Synthesis and characterization of novel ...

Synthesis and characterization of novel benzohydrazide as potential antibacterial agents from natural product vanillin and wintergreen oil Amri Setyawati, Tutik Dwi Wahyuningsih, and Bambang Purwono

Citation: AIP Conference Proceedings 1823, 020121 (2017); doi: 10.1063/1.4978194 View online: http://dx.doi.org/10.1063/1.4978194 View Table of Contents: http://aip.scitation.org/toc/apc/1823/1 Published by the American Institute of Physics

Synthesis and Characterization of Novel Benzohydrazide as Potential Antibacterial Agents from Natural Product Vanillin and Wintergreen Oil 2

Amri Setyawati1a Tutik Dwi Wahyuningsih and Bambang Purwono2 1 2

Chemistry Study Program, Universitas Islam Indonesia, Jl. Kaliurang Km 14,5, Sleman, Yogyakarta, Indonesia. Chemistry Department, Universitas Gadjah Mada, Jl. Kaliurang street Km 4, Sleman, Yogyakarta, Indonesia. a)

Corresponding author: [email protected]

Abstract. A chalcone-like benzohydrazide derivatives (3) has been synthesis from natural resources vanillin (1a) and wintergreen oil (1b). This compound was synthesis as modified natural resource antibacterial agent. Some modification was done to increase the biological activity. Bromide was introduced to the vanillin structure to increase the activity (2a), whereas Hydrazine monohydrate was reacted with wintergreen oil to make new nucleophile (2b). Furthermore, chalcone like benzohydrazide compound was synthesized by stirring 5-bromovanillin (2a) with salicyl hydrazine (2b) at room temperature for 2-3 hours. The product was analyzed by FTIR, GCMS, 1H- and 13C-NMR to confirm its structure. The result showed that 5-bromovanillin, salicyl hydrazine, and benzohydrazide were successfully synthesized with 98, 78, and 33% of yield respectively. The target molecule 3 achieved with yellowish color with m.p. 106-111 °C, 97% purity.

INTRODUCTION Pathogenic microorganisms caused a lot of diseases in this earlier century, especially in third world country[1]. This becomes serious by the emergence of many strains of microorganisms that are resistant to the drug[2]-[3]. In fact, according to the WHO, the pathogen is the major causes of death in the 21st century. Multidrug resistance has also become an important focus of research in Europe and Australia[4]. Therefore, it is necessary to be able to find potential compound as an alternative antimicrobial agent. Chalcone and pyrazoline are nature compounds found in watermelon and now it has a lot synthesized[5]. These compound have many biological activities such as antioxidant[6]-[7], antimicrobial[7]–[13] and antiinflammatory[14]-[15] antifungal[10], antitumor[16], antidepresan[17], antihistamine, and insecticide[18]. Research on chalcone as antimicrobial much focused on a group attached to the aryl groups of chalcone. This proves that the aryl group substituent of chalcone determines its activities. Among the various substituents attached to the aryl Chalcone, bromide is reported as the most significant group increases Chalcone antibacterial activity[12]-[19]. While in the pyrazoline compound, the pyrazoline ring became the main focus of research to improve their activities. This occurs because the C-N and N=N cluster are reported as the key to various biological activity at some compound[7],[20]. This is the main reason why we chose to synthesize benzohydrazide derivatives that similar to chalcone structure by replacing the C=C group on chalcone with the C-N and N-N group to increase its biological activity. In addition, the halogen groups can also be added to increase the activity. Therefore this study is designed to synthesis a benzohydrazide derivatives compound that has a bromide as substituents. Compounds with the azide structure have been reported to have many biological activities such as antioxidants[21]-[22], inhibitors of kinase enzymes[23], anti-cancer[24] and antimicrobial[22], [25], [26]. Some of the benzohydrazide compounds are synthesized from benzohydrazine derivative and benzaldehyde. Designing a synthesis should consider the availability of the precursor. Vanillin and wintergreen oil is a natural compound that is commonly produced from a local source of Indonesia. Vanillin is natural a source of aldehyde and wintergreen oil is

International Conference on Chemistry, Chemical Process and Engineering (IC3PE) 2017 AIP Conf. Proc. 1823, 020121-1–020121-8; doi: 10.1063/1.4978194 Published by AIP Publishing. 978-0-7354-1491-4/$30.00

020121-1

an aromatic ester which is easy to convert as benzohydrazide. In otherwise, vanillin has a hydroxy and methoxy group, while wintergreen oil has a phenolic group. These clusters are reported as an anti-bacterial active agent[27]. Therefore this antibacterial agent synthesis derived from vanillin and wintergreen oil will have a great precursor’s availability while increasing value to local resources.

RESEARCH METHODS General Vanillin powder and wintergreen oil were obtain from traditional market with cap mobil and ika brand respectively, while another material were used from E.Merck with pro analysis (p.a) quality such as 2nitroacetophenone, hydrazine monohydrate, potassium bromate, sodium thiosulphate, sodium hydroxide, hydrochloric acid, glacial acetic acid, ethanol, methanol, n-hexane, ethyl acetate and dimethyl sulfoxide 99.9%. Melting points were measured using Electrothermal 9100 melting point apparatus and are uncorrected. Infrared spectra were recorded with a Shimadzu Prestige-21 FTIR spectrometer using KBr disc. 1H and 13C NMR spectra were obtained on a JEOL JNMECA (500 MHz) spectrometer. Mass spectra were recorded on Agilent GC-6890 and MS-5973 (EI). The purity of the compounds was confirmed by GC and thin layer chromatography.

Experimental Procedure 3-bromo-4-hydroxy-5-methoxybenzaldehyde (2a) A total of 0.6 g (3.6 mmoles) KBrO3 and 15 mL of glacial acetic acid were mixed into Erlenmeyer flask then added by 3 mL of HBr 47% dropwise under stirring. The mixture then added by 1.5 g (10 mmoles) of vanillin and continued for 30 minutes. Followed by the addition of ± 50 mL of ice distilled water and 30 drops of Na 2S2O3 10% (w/v) solution into the mixture. The formed precipitate was filtered and recrystallized with 50% ethanol to get a white powder 2a (98%), m.p. 164 °C, IR (KBr) cm-1: 3333 (OH), 2746 (C-H aldehyde), 1674 (C=O), 1589;1427 (C=C aromatic) and 678 (C-Br), 1H-NMR (500 MHz, CDCl3) δ: 3.90 (s,3H,-OCH3), 7.41 (d,1H,Ar-H), 7.72 (d,1H,Ar-H), 9.77 (s,1H,CHO). 13C-NMR (125 MHz, CDCl3) δ: 29.88 (OCH3), 56-150 (Ar-C), 190.43 (CHO). MS (EI) m/z: 232 (M+2, 81Br, 98%), 230 (M+, 79Br, 100), 217 (M-CH3, 10), 201 (M-CHO, 20), 187 (M-CH3-CO,27), 79 (M-CH3-2CO-Br,45), 51 (C4H7,50). 2-hydroxybenzohydrazide (2b) A solution of 3.8 mL methyl salicylate (25 mmoles) in 10 mL ethanol was added by 2.5 mL hydrazine monohydrate (50 mmoles). The mixture was refluxed for 2.5 hours then concentrated. The product was placed in an ice bath and stirred to form a precipitate. The product was washed with distilled water and dried to get white powder 2b (98), mp 143-146 °C, IR (KBr) cm-1: 3271 (NH), 3055(C-H aromatic), 1643 (C=O), 1527 (C=C), 1134 (C-O), 1087 (C=N), GC: 98.65%. MS (EI) m/z: 152 (M+, 20%), 121 (M-∙N2H3, 100), 93 (M-C2O2N2H3, 30), 65 (MCON2H3, 50). (E)-N'-(3-bromo-4-hydroxy-5-methoxybenzylidene)-2-hydroxybenzohydrazide (3) Benzohydrazine derivative was formed by stirring the 2a product (0.35 g, 1mmol) with was salicyl hydrazine 2b (0.32 g, 2 mmoles) in methanol until the precipitate formed. The precipitate was filtered and washed with ethyl acetate to get 3c powder (33.69%), mp 106-111 °C, IR (KBr) cm-1: 3348 (OH), 3255 (NH), 2939 (CH 3-sim), 2862 (CH3-asim), 1635 (C=O), 1589 (C=N), 1373 (CH 3), 1H-NMR (CDCl3) δ: 3.96 (s,3H,CH3), 6.96-7.91 (Ar-H), 8.18 (Csp2-H). TLC scanner: 97%.

020121-2

FIGURE 1. Synthesis of benzohydrazide derivatives

RESULTS AND DISCUSSION Synthesis 3-bromo-4-hydroxy-5-methoxybenzaldehyde (2a) Bromium is reported to the most dominant halogen group candidates as antibacterial agents against Grampositive and negative bacteria compared to some other halogen groups[28]. In addition, vanillin has m-methoxy substituent and p-hydroxy reported active as an anti-bacterial. Therefore, brominated vanillin would be a good precursor in the synthesis of some antibacterial target compounds. In this study, vanillin is brominated using KBrO 3 as a source of bromide in situ under acidic conditions. This method is more environmentally safe than many other methods that use bromine as a bromide source. The direct use of bromine is avoided because bromine is harmful if it is inhaled or exposed to the skin. The Synthesis of 5-bromovanilin has produced a white powder with 98% of yield and m.p. 164 °C. The FTIR spectra of this result are presented in Figure 5.

FIGURE 2. Chromatogram GC of brominated vanillin.

020121-3

FIGURE 3. Mass Spectra (b) of brominated vanillin.

Peaks at 1666 (C=O) and 3178 cm-1 (OH) at vanillin is shifted into 1674 and 3333 cm-1 after brominated. It also appears a new peak at 678 cm-1 which are the stretching vibration of C-Br. GCMS results (Figure 2 and 3) showed a peak at 19.04 min with molecular ions (M+) 230 and 232 and relative abundance of 1: 1. This molecular ion is equal to its molecular weight of 5-bromovanilin with 79Br and 81Br isotope. 1 H-NMR and 13C-NMR (Figure 4) analysis indicated the synthesis of 5-bromovanillin occur. The value of the coupling constants (J) of 1.3 Hz obtained from 1H-NMR showed aromatic protons on the benzene ring is at the meta position, proving that the bromine group substituted on a carbon atom number 5. The results of 13C-NMR analysis provide information regarding the 8 peaks carbon with different electronic environments. The tops of the peaks include C δ 190.43 ppm (aldehyde), CE δ 109.20 to 149.78 (aromatic), F and G δ 148.64 (C-Ome), 149.78 (C-OH ), B δ 109.20 (C-Br) and A δ 56.33 ppm (CH3).

FIGURE 4. 1H- and 13C-NMR Spectra of brominated vanillin

Synthesis of 2-hydroxybenzohydrazide (2b) The objective of the 2-hydroxybenzohydrazide is to attach hydrazine to salicylic acid and form new nucleophile. This is nucleophilic substitution reaction between hydrazine and methanol anion. Hydrazine is binucleophile molecular that easy attack to carbonyl atom at salicyl structure followed by the broken of methanol anion. the Salicylic hydrazine (2b) is synthesized from wintergreen oil and produced a white powder with a melting point of 143-146 °C, 74% yield with 98.65% of purity. This result is equal with Sharma[9] that reached 77% of yield. FTIR spectra indicate the presence of salicylic hydrazine with strong and sharp absorption at wavenumber 3271 cm-1 of N-H group. The two sharp splitting peaks formed is indicating a primary amine. Other absorption peaks is belong to the methyl salicylate group i.e. 3055 cm-1 (C-H aromatic), 1643 cm-1 (C = O), 1527 cm-1 (C = C), 1134 (C-O) and 1087 cm-1 (C-N).

020121-4

FIGURE 5. FTIR Spectra of brominated vanillin (2a), 2-hydroxybenzohydrazine (2b) and Benzohydrazide derivatives (3).

The synthesized product is analyzed by GCMS (Figure 6 and 7). The results of the chromatogram GC showed two product compounds with 1.35% area for the first peak at tR 23.17 min and 98.65% for the second peak at tR 26.67 min. According to mass spectra, the target compound salicylic hydrazine or 2-hidroksibenzohydrazide emerges as the second peak.

FIGURE 6. Chromatogram GC of 2-hidroxybenzohydrazine.

FIGURE 7. Mass Spectra (b) of 2-hidroxybenzohydrazine.

020121-5

The second peak at tR 26.67 min appears with M+ 152 indicates that this peak is salicylic hydrazine with molecular weight 152 g/mol. This peak then is experienced α fragmentation on the amide bond yield m/z 121 and heterolysis disconnection produce a fragment with m/z 31. Fragments 121 have the highest relative abundance (base peak) because this is the most stable a fragment. Heterolysis disconnection between phenol and carbonyl group produce fragment with m/z 93, following with m/z 65 because CO molecule release.

Synthesis of (E)-N'-(3-bromo-4-hydroxy-5-methoxybenzylidene)-2-hydroxybenzohydrazide (3) Benzohydrazide is a compound with a C=O and the group C-N and N=N group. The impression of this compound is inspired by the properties of chalcone and pyrazoline structure. Both have antibacterial activity, but the mechanisms of action of these molecules are different. Chalcone is reported to be able to interact with the cell wall and also penetrate into cell because its smaller structure. In otherwise, pyrazoline with C-N, N=N and NH2 active group kill bacteria by interacting with the cell wall without being able to penetrate into the cell because the larger structure. By summarizing the active site in one molecule we can maintain the same relative size with Chalcone but have pyrazolinea active sites. As in chalcone formation, benzohydrazide formation is a condensation reaction. The two or more molecules combine to form larger molecules, with or without the release of small molecules such as H 2O. The mechanism of this reaction involves two steps: addition reaction and dehydration step. Chalcone-like benzohydrazide was synthesized from 5-bromovanilin 2b with salicylic hydrazine in methanol under the stirring condition for 2 and 3 hours at room temperature. The Products is formed with yellowish powder, 229-230 °C of melting point and 235238 °C of boiling point, the yield generated from this reaction is 33.69% with 97% purity. FTIR spectra of benzohydrazide (Figure 5) shows a peak at 3356 cm-1 (OH), 3078 cm-1 (Ar-H), 2931 cm-1 (CH3 asymmetric), 2854 cm-1 (CH3 asymmetric), 1365 cm-1 (CH3), 1635 cm-1 (C=O), 1597 cm-1 (C=N), and 3325 cm-1 (NH). The products were subsequently analysed by 1H- and 13C-NMR. Results spectra 1H- and 13C-NMR is presented in Figure 8.

FIGURE 8. 1H- and 13C-NMR Spectra of benzohydrazine derivatives.

The 1H-NMR results of benzohydrazide compound 3 (Figure 8) shows there are 8 types of protons with a total integration of 10 protons. Protons 1 with having triple area than other are integrated as an OCH3 proton. This proton appeared on the most up field areas than others. Proton 2 appears as a doublet with coupling constants Jorto=7.8 Hz couple by proton 5. Proton 3 and 5 appeared as a triplet with coupling constants Jorto=7.1 and 7.8 Hz, which means these carbon are around by two atoms each has 1 H. Proton 4 and 6 appear as duplet with coupling constants Jmeta =1.95 Hz. There are only two protons that have a meta coupling constants, so most likely protons 4 and 6 is experience mutual coupling. Proton 7 appears as a doublet with coupling constants Jorto=8.45 Hz and the last proton 8 appear as a singlet at most down field area.

020121-6

The results of 13C-NMR spectra presented in Figure 6 shows a total of 15 carbons. Peak 15 at δ 167 ppm is characteristic of the carbonyl group. Peak 14, 13 and 11 at δ 160, 150 and 148 ppm is the result of Ar-C-O. Peak 12 at 150 ppm is C=N carbon. The 15-11 peak is carbon that binds to the hetero atom which has the power to attract the electron cloud C so that it appears on the down field area, except for the Ar-C-Br appearing in the relatively up field. Br has a relatively large size compared to C so that the electron cloud enveloped the succession Br C, C-Br consequently appears in the area up the field. Benzohydrazide derivatives spectra showed the same pattern both in the 1H- and 13C-NMR. This indicates the results obtained have the same structure. Referring from the results of the FTIR, 1H- and 13C-NMR spectra concluded that a compound formed is (E)-N'-(3-bromo-4-hydroxy-5-metoxybenzylidine)-2-hydroxybenzohydrazide. This compound was successfully synthesized from natural ingredients vanillin and wintergreen oils with a 33.69% yield.

CONCLUSION Novel compound chalcone-like benzohydrazide derivatives was successfully synthesis from natural resources vanillin and wintergreen oil. Structure elucidation of this molecule show as a (E)-N'-(3-bromo-4-hydroxy-5methoxybenzylidene)-2-hydroxybenzohydrazide (3) results in a yellowish color, 33.69% of yield and a 97% of purity.

ACKNOWLEDGMENTS We thank Ministry of education of Indonesia (DIKTI), for financial support

REFERENCE 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

M. T. Madigan, J. M.Martinko, D. A. Stahl and D. P. Clark, BROCK Biology of Microorganisms (Benjamin Cuming, San Francisco, 2012), pp. 945-977. L. S. P. Moore, R. Freeman, M. J. Gilchrist, M. Gharbi, E. T. Brannigan, H. Donaldson, D. M. Livermore and A. H. Holmes, J. Antimicrob. Chemother. 69, 3409–3422 (2014). J. Reyes, D. Panesso, T. T. Tran, N. N. Mishra, M. R. Cruz, J. M. Munita, K. V. Singh, M. R. Yeaman, B. E. Murray, Y. Shamoo, D. Garsin, A. S. Bayer and C. A. Arias, J. Infect. Dis. 211, 1317–1325 (2015). R. James, L. Upjohn, M. Cotta, S. Luu, C. Marshall, K. Buising and K. Thursky, J. Antimicrob. Chemother. 26, 1912–1918 (2015). T. Eicher, S. Hauptmann and A. Speicher, The Chemistry of Heterocycles: Structures, Reactions, Synthesis, and Applications 3rd, Completely Revised and Enlarged Edition (WILEY-VCH, Weinheim, 2012) pp. 236246. S. Singh, P. K. Sharma, N. Kumar and R. Dudhe, J. Adv. Sci. Res. 2, 37–41 (2011). N. Parmar, S. Teraiya, R. Patel, H. Barad, H. Jajda and V. Thakkar, J. Saudi Chem. Soc. 19, 36–41 (2015). M. S. Karthikeyan, B. S. Holla and N. S. Kumari, Eur. J. Med. Chem. 42, 30–36 (2007). R. Chawla, U. Sahoo, A. Arora, P. C. Sharma and V. Radhakrishnan, Acta Pol. Pharm. Drug Res. 67, 55–61, (2010). R. Gupta, N. Gupta and A. Jain, Indian J. Chem.-Sect. B Org. Med. Chem. 49, 351–355 (2010). B. K. Sarkar, R. Patel and U. Bhadoriya, J. Adv. Pharm. Educ. Res. 1, 243–250 (2011). V. D. Joshi, M. D. Kshirsagar and S. Singhal, Inter. J. Chem. Tech. Res. 4, 971–975 (2012). A. Ahmad, A. Husain and S. A. Khan, J. Saudi Chem. Soc. 10, 219–229 (2014). R. S. Joshi, P. G. Mandhane, S. D. Diwakar, S. K. Dabhade and C. H. Gill, Bioorganic Med. Chem. Lett. 20, 3721–3725 (2010). D. Bardalai and P. Panneerselvam, Int. Res. J. Pharm. App. Sci. 2, 1–8 (2012). R. M. Mohareb, N. N. E. El-sayed and M. A. Abdelaziz, Molecules 17, 8449–8463 (2012). A. Chauhan, P. K. Sharma and N. Kaushik, Int. J. Chem. Tech. Res. 3, 11–17 (2011). M. A. Rahman and A. A. Siddiqui, Int. J. Pharm. Sci. Drug Res. 2, 165–175 (2010). J. Kumar, G. Chawla, U. Kumar and K. Sahu, Med. Chem. Res. 23, 3929–3940 (2014). S. Malladi, A. M. Isloor, S. Isloor, D. S. Akhila and H. K. Fun, Arab. J. Chem. 6, 335–340 (2013).

020121-7

21. H. S. Kareem, N. Nordin, T. Heidelberg, A. Abdul-Aziz, A. Ariffin and R. A. Bunce, Molecules 21, 224-243 (2016). 22. N. Nazarbahjat, N. Nordin, Z. Abdullah, M. A. Abdulla, W. A. Yehye, S. N. A. Halim, C. H. Kee and A. Ariffin, Molecules 19, 11520–11537 (2014). 23. H. C. Wang, X. Q. Yan, T. L. Yan, H. X. Li, Z. C. Wang and H.-L. Zhu, Molecules 21, 1012-1025 (2016). 24. A. Malki, M. Mohsen, H. Aziz, O. Rizk, O. Shaban, M. El-Sayed, Z. Sherif and H. Ashour, Molecules 21, 230255 (2016). 25. R. Maheswari and J. Manjula, Int. J. Appl. Res. 1, 587–592 (2015). 26. S. Y. Hassan, Molecules 18, 2683–2711 (2013). 27. Y. Kim, S. Farrah and R. H. Baney, Int. J. Antimicrob. Agents 6, 4531-4545 (2006). 28. D. P. Gupta, R. S. Bhadauria and V. Soan, Int. J. Pharm. Appl. Sci. 1, 97–99 (2010).

020121-8