Research Journal of Chemical Sciences ISSN 2231-606X Synthesis, Characterization and Antimicrobial screening of some Azo compounds derived from Ethyl vanillin Abstract Pagariya S.K. *, Pathade R.M. and Bodkhe P.S. Department of Chemistry, Vidyabharati Mahavidyalaya, Amravati, 444602, INDIA Available online at: www.isca.in, www.isca.me Received 19 th June 2015, revised 30 th June 2015, accepted 17 th July 2015 Azo compounds were synthesized in excellent yield by diazotization of some substituted aromatic amines using NaNO 2 and concentrated HCl followed by coupling with ethyl vanillin in alkaline medium. These azo compounds were characterized by FTIR and H 1 NMR spectroscopic technique and have been tested against the growth of five gram positive and negative microorganisms in order to assess their antimicrobial activity. Keywords: Azo compounds, ethyl vanillin, diazotization, antimicrobial activity. Introduction Azo compounds are mostly used as dyes due to its various applications in the fields such as textile fibres, colouring of different materials, biomedical studies and organic synthesis 1-2. The azo dyes containing azo linkages have advanced applications in high technology areas like lasers 3, LCD color filters 4. In addition to this, azo dyes were reported to have variety of biological applications like antineoplastics, antidiabetics, antiseptics, anti-inflammatory and other useful chemotherapeutic agents 5-8. Scarlet red and diamazon are the most commonly used azo dyes which are antiseptics. Several azo compounds derived from thymol 9, aspirin 10, paracetamol 11, m-cresol 12, resorcinol 13 and vanillin 14 moieties have been frequently reported and exhibit excellent biological properties. In the present work, we have synthesized four azo compounds derived from ethyl vanillin and characterized by FTIR and H 1 NMR spectral technique. The antimicrobial potential of synthesized azo compounds of ethyl vanillin has been tested against the growth of five gram positive and negative microorganisms using agar well diffusion method. Material and Methods In the present synthesis, chemicals and reagents used were of analytical grade, Merck and Alfa Aesar Company Ltd. The azo compounds were characterized by FTIR and H 1 NMR spectroscopic techniques. The Perkin-Elmer spectrum One FTIR instrument was used for characterization of IR spectra in the form of KBr pallet. Bruker Avance II 400 MHz NMR spectrometer was used for characterization of H 1 NMR spectra of azo compounds using CDCl 3 as a solvent and TMS as an internal standard. The purity of all the compounds was checked by thin layer chromatography and were recrystalised from hot ethanol. The melting points were measured by open capillary method and they are uncorrected. Procedure for the synthesis of azo compounds 9-10: Aromatic amines (0.01 mole) were mixed with 2.5 ml of concentrated HCl. To this resultant solution, 25 gm of crushed ice and 2.5 ml of NaNO 2 (4N) was added with constant stirring. The temperature of the reaction mixture was kept constant up to 0-5 0 C. The diazonium salt solution prepared was added to the alkaline solution of ethyl vanillin drop by drop with constant stirring for 10-25 minutes at constant temperature of 5-10 0 C. The colored precipitate given out was filtered and washed with water number of times. The colored product was recrystallised from hot ethanol. The general reaction scheme for synthesis of azo compounds of ethyl vanillin is shown in figure-1. Antimicrobial Activity: The azo compounds 1a-d were analysed for their antibacterial activity against five gram positive and negative pathogens viz. Escherichia coli, Pseudomonas aeroginosa, Salmonella typhi, Bacillus subtilis and Staphylococcus aureus by using agar well diffusion method 15-16. These compounds were mixed in DMSO to form solutions of concentration 1mg/ml. Sterile discs were dipped in this solutions, dried it and placed on nutrient agar plates spreaded with the bacteria. The plates were further incubated for 24 to 48 hours at 37 0 C and the diameters of zones of inhibition were measured in millimeter. Results and Discussion The data of synthesized azo compounds of ethyl vanillin (i.e. symbols, compounds name, molecular formulae, molecular weights, melting points and percentage yield) are given in table- 1. The FTIR and H 1 NMR spectroscopic data of synthesized azo compounds are illustrated in table-2 and are shown in figure-6 to 13. A total four azo compounds of ethyl vanillin have been synthesized, recrystalised and used separately to study its antimicrobial activity against five gram positive and negative microorganism s viz. Escherichia coli, Pseudomonas aeroginosa, Salmonella typhi, Bacillus subtilis and International Science Congress Association 20
Staphylococcus aureus. The data on antimicrobial activity of azo compounds of ethyl vanillin 1a-d against five pathogens are presented in table-3. From the results it was observed that the azo compounds of ethyl vanillin have showed miraculous antibacterial potential against all five pathogens. The compound 1a showed 10.4 and 9.6 mm zones of inhibition against the test pathogens Escherichia coli and Staphylococcus aureus respectively as shown in figure-2 and do not showed any inhibitory action against Salmonella typhi, Pseudomonas aeroginosa and Bacillus subtilis. The azo compound 1b showed 10.6, 12.7, 11.8 and 9.8 mm zones of inhibition against the test pathogens Escherichia coli, Salmonella typhi, Bacillus subtilis and Staphylococcus aureus respectively shown in figure-3 but do not showed any inhibitory action against Pseudomonas aeroginosa. The azo compound 1c showed 10.3, 13.1, 13.5 and 12.8 mm zones of inhibition against the pathogens Escherichia coli, Salmonella typhi, Bacillus subtilis and Staphylococcus aureus respectively as shown in figure-4 but do not showed any inhibitory action against Pseudomonas aeroginosa. The azo compound 1d showed 13.6, 12.8, 15.6 and 14.4 mm zones of inhibition against the test pathogens Escherichia coli, Salmonella typhi, Pseudomonas aeroginosa and Staphylococcus aureus as shown in figure-5. Compound 1d showed 11.3 mm zone of inhibition against the pathogen Bacillus subtilis. OH C 2 H 5 O NH 2 N=N Cl OH CHO C 2 H 5 O N=N Ar R NaNO 2 +HCl 0-5 o C R 2 5-10 o C 1 CHO 1a-d O 2 N Ar = a b c d Figure-1 The general reaction scheme for synthesis of azo compounds of ethyl vanillin Table-1 The symbols, compounds name, molecular formulae, molecular weights, melting points and percentage yield of synthesized azo compounds of ethyl vanillin Symbols for antimicrobial activity 1a 1b 1c 1d Symbols for FTIR and H 1 NMR 4P 4Q 4R 4S Compounds Name CH 3 3-ethoxy-4-hydroxy-5- (phenyldiazenyl)benzaldehyde 3-ethoxy-4-hydroxy-5-((2- nitrophenyl)diazenyl)benzaldehyde 3-ethoxy-4-hydroxy-5-(ptolyldiazenyl)benzaldehyde 3-ethoxy-4-hydroxy-5-(napthalen-1- yldiazenyl)benzaldehyde Molecular Formulae Molecular Weights Melting points ( 0 C) Yield (%) C 15 H 14 N 2 O 3 270.28 67 67 C 15 H 13 N 3 O 5 315.28 128-130 65 C 16 H 16 N 2 O 3 284.31 64 72 C 19 H 16 N 2 O 3 320.34 127-129 80 International Science Congress Association 21
Symbols for compounds 4P 4Q 4R 4S Table-2 FTIR and H 1 NMR data of synthesized azo compounds of ethyl vanillin Types of spectra FTIR (KBr,cm -1 ) H 1 NMR (δ ppm) FTIR (KBr,cm -1 ) H 1 NMR (δ ppm) FTIR (KBr,cm -1 ) H 1 NMR (δ ppm) FTIR (KBr,cm -1 ) H 1 NMR (δ ppm) FTIR and H 1 NMR spectral data 3362 (OH of Phenol), 2829 (C-H of H-C=O), 1686 (C=O), 1516 (C=C), 1255 (C-O), 1280 (C-N), 1579 (N=N). 1.4 (t, 3H of -CH 3 ), 4.2 (q, 2H of -OCH 2 ), 6.2 (s, 1H of Phenolic -OH), 9.8 (s, 1H of - CHO), 7.0 to 7.5 (m, 7H of Aromatic-H). 3398 (OH of Phenol), 2882 (C-H of H-C=O), 1673 (C=O), 1519 (C=C), 1262 (C-O), 1232 (C-N), 1590 (N=N). 1.5 (t, 3H of -CH 3 ), 4.4 (q, 2H of -OCH 2 ), 4.2 (s, 1H of Phenolic -OH), 9.8 (s, 1H of - CHO), 7.0 to 8.5 (m, 6H of Aromatic-H), 9.9 due to moisture. 3363 (OH of Phenol), 2828 (C-H of H-C=O), 1681 (C=O), 1514 (C=C), 1281 (C-O), 1168 (C-N), 1580 (N=N). 1.5 (t, 3H of -CH 3 ), 4.3 (q, 2H of -OCH 2 ), 6.3 (s, 1H of Phenolic -OH), 9.8 (s, 1H of - CHO), 7.0 to 7.4 (m, 6H of Aromatic-H). 3333 (OH of Phenol), 2842 (C-H of H-C=O), 1682 (C=O), 1515 (C=C), 1261 (C-O), 1231 (C-N), 1592 (N=N). 1.5 (t, 3H of -CH 3 ), 4.1 (q, 2H of -OCH 2 ), 4.3 (s, 1H of Phenolic -OH), 9.8 (s, 1H of - CHO), 7.0 to 8.5 (m, 9H of Aromatic-H). Table-3 Antimicrobial activities of the synthesized azo compounds of ethyl vanillin Microbial species and diameter of zone of inhibition in mm Escherichia coli Salmonella typhi Pseudomonas aeroginosa Bacillus subtilis 1a 10.4 No inhibition No inhibition No inhibition 9.6 1b 10.6 12.7 No inhibition 11.8 9.8 1c 10.3 13.1 No inhibition 13.5 12.8 1d 13.6 12.8 15.6 11.3 14.4 Symbols for compounds Staphylococcus aureus E. coli S. aureus Figure-2 Zone of inhibition of azo compound 1a E. coli S. typhi B. subtilis S. aureus Figure-3 Zone of inhibition of azo compound 1b International Science Congress Association 22
E. coli S. typhi B. subtilis S.aureus Figure-4 Zone of inhibition of azo compound 1c E. coli S. typhi P. aeroginosa S.aureus Figure-5 Zone of inhibition of azo compound 1d 55.4 %T 50 45 40 35 30 25 20 15 10 5 0.0 3729,53 3362,13 2681,40 2539,42 2887,34 2722,35 2797,32 2979,30 2934,29 2829,29 4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0 cm-1 Pagariya-21.sp - 4/21/2014-4P 2331,45 2097,49 1908,49 1782,52 1604,18 1686,7 1674,8 1579,5 1516,7 1477,29 1380,27 1398,26 1439,17 Figure-6 FTIR spectrum of azo compound 1a 1255,9 1280,4 1197,14 1168,4 1115,10 1104,10 1041,15 970,23 890,37 852,27 802,27 734,40 786,21 839,20 585,28 653,27 524,26 633,25 416,47 International Science Congress Association 23
65.4 60 2757,60 55 3398,57 2882,59 2926,58 2843,58 2982,56 530,54 %T 50 45 40 1590,41 1335,48 1041,48 1126,43 1232,42 1441,41 1308,41 590,51 851,45 639,45 878,45 829,45 742,42 35 1673,37 1519,38 31.5 4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0 cm-1 Pagariya-22.sp - 4/21/2014-4Q Figure-7 FTIR spectrum of azo compound 1b 1262,34 33.5 32 30 28 3778,31 415,30 26 2096,27 1907,28 24 2321,24 22 2540,23 %T 20 18 16 14 12 10 8 7.0 3363,17 2887,20 2980,20 2724,20 2934,19 2828,19 2796,20 4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0 cm-1 S K Pagariya-1.sp - 5/12/2015-4R 1681,11 1514,11 1580,11 1396,17 1382,17 1439,14 Figure-8 FTIR spectrum of azo compound 1c 1257,11 1281,10 1168,10 1107,12 1040,13 969,16 889,21 801,18 783,16 837,16 633,16 522,19 584,18 International Science Congress Association 24
18.7 18 16 2351,16 2007,18 1938,18 1871,17 14 3333,14 424,14 12 3054,12 2758,13 %T 10 2882,12 2927,11 2842,11 489,11 2981,10 8 6 4 1165,7 1487,6 1592,5 1515,6 1401,6 1231,5 1442,5 1308,5 1136,5 1283,5 1261,4 1682,4 1041,8 1003,8 878,6 638,6 860,6 741,6 828,6 716,5 795,6 767,5 545,9 574,8 590,8 2.0 4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0 cm-1 S K Pagariya-2.sp - 5/12/2015-4S Figure-9 FTIR spectrum of azo compound 1d 4P 9.8170 7.4245 7.4200 7.4013 7.2680 7.0538 7.0335 6.3361 4.2260 4.2085 4.1910 4.1735 1.4971 1.4797 1.4622 0.0000 BRUKER AVANCE II 400 NMR Spectrometer SAIF Panjab University Chandigarh Current Data Parameters NAME May13-2014-Administrator EXPNO 50 PROCNO 1 F2 - Acquisition Parameters Date_ 20140513 Time 17.32 INSTRUM spect PROBHD 5 mm PABBO BB- PULPROG zg30 TD 65536 SOLVENT CDCl3 NS 8 DS 2 SWH 12019.230 Hz FIDRES 0.183399 Hz AQ 2.7263477 sec RG 287 DW 41.600 usec DE 6.00 usec TE 296.7 K D1 1.00000000 sec TD0 1 ======== CHANNEL f1 ======== NUC1 1H P1 10.90 usec PL1-3.00 db SFO1 400.1324710 MHz F2 - Processing parameters SI 32768 SF 400.1300064 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 1.00 2.06 1.01 1.04 2.20 3.35 13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm Figure-10 H 1 NMR spectrum of azo compound 1a avtar_saifpu@yahoo.co.in International Science Congress Association 25
4Q 14.5427 13.7555 9.9354 9.8171 8.1637 8.1306 8.0701 8.0542 8.0220 7.7855 7.7311 7.7180 7.6995 7.6724 7.6634 7.6590 7.6450 7.6299 7.5760 7.5574 7.5474 7.5176 7.5105 7.4673 7.4470 7.4318 7.4232 7.4190 7.3995 7.2635 7.0520 7.0325 4.2310 4.2137 4.1963 4.1831 4.1787 4.1662 4.1092 4.0919 1.6380 1.6176 1.5321 1.5091 1.5016 1.4916 1.4841 1.4743 1.4666 1.4019 1.2542 1.2431 BRUKER AVANCE II 400 NMR Spectrometer SAIF Panjab University Chandigarh Current Data Parameters NAME May13-2014-Administrator EXPNO 60 PROCNO 1 F2 - Acquisition Parameters Date_ 20140513 Time 17.37 INSTRUM spect PROBHD 5 mm PABBO BB- PULPROG zg30 TD 65536 SOLVENT CDCl3 NS 8 DS 2 SWH 12019.230 Hz FIDRES 0.183399 Hz AQ 2.7263477 sec RG 912 DW 41.600 usec DE 6.00 usec TE 296.7 K D1 1.00000000 sec TD0 1 ======== CHANNEL f1 ======== NUC1 1H P1 10.90 usec PL1-3.00 db SFO1 400.1324710 MHz F2 - Processing parameters SI 32768 SF 400.1300083 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 0.36 0.38 1.05 0.56 4.50 5.47 3.45 1.00 0.68 3.57 1.04 2.16 6.97 2.21 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm Figure-11 H 1 NMR spectrum of azo compound 1b avtar_saifpu@yahoo.co.in 4R 9.8151 7.4239 7.4196 7.4008 7.2702 7.0528 7.0331 6.3759 4.2226 4.2052 4.1877 4.1702 1.4945 1.4770 1.4595 0.0000 BRUKER AVANCE II 400 NMR Spectrometer SAIF Panjab University Chandigarh Current Data Parameters NAME May13-2014-Administrator EXPNO 70 PROCNO 1 F2 - Acquisition Parameters Date_ 20140513 Time 17.42 INSTRUM spect PROBHD 5 mm PABBO BB- PULPROG zg30 TD 65536 SOLVENT CDCl3 NS 8 DS 2 SWH 12019.230 Hz FIDRES 0.183399 Hz AQ 2.7263477 sec RG 512 DW 41.600 usec DE 6.00 usec TE 296.8 K D1 1.00000000 sec TD0 1 ======== CHANNEL f1 ======== NUC1 1H P1 10.90 usec PL1-3.00 db SFO1 400.1324710 MHz F2 - Processing parameters SI 32768 SF 400.1300055 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 1.01 2.03 1.07 1.00 2.15 3.31 13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm Figure-12 H 1 NMR spectrum of azo compound 1c avtar_saifpu@yahoo.co.in International Science Congress Association 26
4S 14.8291 9.9637 9.8137 8.5623 8.5038 8.4831 8.4134 8.1390 8.1352 8.0617 8.0498 8.0430 8.0301 7.9963 7.9716 7.9517 7.9336 7.8555 7.7797 7.7743 7.7272 7.6810 7.6640 7.6403 7.6355 7.6197 7.6001 7.5764 7.5508 7.5420 7.5276 7.5020 7.4987 7.4127 7.3936 7.2588 7.0480 7.0285 4.3539 4.3362 4.2870 4.2766 4.2592 4.2417 4.2239 4.2044 4.1869 4.1694 1.6524 1.6350 1.6178 1.5899 1.5725 1.5551 1.5283 1.5105 BRUKER AVANCE II 400 NMR Spectrometer SAIF Panjab University Chandigarh Current Data Parameters NAME May13-2014-Administrator EXPNO 80 PROCNO 1 F2 - Acquisition Parameters Date_ 20140513 Time 17.49 INSTRUM spect PROBHD 5 mm PABBO BB- PULPROG zg30 TD 65536 SOLVENT CDCl3 NS 8 DS 2 SWH 12019.230 Hz FIDRES 0.183399 Hz AQ 2.7263477 sec RG 812 DW 41.600 usec DE 6.00 usec TE 297.0 K D1 1.00000000 sec TD0 1 ======== CHANNEL f1 ======== NUC1 1H P1 10.90 usec PL1-3.00 db SFO1 400.1324710 MHz F2 - Processing parameters SI 32768 SF 400.1300103 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 1.01 0.98 1.00 1.17 2.32 1.99 4.88 1.55 0.81 0.70 0.49 2.94 4.64 1.30 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm Figure-13 H 1 NMR spectrum of azo compound 1d avtar_saifpu@yahoo.co.in Conclusion The current investigation reveals that, there was a miraculous inhibition of compound 1d shows antibacterial activity against all the tested organisms while the compound 1a was found to be most resistant by the pathogens. The highest zone of inhibition was obtained for 1d against Pseudomonas aeroginosa (15.6 mm) while lowest zone was recorded for 1a against Staphylococcus aureus (9.6 mm). The compounds 1b and 1c shows moderate inhibition of growth against tested pathogens. References 1. Towns A.D., Developments in azo disperse dyes derived from heterocyclic diazo compounds, Dyes and Pigments, 42, 3-28 (1999) 2. Zollinger H., Synthesis, properties and applications of organic dyes and pigments, Color Chemistry, third revised edition,wiley-vch.,weinheim (2003) 3. Gayatri C. and Ramalingam A.,Z-scan determination of the third order optical nonlinerities of an azo dye using dipole: pumped Nd:Yag laser, Optik, 119(9), 409-414 (2008) 4. Sakong C., Kim Y. D., Choi J. H., Yoon C. and Kim J.P., The synthesis of thermally stable red dyes for LCD color filters and analysis of their aggregation and spectral properties, Dyes and Pigments, 88(2),166-173 (2011) 5. Bae J.S., Freeman H.S. and El-Shafei A., Metallization of non-genotoxic direct dyes, Dyes and Pigments, 57(2), 121-129 (2003) 6. Sanjay F.T., Dinesh M.P., Manish P.P. and Ranjan G.P., Synthesis and antibacterial activity of novel pyraazolo [3,4-b] quinoline base heterocyclic azo compounds and their dyeing performance, Saudi Pharm. Journal, 15(1), 48-54 (2007) 7. Child R.G., Wilkinson R.G. and Tomcu-Fucik A., Effect of substrate orientation of adhesion of polymer joints, Chem. Abstr., 87, 6031 (1977) 8. Garg H.G. and Prakash C.J., Preparation of 4-arylazo- 3,5-disubstituted-(2H)-1,2,6-thiadizine1,1-dioxides, Med. Chem.,15(4), 435-436 (1972) 9. Koshti S.M., Sonar J.P., Sonawane A.E., Pawar Y.A., Nagle P.S., Mahulikar P.P. and More D.H., Synthesis of azo compounds containing thymol moiety, Indian J. Chem., 47B, 329-331 (2008) 10. Pathan R.U. and Borul S.B., Synthesis and antimicrobial activity of azo compounds containing drug moiety, Oriental J. Chem., 24(3), 1147-1148 (2008) International Science Congress Association 27
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