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

Thiophenemoiety have attracted many researchers in medicinal field due to its variousbiological activities such as antimicrobial 1, antibacterial 2, antifungal3, anticancer 4, antitubercular 5, antiamoebic 6, antiparasitic 7, anti-inflammatory8, antioxidant 9, anticonvulsant 10, local anaesthetic 11, andanti-arrhythmic 12.                        Pyrimidine derivatives play a vitalrole in many biological aspects and transformation. This ring system is presentin cytosine, adenine, guanine and thiamine, which form a part of ribonucleicacid (RNA) and deoxyribonucleic acid (DNA) and vitamins like vitamin B2(Riboflavin) and vitamin B6 (Pyridoxine) co-enzyme and other purines 13. Pyrimidinenucleus is also present in barbituric acid and its several derivatives likeVeranal which are used as hypnotics 14.              Condensedpyrimidine derivatives have been reported as anti-microbial 15, anti-inflammatory16, anti- HIV 17, anti-tubercular 18, anti-tumor 19, anti-neoplastic 20,anti-malarial 21, diuretic 22, cardiovascular 23 agents and hypnoticdrugs for the nervous system 24, calcium-sensing receptor antagonists 25and also for antagonists of the human A2A adenosine receptor.

26 etc             The heterocyclic fusion of thiophenering with pyrimidine ring system resulted in formation of thienopyrimidine, astructural analogue of biogenic purine, an important class of heterocycliccompounds which possesses broad spectrum of biological activities antibacterial27, antimicrobial 28, antiviral 29, anti-HIV 30, anti-avian influenzavirus (H5N1) 31,  and analgesic 32ect. Among them, thieno2,3-dpyrimidines have been proved useful forcerebral ischemia, malaria, tuberculosis, Alzheimer’s and Parkinson’s diseases.  LITERATURE REVIEWThieno2,3-dpyrimidines                        Alagarsamy etal.,have synthesized novel 2-mercapto-3-substituted-5,6-dimethylthieno2,3-dpyrimidin-4(3H)-ones and reported as anti inflammatory andanalgesic agents 33.               J.E. Tarver Jr.

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et al., havesynthesized 2-((5,6-thieno2,3-dpyrimidin-4-yl)thio)aceticacids and proved to be inhibitors of Notum Pectinacetylesterase 34.                          JiangpingLou et al., discovered compounds witha cyclic thienopyrimidine moiety and an aceto-hydrazone moiety in theirchemical structure and screened cytotoxicity against tumor cell lines A549,HeLa, MDA-MB-231 and HEK-293T 35.              Srujana Muthadi etal., have synthesized a series of novel2-Methyl-4-(1-methyl-1H-1,2,3triazol-4-yl-methoxy)-5,6,7,8-tetrahydra-benzo4,5thieno2,3-dpyrimidinederivatives and screened for in vitro antimicrobial activity 36.

                        M. R. Bhadane etal., have synthesized varioussubstituted thieno(2,3-d)pyrimidines and examined their antioxidant activity byDPPH scavenging method 37.    1,2,3-triazoles            Haideret al., have synthesized a library of benzoxazolinone based 1, 2,3-triazoles using click chemistry approach and screened them for their invitro and in vivo anti-inflammatory activity 38.

            Yempalaet al., have synthesized a series of novel dibenzofuran based 1, 2,3-triazole derivatives using click chemistry approach as in vitro anti-mycobacterialagent against Mycobacterium tuberculosis H37Rv 39.             Duanet al., have synthesized a series of novel 1, 2, 3- triazole-dithiocarbamatehybrids and tested them against four selected human tumor cell lines i.e.MGC-803, MCF- 7, PC-3, EC-109 40.             Abdel-Wahabet al.

, have carried out the synthesis of novel pyrazolyl-1,2,3-triazolesand screened for antimicrobial activity 41.                         Jordaoet 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 acidhydrazides and screened them against Cantagalo virus replication 42.  PresentworkObjectives:Ø  Tosynthesize novel1,2,3-triazole derivatives of thieno2,3-dpyrimidinonesØ  Tocharacterize the derivatives by IR, 1H NMR, 13C NMR, massspectra and also by elemental analysis.Ø  Toexamine in vitro antibacterial activity of compounds by broth dilutionmethod against gram positive and gram negative bacteria.Ø  Todetermine docking studies against Bacillus subtilis purine riboswitchand Escherichia coli TPP riboswitch.             Today, bacterial infection is one of themost complex health issues globally. Because of increasing antibiotic resistantorganisms and also decreasing the discovery of new antimicrobial agents, thereis a pressing need of new antimicrobial agents that are capable of treatingthose strains 43. As a result, increasing efforts have been made to developmulti drug resistant (MDR) strategy 44 by which each drug moiety is designedto bind independently two different biological targets which offer thepossibility to overcome the current resistance 45.

            Amongfused pyrimidines, thieno2,3-dpyrimidineswere  found to exhibit various biologicalactivities. Several thieno2,3-dpyrimidinederivatives were synthesized and proved to be antibacterial agents. Inaddition, 1,2,3-triazoles possess unique features which make them importantpharmacophore in curative medicinal chemistry and they act as bioisosters andlinkers 46. Also they are reliable and quantitative to synthesizeby using Huisgen’s copper catalyzed azide-alkyne cyclo addition (CuAAC) undermild conditions without protecting groups and purification 47.            Inliterature, there are numerous fused 1,2,3-triazole derivatives of thieno2,3-dpyrimidines but 1,2,3-triazole linkedthieno2,3-dpyrimidine derivativesare exceptional in synthesis.

In the light of the above specifics, considerableefforts have been made to develop novel synthetic strategies by joining thieno2,3-d pyrimidinone scaffold and1,2,3-triazole moiety through ester linkage. The synthesized compounds werescreened for their antibacterial activity to obtain novel candidates ofincredible activities with nominal side effects. In order to establish thebinding interactions, docking studies were performed on B. subtilispurine riboswitch and E. coli TPPriboswitch.  Scheme1  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   Methodsand MaterialsChemistry Synthesisof 5,6,7,8-tetrahydro1benzothieno2,3-dpyrimidin-4(3H)-one (2): Concentratedsulfuric acid (4 mL) at 0-5oC was added to a mixture of 2-amino-4,5,6,7tetrahydrobenzobthiophene-3-carbonitrile1 (1 g, 5.

6 mmol) in formic acid (10mL). 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.

     Browncolor 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 ionmode) (m/z): 205 M-H– forC10H10N2OS.Synthesisof 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) andpotassium carbonate (2 g, 15 mmol) in 20 mL acetone was added and stirred for8h.

After completion of reaction mixture, the acetone was evaporated and theprecipitated solid was obtained, washed with cold water and recrystallized withethanol.         Paleyellow 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); 13CNMR (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+ forC14H16N2O3S.Synthesisof 2-(4-oxo-5,6,7,8-tetrahydrobenzo4,5thieno2,3-dpyrimidin-3(4H)-yl)aceticacid (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 temperatureovernight.

After completion of reaction, the reaction mixture was poured intocrushed ice and con. HCl was added till it gets acidified. The formed solid wasfiltered 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.Synthesisof prop-2-yn-1-yl-2-(4-oxo-5,6,7,8-tetrahydrobenzo4,5thieno2,3-dpyrimidin-3(4H)-yl)acetate (5): To a solution ofcompound 4 (2 g, 7.6 mmol) in dryacetone (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 mixturewas stirred overnight at room temperature. The reaction was monitored by TLC.Upon completion of reaction, the reaction mixture was poured into crushed iceand stirred continuously.

The obtained solid was filtered and purified bycolumn 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); 1HNMR (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+, 325M+Na+  for C15H14N2O3S.Synthesisof aryl azides (6a-j)             Anilinederivative (9.

1 mmol) was dissolved in con. HCl (91 mmol) at room temperatureand cooled up to 0 oC, followed by addition of a solution of sodiumnitrite (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 hat room temperature. After completion of reaction, the mixture was extractedwith hexane. The organic layer was dried over anhydrous sodium sulphate andafter evaporation of the solvent the desired azide derivative was obtained.    Generalprocedure 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): Compound5 (0.2 g, 0.7 mmol) was dissolved in2 mL of THF: H2O (1:1) solvent in a 50 mL round bottom flask. Tothis CuSO4.5H2O(0.

17 g, 0.7 mmol) was added and the reaction mixture was stirred for 5 min. Tothis light blue colour reaction mixture sodium ascorbate (0.2 g, 1.

05 mmol) wasadded and stirred for 15 min. The reaction mixture becomes dark yellow. Thenaryl azide (6a-j) (1.05 mmol) wasadded and allowed to stir for 30 min to 1 h.

After completion of reactionmonitored by TLC, the reaction mixture was extracted with ethyl acetate byadding excess water. The combined organic layer was washed with brine solutionand dried over anhydrous sodium sulphate. The solvent was evaporated underreduced pressure and the crude product was purified by silica gel columnchromatography 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).  1HNMR (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-tetrahydrobenzo4,5thieno2,3-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 (esterC=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+, 478.1M+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-dpyrimidin-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); 13CNMR (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):Yellowsolid 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); 1HNMR (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-tetrahydrobenzo4,5thieno2,3-dpyrimidin-3(4H)-yl)acetate (7e): Pale yellowsolid 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); 1HNMR (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-tetrahydrobenzo4,5 thieno 2,3-d pyrimidin-3(4H)-yl)acetate (7f): Light brownsolid 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); 1HNMR (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):Brownsolid 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):YellowSolid 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); 1HNMR (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 = 9.0Hz, 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):Brownsolid 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); 1HNMR (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 (esterC=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 antibacterialactivities of the synthesized compounds was carried out in 96-well micro titerplates using the broth dilution method 48. The bacterial cultures were grownovernight at 37oC and serially diluted to a concentration of 106colony forming units/mL with sterile MHB (Mueller-Hintonbroth) 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 eachcompound 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 bacterialsuspensions and 100 ?L MHB broths to each well, and the plates were incubatedat 37oC for 18-20h. Controls were performed without synthesized compounds and the MIC ofchemical compounds was defined as the lowest concentration of the synthesizedcompounds required for complete inhibition or killing the bacterial inoculu