triazine Derivatives - Wiley Online Library

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Chem Biol Drug Des 2012; 80: 598–604 Research Article

Design, Facile Synthesis, and Antibacterial Activity of Hybrid 1,3,4-thiadiazole-1,3,5-triazine Derivatives Tethered via –S– Bridge Vaibhav Dubey, Manish Pathak, Hans R. Bhat and Udaya P. Singh* Department of Pharmaceutical Sciences, Sam Higginbottom Institute of Agriculture, Technology & Sciences (Formerly-Allahabad Agricultural Institute), Deemed-to-be-University, Allahabad 211007, India *Corresponding author: Udaya P. Singh, [email protected] Some hybrid 1,3,4-thiadiazole-1,3,5-triazine derivatives tethered via –S– bridge were synthesized and characterized with the aid of spectroscopic and elemental analysis. These hybrid conjugates were then investigated for their antibacterial activity against selected Gram-positive and Gramnegative bacteria. Excellent to moderate antibacterial activity was presented by the target compounds. Key words: 1,3,4-thiadiazole, 1,3,5-triazine, antibacterial, hybrid Received 26 March 2012, revised 18 May 2012 and accepted for publication 5 June 2012

Since the discovery of penicillin by Sir Alexander Fleming some eight decades ago, the use of antibiotics has made major contributions to public health. Surprisingly, just 4 years after the drug started being mass-produced, over 50% of micro-organisms were no longer susceptible (1). The rapid growth of antibiotic resistance has resulted in the generation of multidrug-resistant (MDR) pathogens that produce infections that are increasingly difficult to treat with antibiotics currently available. According to Centre for Disease Control Statistics, nearly 2 million patients in the United States acquire an infection in the hospital each year and about 90 000 of those patients die each year as a result of their infectiona. Methicillin-resistant Staphylococcus aureus (MRSA) alone account for the death of 19 000 people a year in the United States, which is more than the death from AIDS (2). Now, we have recently moved into an era of not just multiple resistant bacteria but of totally resistant pathogens, which now include vancomycin-resistant enterococci (3), carbapenem-resistant Acinetobacter baumannii (4,5), vancomycin-resistant MRSA (6), and very recently, New Delhi metallobeta-lactamase-1 (7). Furthermore, heavy investment and far too low returns compared with chronic disease and lifestyle medications has caused to prevent the investment of pharmaceutical 598

industries into the projects related to the development of new antibioticsb. Thus, increased incidence of bacterial resistance to currently available antibiotics together with financial constraints necessitates the discovery and introduction of new, economic, and effective drugs. Nowadays, combination therapies have emerged as a better option to treat MDR pathogens than individual drugs, for example, HAART and ACTs. However, combination therapies have certain disadvantages such as (i) increased expense; (ii) increased risk of adverse effects; (iii) pharmacokinetic intolerability; and (iv) superinfection (8). Molecular hybridization is an innovative approach gaining attentionfrom medicinal chemists owing to resemblance with combination therapies where two diverse pharmacophoric groups were joined covalently through a linker. Enlarged spectrums of activity, less prone to spontaneous mutations and resistance development, dual drug targeting at more than one site and privileged activity when compared to the individual agent are deemed as main possible advantages of this strategy, Figure 1 (9). As a part of our ongoing research on the discovery of economic and potential antimicrobial agents, we are emphasizing on the development of hybrid molecules containing diverse pharmacophoric groups (10–16). In present study, we decided to enlarge our investigation by synthesizing the hybrid analogues of 1,3,4-thiadiazole and 1,3,5-triazine derivatives tethered via mercapto (–S–) bridge. The rationale behind the study is to exemplify the versatility of substituent in terms of antibacterial efficacy with the aim to confirm earlier observations.

Experimental Melting points of synthesized compounds were determined in open capillary tubes Hicon melting point apparatus and are uncorrected. Thin-layer chromatographic analysis (TLC) was carried out to monitor the completion of reaction. The different mobile phases were selected according to the assumed polarity of the products. The spots were visualized by exposure to iodine vapor and UV light. The structures of the intermediate compounds were established on the basis of spectral (FT-IR, mass) and elemental analysis, whereas structures of the title compounds were ascertained on the basis of FT-IR, 1H NMR, mass spectral and elemental analysis. FT-IR (in 2.0

Antibacterial Activity of 1,3,5-triazines

A

B

C

Figure 1: Hybrid molecules, concept, and approaches. per cm, flat, smooth, abex, KBr) spectra were recorded on Biored FTs spectrophotometer. Proton magnetic resonance spectra (1H NMR) were recorded on Bruker Model DRX-400 and 300 MHz NMR spectrometer in CDCl3-d6 and DMSO using tetramethylsilane (TMS) as an internal standard. Chemical shift is reported in parts per million (ppm, d), and signals are described as s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). The FAB mass spectrum was recorded on a THERMO Finnigan LCQ Advantage max ion trap mass spectrometer; samples were introduced into ESI source through Finnigan surveyor autosampler. Elemental analysis was carried out on a Vario EL III CHNOS elemental analyzer, and results were found within €0.4%. Step 1: Synthesis of 5-(3,5-dimethyl-1-phenyl4,5-dihydro-1H-pyrazol-4-ylamino)-1,3,4thiadiazole-2-thiol (6) Procedure of synthesis of 5-amino-1,3,4thiadiazole-2-thiol (3) Anhydrous sodium carbonate (24 g) with carbon disulfide 0.25 mol was added in thiosemicarbazide (0.25 mol) suspended in absolute ethanol. The mixture was warmed with stirring under reflux for 1 h and then further heated on steam bath for 4 h. The completion of reaction was indicated by TLC using toluene ⁄ ethyl acetate ⁄ formic acid (5:4:1) as a mobile phase. The solvent was removed, and the residue was dissolved in water and acidified further with concentrated HCl to afford solid product 3. Yellowish crystals; yield: 89%; mp: 233 C; MW: 133.19; Rf : 0.68 (hexane ⁄ ethyl acetate; 6:4); FT-IR (mmax per cm, KBr): 3410, 3300 (NH primary), 2555 (S-H broad thiol), 1000–1300 (C=N); elemental analysis for C2H3N3S2: Calculated: C, 18.03; H, 2.37; N, 31.54. Found: C, 18.12; H, 2.34; N, 31.62. Chem Biol Drug Des 2012; 80: 598–604

Procedure of diazotization of 5-amino-1,3,4thiadiazole-2-thiol (4) A mixture of 3 (0.01 mol) in concentrated HCl (3 mL) was cooled to 0–5 C under ice followed by dropwise addition of cooled sodium nitrite solution (1.5 g in 10 mL of water) over a period of 10 min. The reaction mixture was then stirred for 30 min to yield diazonium salt intermediate 4.

Procedure for the preparation of 3-(5-mercapto1,3,4-thiadiazol-2-ylimino)pentane-2,4-dione with active methylene (5) To an ice-cold mixture of the active methylene compound, acetylacetone (0.01 mol), and sodium acetate (0.05 mol) in ethanol (50 mL) was added dropwise with stirring a solution of diazonium salt compound 4 (0.01 mol) over 15 min. The stirring was continued for 30 min and the reaction mixture then left for 2 h at room temperature, and the completion of reaction was indicated by TLC using toluene ⁄ ethyl acetate ⁄ formic acid (5:4:1) as a mobile phase. Resulted solid product was collected and recrystallized from ethanol to afford the corresponding imino derivatives. Brownish yellow crystals; yield: 82%; mp: 241 C; MW: 229; Rf : 0.48; FT-IR (mmax per cm, KBr): 2550 (S-H), 1250 (C-N), 1650, 1662 (C=O); elemental analysis for C7H7N3O2S2: Calculated: C, 36.67; H, 3.08; N, 18.33. Found: C, 36.52; H, 3.00; N 18.35.

Procedure for the synthesis of 5-(3,5-dimethyl1-phenyl-4,5-dihydro-1H-pyrazol-4-ylamino)1,3,4-thiadiazole-2-thiol (6) To a solution of 5 (0.01 mol) in glacial acetic acid (30 mL), phenyl hydrazine (0.012 mol) and anhydrous sodium acetate (0.01 mol) 599

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were added. The reaction mixture was heated under reflux for 4 h. Completion of reaction was monitored by TLC using toluene ⁄ ethyl acetate ⁄ formic acid (5:4:1) as a mobile phase. Thus, mixture obtained was poured into ice-cold water and stored in a refrigerator. The obtained crude product was washed with water, dried and recrystallized from methanol. Dark yellowish crystals; yield: 69%; mp: 253C; MW: 305; Rf : 0.46; FT-IR (mmax per cm, KBr): 3305, 3300 (N-H secondary), 3010, 2998 (C-H), 2559 (S-H), 1156, 1685 (C=N), 1625 (C=C), 1610 (C=C aromatic ring), 1450 (C=C aromatic ring), 1250 (C-N); elemental analysis for C13H15N5S2: Calculated: C, 51.12; H, 4.95; N, 22.93. Found: C, 51.10; H, 4.92; N, 22.97.

Step 2: General procedure for the synthesis of 2,4-bis(substituted amine)-6-chloro-1,3,5triazine 9 (a–f) Various substituted anilines 8 (a–f) (0.2 mol) were added into 100 mL of acetone, maintaining temperature at 40–45 C. The solution of 2,4,6-tri chloro-1,3,5-triazine (7) (0.1 mol) in 25 mL acetone was added constantly, stirred for 3 h followed by dropwise addition of NaHCO3 solution (0.1 mol), taking care that reaction mixture does not become acidic. Completion of the reaction was analyzed by TLC utilizing benzene ⁄ ethyl acetate as a mobile phase (9:1). The product was filtered and washed with cold water and recrystallized with ethanol to afford pure compounds 9(a–f).

6-Chloro-N2,N4-bis(4-chlorophenyl)-1,3,5triazine-2,4-diamine 9a Yellow crystals; yield: 75%; mp: 135–137 C; MW: 366.63; Rf: 0.48; FT-IR (mmax cm)1, KBr): 3243.26 (N–H secondary), 2965.46 (C–H broad), 1387.38 (aromatic –C=N); 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 7.32 (d, 4H, 3,5-CH aromatic), 7.08 (d, 4H, 2,6-CH aromatic), 4.82 (s, 2H, NH); elemental analysis for C15H10Cl3N5: Calculated: C, 49.14; H, 2.75; N, 19.10. Found: C, 49.17; H, 2.77; N, 19.15.

6-Chloro-N2,N4-di-p-tolyl-1,3,5-triazine-2,4diamine 9d Pale-yellowish crystals; yield: 78%; mp: 212–214 C; MW: 325.80; Rf: 0.72; FT-IR (mmax per cm, KBr): 3310 (N-H secondary), 3000 (C-H), 1605 (C=C aromatic ring), 1620–1650 (C=C), 1475 (C=C aromatic ring); 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 7.03 (d, 1H, J = 8.2 Hz, Ar-H), 7.06 (d, 1H, J = 5.4 Hz, Ar-H), 7.31 (d, 1H J = 8.2 Hz, Ar-H), 7.23 (d, 1H, Ar-H), 5.24 (br, s, 1H, NH), 2.53 (s, 3H, CH3); elemental analysis for C17H16ClN5: Calculated: C, 62.67; H, 4.95; N, 21.50. Found: C, 62.63; H, 4.98; N, 21.55.

6-Chloro-N2,N4-bis(4-methoxyphenyl)-1,3,5triazine-2,4-diamine 9e Yellow crystals; yield: 88%; mp: 235 C; MW: 357.79; Rf: 0.69; FT-IR (mmax per cm KBr): 3300 (N-H secondary), 3015 (C-H), 1670–1685 (C=O), 1630–1640 (C=C), 1585 (C=C aromatic ring), 1460 (C=C aromatic ring), 1100–1230 (C-N). 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 7.43 (d, 1H, J = 8.68 Hz, Ar-H), 7.52 (d, 1H J = 5.4 Hz, Ar-H), 7.39 (d, 1H J = 8.76 Hz, Ar-H), 7.43 (d, 1H Ar-H), 5.49 (br s, 1H, NH), 3.65 (s, 3H, OCH3). Elemental analysis for C17H16ClN5O2: Calculated: C, 57.07; H, 4.51; N, 19.57. Found: C, 57.02; H, 4.45, N, 19.58.

6-Chloro-N2,N4-bis(4-nitrophenyl)-1,3,5-triazine2,4-diamine 9f Black crystals; yield: 86%; mp: 143–145 C; MW: 387.74; Rf: 0.55; FTIR (mmax per cm KBr): 3289.56 (N–H secondary), 3055.70 (C–H broad), 1548.28–1446.06 (aromatic C=N); 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 7.40 (t, 4H, 4 = CH aromatic), 7.32 (t, 4H, 4 = CH aromatic), 3.62 (d, 2H, 2-NH aromatic); elemental analysis for C15H10ClN7O4: Calculated: C, 46.46; H, 2.60; N, 25.29. Found: C, 46.48; H, 2.65; N, 25.26.

N2,N4-Bis(4-bromophenyl)-6-chloro-1,3,5triazine-2,4-diamine 9b Brownish-black crystals; yield: 81%; mp: 169–170 C; MW: 455.53; Rf: 0.35; FT-IR (mmax per cm, KBr): 3350.62 (N–H secondary), 3015.43 (C–H broad), 1656.15 (C=C stretch); 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 7.26 (d, 4H, 3,5-CH aromatic), 7.06 (d, 4H, 2,6-aromatic), 4.81 (s, 2H, NH); elemental analysis for C15H10Br2ClN5: Calculated: C, 39.55; H, 2.21; N, 15.37. Found: C, 39.53; H, 2.20; N, 15.34.

Step 3: General procedure for synthesis hybrid 3,5-dimethyl-1-phenyl-4,5-dihydro-1H-pyrazole4ylamino-1,3,4-thiadiazole-1,3,5-triazine derivatives tethered via –S– bridge 10 (a–f) 2,4-Bis(substituted amine)-6-chloro-1,3,5-triazine 9(a–f) (0.1 mol) was added into 50 mL of 1,4-dioxane, maintaining temperature at 40–45 C. The solution of 5-(3,5-dimethyl-1-phenyl-4,5-dihydro-1Hpyrazol-4-ylamino)-1,3,4-thiadiazole-2-thiol (6) (0.1 mol) in 35 mL of 1,4-dioxane was added constantly to above solution and stirred for 90 min followed by dropwise addition of K2CO3 (0.1 mol), and this mixture was refluxed at 135–145 C for 9 h. The product was filtered and washed with cold water and recrystallized with ethanol to afford the corresponding pure compounds 10(a-f).

6-Chloro-N2,N4-bis(4-fluorophenyl)-1,3,5triazine-2,4-diamine 9c Reddish-orange crystals; yield: 66%; mp: 124–125 C; MW: 333.72; Rf: 0.42; FT-IR (mmax per cm, KBr): 3054.72 (C–H broad), 1548.58 (C=N), 1448.25 (C–N aromatic); 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 7.68 (d, 4H, 2-CH, aromatic), 7.14 (d, 4H, 2-CH aromatic), 4.84 (t, 2H, -NH amino); elemental analysis for C15H10ClF2N5: Calculated: C, 53.99; H, 3.02; N, 20.99. Found: C, 53.96; H, 3.02; N, 20.95.

N2,N4-Bis(4-chlorophenyl)-6-((5-((3,5-dimethyl-1phenyl-4,5-dihydro-1H-pyrazol-4-yl)amino)-1,3,4thiadiazol-2-yl)thio)-1,3,5-triazine-2,4-diamine 10a Deep yellow crystals; yield: 92%; mp: 304–306 C; MW: 635.59; FTIR (mmax; per cm KBr): 3390–3318 (N–H secondary), 1610 (Ar, -C=C aromatic, Bz), 1540 (C=N stretching), 720, 680; 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 6.90 (m, 2H, Ar-Bz-H, 10,11), 7.16 (m, 2H, Ar-Bz-H, 12,13), 9.15 (s, 1H, Ar-Bz-H, 8), 13.15 (s-1H, Ar-Bz-H, 6),

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8.20–8.18 (d, 1H, J = 8 Hz, Ar-H), 8.14–8.12 (d, 1H, J = 9.2 Hz, ArH), 8.01–7.99 (d, 1H, J = 6.8 Hz, Ar-H); mass: 636 (M + H+); elemental analysis of C28H24Cl2N10S2: C, 52.51; H, 3.81; N, 22.04. Found: C, 52.30; H, 3.95; N, 22.90.

N2,N4-Bis(4-bromophenyl)-6-((5-((3,5-dimethyl-1phenyl-4,5-dihydro-1H-pyrazol-4-yl)amino)-1,3,4thiadiazol-2-yl)thio)-1,3,5-triazine-2,4-diamine 10b Yellowish brown crystals; yield: 89%; mp; 368–370 C; MW: 724.50; FT-IR (mmax per cm, KBr): 3129–3098 (N–H secondary), 1609 (Ar, -C=C aromatic, Bz), 1520 (C=N stretching), 1440, 660; 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 6.96 (d, 2H, J = 8.5 Hz Ar-Bz-H 11,13), 7.29 (dd, 1H, J = 8.5, 2.5 Hz Ar-Bz-H 10), 7.94–7.92 (d, 1H, J = 8.4 Hz Ar-H), 7.76 (s, 1H, NH), 7.66–7.67 (d, 1H, J = 8.8 Hz, ArH), 7.95 (dd, 1H, J = 8.5,2.5 Hz, Ar-Bz-H, 14), 8.64 (s, 1H, Ar-H 8); mass: 725 (M + H+); elemental analysis of C28H24Br2N10S2: C, 46.42; H, 3.34; N, 19.33. Found: C, 46.20; H, 3.48; N, 19.95.

6-((5-((3,5-Dimethyl-1-phenyl-4,5-dihydro-1Hpyrazol-4-yl)amino)-1,3,4-thiadiazol-2-yl)thio)N2,N4-bis(4-fluorophenyl)-1,3,5-triazine-2,4diamine 10c Deep yellow crystals; yield: 76%; mp; 391–393 C; MW: 602.68; FTIR (mmax per cm, KBr): 3310–3104 (N–H secondary), 3010 (C-H stretching) 2490, 1652 (Ar, -C=C stretching, Bz), 1503 (C=N stretching), 810, 610 (Ar-C-F stretching, Bz); 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 7.14 (m, 3H, Ar-Bz-H 14, 15, 16), 7.24 (m, 2H, Ar-Bz-H, 17,18), 7.70 (d, 2H, J = 15.6 Hz, Ar-Bz-H 10,11), 7.89 (d, 2H, J = 8.1 Hz, Ar-Bz-H 23,24), 7.94 (d, 1H, J = 15.6 Hz Ar-Bz-H 11,12), 8.14 (d, 2H, J = 8.4 Hz Ar-Bz-H 21,22); mass: 603 (M + H+); elemental analysis of C28H24F2N10S2: C, 55.80; H, 4.01; N, 23.24. Found: C, 55.25; H, 4.01; N, 23.01.

6-((5-((3,5-Dimethyl-1-phenyl-4,5-dihydro-1Hpyrazol-4-yl)amino)-1,3,4-thiadiazol-2-yl)thio)N2,N4-di-p-tolyl-1,3,5-triazine-2,4-diamine 10d Pale-yellow crystals; yield: 68%; mp; 227–229 C; MW: 594.76; FTIR (mmax per cm, KBr): 3389–3120 (NH stretching, Bz), 3010 (C-H stretching), 1630 (Ar, -C=C stretching, Bz), 1503 (C=N stretching), 800; 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 7.44 (d, 2H, J = 8.5 Hz, Ar-Bz-H 12,13), 7.60 (d, 2H, J = 8.4 Hz, Ar-Bz-H, 15,16), 7.70 (d, 2H, J = 15.6 Hz, Ar-Bz-H 9), 7.80 (d, 2H, J = 3.1 Hz, Ar-BzH 18,19), 7.89 (d, 1H, J = 15.6 Hz Ar-Bz-H 10), 8.14 (d, 2H, J = 8.4 Hz Ar-Bz-H 21,22); mass: 595 (M + H+); elemental analysis of C30H30N10S2: C, 60.58; H, 5.08; N, 23.55. Found: C, 60.25; H, 5.01; N, 23.59.

6-((5-((3,5-Dimethyl-1-phenyl-4,5-dihydro-1Hpyrazol-4-yl)amino)-1,3,4-thiadiazol-2-yl)thio)-N2, N4-bis(4-methoxyphenyl)-1,3,5-triazine-2,4diamine 10e Deep yellow crystals; yield: 82%; mp; 236–238 C; MW: 626.76; FTIR (mmax per cm, KBr): 3117–2990 (N–H secondary), 2930 (C-H Chem Biol Drug Des 2012; 80: 598–604

stretching), 2870, 1645 (Ar, -C=C stretching, Bz), 1504 (C=N stretching), 810, 740. 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 3.84 (s, 3H, OCH3), 6.91 (d, 2H, J = 8.8 Hz, Ar-Bz-H 18,19), 7.33 (d, 1H, J = 15.6 Hz, Ar-Bz-H, 9), 7.44 (d, 2H, J = 8.4 Hz, Ar-Bz-H 21,22), 7.57 (d, 1H, J = 8.8 Hz, Ar-Bz-H 12,13), 7.75 (d, 2H, J = 15.6 Hz ArBz-H 10), 7.92 (d, 2H, J = 8.8 Hz Ar-Bz-H 15,16); mass: 627 (M + H+); elemental analysis of C30H30N10O2S2: C, 57.49; H, 4.82; N, 22.35. Found: C, 57.42; H, 4.80; N, 22.56.

6-((5-((3,5-Dimethyl-1-phenyl-4,5-dihydro-1Hpyrazol-4-yl)amino)-1,3,4-thiadiazol-2-yl)thio)-N2, N4-bis(4-nitrophenyl)-1,3,5-triazine-2,4-diamine 10f Brown crystals; yield: 91%; mp; 221–223 C; MW: 656.70; FT-IR (mmax per cm, KBr): 3390–3180 (N–H secondary), 1610 (Ar, -C=C stretching, Bz), 1508 (C=N stretching), 1435 (C-H stretching, methyl), 769. 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 1.92 (s, 3H, CH3), 1.18 (d, 3H, J = 6.86 CH3), 6.93 (d, 2H, J = 8.7 Hz, Ar-Bz-H 18,19), 7.31 (d, 1H, J = 15.4 Hz, Ar-Bz-H, 9), 7.42 (d, 2H, J = 8.1 Hz, Ar-BzH 21,22), 7.49 (d, 1H, J = 8.6 Hz, Ar-Bz-H 12,13), 7.68 (d, 2H, J = 15.7 Hz Ar-Bz-H 10), 7.82 (d, 2H, J = 8.7 Hz Ar-Bz-H 15,16); mass: 657 (M + H+). Elemental analysis of C28H24N12O4S2: C, 51.21; H, 3.68; N, 25.59. Found: C, 51.02; H, 3.70; N, 25.56.

Result and Discussion Chemistry The synthesis of title 3,5-dimethyl-1-phenyl-4,5-dihydro-1H-pyrazole4ylamino-1,3,4-thiadiazole-1,3,5-triazine derivatives 10(a–f) tethered via –S– bridge was accomplished by three different steps: First step corresponds to synthesis of 5-(3,5-dimethyl-1-phenyl-4,5-dihydro-1H-pyrazol-4-ylamino)-1,3,4-thiadiazole-2-thiol (6) by cyclo-condensation reaction in the presence of phenyl hydrazine from imino derivative, 3-(5-mercapto-1,3,4-thiadiazol-2-ylimino)pentane-2,4-dione (5). Compound 5 was obtained through the involvement of series of steps started from the synthesis of 5-amino-1,3,4-thiadiazole-2-thiol (3) by the reaction between thiosemicarbazide (1) and carbon disulfide (2) in the presence of alcoholic NaOH, in which first sodium hydrazothiocarbonamide dithiocarboxylate (NH2CSNHNHCSSNa) is formed and upon raising the temperature of the reaction, this sodium salt yields the new closed-ring system, compound 3 (17). Compound (3) was further subjected to diazotization to afford its diazonium salt (4) which was again allowed to react with acetyl acetone (active methylene) to generate imino derivative, 3-(5-mercapto-1,3,4-thiadiazol-2-ylimino)pentane-2,4-dione (5). The synthesis of 2,4-bis(substituted phenyl amine)-6-chloro-1,3,5-triazine derivatives was served as step 2. The synthesis was initiated by using 2,4,6-trichloro-1,3,5-triazine (7) through consecutive aromatic nucleophilic substitution (SNAr) reactions under controlled conditions by treating two equivalents of substituted amine derivatives 8(a–f) in the presence of NaHCO3 as activating base. The last step corresponds to the clubbing of 3,5-dimethyl-1-phenyl4,5-dihydro-1H-pyrazole-4ylamino-1,3,4-thiadiazole (6) with di-substituted monochloro-1,3,5-triazines 9(a–f) by means of the nucleophilic 601

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substitution (SNAr) reaction between SH of 1,3,4-thiadiazole (6) and monochlorine of di-substituted-1,3,5-triazines 9(a–f) in the presence of base under vigorous condition to yield title hybrid analogues 10 (a–f), Scheme 1 (Parts 1–3) (10). Both analytical and spectral data of all the compounds are found in full agreement with the proposed structures.

Antibacterial activity As depicted from the Table 1, entire set of target compounds 10(a–f) showed moderate to least activity against the tested Gram-positive and Gram-negative micro-organisms in comparison with cefixime as standard. Compounds with chloro-substitution on phenyl amine position (10a) connected to 1,3,5-triazine core exhibit significant activity against Pseudomonas aeruginosa (6.25 lg ⁄ mL), moderate activity against Bacillus cereus and Escherichia coli (12.5 lg ⁄ mL) and presented no activity at highest test concentration of 100 lg ⁄ mL against the Bacillus subtilis. Upon replacement of chloro to bromo (10b), no significant shift in activity was observed, except against B. cereus (100 lg ⁄ mL). However, considerable increase in the activity was observed in the case of compound 10c having fluoro as substitution against the E. coli, whereas no change in the activity for P. aeruginosa and no activity against the rest of the strains were revealed by the same test compound. Introduction of methyl group (10d) markedly results in decrease and increase in the activity for P. aeruginosa and B. cereus, respectively. Substantial to moderate activity was observed in the case of compound 10e having methoxy as substituent, toward P. aeruginosa and E. coli, respectively. However, substantial to significant activity was observed in the case of analogue having nitro as substituent (10f) for P. aeruginosa and E. coli, respectively. The resultant MIC value for the title compounds was found in good agreement with the results of zone of inhibition (Table 1). It was disclosed from the antibacterial screening that the presence of 5-(3,5-dimethyl-1-phenyl-4,5-dihydro-1H-pyrazol-4-ylamino)-1,3,4-thi-

adiazole-2-thiol was optimally tolerated as pendant substituent on 1,3,5-triazine core. The compounds having halogen atoms on fourth position of phenyl amine 10(a–c) showed prominent activities against P. aeruginosa and E. coli. However, no activity was reported against B. subtilis and B. cereus by the same test compounds. In next instance, replacement of halogen by non-halogen substituents causes a dramatic decrease in the activity for P. aeruginosa, and a further decrease was reported for the rest of strains. Though, it is noteworthy to consider here the effect of halogen atom on the modulation of antibacterial activity, which is further found in agreement with the study carried out by Singh et al. (10). These results suggest the necessity of electron-withdrawing group for the generation and escalation of antibacterial activity. However, ease of penetration of these molecules across the bacterial cell wall because of the modulation of the steric effect on phenyl ring could be acted as a plausible cause of variation in antibacterial profile.

Conclusion As a concluding remark, the present study demonstrates the utility of hybrid 1,3,4-thiadiazole-1,3,5-triazine as a potential lead for ongoing antimicrobial lead discovery program on the basis of considerable bioactivity, less reaction time, cheap, and facile synthesis. Additionally, it was confirmed that the presence of 5-(3,5-dimethyl1-phenyl-4,5-dihydro-1H-pyrazol-4-ylamino)-1,3,4-thiadiazole-2-thiol as a pendent substituent was optimally tolerated on 1,3,5-triazine core. Nevertheless, further studies are needed to obtain novel insight into their structure–activity relationship and confirmation of molecular mechanism through additional experiments. Our studies are in progress and reported subsequently in the future.

Antibacterial Screening Minimum inhibitory concentration All synthesized compounds were screened for their minimum inhibitory concentration (MIC, lg ⁄ mL) against selected Gram-positive

Scheme 1: Part 1, Synthesis of 5-(3,5-dimethyl-1-phenyl-4,5-dihydro-1H-pyrazol-4-ylamino)-1,3,4-thiadiazole-2-thiol (6). Reagents and conditions: (a) Ethanol, Na2CO3, reflux. (b) NaNO2 ⁄ HCl. (c) Acetyl acetone, CH3COONa, ethanol, stirring. (d) Phenyl hydrazine, CH3COONa; Part 2, Synthesis of di-substituted monochloro-1,3,5-triazines 9 (a–f). (e) NaHCO3, 40–45 C; Part 3, Synthesis of hybrid 3,5-dimethyl-1-phenyl-4,5-dihydro-1H-pyrazole-4ylamino-1,3,4-thiadiazole and 1,3,5-triazine derivatives tethered via –S– bridge 10 (a–f) f) Reflux, 120–135C, K2CO3. 602



Table 1: Antibacterial activity of hybrid 1,3,4-thiadiazole-1,3,5-triazine derivatives Minimum inhibitory concentration (lg ⁄ mL)

Zone of Inhibition (in mm)

Percentage of inhibition (in comparison with standard)

Pseudomonas Bacillus Bacillus Escherichia Pseudomonas Bacillus Bacillus Escherichia Pseudomonas Bacillus Bacillus Escherichia Compounds aeruginosa cereus subtilis coli aeruginosa cereus subtilis coli aeruginosa cereus subtilis coli 10a 10b 10c 10d 10e 10f Cefixime

6.25 6.25 6.25 12.5 12.5 12.5 3.125

12.5 100 100 50 100 100 6.25

100 100 100 100 100 100 6.25

12.5 12.5 6.25 6.25 50 6.25 3.125

11 14 12 15 10 13 20

organisms viz. Bacillus subtilis (NCIM-2063), B. cereus (NCIM-2156) and Gram-negative organism viz. Pseudomonas aeruginosa (NCIM2036), E. coli (NCIM-2065), and by the broth dilution method as recommended by the National Committee for Clinical Laboratory Standards (18) with minor modifications. Cefixime was used as standard antibacterial agent. Solutions of the test compounds and reference drug were prepared in dimethyl sulfoxide (DMSO) at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 lg ⁄ mL. Eight tubes were prepared in duplicate with the second set being used as MIC reference controls (16–24 h visual). After sample preparation, the controls were placed in a 37 C incubator and read for macroscopic growth (clear or turbid) the next day. Into each tube, 0.8 mL of nutrient broth was pipetted (tubes 2–7); tube 1 (negative control) received 1.0 mL of nutrient broth and tube 8 (positive control) received 0.9 mL of nutrient. Tube 1, the negative control, did not contain bacteria or antibiotic. The positive control, tube 8, received 0.9 mL of nutrient broth because it contained bacteria but not antibiotic. The test compounds were dissolved in DMSO (100 lg ⁄ mL); 0.1 mL of increasing concentration of the prepared test compounds is serially diluted from tube 2 to tube 7 from highest (100 lg ⁄ mL) to lowest (3.125 lg ⁄ mL) concentration (tubes 2–7 containing 100, 50, 25, 12.5, 6.25, 3.125 lg ⁄ mL). After this process, each tube was inoculated with 0.1 mL of the bacterial suspension whose concentration corresponded to 0.5 McFarland scale (9 · 108 cells ⁄ mL), and each bacterium was incubated at 37 C for 24 h at 150 rpm. The final volume in each tube was 1.0 mL. The incubation chamber was kept humid. At the end of the incubation period, MIC values were recorded as the lowest concentration of the substance that gave no visible turbidity, that is, no growth of inoculated bacteria.

8 10 10 11 6 12 20

1 4 2 1 3 3 20

8 3 6 5 2 1 18

58.3 69.8 62.0 77.4 50.6 68.4 100

41.6 50.3 51.7 56.0 33.3 60.5 100

6.5 25.5 13.6 9.3 19.2 21.4 100

50.7 21.1 36.6 30.4 13.0 6.0 100

agar plate is inoculated by streaking the swab over the entire sterile agar surface (19). The plates containing bacteria were inoculated a disk of cefixime (20 lg) and synthesized compound (20 lg), while the control plate was inoculated with DMSO which shows no inhibition of bacterial growth. Each disk must be pressed down to ensure complete contact with the agar surface. Then, the plates are inverted and placed in an incubator set to 35 C within 15 min after the disks are applied. They were then incubated at 37 C for 24 h, after which the inhibition halo was measured with a milimetric ruler (zone of inhibition). This qualitative screening was performed to verify positive antimicrobial activity of the synthesized compound. These results are further quantified in terms of percentage of inhibition in reference to cefixime as standard and results are shown in Table 1.

Acknowledgments Authors are gratified to SAIF, Central Drug Research Institute, Lucknow, India, for providing spectral data of compounds synthesized herein and SHIATS for providing basic facilities to carry out the project. One of the author, UPS, is also pleased to acknowledge Prof. Ray C. F. Jones, Professor of Organic and Biological Chemistry, Loughborough University, UK, for his critical discussion and suggestions on the chemistry of 1,3,5-triazines.

Conflict of Interest None.

Disk diffusion along with percentage of inhibition in comparison with standard The inoculum can be prepared by making a direct broth or saline suspension of isolated colonies of the same strain from 18- to 24-h Meller–Hinton agar plate. The suspension is adjusted to match the 0.5 McFarland turbidity standard, using saline and a vortex mixer. Optimally, within 15 min after adjusting the turbidity of the inoculum suspension, a sterile cotton swab is dipped into the adjusted suspension, and then afterward, the dried surface of an Chem Biol Drug Des 2012; 80: 598–604

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Notes a

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