Synthetic tactics of new class of 4-aminothieno[2,3-d]pyrimidine-6- carbonitrile derivatives acting as antimicrobial agents

Article abstract of DOI:10.1016/j.ejmech.2013.03.039

Thermal selective reactions were studied on oxothieno[2,3-d]pyrimidine-6- carboxamide 3 with POCl₃ and PCl₅. At 25-50 C, the C ₇-amide rearranges to nitrile furnished compound 4 in 85-90% yield, while at 80-110 C furnished

Full text of DOI:10.1016/j.ejmech.2013.03.039

European Journal of Medicinal Chemistry 64 (2013) 314e320
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthetic tactics of new class of 4-aminothieno[2,3-d]pyrimidine-6-
carbonitrile derivatives acting as antimicrobial agents
,
b
Shrikant B. Kanawade a, Raghunath B. Toche a , Dhanji P. Rajani
*
a Organic Chemistry Research Centre, Department of Chemistry, K.R.T. Arts, B.H. Commerce and A.M. Science College, Gangapur Road, Nashik 422 002,
Maharashtra, India1
b Microcare Laboratory and TRC, Unapani Road, Lal Darwaja, Surat 395 003, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Thermal selective reactions were studied on oxothieno[2,3-d]pyrimidine-6-carboxamide 3 with POCl3
and PCl5. At 25e50 ꢀC, the C7-amide rearranges to nitrile furnished compound 4 in 85e90% yield, while
at 80e110 ꢀC furnished mixture of products 4 and 5 in 28e68% yields. The chloro displacement with
amines in compound 5 yielded 4-aminothieno[2,3-d]pyrimidine-6-carbonitrile derivatives 8(aeh) and
9(aee). Antimicrobial activity of new compounds was studied against several bacteria such as Staphy-
lococcus aureus MTCC-96, Escherichia coli MTCC-443, Pseudomonas aeruginosa MTCC-4 41, Streptococcus
pyogenes MTCC-442 and fungi Aspergillus niger MTCC-282, Aspergillus clavatus MTCC-1323, Candida
albicans MTCC-227 using broth microdilution method. Compounds 4, 8b, 8d, 8e, 8h and 9a showed
promising antibacterial activity compared to ampicillin and compounds 8b, 8h showed better antifungal
activity compared to greseofulvin.
Received 22 October 2012
Received in revised form
10 March 2013
Accepted 21 March 2013
Available online 1 April 2013
Keywords:
Conventional method
Non-conventional green method
Thieno[2,3-d]pyrimidine
4-Chlorothieno[2,3-d]pyrimidine
Ipso substitution
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
(Fig. 1). Consequently, thienopyrimidines have become a well sought
privileged class of compounds in drug discovery programs.
Antibiotics have revolutionized the medical care in the 20th
century. With the discovery of antibiotics, people were convinced
that infectious diseases might someday be wiped out. However, the
emergence of superbugs i.e. pathogenic bacteria that resist the ef-
fects of most powerful antibiotics available today are posing a great
challenge to the field of medicines. Thus scientists are working to
find new ways to defeat bacteria which has become one of the most
important areas of antibacterial research today. In addition, it is
known that antifungal drugs do not have selective activity because
of the biochemical similarity between human cell and fungi forms.
Hence there are many studies focused on antibacterial and anti-
fungal compounds [1e3]. It is well known that pyrimidine and
fused pyrimidine derivatives are of great biological interest, espe-
cially as antiviral, antitumor and antimicrobial agents [4e15].
Among thieno[2,3-d]pyrimidine derivatives, the 3-amino-5,6-
dimethyl-2-[4-(1-phenylmethyl)-1-piperazinyl]thieno[2,3-d]pyrim-
idine-4-(3H)-one I [16] and 4-(4-methyl-1-piperazinyl)-2-methyl-
6,7-dihydro-5H-cyclopenta [4,5]thieno[2,3-d]pyrimidine II [17]
exhibited remarkable affinity and selectivity for the 5-HT3 receptor
Pyrimidine nucleus fused with other heterocycle has been found
to be an integral part of natural products, agrochemicals and vet-
erinary products [18,19]. This class of compounds has also been
found wide applications in the design and discovery of novel
bioactive compounds and drugs [20] such as kinase inhibitors [21],
LHCGR agonists [22], phosphodiesteras inhibitors [23], antifolate
and antimalarial agents [24], blood platelet aggregation inhibitors
[25] and reverse inhibitors of the gastric (Hþ/Kþ) ATPase [26].
On this context, we have previously reported the synthesis and
efficacy of various polysubstituted thiophenes, fused thienopyr-
imidines as very good molluscicidal agents [27]. In pursuance of our
ongoing research in the synthesis and biological activity study of new
class of thienopyrimidines, we are interested to generate small library
based on this vital thieno[2,3-d]pyrimidine scaffold by both base
catalyzed conventionalheating andcatalystfree neatheating method.
2. Results and discussion
2.1. Chemistry
Recently, we have reported the synthesis of 5-amino-3-(4-
chlorophenyl)thiophene-2,4-dicarbo-nitrile [28] 1 and 5-amino-
3-(4-chlorophenyl)thiophene-2,4-dicarboxamide [28] 2 which
* Corresponding author. Tel.: þ91 0253 2571376; fax: þ91 0253 2577341.
E-mail address: raghunath_toche@rediffmail.com (R.B. Toche).
Affiliated to University of Pune, Pune, Maharashtra, India.
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.039

S.B. Kanawade et al. / European Journal of Medicinal Chemistry 64 (2013) 314e320
315
heating in isopropanol (IPA) in presence of catalytic amount of trie-
thylamine (Method C) or by non-conventional catalyst free neat
heating in a sealed tube (Method D) furnished desired compounds 8
and 9 in good yields (Scheme 2). Herein, we keenly observed that
neat reaction was found superior over conventional method as the
former method afforded higher yields of the desired product in a
shorter time. The green approach for displacement of chloro-
functionality by amines, saves organic solvent and catalyst, pre-
vents pollution with increasing atom economy of the reaction
(Tables 2 and 3). The structures of all new compounds 3, 4, 5, 8 and 9
were confirmed from their IR,1H NMR,13C NMR and MS spectral data
given in experimental protocol.
N
O
NH2
N
N
N
N
S
N
N
SCH3
N
S
II
I
Fig. 1. Compounds I and II exhibited remarkable affinity and selectivity for the 5-HT3
receptor.
3. Biology
were prepared using literature procedures [29e32]. These pre-
cursors 1 and 2 were well utilized for the synthesis of an important
intermediate 5-(4-chlorophenyl)-3,4-dihydro-4-oxothieno[2,3-d]
pyridine-6-carboxamide 3 [28] in good yield (Scheme 1).
The MICs of synthesized compounds were carried out by broth
microdilution method as described by Rattan [35]. Antibacterial
activity was screened against two Gram positive (Staphylococcus
aureus MTCC 96, Streptococcus pyogenes MTCC 442) and two Gram
negative (Escherichia coli MTCC 443, Pseudomonas aeruginosa MTCC
441) bacteria by using gentamycin, ampicillin, chloramphenicol, cip-
rofloxacin, norfloxacin as a standard antibacterial agents. Antifungal
activity was screened against three fungal species Candida albicans
MTCC 227, Aspergillus niger MTCC 282 and Aspergillus clavatus MTCC
1323 and nystatin and greseofulvin was used as a standard anti-
fungal agent. In literature various 6-benzyl-2-phenyl-4-amino
(hydroxy, methyl substituted or unsubstituted-1-phenyl)-5,6,7,8-
tetrahydropyridothieno[2,3-d]pyrimidine [36] derivatives revealed
significant antibacterial activity against E. coli. In this article,
our compounds 5-(4-chlorophenyl)-3,4-dihydro-4-oxothieno[2,3-
d]pyrimidine-6-carbonitrile 4 and 5-(4-chlorophenyl)-4-(amino)
thieno[2,3-d]pyrimidine-6-carbonitrile (8b, 8d, 8e, 8h, 9a) de-
rivatives showed good antibacterial activity as well as antifungal
activity compared to ampicillin and greseofulvin respectively. This
antimicrobial screening data is summarized in Table 4.
Reaction of intermediate 3 in phosphorusoxychloride (POCl3) in
presence of phosphorous pentachloride (PCl5) at variable temper-
ature and reaction time afforded 5-(4-chlorophenyl)-3,4-dihydro-
4-oxothieno[2,3-d]pyrimidine-6-carbonitrile 4 and 4-chloro-5-(4-
chlorophenyl)thieno[2,3-d]pyrimidine-6-carbonitrile
5 (Scheme
1) derivatives in 28e90% yields (Table 1), were isolated by col-
umn chromatography (Silica Gel 60e120 mesh). Interestingly, on
heating above reaction mixture at 25e50 ꢀC, primary amide rear-
ranged to give nitrile 4 as a sole product, while at elevated tem-
perature at 80e110 ꢀC, along with compound 5 as a major product
compound 4 was also formed as minor product (Scheme 1). It
means, at low temperature only primary amide react with POCl3
while at higher temperature along with primary amide, secondary
amide also react. The lactumelactim tautomerism occurred at
higher temperature give evidence to the formation of target
molecule (TM) 5 as major product (Table 1).
The C4-substituent played a key role in displaying higher phar-
macological activities to thieno-pyrimidine derivatives [16,17,33],
hence we focused on the ‘C4’ position of TM 5 as target site for gen-
eration of library of new thienopyrimidines. Numerous reports are
available for the introduction of aliphatic and aromatic amines at the
C4-position [34] of thieno[2,3-d]pyrimidine ring. Herein, we report
thesubstitution ofsubstituted anilinesandpiperazines at C4position
of TM 5, by different strategy. The C4-SNAr Cl-displacement in com-
pound 5 with various amines was successfully done by conventional
4. Conclusion
Thermal selective reaction of oxothieno[2,3-d]pyrimidine-
carboxamidewith POCl3 gave rearrangementof primaryamide group
to nitrile at lower temperature, while at higher temperature formed
SNAr chloro substitution by lactumelactim tautomerism along with
Cl
POCl3, PCl5
O
H
25-50°C
85-90 %
N
Cl
Cl
N
NC
Method A
S
4
O
H
HCOOH
CN
NH2
N
Reflux, 12 h
80%
H2N
N
NC
S
S
1
Cl
O
3
Cl
POCl3, PCl5
N
HCONH2
Reflux, 5h
76%
+
4
Conc. H2SO4
rt, 6h
Method B
90%
Cl
80-110°C
28-68%
N
NC
S
5
O
NH2
NH2
TM
H2N
S
O
2
Scheme 1. Synthetic strategy for the synthesis of target molecule, 5.

316
S.B. Kanawade et al. / European Journal of Medicinal Chemistry 64 (2013) 314e320
Cl
Cl
Cl
amine
R1 R2
Method C
R3-NH2
N
H
+
N
or
N
N
or
Method D
NC
7a-e
6a-h
TP
S
N
NC
S
5
8a-h/ 9a-e
TM
Method C = Isopropanol, Et3N:, reflux
Method D = Neat heat 120-130°C
Scheme 2. Synthesis of new derivatives of 4-aminothieno[2,3-d]pyrimidine-6-carbonitrile derivatives.
rearranged product. The environmentally benign neat heat reaction
has been successfully utilized for the synthesis of new class of 4-
aminothieno[2,3-d]pyrimidine-6-carbonitrile derivatives with good
to excellent yields in shorter time compared to conventional reaction
conditions using solvent and base afforded quite good yieldsin longer
time. Compounds 4, 8b, 8d, 8e, 8h and 9a showed promising anti-
bacterial activity compared to ampicillin and compounds 8b, 8h
showed better antifungal activity compared to greseofulvin.
Mycobacterium tuberculosis H37Rv strain by using Lowensteine
Jensen medium (conventional method) as described by Rattan [35].
5.1. 5-Amino-3-(4-chlorophenyl)thiophene-2,4-dicarbonitrile (1)
This compound was synthesized by the known literature
method [12e14], recrystallized from water/DMF (4:2). Obtained
yield 2.12 g (82%), mp 292e294 ꢀC [Lit. mp 293e294 ꢀC].
5. Experimental
5.2. 5-Amino-3-(4-chlorophenyl)thiophene-2,4-dicarboxamide (2)
Melting points were determined on a Gallenkamp melting point
apparatus. The 1H (300 MHz) and 13C (75 MHz) NMR spectra were
recorded on a Varian NMR Mercury 300 spectrometer. Chemical
shifts were reported in ppm relative to tetramethylsilane (TMS), and
multiplicities are given as s (singlet), bs (broad singlet), d (doublet),
t (triplet), q (quartet), or m (multiplet). Infrared spectra were
recorded as KBr pellets on a Shimadzu FTIR-408 spectrophotometer.
Mass spectrawere recorded on a Shimadzu LC-MS: EI QP 2010A mass
spectrometer with an ionization potential of 70 eV. Elemental ana-
lyses (C, H and N) were performed onThermo Finnigan Eager 300 EA
1112 series analyzer. Reactions were monitored by thin layer chro-
matography (TLC), carried out on 0.2 mm silica gel 60 F254 (Merck)
plates using UV light (254 and 366 nm) fordetection and compounds
were purified by column chromatography by using silica gel of 5e
This compound is also synthesized by the literature procedure
[28] and recrystallized from ethanol:DMF (8:2). Obtained yield
2.59 g (88%), mp 200e201 ꢀC [Lit. mp 200e202 ꢀC].
5.3. 5-(4-Chlorophenyl)-3,4-dihydro-4-oxothieno[2,3-d]
pyrimidine-6-carboxamide (3)
(Method A): 5-Amino-3-(4-chlorophenyl)thiophene-2,4-dicarbo-
nitrile 1 (2.59 g, 0.01 mol) in formic acid (5 mL) was refluxed for 12 h
(TLC check, chloroform:methanol, 8:1). On cooling to room temper-
ature the obtained solid product 3 was collected by vacuum filtration,
washed thoroughly with water, dried and purified by column chro-
matography eluting with chloroform:methanol (8:1) to give pale
green crystals. Obtained yield 2.44 g (80%) [28].
20
m
m (Merck, 60e120 mesh). Column dimension is 39 ꢁ 2 cm2 and
(Method B): 5-Amino-3-(4-chlorophenyl)thiophene-2,4-dicarbo-
xamide, 2 (2.95 g, 0.01 mol) was dissolved in formamide (5 mL). The
resulting solution was heated at reflux temperature for 5 h (chlor-
oform:methanol, 8:1), and then it was allowed to stand at room
temperature. The dark green crystals that formed was collected by
vacuum filtration and washed with sufficient amount of cold meth-
anol to give pale green crystals. Obtained yield 2.32 g (76%).
Mp 265e266 ꢀC; IR (KBr): 1566, 1635 (Ar C]C), 1697 (C]O),
elution volume used is about 200e400 mL for each product where
necessary. Common reagent-grade chemicals are either commer-
cially available and were used without further purification or were
prepared by standard literature procedures. All MTCC cultures were
collected from Institute of Microbial Technology, Chandigarh and
tested against above mentioned known drugs. Mueller Hinton broth
was used as nutrient medium to grow and dilute the drug suspen-
sion for the test. Inoculums’ size for test strain was adjusted to
108 CFU (Colony Forming Unit) per milliliter by comparing the
turbidity. DMSO was used as diluents to get desired concentration of
drugs to test upon standard bacterial strains. Each set of antimi-
crobial assay experiment was run three times and the average MIC’s
were calculated. MIC of compounds was determined against
3307 (NH), cmꢂ1 1H NMR (300 MHz, DMSO-d6):
; d 6.59 (bs, 1H,
1ꢀamide NH2), 7.38 (d, J ¼ 8.4 Hz, 2H, ArH), 7.45 (d, J ¼ 8.4 Hz, 2H,
ArH), 7.62 (bs, 1H, 1ꢀamide NH2), 8.17 (s, 1H, C2H), 12.49 (bs, 1H,
2ꢀamide NH); 13C NMR (75 MHz, DMSO-d6):
d 122.70, 127.65,
130.96, 131.64, 132.69, 132.87, 136.44, 147.48, 157.19, 162.72,164.06;
MS (EI): m/z 304.30 (M ꢂ 1, 60%), 306.10 (M þ 1, 20%). Anal. calcd.
for C13H8ClN3O2S (305.00): C, 51.07; H, 2.64; N, 13.74. Found: C,
51.19; H, 2.79; N, 13.59.
Table 1
Temperature dependent yields of compounds 4 and 5.
Entry
Temperature ꢀC
Time h
Yielda
%
5.4. Synthesis of 5-(4-chlorophenyl)-3,4-dihydro-4-oxothieno[2,3-
d]pyrimidine-6-carbonitrile (4) and 4-chloro-5-(4-chlorophenyl)
thieno[2,3-d]pyrimidine-6-carbonitrile (5)
Compd 4
Compd 5
1
2
3
4
25
50
80
18
90
85
64
36
e
8.0
1.0
0.5
e
28
68
5-(4-Chlorophenyl)-3,4-dihydro-4-oxothieno[2,3-d]pyrimidine-
6-carboxamide, 3 (3.05 g, 0.01 mol) was stirred in POCl3 (15 mL) and
PCl5 (1.0 g) at room temperature for 18 h. The excess POCl3 was then
110
a
Isolated yields after column chromatography.

S.B. Kanawade et al. / European Journal of Medicinal Chemistry 64 (2013) 314e320
317
Table 2
Synthesis of new 4-aminothieno[2,3-d]pyrimidine derivatives by using secondary amines.
Entry
Secondary amine NHR1R2
Product
Method C
Method D
Yielda
%
Time h
10
Yielda
%
Time h
TP
H
N
N
1
55
71
4
5a
3a
TP
N
H
N
2
3
4
62
60
55
12
13
11
73
68
65
2.5
5b
3b
TP
N
H
N
3.5
O
O
5c
3c
TP
N
H
N
2
N
H
N
H
5d
3d
TP
N
H
N
5
62
16
70
3
N
N
CO2Et
5e
CO2Et
3e
TP
H
N
N
N
N
6
50
14
67
5
Cl
Cl
Cl
Cl
5f
3f
TP
N
H
N
N
N
7
52
15
65
7
N
S
N
S
5g
3g
TP
N
H
N
N
N
8
55
18
66
6
O
O
O
O
O
O
5h
3h
*TP e Thienopyrimidine.
a
Isolated yields after column chromatography.

318
S.B. Kanawade et al. / European Journal of Medicinal Chemistry 64 (2013) 314e320
Table 3
Table 4
Synthesis of new 4-aminothieno[2,3-d]pyrimidine derivatives by using primary
In vitro antimicrobial activity of newly synthesized compounds 2 to 9e as MICs
amines.
(m
g/mL).
Entry
Primary
Product
Method C
Method D
Compd
Gram positive
bacteria
Gram negative
bacteria
Fungi
amine R3-NH2
Yielda Time Yielda Time
%
h
%
h
E.C.
P.A.
S.A.
S.P.
C.A.
A.N.
A.C.
MTCC
443
MTCC
441
MTCC
96
MTCC
442
MTCC
227
MTCC
282
MTCC
1323
TP
NH2
N
1
53
16
68
4.5
H
6a
2
3
4
5
8a
8b
8c
8d
8e
8f
8g
8h
9a
9b
9c
9d
200
125
100
200
200
250
125
125
62.5
200
100
100
250
125
100
200
250
0.05
100
50
250
200
62.5
250
250
250
200
250
100
100
250
250
250
62.5
100
200
200
1
250
200
200
100
200
125
250
62.5
250
250
200
100
100
200
200
250
250
0.25
250
50
200
200
250
250
200
200
250
100
250
250
200
125
125
100
250
125
250
0.5
500
>1000
500
>1000
250
>1000
500
5a
>1000
1000
>1000
>1000
>1000
1000
1000
500
>1000
1000
1000
>1000
500
>1000
>1000
n.t.
n.t.
n.t.
n.t.
n.t.
100
100
>1000
1000
>1000
>1000
>1000
>1000
1000
1000
>1000
1000
1000
>1000
500
>1000
>1000
n.t.
n.t.
n.t.
n.t.
n.t.
100
100
H
N
NH2
1000
1000
250
1000
1000
>1000
1000
500
250
1000
500
>1000
>1000
>1000
n.t.a
TP
2
54
20
60
3
Cl
Cl
6b
5b
3
50
60
13
15
66
70
4
NH2
NH
TP
Cl
Cl
5c
6c
NH2
H
N
9e
TP
Std.1
Std.2
Std.3
Std.4
Std.5
Std.6
Std.7
4
3.5
e
100
50
50
10
n.t.
n.t.
n.t.
n.t.
n.t.
100
500
CH3
CH3
50
25
10
n.t.
n.t.
6d
5d
25
10
n.t.
n.t.
50
10
n.t.
n.t.
H
N
n.t.
TP
Std.1 Gentamycin; Std.2 Ampicillin; Std.3 Chloramphenicol; Std.4 Ciprofloxacin;
Std.5 Norfloxacin; Std.6 Nystatin; Std.7 Greseofulvin E.C.: Escherichia coli, P.A.:
Pseudomonas aeruginosa, S.A.: Staphylococcus aureus, S.P.: Streptococcus pyogenes,
C.A.: Candida albicans, A.N.: Aspergillus niger, A.C.: Aspergillus clavatus.
5
53
18
62
5
H3C
NH2
CH3
6e
a
n.t. not tested.
5e
*TP e Thienopyrimidine.
a
5.5. General procedure for the synthesis of 5-(4-chlorophenyl)-4-
aminothieno[2,3-d]pyrimidine-6-carbonitrile derivatives 8aeh and
9aee
Isolated yields after column chromatography.
distilled off under vacuum and obtained residue was stirred in ice
water (500 mL), neutralized with saturated sodium carbonate
(50 mL) and filtered. This separated solid residue was purified by
column chromatography by using chloroform:methanol (9:1) as an
eluent. To optimize the reaction condition, the reaction mixture was
heated at 50, 80 and 110 ꢀC for time interval of 8.0, 1.0 and 0.5 h
respectively and worked out as described above. The yields of
products 4 and 5 are as shown in Table 1.
(Method C): 4-Chloro-5-(4-chlorophenyl)thieno[2,3-d]pyrimi-
dine-6-carbonitrile, 5 (0.31 g, 0.001 mol) and the appropriate pri-
mary (0.002 mol) and/or secondary amines (0.0011 mol) were
refluxed in isopropanol (10 mL) by using catalytic amount of trie-
thylamine for about 10e22 h (TLC check, chloroform:methanol, 9:1).
The mixture was then cooled to room temperature and excess iso-
propanol was distilled off under vacuum, and the resulting semisolid
was triturated with cold methanol. The solid product precipitated
was collected by suction filtration. All these compounds were puri-
fied by column chromatography using chloroform:methanol (9:1) as
an eluent and yields are given in Tables 2 and 3.
5.4.1. 5-(4-Chlorophenyl)-3,4-dihydro-4-oxothieno[2,3-d]pyrimidine-
6-carbonitrile (4)
Pale yellow amorphous solid, mp 324e325 ꢀC; IR (KBr): 1587,
1630 (Ar C]C), 1679 (C]O), 2214 (CN), 3190 cmꢂ1 1H NMR
;
(300 MHz, DMSO-d6):
d
7.54 (d, J ¼ 8.7 Hz, 2H, ArH), 7.57 (d,
(Method D): The same reaction mixture as in above method
(Method C) without using the solvent isopropanol in (Method D) was
heated in a sealed hard glass tube at 120e130 ꢀC for 2e7 h. The
resulting semisolid was triturated with cold methanol (5 mL) and
stirred overnight. The solid product precipitated was collected by
suction filtration and washed with appropriate cold methanol. All
these compounds were purified by column chromatography eluting
with chloroform:methanol (9:1). (Yields are given in Tables 2 and 3.)
J ¼ 8.7 Hz, 2H, ArH), 8.34 (s, 1H, C2H), 12.87 (bs, 1H, NH); MS (EI):
m/z 286.10 (M ꢂ 1, 100%), 288.05 (M þ 1, 30%). Anal. calcd. for
C
13H6ClN3OS (286.99): C, 54.27; H, 2.10; N, 14.60. Found: C, 53.98;
H, 2.24; N, 14.32.
5.4.2. 4-Chloro-5-(4-chlorophenyl)thieno[2,3-d]pyrimidine-6-
carbonitrile (5)
Yellow crystalline solid, mp 240e241 ꢀC; IR (KBr): 1537, 1621 (Ar
C]C), 2219 (CN) cmꢂ1
;
1H NMR (300 MHz, CDCl3):
d
7.40 (d,
J ¼ 8.7 Hz, 2H, ArH), 7.53 (d, J ¼ 8.7 Hz, 2H, ArH), 9.01 (s, 1H, C2H);
13C NMR (75 MHz, CDCl3): 109.16, 113.07, 120.81, 128.21, 129.69,
5.5.1. 5-(4-Chlorophenyl)-4-(diethylamino)thieno[2,3-d]
pyrimidine-6-carbonitrile (8a)
d
Light yellow crystalline solid, mp 166e168 ꢀC; IR (KBr): 1527,
130.63, 135.59, 154.62, 156.51, 156.70, 168.52; MS (EI): m/z 306.00
(M þ 1, 100%), 308.00 (M þ 3, 63.5%), 310 (M þ 5, 10.6%), Anal. calcd.
for C13H5Cl2N3S (304.96): C, 51.00; H, 1.65; N, 13.72. Found: C,
50.88; H, 1.44; N, 13.96.
1621 (Ar C]C), 2210 (CN), 3321 (NH) cmꢂ1 1H NMR (300 MHz,
;
CDCl3):
d
0.92 (t, J ¼ 7.2 Hz, 6H, 2CH3), 3.17 (q, J ¼ 7.2 Hz, 4H, 2CH2),
7.43 (d, J ¼ 8.4 Hz, 2H, ArH), 7.48 (d, J ¼ 8.4 Hz, 2H, ArH), 8.56 (s, 1H,
C2H); MS (EI): m/z 342.10 (Mþ, 50%), 344.12 (M þ 2, 16.5%). Anal.

S.B. Kanawade et al. / European Journal of Medicinal Chemistry 64 (2013) 314e320
319
calcd. for C17H15ClN4S (342.07): C, 59.56; H, 4.41; N,16.34. Found: C,
59.25; H, 4.60; N, 16.12.
NMR (75 MHz, DMSO-d6):
d
49.01, 113.92, 120.63, 123.39, 124.05,
127.66, 129.42, 129.98, 131.18, 136.11, 144.69, 152.82, 154.50, 161.36,
162.83, 170.21; MS (EI): m/z 489.10 (M þ 1, 100%), 491.00 (M þ 3,
40%). Anal. calcd. for C24H17ClN6S2 (488.06): C, 58.95; H, 3.50; N,
17.19; Found: C, 59.19; H, 3.23; N, 17.46.
5.5.2. 5-(4-Chlorophenyl)-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine-
6-carbonitrile (8b)
Light yellow amorphous solid, mp 170e171 ꢀC; IR (KBr): 1537,
1629 (Ar C]C), 2208 (CN) cmꢂ1; 1H NMR (300 MHz, CDCl3):
d
1.24e
5.5.8. 5-(4-Chlorophenyl)-4-(2,3-dihydrobenzo[b][1,4]dioxin-3-
yl)(piperazin-1-yl)methanone-thieno[2,3-d]pyrimidine-6-
carbonitrile (8h)
1.46 (m, 6H, 3CH2), 3.16e3.19 (m, 4H, 2CH2), 7.43 (d, J ¼ 8.4 Hz, 2H,
ArH), 7.51 (d, J ¼ 8.4 Hz, 2H, ArH), 8.58 (s, 1H, C2H); MS (EI): m/z
355.10 (M þ 1, 100%), 357.10 (M þ 3, 35%). Anal. calcd. for
Colorless amorphous solid, mp 212e213 ꢀC; IR (KBr): 1529, 1625
C
18H15ClN4S (354.07): C, 60.92; H, 4.26; N, 15.79; Found: C, 60.68;
(Ar C]C), 1654 (C]O), 2208 (CN) cmꢂ1; 1H NMR (300 MHz, DMSO-
H, 3.99; N, 15.47.
d6):
d
3.69e4.41 (m, 8H, 4CH2), 5.12 (t, J ¼ 4.5 Hz, 1H, OCH), 5.26 (d,
J ¼ 4.5 Hz, 2H, CH2), 6.85e6.92 (m, 4H, ArH), 7.64 (d, J ¼ 8.4 Hz, 2H,
ArH), 7.71 (d, J ¼ 8.4 Hz, 2H, ArH), 8.72 (s, 1H, C2H). MS (EI): m/z
518.10 (M þ 1, 100%), 520.05 (M þ 3, 35%). Anal. calcd. for
5.5.3. 5-(4-Chlorophenyl)-4-morpholinothieno[2,3-d]pyrimidine-
6-carbonitrile (8c)
Dark yellow amorphous solid, mp 199e200 ꢀC; IR (KBr): 1271
C26H20ClN5O3S (517.10): C, 60.29; H, 3.89; N, 13.52; Found: C, 60.10;
(CeO), 1533, 1633 (Ar C]C), 2206 (CN) cmꢂ1
;
1H NMR (300 MHz,
H, 3.68; N, 13.71.
CDCl3):
d
3.21e3.39 (m, 8H, 4CH2), 7.44 (d, J ¼ 8.2 Hz, 2H, ArH),
7.57 (d, J ¼ 8.2 Hz, 2H, ArH), 8.65 (s, 1H, C2H); MS (EI): m/z 357.10
(M þ 1, 100%), 359.05 (M þ 2, 36%). Anal. calcd. for C17H13ClN4OS
(356.05): C, 57.22; H, 3.67; N, 15.70; S, 8.99; Found: C, 57.38; H,
3.51; N, 15.48.
5.5.9. 4-(Benzylamino)-5-(4-chlorophenyl)thieno[2,3-d]pyrimidine-
6-carbonitrile (9a)
Yellow amorphous solid, mp 100e102 ꢀC; IR (KBr): 1577, 1620
(Ar C]C), 2212 (CN), 3035, 3417 (NH) cmꢂ1 1H NMR (300 MHz,
;
CDCl3): d 4.59 (s, 2H, CH2), 5.22 (bs,1H, NH), 7.06e7.32 (m, 5H, ArH),
5.5.4. 5-(4-Chlorophenyl)-4-(piperazin-1-yl)thieno[2,3-d]pyrimidine-
6-carbonitrile (8d)
7.38 (d, J ¼ 8.4 Hz, 2H, ArH), 7.45 (d, J ¼ 8.4 Hz, 2H, ArH), 8.60 (s, 1H,
C2H). MS (EI): m/z 377.10 (M þ 1, 100%), 379.10 (M þ 3, 32%). Anal.
calcd. for C20H13ClN4S (376.05): C, 63.74; H, 3.48; N,14.87; Found: C,
63.50; H, 3.56; N, 14.96.
Off white amorphous solid, mp 230e231 ꢀC; IR (KBr): 1533,1628
(Ar C]C), 2212 (CN), 3033, 3475 (NH) cmꢂ1 1H NMR (300 MHz,
;
DMSO-d6):
d 2.89 (m, 4H, 2CH2), 3.21 (m, 4H, 2CH2), 3.65 (bs, 1H,
NH), 7.60 (d, J ¼ 8.1 Hz, 2H, ArH), 7.69 (d, J ¼ 8.1 Hz, 2H, ArH), 8.68 (s,
1H, C2H); MS (EI): m/z 355.10 (Mþ, 100%), 357.13 (M þ 2, 33%). Anal.
calcd. for C17H14ClN5S (355.07): C, 57.38; H, 3.97; N, 19.68. Found: C,
57.60; H, 4.12; N, 19.72.
5.5.10. 4-(2-Chlorophenylamino)-5-(4-chlorophenyl)thieno[2,3-d]
pyrimidine-6-carbonitrile (9b)
Pale green amorphous solid, mp 178e180 ꢀC; IR (KBr): 1626 (Ar
C]C), 2214 (CN), 3029, 3066 (NH) cmꢂ1 1H NMR (300 MHz,
;
CDCl3):
d 7.04e7.34 (m, 4H, ArH), 7.46 (bs, 1H, NH), 7.53 (d,
5.5.5. Ethyl-4-(5-(4-chlorophenyl)-6-cyanothieno[2,3-d]pyrimidin-
4-yl)piperazine-1- carboxylate (8e)
J ¼ 8.4 Hz, 2H, ArH), 7.63 (d, J ¼ 8.4 Hz, 2H, ArH), 8.73 (s, 1H, C2H).
MS (EI): m/z 397.10 (M þ 1, 100%), 399.10 (M þ 3, 57%), 401.05
(M þ 5, 9%). Anal. calcd. for C19H10Cl2N4S (396.00): C, 57.44; H, 2.54;
N, 14.10; Found: C, 57.73; H, 2.66; N, 13.94.
Light yellow crystalline solid, mp 164e166 ꢀC; IR (KBr): 1125 (Ce
O), 1533, 1622 (Ar C]C), 1718 (C]O), 2208 (CN) cmꢂ1 1H NMR
;
(300 MHz, DMSO-d6):
d
1.02 (m, J ¼ 11.7 Hz, 2H, CH2), 1.14 (t,
J ¼ 7.2 Hz, 3H, CH3), 1.53 (m, J ¼ 11.7 Hz, 2H, CH2), 2.71 (m,
J ¼ 11.7 Hz, 2H, CH2), 3.63 (m, J ¼ 11.7 Hz, 2H, CH2), 4.00 (q,
J ¼ 7.2 Hz, 2H, OCH2), 7.59 (d, J ¼ 8.7 Hz, 2H, ArH), 7.67 (d, J ¼ 8.7 Hz,
5.5.11. 4-(3-Chlorophenylamino)-5-(4-chlorophenyl)thieno[2,3-d]
pyrimidine-6-carbonitrile (9c)
Brown amorphous solid, mp 160e161 ꢀC; IR (KBr): 1623 (Ar C]
2H, ArH), 8.65 (s, 1H, C2H); 13C NMR (75 Hz, DMSO-d6):
d
14.07,
C), 2210 (CN), 3032, 3067 (NH) cmꢂ1 1H NMR (300 MHz, CDCl3):
;
26.96, 40.20, 48.62, 60.52,103.28, 113.76, 114.06, 129.21, 129.94,
d
6.89e7.70 (m, 4H, ArH), 7.19 (bs, 1H, NH), 7.23 (d, J ¼ 8.4 Hz, 2H,
131.20, 135.79, 144.86, 154.49, 161.28, 170.08, 173.67. MS (EI): m/z
ArH), 7.55 (d, J ¼ 8.4 Hz, 2H, ArH), 8.72 (s, H, C2H). MS (EI): m/z
397.05 (M þ 1, 100%), 399.10 (M þ 3, 65%), 401.10 (M þ 5, 10%). Anal.
calcd. for C19H10Cl2N4S (396.00): C, 57.44; H, 2.54; N, 14.10; Found:
C, 57.75; H, 2.68; N, 13.91.
427.10 (Mþ, 100%), 429.10 (M
C
þ
2, 40%). Anal. calcd. for
20H18ClN5O2S (427.09): C, 56.14; H, 4.24; N, 16.37; Found: C, 56.34;
H, 4.52; N, 16.15.
5.5.6. 5-(4-Chlorophenyl)-4-(4-(2,3-dichlorophenyl)piperazin-1-yl)
thieno[2,3-d]pyrimidine-6-carbonitrile (8f)
5.5.12. 4-(o-Tolylamino)-5-(4-chlorophenyl)thieno[2,3-d]pyrimidine-
6-carbonitrile (9d)
Off white amorphous solid, mp 206e208 ꢀC; IR (KBr): 1533,1627
Pale brown amorphous solid, mp 200e202 ꢀC; IR (KBr): 1560,
(Ar C]C), 2208 (CN) cmꢂ1; 1H NMR (300 MHz, CDCl3):
d 2.79e2.81
1612 (Ar C]C), 2208 (CN), 2966 (CeH), 3024, 3403 (NH) cmꢂ1; 1H
(m, J ¼ 4.8 Hz, 4H, 2CH2), 3.33e3.35 (m, J ¼ 4.8 Hz, 4H, 2CH2), 6.57e
7.23 (m, 3H, ArH), 7.45 (d, J ¼ 8.4 Hz, 2H, ArH), 7.52 (d, J ¼ 8.4 Hz, 2H,
ArH), 8.63 (s, 1H, C2H); MS (EI): m/z 500.00 (M þ 1, 100%), 502.00
(M þ 3, 90%), 504.15 (M þ 5, 30%), 505.80 (M þ 7, 5%). Anal. calcd.
for C23H16Cl3N5S (499.02): C, 55.16; H, 3.22; N, 13.98; Found: C,
55.37; H, 3.38; N, 14.12.
NMR (300 MHz, DMSO-d6): d 1.85 (s, 3H, CH3), 7.19e7.62 (m, 4H,
ArH), 7.36 (bs, 1H, NH), 7.72 (d, J ¼ 8.7 Hz, 2H, ArH), 7.76 (d,
J ¼ 8.7 Hz, 2H, ArH), 8.59 (s, H, C2H). MS (EI): m/z 377.10 (M þ 1,
100%), 379.05 (M þ 3, 31%). Anal. calcd. for C20H13ClN4S (376.05): C,
63.74; H, 3.48; N, 14.87; Found: C, 63.67; H, 3.70; N, 14.78.
5.5.13. 4-(m-Tolylamino)-5-(4-chlorophenyl)thieno[2,3-d]pyrimidine-
6-carbonitrile (9e)
5.5.7. 4-(4-(Benzo[d]isothiazol-3-yl)piperazin-1-yl)-5-(4-
chlorophenyl)thieno[2,3-d]pyrimidine-6-carbonitrile (8g)
Yellow amorphous solid, mp 184e186 ꢀC; IR (KBr): 1529, 1630
Pale green amorphous solid, mp 130e132 ꢀC; IR (KBr): 1568,
1608 (Ar C]C), 2212 (CN), 2968 (CeH), 3029, 3400 (NH) cmꢂ1; 1H
NMR (300 MHz, CDCl3): d 2.33 (s, 3H, CH3), 6.92e7.24 (m, 4H, ArH),
(Ar C]C), 2208 (CN) cmꢂ1
;
1H NMR (300 MHz, DMSO-d6):
d
3.24
(m, 4H, 2CH2), 3.46 (m, 4H, 2CH2), 7.55 (d, J ¼ 9.0 Hz, 2H, ArH), 7.80
7.18 (bs, 1H, NH), 7.56 (d, J ¼ 8.7 Hz, 2H, ArH), 7.67 (d, J ¼ 8.7 Hz, 2H,
(d, J ¼ 9.0 Hz, 2H, ArH), 7.47e7.76 (m, 4H, ArH), 8.66 (s, 1H, C2H); 13
C
ArH), 8.69 (s, 1H, C2H). MS (EI): m/z 377.05 (M þ 1, 100%), 379.10

320
S.B. Kanawade et al. / European Journal of Medicinal Chemistry 64 (2013) 314e320
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Acknowledgments
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Authors especially thank CSIR, New Delhi for financial support
and University of Pune for providing spectral data. Authors also
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and special thanks to Microcare Laboratory, Surat (India) for eval-
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