Pseudomonas sp.

Molecules 2012, 17
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On the other hand, when different concentrations of compound 9a were used, it was exhibited a
moderate antibacterial activity, but it exhibited very good antibacterial activity at higher concentrations
(3× and 4×) (Table 4), while different concentrations of compounds 5a and 10a exhibited very good
antifungal activities (2× and 3×) (Table 5).
Table 4. Antibacterial activity of compound 9a at different concentrations.
Bacteria
Comp.
No.
Conc.
Strep. sp
Gram − ve
Gram + ve
B.s
20
23
24
25
S.l
21
22
24
27
E.c
19
23
24
25
Ps
20
22
26
26
1×
2×
3×
4×
20
23
25
25
9a
Where × = 10 μg.
Table 5. Antifungal activity of compounds 5a and 10a at different concentrations.
Comp.
No.
Conc.
Fungi
Pen. sp
A.n
17
18
19
20
15
16
18
20
C. a R.i
1×
2×
3×
4×
1×
2×
3×
4×
16
18
20
22
16
18
20
22
16
19
20
20
13
18
20
20
17
19
21
21
14
17
20
21
5a
10a
Where × = 10 μg.
3. Experimental
3.1. Chemistry
Melting points were measured using Electrothermal 9100 digital melting point apparatus
(Electrothermal, Essex, UK) and are uncorrected. IR spectra were recorded on a Perkin-Elmer 1600
1
FTIR (Perkin-Elmer, Downers Grove, IL, USA) in KBr discs. H- and 13C-NMR spectra were
measured on a Jeol 5000 MHz spectrometer (Jeol, Tokyo, Japan) in DMSO-d6, and chemical shifts
were recorded in δ ppm relative to the internal standard TMS. The Mass spectra were run at 70 eV
with a Finnigan SSQ 7000 spectrometer (Madison, WI, USA) using EI and the values of m/z are
indicated in Dalton. Elemental analyses were performed on a Perkin-Elmer 2400 analyzer
(Perkin-Elmer) and were found within ±0.4% of the theoretical values. All reactions were followed by
TLC (Silica gel, Aluminum Sheets 60 F254, Merck, Darmstadt, Germany). Starting material 2 was
prepared from citrazinic acid (1) according to published procedures [1,19]. Antimicrobial screening was
carried out in Department of Microbial Chemistry, National Research Center, Cairo, Egypt.
1-(2-Chloro-6-ethoxypyridin-4-yl)-3-(substituted phenyl)prop-2-en-1-ones 3a–c. A mixture of
2-chloro-6-ethoxy-4-acetylpyridine (2) [19] (1 mmol) and an aromatic aldehyde, namely, 4-flouro-,
4-chloro- or 2,4-dichlorobenzaldehyde (1 mmol) in absolute ethanol (30 mL) was refluxed in the

Molecules 2012, 17
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presence of a mixture of TEA/DEA (3 mL, 1:1 v:v) for 6 h. The reaction mixture was concentrated
under reduced pressure, the obtained solid was filtered off, washed with ether, dried and crystallized
from the proper solvents to afford the corresponding acryloyl derivatives 3a–c, respectively.
1-(2-Chloro-6-ethoxypyridin-4-yl)-3-(4-fluorophenyl)prop-2-en-1-one (3a). IR (KBr, cm−1):  1679
(C=O), 1607 (C=C); 1H-NMR: δ 1.32 (t, 3H, CH3, J = 6.95 Hz), 3.81 (q, 2H, CH2, J = 6.95 Hz), 6.65
(d, 1H, CH-olefinic-H, J = 14.60 Hz), 6.98 (d, 1H, CH-olefinic-H, J = 14.60 Hz), 7.28–7.96 (m, 6H,
13
4 Ph-H + 2 pyr-H); C-NMR: 13.68, 64.32, 104.95, 109.56, 114.72, 121.30, 129.86, 130.05, 144.65,
145.84, 146.50, 160.95, 164.96, 186.50; MS, m/z (%): 306 (M+, 15), 184 (100); Elemental analysis for
C16H13ClFNO2 (305.73): calcd.: C, 62.86; H, 4.29; Cl, 11.60; N, 4.58. found: C, 62.80; H, 4.26; Cl,
11.55; N, 4.52.
1-(2-Chloro-6-ethoxypyridin-4-yl)-3-(4-chlorophenyl)prop-2-en-1-one (3b). IR (KBr, cm−1):  1682
(C=O), 1610 (C=C); 1H-NMR: δ 1.33 (t, 3H, CH3, J = 6.95 Hz), 3.92 (q, 2H, CH2, J = 6.95 Hz), 6.58
(d, 1H, CH-olefinic-H, J = 14.60 Hz), 7.05 (d, 1H, CH-olefinic-H, J = 14.60 Hz), 7.12–7.88 (m, 6H,
13
4 Ph-H + 2 pyr-H); C-NMR: 13.86, 64.26, 105.78, 109.62, 121.12, 126.86, 128.25, 132.85, 132.96,
144.68, 145.78, 146.65, 164.84, 186.86; MS, m/z (%): 322 (M+, 8), 165 (100); Elemental analysis for
C16H13Cl2NO2 (322.18): calcd.: C, 59.65; H, 4.07; Cl, 22.01; N, 4.35. found: C, 59.60; H, 4.00; Cl,
21.96; N, 4.30.
1-(2-Chloro-6-ethoxypyridin-4-yl)-3-(2,4-dichlorophenyl)prop-2-en-1-one (3c). IR (KBr, cm−1):
1
 1678 (C=O), 1612 (C=C); H-NMR: δ 1.28 (t, 3H, CH3, J = 6.95 Hz), 3.86 (q, 2H, CH2, J = 6.95
Hz), 6.46 (d, 1H, CH-olefinic-H, J = 14.60 Hz), 7.10 (d, 1H, CH-olefinic-H, J = 14.60 Hz), 7.25–7.76
13
(m, 5H, 3 Ph-H + 2 pyr-H); C-NMR: 13.92, 64.30, 105.96, 109.46, 121.21, 125.69, 128.78, 129.56,
130.85, 132.05, 133.65, 144.86, 145.88, 146.54, 164.78, 187.05; MS, m/z (%): 356 [M+,10], 199 [100,
base peak]; Elemental analysis for C16H12Cl3NO2 (356.63): calcd.: C, 53.89; H, 3.39; Cl, 29.82; N,
3.93. found: C, 53.83; H, 3.34; Cl, 29.80; N, 3.88.
6-(2-Chloro-6-ethoxypyridin-4-yl)-4-(substituted phenyl)-1,2-dihydro-2-thioxopyridine-3-carbonitriles
4a–c. A mixture of 3a–c (1 mmol), 2-cyanothioacetamide (0.10 g, 1 mmol) and ammonium acetate
(0.6 g, 8 mmol) in absolute ethanol (30 mL) was refluxed for 5 h. After cooling, the formed product
was collected by filtration, washed with ethanol, dried and crystallized from the proper solvents to give
the corresponding thioxopyridine derivatives 4a–c, respectively.
6-(2-Chloro-6-ethoxypyridin-4-yl)-4-(4-fluorophenyl)-1,2-dihydro-2-thioxopyridine-3-carbonitrile (4a).
1
IR (KBr, cm−1):  3330 (NH), 2210 (CN), 1218 (C=S); H-NMR: δ 1.30 (t, 3H, CH3, J = 6.95 Hz),
3.90 (q, 2H, CH2, J = 6.95 Hz), 6.95–7.78 (m, 6H, 4 Ph-H + 2 pyr-H), 8.46 (s, 1H, pyr-5'-H), 9.24 (s,
13
1H, NH exchangeable with D2O); C-NMR: 13.66, 63.98, 103.66, 103.88, 107.89, 108.55, 114.58,
116.02, 127.50, 128.04, 145.48, 148.60, 160.56, 161.76, 164.65, 167.47, 168.05; MS, m/z (%): 386
[M+,24], 135 [100, base peak]; Elemental analysis for C19H13ClFN3OS (385.84): calcd.: C, 59.14; H,
3.40; Cl, 9.19; N, 10.89; S, 8.31. found: C, 59.10; H, 3.35; Cl, 9.14; N, 10.85; S, 8.28.
6-(2-Chloro-6-ethoxypyridin-4-yl)-4-(4-chlorophenyl)-1,2-dihydro-2-thioxopyridine-3-carbonitrile (4b).
1
IR (KBr, cm−1):  3356 (NH), 2215 (CN), 1210 (C=S); H-NMR: δ 1.34 (t, 3H, CH3, J = 6.95 Hz),

Molecules 2012, 17
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3.86 (q, 2H, CH2, J = 6.95 Hz), 7.12–7.80 (m, 6H, 4 Ph-H + 2 pyr-H), 8.52 (s, 1H, pyr-5'-H), 9.18 (s,
13
1H, NH exchangeable with D2O); C-NMR: 13.92, 64.12, 103.96, 104.04, 108.14, 108.86, 115.82,
127.66, 128.10, 129.68, 132.67, 145.56, 148.72, 160.77, 164.58, 167.55, 167.86; MS, m/z (%): 402
[M+,32], 211 [100, base peak]; Elemental analysis for C19H13Cl2N3OS (402.29): calcd.: C, 56.73; H,
3.26; Cl, 17.63; N, 10.45; S, 7.97. found: C, 56.68; H, 3.20; Cl, 17.60; N, 10.40; S, 7.92.
6-(2-Chloro-6-ethoxypyridin-4-yl)-4-(2,4-dichlorophenyl)-1,2-dihydro-2-thioxopyridine-3-carbonitrile
1
(4c).  3348 (NH), 2218 (CN), 1212 (C=S); H-NMR: δ 1.32 (t, 3H, CH3, J = 6.95 Hz), 3.78 (q, 2H,
CH2, J = 6.95 Hz), 6.98–7.68 (m, 5H, 3 Ph-H + 2 pyr-H), 8.64 (s, 1H, pyr-5'-H), 9.34 (s, 1H, NH
13
exchangeable with D2O); C-NMR: 14.14, 64.18, 103.88, 104.08, 108.22, 108.92, 115.76, 125.98,
128.56, 129.16, 131.86, 132.15, 134.86, 145.64, 148.80, 161.24, 164.32, 167.45, 168.18; MS, m/z (%):
436 [M+,14], 279 [100, base peak]; Elemental analysis for C19H12Cl3N3OS (436.74): calcd.: C, 52.25;
H, 2.77; Cl, 24.35; N, 9.62; S, 7.34. found: C, 52.20; H, 2.71; Cl, 24.30; N, 9.57; S, 7.28.
Ethyl 2-(6-(2-chloro-6-ethoxypyridin-4-yl)-3-cyano-4-(substituted phenyl)pyridin-2-ylthio)acetates 5a–c.
To a mixture of 4a–c (1 mmol) and anhydrous K2CO3 (0.18 g, 1 mmol) in N-dimethylformamide (25 mL)
was stirred at room temperature for 2 h, ethyl chloroacetate (0.18 g, 1.5 mmol) was added with stirring.
The reaction mixture was heated at 60 °C for 2 h and after cooling poured into ice. The solid formed
was collected by filtration, washed with water, dried and crystallized from the proper solvents to afford
the corresponding pyridinethioacetate derivatives 5a–c, respectively.
Ethyl 2-(6-(2-chloro-6-ethoxypyridin-4-yl)-3-cyano-4-(4-fluorophenyl)pyridin-2-ylthio)acetate (5a). IR
1
(KBr, cm−1):  2219 (CN), 1735 (C=O, ester); H-NMR: δ 1.28, 1.32 (2t, 6H, 2 CH3), 3.68, 3.86 (2q,
4H, 2 CH2), 4.38 (s, 2H, S–CH2), 7.16–7.82 (m, 6H, 4 Ph-H + 2 pyr-H), 8.18 (s, 1H, pyr-5'-H);
13C-NMR: 13.65, 14.05, 32.04, 59.86, 64.08, 101.36, 101.57, 102.85, 115.02, 116.75, 117.02, 128.74,
132.58, 145.65, 151.56, 153.65, 157.08, 162.15, 163.56, 163.94, 168.90; MS, m/z (%): 472 [M+,12],
426 [100, base peak]; Elemental analysis for C23H19ClFN3O3S (471.93): calcd.: C, 58.54; H, 4.06; Cl,
7.51; N, 8.90; 17; S, 6.79. found: C, 58.48; H, 4.00; Cl, 7.45; N, 8.84; 17; S, 6.72.
Ethyl 2-(6-(2-chloro-6-ethoxypyridin-4-yl)-3-cyano-4-(4-chlorophenyl)pyridin-2-ylthio)acetate (5b).
1
IR (KBr, cm−1):  2222 (CN), 1732 (C=O, ester); H-NMR: δ 1.29, 1.32 (2t, 6H, 2 CH3), 3.56, 3.84
(2q, 4H, 2 CH2), 4.42 (s, 2H, S–CH2), 7.10–7.72 (m, 6H, 4 Ph-H + 2 pyr-H), 8.64 (s, 1H, pyr-5'-H);
13C-NMR: 13.78, 14.15, 32.18, 60.05, 64.18, 101.48, 101.68, 102.74, 116.88, 117.02, 127.54, 128.12,
129.57, 133.45, 145.56, 150.96, 153.64, 157.18, 163.72, 164.05, 170.04; MS, m/z (%): 488 [M+,32],
120 [100, base peak]; Elemental analysis for C23H19Cl2N3O3S (488.38): calcd.: C, 56.56; H, 3.92; Cl,
14.52; N, 8.60; S, 6.57. found: C, 56.50; H, 3.88; Cl, 14.47; N, 8.55; S, 6.51.
Ethyl 2-(6-(2-chloro-6-ethoxypyridin-4-yl)-3-cyano-4-(2,4-dichlorophenyl)pyridin-2-ylthio)acetate (5c).
1
IR (KBr, cm−1):  2218 (CN), 1735 (C=O, ester); H-NMR: δ 1.26, 1.30 (2t, 6H, 2 CH3), 3.58, 3.78
(2q, 4H, 2 CH2), 4.36 (s, 2H, S–CH2), 7.12–7.65 (m, 5H, 3 Ph-H + 2 pyr-H), 8.56 (s, 1H, pyr-5'-H);
13C-NMR: 13.84, 14.18, 32.18, 59.92, 64.18, 100.98, 101.59, 102.66, 116.82, 117.06, 125.86, 128.48,
129.24, 131.92, 132.24, 134.74, 145.58, 151.08, 153.72, 157.22, 163.88, 164.15, 168.84; MS, m/z (%):

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523 [M+,8], 247 [100, base peak]; Elemental analysis for C23H18Cl3N3O3S (522.83): C, 52.84; H, 3.47;
Cl, 20.34; N, 8.04; S, 6.13. found: C, 52.78; H, 3.40; Cl, 20.28; N, 8.00; S, 6.07.
Ethyl
3-amino-6-(2-chloro-6-ethoxypyridin-4-yl)-4-(substituted
phenyl)thieno[2,3-b]pyridine-2-
carboxylates 6a–c. A mixture of 5a–c (1 mmol) in sodium methoxide solution (20 mL, 2%) was
refluxed for 1 h on a water bath at 70 °C with stirring. The reaction mixture was evaporated under
reduced pressure, the obtained residue was dissolved in CH2Cl2, washed with H2O, 10 mL 1 N HCl
and then with water. The solvent was dried over anhydrous CaCl2, evaporated under reduced pressure,
and the obtained product was crystallized to afford from the proper solvents to afford the
corresponding ethyl thienopyridinecarboxylates 6a–c, respectively.
Ethyl 3-amino-6-(2-chloro-6-ethoxypyridin-4-yl)-4-(4-fluorophenyl)thieno[2,3-b]pyridine-2-carboxyl-
1
ate (6a). IR (KBr, cm−1):  3443 (NH2), 1742 (C=O, ester); H-NMR: δ 1.30, 1.34 (2t, 6H, 2 CH3),
3.72, 4.06 (2q, 4H, 2 CH2), 4.36 (s, 2H, NH2 exchangeable with D2O), 7.24–7.75 (m, 6H, 4 Ph-H + 2
13
pyr-H), 8.35 (s, 1H, pyr-5'-H); C-NMR: 13.95, 14.16, 60.24, 64.18, 101.58, 103.02, 115.16, 118.35,
120.76, 122.15, 128.66, 132.64, 134.12, 145.72, 149.65, 151.64, 154.57, 155.75, 160.12, 162.65,
164.12; MS, m/z (%): 472 [M+,26], 317 [100, base peak]; Elemental analysis for C23H19ClFN3O3S
(471.93): calcd.: C, 58.54; H, 4.06; Cl, 7.51; N, 8.90; S, 6.79. found: C, 58.48; H, 4.00; Cl, 7.45; N,
8.86; S, 6.71.
Ethyl 3-amino-6-(2-chloro-6-ethoxypyridin-4-yl)-4-(4-chlorophenyl)thieno[2,3-b]pyridine-2-carboxyl-
1
ate (6b). IR (KBr, cm−1):  3452 (NH2), 1737 (C=O, ester); H-NMR: δ 1.26, 1.31 (2t, 6H, 2 CH3),
3.78, 4.10 (2q, 4H, 2 CH2), 4.48 (s, 2H, NH2 exchangeable with D2O), 7.24–7.82 (m, 6H, 4 Ph-H + 2
13
pyr-H), 8.72 (s, 1H, pyr-5'-H); C-NMR: 14.08, 14.25, 60.15, 64.10, 100.42, 103.64, 118.05, 121.16,
122.25, 127.66, 128.44, 133.45, 133.95, 134.50, 146.02, 149.75, 151.18, 154.65, 156.05, 159.64,
164.15; MS, m/z (%): 488 [M+,8], 332 [100, base peak]; Elemental analysis for C23H19Cl2N3O3S
(488.38): calcd.: C, 56.56; H, 3.92; Cl, 14.52; N, 8.60; S, 6.57. found: C, 56.50; H, 3.88; Cl, 14.46; N,
8.55; S, 6.50.
Ethyl
3-amino-6-(2-chloro-6-ethoxypyridin-4-yl)-4-(2,4-dichlorophenyl)thieno[2,3-b]pyridine-2-
1
carboxylate (6c). IR (KBr, cm−1):  3456 (NH2), 1735 (C=O, ester); H-NMR: δ 1.30, 1.33 (2t, 6H,
2 CH3), 3.82, 4.15 (2q, 4H, 2 CH2), 4.56 (s, 2H, NH2 exchangeable with D2O), 7.08–7.68 (m, 5H,
13
3 Ph-H + 2 pyr-H), 8.62 (s, 1H, pyr-5'-H); C-NMR: 14.12, 14.26, 59.98, 64.32, 100.86, 102.72,
118.02, 121.06, 122.00, 126.16, 128.87, 129.36, 132.18, 134.05, 135.44, 136.74, 145.64, 149.85,
151.38, 154.72, 155.43, 160.04, 164.25; MS, m/z (%): 523 [M+,6], 177 [100, base peak]; Elemental
analysis for C23H18Cl3N3O3S (522.83): calcd.: C, 52.84; H, 3.47; Cl, 20.34; N, 8.04; S, 6.13. found: C,
52.77; H, 3.42; Cl, 20.30; N, 7.97; S, 6.08.
3-Amino-6-(2-chloro-6-ethoxypyridin-4-yl)-4-(substituted-phenyl)thieno[2,3-b]pyridine-2-carbox-amides
7a–c. A current of ammonia gas was passed through a suspension of 6a–c (1 mmol) in absolute ethanol
(100 mL), at 0 °C till saturation. The reaction mixture was left overnight at −4 °C, evaporated under
reduced pressure, the residue obtained was triturated with n-hexane, the formed solid was filtered off,

Molecules 2012, 17
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washed with water and crystallized from the proper solvents to give the corresponding thienopyridine
carboxamides 7a–c, respectively.
3-Amino-6-(2-chloro-6-ethoxypyridin-4-yl)-4-(4-fluorophenyl)thieno[2,3-b]pyridine-2-carboxamide
(7a). IR (KBr, cm−1):  3460–3380 (NH2), 1675 (C=O, amide); 1H-NMR: δ 1.32 (t, 3H, CH3), 3.85 (q,
2H, CH2), 4.46, 6.85 (2s, 4H, 2 NH2 exchangeable with D2O), 7.12–7.68 (m, 6H, 4 Ph-H + 2 pyr-H),
8.56 (s, 1H, pyr-5'-H); 13C-NMR: 14.06, 64.28, 100.96, 102.22, 115.36, 120.82, 122.45, 128.37,
128.46, 132.84, 137.15, 145.82, 149.65, 152.00, 154.74, 157.75, 161.55, 162.76, 164.30; MS, m/z (%):
443 [M+,8], 332 [100, base peak]; Elemental analysis for C21H16ClFN4O2S (442.89): calcd.: C, 56.95;
H, 3.64; Cl, 8.00; N, 12.65; S, 7.24. found: C, 56.90; H, 3.60; Cl, 7.940; N, 12.60; S, 7.19.
3-Amino-6-(2-chloro-6-ethoxypyridin-4-yl)-4-(4-chlorophenyl)thieno[2,3-b]pyridine-2-carboxamide
(7b).  3456–3378 (NH2), 1672 (C=O, amide); 1H-NMR: δ 1.30 (t, 3H, CH3), 3.82 (q, 2H, CH2), 4.44,
6.88 (2s, 4H, 2 NH2 exchangeable with D2O), 7.32–7.68 (m, 6H, 4 Ph-H + 2 pyr-H), 8.68 (s, 1H,
13
pyr-5'-H); C-NMR: 13.68, 64.12, 100.00, 102.04, 121.24, 122.12, 127.85, 128.38, 128.55, 134.05,
135.15, 137.05, 146.12, 149.65, 151.00, 154.36, 156.14, 161.42, 164.04; MS, m/z (%): 459 [M+,25],
287 [100, base peak]; Elemental analysis for C21H16Cl2N4O2S (459.34): calcd.: C, 54.91; H, 3.51; Cl,
15.44; N, 12.20; S, 6.98. found: C, 54.86; H, 3.45; Cl, 15.39; N, 12.16; S, 6.92.
3-Amino-6-(2-chloro-6-ethoxypyridin-4-yl)-4-(2,4-dichlorophenyl)thieno[2,3-b]pyridine-2-carboxamide
1
(7c). IR (KBr, cm−1):  3456 (NH2), 1735 (C=O, ester); H-NMR: δ 1.28 (t, 3H, CH3), 3.86 (q, 2H,
CH2), 4.54, 6.76 (2s, 4H, 2 NH2 exchangeable with D2O), 7.12–7.73 (m, 5H, 3 Ph-H + 2 pyr-H), 8.48
13
(s, 1H, pyr-5'-H); C-NMR: 14.12, 64.33, 101.04, 102.84, 121.32, 122.40, 126.24, 128.65, 128.75,
129.72, 132.32, 135.12, 136.66, 137.22, 145.56, 149.55, 151.22, 154.44, 157.12, 161.26, 164.57; MS,
m/z (%): 494 [M+,12], 320 [100, base peak]; Elemental analysis for C21H15Cl3N4O2S (493.79): calcd.:
C, 51.08; H, 3.06; Cl, 21.54; N, 11.35; S, 6.49. found: C, 51.00; H, 3.00; Cl, 21.50; N, 11.30; S, 6.44.
7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(substituted-phenyl)-2-methyl-4H-pyrido[3',2':4,5]thieno[3,2-d]-
[1,3]-oxazin-4-ones 9a–c. A mixture of 6a–c (1 mmol) in ethanolic NaOH (100 mL, 5%) was heated
under reflux for 4 h. The solvent was evaporated under reduced pressure, the obtained sodium salt 8a–c
was dissolved in acetic anhydride (100 mL) and refluxed it for 6 h. The reaction mixture was
concentrated and allowed to cool, poured onto ice water, the obtained solid was collected by filtration,
washed with water, dried and crystallized from the proper solvents to afford the corresponding
thienooxazinopyridine derivatives 9a–c, respectively.
7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(4-fluorophenyl)-2-methyl-4H-pyrido[3',2':4,5]thieno[3,2-d]-[1,3]
1
oxazin-4-one (9a). IR (KBr, cm−1):  1750 (C=O); H-NMR: δ 1.30 (t, 3H, CH3), 2.03 (s, 3H, CH3),
13
3.78 (q, 2H, CH2), 7.04–7.58 (m, 6H, 4 Ph-H + 2 pyr-H), 8.62 (s, 1H, pyr-5'-H); C-NMR: 14.10,
18.98, 64.30, 100.86, 101.68, 116.02, 120.80, 125.85, 128.42, 132.78, 134.46, 135.35, 145.72, 150.05,
151.75, 154.90, 155.25 158.62, 162.70, 164.08, 165.25; MS, m/z (%): 468 [M+,6], 217 [100, base
peak]; Elemental analysis for C23H15ClFN3O3S (467.89): calcd.: C, 59.04; H, 3.23; Cl, 7.58; N, 8.98;
S, 6.85. found: C, 58.96; H, 3.18; Cl, 7.52; N, 8.90; S, 6.80.

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7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(4-chlorophenyl)-2-methyl-4H-pyrido[3',2':4,5]thieno[3,2-d]-[1,3]
1
oxazin-4-one (9b). IR (KBr, cm−1):  1745 (C=O); H-NMR: δ 1.28 (t, 3H, CH3), 2.01 (s, 3H, CH3),
13
3.89 (q, 2H, CH2), 7.23–7.65 (m, 6H, 4 Ph-H + 2 pyr-H), 8.42 (s, 1H, pyr-5'-H); C-NMR: 14.08,
21.60, 64.18, 100.55, 101.45, 121.12, 125.68, 128.12, 128.96, 133.05, 134.58, 135.32, 135.72, 145.92,
149.75, 151.84, 154.86, 155.14, 158.70, 164.12, 165.18; MS, m/z (%): 484 [M+,15], 156 [100, base
peak]; Elemental analysis for C23H15Cl2N3O3S (484.35): calcd.: C, 57.03; H, 3.12; Cl, 14.64; N, 8.68;
S, 6.62. found: C, 56.95; H, 3.10; Cl, 14.60; N, 8.63; S, 6.58.
7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(2,4-dichlorophenyl)-2-methyl-4H-pyrido[3',2':4,5]thieno[3,2-d]-
1
[1,3]oxazin-4-one (9c). IR (KBr, cm−1):  1750 (C=O); H-NMR: δ 1.30 (t, 3H, CH3), 2.00 (s, 3H,
13
CH3), 3.82 (q, 2H, CH2), 7.21–7.68 (m, 5H, 3 Ph-H + 2 pyr-H), 8.54 (s, 1H, pyr-5'-H); C-NMR:
14.10, 19.18, 64.22, 100.28, 101.15, 121.16, 125.56, 126.46, 128.58, 129.80, 132.44, 134.34, 135.18,
135.45, 136.73, 145.88, 149.72, 151.69, 154.78, 155.18, 159.06, 164.18, 165.32; MS, m/z (%): 519
[M+,8], 320 [100, base peak]; Elemental analysis for C23H14Cl3N3O3S (518.79): calcd.: C, 53.25; H,
2.72; Cl, 20.50; N, 8.10; S, 6.18. found: C, 53.18; H, 2.68; Cl, 20.45; N, 8.00; S, 6.12.
7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(substituted-phenyl)-2-methylpyrido[3',2':4,5]thieno[3,2-d]-
pyrimidin-4(3H)-ones 10a–c
A mixture of 9a–c (1 mmol) and ammonium acetate (0.6 g, 8 mmol) in glacial acetic acid (100 mL)
was heated under reflux for 6 h. The reaction mixture was evaporated under reduced pressure, the
residue was triturated with cooled water, the solid formed was collected by filtration, washed with
water, dried and crystallized from the proper solvents to afford the corresponding thienopyrimidino-
pyridine 0.30 g (70%) 10a–c, respectively.
7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(4-fluorophenyl)-2-methylpyrido[3',2':4,5]thieno[3,2-d]-pyrimidin-
1
4(3H)-one (10a). IR (KBr, cm−1):  3420 (NH), 1650 (C=O); H-NMR: δ 1.28 (t, 3H, CH3), 2.32 (s,
3H, CH3), 3.86 (q, 2H, CH2), 7.12–7.64 (m, 6H, 4 Ph-H + 2 pyr-H), 8.58 (s, 1H, pyr-5'-H), 9.26 (s, 1H,
NH exchangeable with D2O); 13C-NMR: 14.08, 24.98, 64.42, 101.02, 102.54, 116.15, 121.04, 126.14,
128.85, 132.86, 136.76, 137.05, 145.88, 150.15, 151.98, 154.10, 154.86, 157.25, 160.03, 162.99,
164.28; MS, m/z (%): 467 [M+,18], 156 [100, base peak]; Elemental analysis for C23H16ClFN4O2S
(466.91): calcd.: C, 59.16; H, 3.45; Cl, 7.59; N, 12.00; S, 6.87. found: C, 59.10; H, 3.38; Cl, 7.52; N,
11.94; S, 6.83.
7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(4-chlorophenyl)-2-methylpyrido[3',2':4,5]thieno[3,2-d]-pyrimidin-
1
4(3H)-one (10b). IR (KBr, cm−1):  3438 (NH), 1649 (C=O); H-NMR: δ 1.31 (t, 3H, CH3), 2.24 (s,
3H, CH3), 3.78 (q, 2H, CH2), 7.33–7.72 (m, 6H, 4 Ph-H + 2 pyr-H), 8.62 (s, 1H, pyr-5'-H), 9.32 (s, 1H,
NH exchangeable with D2O); 13C-NMR: 13.98, 25.04, 64.53, 100.12, 101.36, 121.13, 126.45, 128.15,
129.05, 133.76, 135.99, 136.88, 145.76, 146.05, 149.85, 151.90, 154.16, 154.92, 157.48, 159.73,
164.36; MS, m/z (%): 483 [M+,18], 326 [100, base peak]; Elemental analysis for C23H16Cl2N4O2S
(483.36): calcd.: C, 57.15; H, 3.34; Cl, 14.67; N, 11.59; S, 6.63. found: C, 57.10; H, 3.28; Cl, 14.62; N,
11.53; S, 6.58.
7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(2,4-dichlorophenyl)-2-methylpyrido[3',2':4,5]thieno[3,2-d]-pyrimidin-
4(3H)-one (10c). IR (KBr, cm−1):  3465 (NH), 1653 (C=O); 1H-NMR: δ 1.26 (t, 3H, CH3), 2.12 (s, 3H,

Molecules 2012, 17
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CH3), 3.80 (q, 2H, CH2), 7.21–7.70 (m, 5H, 3 Ph-H + 2 pyr-H), 8.72 (s, 1H, pyr-5'-H), 9.48 (s, 1H, NH
exchangeable with D2O); 13C-NMR: 13.92, 24.87, 64.42, 99.96, 101.02, 120.33, 126.32, 126.64,
128.36, 129.72, 132.88, 135.09, 136.64, 136.84, 145.86, 146.13, 149.77, 151.92, 153.96, 154.66,
157.68, 160.02, 164.48; MS, m/z (%): 518 [M+,5], 145 [100, base peak]; Elemental analysis for
C23H15Cl3N4O2S (517.81): calcd.: C, 53.35; H, 2.92; Cl, 20.54; N, 10.82; S, 6.19. found: C, 53.30; H,
2.87; Cl, 20.50; N, 10.79; S, 6.14.
7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(4-fluorophenyl)-2,3-dimethylpyrido[3',2':4,5]thieno[3,2-d]pyrimidin-
4(3H)-ones 11a–c. A solution of 10a–c (1 mmol) in DMF (20 mL) was stirred with anhydrous K2CO3
(0.19 g, 1 mmol) for 10 min at room temperature, then methyl iodide (0.28 g, 2 mmol) in DMF (5 mL)
were added. The reaction mixture was heated at 60 °C for 4 h, after cooling, poured into ice water, and
the formed precipitate was filtered off, washed with water, dried and crystallized from the proper
solvents to afford the corresponding thieno-N-methylpyrimidinopyridines 11a–c, respectively.
7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(4-fluorophenyl)-2,3-dimethylpyrido[3',2':4,5]thieno[3,2-d]pyrimidin-
1
4(3H)-one (11a). IR (KBr, cm−1):  1668 (C=O); H-NMR: δ 1.28 (t, 3H, CH3), 2.32, 3.10 (2s, 6H,
13
2 CH3), 3.78 (q, 2H, CH2), 7.08–7.68 (m, 6H, 4 Ph-H + 2 pyr-H), 8.62 (s, 1H, pyr-5'-H); C-NMR:
14.00, 22.14, 26.06, 64.15, 100.10, 101.32, 116.18, 120.34, 126.34, 128.95, 132.90, 136.42, 145.76,
146.15, 149.85, 151.80, 154.02, 154.77, 157.36, 159.63, 162.76, 164.30; MS, m/z (%): 481 [M+,4], 98
[100, base peak]; Elemental analysis for C24H18ClFN4O2S (480.94): calcd.: C, 59.94; H, 3.77; Cl, 7.37;
N, 11.65; S, 6.67. found: C, 59.88; H, 3.72; Cl, 7.33; N, 11.60; S, 6.61.
7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(4-chlorophenyl)-2,3-dimethylpyrido[3',2':4,5]thieno[3,2-d]-pyrimidin-
4(3H)-one (11b). IR (KBr, cm−1):  1670 (C=O); 1H-NMR: δ 1.29 (t, 3H, CH3), 2.18, 3.06 (2s, 6H, 2
13
CH3), 3.82 (q, 2H, CH2), 7.28–7.68 (m, 6H, 4 Ph-H + 2 pyr-H), 8.78 (s, 1H, pyr-5'-H); C-NMR:
13.86, 22.00, 26.28, 64.14, 99.58, 100.12, 120.56, 126.76, 128.00, 128.95, 133.45, 135.85, 136.56,
145.32, 146.75, 149.80, 150.87, 153.78, 154.65, 157.45, 160.02, 164.28; MS, m/z (%): 497 [M+,19],
162 [100, base peak]; Elemental analysis for C24H18Cl2N4O2S (497.39): calcd.: C, 57.95; H, 3.65; Cl,
14.26; N, 11.26; S, 6.45. found: C, 57.90; H, 3.59; Cl, 14.22; N, 11.20; S, 6.40.
7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(2,4-dichlorophenyl)-2,3-dimethylpyrido[3',2':4,5]thieno[3,2-d]-
1
pyrimidin-4(3H)-one (11c). IR (KBr, cm−1):  1667 (C=O); H-NMR: δ 1.28 (t, 3H, CH3), 2.22, 2.96
(2s, 6H, 2 CH3), 3.80 (q, 2H, CH2), 7.24–7.70 (m, 5H, 3 Ph-H + 2 pyr-H), 8.65 (s, 1H, pyr-5'-H);
13C-NMR: 13.90, 22.01, 26.48, 64.08, 99.86, 100.09, 120.60, 126.45, 126.58, 128.25, 129.52, 132.66,
135.14, 136.60, 136.78, 145.42, 146.78, 149.82, 151.14, 153.88, 154.72, 157.48, 159.72, 164.20; MS,
m/z (%): 532 [M+,19], 252 [100, base peak]; Elemental analysis for C24H17Cl3N4O2S (531.84): calcd.:
C, 54.20; H, 3.22; Cl, 20.00; N, 10.53; S, 6.03. found: C, 54.15; H, 3.16; Cl, 19.85; N, 10.48; S, 6.00.
3.2. Antimicrobial Screening Media
The following media were used:
1. PDA medium: this medium was used for fungi cultivation. It consists of 4 g dextrose/L
potatoes extract.

Molecules 2012, 17
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2. Czapek Dox medium: it consists of 10 g glucose, 2 g KNO3, 1g K2HPO4, 0.5 g KCl, 0.5 g
MgSO4, and 0.05 g ferrous sulphate/L distilled water. This medium is specialized for
bacteria cultivation.
3. Medium 3: it consists of 10 glucose, 5 g peptone, 3 yeast extract, and 3 malt extract. It was used
for yeast cultivation.
4. Conclusions
A series of newly compounds 3–11 were prepared using citrazinic acid (2,6-dihydroxyisonicotinic
acid) as a starting material. The obtained derivatives were screening as antimicrobial and antifungal
agents. Four of the synthesized compounds 5a, 7b, 9b and 10b exhibited potent antibacterial and
antifungal bioactivity compared with streptomycin and fusidic acid used as reference drugs. The other
tested compounds were found to exhibit moderate to low antibacterial activity. On the other hand when
higher concentrations (3× and 4×) of compound 9a, which exhibited a moderate antibacterial activity,
were used, this compound exhibited very good antibacterial activity. While different concentrations of
compounds 5a and 10a exhibited a very good antifungal activity (2× and 3×).
Acknowledgments
The authors extend their appreciation to the Deanship of Scientific Research at King Saud
University for funding the work through the research group project No. RGP-VPP-172.
References
1. Amr, A.E. Synthesis of some heterocyclic compounds as potential antimicrobial agents using
2,6-diacetylpyridine as synthon. Indian J. Heterocycl. Chem. 2000, 10, 49–58.
2. Attia, A.; Abdel-Salam, O.I.; Amr, A.E.; Stibor, I.; Budesinsky, M. Synthesis and antimicrobial
activity of some new chiral bridged macrocyclic pyridines. Egypt. J. Chem. 2000, 43, 187–201.
3. Nehad, A.A.; Amr, A.E.; Alhusien, A.I. Synthesis, reactions and pharmacological screening of
heterocyclic derivatives using nicotinic acid as a natural synthon. Monatsh. Chem. 2007, 138,
559–567.
4. Amr, A.E.; Hegab, M.I.; Ibrahim, A.A.; Abdulla, M.M. Synthesis and reactions of some fused
oxazinone, pyrimidinone, thiopyrimidnone and triazinone derivatives with thiophene ring as
analgesic, anticonvulsant and antiparkinsonian agents. Monatsh. Chem. 2003, 134, 1395–1409.
5. Amr, A.E.; Mohamed, A.M.; Mohamed, S.F.; Abdel-Hafez, N.A.; Hammam, A.G. Anticancer
activities of some newly synthesized pyridine, pyrane and pyrimidine derivatives. Bioorg. Med. Chem.
2006, 14, 5481–5488.
6. Abou-Ghalia, M.H.; Amr, A.E. Synthesis and investigation of a new cyclo-(Nα-
dipicolinoyl)pentapeptide of a breast and CNS cytotoxic activity and an ionophoric specifity.
Amino Acids 2004, 26, 283–289.
7. Hammam, A.G.; Fahmy, A.F.M.; Amr, A.E.; Ashraf, M.M. Synthesis of novel tricyclic
heterocyclic compounds as potential anticancer agents using chromanone and thiochromanone as
synthons. Indian J. Chem. Sect. B 2003, 42B, 1985–1993.

Molecules 2012, 17
13655
8. Chakrabarti, J.K.; Horsman, L.; Hotten, T.M.; Pullar, I.A.; Tupper, D.E.; Wright, F.C.
4-Piperazinyl-10H-thieno[2,3-b][1,5]benzodiazepines as potential neuroleptics J. Med. Chem.
1980, 23, 878–884.
9. Ram, V.J.; Pandey, H.K.; Vlietinck, A.J. Thieno[2,3-d]pyrimidines as potential chemotherapeutic
agents. II. J. Heterocycl. Chem. 1981, 18, 1277–1280.
10. Fahmy, H.H.; El-Eraqy, W. Synthesis and evaluation of the analgesic and antiinflammatory
activities of O-substituted salicylamides. Arch. Pharm. Res. 2001, 24, 171–179.
11. De Clercq, E. Potential of bromovinyldeoxyuridine in anticancer chemotherapy. Anticancer Res.
1986, 6, 549–556.
12. De Clercq, E.; Desgranges, C.; Herdewijn, P.; Sim, I.S.; Jones, A.S.; McLean, M.J.; Walker, R.T.
Synthesis and antiviral activity of (E)-5-(2-bromovinyl)uracil and (E)-5-(2-bromovinyl)uridine.
J. Med. Chem. 1986, 29, 213–217.
13. Braña, M.F.; Castellano, J.M.; Morán, M.; Pérez, de Vega M.J.; Romerdahl, C.R.; Qian, X.D.;
Bousquet, P.; Emling, F.; Schlick, E.; Keilhauer, G. Bis-naphthalimides: A new class of antitumor
agents. Anticancer Drug Des. 1993, 8, 257–268.
14. Amr, A.E.; Sabry, N.M.; Abdalla, M.M. Synthesis, reactions and antiinflammatory activity of
heterocyclic derivatives system fused to a thiophene moiety using citrazinic acid as synthon.
Monatsh. Chem. 2007, 138, 699–707.
15. Ghozlan, S.A.S.; Al-Omar, M.A.; Amr, A.E.; Khalil, K.; Abd El-Wahab, A.A. Synthesis and
antimicrobial activity of some heterocyclic 2,6-bis(substituted)-1,3,4-thiadiazolo-, oxadiazolo-
and oxathiazolidinopyridine derivatives from 2,6-pyridine dicarboxylic acid dihydrazide.
J. Heterocycl. Chem. 2011, 48, 1103–1110.
16. Al-Omar, M.A.; Amr, A.E. Synthesis of some new pyridine-2,6-carboxamide-derived Schiff
Bases as potential antimicrobial agents. Molecules 2010, 15, 4711–4721.
17. Al-Salahi, R.A.; Al-Omar, M.A.; Amr, A.E. Synthesis of chiral macro-cyclic or linear pyridine
carboxamides from pyridine-2,6-dicarbonyl dichloride as anti-microbial agents. Molecules 2010,
15, 6588–6597.
18. Abdel Fattah, Y.; Amr, A.E.; Mohamed, S.F.; Abdalla, M.M.; Al-Omar, M.A.; Shafik, S.H.
Cytotoxicity and anti-HIV evaluations of some new synthesized quinazoline and thioxo-
pyrimidine derivatives using 4-(thiophen-2-yl)-3,4,5,6-tetrahydro-benzo[h]quinazoline-2(1H)-
thione as synthon. J. Chem. Sci. 2012, 124, 693–702.
19. Attia, A.; Abdel-Salam, O.I.; Abo-Ghalia, M.H.; Amr, A.E. Chemical and biological reactivity of
newly synthesized 2-chloro-6-ethoxy-4-acetylpyridine. Egypt. J. Chem. 1995, 38, 543–553.
20. Furtado, G.L.; Medeiros, A.A. Single-disk diffusion testing (Kirby-Bauer) of susceptibility of
Proteus mirabilis to chloramphenicol: Significance of the intermediate category. J. Clin. Microbiol.
1980, 12, 550–553.
Sample Availability: Samples of the compounds are available from the authors.
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