Thiophene of benzoxazolinone based 1, 2, 3-triazoles using

Thiophene
moiety have attracted many researchers in medicinal field due to its various
biological activities such as antimicrobial 1, antibacterial 2, antifungal
3, anticancer 4, antitubercular 5, antiamoebic 6, antiparasitic 7, anti-inflammatory
8, antioxidant 9, anticonvulsant 10, local anaesthetic 11, and
anti-arrhythmic 12.

           

            Pyrimidine derivatives play a vital
role in many biological aspects and transformation. This ring system is present
in cytosine, adenine, guanine and thiamine, which form a part of ribonucleic
acid (RNA) and deoxyribonucleic acid (DNA) and vitamins like vitamin B2
(Riboflavin) and vitamin B6 (Pyridoxine) co-enzyme and other purines 13. Pyrimidine
nucleus is also present in barbituric acid and its several derivatives like
Veranal which are used as hypnotics 14.

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            Condensed
pyrimidine derivatives have been reported as anti-microbial 15, anti-inflammatory
16, anti- HIV 17, anti-tubercular 18, anti-tumor 19, anti-neoplastic 20,
anti-malarial 21, diuretic 22, cardiovascular 23 agents and hypnotic
drugs for the nervous system 24, calcium-sensing receptor antagonists 25
and also for antagonists of the human A2A adenosine receptor. 26 etc

 

            The heterocyclic fusion of thiophene
ring with pyrimidine ring system resulted in formation of thienopyrimidine, a
structural analogue of biogenic purine, an important class of heterocyclic
compounds which possesses broad spectrum of biological activities antibacterial
27, antimicrobial 28, antiviral 29, anti-HIV 30, anti-avian influenza
virus (H5N1) 31,  and analgesic 32
ect. Among them, thieno2,3-dpyrimidines have been proved useful for
cerebral ischemia, malaria, tuberculosis, Alzheimer’s and Parkinson’s diseases.

 

 

LITERATURE REVIEW

Thieno2,3-dpyrimidines

           

            Alagarsamy et
al.,
have synthesized novel 2-mercapto-3-substituted-5,6-dimethyl
thieno2,3-dpyrimidin-4(3H)-ones and reported as anti inflammatory and
analgesic agents 33.

 

 

            J.
E. Tarver Jr. et al., have
synthesized 2-((5,6-thieno2,3-dpyrimidin-4-yl)thio)acetic
acids and proved to be inhibitors of Notum Pectinacetylesterase 34.

 

           

            Jiangping
Lou et al., discovered compounds with
a cyclic thienopyrimidine moiety and an aceto-hydrazone moiety in their
chemical structure and screened cytotoxicity against tumor cell lines A549,
HeLa, MDA-MB-231 and HEK-293T 35.

 

            Srujana Muthadi et
al., have synthesized a series of novel
2-Methyl-4-(1-methyl-1H-1,2,3
triazol-4-yl-methoxy)-5,6,7,8-tetrahydra-benzo4,5thieno2,3-dpyrimidine
derivatives and screened for in vitro antimicrobial activity 36.

           

            M. R. Bhadane et
al., have synthesized various
substituted thieno(2,3-d)pyrimidines and examined their antioxidant activity by
DPPH scavenging method 37.

 

 

 

1,2,3-triazoles

            Haider
et al., have synthesized a library of benzoxazolinone based 1, 2,
3-triazoles using click chemistry approach and screened them for their in
vitro and in vivo anti-inflammatory activity 38.

            Yempala
et al., have synthesized a series of novel dibenzofuran based 1, 2,
3-triazole derivatives using click chemistry approach as in vitro anti-mycobacterial
agent against Mycobacterium tuberculosis H37Rv 39.

            Duan
et al., have synthesized a series of novel 1, 2, 3- triazole-dithiocarbamate
hybrids and tested them against four selected human tumor cell lines i.e.
MGC-803, MCF- 7, PC-3, EC-109 40.

            Abdel-Wahab
et al., have carried out the synthesis of novel pyrazolyl-1,2,3-triazoles
and screened for antimicrobial activity 41.

           

            Jordao
et al., have synthesized N-amino-1,2,3-triazole derivatives i.e.
1-(4-substituted-phenylamino)-5-methyl- 1H-1,2,3-triazole-4-carboxylic acid
hydrazides and screened them against Cantagalo virus replication 42.

 

Present
work

Objectives:

Ø  To
synthesize novel1,2,3-triazole derivatives of thieno2,3-dpyrimidinones

Ø  To
characterize the derivatives by IR, 1H NMR, 13C NMR, mass
spectra and also by elemental analysis.

Ø  To
examine in vitro antibacterial activity of compounds by broth dilution
method against gram positive and gram negative bacteria.

Ø  To
determine docking studies against Bacillus subtilis purine riboswitch
and Escherichia coli TPP riboswitch.

 

            Today, bacterial infection is one of the
most complex health issues globally. Because of increasing antibiotic resistant
organisms and also decreasing the discovery of new antimicrobial agents, there
is a pressing need of new antimicrobial agents that are capable of treating
those strains 43. As a result, increasing efforts have been made to develop
multi drug resistant (MDR) strategy 44 by which each drug moiety is designed
to bind independently two different biological targets which offer the
possibility to overcome the current resistance 45.

            Among
fused pyrimidines, thieno2,3-dpyrimidines
were  found to exhibit various biological
activities. Several thieno2,3-dpyrimidine
derivatives were synthesized and proved to be antibacterial agents. In
addition, 1,2,3-triazoles possess unique features which make them important
pharmacophore in curative medicinal chemistry and they act as bioisosters and
linkers 46. Also they are reliable and quantitative to synthesize
by using Huisgen’s copper catalyzed azide-alkyne cyclo addition (CuAAC) under
mild conditions without protecting groups and purification 47.

            In
literature, there are numerous fused 1,2,3-triazole derivatives of thieno2,3-dpyrimidines but 1,2,3-triazole linked
thieno2,3-dpyrimidine derivatives
are exceptional in synthesis. In the light of the above specifics, considerable
efforts have been made to develop novel synthetic strategies by joining thieno
2,3-d pyrimidinone scaffold and
1,2,3-triazole moiety through ester linkage. The synthesized compounds were
screened for their antibacterial activity to obtain novel candidates of
incredible activities with nominal side effects. In order to establish the
binding interactions, docking studies were performed on B. subtilis
purine riboswitch and E. coli TPP
riboswitch.

 

Scheme
1

 

 

Here:     6a,
7a: R = 4-CH3                             6f, 7f: R = 3-F

              6b, 7b: R = 4-Cl                                   6g, 7g: R = 3-NO2

              6c, 7c: R = H                                                 6h, 7h:
R = 4-NO2

              6d, 7d: R = 2,4,5-tri Cl                        6i, 7i:
R = 4-Cl, 2-OCH3, 6-NO2

              6e, 7e: R = 4-OCH3                                               6j,
7j: R = 2-Cl, 5-NO2

 

 

 

Methods
and Materials

Chemistry

Synthesis
of 5,6,7,8-tetrahydro1benzothieno2,3-dpyrimidin-4(3H)-one (2): Concentrated
sulfuric acid (4 mL) at 0-5oC was added to a mixture of 2-amino-4,5,6,7
tetrahydrobenzobthiophene-3-carbonitrile
1 (1 g, 5.6 mmol) in formic acid (10
mL). The mixture was re?uxed for 2 h and allowed to cool at room temperature
(rt). Then the mixture was poured into crushed ice. The separated solid was
?ltered and puri?ed by recrystallization with ethanol.

     Brown
color solid Yield 89%; mp 190-192oC; IR (KBr) (?maxcm-1):
2836 (-CH), 1651 (C=O), 1581 (C=N); 1H NMR (400 MHz, DMSO-  d6):
? 1.78-1.85 (m, 4H, CH2), 2.77
(t, J = 5.2 Hz, 2H, CH2), 2.91 (t, J = 5.6 Hz, 2H, CH2),
8.02 (s, 1H, CH-pyrimidine), 12.32 (br s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6): ? 21.7,
22.4, 24.4, 25.2, 122.6, 130.7, 132.0, 144.7, 157.6, 162.3; LCMS (negative ion
mode) (m/z): 205 M-H– for
C10H10N2OS.

Synthesis
of ethyl 2-(4-oxo-5,6,7,8-tetrahydrobenzo4,5thieno2,3-dpyrimidin-3(4H)-yl)acetate
(3): A solution of compound 2 (2 g, 10 mmol), ethyl bromo acetate (1.3 mL, 12 mmol) and
potassium carbonate (2 g, 15 mmol) in 20 mL acetone was added and stirred for
8h. After completion of reaction mixture, the acetone was evaporated and the
precipitated solid was obtained, washed with cold water and recrystallized with
ethanol.    

    Pale
yellow color solid Yield: 83%; mp 192-194oC; IR (KBr) (?max,
cm-1): 3115 (=CH), 1736 (ester C=O), 1668 (amide C=O), 1609
(C=N); 1H NMR (400 MHz, DMSO-d6):
? 1.28 (t, J =
8.0 Hz, 3H, CH2CH3), 1.81-1.87 (m,
4H, 2CH2), 2.82 (t, J = 5.6 Hz, 2H, CH2), 2.93 (t,
J = 6.0 Hz, 2H, CH2), 4.24 (q, J = 8.0 Hz, 2H, CH2CH3),
4.84 (s, 2H, CH2CO), 8.36 (s, 1H, CH-pyrimidine); 13C
NMR (100 MHz, DMSO-d6): ?
13.0, 21.1, 21.8, 24.2, 24.5, 45.9, 61.1, 121.5, 130.6, 133.5, 144.3,
156.4, 161.1, 166.3; LCMS (m/z): 293 M+H+, 315 M+Na+ for
C14H16N2O3S.

Synthesis
of 2-(4-oxo-5,6,7,8-tetrahydrobenzo4,5thieno2,3-dpyrimidin-3(4H)-yl)acetic
acid (4): To a solution of compound 3 (2 g, 6.8 mmol) in methanol, sodium hydroxide (0.68 g, 17 mmol)
solution in water was added and subjected to stirring at room temperature
overnight. After completion of reaction, the reaction mixture was poured into
crushed ice and con. HCl was added till it gets acidified. The formed solid was
filtered and purified by recrystallization with ethanol.

 White Solid Yield: 80%; mp 130-133oC;
IR (KBr) (?max, cm-1): 3615 (OH), 3115 (=CH), 1716
(ester C=O), 1668 (amide C=O), 1558   (C=N);
1H NMR (400 MHz, CDCl3): ? 1.74-1.81
(m, 4H, 2CH2), 2.71 (t, J = 4.7 Hz, 2H, CH2), 2.86
(t, J = 5.8 Hz, 2H, CH2), 4.7 (s, 2H, CH2), 8.06
(s, 1H, pyrimidine-CH), 12.90 (s, 1H, COOH); 13C NMR (100 MHz, CDCl3):
? 20.6, 21.3, 23.3, 24.0, 55.7, 116.6, 123.4, 144.0, 145.5, 151.3,
163.7, 166.7; LCMS (m/z): 265 M+H+
for C12H12N2O3S.

Synthesis
of prop-2-yn-1-yl-2-(4-oxo-5,6,7,8-tetrahydrobenzo4,5thieno2,3-dpyrimidin-3(4H)-yl)acetate (5): To a solution of
compound 4 (2 g, 7.6 mmol) in dry
acetone (20 mL) were added anhydrous potassium carbonate (2.12 g, 11.4 mmol)
and 3-bromoprop-1-yne (1.04 mL, 11.4 mmol) successively. The resultant mixture
was stirred overnight at room temperature. The reaction was monitored by TLC.
Upon completion of reaction, the reaction mixture was poured into crushed ice
and stirred continuously. The obtained solid was filtered and purified by
column chromatography using ethyl acetate and hexane as eluent.

   Yellow Solid Yield:
80%; mp 110-112oC; IR (KBr) (?max, cm-1): 3244
(?CH), 3125 (=CH), 2124 (C?C), 1719 (ester C=O), 1645 (amide C=O), 1618 (C=N); 1H
NMR (400 MHz, CDCl3): ?
1.83-1.88 (m, 4H, 2CH2), 2.53 (s, 1H, ?CH), 2.81 (t, J = 6.1 Hz, 2H, CH2), 3.00
(t, J = 5.6 Hz, 2H, CH2),
4.72 (s, 2H, CH2-C?CH), 4.81 (s, 2H, N-CH2-C=O),
7.85 (s, 1H, pyrimidine-CH); 13C NMR (100 MHz, CDCl3): ? 22.2, 22.8, 25.2, 25.5, 46.8, 53.3,
75.8, 76.4, 128.8, 131.7, 145.0, 149.2, 156.7, 157.4, 166.6; LCMS (m/z): 303.1 M+H+, 325
M+Na+  for C15H14N2O3S.

Synthesis
of aryl azides (6a-j)

            Aniline
derivative (9.1 mmol) was dissolved in con. HCl (91 mmol) at room temperature
and cooled up to 0 oC, followed by addition of a solution of sodium
nitrite (45.5 mmol). The reaction mixture was stirred for 10 min at 0-5 oC.
Sodium azide (27.3 mmol) was added and the mixture was further stirred for 2 h
at room temperature. After completion of reaction, the mixture was extracted
with hexane. The organic layer was dried over anhydrous sodium sulphate and
after evaporation of the solvent the desired azide derivative was obtained.

   

General
procedure for the synthesis of (1-(substituted phenyl)-1H – 1,2,3-triazol-4-yl)methyl-2-(4-oxo-5,6,7,8-tetrahydrobenzo4,5thieno2,3-dpyrimidin-3(4H)-yl)acetate derivatives
(7a-j): Compound
5 (0.2 g, 0.7 mmol) was dissolved in
2 mL of THF: H2O (1:1) solvent in a 50 mL round bottom flask. To
this CuSO4.5H2O
(0.17 g, 0.7 mmol) was added and the reaction mixture was stirred for 5 min. To
this light blue colour reaction mixture sodium ascorbate (0.2 g, 1.05 mmol) was
added and stirred for 15 min. The reaction mixture becomes dark yellow. Then
aryl azide (6a-j) (1.05 mmol) was
added and allowed to stir for 30 min to 1 h. After completion of reaction
monitored by TLC, the reaction mixture was extracted with ethyl acetate by
adding excess water. The combined organic layer was washed with brine solution
and dried over anhydrous sodium sulphate. The solvent was evaporated under
reduced pressure and the crude product was purified by silica gel column
chromatography using hexane ethyl acetate as eluent.  

 (1-(p-Tolyl)-1H-1,2,3-triazol-4-yl)methyl-2-(4-oxo-5,6,7,8-tetrahydrobenzo4,5thieno2,3-dpyrimidin-3(4H)-yl)acetate
(7a):
Light brown solid Yield 70 mg 83%, mp 120-125oC. IR (KBr) ?, cm-1:
3122 (= CH), 2922 (-CH), 1735 (ester C=O), 1676 (amide C=O), 1611 (C=N).  1H
NMR (400 MHz, CDCl3): ?
1.81-1.89 (m, 4H, 2CH2), 2.43 (s, 3H, CH3), 2.78 (t, J = 5.5 Hz, 2H, CH2), 2.98
(t, J = 5.5 Hz, 2H, CH2),
4.71 (s, 2H, CH2), 5.44 (s, 2H, CH2-triazole), 7.31 (d, J = 8.0 Hz, 2H, Ar-H), 7.61 (d, J = 8.0 Hz, 2H, Ar-H), 7.90 (s, 1H,
triazole-CH), 8.11 (s, 1H, pyrimidine-CH); 13C NMR (100 MHz, CDCl3):
? 22.1, 30.2, 30.8, 33.2, 33.6, 54.9,
70.1, 112.5, 116.9,
123.9, 130.6, 142.6, 145.2, 148.0, 153.3, 159.0, 161.4, 165.5, 170.2, 174.9;
LCMS (m/z): 436.1 M+H+,
458.2 M+Na+ for C22H21N5O3S.
Elem. Anal. for C22H21N5O3S:
(Calc) C, 60.68; H, 4.86; N, 16.08; S, 7.36. Found: C, 60.45; H, 4.52; N,
15.75; S, 7.62.

       (1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl-2-(4-oxo-5,6,7,8-tetrahydrobenzo4,5thieno
2,3-d pyrimidin-3(4H)-yl)acetate (7b):  Light brown solid Yield: 80%; mp 155-157oC;
IR (KBr) (?max cm-1): 3107 (=CH), 2924 (-CH), 1740 (ester
C=O), 1669 (amide C=O), 1605 (C=N); 1H NMR (400 MHz, CDCl3):
? 1.81-1.89 (m, 4H, 2CH2),
2.78 (t, J = 6.0

Hz, 2H, CH2),
2.99 (t, J = 6.0 Hz, 2H, CH2),
4.71 (s, 2H, CH2), 5.43 (s, 2H, CH2-triazole), 7.45 (d, J = 8.1 Hz, 2H, Ar-H), 7.65 (d, J = 8.1 Hz, 2H, Ar-H), 7.89 (s, 1H,
triazole-CH), 8.01 (s, 1H, pyrimidine-CH); 13C NMR (100 MHz, CDCl3):
?  26.9, 27.5, 29.9, 30.2, 52.1, 63.7,
125.2, 126.9, 127.2, 133.6, 134.4, 136.2, 139.5, 141.5, 147.5, 149.9, 162.2,
167.0, 172.1. LCMS (m/z): 456.1M+H+, 478.1
M+Na+ for C21H18N5O3SCl.
Elem. Anal. for C21H18N5O3SCl:
(Calc) C, 55.32; H, 3.98; N, 15.36; S, 7.03. Found: C, 54.95;
H, 3.58; N, 15.50; S, 6.87.

(1-Phenyl-1H-1,2,3-triazol-4-yl)methyl-2-(4-oxo-5,6,7,8-tetrahydrobenzo4,5thieno2,3-d
pyrimidin-3(4H)-yl) acetate (7c): Brown solid Yield:
70%; mp 110-111 oC; IR (KBr) (?max cm-1): 3116
(=CH), 2921 (-CH), 1735 (ester C=O), 1665 (amide C=O), 1615 (C=N); 1H NMR (400 MHz, DMSO): ? 1.72-1.80 (m, 4H, 2CH2),
2.75 (t, J = 5.5 Hz, 2H, CH2),
2.83 (t, J = 5.5 Hz, 2H, CH2),
4.85 (s, 2H, CH2), 5.36 (s, 2H, CH2-triazole), 7.49-7.63
(m, 3H, Ar-H), 7.89 (d, J = 7.9 Hz,
2H, Ar-H), 8.10 (s, 1H, triazole-CH), 8.59 (s, 1H, pyrimidine-CH); 13C
NMR (100 MHz, CDCl3): ?
22.1, 22.7, 25.1, 25.5, 47.3, 59.0, 120.4, 122.1, 122.4, 128.8, 129.7, 131.4,
134.7, 136.7, 142.8, 145.1, 157.4, 162.2, 167.3; LCMS (m/z): 422 M+H+ for C21H19N5O3S.
Elem. Anal. for C21H19N5O3S:
(Calc) C, 59.84; H, 4.54; N, 16.62; S, 7.61. Found: C, 61.18; H, 4.55; N,
16.67; S, 7.40. 

(1-(2,4,5-Trichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl-2-(4-oxo-5,6,7,8-tetrahydrobenzo4,5thieno2,3-dpyrimidin-3(4H)-yl)acetate
(7d):
Yellow
solid Yield: 60%; mp 170-172 oC; IR (KBr) (?max cm-1):
3115.3 (=CH), 2915 (-CH), 1732 (ester C=O), 1664 (amide C=O), 1598 (C=N); 1H
NMR (400 MHz, CDCl3): ?
1.81-1.89 (m, 4H, 2CH2), 2.79 (t, J = 5.5 Hz, 2H, CH2), 2.95 (t, J = 6.1 Hz, 2H, CH2), 4.71 (s, 2H, CH2), 5.46
(s, 2H, CH2-triazole), 7.71 (s, 1H, Ar-H), 7.81 (s, 1H, Ar-H), 7.92
(s, 1H, triazole-CH), 8.14 (s, 1H, pyrimidine-CH); 13C NMR (100 MHz,
CDCl3): ? 22.7, 23.5,
23.9, 34.6, 42.5, 62.7, 117.6, 119.1, 125.4, 128.2, 131.8, 131.9, 132.8, 135.2,
139.4, 142.7, 144.4, 147.6, 155.5, 161.6, 169.5; LCMS (m/z): 524 M+H+ for 
C21H16N5O3SCl3.
Elem. Anal. for C21H16N5O3SCl3:
(Calc) C, 48.06; H, 3.07; N, 13.35; S, 6.11. Found: C, 47.84; H, 2.76; N,
13.18; S, 6.28.

(1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl-2-(4-oxo-5,6,7,8-tetrahydrobenzo
4,5thieno2,3-dpyrimidin-3(4H)-yl)acetate (7e): Pale yellow
solid Yield: 90%; mp 180-185oC; IR (KBr) (?max cm-1):
3111 (=CH), 2926 (-CH), 1741 (ester C=O), 1672 (amide C=O), 1609 (C=N); 1H
NMR (400 MHz, CDCl3): ?
1.81-1.89 (m, 4H, 2CH2), 2.78 (t, J = 6.0 Hz, 2H, CH2), 2.98 (t, J = 6.0 Hz, 2H, CH2), 3.89 (s, 3H, -OCH3),
4.70 (s, 2H, CH2), 5.43 (s, 2H, CH2-triazole), 7.02 (d, J = 9.0, 2H, Ar-H), 7.64 (d, J = 9.0 Hz, 2H, Ar-H), 7.86 (s, 1H,
triazole-CH), 8.03 (s, 1H, pyrimidine-CH); 13C NMR (100 MHz, CDCl3):
?  24.1, 24.7, 27.1, 27.4, 32.8, 49.3,
60.9, 122.4, 124.1, 124.4, 130.8, 131.6, 133.4, 136.7, 138.7, 144.7, 147.1,
159.4, 164.2, 169.3. LCMS (m/z):
452.2 M+H+, 474.1 M+Na+ for C22H21N5O4S.
Elem. Anal. for C22H21N5O4S:
(Calc) C, 58.53; H, 4.69; N, 15.51; S, 7.10. Found: C, 58.16; H, 4.90; N,
15.17; S, 6.87.

 (1-(3-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl-2-(4-oxo-5,6,7,8-tetrahydrobenzo
4,5 thieno 2,3-d pyrimidin-3(4H)-yl)acetate (7f): Light brown
solid Yield: 70%; mp 130-132oC; IR (KBr) (?max cm-1):
3120 (=CH), 2920 (-CH), 1734 (ester C=O), 1679 (amide C=O), 1614 (C=N); 1H
NMR (400 MHz, DMSO): ? 1.72-1.80 (m,
4H, 2CH2), 2.74 (t, J =
5.5 Hz, 2H, CH2), 2.82 (t, J
= 5.2 Hz, 2H, CH2), 4.85 (s, 2H, CH2), 5.35 (s, 2H, CH2-triazole),
7.61-7.69 (m, 1H, Ar-H), 7.78-7.86 (m, 2H, Ar-H), 8.07 (s, 1H, Ar-H), 8.33 (s,
1H, triazole-CH), 8.60 (s, 1H, pyrimidine-CH ); 13C NMR (100 MHz,
CDCl3): ? 23.8, 24.5,
26.8, 27.2, 48.5, 63.7, 110.1, 113.4, 117.5, 124.2, 124.4, 129.6, 133.3, 136.2,
138.7, 146.2, 146.9, 159.1, 163.8, 168.4, 168.9; LCMS (m/z): 440 M+H+ for C21H18N5O3SF.
Elem. Anal. for C21H18N5O3SF.:
(Calc) C, 57.39; H, 4.13; N, 15.94; S, 7.30. Found: C, 57.75; H, 4.04; N,
15.98; S, 7.21.

(1-(3-Nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl-2-(4-oxo-5,6,7,8-tetrahydrobenzo4,5thieno2,3-dpyrimidin-3(4H)-yl)acetate
(7g):
Brown
solid Yield: 90%; mp 135-140oC; IR (KBr) (?max cm-1):
3113 (=CH), 2932 (-CH), 1731 (ester C=O), 1670 (amide C=O), 1608 (C=N), 1527
(NO2); 1H NMR

(400 MHz, CDCl3): ? 1.79-1.88 (m, 4H, 2CH2), 2.78 (t, J = 6.0 Hz, 2H, CH2), 2.95
(t, J = 6.1 Hz, 2H, CH2),
4.71 (s, 2H, CH2), 5.47 (s, 2H, CH2-triazole), 7.75
(t,  J
=8.15Hz, 1H, Ar-H), 7.89 (s, 1H, triazole-CH), 8.19-8.21 (m, 1H), 8.31-8.33 (m,
2H), 8.64 (s, 1H, pyrimidine-CH ); 13C NMR (100 MHz, CDCl3):
? 22.6, 23.2, 25.6, 25.9, 47.3, 62.5,
106.3, 108.8, 112.3, 116.3, 123.0, 123.2, 132.1, 134.9, 137.5, 145.7, 157.8,
162.6, 167.7, 173.7, 175.1; LCMS (m/z):
467.1 M+H+, 489.2 M+Na+ for C21H18N6O5S.
Elem. Anal. for C21H18N6O5S:
(Calc) C, 54.07; H, 3.89; N, 18.02; S, 6.87. Found: C, 54.10; H, 4.06; N,
17.72; S, 6.46.

 (1-(4-Nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl-2-(4-oxo-5,6,7,8-tetrahydrobenzo4,5thieno2,3-dpyrimidin-3(4H)-yl)acetate
(7h):
Yellow
Solid Yield: 70%; mp 155-156oC; IR (KBr) (?max cm-1):
3112.8 (=CH), 2939 (-CH), 1738 (ester C=O), 1675 (amide C=O), 1606 (C=N); 1H
NMR (400 MHz, CDCl3): ?
1.80-1.90 (m, 4H, 2CH2), 2.79 (t, J = 6.0 Hz, 2H, CH2), 2.99 (t, J = 6.0 Hz, 2H, CH2), 4.70 (s, 2H, CH2), 5.44
(s, 2H, CH2-triazole), 7.02 (d, J
= 9.0 Hz, 2H, Ar-H), 7.64 (d, J = 9.0
Hz, 2H, Ar-H), 7.86 (s, 1H, triazole-CH), 
8.04 (s, 1H, pyrimidine-CH); 13C NMR (100 MHz, CDCl3):
? 21.7, 23.1, 23.5, 27.5, 41.5, 60.5,
117.6, 119.1, 120.9, 123.9, 125.4, 139.4, 142.9, 144.4, 147.6, 148.2, 155.5,
161.6, 169.5; LCMS (m/z): 467.1 M+H+,
489.2 M+Na+ for  C21H18N6O5S.
Elem. Anal. for C21H18N6O5S:
(Calc) C, 54.07; H, 3.89; N, 18.02; S, 6.87. Found: C, 54.37;
H, 4.17; N, 17.75; S, 6.48.

(1-(4-Chloro-2-methoxy-6-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl-2-(4-oxo-5,6,7,8-tetrahydrobenzo4,5thieno2,3-dpyrimidin-3(4H)-
yl)acetate (7i):
Brown
solid Yield: 70%; mp 150-155oC; IR (KBr) (?max cm-1):
3114 (=CH), 2939 (-CH), 1730 (ester C=O), 1672 (amide C=O), 1610 (C=N); 1H
NMR (400 MHz, CDCl3): ?
1.79-1.89 (m, 4H, 2CH2), 2.77 (t, J = 5.8 Hz, 2H, CH2), 2.96 (t, J = 5.9 Hz, 2H, CH2), 3.93 (s, 3H, -OCH3),
4.72 (s, 2H, CH2), 5.44 (s, 2H, CH2-triazole), 7.14 (s,
1H, Ar-H), 7.49 (s, 1H, Ar-H), 7.86 (s, 1H, triazole-CH), 8.26 (s, 1H,
pyrimidine-CH); 13C NMR (100 MHz, CDCl3): ? 22.5, 23.0, 23.7, 25.4, 42.5, 55.8,
71.5, 107, 116.5, 117.6, 119.1, 121.2, 125.4, 136.2, 139.4, 144.4, 147.6, 149.1,
155.5, 156.4, 161.6, 169.5; LCMS (m/z):
531.1 M+H+ for C22H19N6O6SCl.
Elem. Anal. for C22H19N6O6SCl:
(Calc) C, 49.77; H, 3.61; N, 15.83; S, 6.04. Found: C, 50.04; H, 3.98; N,
15.63; S, 5.99.

(1-(2-Chloro-5-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl-2-(4-oxo-5,6,7,8-tetrahydrobenzo4,5thieno2,3-dpyrimidin-3(4H)-yl)acetate
(7j): Brown solid Yield: 60%; mp 170-180oC; IR
(KBr) (?max cm-1): 3108.6 (=CH), 2938 (-CH), 1751 (ester
C=O), 1676 (amide C=O), 1610 (C=N); 1H NMR (400 MHz, CDCl3):
? 1.79-1.89 (m, 4H, 2CH2),
2.78 (t, J = 6.1 Hz, 2H, CH2),
2.95 (t, J = 6.1 Hz, 2H, CH2),
4.71 (s, 2H, CH2), 5.48 (s, 2H, CH2-triazole), 7.81 (d, J = 8.8 Hz, 1H, Ar-H), 7.87 (s, 1H,
triazole-CH), 8.21 (s, 1H, pyrimidine-CH), 8.35 (d, J = 6.2 Hz, 1H, Ar-H), 8.57 (d, J
= 2.6 Hz, 1H, Ar-H); 13C NMR (100 MHz, CDCl3): ? 21.5, 22.5, 23.7, 37.5, 42.3, 61.5,
109.0, 117.6, 119.1, 125.4, 127.3, 129.7, 133.3, 135.6, 138.6, 139.4, 144.4,
147.6, 155.6, 161.6, 169.5; LCMS (m/z):
501.1 M+H+, 523.1 M+Na+ for C21H17N6O5SCl.
Elem. Anal. for C21H17N6O5SCl:
(Calc) C, 50.35; H, 3.42; N, 16.78; S, 6.40. Found: C, 50.40; H, 3.69; N,
16.64; S, 6.52.

In vitro antibacterial activity assay

            Evaluation of antibacterial
activities of the synthesized compounds was carried out in 96-well micro titer
plates using the broth dilution method 48. The bacterial cultures were grown
overnight at 37oC and serially diluted to a concentration of 106
colony forming units/mL with sterile MHB (Mueller-Hinton
broth) medium. A different synthesized compounds (1, 2.5, 5, 10, 25, 50, 75,
100 µg/mL) were diluted with MHB medium was performed in triplicate for each
compound in a sterile 96-well plate to a final volume of 50 ?L in each well.
The final volume was adjusted to 250?L by adding an aliquot of 100 ?L bacterial
suspensions and 100 ?L MHB broths to each well, and the plates were incubated
at 37oC for 18-20
h. Controls were performed without synthesized compounds and the MIC of
chemical compounds was defined as the lowest concentration of the synthesized
compounds required for complete inhibition or killing the bacterial inoculu