C

urre

nt O

rgan

ic S

ynth

esis

���������������������

���2��89:;.8::9.?@:8

���������������������

��������������

��������������

Send Orders for Reprints to reprints@benthamscience.ae

Current Organic Synthesis, 2018, 15, 1161-1170

1161

RESEARCH ARTICLE

Synthesis, Antimicrobial Evaluation of Some New 1, 3, 4-Thiadiazoles and 1, 3, 4-Thiadiazines

Ameen Ali Abu-Hashem1, 2,* and Rasha A. M. Faty3

1Photochemistry Department (Heterocyclic Unit), National Research Centre, 12622, Dokki, Giza, Egypt; 2Chemistry Department, Faculty of Science, Jazan University, 2097 Jazan, KSA; 3Department of Chemistry, Faculty of Science, Cairo University, Giza, Egypt

A R T I C L E H I S T O R Y

Received: October 04, 2017 Revised: June 14, 2018 Accepted: July 12, 2018

DOI: 10.2174/1570179415666180720114547

Abstract: Background: 1, 3, 4-thiadiazoles, 1, 3, 4-thiadiazines and thienopyrimidines have newly attracted attention due to their forceful pharmacological activities. They showed antimicrobial, antiviral, analgesic and anti-inflammatory properties.

Objective: The aim of this research is to synthesize new thiadiazolothienopyrimidines (2-10), thienopyrimido-thiadiazines (11-15), quinoxaline-thienopyrimidinones (16) and thienopyrimido- thiadiazinoquinoxalinones (17) via effectual high yield procedure for assessing their antimicrobial activity.

Method: A series of new 1, 3, 4-thiadiazolothienopyrimidines, thienopyrimidothiadiazines and thienopyrimido-thiadiazinoquinoxalinones was prepared from 6-acetyl-3-amino-5-methyl-2-thioxo-2, 3-dihydrothieno [2, 3-d] pyrimidin-4(1H)-one (1) as the beginning material.

Results: The 1, 3, 4-thiadiazoles, 1, 3, 4-thiadiazines derivatives (1-17) were synthesized in adequate to good yields (60-85%) in a stepwise effectual procedure under condition. The chemical structures of these new com-pounds were confirmed via many spectroscopic techniques as UV, IR, NMR, mass spectra and elemental analysis. In vitro, antimicrobial was evaluated for the synthesized compounds using minimal inhibitory concen-tration of these compounds against bacteria and fungi.

Conclusion: The 1, 3, 4-thiadiazole and 1, 3, 4-thiadiazine derivatives (15-17) exhibited higher antimicrobial activity (Gram-positive, Gram-negative bacteria and fungi) compared with the standard antibiotic drugs; Levofloxacin (Tavanic) and Nystatin.

Keywords: 1, 3, 4-Thiadiazoles, 1, 3, 4-thiadiazines, thienopyrimidines, spiro-cyclohexane, quinoxalines, antimicrobial activity.

1. INTRODUCTION Recently, 1, 3, 4-thiadiazole and 1, 3, 4-thiadiazine moieties

have drawn much attention for their great biological importance. Compounds containing such moieties have been therapeutically used for the treatment of pains, inflammations and hypertension [1, 2]. Moreover, they have pharmacological properties as antimicro-bial [3], analgesic [4], antiviral and anti-inflammatory properties [5, 6]. However, the thiadiazole moiety, containing (N=C-S-) group, exhibits different biological activities such as being antidepressant, antioxidant, anti-cancer, anti-inflammatory, antimicrobial, anti-convulsant [7], cytotoxic [8], anti-tubercular [9] and anxiolytic activities [10]. It was found that thiadiazines fused with triazoles exhibit antimicrobial [11-15], anti-tubercular [15], anti-inflam-matory and anti-molluscicidal [16], antitumor activities [17], antivi-ral activities against herpes simplex virus (HSV-1) and Japanese Encephalitis Virus (JEV) [17-19]. Also, thienopyrimidine deriva-tives display potent pharmacological activities such as treatment of reproductive diseases disorders and cancers [20], antitumor [21], inhibitors of cancer cell proliferation [22], treatment for the inhibi-tion of tumor growth [23], anti-proliferative activity [24], antioxi-dant and antibacterial [25].

*Address correspondence to this author at the Photochemistry Department (Heterocyc-lic Unit), National Research Centre, 12622 Dokki, Giza, Egypt and Chemistry Depart-ment, Faculty of Science, Jazan University, 2097 Jazan, Saudi Arabia; Tel: +2-01225211700, + 966-591363915, Fax: + 202 33370931, + 966-73245-212; E-mail: aminaliabuhashem@yahoo.com

Moreover, when thienopyrimidines are combined with pyra-zole, oxadiazole or thiadiazole rings, they showed important anti-inflammatory activity [26]. In addition, thiophene nucleus can be used in a general diversity of antimicrobial [27], antioxidant and antitumor activities [28]. In a continuation of our previous work on the synthesis of biologically active heterocyclic compounds [29-35], we hereby report on new synthetic procedures for the prepara-tion of some new 1,3,4-thiadiazole and 1,3,4-thiadiazine derivatives fused to several heterocyclic systems.

2. MATERIALS AND METHODS 2.1. Materials

All materials used were obtained from Sigma Aldrich.

2.2. EquipmentsAll melting points are in degree centigrade (uncorrected) and

were determined on Gallenkamp electric melting point apparatus. TLC analysis was carried out on silica gel 60 F254 precoated alumi-num sheets. The IR spectra were recorded (KBr) on a Perkin–Elmer 1430 spectrometer (�, cm-1) in National Research Center, Egypt. 1Hand 13C-NMR Spectra were measured on JEOL-ECA 500 and JEOL JNM-LA-400 FT NMR Spectrometers at 500, 125 MHz, respectively, using tetramethylsilane (TMS) as an internal reference and DMSO-d6 as the solvent at the Microanalytical Center in Na-tional Research Center, Egypt. The mass spectra (EI) were recorded

1875-6271/18 $58.00+.00 © 2018 Bentham Science Publishers

1162 Current Organic Synthesis, 2018, Vol. 15, No. 8 Abu-Hashem and Faty

on GCMS-QP 1000 EX (Shimadzu) at National Research Center, Egypt. Elemental analyses (C, H and N) were carried out at the Microanalytical Center in National Research Center, Egypt. The elemental analyses were found to agree favorably with the calcu-lated values.

2.2.1. 6-Acetyl-7-methyl-3a,4-dihydro-8H-[1,3,4]thiadiazolo[3,2- a]thieno[2,3-d]pyrimidin-8-one(2)

A mixture of 1 (2.55g, 10 mmol) in formic acid (20 mL) was heated and refluxed for 10-12 hours and monitored using TLC. The deposited precipitate was filtered off, and crystallized from metha-nol as white crystals in 78% yield, MP 347-349 °C; IR (�max,cm-1)KBr: 3355 (br, NH), 3035 (CH aryl), 2922 (CH alkyl),1700,1680 (2CO); 1H NMR (DMSO-d6) � 2.85 (s, 3H, CH3), 3.05 (s, 3H, CH3), 5.80 (s, 1H, CH, thiadiazole), 6.80 (br, 1H, NH, D2O ex-changeable), 8.01 (s, 1H, CH, thiadiazole ); 13C NMR (DMSO-d6) �18.1, 28.4 (2CH3), 77.4,118.8 (pyrimidine C), 136.2 (thiophene C), 152.1 (thiadiazole C),155.6, 158.8 (thiophene C),166.9, 180.5 (2CO); MS (70 ev, %) m/z 267 (M+, 100%); Anal. Calc. for C10H9N3O2S2 (267.32): C, 44.93; H, 3.39; N, 15.72. Found: C, 44.85; H, 3.32; N, 15.80.

2.2.2. 6-Acetyl-7-methyl-8H-[1,3,4]thiadiazolo[3,2-a]thieno[2,3-d]pyrimidine-2,8(1H)-dione (3)

Method a: A mixture of 1 (2.55g, 10 mmol) in methanol (50 mL) with sodium methoxide (10 mmol) and ethyl chloroformate (0.95 mL, 10 mmol) was added. The reaction mixture was refluxed for 8-10 h, then evaporated under vacuum, the solid obtained washed with H2O and recrystallized from dioxane.

Method b: A mixture of 1 (2.55g, 10 mmol) and urea (0.6 g, 10 mmol) was fused at 270°C for 10 h. The reaction mixture was cooled and added to a solution of sodium hydroxide (5%, 20 mL), then filtered and the filtrate acidified with dilute HCl. The solid product recrystallized from DMF as white powder in 75% yield, MP > 350°C (melted); IR (�max,cm-1) KBr: 3352 (br, NH), 2920 (CH alkyl), 1701, 1682, 1673 (3CO); 1H NMR (DMSO-d6) � 2.82 (s, 3H, CH3), 3.03 (s, 3H, CH3), 9.55 (br, NH, D2O exchangeable); 13C NMR (DMSO-d6) � 18.5, 28.2 (2CH3), 118.8 (pyrimidine C), 138.7 (thiophene C),151.8 (thiadiazole C), 154.9, 159.5 (thiophene C), 163.2, 166.8, 182.4 (3CO); MS (70ev,%) m/z 281 (M+, 90%); Anal. Calc. for C10H7N3O3S2 (281.30): C, 42.70; H, 2.51; N, 14.94. Found: C, 42.77; H, 2.58; N, 14.90.

2.2.3. 6-Acetyl-2-mercapto-7-methyl-8H-[1,3,4]thiadiazolo[3,2-a]thieno[2,3-d]pyrimidin-8-one(4)

Method a: To a solution of 1 (2.55g, 10 mmol) in ethanolic KOH solution (10 mL ethanol and 1 g, KOH) and Carbon disul-phide (2 mL) was added dropwise with stirring and refluxed for 2-4 h. The solvent was cooled and then product was extracted with dry methanol, which was then poured onto ice and acidified with dil. HCl. The solid product obtained was washed thoroughly with water and recrystallized from methanol.

Method b: A mixture of 3 (2.81g, 10 mmol) and P2S5 (2.22g, 10 mmol) in dry pyridine with refluxed for 10-12 h was monitored (TLC). The solid formed was collected by filtration, washed with water and ethanol, and dried. The product was recrystallized from methanol as yellow crystals in 70% yield, MP 288-290°C (melted); IR (�max,cm-1) KBr: 2930 (CH alkyl),1708,1685(2CO), 1662 (C=N), 1605 (C=C), 1525 (C-N stretching),1145 (CS); 1H NMR (DMSO-d6) � 2.86 (s, 3H, CH3), 3.08 (s, 3H, CH3), 13.55 (br,1H,SH); 13C-NMR (DMSO-d6) � 19.1, 28.5 (2CH3), 119.6 (pyrimidine C), 139.2 (thiophene C),152.2,157.5 (thiadiazole C), 158.1, 159.9 (thiophene C),165.4,180.2 (2CO); MS (70 ev,%) m/z 297 (M+, 84%); Anal. Calc. for C10H7N3O2S3 (297.37): C, 40.39; H, 2.37; N, 14.13. Found: C, 40.45; H, 2.41; N, 14.18.

2.2.4. 6-Acetyl-2,7-dimethyl-8H-[1,3,4]thiadiazolo[3,2-a]thieno[ 2,3-d]pyrimidin-8-one(5) and 6-acetyl-7-methyl-2-phenyl -8H-[1,3,4]thiadiazolo[3,2-a]thieno[2,3-d]pyrimidin-8-one (6): Gen-eral procedure

A mixture of 1 (2.55g, 10 mmol) and oxalic acid or benzoic acid (10 mmol) in POCl3 (40 mL) was refluxed for 8-12 h. The reaction mixture was slowly poured into crushed ice with stirring and neutralized with NaHCO3. The solid product was precipitated off filtered and washed with 100 ml water, recrystallized from the proper solvent to give 5 and 6, respectively.

2.2.5. 6-Acetyl-2,7-dimethyl-8H-[1,3,4]thiadiazolo[3,2-a]thieno [2,3-d]pyrimidin-8-one(5)

Compound 5 was obtained from the reaction of 1 (2.55g, 10 mmol) and oxalic acid (0.9g, 10 mmol), crystallized from benzene as brownish crystals 73% yield, MP > 350°C (melted); IR (�max, cm-1) KBr: 2935 (CH alkyl),1710,1680 (2CO), 1655 (C=N), 1608 (C=C), 1527 (C-N stretching), 1155(CS); 1H NMR (DMSO-d6) �1.22 (s, 3H, CH3), 2.83 (s, 3H, CH3), 3.06 (s, 3H, CH3); 13C NMR (DMSO-d6) � 19.4, 25.1, 28.2 (3C, 3CH3), 119.8 (pyrimidine C), 139.5 (thiophene C), 154.6, 157.8 (thiadiazole C),159.7, 161.8 (thiophene C), 166.2, 181.5 (2CO); MS (70 ev, %) m/z 279 (M+,90%); Anal. Calc. for C11H9N3O2S2 (279.33): C, 47.30; H, 3.25; N, 15.04. Found: C, 47.38; H, 3.29; N, 15.11.

2.2.6. 6-Acetyl-7-methyl-2-phenyl-8H-[1,3,4]thiadiazolo[3,2-a] thieno[2,3-d]pyrimidin-8-one(6)

The compound 6 was obtained from the reaction of 1 (2.55g, 10 mmol) and benzoic acid (1.22g, 10 mmol), crystallized from hexane as yellowish crystals 76% yield, MP > 350°C (melted); IR (�max, cm-1) KBr: 3040 (CH aryl), 2932 (CH alkyl), 1709,1681 (2CO), 1658 (C=N), 1609 (C=C), 1526 (C-N stretching), 1153 (CS); 1HNMR (DMSO-d6) � 2.80 (s, 3H, CH3), 3.02 (s, 3H, CH3), 7.50 -7.70 (m, 5H, Ar); 13C NMR (DMSO-d6) � 19.1, 28.1 (2CH3), 119.4 (pyrimidine C), 128.5, 129.1, 130.7, 131.2 (Ar-C), 139.3 (thiophene C), 144.2, 155.9 (thiadiazole C),158.8, 160.7 (thiophene C), 164.5, 182.6 (2CO); MS (70 ev, %) m/z 341 (M+, 80%); Anal. Calc. for C16H11N3O2S2 (341.40): C, 56.29; H, 3.25; N, 12.31. Found: C, 56.21; H, 3.20; N, 12.38.

2.2.7. Ethyl 2-(6-Acetyl-7-methyl-8-oxo-8H-[1,3,4]thiadiazolo[3, 2-a]thieno[2,3-d] pyrimidin-2-yl) acetate (7)

A mix of 1 (2.55g, 10 mmol) and ethyl cyanoacetate (1.06 mL, 10 mmol) in polyphosphoric acid (PPA, 15 mL) was stirred with temperature of 60-80°C for 8-10 h and then allowed to cool to room temperature and finally poured into cold water (100 mL) and neu-tralized by sodium bicarbonate. The solid product that precipitated was filtered off, washed with water and dried. Then recrystallized from dioxane as Yellow crystals in 65% yield, MP 310-312°C; IR (�max,cm-1) KBr: 2940 (CH alkyl), 1775, 1710, 1684 (3CO), 1668 (C=N), 1600 (C=C), 1523 (C-N stretching), 1140 (CS); 1H NMR (DMSO-d6) � 1.25 (t, 3H, J = 6.45 Hz, CH3), 2.84 (s, 3H, CH3),3.01 (s, 3H, CH3), 3.12 (s, 2H, CH2), 4.21 (q, 2H, J = 6.55 Hz, CH2); 13C NMR (DMSO-d6) � 18.4, 21.2, 28.1 (3CH3), 36.3, 62.5 (2CH2), 119.3 (pyrimidine C), 139.5 (thiophene C), 153.1, 157.8 (thiadiazole C), 158.7, 160.5 (thiophene C), 164.2, 169.1, 184.6 (3CO); MS (70 ev, %) m/z 351 (M+, 75%); Anal. Calc. for C14H13N3O4S2 (351.39): C, 47.85; H, 3.73; N, 11.96. Found: C, 47.90; H, 3.79; N, 11.90.

2.2.8. 6'-Acetyl-7'-methyl-1'H, 8'H-spiro [cyclohexane-1,2'-[1, 3, 4] thiadiazolo[3,2-a]thieno[2,3-d] pyrimidin]-8'-one(8)

A mixture of 1 (2.55g, 10 mmol), cyclohexanone (1.03 mL, 10 mmol) and triethyl-amine (1.01 mL, 10 mmol) in dimethylforma-mide (30 mL) was refluxed for 14-16 h. The reaction mixture was allowed to cool to room temperature and poured into cold water (100 mL). The deposited precipitate was filtered off, dried and crys-tallized from dioxane as white crystals in 68% yield, MP 238-240

Synthesis, Antimicrobial Evaluation of Some New 1, 3, 4-Thiadiazoles Current Organic Synthesis, 2018, Vol. 15, No. 8 1163

°C; IR (�max,cm-1) KBr: 3350-3280 (br, NH), 2945 (CH alkyl), 1712, 1688 (2CO), 1662 (C=N), 1603 (C=C), 1530 (C-N stretch-ing), 1144 (CS); 1H NMR (DMSO-d6) � 1.41-1.54 (m, 6H, 3CH2), 2.15 (t, 4H, 2CH2), 2.80 (s, 3H, CH3), 3.06 (s, 3H, CH3), 10.15 (br, NH, D2O exchangeable); 13C NMR (DMSO-d6) � 18.1(CH3), 24.6, 25.5 (3CH2), 28.3 (CH3), 33.4 (2CH2), 74.6 (Spiro carbon), 118.8 (pyrimidine C), 138.9 (thiophene C), 158.1 (thiadiazole C), 159.5, 161.7 (thiophene C), 165.3, 185.2 (2CO); MS (70 ev, %) m/z 335 (M+, 72%); Anal. Calc. for C15H17N3O2S2 (335.44): C, 53.71; H, 5.11; N, 12.53. Found: C, 53.79; H, 5.20; N, 12.58.

2.2.9. 6-Acetyl-2-(4-substituted-phenyl)-7-methyl-1,2-dihydro-8H-[1,3,4]thiadiazolo[3,2-a]thieno[2,3-d]pyrimidin-8-one (9a-c): Ge-neral procedure

A mixture of 1 (2.55g, 10 mmol), appropriate aromatic alde-hyde (10 mmol), in dimethylformamide (35 mL) and a catalytic amount of p-toluene sulfonic acid (0.2 g) was heated under reflux for 15-18 h, The solution was allowed to cool to room temperature and poured into crushed ice with stirring The obtained solid was filtered off and recrystallized from the proper solvent.

2.2.10. 6-Acetyl-7-methyl-2-phenyl-1,2-dihydro-8H-[1,3,4]thiadi- azolo[3,2-a]thieno[2,3-d]pyrimidin-8-one(9a)

Compound 9a was obtained from the reaction of 1 (2.55g, 10 mmol) and benzaldehyde (1.06 g, 10 mmol), crystallized from methanol as brownish crystals 66% yield, MP > 350°C (melted); IR (�max, cm-1) KBr: 3355 (br, NH), 3045 (CH aryl), 2930 (CH alkyl), 1706,1680 (2CO), 1652 (C=N), 1601 (C=C), 1525 (C-N stretch-ing), 1150 (CS); 1H NMR (DMSO-d6) � 2.82 (s, 3H, CH3), 3.06 (s, 3H, CH3), 7.22-7.38 (m, 5H, Ar), 10.85 (br, NH, D2O exchange-able); 13C NMR (DMSO-d6) � 18.4, 28.5 (2CH3), 67.8 (thiadiazole C),119.7 (pyrimidine C), 125.6, 127.3, 128.9, 138.8 (Ar-C), 139.6 (thiophene C), 156.3 (thiadiazole C), 157.8, 160.4 (thiophene C), 163.2, 181.5 (2CO); MS (70 ev, %) m/z 343 (M+, 92%); Anal. Calc. for C16H13N3O2S2 (343.42): C, 55.96; H, 3.82; N, 12.24. Found: C, 55.90; H, 3.89; N, 12.30.

2.2.11. 6-Acetyl-2-(4-chlorophenyl)-7-methyl-1,2-dihydro-8H-[1, 3,4]thiadiazolo[3,2-a]thieno[2,3-d]pyrimidin-8-one(9b)

The compound was obtained from the reaction of 1 (2.55g, 10 mmol) and 4-chloro benzaldehyde (1.4 g, 10 mmol), crystallized from ethanol as white crystals 74% yield, MP > 350 °C; IR (�max, cm-1) KBr: 3360 (br, NH), 3043 (CH aryl), 2929 (CH alkyl), 1705, 1682 (2CO), 1654 (C=N), 1604 (C=C), 1528 (C-N stretching), 1151 (CS); 1H NMR (DMSO-d6) � 2.83 (s, 3H, CH3), 3.07 (s, 3H, CH3), 5.75 (s, 1H, CH, thiadiazole), 7.24 (d,d, 2H, J = 7.55 Hz, Ar), 7.40 (d,d, 2H, J = 7.52 Hz, Ar), 10.80 (br, NH, D2O exchangeable); 13C NMR (DMSO-d6) � 18.5, 28.6 (2CH3), 67.6 (thiadiazole C), 119.5 (pyrimidine C), 128.7, 129.2, 131.9, 139.5 (Ar-C), 139.9 (thiophene c), 156.8 (thiadiazole C), 158.7, 161.2 (thiophene C), 164.5, 184.7 (2CO); MS (70 ev, %) m/z 377 (M+, 95%); Anal. Calc. for C16H12ClN3O2S2 (377.86): C, 50.86; H, 3.20; N, 11.12. Found: C, 50.92; H, 3.28; N, 11.19.

2.2.12. 6-Acetyl-2-(4-methoxyphenyl)-7-methyl-1,2-dihydro-8H-[1,3,4]thiadiazolo[3,2-a]thieno[2,3-d]pyrimidin-8-one(9c)

The compound was obtained from the reaction of 1 (2.55g, 10 mmol) and 4-anisaldehyde (1.36 g, 10 mmol), crystallized from dioxane as yellowish crystals 67% yield, MP > 350°C (melted); IR (�max, cm-1) KBr: 3358 (br, NH), 3041 (CH aryl), 2928 (CH alkyl), 1703, 1680 (2CO), 1650 (C=N), 1600 (C=C), 1526 (C-N stretch-ing), 1150 (CS); 1H NMR (DMSO-d6) � 2.81 (s, 3H, CH3), 3.08 (s, 3H, CH3), 3.85 (s, 3H, CH3), 5.72 (s, 1H, CH, thiadiazole), 7.03 (d,d, 2H, J = 7.58 Hz, Ar), 7.85 (d,d, 2H, J = 7.54 Hz, Ar), 10.88 (br, NH, D2O exchangeable); 13C NMR (DMSO-d6) � 18.3, 28.2, 56.4 (3CH3), 67.7 (thiadiazole), 119.4 (pyrimidine C), 121.1, 128.8, 134.4, 152.5 (Ar-C), 139.3 (thiophene C), 155.6 (thiadiazole C), 157.5, 160.9 (thiophene C), 162.8, 185.1 (2CO); MS (70 ev, %) m/z

373 (M+, 80%); Anal. Calc. for C17H15N3O3S2 (373.44): C, 54.68; H, 4.05; N, 11.25. Found: C, 54.60; H, 4.11; N, 11.31.

2.2.13. 6-Acetyl-2-(substituted-amino)-7-methyl-8H-[1,3,4]thiadi- azolo[3,2-a]thieno[2,3-d]pyrimidin-8-one(10a,b): General Proce-dure

A solution of 1 (2.55g, 10 mmol) and alkyl/aryl isothiocyanate (10 mmol) in anhydrous dimethylformamide (40 mL) was heated and refluxed with stirring for 8-10 h (TLC). The solution was al-lowed to cool to room temperature and poured into crushed ice with stirring. The deposited precipitate was washed with H2O and fil-tered off, dried and crystallized from the proper solvent.

2.2.14. 6-Acetyl-2-(ethylamino)-7-methyl-8H-[1,3,4]thiadiazolo [3, 2-a]thieno[2,3-d]pyrimidin-8-one (10a)

The compound was obtained from the reaction of 1 (2.55g, 10 mmol) and ethyl-isothoicyanate (0.98 mL, 10 mmol), crystallized from dioxane as yellow crystals 61% yield, MP > 350°C (melted); IR (�max, cm-1) KBr: 3354 (br, NH), 2927 (CH alkyl), 1704, 1685 (2CO), 1652 (C=N), 1602 (C=C), 1524 (C-N stretching), 1149 (CS); 1H NMR (DMSO-d6) � 1.21 (t, 3H, J = 6.62 Hz, CH3), 2.80 (s, 3H, CH3), 3.02 (s, 3H, CH3), 4.01 (q,2H, J = 6.65 Hz, CH2),12.60 (br, NH, D2O exchangeable); 13C NMR (DMSO-d6) � 18.2, 22.4, 28.7 (3CH3), 35.7 (CH2), 119.4 (pyrimidine C), 139.2 (thio-phene C), 154.3, 156.2 (thiadiazole C), 158.9,162.4 (thiophene C), 166.2, 185.6 (2CO). MS (70 ev, %) m/z 308 (M+, 80%); Anal. Calc. for C12H12N4O2S2 (308.37): C, 46.74; H, 3.92; N, 18.17. Found: C, 46.79; H, 3.98; N, 18.22.

2.2.15. 6-Acetyl-7-methyl-2-(phenylamino)-8H-[1,3,4]thiadiazolo [3,2-a]thieno[2,3-d]Pyrimidin-8-one(10b)

The compound was obtained from the reaction of 1 (2.55g, 10 mmol) and phenylisothiocyanate (1.19 mL, 10 mmol), crystallized from benzene as yellowish crystals 65% yield, MP > 350°C (melted); IR (�max, cm-1) KBr: 3362 (br, NH), 3043 (CH aryl), 2925 (CH alkyl), 1706, 1682 (2CO), 1651 (C=N), 1600 (C=C), 1523 (C-N stretching), 1145 (CS); 1H NMR (DMSO-d6) � 2.86 (s, 3H, CH3),3.08 (s, 3H, CH3), 7.01-7.15 (m, 5H, Ar), 11.10 (br, NH, D2O ex-changeable); 13C NMR (DMSO-d6) � 18.1, 28.3 (2CH3), 119.5 (pyrimidine C), 121.2, 123.4, 129.7, 137.8 (Ar-C), 139.5 (thiophene C), 155.7, 158.5 (thiadiazole C), 159.1, 161.5 (thiophene C), 164.5, 184.8 (2CO); MS (70 ev, %) m/z 356 (M+, 85%); Anal. Calc. for C16H12N4O2S2 (356.42): C, 53.92; H, 3.39; N, 15.72. Found: C, 53.85; H, 3.32; N, 15.79.

2.2.16. 7-Acetyl-8-methyl-2,3-diphenyl-1H,9H-thieno[2',3':4,5] pyrimido[2,1-b][1,3,4]thiadiazin-9-one (11)

Method a: To mix of 1 (2.55g, 10 mmol) and benzoin (2.12g, mmol) in (35 mL) Polyphosphoric Acid (PPA) was heated under refluxe for 8-12h (TLC). The solution was cooled to room tempera-ture and poured into ice-water. The resultant solid was filtered off, washed with H2O and dried over calcium chloride in vacuum, re-crystallized.

Method b: A solution of 1 (2.55g, 10 mmol) and benzoin (2.12g, 10 mmol) in (30 mL) potassium ethoxide (0.56 g, potassium hydroxide in (40 mL) absolute EtOH, 10 mmol) was heated and refluxed for 12-15 h (TLC). The mixture was cooled at the tempera-ture of the room, poured into ice-water and neutralized with dilute HCl. The separated solid was filtered off, dried and recrystallized from methanol / petroleum ether as brownish crystals in 76% yield, MP > 350°C (melted); IR (�max, cm-1) KBr: 3370 (br, NH), 3044 (CH aryl), 2926 (CH alkyl), 1702,1684 (2CO), 1654 (C=N), 1606 (C=C), 1527 (C-N stretching), 1148 (CS); 1H NMR (DMSO-d6) �2.88 (s, 3H, CH3), 3.09 (s, 3H, CH3), 7.18-7.85 (m, 10H, Ar), 11.60 (br, 1H, NH, D2O exchange- able); 13C NMR (DMSO-d6) � 18.2, 28.5 (2CH3), 97.2 (thiadiazine C), 119.6 (pyrimidine C), 127.5, 127.6, 128.1, 128.3, 128.5, 128.6, 130.7, 134.3 (Ar-C), 139.2 (thio-phene C), 144.1, 156.4 (thiadiazine C), 158.6, 161.8 (thiophene C),

1164 Current Organic Synthesis, 2018, Vol. 15, No. 8 Abu-Hashem and Faty

165.7, 186.2 (2CO); MS (70 ev, %) m/z 431 (M+, 100%); Anal. Calc. for C23H17N3O2S2 (431.53): C, 64.02; H, 3.97; N, 9.74. Found: C, 64.12; H, 3.90; N, 9.80.

2.2.17. 2-((6-Acetyl-3-amino-5-methyl-4-oxo-3,4-dihydrothieno [2, 3-d]pyrimidin-2-yl)thio)acetic Acid (12)

A mixture of 1 (2.55 g, 10 mmol), sodium acetate (0.82 g, 10 mmol) and chloroacetic acid (0.94 g, 10 mmol) in absolute ethanol (35 mL) was heated under reflux for 14-16 h (TLC). After cooling, the solvent was removed under reduced pressure, the residue was dissolved in water (30 mL) and the final product was extracted with chloroform (CHCl3, 40 mL). The solid product washed with H2O / C2H5OH and dried over Na2SO4 and recrystallized from chloroform as yellowish crystals in 80% yield, MP 258-260 °C; IR (�max, cm-1)KBr: 3425-3145 (br, OH and NH2), 3045 (CH aryl), 2930 (CH alkyl), 1770, 1707, 1681 (3CO), 1652 (C=N), 1603 (C=C), 1529 (C-N stretching), 1153 (CS); 1H NMR (DMSO-d6) � 2.85 (s, 3H, CH3), 3.04 (s, 3H, CH3), 3.92 (s, 2H, CH2), 6.10 (br,2H, NH2, D2Oexchangeable), 13.05 (br,1H, OH, D2O exchangeable); 13C NMR (DMSO-d6) � 18.3, 28.6 (2CH3), 33.1(CH2), 120.2 (pyrimidine C), 139.4 (thiophene C), 155.6 (pyrimidine C), 157.4, 159.9 (thiophene C), 163.8, 175.1, 184.5 (3CO); MS (70 ev, %) m/z 313 (M+, 100%); Anal. Calc. for C11H11N3O4S2 (313.35): C, 42.16; H, 3.54; N, 13.41.Found: C, 42.10; H, 3.59; N, 13.48.

2.2.18. 7-Acetyl-8-methyl-3-hydro-1H,9H-thieno[2',3':4,5] pyri-mido[2, 1-b][1,3,4]thiadiazine-2,9-dione (13)

Method a: A mixture of 12 (3.13 g, 10 mmol) and phosphoryl chloride. (20 mL) was heated under reflux for 6-8 h. After cooling, the solvent was removed under reduced pressure and the solid resi-due formed was cooled by ice-H2O and neutralized with ammonia solution. The precipitated that formed was filtered off and recrystal-lized.

Method b: To a mixture of 1 (2.55 g, 10 mmol), in dioxane (45 mL) and trimethylamine (0.94 mL, 10 mmol), ethyl bromoacetate (1.10 mL, 10 mmol) was added. The mixture was refluxed for 6-8 (TLC) h. The formed precipitate after cooling was filtered off, washed with water, dried and recrystallized from DMF as yellow crystals in 70% yield, MP > 350°C (melted); IR (�max, cm-1) KBr: 3375 (br, NH), 3048 (CH, aryl), 2932 (CH, alkyl), 1715, 1709, 1685 (3CO), 1650 (C=N),1601 (C=C), 1524 (C-N stretching), 1158 (CS); 1H NMR (DMSO-d6) � 2.81 (s, 3H, CH3), 3.01 (s, 3H, CH3),3.80 (s, 2H, CH2), 12.40 (br, 1H, NH, D2O exchangeable); 13CNMR (DMSO-d6) � 18.5, 28.8 (2CH3), 34.6 (CH2), 120.3 (pyrimidine C), 139.6 (thiophene C), 155.8 (pyrimidine C), 158.1, 160.4 (thiophene C), 164.2, 171.3, 185.1, (3CO); MS (70 ev, %) m/z 295 (M+, 90%); Anal. Calc. for C11H9N3O3S2 (295.33): C, 44.74; H, 3.07; N, 14.23. Found: C, 44.82; H, 3.10; N, 14.28.

2.2.19. 7-Acetyl-2-(subsitituted)-8-methyl-3H,9H-thieno[2',3':4,5] pyrimido[2,1-b][1,3,4]thiadiazin-9-one (14a) and (14b)

General Procedure: A mix of 1 (2.55 g, 10 mmol), and chloroacetone (0.82 mL, 10 mmol) or phenacyl bromides (1.99 g, 10 mmol) in absolute ethanol (45 ml) was stirred under refluxed for 10-12 h (TLC). The reaction mix was allowed to cool to 0°C for 12 -16 h. the deposited solid precipitated was filtered off, washed with water / ethanol, dried and recrystallized from proper solvent.

2.2.20. 7-Acetyl-2,8-dimethyl-3H,9H-thieno[2',3':4,5]pyrimido [2,1-b][1,3,4]thiadiazin-9-one(14a)

The compound was obtained from the reaction of 1 (2.55g, 10 mmol) and chloroacetone (0.82 mL, 10 mmol), crystallized from methanol as white crystals 68% yield, MP > 350°C (melted); IR (�max, cm-1) KBr: 3040 (CH aryl), 2927 (CH alkyl), 1705, 1684 (2CO), 1652 (C=N), 1601 (C=C), 1527 (C-N stretching), 1152 (CS); 1H NMR (DMSO-d6) � 2.10 (s, 3H, CH3), 2.85 (s, 3H, CH3),3.08 (s, 3H, CH3), 3.84 (s, 2H, CH2); 13C NMR (DMSO-d6) � 18.1, 21.5 (2CH3), 26.2 (CH2), 28.6 (CH3), 120.1 (pyrimidine C), 139.5

(thiophene C), 155.5, 158.2 (thiadiazine C), 159.8, 161.7 (thiophene C), 165.3, 183.4, (2CO); MS (70 ev, %) m/z 293 (M+, 86%); Anal. Calc. for C12H11N3O2S2 (293.36): C, 49.13; H, 3.78; N, 14.32. Found: C, 49.19; H, 3.70; N, 14.38.

2.2.21. 7-Acetyl-8-methyl-2-phenyl-3H,9H-thieno[2',3':4,5] pyri-mido[2,1-b][1,3,4]thiadiazin-9-one(14b)

Compound 14b was obtained from the reaction of 1 (2.55g, 10 mmol) and phenacyl bromides (1.99 g, 10 mmol), crystallized from hexane as yellow crystals 60% yield, MP > 350°C (melted); IR (�max, cm-1) KBr: 3042 (CH aryl), 2925 (CH alkyl), 1708, 1682 (2CO),1654 (C=N), 1600 (C=C), 1525 (C-N stretching),1154 (CS); 1H NMR (DMSO-d6) � 2.87 (s, 3H, CH3), 3.06 (s, 3H, CH3), 3.83 (s, 2H, CH2), 7.52-7.95(m, 5H, Ar); 13C NMR (DMSO-d6) � 18.6 (CH3), 26.4 (CH2), 28.5 (CH3), 120.3 (pyrimidine C), 128.1, 128.7, 131.4, 134.6 (Ar-C), 139.7 (thiophene C), 155.8, 158.5 (thiadiazine C), 159.9, 161.8 (thiophene C), 164.6, 183.5, (2CO); MS (70 ev, %) m/z 355 (M+, 84%); Anal. Calc. for C17H13N3O2S2 (355.43): C, 57.45; H, 3.69; N, 11.82. Found: C, 57.40; H, 3.61; N, 11.89.

2.2.22. 7-Acetyl-2-amino-8-methyl-3H,9H-thieno[2',3':4,5] pyri-mido[2,1-b][1,3,4]thiadiazin-9-one(15)

A mix of 1 (2.55g, 10 mmol), chloroacetonitrile (0.63 mL, 10 mmol) and sodium acetate (1.57 g, 10 mmol) in absolute ethanol (40 mL) was heated under reflux for 12-14 h and then allowed to cool to room temperature, poured into cold water (100 ml). The solid product that precipitated was filtered off, washed with water and The recrystallized from methanol as Yellowish crystals in 62% yield, MP > 350°C; IR (�max, cm-1) KBr: 3395 (br, NH2), 3040 (CH aryl), 2935 (CH alkyl), 1711, 1688 (2CO), 1652 (C=N), 1606 (C=C), 1527 (C-N stretching), 1153(CS); 1H NMR (DMSO-d6) �2.83 (s, 3H, CH3), 3.08 (s, 3H, CH3), 3.92 (s, 2H, CH2), 6.55 (br, 2H, NH2, D2O exchangeable); 13C NMR (DMSO-d6) � 18.9, 28.2 (2CH3), 32.5 (CH2), 120.1 (pyrimidine C), 139.2 (thiophene C),145.8, 156.4 (thiadiazine C), 158.7, 161.2 (thiophene C), 165.1, 187.3, (2CO); MS (70 ev, %) m/z 294 (M+, 82%); Anal. Calc. for C11H10N4O2S2 (294.35): C, 44.89; H, 3.42; N, 19.03. Found: C, 44.80; H, 3.49; N, 19.10.

2.2.23. 6-Acetyl-3-amino-2-((3-chloroquinoxalin-2-yl)thio)-5-met-hylthieno[2,3-d]pyrimidin-4(3H)-one (16)

A solution of 1 (2.55g, 10 mmol), 2,3-dichloroquinoxaline (1.99 g, 10 mmol) and triethyl amine (TEA, 0.5 mL) as a catalyst in absolute ethanol (35 mL) was heated under refluxed for 12-16 h (TLC). After cooling the solid product was collected by filtration, washed with water and ethanol, dried and recrystallized from DMF as Yellow crystals in 85% yield, MP 273-275°C (melted); IR (�max, cm-1) KBr: 3412 (br, NH2), 3048 (CH aryl), 2933 (CH alkyl), 1710, 1686 (2CO), 1650 (C=N), 1602 (C=C), 1525 (C-N stretching), 1149(CS); 1H NMR (DMSO-d6) � 2.81 (s, 3H, CH3), 3.06 (s, 3H, CH3), 6.10 (br, 2H, NH2, D2O exchangeable), 7.65-7.82 (m,4H,Ar-H); 13C NMR (DMSO-d6) � 18.5, 28.4 (2CH3), 119.8 (pyrimidine C), 128.3, 129.2, 129.9, 130.5, 136.4, 138.9 (Ar-C), 139.6 (thio-phene C), 144.8, 152.7 (pyrazine C), 156.4 (pyrimidine C), 158.2, 160.9 (thiophene C), 162.5, 185.1, (2CO); MS (70 ev, %) m/z 417 (M+, 100%); Anal. Calc. for C17H12ClN5O2S2 (417.89): C, 48.86; H, 2.89; N, 16.76. Found: C, 48.94; H, 2.81; N, 16.70.

2.2.24. 2-Acetyl-3-methyl-4H,6H-thieno[2'',3'':4',5']pyrimido[2', 1': 2,3][1,3,4]thiadiazino[5,6-b]quinoxalin-4-one (17)

Method a: A mix of 16 (4.17g, 10 mmol) and triethyl amine (0.5 mL) in dimethyl- formamide (DMF, 40 mL) was heated under reflux for 20-24 h (TLC). The solution was cooled, the product formed was filtered off, washed with ethanol, dried and recrystal-lized to give compound 17.

Method b: To a mix of 1 (2.55g, 10 mmol), 2,3-dichloro qui-noxaline (1.99 g, 10 mmol) with triethyl amine (0.5 mL) as a cata-lyst in DMF (35 mL). The reaction solution was heated under reflux

Synthesis, Antimicrobial Evaluation of Some New 1, 3, 4-Thiadiazoles Current Organic Synthesis, 2018, Vol. 15, No. 8 1165

for 40-48 h (TLC), then allowed to cool to room temperature. The solid obtained was filtered off, washed with water / ethanol, dried and recrystallized from benzene as Yellowish crystals in 75% yield, MP > 350°C (melted); IR (�max, cm-1) KBr: 3355 (br, NH), 3035 (CH aryl), 2930 (CH alkyl), 1708, 1682 (2CO), 1656(C=N), 1606 (C=C), 1527 (C-N stretching), 1148(CS); 1H NMR (DMSO-d6) � 2.83 (s, 3H, CH3), 3.07 (s, 3H, CH3), 7.66-7.83(m, 4H, Ar-H), 11.85 (br, NH, D2O exchangeable); 13C NMR (DMSO-d6) � 18.4, 28.1 (2CH3), 120.3 (pyrimidine C), 125.2, 126.1,127.5, 128.8, 135.6, 137.7 (Ar-C), 139.5 (thiophene), 144.7, 150.1 (pyrazine C), 155.8 (pyrimidine C), 157.4, 160.5 (thiophene C), 163.9, 186.2, (2CO); MS (70 ev, %) m/z 381 (M+, 90%); Anal. Calc. for C17H11N5O2S2 (381.43): C, 53.53; H, 2.91; N, 18.36. Found: C, 53.61; H, 2.98; N, 18.30.

3. BIOLOGICAL SCREENING Antimicrobial activity of the newly synthesized compounds was

tested in vitro against the next bacteria: Gram-positive bacte-ria(+Ve) Rhodopseudomonas fp., Bacillus cereus and Micrococcus luteus, Gram-negative bacteria (-Ve) E. coli (HD 701), Salmonella typhi and the fungi Alternaria alternate, Aspergillus flavus(ATCC-

24556), Candida albicans (ATCC 15056) and Curvularia lunata. The newly prepared compounds were dissolved in dimethylsulfox-ide and tested for antimicrobial activity via the agar disk dissemina-tion method taking Levofloxacin and Nystatin [36] as mention drugs for bacteria and fungi, respectively. A solution of 100 �g mL–1 of the test compound was practical and microplate-wells, 1 cm in diameter, were used. Areas of inhibition were measured with cali-pers or automated scanners and were paralleled with those of the standards. Levofloxacin (tavanic) (0.15 �mol mL–1) and Nystatin (0.037 �mol mL–1) were used as citation drugs for antibacterial and antifungal activity, respectively. Compound-impregnated disks were placed on an agar plate containing a standard deferment of microorganisms. The platter was protected for 24 h at 37°C. For the assessment of Minimal Inhibitory Concentration (MIC) by the se-rial plate dilution way [36], 5 mg of each test compounds were dissolved in 1 mL of DMSO separately to prepare stock solutions. Serial dilutions were prepared from each stock solution. The plates were incubated at 37°C for 24 h. MIC is defined as the lowest con-centration (�mol mL–1) of the test compound that results in no visi-ble growth on the plates. DMSO was used as a solvent control to ensure that the solvent had no effect on bacterial growth. The re-sults are shown in (Tables 1 and 2).

Table 1. Minimal inhibitory concentration of the prepared compounds against bacteria.

MIC (�mol L–1)

Gram-positive bacteria Gram-negative bacteria Organism

Compd. Rhodopseudomonas fp. Bacillus cereus Micrococus luteus E. coli

(HD 701)

Salmonella typhi

1 36 38 39 40 41

2 34 35 36 36 38

3 30 32 30 31 33

4 24 25 23 24 26

5 26 27 26 25 29

6 20 22 21 20 23

7 14 15 16 15 17

8 6 6 8 6 6

9a 18 19 20 19 21

9b 15 16 18 16 18

9c 16 17 19 18 19

10a 5 4 7 5 4

10b 6 5 7 6 5

11 7 8 9 7 8

12 10 11 13 12 13

13 12 14 15 14 15

14a 9 10 12 10 11

14b 8 9 10 8 9

15 5 4 6 4 3

16 5 4 5 3 2

17 4 3 4 2 1

levofloxacin 5 4 4 3 2

Negative control

NI NI NI NI NI

DMSO is a negative control; also the solvent for test compounds and the reference drug. NI – No inhibition

1166 Current Organic Synthesis, 2018, Vol. 15, No. 8 Abu-Hashem and Faty

4. RESULTS AND DISCUSSION

4.1. Chemistry The reactivity of 6-acetyl-3-amino-5-methyl-2-thioxo-2, 3-

dihydrothieno [2, 3-d]pyrimidin-4(1H)-one (1) [28], towards car-boxylic acid derivatives was studied. Thus, heating under reflux compound 1 with formic acid resulted in cyclization via elimination of two molecules of water to give the 6-acetyl-7-methyl-3a,4-dihydro-8H-[1,3,4] thiadiazolo[3,2-a] thieno[2,3-d]pyrimidin-8-one (2). The IR spectrum of 2 displayed absorption bands at � 3355 cm-1(NH), 1700, 1680 cm-1 (2C=O). Its 1H-NMR (DMSO-d6,� ppm) spectra showed two singlet signals at 2.85 and 3.05 for two CH3groups, two singlet signals at 5.80 and 8.01 for the two protons of thiadiazole ring and one broad singlet signals at 6.80 for NH group (D2O exchangeable). Compound 1 underwent cycloaddition upon fusion with urea at 270°C, to produce the 6-acetyl-7-methyl-8H-[1,3,4]thiadiazolo[3,2-a]thieno[2,3-d]pyrimidine-2,8(1H)-dione(3).The same product was obtained, through the reaction of 1 with ethyl chloroformate in the presence of sodium methoxide, in a good yield.1H NMR spectrum of 3 showed two singlet signals at � 2.82 and 3.03 ppm for two methyl groups and one singlet signal at � 9.55 ppm for NH proton (D2O exchangeable). Its mass spectrum showed the molecular ion peak at m/z 281 (90%). Treatment of 3 with P2S5in dry pyridine resulted in the formation of 6-acetyl-2-mercapto-7-

methyl-8H-[1,3,4]thiadiazolo[3,2-a] thieno[2,3-d]pyrimidin-8-one (4). The latter compound could be obtained via the reaction of 1with carbon disulphide in the presence of ethanolic KOH solution. Mass spectrum for compound 4 gave the molecular ion peak at m/z 297 (84%), that corresponds to the molecular formula C10H7N3O2S3of the assigned structure. On the other hand, reaction of 1 with ox-alic acid or benzoic acid in POCl3 led to the formation of the corre-sponding 6-acetyl-2,7-dimethyl-8H-[1,3,4]thiadia- zolo [3,2-a]thieno[2,3-d]pyrimidin-8-one (5) and 6-acetyl-7-methyl-2-phenyl-8H-[1,3,4]thiadiazolo[3,2-a]thieno[2,3-d] pyrimidin-8-one (6), respectively. IR and 1H-NMR spectra of 5, 6 revealed the ab-sence of NH2 group. Compound 1 reacted with ethyl cyanoacetate in polyphosphoric acid to afford ethyl 2-(6-acetyl-7-methyl-8-oxo-8H-[1,3,4]thiadiazolo[3,2-a]thieno [2,3-d] pyrimidin-2-yl)acetate (7) in good yield (Scheme 1). The 1H-NMR spectrum of 7 showed a triplet signal at � 1.25 ppm for CH3 group (J = 6.45 Hz), three sin-glet signals at � 2.84, 3.01, 3.12 for two CH3 and one CH2 group and one quartet signal at � 4.21 ppm for CH2 group (J = 6.55 Hz). Its mass spectrum showed the molecular ion peak at m/z 351 (75%). All spectral data and elemental analysis were compatible with the proposed structures (Experimental, Scheme 1).

Moreover, compound 1 reacted with cyclohexanone to produce the corresponding spiro compound 6'-acetyl-7'-methyl-1'H,8'H-

Table 2. Minimal inhibitory concentration of the prepared compounds against fungi.

MIC (�mol L–1)

Organism Compd.

Alternaria alternata

Aspergillus flavus

Candida albicans

Curvularia lunata

1 38 42 44 46

2 35 37 38 39

3 32 34 32 34

4 25 25 24 23

5 28 29 28 27

6 22 21 20 21

7 15 14 13 16

8 9 8 7 8

9a 19 18 17 19

9b 16 15 14 17

9c 17 16 15 18

10a 7 6 5 4

10b 8 7 6 7

11 10 9 8 9

12 13 12 11 13

13 14 13 12 15

14a 12 11 10 12

14b 11 10 9 10

15 6 5 5 3

16 4 3 4 2

17 3 2 2 1

Nystatin 3 2 2 1

Negative control

NI NI NI NI

DMSO is a negative control, also the solvent for test compounds and the reference drug. NI -No inhibition

Synthesis, Antimicrobial Evaluation of Some New 1, 3, 4-Thiadiazoles Current Organic Synthesis, 2018, Vol. 15, No. 8 1167

spiro[cyclohexane-1,2'-[1,3,4]thiadiazolo[3, 2-a]thieno[2,3-d]pyri-midin]-8'-one (8). The IR spectrum of (8) showed the absorption bands at � 3350 cm-1 for the (NH), 1712,1688 cm-1 for the two car-bonyl group. Its 1H-NMR spectra exhibited a singlet signal at �10.15 ppm for one proton of NH (D2O exchangeable), multiple signals at 1.41-1.54 (m, 6H, 3CH2) and 2.15 (t, 4H, 2CH2). The 13C-NMR of 8 showed signals at � 74.6 ppm for one spiro carbon atom. Treated compound 1 with some aromatic aldehydes namely, ben-zaldehyde, 4-chloro- benzaldehyde and 4-anisaldehyde in the pres-ence of p-toluene sulphonic acid yielded 6-acetyl-2-(4-substituted-phenyl)-7-methyl-1,2-dihydro-8H-[1,3,4]thia- diazolo[3,2-a]thieno [2,3-d]pyrimidin-8-one 9a-c derivatives in good yields. IR spectra of (9a-c) showed absorption of a broadband at � 3360-3355 cm-1corresponding to (NH) group and 1706-1703 and 1682-1680 cm-1for two carbonyl groups. Furthermore,6-acetyl-3-amino-5-methyl-2-thioxo-2, 3-dihydrothieno[2,3-d]pyrimidin-4(1H)-one (1) was refluxed with ethyl /phenyl isothio- cyanates in dimethylformamide to give 6-acetyl-2-(ethyl amino)-7-methyl-8H-[1,3,4] thiadia- zolo[3,2-a]thieno[2,3-d] pyrimidin-8-one and 6-acetyl-7-methyl-2-(phenylamino)-8 H-[1,3,4]thiadiazolo[3,2-a]thieno [2,3-d]pyri-midin-8-one (10 a, b) respectively. The 1H NMR spectra of (10a),as an example, revealed the disappearance of NH2 and SH signals and appearance of new singlet signal at � 12.60 characteristics for alkyl amino proton (NH, D2O exchangeable). Assignment of the newly synthesized compounds was based on elemental analyses, IR, 1H, 13C NMR, and mass spectral data (Experimental, Scheme 2).

Compound 1 condensed with benzoin in potassium ethoxide so-lution and yielded 7-acetyl-8-methyl-2,3-diphenyl-1H,9H-thi-

eno[2',3':4,5]pyrimido[2,1-b][1,3,4]thiadiazin-9-one (11).IR spectra of (11) shown the absorption bands at � 3370 cm-1 for the (NH), 1702,1684 cm-1 for the two carbonyl group,1654 cm-1 for the (C=N), 1148 cm-1 for the (C-S). 1H-NMR spectra of (11) exposed one singlet signal at � 11.60 ppm for one proton of the (NH) group. On the other hand, treatment of compound 1 with chloroacetic acid, in absolute ethanol and sodium acetate, gave product 2-[(6-acetyl-3-amino-5-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl) thio] acetic acid (12) (Scheme 3). The latter compound could be cyclized into 7-acetyl-8-methyl-3-hydro-1H,9H-thieno [2',3':4,5]py-rimido[2,1-b][1,3,4]thiadiazine-2,9-dione (13) in boiling phos-phoryl chloride. We obtained the same compound 13 by refluxing of compound 1 with ethyl bromoacetate in dioxane and trimethy-lamine as a catalyst. The IR spectrum for 12 showed the absorption bands at � 3425-3145 cm-1 for the OH and NH2 groups and three stretching bands at 1770, 1707 and 1681 corresponding to 3 CO groups. Its 1H NMR spectrum showed one singlet at � 6.10 ppm for two protons of NH2 group (D2O exchangeable) and a broad singlet at � 13.05 ppm corresponding to OH group (D2O exchangeable). The 1H NMR spectra of (13), revealed the disappearance of NH2and OH signals and appearance of a new broad singlet at � 12.40 ppm confirming the presence of the NH group (D2O exchangeable). Moreover, the amino-thione compound 1, reacted with each of chloroacetone, phenacyl bromide and chloro acetonitrile in reflux-ing ethanol to produce 7-acetyl-2-(substituted)-8-methyl-3H,9H-thieno[2',3':4,5]pyrimido[2,1-b][1,3,4] thiadiazin-9-one (14a,b) and 7-acetyl-2-amino-8-methyl-3H,9H-thieno[2',3':4,5]pyrimido[2,1-b] [1,3,4]thiadiazin-9-one (15), respectively, The 1H NM R spectrum for 14a, as an example, exposed the presence of signals � 2.10,

Scheme 1. Synthesis of 1, 3, 4-thiadiazole-thienopyrimidine derivatives. Reagents and conditions: Compound (1) with several reagents; aliphatic and aromatic acid derivatives, ethyl chloroformate, ethyl cyanoacetate, urea, CS2, reflux for 2-12 h, 65-78% yield.

S N

N

O

SH

NH2H3C

H3C

O

1

10-12 hS NH

N

O

S

NH3C

H3C

O H

O

OH

2

Cl OEt

O

CH3ONa, 8-10 h

S N

N

O

S

HN

H3C

H3C

O

3

OH2N NH2

O

NaOH / HCl

O O

HO OH

POCl3 , 8-12 hS N

N

O

S

NH3C

H3C

OCOOH

S N

N

O

S

NH3C

H3C

OCH3

5

POCl3

O

HO

POCl3 , 8-12 h S N

N

O

S

NH3C

H3C

O

6

CNEtO

O

PPA S N

N

O

S

NH3C

H3C

O OEt

O

7

S N

N

O

S

NH3C

H3C

OSH

CS2 / KOH

4

P2S5 / Pyridine

EtOH, 2-4 h

78 %

75 %

200-1000 °C,10 h

70 %8-12 h

73%

76%

60-80 °C,8-10 h

65 %

10-12 h

1168 Current Organic Synthesis, 2018, Vol. 15, No. 8 Abu-Hashem and Faty

Scheme 2. Synthesis of spiro(cyclohexane) and 1, 3, 4-thiadiazolothienopyrimidine derivatives. Reagents and conditions: Compound (1) with many reagents; cyclohexanone, aromatic aldehydes derivatives, ethyl /phenyl isothiocyanates, refluxed for 8-18 h, 61-74% yield.

Scheme 3. Synthesis of thienopyrimidine-1, 3, 4-thiadiazine derivatives, reagents and conditions: Various reagents; benzoin, chloroacetic acid, ethyl bromoace-tate, chloroacetone, phenacyl bromide, chloro acetonitrile, 2, 3-dichloroquinoxaline, refluxed for 6-48 h, 60-85% yield.

2.85, 3.08 and 3.84 ppm corresponding to three CH3 and CH2groups. Elemental analyses, as well as spectroscopic data, were in

agreement with the proposed structure (Experimental). Finally, compound 1 reacted with 2,3-dichloroquinoxaline, in refluxing

S N

N

O

SH

NH2H3C

H3C

O

1

a, Ar = Ph b, Ar = 4-chlorophenyl c, Ar = 4-methoxyphenyl

DMF / 8-10 h

S N

N

O

S

NH3C

H3C

ONH

R

CNS R

a, R = C2H5; b, R = C6H5

P-Toluene sulfonic acid S N

N

O

S

HN

H3C

H3C

OAr

Ar H

O

DMF, 15-18 h

O

S N

N

O

S

HN

H3C

H3C

O

DMF / N(C2H5)38

9a-c

10a,b

14-16 h68%

a,66%; b,74%; c,67%

a,61%; b,65%

S N

N

O

SH

NH2H3C

H3C

O

1

RO

XO

OBrH3C

S N

N

O

S

NH3C

H3C

O

S N

N

O

S

HN

H3C

H3C

O

R

O

Ph

OHO

Ph

S N

N

O

S

HN

H3C

H3C

O Ph

Ph

N

N

Cl

Cl

S N

N

O

S

HN

H3C

H3C

O

N

N

EtOH, 10-12 h

EtOH / KOH or PPA DMF / TEA,40-48 h

ClCH2COOH

CH3COONa / EtOH

N(CH3)3 / Dioxane, 6-8 h

11

13

14a, 68%; 14b,60%

17

ClCH2CN

NaOAc / EtOH S N

N

OH3C

H3C

O

S

N NH2

15

S N

N

O

S

NH2H3C

H3C

O

O

OH

POCl3

12

14a, X= Cl, R=CH3 : 14b, X= Br, R=Ph

S N

N

O

S

NH2H3C

H3C

O

N

N

16

Cl

N

N

Cl

Cl

Ethanol / TEA,12-16 h

DMF / TEA,

8-15 h

76 %

14-16 h

80 %

6-8 h

70 %12-14 h

62 %

20-24 h

85 %

75 %

Synthesis, Antimicrobial Evaluation of Some New 1, 3, 4-Thiadiazoles Current Organic Synthesis, 2018, Vol. 15, No. 8 1169

ethanol, in the presence of a catalytic amount of triethylamine for 12h and led to the formation of 6-acetyl-3-amino-2-[(3-chloro-quinoxalin-2-yl) thio]-5-methylthieno [2, 3-d]pyrimidin-4(3H)-one (16), which on refluxing for 24h in DMF gave the corresponding 2-acetyl-3-methyl-4H,6H-thieno[2'',3'':4',5']pyrimido[2',1':2,3][1,3,4] thiadiazino[5,6-b] quinoxalin-4-one (17). In another way compound, 17 was obtained in high yield by refluxing for 40h a mix of 1 and 2, 3-dichloroquinoxaline in DMF. All spectral data and elemental analysis were compatible with the proposed struc-tures (Experimental, Scheme 3).

6. BIOLOGICAL ACTIVITY

6.1. Antimicrobial Screening and SAR The compounds showed satisfactory results for their elemental

and spectral analysis. Thus, all new compounds were tested in vitro for antimicrobial properties; all results are represented in Tables 1 and 2. Most of these compounds revealed good antimicrobial activ-ity, analogous to that of Levofloxacin (MIC= 2-5 �mol mL–1). Some of these compounds (8,10a, 10b, 15, 16 and 17) exhibited strong antimicrobial activity towards Gram-positive (Rhodopseu-domonas fp., Bacillus cereus and Micrococcus luteus) and Gram-negative (E. coli (HD 701) and Salmonela typhi) bacteria with MIC values (1- 8) �mol mL–1. Also, the same compounds displayed higher antifungal activity and specifically compound 17 with a MIC of (1- 3) �mol mL–1 than Nystatin (MIC 1-3 �mol mL–1). The fungi used are Alternaria alternate, Aspergillus flavus (ATCC-24556), Candida albicans (ATCC 15056) and Curvularia lunata. While, some compounds (11, 12, 13, 14a and 14b) showed moderate level antimicrobial activity when compared with the Tavanic (MIC=2-5 �mol mL–1) and the same compounds exhibited suitable antifungal activity when compared with the Nystatin (MIC 1-3 �mol mL–1). These compounds contain several functional groups that have a positive effect on the bacteria and fungi. Also, the rest of these new compounds showed slightly activities compared with the antimicrobial activity of the standard drugs; Levofloxacin (Tavanic) and Nystatin as shown in Tables (1, 2).

CONCLUSION A successful synthetic strategy of fused 1, 3, 4-thiadiazole or 1,

3, 4-thiadiazine with thienopyrimidines and organic heterocyclic compounds was developed. These newly synthesized compounds, 1,3,4-thiadiazolo[3,2-a]thieno[2,3-d] pyrimidine (2-10), thieno[2, '3':4,5]pyrimido[2, 1-b][1,3,4] thiadiazin (11-15), 6-acetyl-3-amino-2-[(3-chloroquinoxalin-2-yl)thio]-5-methylthieno[2,3-d]pyrimidin-4(3H)-one (16) and 2-acetyl-3-methyl-4H,6H-thieno[2'',3'':4',5'] pyrimido [2', 1':2,3][1,3,4]thiadiazino[5,6-b]quinoxalin-4-one (17),ex-hibited strong antimicrobial activity.

CONSENT FOR PUBLICATION Not applicable.

CONFLICT OF INTEREST The authors declare no conflict of interest, financial or other-

wise.

ACKNOWLEDGEMENTS The author is extremely grateful to Department of Chemistry of

Natural and Microbial Products, National Research Center for help-ing us to screen the newly synthesized compounds for antimicrobial activity. The presented work was supported by the Department of Photochemistry (Heterocyclic unit); Chemical Industries Research Division, National Research Centre in Cairo, Egypt.

REFERENCES[1] Ahmad, T.; Singh, A.K.; Jaiswal, N.; Singh, D. Synthesis and pharmacologi-

cal activity of 1, 3, 4-thiadiazole derivatives: A review. Int. Res. J. Pharm., 2012, 3, 70-82.

[2] Clerici, F.; Pocar, D.; Guido, M.; Loche, A.; Perlini, V.; Brufani, M. Synthe-sis of 2-amino-5-sulfanyl-1, 3, 4-thiadiazole derivatives and evaluation of their antidepressant and anxiolytic activity. J. Med. Chem., 2001, 44, 931-936.

[3] Goksen, U.S.; Kelekci, N.G.; Goktas, O.; Koysal, Y.; Kilic, E.; Isik, S.; Aktay, G.; Ozalp. 1-Acylthiosemicarbazides, 1, 2, 4-triazole-5(4H)-thiones, 1, 3, 4-thiadiazoles and hydrazones containing 5-methyl-2-benzoxazolinones: synthesis, analgesic-antiinflam matory and antimicrobial activities. Bioorg. Med. Chem., 2007, 15, 5738 -5751.

[4] Turan- Zitouni, G.; Kaplancikli, Z. A.; Erol, K.; Kilic, F. S. Synthesis and analgesic activity of some triazoles and triazolothiadiazines. Farmaco, 1999,54, 218-223.

[5] Isloor, A.M.; Kalluraya, B.; Shetty, P. Regioselective reaction: synthesis, characterization and pharmacological studies of some new Mannich bases derived from 1, 2, 4-triazoles. Eur. J. Med. Chem., 2009, 44, 3784-3787.

[6] Abadi, A.H.; Hegazy, G.H.; El-Zaher, A.A. Synthesis of novel 4-substituted-7-trifluoro- methylquinoline derivatives with nitric oxide releasing properties and their evaluation as analgesic and anti-inflammatory agents. Bioorg. Med. Chem., 2005, 13, 5759-5765.

[7] Hu, Y.; Li, C.Y.; Wang, X.M.; Yang, Y.H.; Zhu, H.L. 1, 3, 4-Thiadiazole: synthesis, reactions, and applications in medicinal, agricultural, and materials chemistry. Chem. Rev., 2014, 114, 5572-5610.

[8] Padmavathi, V.; Reddy, G.S. ; Padmaja, A. ; Kondaiah, P. Synthesis, antimi-crobial and cytotoxic activities of 1, 3, 4-oxadiazoles, 1, 3, 4-thiadiazoles and 1, 2, 4-triazoles. Eur. J. Med. Chem., 2009, 44, 2106-2112.

[9] Kolavi,G.; Hegde, V.; Khazi, I. A.; Gadad, P. Synthesis and evaluation of antitubercular activity of imidazo[2,1-b][1,3,4] thiadiazole derivatives. Bioorg. Med. Chem., 2006, 14, 3069-3080.

[10] Matysiak, J. Biological and pharmacological activities of 1, 3, 4-thiadiazole based compounds. Mini-Rev. Med. Chem., 2015, 15, 762-775.

[11] Badr, S.M.I.; Barwa, R.M. Synthesis of some new [1,2,4]triazolo[3,4-b][1,3,4] thiadiazines and [1,2,4]triazolo[3,4-b][1,3,4] thiadiazoles starting from 5-nitro-2-furoic acid and evaluation of their antimicrobial activity. Bioorg. Med. Chem., 2011, 19, 4506-4512.

[12] Prakash, O.; Aneja, D.K.; Hussain, K, Aneja, K.R. Synthesis and biological evaluation of dihydroindeno and indeno [1, 2-e] [1,2,4] triazolo [3,4-b][1, 3, 4] thiadiazines as antimicrobial agents. Eur. J. Med. Chem., 2011, 46, 5065-5073.

[13] Mohammad, R.; Ali, S.; Mahsa, R.; Mehdi, B. Synthesis and antibacterial evaluation of new heterocyclic system:[1,2,4] triazolo-[3,4:6,1]pyridazi no-[4, 3-e][1,3,4] thiadiazine. Heter. Comm., 2012, 18, 39-42.

[14] Altintop, M.; Kaplancikli, Z.A.; Zitouni, G.T.; Özdemir, A.; ��can, G.; Akal�n, G.; Y�ld�r�m, �. Synthesis and anticandidal activity of new triazolothiadiazine derivatives. Eur. J. Med. Chem., 2011, 46, 5562-5566.

[15] Kumar, G.V.S.; Prasad, Y.R.; Mallikarjuna, B.P.; Chandrashekar, S.M. Synthesis and pharmacological evaluation of clubbed isopropylthiazole de-rived triazolothiadiazoles, triazolothiadia- zines and mannich bases as poten-tial antimicrobial and anti-tubercular agents. Eur. J. Med. Chem., 2010, 45, 5120-5129.

[16] El Shehry, M.F.; Abu-Hashem, A.A.; El-Telbani, E.M. Synthesis of 3-((2, 4-di chlorophenoxy) methyl)-1, 2, 4-triazolo (thiadiazoles and thiadiazines) as anti-inflammatory and molluscicidal agents. Eur. J. Med. Chem., 2010, 45, 1906-1911.

[17] Al-Masoudi, N.A.; Al-Soud, Y.A. New sulphonamide and carboxamide derivatives of acyclic C-nucleosides of triazolothiadiazole and the thiadiazine analogues. Synthesis, anti-HIV, and antitumor activities. Part 2. Nucleosides, Nucleotides and Nucleic Acids, 2008, 27, 1034-1044.

[18] Holla, B.S.; Akberali, P.M.; Shivananda, M.K. Studies on nitrophenylfuran derivatives: part XII. Synthesis, characterization, antibacterial and antiviral activities of some nitrophenyl furfurylidene-1, 2, 4-triazolo[3,4-b]-1, 3, 4- thiadiazines. Farmaco, 2001, 56, 919-927.

[19] Dutta, K.; Rangarajan, P.N.; Vrati, S.; Basu, A. Japanese encephalitis: patho-genesis, prophylactics and therapeutics. Curr. Sci., 2010, 98, 326-334.

[20] Guo, Z.; Chen, Y.; Wu, D.; Zhu, Y.F.; Struthers, R.S.; Saunders, J.; Xie, Q.; Chen, C. Synthesis and structure-activity relationships of thieno[2,3-d]pyrimidine-2,4-dione derivatives as potent GnRH receptor antagonists. Bioorg. Med. Chem. Lett., 2003, 13, 3617-3622.

[21] Gangjee, A.; Qiu, Y.; Kisliuk, R.L. Synthesis of classical and nonclassical 2-amino-4-oxo-6-benzylthieno [2, 3-d] pyrimidines as potential thymidylate synthase inhibitors. J. Heterocycl. Chem., 2004, 41, 941-946.

[22] Jennings, L.D.; Kincaid, S.L.; Wang, Y.D.; Krishnamurthy, G.; Beyer, C.F.; Mc- Ginnis, J. P.; Miranda, M.; Discafani, C.M.; Rabindran, S.K. Parallel synthesis and biological evaluation of 5,6,7,8-tetra hydrobenzothieno[2,3-d]pyrimidin-4(3H)-one cytotoxic agents selective for p21-deficient cells. Bioorg. Med. Chem. Lett., 2005, 15, 4731-4735.

[23] Horiuchi, T.; Chiba, J.; Uoto, K.; Soga, T. Discovery of novel thieno [2, 3-d]pyrimidin-4-yl hydrazone-based inhibitors of Cyclin D1-CDK4: Synthesis, biological evaluation, and structure-activity relationships. Bioorg. Med. Chem. Lett., 2009, 19, 305-308.

1170 Current Organic Synthesis, 2018, Vol. 15, No. 8 Abu-Hashem and Faty

[24] Rheault, T.R.; Caferro, T.R.; Dickerson, S.H.; Donaldson, K.H.; Gaul, M.D.; Goetz, A.S. Thienopyrimidine-based dual EGFR/ErbB-2 inhibitors. Bioorg. Med. Chem. Lett., 2009, 19, 817-820.

[25] Nagaraju, K.; Harikrishna, N.; Vasu, K.; Rao, C.V. Synthesis and biological activity of novel bis and mono heterocycles of thienopyrimidine derivatives. Indo American J. Pharm. Res., 2015, 5, 1604-1612.

[26] Venkanagouda, G.; Rashmi, P.; Shantharam, U.; Kuntal, H.; Lax-mivenkatesh, G.N. Synthesis of novel thienopyrimidines and evaluation for their anti-inflammatory activity. J. Chem. Pharm. Res., 2012, 4, 3100-3106.

[27] Abu-Hashem, A.A., Abu-Zied, K.M.; El-Shehry, M.F. Synthetic utility of bifunctional thiophene derivatives and antimicrobial evaluation of the newly synthesized agents. Monatsh. Chem., 2011,142, 539-545.

[28] Abu-Hashem, A.A.; El-Shehry, M.F.; Badria, F.A. Design and synthesis of novel thiophenecarbo hydrazide, thienopyrazole and thienopyrimidine de-rivatives as antioxidant and antitumor agents. Acta Pharm., 2010, 60,311-323.

[29] Abu-Hashem, A.A.; Hussein, H. A.R.; Abu-zied, K.M. Synthesis of novel 1, 2, 4-triazolo pyrimidines and their evaluation as antimicrobial agents. Med. Chem. Res., 2016, 26, 120-130.

[30] Abu-Hashem, A.A.; Badria, F.A. Design, synthesis of novel thiourea and pyrimidine derivatives as potential antitumor Agents. J. Chin. Chem. Soc., 2015, 62,506-512.

[31] Abu-Hashem, A.A.; Hussein, H.A. Synthesis, antitumor of new pyrimidine and caffeine derivatives. Lett. Drug Des. Discov., 2015, 12, 471-478.

[32] Abu-Hashem, A. A. Synthesis and reaction of novel spiro -pyrimidine de-rivatives. J Heterocyc. Chem. 2014, 51, 1020-1026.

[33] Abu-Hashem, A.A.; Yousef, M.M. Synthesis of new visnagen and khellin furochromone pyrimidine derivatives and their anti-inflammatory and anal-gesic activity. Molecules 2011, 16, 1956-1972.

[34] Abu-Hashem, A.A.; Gouda, M.A.; Badria, F.A. Synthesis of some new pyrimido [2�, 1�: 2, 3]thiazolo[4,5-b]quinoxaline derivatives as anti-inflam-matory and analgesic agents. Eur. J. Med. Chem., 2010, 45, 1976-1981.

[35] Faty, R.A.M.; Youssef, A.M.S. Oxothioxopyridine carbonitriles as Precur-sors for thiazolopyridines, pyrazolopyridotriazines and pyridothiazolopyrimi dines. Curr. Org. Chem., 2009, 13, 1577-1584.

[36] Hafez, H.N.; El-Gazzar, A.B.A.; Zaki, M.E.A. Simple approach to thieno [3, 2-d] pyrimidines as new scaffolds of antimicrobial activities. Acta Pharm., 2016, 66, 331-351.

Synthesis, Antimicrobial Evaluation of Some New 1, 3, 4-Thiadiazoles and 1, 3, 4-ThiadiazinesAbstract: BackgroundObjective:Method:Results:Conclusion:Keywords:1. INTRODUCTION2. MATERIALS AND METHODSTable 1.3. BIOLOGICAL SCREENINGHTable2.4. RESULTS AND DISCUSSIONScheme 1.Scheme 2.Scheme 3.6. BIOLOGICAL ACTIVITYCONCLUSIONCONSENT FOR PUBLICATIONCONFLICT OF INTERESTACKNOWLEDGEMENTSREFERENCES