Synthesis and antioxidant activity of 1,3,4-oxadiazole tagged thieno[2,3-d]pyrimidine derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.10.007

This study represents the synthesis of a new series of N-substituted phenyl-5-methyl-6-(5-(4-substituted phenyl)-1,3,4-oxadiazol-2-yl)thieno[2,3-d] pyrimidin-4-amine derivatives (4a-l) and substituted phenylamino-5- methylthieno[2,3-d]pyrimidine-6-carboxylic acid derivatives (3a-d). The newly synthesized compounds were characterized by ¹H NMR, ¹³C NMR, LC-MS and IR analyses. All these novel compounds were screened for their in vitro antioxidant activity by employing DPPH, hydrogen peroxide, and nitric oxide radical scavenging assays. Compounds 4k, 4j, 4d, and 4e showed significant radical scavenging due to the presence of electron donating substituent on both sides of the thienopyrimidine ring enhances the activity and electron withdrawing groups like nitro decrease.

Full text of DOI:10.1016/j.ejmech.2012.10.007

European Journal of Medicinal Chemistry 58 (2012) 340e345
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antioxidant activity of 1,3,4-oxadiazole tagged thieno[2,3-d]
pyrimidine derivatives
*
Y. Kotaiah, N. Harikrishna, K. Nagaraju, C. Venkata Rao
Department of Chemistry, Sri Venkateswara University, Tirupati 517 502, Andhra Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
This study represents the synthesis of a new series of N-substituted phenyl-5-methyl-6-(5-(4-substituted
phenyl)-1,3,4-oxadiazol-2-yl)thieno[2,3-d]pyrimidin-4-amine derivatives (4ael) and substituted phe-
nylamino-5-methylthieno[2,3-d]pyrimidine-6-carboxylic acid derivatives (3aed). The newly synthesized
compounds were characterized by 1H NMR, 13C NMR, LCeMS and IR analyses. All these novel compounds
were screened for their in vitro antioxidant activity by employing DPPH, hydrogen peroxide, and nitric
oxide radical scavenging assays. Compounds 4k, 4j, 4d, and 4e showed significant radical scavenging due
to the presence of electron donating substituent on both sides of the thienopyrimidine ring enhances the
activity and electron withdrawing groups like nitro decrease.
Received 6 July 2012
Received in revised form
4 October 2012
Accepted 4 October 2012
Available online 12 October 2012
Keywords:
Thiophene
Thieno[2,3-d]pyrimidines
1,3,4-Oxadiazoles synthesis
Anti-oxidant activity
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
Furthermore, substituted 1,3,4-oxadiazole derivatives also have
been reported to show broad spectrum of biological activities
Several types of pyrimidines and thienopyrimidines have
provoked much interest due to their valuable pharmacological
properties such as antiviral [1], antioxidant [2] and antimalarial [3,4].
The antioxidants that scavenge reactive oxygen species may be of
great value in preventing the onset and propagation of oxidative
diseases like autoimmune diseases, cardiovascular diseases, neuro-
vascular diseases [5] and neurodegenerative changes associated with
aging [6]. The homeostatic balance between the reactive oxygen
species (ROS) and endogenous antioxidants is important in main-
taining healthy tissues. Excessive ROS states are important in
diseases such as acute respiratory distress syndrome and idiopathic
pulmonary fibrosis [7]. Most living organisms possess enzymatic and
non enzymatic defense systems against excessive production of the
reactive oxygen species. However, different external factors (smoke,
diet, alcohol and some drugs) and aging decrease the efficiency of
such protecting systems, resulting in disturbances of the redox
equilibrium established under healthy conditions [8]. In addition, the
pyrimidine based derivatives like thieno[2,3-d]pyrimidines have
fascinated importance in medicinal chemistry, exhibiting pharma-
cological and therapeutic properties such as antidepressant [9],
antibacterial [10e12], antifungal [13,14], anti-inflammatory [15,16],
antiplatelet [17], antihypertensive [18], herbicidal [19] and plant
growth regulatory [20] properties.
including anti-inflammatory, analgesic and ulcerogenicity [21]
antioxidant agents [22]. Hence, the development of physiologi-
cally highly potent fused pyrimidines and 1,3,4-oxadiazole
substituted fused pyrimidines has been of great interest in the
synthesis as well as in the field of medicinal chemistry.
Over view of literature survey, the importance of thieno[2,3-d]
pyrimidines and 1,3,4-oxadiazoles individually in the biological
systems led to assimilate of these two moieties may show syner-
gistic effect. In the present work we report the details of the
synthesis of novel 1,3,4-oxadiazole tagged thieno[2,3-d]pyrimidine
derivatives and in vitro antioxidant properties of them.
2. Results and discussion
2.1. Chemistry
Compound ethyl 5-amino-4-cyano-3-methylthiophene-2-
carboxylate (1) was synthesized via single step process of ethyl
acetoacetate, malononitrile, elemental sulfur in DMF and catalytic
amount of imidazole by the reaction of Gewald method [23].
It was then converted into ethyl (halo substituted phenylamino)-
5-methylthieno[2,3-d]pyrimidine-6-carboxylate (2aed) by cycli-
zation with suitable triethyl orthoformate in the presence of few
drops of acetic acid as a catalyst and appropriate substituted
anilines to a good yield [24,25]. Compound 2 was treated with aq.
* Corresponding author. Tel.: þ91 877 2289303; fax: þ91 877 2249532.
E-mail address: cvrsvu@gmail.com (C. Venkata Rao).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.007

Y. Kotaiah et al. / European Journal of Medicinal Chemistry 58 (2012) 340e345
341
NaOH and MeOH medium to give 4-(substitutedphenylamino)-
5-methylthieno[2,3-d]pyrimidine-6-carboxylic acid derivatives
(3aed). Further compound 3 on treatment with appropriate
substituted arylacidhydrazides in the presence of the POCl3
yielded the title compounds 4ael in good to excellent yields
(Scheme 1). The structures of newly synthesized compounds
3aed & 4ael were confirmed on the basis of 1H NMR, 13C NMR,
LCeMS and IR spectrometry analysis.
All the synthesized intermediates and titled chemical entities gave
satisfactory analyses for the proposed structures on the basis of their
spectral data. The formation of intermediates 2(aed)wereconfirmed by
IR spectra which showed characteristic absorption bands in the range
between 1551e1562 cmꢀ1 and 3417e3428 cmꢀ1 due to C]N and NH
stretchings respectively and the 1H NMR spectral data showed singlets
4e showed good radical scavenging activity in all three methods
due to the presence of mild electron donating groups such as di-
fluoro, fluoro and chloro-fluoro groups attached to the benzene
rings when compared with the standard drug ascorbic acid.
Compounds 4a, 4b, 4g and 4h showed moderate antioxidant
activity whereas the other compounds 4c, 4i and 4f displayed mild
activity. In general, it was observed that halo substituted and
unsubstituted compounds 4k, 4j, 4d and 4e exhibited greater
activity when compared with the respective nitro substituted
compounds. The IC50 value of the standard ascorbic acid in DPPH
method was found to be 15.11
values of the compounds 4k, 4j, 4d and 4e were found to be 16.35,
16.91, 17.25 and 17.70 g/ml, respectively. Further Tables 1e3
mg/ml at 25 mg/ml whereas the IC50
m
indicate that radical scavenging activity in DPPH, nitric oxide and
hydrogen peroxide methods increases with concentration.
in the range between at d 7.39e8.38 ppm and d 8.57e8.60 ppm for NH
and CH groups respectively. Further, the appearance of the broad singlet
in the range 13.54e13.58 ppm confirmed the transformation of
ester 2(aed) to carboxylic acid 3(aed). The IR spectra of compounds
4ael revealed characteristic absorption bands at 1522e1556 cmꢀ1 for
C]N, 1602e1641 cmꢀ1 for C]C, 2915e2986 cmꢀ1 for thiopheneeCH3
and 3051e3486 cmꢀ1 corresponding to NH stretching vibrations. In the
4. Conclusion
In conclusion, a new class of 1,3,4-oxadiazole tagged thieno[2,3-
d]pyrimidines derivatives were prepared from simple starting
material and substituted arylacidhydrazides in good yields and
studied for their antioxidant activity. It was observed that the
compounds having thieno[2,3-d]pyrimidines in combination with
1,3,4-oxadiazoles exhibited greater antioxidant activity. The
investigation of antioxidant screening data reveals that among the
12 compounds screened, compounds 4k, 4j, 4d and 4e showed
excellent, almost equivalent to that of standards the remaining
compounds showed moderate to mild inhibition activity. The
presence of electron donating substituent on both sides of the
thienopyrimidine ring enhances the activity and electron with-
drawing groups like nitro decrease.
1H NMR spectra displayed peaks in the range
of thiophene, 8.21e8.92 ppm for pyrimidineeNH,
d
3.05e3.28 ppm for eCH3
8.60e9.00 ppm for
d
d
CeH groups, and other prominent peaks also appeared in the aromatic
region corresponding to both the phenyl rings which are attached to
thienopyrimidine and oxadiazole rings. The 13C NMR and LCeMS
spectral data have provided further support for the confirmation of
the structures of the synthesized compounds, which are presented in
the Experimental section.
3. Pharmacological assay
3.1. Antioxidant testing
5. Experimental section
Compounds 4ael are tested for in vitro antioxidant property
by 1,1-diphenylpicrylhydrazyl (DPPH) [26,27], nitric oxide (NO)
[28,29] and hydrogen peroxide (H2O2) [30] methods which were
summarized in Tables 1e3 respectively. Compounds 4k, 4j, 4d and
5.1. Chemistry
Melting points were determined in open capillaries on a Mel-
Temp apparatus and are uncorrected. All the reactions were
R
R
R
CN
H2N
HN
HN
CN
a
b
N
N
EtOOC
NH2
OEt
AcOH
S
EtOOC
N
S
EtOOC
HOOC
N
N
S
S
1
2a-d
3a-d
2a, R= 4-Cl
3a, R= 4-Cl
3b, R= 4-F
3c, R= 3-Cl,4-F
3d, R=2,4-di-F
2b, R= 4-F
2c, R= 3-Cl,4-F
2d, R=2,4-di-F
R
CONHNH2
c
HN
R1
N
N N
O
N
S
R1
4a-l
4
a
b
c
d
e
f
g
h
i
j
k
l
4–Cl
4–Cl
4–Cl
4–F
4–F
4–F
3–Cl,
4–F
4–H
3–Cl,
4–F
3–Cl, 2,4–diF 2,4–diF 2,4–diF
4–F
R
4–H
4–Cl 4–NO2 4–H
4–Cl
4–NO2
4–Cl 4–NO2
4–H
4–Cl
4–NO2
R1
Scheme 1. Reagents and conditions: a: HC(OEt)3, (5e10 equiv), reflux, 6 h, AcOH, halo substituted anilines, 4 h, reflux; b: NaOH, MeOH, RT, 16 h; c: POCl3, substituted arylacidhydrazides.

342
Y. Kotaiah et al. / European Journal of Medicinal Chemistry 58 (2012) 340e345
Table 1
The in vitro antioxidant activity of 4ael in DPPH method.
Compound
Concentration (
25
mg/ml)
50
75
100
IC50
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
69.32 ꢂ 1.06
64.70 ꢂ 1.44
52.74 ꢂ 1.73
72.46 ꢂ 0.36
70.59 ꢂ 0.26
59.73 ꢂ 1.17
68.49 ꢂ 1.26
63.87 ꢂ 0.71
48.65 ꢂ 0.60
73.89 ꢂ 0.19
76.43 ꢂ 0.28
64.61 ꢂ 1.05
82.68 ꢂ 0.12
e
71.56 ꢂ 0.87
68.41 ꢂ 1.21
58.25 ꢂ 1.02
75.83 ꢂ 0.60
74.21 ꢂ 0.43
64.48 ꢂ 1.24
72.84 ꢂ 1.41
67.93 ꢂ 1.32
52.86 ꢂ 1.24
77.42 ꢂ 0.45
79.66 ꢂ 0.49
69.51 ꢂ 1.40
83.52 ꢂ 0.38
e
76.84 ꢂ 1.32
73.84 ꢂ 1.56
61.37 ꢂ 0.92
78.36 ꢂ 0.70
77.85 ꢂ 0.65
68.94 ꢂ 0.88
74.93 ꢂ 1.58
71.48 ꢂ 1.29
56.63 ꢂ 0.55
79.63 ꢂ 0.46
83.29 ꢂ 0.61
73.92 ꢂ 0.82
85.52 ꢂ 0.44
e
81.26 ꢂ 1.05
77.52 ꢂ 0.95
67.81 ꢂ 1.60
83.13 ꢂ 0.86
81.92 ꢂ 0.70
72.16 ꢂ 0.95
79.25 ꢂ 1.04
76.64 ꢂ 1.34
61.93 ꢂ 0.82
83.85 ꢂ 0.70
85.76 ꢂ 0.78
77.83 ꢂ 1.63
87.22 ꢂ 0.53
e
18.03 ꢂ 1.07
19.31 ꢂ 1.13
23.70 ꢂ 1.47
17.25 ꢂ 0.52
17.70 ꢂ 0.61
20.92 ꢂ 0.78
18.25 ꢂ 1.10
19.57 ꢂ 1.23
25.69 ꢂ 1.10
16.91 ꢂ 0.60
16.35 ꢂ 0.28
19.34 ꢂ 1.07
15.11 ꢂ 0.44
e
Ascorbic acid
Blank
(e) Showed no scavenging activity. Values were the means of three replicates ꢂ SD.
monitored by thin layer chromatography (TLC) on precoated silica
gel 60 F254 (mesh); spots were visualized with UV light. Merck
silica gel (60e120 mesh) was used for column chromatography.
The IR spectra were recorded on a PerkineElmer BX1 FTIR Spec-
trophotometer as KBr pellets and the wave numbers were given in
was monitored by TLC). The reaction was then concentrated in
vacuo. The obtained residue was dissolved in AcOH as a solvent to
this added (13.47 mmol) of appropriate halo substituted anilines
and refluxed for 4 h. After completion of the starting materials total
reaction mixture was cooled to room temperature for 2 h then
poured into ice cold water and stirred for 15 min, the product was
separated by filtration and washed with water, dried well and
recrystallized from chloroform and n-hexane to give compounds
2(aed) in good yield.
cmꢀ1 1H NMR (400 MHz), and 13C NMR (100 MHz) spectra were
.
recorded on a Bruker AMX 400 MHz NMR spectrometer in
CDCl3/DMSO-d6 solution using TMS as an internal standard. All
chemical shifts are reported in
d (ppm) using TMS as an internal
standard. The mass spectra were recorded on Agilent 1100 LC/MSD
instrument with method API-ES at 70 eV. The microanalyses were
performed on a PerkineElmer 240C elemental analyzer. The anti-
oxidant property was carried out by using Shimadzu UV-2450
spectrophotometer.
5.1.2.1. Ethyl 4-(4-chlorophenylamino)-5-methylthieno[2,3-d]pyrim-
idine-6-carboxylate (2a). Yield 81%; white crystalline solid
m.p. ¼ 174e176 ꢁC; IR (KBr)
n
(cmꢀ1): 3424 (NH), 2922 (thiophenee
CH3), 1708 (C]O), 1561 (C]N); 1H NMR (CDCl3, 400 MHz)
d 1.41
(t, 3H, CH2eCH3), 3.10 (s, 3H, thiopheneeCH3), 4.39 (q, 2H, eCH2e
CH3), 7.36 (d, 2H, AreH, J ¼ 8.0 Hz), 7.43 (s, 1H, NeH), 7.62 (d, 2H,
AreH, J ¼ 8.0 Hz), 8.57 (s, 1H, CeH); 13C NMR (CDCl3, 100 MHz);
5.1.1. Synthesis of ethyl 5-amino-4-cyano-3-methylthiophene-2-
carboxylate (1)
The starting ethyl 5-amino-4-cyano-3-methylthiophene-2-
carboxylate (1) was prepared according to Gewald synthetic
procedure [23].
d 14.26, 15.83, 61.48, 114.93, 117.32, 120.64, 125.86, 134.48, 139.94,
155.13, 157.45, 160.83, 162.25 and 166.18; LCeMS (negative ion
mode): m/z 346 (M ꢀ H)ꢀ for C16H14ClN3O2S.
5.1.2. General procedure for the synthesis of ethyl 4-(substituted
phenylamino)-5-methylthieno[2,3-d]pyrimidine-6-carboxylate (2aed)
5.1.2.2. Ethyl 4-(4-fluorophenylamino)-5-methylthieno[2,3-d]pyrim-
idine-6-carboxylate (2b). Yield 78%; white solid m.p. ¼ 186e188 ꢁC;
A
mixture of ethyl 5-amino-4-cyano-3-methylthiophene-
IR (KBr) n
(cmꢀ1): 3428 (NH), 2920 (thiopheneeCH3), 1706 (C]O),
2-carboxylate (1) (2.0 g 9.5 mmol) was dissolved in dry toluene as
a solvent to this added triethyl orthoformate (4.6 ml, 28.08 mmol)
and catalytic amount of AcOH were refluxed for 4 h (the reaction
1562 (C]N); 1H NMR (CDCl3, 400 MHz)
d 1.50 (t, 3H, CH2eCH3),
3.12 (s, 3H, thiopheneeCH3), 4.34 (q, 2H, eCH2eCH3), 7.32 (d, 2H,
AreH, J ¼ 8.0 Hz), 7.48 (s, 1H, NeH), 7.64 (d, 2H, AreH, J ¼ 8.0 Hz),
Table 2
The in vitro antioxidant activity of 4ael in nitric oxide (NO) method.
Compound
Concentration (
25
mg/ml)
50
75
100
IC50
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
71.26 ꢂ 0.88
68.39 ꢂ 0.90
60.98 ꢂ 1.52
73.06 ꢂ 0.26
71.84 ꢂ 0.17
62.04 ꢂ 1.41
72.90 ꢂ 0.85
64.37 ꢂ 1.18
53.64 ꢂ 1.39
75.23 ꢂ 0.26
78.19 ꢂ 0.28
63.57 ꢂ 1.32
84.72 ꢂ 0.18
e
74.84 ꢂ 1.07
70.61 ꢂ 1.39
63.47 ꢂ 1.21
78.14 ꢂ 0.45
76.29 ꢂ 0.35
66.83 ꢂ 1.56
74.06 ꢂ 0.94
69.22 ꢂ 1.57
56.49 ꢂ 1.24
79.53 ꢂ 0.37
83.28 ꢂ 0.47
71.42 ꢂ 1.24
85.96 ꢂ 0.36
e
77.14 ꢂ 1.41
74.18 ꢂ 0.95
67.56 ꢂ 1.31
82.44 ꢂ 0.62
79.65 ꢂ 0.54
70.92 ꢂ 0.78
78.25 ꢂ 1.06
74.69 ꢂ 1.41
60.03 ꢂ 0.71
82.64 ꢂ 0.57
84.62 ꢂ 0.61
76.22 ꢂ 1.04
88.32 ꢂ 0.52
e
82.93 ꢂ 1.27
79.42 ꢂ 1.21
72.36 ꢂ 0.71
84.87 ꢂ 0.79
83.59 ꢂ 0.69
73.89 ꢂ 1.07
82.36 ꢂ 1.41
79.53 ꢂ 0.71
64.28 ꢂ 1.08
85.44 ꢂ 0.72
86.18 ꢂ 0.80
78.96 ꢂ 0.79
90.41 ꢂ 0.69
e
17.54 ꢂ 0.95
18.27 ꢂ 1.05
20.49 ꢂ 0.94
17.10 ꢂ 0.91
17.39 ꢂ 1.15
20.14 ꢂ 0.97
17.14 ꢂ 0.56
19.41 ꢂ 1.23
23.30 ꢂ 1.09
16.61 ꢂ 0.56
15.98 ꢂ 0.77
19.66 ꢂ 1.12
14.75 ꢂ 0.58
e
Ascorbic acid
Blank
(e) Showed no scavenging activity. Values were the means of three replicates ꢂ SD.

Y. Kotaiah et al. / European Journal of Medicinal Chemistry 58 (2012) 340e345
343
Table 3
The in vitro antioxidant activity of 4ael in hydrogen peroxide (H2O2) method.
Compound
Concentration (
25
mg/ml)
50
75
100
IC50
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
61.06 ꢂ 0.86
58.29 ꢂ 1.12
51.37 ꢂ 1.16
66.28 ꢂ 1.12
64.12 ꢂ 0.27
53.08 ꢂ 0.89
60.26 ꢂ 1.06
61.94 ꢂ 1.32
45.02 ꢂ 0.88
63.87 ꢂ 0.30
68.42 ꢂ 0.28
54.38 ꢂ 1.19
76.41 ꢂ 0.18
e
63.84 ꢂ 1.58
62.31 ꢂ 1.17
54.03 ꢂ 0.86
69.53 ꢂ 1.31
67.44 ꢂ 0.64
56.61 ꢂ 1.39
63.48 ꢂ 1.27
64.51 ꢂ 1.18
47.38 ꢂ 1.17
65.49 ꢂ 0.46
71.68 ꢂ 0.45
58.07 ꢂ 0.88
78.60 ꢂ 0.33
e
68.22 ꢂ 1.07
65.74 ꢂ 1.47
58.12 ꢂ 0.97
71.68 ꢂ 0.60
69.81 ꢂ 0.69
59.95 ꢂ 0.78
67.84 ꢂ 1.57
68.22 ꢂ 1.07
50.44 ꢂ 1.27
68.97 ꢂ 0.65
74.51 ꢂ 0.61
62.75 ꢂ 1.49
82.39 ꢂ 0.61
e
71.59 ꢂ 1.37
69.80 ꢂ 1.55
63.72 ꢂ 0.63
74.19 ꢂ 0.71
72.59 ꢂ 0.80
64.20 ꢂ 1.05
70.93 ꢂ 0.83
71.86 ꢂ 1.57
53.61 ꢂ 1.39
72.52 ꢂ 0.78
79.66 ꢂ 0.82
66.19 ꢂ 1.00
86.59 ꢂ 0.71
e
20.47 ꢂ 1.23
21.22 ꢂ 1.07
24.33 ꢂ 1.04
18.85 ꢂ 0.60
19.49 ꢂ 0.26
23.54 ꢂ 0.42
20.74 ꢂ 0.52
20.18 ꢂ 0.78
27.76 ꢂ 0.64
19.57 ꢂ 1.26
18.26 ꢂ 0.95
22.98 ꢂ 0.69
16.35 ꢂ 0.27
e
Ascorbic acid
Blank
(e) Showed no scavenging activity. Values were the means of three replicates ꢂ SD.
8.60 (s, 1H, CeH); 13C NMR (CDCl3, 100 MHz):
d
14.14, 16.76, 61.11,
(br s, 1H, eCOOH); 13C NMR (DMSO-d6,100 MHz):
d
16.73, 115.48,
103.54, 111.26, 119.58, 122.83, 124.64, 128.84, 141.97, 154.37, 158.15,
163.16 and 171.72; LCeMS (negative ion mode): m/z 330 (M ꢀ H)ꢀ
for C16H14FN3O2S.
119.87, 122.69, 124.53, 135.18, 140.06, 152.19, 153.34, 155.73, 157.47
and 164.96; LCeMS (positive ion mode): m/z 320 (M þ H)þ for
C14H10ClN3O2S.
5.1.2.3. Ethyl 4-(3-chloro-4-fluorophenylamino)-5-methylthieno
[2,3-d]pyrimidine-6-carboxylate (2c). Yield 72%; pale-yellow solid
5.1.3.2. 4-(4-Fluorophenylamino)-5-methylthieno[2,3-d]pyrimidine-
6-carboxylic acid (3b). Yield 88%; white solid m.p. ¼ 260e262 ꢁC; IR
m.p. ¼154e156 ꢁC; IR (KBr)
n
(cmꢀ1): 3417 (NH), 2923 (thiophenee
(KBr) n
(cmꢀ1): 3430 (NH), 2920 (thiopheneeCH3), 1713 (C]O),
CH3), 1708 (C]O), 1557 (C]N); 1H NMR (CDCl3, 400 MHz)
d
1.42
1575 (C]N); 1H NMR (DMSO-d6, 400 MHz)
d 3.05 (s, 3H,
(t, 3H, eCH2eCH3), 3.11 (s, 3H, thiopheneeCH3), 4.40 (q, 2H, eCH2e
CH3), 7.19 (t, 1H, AreH, J ¼ 9.0 Hz), 7.39 (s, 1H, NeH), 7.45 (t, 1H, Are
thiopheneeCH3), 7.24 (t, 2H, AreH, J ¼ 8.0 Hz), 7.64 (q, 2H, AreH,
J ¼ 4.0 Hz), 8.49 (s, 1H, N-H), 8.81 (s, 1H, CeH), 13.55 (br s, 1H, e
H, J ¼ 8.0 Hz), 7.86 (d, 1H, AreH, J ¼ 4.0 Hz), 8.58 (s, 1H, CeH); 13
C
COOH); 13C NMR (DMSO-d6, 100 MHz):
d 15.26, 117.54, 123.70,
NMR (CDCl3, 100 MHz):
d
14.29, 16.14, 60.89, 113.42, 116.31, 117.84,
124.84, 128.38, 137.93, 138.86, 154.52, 156.48, 163.31, 166.93 and
119.62, 122.45, 140.79, 144.16, 149.49, 151.60, 154.71, 158.36, 159.24
169.26; LCeMS (positive ion mode): m/z 304 (M þ H)þ for
and 169.86; LCeMS (negative ion mode): m/z 365 (M ꢀ H)ꢀ for
C14H10FN3O2S.
C
16H13ClFN3O2S.
5.1.3.3. 4-(3-Chloro-4-fluorophenylamino)-5-methylthieno[2,3-d]
pyrimidine-6-carboxylic acid (3c). Yield 84%; white solid
5.1.2.4. Ethyl
pyrimidine-6-carboxylate (2d). Yield 82%; white solid, m.p. ¼ 192e
194 ꢁC; IR (KBr) (cmꢀ1): 3420 (NH), 2926 (thiopheneeCH3), 1710
(C]O),1552 (C]N); 1H NMR (CDCl3, 400 MHz)
1.33 (t, 3H, eCH2e
4-(2,4-difluorophenylamino)-5-methylthieno[2,3-d]
m.p.
(thiopheneeCH3), 1692 (C]O), 1562 (C]N); 1H NMR (DMSO-d6,
400 MHz) 3.04 (s, 3H, thiopheneeCH3), 7.44 (t, 1H, AreH,
¼
305e307 ꢁC; IR (KBr) (cmꢀ1): 3430 (NH), 2927
n
n
d
d
CH3), 3.00 (s, 3H, thiopheneeCH3), 4.32 (q, 2H, eCH2eCH3), 7.19
(m, 1H, AreH, J ¼ 4.0 Hz), 7.36 (m, 1H, AreH, J ¼ 8.0 Hz), 7.63 (q, 1H,
AreH, J ¼ 8.0 Hz), 8.38 (s, 1H, NeH), 8.59 (s, 1H, CeH); 13C NMR
J ¼ 8.0 Hz), 7.64 (t, 1H, AreH, J ¼ 4.0 Hz), 7.92 (d, 1H, AreH,
J ¼ 4.0 Hz), 8.52 (s, 1H, NeH), 8.70 (s, 1H, CeH), 13.56 (br s, 1H, e
COOH); 13C NMR (DMSO-d6,100 MHz):
d 16.18,115.94,117.28,118.56,
(CDCl3, 100 MHz):
d
14.16,15.38, 61.07,103.84,106.46,107.91,114.66,
121.72, 124.61, 139.83, 147.14, 148.69, 151.37, 154.48, 158.14, 161.62
118.74, 130.89, 144.93, 148.68, 152.58, 154.16, 156.42, 158.73 and
and 164.81; LCeMS (positive ion mode): m/z 338 (M þ H)þ for
162.58; LCeMS (negative ion mode): m/z 348 (M ꢀ H)ꢀ for
C14H9ClFN3O2S.
C
16H13F2N3O2S.
5.1.3.4. 4-(2,4-Difluorophenylamino)-5-methylthieno[2,3-d]pyrimi-
dine-6-carboxylic acid (3d). Yield 81%; white crystalline solid
5.1.3. General procedure for the synthesis of 4-(substituted
phenylamino)-5-methylthieno[2,3-d]pyrimidine-6-carboxylic acid
(3aed)
Compounds 2(aed) was dissolved in MeOH/H2O (8 ml:4 ml),
and 15% v/v NaOH aq (2 ml) was added. Stirring was continued for
16 h at rt, then CHCl3 was added. The aqueous layer was acidified
with 1 N HCl, stirred for 15 min, the product was separated by
vacuum filtration and washed with water, dried well and recrys-
tallized from chloroform and methanol to give compounds 3(aed)
in good yield.
m.p.
(thiopheneeCH3), 1712 (C]O), 1565 (C]N); 1H NMR (DMSO-d6,
400 MHz) 3.00 (s, 3H, thiopheneeCH3), 7.15 (t, 1H, AreH,
¼
294e296 ꢁC; IR (KBr) (cmꢀ1): 3417 (NH), 2926
n
d
J ¼ 8.0 Hz), 7.37 (t, 1H, AreH, J ¼ 8.0 Hz), 7.65 (q, 1H, AreH,
J ¼ 8.0 Hz), 8.38 (s, 1H, NeH), 8.56 (s, 1H, CeH), 13.54 (br s, 1H, e
COOH); 13C NMR (DMSO-d6,100 MHz):
d 14.47, 109.25, 115.62,
118.58, 126.93, 141.86, 147.72, 151.35, 153.48, 158.74, 159.28, 161.39,
164.18 and 169.06; LCeMS (positive ion mode): m/z 322 (M þ H)þ
for C14H9F2N3O2S.
5.1.3.1. 4-(4-Chlorophenylamino)-5-methylthieno[2,3-d]pyrimidine-
5.1.4. General procedure for the synthesis of (4ael)
6-carboxylic acid (3a). Yield 85%; off-white solid, m.p. ¼ 316e
An equimolar mixture of 4-(substituted phenylamino)-5-
methylthieno[2,3-d]pyrimidine-6-carboxylic acids 3(aed) (0.01 mol)
and appropriate substituted aromatic carboxylic acid hydrazide
(0.01 mol) in phosphorus oxychloride (20 ml) was refluxed for 5 h. The
reaction mixture was cooled to room temperature and then gradually
318 ꢁC; IR (KBr)
n
(cmꢀ1): 3450 (NH), 2922 (thiopheneeCH3),
1700 (C]O), 1548 (C]N); 1H NMR (DMSO-d6, 400 MHz)
d 3.05
(s, 3H, thiopheneeCH3), 7.44 (d, 2H, AreH, J ¼ 8.0 Hz), 7.69
(d, 2H, AreH, J ¼ 8.0 Hz), 8.51 (s, 1H, CeH), 8.68 (s, 1H, NeH), 13.58

344
Y. Kotaiah et al. / European Journal of Medicinal Chemistry 58 (2012) 340e345
poured on to crushed ice with stirring. The mixture was allowed
standing overnight and the solid separated out was filtered, treated
with dilute sodium hydroxide solution and washed thoroughly with
cold water, well dried and which was crystallized with chloroform and
hexane to give the title compounds 4(ael) in good yield.
5.1.4.6. N-(4-Fluorophenyl)-5-methyl-6-(5-(4-nitrophenyl)-1,3,4-
oxadiazol-2-yl)thieno-[2,3-d]pyrimidin-4-amine (4f). Brown solid
(80%); m.p. ¼ 231e233 ꢁC; IR (KBr)
n
(cmꢀ1): 3254 (NH), 2916
(thiopheneeCH3), 1606 (C]C), 1554 (C]N); 1H NMR (CDCl3,
400 MHz)
d 3.36 (s, 3H, thiopheneeCH3), 7.41 (t, 2H, AreH,
J ¼ 8.0 Hz), 7.59 (q, 2H, AreH, J ¼ 4.0 Hz), 7.71 (d, 2H, AreH,
5.1.4.1. N-(4-Chlorophenyl)-5-methyl-6-(5-phenyl-1,3,4-oxadiazol-
J ¼ 8.0 Hz), 7.96 (dd, 2H, AreH, J ¼ 8.0 Hz), 8.35 (s, 1H, NeH),
2-yl)thieno[2,3-d]pyrimidin-4-amine (4a). Brown solid (80%);
8.86 (s, 1H, CeH); 13C NMR (CDCl3, 100 MHz):
d 17.56, 116.42,
m.p.
(thiopheneeCH3), 1605 (C]C), 1550 (C]N); 1H NMR (DMSO-d6,
400 MHz) 3.14 (s, 3H, thiopheneeCH3), 7.46 (d, 2H, AreH,
¼
248e250 ꢁC; IR (KBr)
n
(cmꢀ1): 3486 (NH), 2915
116.63, 117.49, 123.60, 127.66, 131.92, 135.48, 137.83, 137.64, 139.12,
149.08, 151.74, 153.63, 157.29, 162.48 and 166.27; LCeMS (positive
ion mode): m/z 449 (M þ H)þ for C21H13FN6O3S.
d
J
¼
8.0 Hz), 7.64e7.72 (m, 5H, AreH), 8.10 (d, 2H, AreH,
J ¼ 8.0 Hz), 8.53 (s, 1H, NeH), 8.84 (bs, 1H, CeH); 13C NMR
5.1.4.7. N-(3-Chloro-4-fluorophenyl)-5-methyl-6-(5-phenyl-1,3,4-
oxadiazol-2-yl)thieno[2,3-d]pyrimidin-4-amine (4g). Pale yellow
(DMSO-d6, 100 MHz); d 14.03, 115.46, 116.28, 120.89, 123.06, 124.74,
125.87, 128.20, 129.46, 131.36, 135.99, 142.15, 147.57, 154.95, 158.26,
160.78 and 168.91; LCeMS (negative ion mode): m/z 418 (M ꢀ H)ꢀ
for C21H14ClN5OS.
solid (70%); m.p. ¼ 214e216 ꢁC; IR (KBr)
n
(cmꢀ1): 3248 (NH), 2934
(thiopheneeCH3), 1614 (C]C), 1547 (C]N); 1H NMR (DMSO-d6,
400 MHz)
d 3.05 (s, 3H, thiopheneeCH3), 7.48 (t, 1H, AreH,
J ¼ 8.0 Hz), 7.54e7.68 (m, 5H, AreH), 7.70 (dd, 1H, AreH,
5.1.4.2. N-(4-Chlorophenyl)-6-(5-(4-chlorophenyl)-1,3,4-oxadiazol-
J ¼ 4.0 Hz), 7.96 (dd, 1H, AreH, J ¼ 4.0 Hz), 8.57 (s, 1H, NeH),
2-yl)-5-methylthieno[2,3-d]pyrimidin-4-amine (4b). Pale yellow
8.75 (s, 1H, CeH); 13C NMR (DMSO-d6, 100 MHz):
d 17.69, 116.32,
solid (85%); m.p. ¼ 232e234 ꢁC; IR (KBr)
n
(cmꢀ1): 3256 (NH), 2922
116.55, 117.02, 118.96, 120.54, 126.71, 127.44, 128.51, 129.48, 132.16,
137.27, 141.84, 151.94, 152.61, 153.76, 157.23, 162.09 and 163.71; LCe
MS (positive ion mode): m/z 438 (M þ H)þ for C21H13ClFN5OS.
(thiopheneeCH3), 1602 (C]C), 1556 (C]N); 1H NMR (CDCl3,
400 MHz)
d 3.25 (s, 3H, thiopheneeCH3), 7.40 (d, 2H, AreH,
J ¼ 8.0 Hz), 7.55 (d, 2H, AreH, J ¼ 8.0 Hz), 7.65 (d, 2H, AreH,
J ¼ 8.0 Hz), 8.09 (d, 2H, AreH, J ¼ 8.0 Hz), 8.60 (s, 1H, NeH), 8.92
5.1.4.8. N-(3-Chloro-4-fluorophenyl)-6-(5-(4-chlorophenyl)-
1,3,4-oxadiazol-2-yl)-5-methylthieno[2,3-d]pyrimidin-4-amine (4h).
(br s, 1H, CeH); 13C NMR (CDCl3, 100 MHz):
d 14.10, 116.84, 117.47,
121.80, 123.46, 128.40,128.75,129.26, 129.68,130.31,132.75,136.20,
138.62, 155.02, 156.66, 161.02 and 163.38; LCeMS (negative ion
mode): m/z 452 (M ꢀ H)ꢀ for C21H13Cl2N5OS.
Yellow solid (77%); m.p. ¼ 254e256 ꢁC; IR (KBr)
n
(cmꢀ1): 3196
(NH), 2930 (thiopheneeCH3), 1641 (C]C), 1548 (C]N); 1H NMR
(DMSO-d6, 400 MHz) d 3.19 (s, 3H, thiopheneeCH3), 7.52 (t, 1H, Are
H, J ¼ 8.0 Hz), 7.72 (m, 1H, AreH), 7.76 (d, 2H, AreH, J ¼ 8.0 Hz), 8.01
5.1.4.3. N-(4-Chlorophenyl)-5-methyl-6-(5-(4-nitrophenyl)-1,3,4-
(dd, 1H, AreH, J ¼ 4.0 Hz), 8.16 (d, 2H, AreH, J ¼ 8.0 Hz), 8.60 (s, 1H,
oxadiazol-2-yl)thieno[2,3-d]pyrimidin-4-amine (4c). Yellow solid
NeH), 8.90 (s, 1H, CeH); 13C NMR (DMSO-d6, 100 MHz)
d 16.16,
(78%); m.p. ¼ 210e212 ꢁC; IR (KBr)
n
(cmꢀ1): 3221 (NH), 2926
115.67, 116.64, 117.48, 118.78, 121.91, 123.96, 128.53, 129.66, 135.23,
135.87, 137.02, 142.68, 149.22, 152.71, 154.62, 156.52, 162.87 and
167.43; LCeMS (negative ion mode): m/z 470 (M ꢀ H)ꢀ for
(thiopheneeCH3), 1605 (C]C), 1528 (C]N); 1H NMR (CDCl3,
400 MHz)
d 3.28 (s, 3H, thiopheneeCH3), 7.41 (d, 2H, AreH,
J ¼ 8.0 Hz), 7.51 (d, 2H, AreH, J ¼ 8.0 Hz), 7.65 (d, 2H, AreH,
C21H12Cl2FN5OS.
J ¼ 8.0 Hz), 8.48 (dd, 2H, AreH, J ¼ 8.0 Hz), 8.62 (s, 1H, NeH),
8.97 (br s, 1H, CeH); 13C NMR (CDCl3, 100 MHz):
d
16.14, 116.26,
5.1.4.9. N-(3-Chloro-4-fluorophenyl)-5-methyl-6-(5-(4-nitrophenyl)-
1,3,4-oxadiazol-2-yl)thieno[2,3-d]pyrimidin-4-amine (4i). Yellow solid
117.68, 122.36, 124.16, 124.42, 128.75, 129.22, 129.68, 130.86, 135.42,
138.62, 146.64, 155.34, 156.12, 163.48, and 167.10; LCeMS (negative
ion mode): m/z 463 (M ꢀ H)ꢀ for C21H14ClN5OS.
(81%); m.p. ¼ 239e241 ꢁC; IR (KBr)
n
(cmꢀ1): 3051 (NH), 2959
(thiopheneeCH3), 1614 (C]C), 1531 (C]N); 1H NMR (DMSO-d6,
400 MHz)
d
3.28 (s, 3H, thiopheneeCH3), 7.20 (t,1H, AreH, J ¼ 8.0 Hz),
5.1.4.4. N-(4-Fluorophenyl)-5-methyl-6-(5-phenyl-1,3,4-oxadiazol-
7.50 (m, 1H, AreH), 7.89 (dd, 1H, AreH, J ¼ 4.0 Hz), 8.49 (d, 2H, AreH,
2-yl)thieno[2,3-d]pyrimidin-4-amine (4d). Brown solid (72%);
J ¼ 8.0 Hz), 8.55 (dd, 2H, AreH, J ¼ 8.0 Hz), 8.63 (s, 1H, NeH), 9.00 (s,
m.p.
(thiopheneeCH3), 1612 (C]C), 1522 (C]N); 1H NMR (CDCl3,
400 MHz) 3.08 (s, 3H, thiopheneeCH3), 7.36 (t, 2H, AreH,
¼
228e230 ꢁC; IR (KBr)
n
(cmꢀ1): 3224 (NH), 2925
1H, CeH); 13C NMR (DMSO-d6,100 MHz)
d 16.87,115.38,116.01,117.59,
118.32, 122.02, 124.72, 128.37, 130.63, 132.54, 132.61, 139.81, 148.87,
149.52, 153.73, 15.21, 157.22, 162.19 and 163.37; LCeMS (negative ion
mode): m/z 481 (M ꢀ H)ꢀ for C21H12ClFN6O3S.
d
J ¼ 8.0 Hz), 7.54 (q, 2H, AreH, J ¼ 4.0 Hz), 7.61e7.74 (m, 5H, Are
H), 8.21 (s, 1H, NeH), 8.60 (s, 1H, CeH); 13C NMR (CDCl3,
100 MHz):
d
17.70, 116.49, 117.28, 118.50, 126.98, 127.04, 128.54,
5.1.4.10. N-(2,4-Difluorophenyl)-5-methyl-6-(5-phenyl-1,3,4-
oxadiazol-2-yl)thieno[2,3-d]pyrimidin-4-amine (4j). Brown solid
129.86, 132.39, 137.64, 139.58, 153.88, 155.41, 158.59, 158.71, 161.69
and 163.02; LCeMS (negative ion mode): m/z 402 (M ꢀ H)ꢀ for
(76%); m.p. ¼ 261e263 ꢁC; IR (KBr)
n
(cmꢀ1): 3251 (NH), 2926
C
21H14FN5OS.
(thiopheneeCH3), 1610 (C]C), 1535 (C]N); 1H NMR (CDCl3,
400 MHz)
d 3.18 (s, 3H, thiopheneeCH3), 7.20 (m, 1H, AreH,
5.1.4.5. 6-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-N-(4-fluo-
J ¼ 4.0 Hz), 7.38 (m, 1H, AreH, J ¼ 4.0 Hz), 7.63 (q, 1H, AreH,
rophenyl)-5-methylthieno[2,3-d]pyrimidin-4-amine
(4e). Brown
J ¼ 8.0 Hz), 7.84e7.91 (m, 5H, AreH), 8.76 (s, 1H, NeH), 8.92 (s,
solid (76%); m.p. ¼ 246e248 ꢁC; IR (KBr)
n
(cmꢀ1): 3198 (NH), 2986
1H, CeH); 13C NMR (CDCl3, 100 MHz)
d 16.15, 114.81, 116.63, 117.51,
(thiopheneeCH3), 1610 (C]C), 1525 (C]N); 1H NMR (CDCl3,
118.97, 123.86, 126.72, 127.46, 128.51, 129.48, 132.21, 135.87, 147.35,
152.73, 154.54, 155.14, 156.46, 162.97 and 163.59; LCeMS (positive
ion mode): m/z 422 (M þ H)þ for C21H13F2N5OS.
400 MHz)
d 3.12 (s, 3H, thiopheneeCH3), 7.32 (t, 2H, AreH,
J ¼ 8.0 Hz),7.56 (q, 2H, AreH, J ¼ 4.0 Hz), 7.64 (d, 2H, AreH,
J ¼ 8.0 Hz), 7.98 (d, 2H, AreH, J ¼ 8.0 Hz), 8.24 (s, 1H, NeH), 8.78
(s, 1H, CeH); 13C NMR (CDCl3, 100 MHz):
d
15.91, 114.81, 116.63,
5.1.4.11. 6-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-N-
118.77, 123.93, 128.58, 129.54, 132.21, 135.01, 137.83, 138.68, 148.72,
152.28, 153.31, 157.09, 162.29 and 167.10; LCeMS (positive ion
mode): m/z 438 (M þ H)þ for C21H13ClFN5OS.
(2,4-difluorophenyl)-5-methylthieno-[2,3-d]pyrimidin-4-amine (4k).
White solid (81%); m.p. ¼ 248e250 ꢁC; IR (KBr)
n
(cmꢀ1): 3244
(NH), 2921 (thiopheneeCH3), 1608 (C]C), 1526 (C]N); 1H NMR

Y. Kotaiah et al. / European Journal of Medicinal Chemistry 58 (2012) 340e345
345
(CDCl3, 400 MHz)
d
3.25 (s, 3H, thiopheneeCH3), 7.18 (m, 1H, AreH,
chromatophore was measured at 546 nm. Nitric oxide scavenging
activity was calculated using Eq. (1).
J ¼ 4.0 Hz), 7.28 (m, 1H, AreH, J ¼ 4.0 Hz), 7.58 (q, 1H, AreH,
J ¼ 8.0 Hz), 7.68 (d, 2H, AreH, J ¼ 8.0 Hz), 7.74 (d, 2H, AreH,
J ¼ 8.0 Hz), 8.48 (s, 1H, NeH), 8.69 (s, 1H, CeH); 13C NMR (CDCl3,
6.4. Hydrogen peroxide (H2O2) scavenging activity
100 MHz):
d 17.70, 110.92, 115.04, 117.51, 119.26, 123.69, 124.72,
127.81, 129.47, 132.63, 135.94, 137.14, 149.08, 152.91, 155.32, 156.82,
160.10, 163.26 and 164.91; LCeMS (positive ion mode): m/z 456
(M þ H)ꢀ for C21H12ClF2N5OS.
The H2O2 scavenging ability of the test compound was deter-
mined according to the method of Ruch et al. A solution of H2O2
(40 mM) was prepared in phosphate buffer (pH 7.4). 25, 50, 75 and
100 mg/ml concentrations of the test compounds in 3.4 ml phos-
5.1.4.12. N-(2,4-Difluorophenyl)-5-methyl-6-(5-(4-nitrophenyl)-1,3,4-
oxadiazol-2-yl)thieno-[2,3-d]pyrimidin-4-amine (4l). Yellow solid
phate buffer were added to H2O2 solution (0.6 ml, 40 mM). The
absorbance value of the reaction mixture was recorded at 230 nm.
The percent of scavenging of H2O2 was calculated using Eq. (1).
(77%); m.p. ¼ 226e228 ꢁC; IR (KBr)
n
(cmꢀ1): 3198 (NH), 2952
(thiopheneeCH3), 1620 (C]C), 1526 (C]N); 1H NMR (DMSO-d6,
400 MHz)
d
3.28 (s, 3H, thiopheneeCH3), 7.24 (t,1H, AreH, J ¼ 8.0 Hz),
Acknowledgments
7.33 (m, 1H, AreH, J ¼ 4.0 Hz), 7.64 (q, 1H, AreH, J ¼ 8.0 Hz), 8.34 (d,
2H, AreH, J ¼ 8.0 Hz), 8.42 (dd, 2H, AreH, J ¼ 8.0 Hz), 8.71 (s, 1H, Ne
Authors are thankful to Prof. Ch. Appa Rao, Department of
Biochemistry, Sri Venkateswara University, Tirupati, India for
providing resource to carry out antioxidant activity. Also, one of the
authors (YK) is grateful acknowledge to UGC-BSR for financial
assistance.
H), 8.96 (s, 1H, CeH); 13C NMR (CDCl3, 100 MHz):
d 17.53, 116.34,
116.65, 117.07, 118.77, 123.04, 128.58, 129.49, 131.94, 132.21, 139.61,
147.05, 149.58, 153.35, 153.80, 156.21, 162.10, 163.74 and 165.61; LCe
MS (negative ion mode): m/z 465 (M ꢀ H)ꢀ for C21H12F2N6O3S.
6. Pharmacological screening
References
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Compounds 4ael are tested for antioxidant property by DPPH,
NO and H2O2 methods.
6.2. DPPH radical scavenging activity
The hydrogen atom or electron donation ability of the
compounds was measured from the bleaching of the purple colored
methanol solution of 1,1-diphenyl-1-picrylhydrazyl (DPPH). The
spectrophotometric assay uses the stable radical DPPH as a reagent.
1 ml of various concentrations of the test compounds (25, 50, 75,
100 and 250 mg/ml) in methanol was added to 4 ml of 0.004% (w/v)
methanol solution of DPPH. After a 30 min incubation period at
room temperature, the absorbance was read against blank at
517 nm. The percent of inhibition (I %) of free radical production
from DPPH was calculated by the following equation
% of scavenging ¼ ½ðA control ꢀ A sampleÞ=A blankꢃ ꢄ 100 (1)
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where A control is the absorbance of the control reaction (con-
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