Design, synthesis and biological evaluation of thiophene based pyrimidin-4-one derivatives as new type of antimicrobial agents

Article abstract of DOI:10.14233/ajchem.2016.19861

In the present investigation, a novel series of 3-(benzylidene-amino)-2-methyl-5-phenyl-3H-thieno [2,3-d]pyrimidin-4-one and its derivatives (5a-i) were synthesized by condensation reaction from the final intermediate, 3-amino-2-methyl-5-phenyl-3H-thieno[

Full text of DOI:10.14233/ajchem.2016.19861

Asian Journal of Chemistry; Vol. 28, No. 9 (2016), 1997-2000
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2016.19861
Design, Synthesis and Biological Evaluation of Thiophene Based
Pyrimidin-4-one Derivatives as New Type of Antimicrobial Agents
1
1
2
1,*
B. PULLARAO , S.D. KHASIM SHARIF , D. RAVI KUMAR and D. RAMACHANDRAN
1Department of Chemistry, Acahrya Nagarjuna University, Nagarjuna Nagar, Guntur-522 510, India
2Department of Chemistry, Krishna University, Dr. M.R.A.R. P.G. Centre, Nuzvid-521 201, India
*Corresponding author: E-mail: pullarao83@gmail.com
Received: 27 January 2016;
Accepted: 4 April 2016;
Published online: 1 June 2016;
AJC-17930
In the present investigation, a novel series of 3-(benzylidene-amino)-2-methyl-5-phenyl-3H-thieno [2,3-d]pyrimidin-4-one and its derivatives
(5a-i) were synthesized by condensation reaction from the final intermediate, 3-amino-2-methyl-5-phenyl-3H-thieno[2,3-d]pyrimidin-4-
one (4). The synthesis of the title compounds commenced from commercially available 2-amino-4-phenyl-thiophene-3-carboxylic acid
ethyl ester (1) and by involving 2-amino-4-phenyl-thiophene-3-carboxylic acid (2) and 2-methyl-5-phenyl-thieno[2,3-d][1,3]oxazin-4-
one (3) as reactive intermediates. The chemical structures of synthesized compounds were characterized by IR, 1H NMR, mass spectral
data and elemental analysis. The final compounds were used to screen for their antifungal activity against two strains of fungi.
Keywords: Thiophene, Pyrimidin-4-ones, Antimicrobial activities.
NMR spectra were recorded on aVarian 300 MHz spectrometer
for 1H NMR. The chemical shifts were reported as ppm down
field using TMS as an internal standard. Mass spectra were
recorded on a VG-Micromass 7070H spectrometer operating
at 70 eV.
Synthesis of 2-amino-4-phenyl-thiophene-3-carboxylic
acid (2): A mixture of ethyl 2-amino-4-phenyl-thiophene-3-
carboxylic acid ethyl ester (1) (0.01 mol) and sodium
hydroxide (1.25 g) in ethanol (15 mL) was heated at reflux
temperature for 4 h. After completion of the reaction (moni-
tored by the TLC), the reaction mixture was cooled and poured
in ice-cold water (25 mL), then acidified with conc. HCl to get
crude solid and it filtered, dried and recrystallized from ethanol
to achieve pure 2-amino-4-phenyl-thiophene-3-carboxylic acid
(2).
Synthesis of 2-methyl-5-phenyl-thieno[2,3-d][1,3]oxazin-
4-one (3): A solution of 2-amino-4-phenyl-thiophene-3-
carboxylic acid (2) (0.01 mol) and acetic anhydride (5 mL)
was refluxed for 6 h with constant stirring. After accomplish-
ment of the reaction (examined by the TLC), the mixture was
cooled, poured into ice-cold water to form crude product. It is
collected by filtration, washed with cold water, dried and
recrystallized from ethyl acetate to achieve 2-methyl-5-phenyl-
thieno[2,3-d][1,3]oxazin-4-one (3).
INTRODUCTION
Pyrimidines represent an important class of heterocycles
and their structural framework is not only a key constituent of
nucleic bases, alkaloids and numerous pharmacophores with
variety of potent biological activities. Pyrimidines occupy a
distinct and unique place in medicine, large array of pyrimidine
non-nucleoside derivatives possess a variety of pharmacolo-
gical properties include anticancer [1], antiviral [2], antibac-
terial [3], antifungal [4], antiprotozoal [5], antihypertensive
[6], antihistaminic [7], anti-inflammatory [8] and central
nervous activities [9]. In view of the above mentioned findings
and following our work on the synthesis of new heterocyclic
compounds we here are aimed at reporting the preparation of
a new class of 3-(benzylidene-amino)-2-methyl-5-phenyl-3H-
thieno [2,3-d]pyrimidin-4-one and its derivatives (5a-i) by
involving 2-amino-4-phenyl-thiophene-3-carboxylic acid ethyl
ester (1) as starting material.
EXPERIMENTAL
All the reagents and solvents were used as purchased
without further purification. Melting points were determined
on a Fisher-Johns melting point apparatus and are uncorrected.
Crude products were purified by column chromatography on
silica gel of 60-120 mesh. IR spectra were obtained on a Perkin-
Elmer BX serried FTIR 5000 spectrometer using KBr pellet.
Synthesis of 3-amino-2-methyl-5-phenyl-3H-thieno-
[2,3-d]pyrimidin-4-one (4): A mixture of 2-methyl-5-phenyl-

1998 Pullarao et al.
Asian J. Chem.
thieno[2,3-d][1,3]oxazin-4-one (3) (0.01 mL) and hydrazine
hydrate (0.05 mol) in ethanol (10 mL) was prepared at room
temperature. Then the reaction mixture was heated at reflux
temperature on uniform stirring for 6 h. After achievement of
the reaction (scanned by the TLC), the resulted mixture cooled
and precipitated after poured in ice-cold water. The crude
product was filtered and washed with cold water, dried and
recrystallized with ethanol to get pure 3-amino-2-methyl-5-
phenyl-3H-thieno[2,3-d]pyrimidin-4-one (4).
2-Methyl-3-[(2-methyl-benzylidene)amino]-5-phenyl-
3H-thieno[2,3-d]pyrimidin-4-one (5b): Yield: 74 %, m.p.:
144-146 °C, IR (KBr, νmax, cm-1): 3064 (C-H, Ar), 2974 (C-H,
CH3), 1668 (C=O), 1632 (C=C, Ar), 1588 (C=N), 848 (C-S);
1H NMR (300 MHz, DMSO-d6): δ 7.77 (s, 1H, thiophene),
7.73-7.50 (m, 9H, Ar-H), 7.48 (s, 1H, N=CH), 2.66 (s, 3H,
CH3), 2.45 (s, 3H, CH3). MS: 359 m/z (M+). Elemental analysis:
Calculated for C21H17N3OS: C 70.17, H 4.77, N 11.69, S 8.92.
Found: C 68.74, H 4.58, N 11.05, S 8.41.
Synthesis of 3-(benzylidene-amino)-2-methyl-5-
phenyl-3H-thieno [2,3-d]pyrimidin-4-one (5a): A solution
of 3-amino-2-methyl-5-phenyl-3H-thieno[2,3-d]pyrimidin-4-
one (4) (0.01 mol) and benzaldehyde (0.01 mol) in acetic acid
(10 mL) was prepared at ambient temperature. Then the reaction
mixture is stirred uniformly for 14 h at reflux temperature.
After fulfillment of the reaction (examined by the TLC), the
mixture was cooled and poured in ice-cold water and it is
collected by filtration, washed with cold water, recrystallized
from ethyl acetate to obtain 3-(benzylidene-amino)-2-methyl-
5-phenyl-3H-thieno[2,3-d]pyrimidin-4-one (5a). Similar pro-
cedure is followed to prepare other compounds of this series
5b-i.
2-Methyl-3-[(2-methoxy-benzylidene)amino]-5-
phenyl-3H-thieno[2,3-d]pyrimidin-4-one (5c): Yield: 75 %,
m.p.: 158-160 °C, IR (KBr, νmax, cm-1): 3060 (C-H, Ar), 2944
(C-H, CH3), 1665 (C=O), 1640 (C=C, Ar), 1581 (C=N), 1235
(C-O), 855 (C-S); 1H NMR (300 MHz, DMSO-d6): δ 7.83 (s,
1H, thiophene), 7.70-7.53 (m, 9H,Ar-H), 7.51 (s, 1H, N=CH),
2.62 (s, 3H, OCH3), 2.54 (s, 3H, CH3). MS: 375 m/z (M+).
Elemental analysis: Calculated for C21H17N3O2S: C 67.18, H
4.56, N 11.19, S 8.54. Found: C 65.47, H 4.39, N 10.84, S 8.30.
2-Methyl-3-[(2-chloro-benzylidene)amino]-5-phenyl-
3H-thieno[2,3-d]pyrimidin-4-one (5d): Yield: 69 %, m.p.:
120-122 °C, IR (KBr, νmax, cm-1): 3048 (C-H, Ar), 2968 (C-H,
CH3), 1665 (C=O), 1644 (C=C, Ar), 1580 (C=N), 859 (C-S);
1H NMR (300 MHz, DMSO-d6): δ7.74 (s, 1H, thiophene), 7.72-
7.49 (m, 9H, Ar-H), 7.55 (s, 1H, N=CH), 2.61 (s, 3H, CH3).
MS: 379 m/z (M+). Elemental analysis: Calculated for
C20H14N3OSCl: C 63.24, H 3.71, Cl 9.33, N 11.06, S 8.44.
Found: C 61.36, H 3.58, Cl 9.06, N 10.75, S 8.19.
2-Methyl-3-[(4-chloro-benzylidene)amino]-5-phenyl-
3H-thieno[2,3-d]pyrimidin-4-one (5e): Yield: 66 %, m.p.:
114-116 °C, IR (KBr, νmax, cm-1): 3058 (C-H, Ar), 2965 (C-H,
CH3), 1658 (C=O), 1648 (C=C, Ar), 1584 (C=N), 865 (C-S);
1H NMR (300 MHz, DMSO-d6): δ 7.82 (s, 1H, thiophene),
7.79-7.58 (m, 5H, Ar-H), 7.54 (d, 2H, J = 7.0 Hz, Ar-H), 7.51
(s, 1H, N=CH), 7.40 (d, 2H, J = 7.0 Hz, Ar-H), 2.66 (s, 3H,
CH3). MS: 379 m/z (M+). Elemental analysis: Calculated for
C20H14N3OSCl: C 63.24, H 3.71, Cl 9.33, N 11.06, S 8.44.
Found: C 61.36, H 3.58, Cl 9.06, N 10.75, S 8.19.
2-Methyl-3-[(2-bromo-benzylidene)amino]-5-phenyl-
3H-thieno[2,3-d]pyrimidin-4-one (5f): Yield: 72 %, m.p.:
150-152 °C, IR (KBr, νmax, cm-1): 3064 (C-H, Ar), 2956 (C-H,
CH3), 1665 (C=O), 1642 (C=C, Ar), 1580 (C=N), 861 (C-S);
1H NMR (300 MHz, DMSO-d6): δ 7.85 (s, 1H, thiophene),
7.79-7.51 (m, 9H, Ar-H), 7.53 (s, 1H, N=CH), 2.68 (s, 3H,
CH3). MS: 424 m/z (M+). Elemental analysis: Calculated for
C20H14N3OSBr: C 56.61, H 3.33, Br 18.83, N 9.90, S 7.56.
Found: C 54.69, H 3.26, Br 18.08, N 9.67, S 7.48.
2-Methyl-3-[(4-bromo-benzylidene)amino]-5-phenyl-
3H-thieno[2,3-d]pyrimidin-4-one (5g): Yield: 67 %, m.p.:
132-134 °C, IR (KBr, νmax, cm-1): 3045 (C-H, Ar), 2971 (C-H,
CH3), 1668 (C=O), 1640 (C=C, Ar), 1584 (C=N), 868 (C-S);
1H NMR (300 MHz, DMSO-d6): δ 7.85 (s, 1H, thiophene),
7.69-7.38 (m, 5H, Ar-H), 7.49 (d, 2H, J = 7.3 Hz, Ar-H), 7.42
(s, 1H, N=CH), 7.21 (d, 2H, J = 7.3 Hz, Ar-H), 2.47 (s, 3H,
CH3). MS: 424 m/z (M+). Elemental analysis: Calculated for
C20H14N3OSBr: C 56.61, H 3.33, Br 18.83, N 9.90, S 7.56.
Found: C 54.69, H 3.26, Br 18.08, N 9.67, S 7.48.
Spectral data
2-Amino-4-phenyl-thiophene-3-carboxylic acid (2):
Yield: 68 %, m.p.: 123-125 °C, IR (KBr, νmax, cm-1): 3314
(N-H, NH2), 3036 (C-H, Ar), 1718 (C=O), 1654 (C=C, Ar),
1
1272 (C-O), 865 (C-S); H NMR (300 MHz, DMSO-d6): δ
11.84 (s, 1H, COOH), 7.82 (s, 1H, thiophene), 7.70-7.45 (m,
5H, Ar-H), 7.36 (s, 2H, NH2). MS: 219 m/z (M+). Elemental
analysis: Calculated for C11H9NO2S: C 60.26, H 4.14, N 6.39,
S 14.62. Found: C 58.65, H 4.08, N 6.12, S 14.14.
2-Methyl-5-phenyl-thieno[2,3-d][1,3]oxazin-4-one (3):
Yield: 70 %, m.p.: 131-133 °C, IR (KBr, νmax, cm-1): 3030
(C-H, Ar), 2970 (C-H, CH3), 1742 (C=O), 1660 (C=C, Ar),
1
1570 (C=N), 1269 (C-O), 854 (C-S); H NMR (300 MHz,
DMSO-d6): δ 7.77 (s, 1H, thiophene), 7.75-7.52 (m, 5H, Ar-H),
2.74 (s, 3H, CH3). MS: 243 m/z (M+). Elemental analysis:
Calculated for C13H9NO2S: C 64.18, H 3.73, N 5.76, S 13.18.
Found: C 62.65, H 3.54, N 5.61, S 12.98.
3-Amino-2-methyl-5-phenyl-3H-thieno[2,3-d]pyrimidin-
4-one (4):Yield: 77 %, m.p.: 154-156 °C, IR (KBr, νmax, cm-1):
3235 (N-H, NH2), 3010 (C-H, Ar), 2958 (C-H, CH3), 1685
(C=O), 1616 (C=C), 1597 (C=N), 862 (C-S); 1H NMR (300
MHz, DMSO-d6): δ 7.85 (s, 2H, NH2), 7.79-7.55 (m, 5H,
Ar-H), 7.73 (s, 1H, thiophene), 2.81 (s, 3H, CH3). MS: 257
m/z (M+). Elemental analysis: Calculated for C13H11N3OS: C
60.68, H 4.31, N 16.33, S 12.46. Found: C 58.67, H 4.21, N
15.87, S 12.12.
3-(Benzylidene-amino)-2-methyl-5-phenyl-3H-thieno-
[2,3-d]pyrimidin-4-one (5a):Yield: 71 %, m.p.: 160-162 °C,
IR (KBr, νmax, cm-1): 3048 (C-H, Ar), 2966 (C-H, CH3), 1672
1
(C=O), 1625 (C=C, Ar), 1584 (C=N), 858 (C-S); H NMR
(300 MHz, DMSO-d6): δ 7.80 (s, 1H, thiophene), 7.71-7.42
(m, 10H, Ar-H), 7.52 (s, 1H, N=CH), 3.79 (s, 3H, CH3). MS:
345 m/z (M+). Elemental analysis: Calculated for C20H15N3OS:
C 69.54, H 4.38, N 12.17, S 9.28. Found: C 67.36, H 4.28, N
11.87, S 9.09.
2-Methyl-3-[(2-nitro-benzylidene)amino]-5-phenyl-
3H-thieno[2,3-d]pyrimidin-4-one (5h): Yield: 73 %, m.p.:

Vol. 28, No. 9 (2016)
Design, Synthesis and Biological Evaluation of Thiophene Based Pyrimidin-4-one Derivatives 1999
147-149 °C, IR (KBr, νmax, cm-1): 3066 (C-H, Ar), 2954 (C-H,
RESULTS AND DISCUSSION
CH3), 1663 (C=O), 1649 (C=C,Ar), 1577 (C=N), 1542 (N=O),
1
We have designed, synthesized and characterized a novel
series of compounds, 3-(benzylidene-amino)-2-methyl-5-
phenyl-3H-thieno [2,3-d]pyrimidin-4-one and its derivatives
(5a-i) in good yields. The synthesis of title compounds com-
menced from commercially available 2-amino-4-phenyl-
thiophene-3-carboxylic acid ethyl ester (1) and by including
2-amino-4-phenyl-thiophene-3-carboxylic acid (2), 2-methyl-
5-phenyl-thieno[2,3-d][1,3]oxazin-4-one (3) and 3-amino-2-
methyl-5-phenyl-3H-thieno[2,3-d]pyrimidin-4-one (4) as
intermediates (Scheme-I).
855 (C-S); H NMR (300 MHz, DMSO-d6): δ 7.80 (s, 1H,
thiophene), 7.71-7.37 (m, 9H, Ar-H), 7.59 (s, 1H, N=CH),
2.62 (s, 3H, CH3). MS: 390 m/z (M+). Elemental analysis:
Calculated for C20H14N4O3S: C 61.53, H 3.61, N 14.35, S 8.21.
Found: C 59.87, H 3.52, N 13.98, S 8.07.
2-Methyl-3-[(4-nitro-benzylidene)amino]-5-phenyl-
3H-thieno[2,3-d]pyrimidin-4-one (5i): Yield: 66 %, m.p.:
131-133 °C, IR (KBr, νmax, cm-1): 3055 (C-H, Ar), 2948 (C-H,
CH3), 1645 (C=O), 1653 (C=C,Ar), 1584 (C=N), 1548 (N=O),
1
863 (C-S); H NMR (300 MHz, DMSO-d6): δ 7.79 (s, 1H,
The initial intermediate 2 has been prepared from raw
material 1 on hydrolysis reaction in presence of catalytic
amount sodium hydroxide in ethanol solvent under reflux for
4 h on uniform stirring. The next intermediate 3 was afforded
from the cyclization followed by condensation between
compound 2 with acetic anhydride at reflux temperature on
constant stirring for 6 h. Then compound 3 is turned into final
intermediate 4 on condensation reaction with hydrazine
hydrate in ethanol solvent under reflux for 6 h with constant
stirring. Finally, the target compounds, 3-(benzylidene-amino)-
2-methyl-5-phenyl-3H-thieno [2,3-d]pyrimidin-4-one and its
derivatives (5a-i) were synthesized in good yields from the
condensation of compound 4 with different aromatic aldehydes
in acetic acid on reflux for 14-18 h on steady stirring. The
chemical structures of the newly prepared compounds were
confirmed by their IR, 1H NMR, mass spectral data and elemental
analysis.
Synthesized compounds 5a-i performed significant to
moderate antifungal activity with a degree of variation at
both 0.05 mL (µg) and 0.1 mL (100 µg) concentration level.
Compounds 5d and 5e exhibited high activity and compounds
5h and 5i possessed maximum activity (Fig. 1). The outstanding
properties of this new class of antifungal substances deserve
further investigation in order to clarify the mode of action at
molecular level, responsible for the activity observed. More
extensive study is also warranted to determine additional
physico-chemical and biological parameters to have a deeper
thiophene), 7.72-7.41 (m, 5H, Ar-H), 7.57 (d, 2H, J = 7.6 Hz,
Ar-H), 7.37 (s, 1H, N=CH), 7.19 (d, 2H, J = 7.6 Hz, Ar-H),
2.54 (s, 3H, CH3). MS: 390 m/z (M+). Elemental analysis:
Calculated for C20H14N4O3S: C 61.53, H 3.61, N 14.35, S 8.21.
Found: C 59.87, H 3.52, N 13.98, S 8.07.
Antifungal activity: Compounds, 3-(benzylidene-amino)-
2-methyl-5-phenyl-3H-thieno[2,3-d]pyrimidin-4-one and its
derivatives (5a-i) were screened for their antifungal activity
against two fungal organisms such as Aspergillus niger and
Pencilium chrosogenum in dimethyl sulfoxide (DMSO) by
broth dilution method [10]. Potato dextrose agar (Hi-media)
was dissolved and distributed in 25 mL quantities in 100 mL
conical flasks and were sterilized in an autoclave at 121 °C
(151 lbs/sq.in) for 20 min. The medium was inoculated at 1 %
level using 48 h old cultures of the test organism mentioned
above aseptically in to sterile petridish and allowed to set at
room temperature for about 0.5 h. In size of 4 inches petridish,
four cups of 8 mm diameter at equal distance were made in
each plate. In each plate, one cup was used for control i.e.
dimethyl sulfoxide (DMSO), other for standard fluconazole
with 100 µg/mL. Other two cups with concentrations of test
compound i.e. 50 and 100 µL solutions. The plates thus
prepared were left for 1.5 h in refrigerator for diffusion. After
incubation for 48 h at 25 °C, plates were examined for inhibition
zones. The experiments were performed in duplicate and the
average diameters of the zones of inhibition measured were
recorded.
O
O
O
O
OH
NH2
O
N
(i)
(ii)
S
1
NH2
S
S
N
2
3
(iii)
O
O
N
NH2
N
N
(iv)
S
4
N
S
R
5a-i
Scheme-I: (i) NaOH, EtOH, reflux, 4 h; (ii)Ac2O, reflux, 6 h; (iii) NH2NH2.H2O, EtOH, reflux, 6 h; (iv)ArCHO,AcOH, reflux, 14-16 h; 5a-i
a) H; b) 2-CH3; c) 2-OCH3; d) 2-Cl; e) 4-Cl; f) 2-Br; g) 4-Br; h) 2-NO2; i) 4-NO2

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Flu – Fluconazole
30
Aspergillus niger 50 µL
Aspergillus niger 100 µL
Pencilium chrysogenum 50 µL
Pencilium chrysogenum 100 µL
25
20
15
10
5
7. S.A. Rahaman, Y. Rajendra Pasad, P. Kumar and B. Kumar, Saudi
Pharm. J., 17, 255 (2009).
0
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Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria,
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242 (1982).
Flu
5a
5b
5c
5d
5e
5f
5g
5h
5i
Fig. 1
insight into structure-activity relationship and to optimize the
effectiveness of this series of molecules.
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