Content area
The product was again washed with water and purified by recrystallization using ethanol.The compounds 8, 9 & 10 were also prepared using the same procedure by heating the mixture of triazole (2mol) and dicarboxylic acids like oxylic acid, succinic acid and tartaric acid respectively (1mol) for about 7 hrs. 7a25 IR (KBr) cm-1:3445 (NH2), 3069 (aromatic C-H str), 1640(NH bending), 1622 (C = N), 1583, 1556, 1508 (C = C ring str), 1402 (C - N), the peak at 1303 for C = S of parent triazole 6 is disappeared, 896 & 881(substituted phenyl &heteroaryl rings) 698 (C - S), 678 (C - Br), 547 (C - C1) 'HNMR: 8 4.20 (2H, S, NH2), 7.45 - 8.60 (4H, m, 2-aryl & 2-heteroaryl protons) ' Mass: m/z = 489 (m+). Majority of the compounds of this series appeared to be novel antibacterial & antifungal agents and active ones can be used as antibacterial & antifungal agents in various forms like capsules, tablets, power, ointment, lotions, creams etc after carrying out the toxicity studies. Acknowledgement The authors thanks to the Sri S. R. Reddy, Chairman NET Medical College & Research Centre, Raichur and Department of Microbiology, NET Medical College & Research Centre, Raichur for providing the facilities to carry out antibacterial & antifungal activity.The authors also express their thanks to the Department of Microbiology at the Maratha Mandal's Nathaji Rao, Dental College and Research Centre, Belgaum for their help in carrying out antitubercular and anticancer activity.
Abstract:
3-(2-amino-3,5-dibromophnyl) - 4- amino 5- mercapto - 1,2,4 - triazole (6) when condensed with substituted aryl/aryloxy/ hetero aryl acids in presence of pocl3 gave 3 -(2-amino-3,5 dibromophenyl)-6-substituted (3,4-b) (1,3,4) triazolothiadiazoles (7a1 - a25).The compound 6 when condensed with dicarboxylic acids like oxalic acid, succinic acid and tartaric acid in presence of pocl3 yielded 6,6 bis- 3-(2-amino-3,5 - dibromo phenyl) - 1,2,4 triazolo (3,4-b) (1,3,4) thia diazoles (8,9 & 10). The structures of the newly synthesized compounds have been established on the basis of their spectral data. The broad spectrum of activity exhibited by triazoles and their N-bridged analogues prompted us to evaluate their antibacterial, antifungal, antitubercular and anticancer activity. Few compounds showed significant antitubercular and anticancer activity. Majority of the compounds exhibited excellent antibacterial and antifungal activity.
Key Words: Triazolothiadiazole, antimicrobial, antitubercular, anticancer.
Introduction
Heterocyclic compounds containing 1,2,4-triazole and thiadiazole nucleus possess a diversity of useful biological effects. Further it was also reported that substituted 1,2,4 triazoles and their Nbridged heterocycles have received considerable attention during past two decades as they are endowed with variety of biological activities and have a wide range of therapeutic properties1-4. Various 3- substituted 4-amino 5-mercapto -1,2,4-triazoles have been studied extensively for the past several years because of their broad spectrum of biological activity and variety of medicinal applications5-8. The amino and mercapto groups of these compounds serve as readily accessible ncleophilic centres for the preparation of N-bridged heterocycles. The 1,3,4-thiadiazoles which display diverse biological activities, possible due to the presence of toxophoric>N-C-S moiety. The 1,2,4 triazolo-(3,4 -b) - 1,3,4 thiadiozole derivatives obtained by fusing biolabile 1,2,4trizole and 1,3,4-thiodiazole rings together are reported to possess antibacterial, antifungal, antitubercular, anti-inflammatory, analgesic, antiviral plant growth regulatory properties. Further it has been reported that many biological active natural and synthetic products have interesting molecular symmetry. Recently some bistriazole derivatives endowed with excellent biological activity have also been reported9-10. Prompted by the above facts and as part of our programme aimed at developing new biologically active compounds, we designed the synthesis and biological evaluation of series of novel 3-(2-amino-3,5- dibromo phenyl)-6-substituted (3,4b) (1, 3, 4) - triazole thiadiozoles (7a1 - a25) and 6,6 -bis-3(2- amino-3,5-dibromo phenyl)-1,2,4-triazolo (3,4b) (1, 3, 4) thiadiazoles8-11. (8,9 & 10) Apart from their chemical interest these compounds could also be a subject of studies as pharmacological agents.
Experimental:
Melting points were determined using open capillary tubes in paraffin oil bath Polmon Digital Melting point. Apparatus (model MP 96). Purity of the compounds was checked by TLC on precoated silica gel sheets obtained from Merck, Germany. Visualization of the spots on TLC plates was achieved either by exposure to Iodine vapour or UV light. Reaction complation and purity was monitored by TLC. IR spectra in KBr (cm-1) were recorded on Perkin Elmer Infrared spectrophotometer (Model Spectrum 100) with Nacl optics. Samples were scanned in KBr pellets.!HNMR spectra were recorded on BrukerAvance 300 MHz and 400 MHz instrument using TMS as internal standard (chemical shifts are expressed in 5 ppm). Mass spectra were recorded on Perkin Elmer PE SCIEX - API - 2000 Mass spectrophotometer.
The parent triazole 3-(2-amino-3,5 -dibromo phenyl)-4-amino5-mercapto 1,2,4 - triazole was prepared by 2 methods and comparative study of the 2 metods is carried out.
Method A
Preparation of 3, 5 - dibromoanthranilic acid hydrazide (3)
3,5-Dibromo methyl anthranilate (0.01 mol) in ethanol (30ml) and hydrazine hydrate (99%, 0.03 mol) was refluxed for 8 hrs. The mixture was cooled, the product obtained was filtered and was crystallised from ethanol (Yield 88%).
Preparation of potassium dithiocarbazinate (4)
A mixture of 3,5-dibromoanthranilic acid hydrazide (0.01mol), KOH (0.03mol) were added to anhydrous alcohol (70ml) and CS2 and was stirred for 12 hrs. The solid product was filtered, dried, washed with either and directly used for the preparation of triazole.
Preparation of 3- (2 -amino-3,5- dibromo phenyl)-4-amino5mercapto, 1, 2,4-triazole (6)
The mixture of dithiocarbazinate (4) and hydrazine hydrate (99%) in the ratio 1:3 was heated at 155 °c till H2S was evolved. The product was added to water and acidified with hydrochloric acid (25%). The required triazole (6) that is separated was purified by crystallization using ethanol (yield 70%, mp 78-80°C).
Method B:
The well triturated mixture of 3,5-dibromoanthranilic acid and thiocarbohydrazide in equimolar proportion was fused for 2 hrs. It was cooled, washed with sodium bicarbonate 5% solution, again washed with water and the dried compound was recrystallized from ethanol. [Yield 85%, mp 78°-80°].
6 IR (KBr) Vcm-1 : 3465, 3349 (NH2), 3074 (aromatic C-H str), 1612 (C=N), 1571, 1531 & 1451 (C=C ring str), 1303 (C=S), 879 (substituted phenyl ring), 690 (C-Br),'HNMR 53.04 (1H, S, SH), 3.65 - 3.89 (2H, S, NH2), 6.17 (2H, S, 2H of N - NH2), 7.94 - 9.02 (2H, m, Ar-H), mass m/z366 (m+), 368 (m+ + 2) other important fragment ions are at m/z= 320, 292, 278 (base peak), 252, 198, 149, etc.
Preparation of 3 (2-amino-3,5-dibromo phenyl)-6substituted (3,4b) (1,3,4) - triazolethiadiazoles (7a1 - a25) and 6, 6 bis-3 (2-amino-3,5- dibromo phenyl) -1,2,4-triazolo (3,4b) (1,3,4) thiadiazoles (8, 9 and 10)
Equimolar mixture of triazole (6) and carboxylic acid in POCl3 (10ml) was heated for 7 hrs. Excess of POCl3 was removed by distillation under vacuum. The product obtained was dissolved in water and treated with sodium bicarbonate solution (5%) to remove unreacted acid. The product was again washed with water and purified by recrystallization using ethanol.The compounds 8, 9 & 10 were also prepared using the same procedure by heating the mixture of triazole (2mol) and dicarboxylic acids like oxylic acid, succinic acid and tartaric acid respectively (1mol) for about 7 hrs. The purity of the compounds was established by TLC silcagel G plates using n-hexane and ethyl acetate (1:1) as elute and observed in UV light the compounds thus prepared are listed in table - 1.
7a2 IR, (KBr), cm-1: 3467 and 3354 (NH2), 3070 (aromatic C-H str). 1640 (NH bending of NH2), 1616 (C=N), 1593, 1567, 1525 & 1488, (C=C ring str), 1402 (C-N), 1280 (N-N=C). The peak at 1303 for C=S of parent triazole 6 is disappeared , 875 and 843 (substituted phenyl rings), 680 (C-S), 656 (C m- Br), 556 (CC1),'HNMR 83.87 (2H, s, NH2), 7B - 9.01 (6H, m, Ar - H), mass : m/2 488 (mt)
7a4 IR (KBr) cm-1: 3460 (NH2), 3092 aromatic C - H str, 1640 (N - H bending of NH2), 1624 (C = N), 1590, 1580, 1541 (C = C ring str), 1541 and 1345 (NO2), 1437 (C - N), the peak at 1303 for C = S of parent triazole 6 is disappeared, 1280 (N - N = C), 895 & 881 (substituted phynl rings), 680 (C - S), 656 (C - Br),556 (C - C1). 'HNMR: 8 3.87 (2H, S, NH2) 7.13 - 8.92 (6H, m, Ar - H), mass : m/z 543 (m+).
7a5 IR (KBr), cm-1: 3420 (NH2), 3068 (aromatic C - H str), 2953 and 2867 C - H str of -CH3, 2929 & 2830 C - Hstr of >C H2, 1596, 1556, 1508 &1463 (C = C ring str), 1314 (C - N), 1278 (>N - N = C).The peak at 1303 for C =S of parent triazole 6 is disappeared, 870, 848 (substituted phenyl rings) 595 (C - Br), 'HNMR : 8 0.89 - 0.92 (6H, d, 2XCH3 of -CH (CH3)2), 1.56 - 1.66 (3H, d, CH3 of (CH-CH3), 1.81-1.94 (1H, m CH of - CH (CH3)2, 2.31 - 2.5 (2H, S, CH2 of CH2 - CH (CH3)2, 3.19 -3.9 (1H, m, CH of CH - CH3), 4.1 (2H, S, NH2), 7.14 - 8.5 (6H, m, Ar-H), mass : m/z 535 (m+) & 537 (m++2)
7a6 IR (KBr) cm-1: 3471& 3468 (NH2), 3071 (aromatic C-H str), 1602 (C = N and C = C), 1362 (C - N), 1277, (> N - N = C), the peak at 1303 for C = S of parent triazole 6 is disappeared, 871 substituted phenyl ring, 682 (C-S), 666 (C- Br), 'HNMR : 8 4.20 (4H, S, 2X NH2),7.12 - 8.70 C(H, m, Ar - H), mass m/z 626 (m+) & 628 (m++2).
7a9 IR (KBr) cm-1: 3366 (NH2), 3063 (aromatic C-H str), 1596 (C=N), 1584, 1563, 1495 (C=C ring str), 1371 (C-N), 1276 (> N - N=C), the peak at 1303 for C=S of parent triazole 6 is disappeared. 874 & 724 (substituted phenyl rings), 699 (C-S), 630 (C-Br),'HNMR: 8 4.25 (2H, S, NH2), 3.80 (1H, S, >CH) 7.00 - 8.52 (12H, m, Ar-H). Mass: m/z 543 (m+).
7a10 IR (KBr) cm-1: 3473 &3371 (NH2), 3243 (NH), 3073 (aromatic C-H str), 2950 & 2843 (C - H str of CH3 asymmetric and symmetric, 1640 (NH-bending) 1613 (C = N), 1575, 1507, 1457 (C= Cring str), 1433 & 1366 (C-H bending of CH3 asymmetric and symmetric). The peak at 1303 for C=S of parent triazole 6 is disappeared, 1277 (>N-N=C), 875 & 725 (substituted phenyl rings), 680 (C-S), 608 (C-Br), 'HNMR:82.33 - 2.35 (6H, S, 2X CH3), 4.98 - 5.30 (2H, bb, NH2) 7.06 - 8.41 (9H, m, Ar - H), 9.14 (1H, S, NH), Mass, m/z572 (m+), other important fragment ion peaks are observed at m/z294, 219, 131, 296.8 & 78.7.
7a11 IR (KBr) cm-1: 3472, 3370 (NH2), 3073 (aromatic C - H str), 1599 (C=N), 1599, 1572, 1532, 1508 (C = C ring str), 1532 & 1348 (NO2), the peak at 1303 for C=S of parent triazole 6 is disappeared, 1277 (N-N=C), 870, 787 (substituted phenyl rings), 681 (C-S), 616 (C-Br),'HNMR : 8 3.86 (2H, S, NH2), mass : m/z513 (m+). Other important fragment ion peaks are observed at m/z 304, 149 & 105.
7a19 IR (KBr) cm-1: 3445 (NH2), 3070 (aromatic C-H str), 2950 & 2858 (C-H str of CH3 and OCH2), 1607 (C=N), 1586, 1555, 1508 (C =C ring str), 1435 and 1340 (C-H bending of CH3 & OCH2) 1308 (C-N), the peak at 1303 for C=S of parent triazole 6 is disappeared. 1121 (C-O-C), 875 & 816 (substituted phenyl rings) 659 (C-Br)? 'HNMR: 8 1.60( 3H, S, CH3), 3.85 (2H, S, OCH2), 4.25 (2H, S, NH2), 7.20 - 8.55 (6H, m, Ar-H) Mass: m/z497 (m+). The other important fragment ion peaks are observed at m/z 376, 138, 97, 89 & 59.
7a21 IR (KBr) cm-1: 3472 (NH2), 3073 (aromatic C-H str), 2947 & 2851 (C-H str of OCH2 asymmetric and symmetric), 1638 (NH bending), 1613 (C=N), 1592, 1558 1507 (C=C ring str), 1558 & 1341 (NO2), 1457 & 1341 (C-H bending of OCH2 asymmetric and symmetric), 1277 (N -N = C), the peak at 1303 for C = S of parent triazole 6 is disappeared, 1110 (C-O-C), 869 & 824 (substituted phenyl rings) , 705 (C-S), 680 (C-Br), 'HNMR : 8 3.80 (2H, S, OCH2) 4.30 (2H, S, NH2), 7.20 - 8.50 (6H, m, Ar-H)? Mass: m/z 524 (m+), the other important fragment ion peaks are observed at m/z 339, 218, 138, 97, 89 & 59.
7a25 IR (KBr) cm-1:3445 (NH2), 3069 (aromatic C-H str), 1640(NH bending), 1622 (C = N), 1583, 1556, 1508 (C = C ring str), 1402 (C - N), the peak at 1303 for C = S of parent triazole 6 is disappeared, 896 & 881(substituted phenyl &heteroaryl rings) 698 (C - S), 678 (C - Br), 547 (C - C1) 'HNMR: 8 4.20 (2H, S, NH2), 7.45 - 8.60 (4H, m, 2-aryl & 2-heteroaryl protons) ' Mass: m/z = 489 (m+).
Biological activities.
Antibacterial & antifungal activities,
The invitro antibacterial & antifungal activities of the synthesized compounds 7a1 - a2s, 8, 9 & 10 was carried out by well diffusion method by punching template on Mullar Hinton agar well size of 6mm was made on agar with a holding capacity of 50 pl. Three standard bacterial strainsviz E.coli(ATC NO 8739), P.aeruginosa (ATCC NO 9027) and S.aureus (ATCC NO 6538) were used for this purpose. Inacolum size of these standard strains were matched with 0.5 Mac Farlands comparator to get 1.5 X 105organisms/ml. Lawn culture is made on mullar Hinton agar plate with standard strain. Known quantity of each sample was dissolved in 1000ml. DMSO solvent in a sterile screw capped Bijou bottle. 50 ml of solvent dissolved sample was charged into the wells of inoculated mullar Hinton Agar Incubation of plates was done for 12 hrs at 37°c & later looked for Zone of inhibition around the well. The diameter of inhibitory zone was measured and recorded in millimeter and concentration of test and standard were taken at 10 mg/ml.
The compounds were also screened for their antifungal activity by preparing the solution of compounds in DMSO solvent of concentrations 75, 50, 25 & 10pg/ml. the selected few compounds of having significant activity were further screened for their antifungal activity following minimum inhibition concentration MIC procedure against the organism. Afumigatus ATCC NO 13073 using fluconazole as a reference standard.9 dilutions of each drug were prepared (with brain heart infusion) BHI for MIC. In the initial tube 20 pl of drug was added in to the 380pl of BHI broth for dilutions 200 pl of BHI broth was added in to the next 9 tubes separately. Then from the initial tube 200pl was transferred to the first tube containing 20pl of BHI broth. This was considered as 10-1 dilution. From 10-1 diluted tube 200pl was transferred to second tube to make 10-2dilution.The serial dilution was repeated up to 10-9 dilution for each drug. From the maintained stock cultures of required organisms, 5pl was taken and added into 2ml of BHI broth. In each serially diluted tube 200pl of above culture suspension was added. The tubes were incubated for 24 hrs and observed for turbidity.
Among the triazole derivatives screened for antibacterial activity,it was observed that majority of triazole derivatives exhibited moderate to excellent activity against the organism E.Coli in comparison with the standard drugs Ciprofloxacin, Gentamycin & Tobramycin. It was found that the compounds 7a1, 7a5, 7a12, 7a13, 7a15, 7a16, 7a20& 7a24 have exhibited significant activity in comparision with the standard Ciprofloxacin.
It was observed in general that triazolethiadiazole derivatives with the substituents at 6th position like 2 - amino - 3, 4 - dibromo phenyl, 4 - isobutyl phenyl ethyl , 2 - chlorophenyl, 2, 4 - dichlorophenyl methyl & a bistriazolothiadiazole derivative bearing CHOH showed much significant activity. Perhaps the electron with drawing group's at 6th position contributes in enhancing the antibacterial activity.
Antifungal screening result indicates that majority of the triazolothiadiazoles except 7a8, 7a18, 7a19, & 09 possess significant activity. The presence of N - C - S linkage in the fused system may be responsible for better antifungal activity. The antifungal activity of triazolothiadiazole derivatives is much beeter than bis derivatives (8, 9 & 10). MIC result also indicates that the representative compounds of the series tested exhibit significant antifungal activity even at lower concentration. The compounds 7a1, 7a3, 7a5, 7a6, 7a7, 7a9, 7a14, 7a15& 7a16 showed activity even at 8pl/ml. the compounds 7a3, 7a5& 7a7 showed activity at 4pg/ml.
Majority of the compounds of this series appeared to be novel antibacterial & antifungal agents and active ones can be used as antibacterial & antifungal agents in various forms like capsules, tablets, power, ointment, lotions, creams etc after carrying out the toxicity studies.
Antitubercular activity14
The evaluation of antitubercular activity was performed using the standard strain mycobacterium tuberculosis H37 RV & middlebrook 7H - 9 broths following the standard procedure. The growth of mycobacterium tuberculosis strain (100000 organisms/ml) was measured after a period of 3 weeks Streptomycin & Pyrazinamide were used as standards.
The parent triazole (6) and its few representative triazolothiadiazole derivatives 7a1, 7a3, 7a11, 7a15, 7a16, 7a19, 7a21, 7a22 & 7a24 were screened against mycobacterium tuberculosis H37 RV at (Concentrations 5, 10 & 25 pg/ml. The compound 7a24 should equipotent activity in comparison with standard drugs at all the concentrations. The compounds 6, 7a1, 7a11, 7a19, & 7a22 showed activity at 10 & 25pg/ml concentrations. Perhaps the heterocyclic moiety as substituent at 6th position of triazolo thiadiazole ring system may be responsible for antitubercular property.
Acknowledgement
The authors thanks to the Sri S. R. Reddy, Chairman NET Medical College & Research Centre, Raichur and Department of Microbiology, NET Medical College & Research Centre, Raichur for providing the facilities to carry out antibacterial & antifungal activity.The authors also express their thanks to the Department of Microbiology at the Maratha Mandal's Nathaji Rao, Dental College and Research Centre, Belgaum for their help in carrying out antitubercular and anticancer activity.
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