DOI: 10.17344/acsi.2015.2124
Acta Chim. Slov. 2016, 63, 173-179
173
Scientific paper
Synthesis and Antimicrobial Activity of Bis[4-methoxy-3-
(6-aryl-7^-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-3-yl) phenyl] methanes and Bis[(triazolo[3,4-#]thiadiazepin-3-yl)
phenyl]methanes
Avula Srinivas*
Department of Chemistry, Vaagdevi Degree & PG College, Kishanpura, Warangal, Telangana, India 506001 * Corresponding author: E-mail: asvas1978@gmail.com Received: 14-11-2015
Abstract
A series of novel bis[4-methoxy-3-(6-aryl-7_ff-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-3-yl)phenyl]methanes and bis[(triazolo[3,4-b]thiadiazepin-3-yl)phenyl]methanes (5a-e and 6a-e) has been synthesized and characterized by IR, and 13C NMR, MS and elemental analysis. All the newly synthesized compounds were screened for their antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Klobsinella aerogenes and Chromobacterium violaceum and antifungal activity against Candida albicans, Aspergillus fumigatus, Trichophyton rubrum and Trichophyton menta-grophytes. Compounds 5b, 5d, 5e, 6b, 6c and 6e exhibited potent activity against the tested bacteria and fungi, and emerged as potential molecules for further development.
Keywords: Bis[4-methoxy-3-(6-aryl-7H-[ 1,2,4]triazolo[3,4-b][ 1,3,4]thiadiazin-3-yl)phenyl]methanes, bis[(triazolo [3,4-b]thiadiazepin-3-yl)phenyl]methanes, organic synthesis, antibacterial activity, antifungal activity.
1. Introduction
Heterocyclic compounds represent one of the most active clasess of compounds posessing a wide spectrum of biological activities, including antibacterial, antifungal, and other biological activities.1-6 The biological activities of various 1,2,4-triazole derivatives and their ^-bridged heterocyclic analogs have been widely investigated as antitumor,7 antiviral,8 anti-inflammatory,9 analgesic,10 and antidepresant.11 It is interesting to use 1,2,4-triazole derivatives as precusors in the synthesis of some important biologically active heterocycles,12-15 which constitute an important class of organic compounds with diverse biological activities, including antiparasitic, analgesic, antibacterial and anti-inflammatory activities.16-21 In addition, it was reported that triazole fused with a six-membered ring system is also found to possess diverse applications in the field of medicine.22-25 The commonly known systems are triazolo-pyridines,26 triazolo-pyridazines,27 tria-zolo-pyrimidines,28 triazolo-triazines,29 triazolo-pyrazi-nes,30 triazolo- triazenes,31 a few monomeric triazolo-thia-diazine,31 and triazolo-thiadiazepines,32 although there are
not many triazoles fused to thiadiazines and thiadiazepi-nes, there is a number of them that are incorporated into a wide variety of therapeutically important compounds possessing a broad spectrum of biological activities.33-37
In recent years attention has been increasingly paid to the synthesis of bis-heterocyclic compounds which exhibit various biological activities,38-41 including antibacterial, fungicidal, tuberculostatic and plant-growth regulating properties. Further development during the recent years42 indicates that the bis-heterocyclic compounds display much better antibacterial activity than the monomeric counterparts.
Owing to the immense importance and varied bioac-tivities exhibited by triazolo-thiadiazines and thiadiazepi-nes and in continuation of our work on biologically active heterocycles,43-51 we were stimulated to integrate thiadia-zines moieties in a triazole framework, since these systems possess well documented antimicrobial activity. In this connection, some bis-heterocyclic compounds such as bis-triazolo thiadiazines and triazolo-thiadiazepines have been synthesized and evaluated for their antibacterial and antifungal activity. For the synthesis of target com-
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pounds, 4-amino-1, 2, 4-triazol-3-thione is used as an intermediate because the amino and mercapto groups are appropriate nucleophile centers for the synthesis of fused heterocyclic compounds.
2. Results and Discussion
5,5'-Methylenebis(2-hydroxybenzoic acid) (2), required for the synthesis of the title compounds, was prepared according to the procedure described in the literatu-re.52 Compound 2 on reaction with methyl iodide, in the presence of aq. KOH at 80 °C, furnished 5-(3-formyl-4-methoxybenzyl)-2-methoxybenzoic acid (3). The condensation of 3 with thiocarbohydrazide at melt temperature for 3 h afforded bis[4-methoxy-3-[4-amino-5-sulfanyl-4H-1,2,4-triazol-3-yl]phenyl]methane (4) as a yellow solid (Scheme 1). IR spectrum of 4 showed two absorption bands in the region of 3335-3235 and 2596 cm-1 assigned to NH2 and SH groups, two absorption bands at1554 and 1512 cm-1 attributable to C=N vibrations, providing a strong evidence for the formation of a triazole ring. 1H NMR spectrum of 4 showed two signals at 5 2.17 and 5.47 ppm corresponding to -SH and -NH2 protons, respectively. The aromatic protons appeared in the region 5 6.62-9.93 ppm in accord with the structure. 13C NMR spectrum of 4 showed signals at 5 156.6 and 134.4 ppm corresponding to the 3-C and 5-C of the triazole moiety, respectively. The other signals observed were at the expected chemical shifts with appropriate integrals. In addition, elemental analysis is also consistent with the structure proposed for 4.
Compound 4 on reaction with a variety of phenacyl-bromides and 1,3-diaryl-2-propanone derivatives in etha-nol at reflux produced bis[4-methoxy-3-(6-aryl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-3-yl)phenyl]met-hanes 5a-e and bis[4-methoxy-3-(6,8-diaryl)-7,8-dihy-dro[ 1,2,4] triazolo[ 3,4-b ][ 1,3,4] thiadiazepin-3-yl)phenyl]methanes 6a-e in 65-82% yield (Scheme 1). The elemental analyses, IR, 1H and 13C NMR and MS spectral data are consistent with the assigned structures. In the IR spectra of compounds 5a-e and 6a-e the absence of absorption bands due to -SH and -NH2 stretching frequencies of parent compound 4 revealed the fusing between compound 4 and various phenacylbromides and 1,3-diaryl-2-propanones. Appearance of three absorption bands at 1595, 1557 and 1520 cm-1 (attributable to C=N vibrations) provides a strong evidence for the fusion of the triazole ring. In the 1H NMR spectra of compounds 5a-e and 6a-e the disappearance of signals at 5 2.17 and 5.47 ppm (due to SH and NH2 protons of compound 4) supports the involvement of these groups in the formation of the thiadiazole ring. In the 13CNMR spectra the signals of triazolo-thiadiazole ring were observed at 5 158.7, 149.0 and 138.2 ppm, respectively. The other signals were observed at the expected chemicals hifts with appropriate
integrals. Elemental analyses are also consistent with the structures proposed for compounds 5a-e and 6a-e.
3. Antibacterial Evaluation
All the newly synthesized compounds 5a-e and 6a-e were screened for their antibacterial activity against Gram-positive bacteria viz. Bacillus subtilis (MTCC 441), Staphylococcus aureus (MTCC 96), and Gram-negative bacteria viz. Klobsinella aerogenes (MTCC 39) and Chro-mobacterium violaceum (MTCC2656) by disc diffusion method.53 For the antibacterial assay, standard inoculums (1-2 x 107 c.f.u/mL 0.5 McFarland standards) were introduced onto the surface of sterile agar plates, and a sterile glass spreader was used for even distribution of the inoculums. The discs measuring 6.26 mm in diameter were prepared from Whatman no. 1 filter paper and sterilized by dry heat at 140 °C for 1 h. The sterile discs previously soaked in a known concentration of the test compounds were placed in the nutrient agar medium. The plates were inverted and incubated for 24 h at 37 °C. The inhibition zones were measured and compared with the standard drug streptomycin. The zone of inhibition data are presented in Table 1. The antibacterial screening revealed that all the tested compounds 5a-e showed moderate to good inhibition towards all the tested strains. Compounds 5b, 5d, 5e, 6b, 6c and 6e exhibited potent inhibitory activity compared to the standard drug at the tested concentrations.
4. Antifungal Evaluation
Compounds 5a-e and 6a-e were also evaluated for in vitro antifungal activity against four fungi viz. Candida albicans (ATCC 10231), Aspergillus fumigates (HIC 6094), Trichophyton rubrum (IFO 9185) and Trichophyton mentagrophytes (IFO 40996) by agar diffusion method.52 For the antifungal assay Sabourands agar media was prepared by dissolving peptone (1 g), D-glucose (4 g) and agar (2 g) in distilled water (100 mL) and adjusting the pH to 5.7. Normal saline was used to make a suspension of spore of fungal strain for lawning. A loopful of particular fungal strain was transferred to 3 mL saline to get a suspension of corresponding species. 20 mL of agar media was poured into each petri-dish, excess of suspension was decanted and the plates were dried by placing in an incubator at 37 °C for 1 h. Using agar punch, wells were made and each well was labeled. A control was also prepared in triplicate and maintained at 37 °C for 3-4 days. The C. albicans was grown for 48 h at 28 °C in YPD broth (1% yeast extract, 2% peptone and 2% dextrose), harvested by centrifugation and then washed twice with sterile distilled water. A. fumigatus, T. rubrum and T. men-tagrophytes were plated in potato dextrose agar (PDA) (Difco) and incubated at ted at 28 °C for two weeks. Spo-
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res were washed three times with sterile distilled water and re-suspended in distilled water to obtain an initial inoculums size of 105 spores/mL. The zones of inhibition were determined and compared with the standard drug amphotericin B (Table 2). Results of antifungal activity showed that most of the new compounds, i.e. 5b, 5d, 5c, 6b, 6c and 6e were active with moderate to good activity.
5. Experimental
Commercial grade reagents were used as supplied. Solvents except analytical reagent grade were dried and purified according to literature when necessary. Reaction progress and purity of the compounds were checked by thin-layer chromatography (TLC) on pre-coated silica gel F254 plates from Merck and were visualized either by exposure to UV light or dipping in 1% aqueous KMnO4 solution. Silica gel chromatographic columns (60-120 mesh) were used for separations. All melting points are uncorrected and measured using Fisher-Johns apparatus. IR spectra were recorded as KBr disks on a Perkin-Elmer FT IR spectrometer. The 1H NMR and 13C NMR spectra were recorded on a Varian gemini spectrometer (300 MHz for 1H and 75 MHz for 13C). Chemical shifts are reported as 5 ppm against TMS as internal reference and coupling constants (J) are reported in Hz units. Mass spectra were recorded on a VG micro mass 7070H spectrometer. Elemental analyses (C, H, N) determined by a Perkin-elmer 240 CHN elemental analyzer were within ±0.4% of theoretical.
7.50 (d, J = 9.1 Hz, 2H, ArH), 9.93 (s, 2H, ArH); 13C NMR (DMSO-d6): 5 42.0, 56.1, 118.2, 124.2, 129.8, 132.7, 134.4, 153.0, 155.1, 156.6; Anal. Calcd for C19H20N8O2S2: C, 49.99; H, 4.42; N, 24.54. Found: C, 491.93; H, 4.45; N, 24.48. MS: m/z 456 (M+).
General procedure for synthesis of bis[4-methoxy-3-(6-aryl[1,2,4]triazolo[3,4-£][1,3,4]thiadiazol)phenyl] methanes 5a-e: A mixture of compound 4 (0.01 mol) and the corresponding phenacylbromide (0.02 mol) in absolute ethanol (20 mL), was refluxed for 6 h. The reaction mixture was concentrated and cooled to room temperature, and the remaining solvent was removed under reduced pressure, then diethyl ether (25 mL) was added and the reaction mixture was left at 0 °C for overnight. The precipitated solid was filtered off; the crude product thus obtained was purified by column chromatography on silica gel with hexane-ethyl acetate as eluent to afford the pure compounds 5a-e.
Bis[4-methoxy-3-(6-phenyl-7#-[1,2,4]triazolo[3,4-£] [1,3,4]thiadiazin-3-yl)phenyl]methane (5a): Yield 82
%; mp 230-232 °C; IR (KBr): vmax 2926, 1614, 1592, 1441, 1068, 796 cm-1; 1H NMR (DMSO-d6, 300 MHz): 5 9.42 (s, 2H, Ar-H), 6.95-7.60 (m, 12H, Ar-H), 6.51 (d, J = 9.1 Hz, 2H, Ar-H), 4.22 (s, 2H, CH2), 3.89 (s, 6H, OCH3), 3.56 (s, 4H, CH2-S); 13C NMR (DMSO-d6, 75 MHz): 5 162.3, 156.6, 154.3, 143.7, 135.1, 132.7, 131.3, 130.7, 129.7, 128.4, 127.9, 123.7, 117.6, 56.1, 42.0, 31.6; Anal. Calcd for C35H28N8S2: C, 64.01; H, 4.30; N, 17.0. Found: C, 63.96; H, 4.33; N, 16.95. MS: m/z 656 (M+).
Preparation of 5-(3-formyl-4-methoxybenzyl)-2-met-hoxybenzoic acid (3): To a solution of 2 (0.01 mol) and K2CO3 (0.04 mol) in DMF (16 mL), Mel (0.03 mol) was added. The reaction mixture was stirred for 12 h at room temperature (TLC, EtOAc : petroleum ether, 2:1). The mixture was poured in water (30 mL), and extracted with Et2O (3 x 20 mL). Washing the organic phase with 2M NaOH solution, drying over Na2SO4 and evaporation of solvent gave compound 3.
Preparation of bis[4-methoxy-3-[4-amino-5-sulfanyl-4ff-1,2,4-triazole-3-yl]phenyl]methane (4): A mixture of compound 3 (0.01 mol) and thiocarbohydrazide (0.02 mol) was heated until the contents melted. The reaction was maintained at this temperature for 3 h. The fused mass thus obtained was treated with sodium bicarbonate solution to dissolve the unreacted compound 3. It was then washed with water and collected by filtration. The product was recrystallized from a mixture of dioxane and ethanol to afford the compound 4 as yellow solid; yield 81%; mp 210-212 °C; IR (KBr) v 3335-3235, 3072, 2596, 1554, 1512, 1314, 1070 cm-1; 1H NMR (DMSO-d6): 5 2.17 (bs, 2H, SH), 3.81 (s, 6H, OCH3), 4.00 (s, 2H, CH2), 5.47 (bs, 4H, NH2), 6.62 (d, J = 9.1 Hz, 2H, ArH),
Bis[4-methoxy-3-(6-(4-methoxyphenyl)-7fl-[1,2,4]tria-zolo[3,4-£][1,3,4]thiadiazin-3-yl)phenyl]methane (5b):
Yield 79%; mp 211-213 °C; IR (KBr): vmax 3035, 1620, 1596, 1535, 1070, 746 cm-1; 1H NMR (DMSO-d6, 300 MHz): 5 9.42 (s, 2H, Ar-H), 7.37 (d, J = 9.1 Hz6, 2H, Ar-H), 7.16 (d, J = 8.6 Hz, 4H, Ar-H), 6.92 (d, J = 8.6 Hz, 4H, Ar-H), 6.51 (d, J = 9.1 Hz, 2H, Ar-H), 4.02 (s, 2H, CH2), 3.89 (s, 6H, OCH3), 3.79 (s, 6H, OCH3), 3.59 (s, 9H, CH2-S); 13C NMR (DMSO-d6, 75 MHz): 5 162.3, 158.6, 156.9, 154.1, 143.7, 135.0, 133.4, 132.6, 131.3, 127.1, 123.7, 117.6, 113.2, 56.1, 48.7, 42.0. Anal. Calcd for C37H32N8O4S2: C, 62.00; H, 4.50; N, 15.63. Found: C, 62.04; H, 4.44; NT, 15.58. MS: m/z 716 (M+).
Bis[4-methoxy-3-(6-(4-hydroxyphenyl)-7fl-[1,2,4]tria-zolo[3,4-£][1,3,4]thiadiazin-3-yl)phenyl]methane (5c):
Yield 69%; mp 241-243 °C; IR (KBr): vmax 3310, 3035, 1618, 1596, 1535, 1069, 746 cm-1; 1H NMR (DMSO-d6, 300 MHz): 5 9.42 (s, 2H, Ar-H), 7.37 (d, J = 9.1Hz, 2H, Ar-H), 7.20 (d, J = 8.6 Hz, 4H, Ar-H), 6.67 (d, J = 8.6 Hz, 4H, Ar-H), 6.51 (d, J = 9.1 Hz, 2H, Ar-H), 4.02 (s, 2H, CH2), 3.90 (s, 6H, OCH3), 3.59 (s, 4H, CH2-S); 13C NMR (DMSO-d6, 75 MHz): 5 162.3, 161.4, 157.6, 154.3, 143.6, 136.1, 135.3, 132.6, 131.3, 125.0, 123.9, 117.6, 115.1,
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56.2, 42.0; Anal. Calcd for C35H28N8O4S2: C, 61.03; H, 4.10; N, 16.27. Found: C, 61.(30; H, 4.04; N, 16.30; MS: m/z 687 (M+ - 1).
Bis[4-methoxy-3-(6-(4-nitrophenyl)-7fl-[1,2,4]triazo-lo[3,4-£][1,3,4]thiadiazin-3-yl)phenyl]methane (5d):
Yield 84%; mp 271-273 °C; IR (KBr): vmax 3035, 1618, 1595, 1532, 1370, 1069, 750 cm-1; 1H NMR (DMSO-d6, 300 MHz): 5 9.42 (s, 2H, Ar-H), 8.40 (d, J = 8.4 Hz, 4H, Ar-H), 7.82 (d, J = 8.4 Hz, 4H, Ar-H), 7.37 (d, J = 9.1 Hz, 2H, Ar-H), 6.51 (d, J = 9.1 Hz, 2H, Ar-H), 4.02 (s, 2H, CH2), 3.90 (s, 6H, OCH3), 3.61 (s, 4H, CH2-S); 13C NMR (DMSO-d6, 75 MHz): 5 162.2, 155.9, 154.1, 147.3, 143.6, 137.9, 135.1, 132.9, 132.1, 131.3, 129.4, 123.2, 117.6, 56.1, 42.0, 31.6; Anal. Calcd for C35H26N10O6S2: C, 56.29; H, 3.51; N, 18.67. Found: C, 56.23; H, 3.469; N, 18.71; MS: m/z 746 (M+).
Bis[4-methoxy-3-(6-(3,4-dichlorophenyl)-7#-[1,2,4] triazolo[3,4-£][1,3,4]thiadiazin-3-yl)phenyl]methane
(5e): Yield 69%; mp 226-228 °C; IR (KBr): vmax 3031, 1624, 1590, 1457, 1069, 750, 680 cm-1; ^if NMR (DMSO-d6, 300 MHz): 5 9.42 (s, 2H, Ar-H), 8.00 (d, J = 7.6 Hz, 2H, Ar-H), 7.62 (s, 2H, Ar-H), 7.37-7.26 (m, 4H, Ar-H), 6.82 (d, J = 9.1 Hz, 2H, Ar-H), 4.00 (s, 2H, CH2), 3.88 (s, 6H, OCH3), 3.81 (s, 4H, CH2-S); 13C NMR (DMSO-d6, 75 MHz): 5 160.0, 157.0, 154.2, 143.7, 136.6, 135.1, 133.2, 132.0, 132.0, 131.3, 130.3, 129.6, 128.7, 123.8, 117.6, 62.0, 42.0, 33.0; Anal. Calcd for C35H24Cl4N8O2S2: C, 52.91; H, 3.04; N, 14.10. Found: C, 523.86; H, 3.00; N, 14.05; MS: m/z 794 (M+).
General procedure for the synthesis of bis[4-methoxy-3-(6,8-diaryl)-7,8-dihydro[1,2,4]triazolo[3,4-6][1,3,4] thiadiazepin-3-yl)phenyl]methanes 6a-e: To a mixture of 4 (0.01 mol) and the corresponding 1,3-diaryl-2-propa-none derivative (0.02 mol) in ethanol (50 mL) a few drops of glacial acetic acid was added and the reaction mixture refluxed for 5 h. At the end of the reaction, the ethanolic solution was concentrated to half of its volume under reduced pressure. The solid that separated from the concentrate was filtered and purified by column chromatography on silica gel with petrolether-ethyl acetate as eluent to afford the pure compounds 6a-e.
Bis[4-methoxy-3-(6,8-diphenyl)-7,8-dihydro[1,2,4] triazolo[3,4-£][1,3,4]thiadiazepin-3-yl)phenyl]metha-
ne (6a): Yield 72 %; mp 210-212 °C; IR (KBr): vmax 3065, 1619, 1595, 1335, 1070, 750 cm-1; 1H NMR (DM-SO-d6, 300 MHz): 5 9.32 (s, 2H, Ar-H), 7.69-7.66 (m, 6H, Ar-H), 7.50-7.35 (m, 12H, Ar-H), 7.24-7.16 (m, 4H, Ar-H), 6.69 (d, J = 9.1 Hz, 2H, Ar-H), 4.98 (dd, JXA = 11.4, JXB = 5.1 Hz, 2H, Hx), 4.00 (s, 2H, CH2), 3.91 (s, 6H, OCH3), 2.31 (dd, JAB = 12.7, JAX = 11.4 Hz, 2H, HA), 2.13 (dd, JBX = 5.1, JBA = 12.7 Hz, 2H, HB); 13C NMR (DMSO-d6, 75 MHz): 5 170.0, 158.7, 152.9,152.4, 144.0,
142.6,	133.7, 131.6, 130.5, 129.8, 129.0, 128.3, 127.7, 127.1, 126.4, 122.3, 118.1, 56.1, 55.3, 43.3, 42.0; Anal. Calcd for C49H40N8O2S2: C, 70.31; H, 4.82; N, 13.39. Found: C, 70.26; H, 4.85; N, 13.32; MS: m/z 838 (M+).
Bis[4-methoxy-3-(6-phenyl-8-(4-chlorophenyl)-7,8-dihydro[1,2,4]triazolo[3,4-£][1,3,4]thiadiazepin-3-yl) phenyl]methane (6b): Yield 76%; mp 223-225 °C; IR (KBr): vmax 2943, 1621, 1599, 1513, 1034, 771, 694 cm-1; 1H NMfUDMSO-dg, 300 MHz): 5 9.50 (s, 2H, Ar-H), 7.90 (d, J = 8.3 Hz, 4H, Ar-H), 7.40-7.35 (m, 12H, Ar-H), 7.00 (d, J = 8.3 Hz, 4H, Ar-H), 6.90 (d, J = 9.1 Hz, 2H, Ar-H), 5.80 (dd, JXA = 11.4, JXB = 5.1 Hz, 2H, HX), 3.95 (s, 2H, CH2), 3.91 (s, 6H, OCH3), 2.31 (dd, JAB = 12.7, JAX = 11.4 Hz, 2H, Ha), 2.14 (dd, JBX = 5.1, JBA = 12.7 Hz, 2H, Hb); 13C NMR (DMSO-d6, 75 MHz): 5 170.0,
158.7,	152.4, 150.0, 144.0, 142.6, 137.1, 133.7, 131.6, 130.3, 129.8, 129.7, 129.0, 128.3, 127.7, 122.3, 118.1, 56.1, 55.3, 43.3, 42.0; Anal. Calcd for C49H38Cl2N8O2S2: C, 64.97; H, 4.23; N, 12.37. Found: C, 641.93; H, 4.19; N, 12.33; MS: m/z 906 (M+).
Bis[4-methoxy-3-(6-phenyl-8-(4-methoxyphenyl)-7,8-dihydro[1,24]triazolo[3,4-£][1,3,4]thiadiazepin-3-yl) phenyl]methane (6c): Yield 81%; mp 241-243 °C. IR(KBr): vmax 3061, 1620, 1596, 1534, 1070, 750 cm-1; 1H NMR (DMSO-d6, 300 MHz): 5 9.32 (s, 2H, Ar-H), 7.50-7.35 (m, 12H, Ar-H), 7.11 (d, J = 8.4 Hz, 4H, Ar-H), 6.72 (d, J = 8.4 Hz, 4H, Ar-H), 6.69 (d, J = 9.1 Hz, 2H, Ar-H), 4.75 (dd, JXA = 11.4, JXB = 5.1 Hz, 2H, HX), 4.00 (s, 2H, CH2), 3.91 (s, 6H, OCH3), 3.72 (s, 6H, OCH3), 2.30 (dd, JAB = 12.7, JAX = 11.4 Hz, 2H, H(), 2.14 (dd, JBX = 5.1, JBB = 12.7 Hz, 2H, Hb); 13C NMR (DMSO-d6, 75 MHz): 5 170.1, 161.4, 153.4, 144.3, 143.7, 142.6, 133.7,
131.6,	129.8, 129.0, 128.4, 127.7, 122.3, 118.1, 114.5, 58.6, 56.7, 56.0, 55.2, 43.4, 42.0; Anal. Calcd for C51H44N8O4S2: C, 68.28; H, 4.94; N, 12.49. Found: C, 685.25; H, 450; N, 12.53; MS: m/z 898 (M+).
Bis[4-methoxy-3-(6-phenyl-8-(3-nitrophenyl)-7,8-dihydro[1,2,4]triazolo[3,4-£][1,3,4]thiadiazepin-3-yl) phenyl]methane (6d): Yield 69%; mp 238-240 °C; IR (KBr): vmax 3059, 1618, 1595, 1530, 1370, 1068, 750 cm-1; 1H NMR (DMSO-d6, 300 MHz): 5 9.32 (s, 2H, Ar-H), 8.47 (d, J = 7.7 Hz, 2H, Ar-H), 8.00 (s, 2H, Ar-H), 7.50-7.35 (m, 12H, Ar-H), 7.40-7.30 (m, 12H, Ar-H), 6.69 (d, J = 9.1 Hz, 2H, Ar-H), 4.75 (dd, JXA = 11.4, JXB = 5.1 Hz, 2H, HX), 4.00 (s, 2H, CH2), 3.91 (s, 6H, OCH3), 2.30 (dd, JAB = 12.7, JAX = 11.4 Hz, 2H, HA), 2.13 (dd, JBX = 5.1, JBA = 12.7 Hz, 2H, HB); 13C NMR (DMSO-d6, 75 MHz): 5 169.8, 157.9, 153.5, 147.9,
143.7,	142.4, 138.3, 134.4, 135.8, 131.6, 130.1, 129.8, 129.0, 128.3, 127.6, 125.0, 122.3, 121.4, 118.1, 56.1, 55.3, 43.3, 42.0; Anal. Calcd for C49H38N10O6S2: C, 63.49; H, 4.13; N, 15.11. Found: C, 633.50; H, 4.06; N, 15.06; MS: m/z 928 (M+).
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Bis[4-methoxy-3-(6-phenyl-8-(3-hydroxy-4-methoxyp-henyl)-7,8-dihydro[1,2,4]triazolo[3,4-£][1,3,4]thia-diazepin-3-yl)phenyl]methone (6e): Yield 74%; mp 249-251 °C; IR (KBr): vmax 3438, 3031, 2925, 1621, 1590, 1513, 1275, 1024, 750 cm-1; 1H NMR (DMSO-d6, 300 MHz): 5 9.40 (s, 2H, Ar-H), 7.50-7.35 (m, 12H, Ar-H), 7.18 (d, J = 8.3 Hz, 2H, Ar-H), 7.11 (s, 2H, Ar-H), 6.69-6.58 (m, 4H, Ar-H), 4.76 (dd, JXA = 11.4, JXB = 5.1 Hz, 2H, HX), 4.49 (s, 2H, OH), 4.00 (s, 2H, CH2), 3.91 (s, 6H, OCH3), 3.76 (s, 6H, OCH3), 2.31 (dd, JAB = 12.7, JAX = 11.4 Hz, 2H, Ha), 2.13 (dd, JBX = 5.1, JBA = 12.7 Hz, 2H, Hb); 13C NMR (DMSO-d6, 75 MHz): 5 169.8, 158.4, 152.7, 150.1, 147.6, 143.8, 142.1, 133.9, 131.6, 129.7, 129.2, 127.9, 127.2, 123.0, 122.3, 118.1, 117.0, 116.4, 112.1, 59.6, 56.1, 55.7, 43.3, 42.0; Anal. Calcd for C51H44N8O6S2: C, 65.93; H, 4.77; N, 12.06. Found: C, 655.88; H, 4.81; N, 12.05; MS: m/z 930 (M+ - 1).
Table 1. Antibacterial activity of 5a-e and 6a-e
Compound	Zone of inhibition in mm"
B.	S.	K.	C.
subtilis aureus aerogenes violaceum
5a	11	12	10	12
5b	22	24	25	26
5c	12	10	12	8
5d	24	24	22	26
5e	24	24	24	25
6a	8	10	11	6
6b	20	22	20	22
6c	20	21	22	22
6d	8	11	11	12
6e	24	22	25	22
Streptomycin	25	30	30	30
a Streptomycin (50 |g / disc) was used as positive reference and compounds 5a-e and 6a-e (50 |g / disc) were screened.
Scheme 1. Synthetic route to methylene bis[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles and bis[4-methoxy-3-(6,8-diaryl)-7,8-dihydro[1,2,4]triazolo [3,4- b][ 1,3,4] thiadiazepinyl)phenyl] methane
Compounds
5.	Ar =	C6H5-	4-MeO-C6H4-	4-HO-C6H4-	4-NO2-C6H4- 3,4-di-Cl-C6H3-
6.	Ar' =	C6H5-	4-MeO-C6H4-	4-HO-C6H4-	4-NO2-C6H4- 3,4-di-Cl-C6H3-
Reagents and conditions: (i) CH2O, H2SO4, reflux; (ii) Mel, K2CO3, DMF, rt; (iii) (iii) Thiocarbohydrazide, heat; (iv) PhCOCH3, EtOH, reflux. (v) PhCOCH=CHArl, reflux.
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b
d
a
c
e
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Table 2. Antifungal activity of 5a-e and 6a-e
Compound
(|ig/mL)a
C. albicans mentagrophytes
Minimum inhibition concentration (MIC) A. fumigatus	T. rubrumT .
5a 10	12	14	16
5b 22	25	15	20
5c 10	10	12	10
5d 24	22	12	22
5e 26	20	22	23
6a 16	12	10	8
6b 25	22	20	23
6c 21	24	19	25
6d 12	10	10	14
6e 24	25	20	22
Streptomycin	30	30	25
25
a Amphotericin (100 |g / disc) was used as positive reference and compounds 5a-e and 6a-e (100 |g / disc) were screened.
6. Conclusions
A new series of bis[4-methoxy-3-(6-aryl[1,2,4] triazolo[3,4-è][1,3,4]thiadiazol)phenyl]methanes and bis[(triazolo[3,4-è]thiadiazepin-3-yl)phenyl]methanes 5a-e and 6a-e has been synthesized and evaluated for their antimicrobial activity against various bacterial and fungal strains. The screened compounds 5b, 5d, 5e, 6b, 6c and 6e exhibited potent antimicrobial activity compared to standard drug at the tested concentrations. Most of the other compounds also showed appreciable activity against the tested bacteria and fungi, and emerged as potential molecules for further development.
7. Acknowledgements
The author is thankful to the Director, CSIR-Indian Institute of Chemical Technology, Hyderabad, India, for providing research facilities and CSIR-New Delhi for the Reseach Associate fellowship.
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Povzetek
Pripravili smo novo serijo bis[4-metoksi-3-(6-aril-7^-[1,2,4]triazolo[3,4-b][1,3,4]tiadiazin-3-il)fenil]metanov in bis[(triazolo[3,4-b]tiadiazepin-3-il)fenil]metanov (5a-e in 6a-e) ter nove spojine karakterizirali z IR, 1H in 13C NMR, MS ter elementno analizo. Vsem novim spojinam smo določili tudi antibakterijsko aktivnost proti Bacillus subtilis, Staphylococcus aureus, Klobsinella aerogenes in Chromobacterium violaceum ter aktivnost proti glivam Candida albicans, Aspergillus fumigatus, Trichophyton rubrum in Trichophyton mentagrophytes. Spojine 5b, 5d, 5e, 6b, 6c in 6e so pokazale veliko aktivnost proti testiranim bakterijam in glivam ter so se izkazale za potencialne molekule za nadaljnji razvoj.
Srinivas: Synthesis and Antimicrobial Activity of ...