Synthesis , Characterization and Antibacterial Activity of Biologically Important Vanillin Related Hydrazone Derivatives

Hydrazone derivatives of vanillin are found to possess anti-bacterial activities. Based on higher bio-activity of hydrazones, new hydrazone derivatives were synthesized from Piperdine-4-carboxylicacid methyl ester (1). The compounds 1-pyrimidine-2-yl piperidine-4-carboxylicacid(4-hydroxy-3-methoxy benzylidine)-hydrazide (10), 1-pyrimidine-2-yl piperidine-4-carboxylicacid (3,4-dimethoxy benzylidine) hydrazide (11), 1-pyrimidine-2-yl piperidine-4-carboxylicacid(4-butoxy-3-methoxy benzylidine)-hydrazide (12), 1-pyrimidine-2-yl piperidine-4-carboxylicacid(3-methoxy-4(2-methoxy ethoxy) benzylidine)-hydrazide (13) were synthesized, purified and characterized by HNMR, CNMR, LCMS, FT-IR and HPLC techniques. The synthesized hydrazone derivatives were further checked for anti-bacterial activities by paper disc diffusion method against Pseudomonas aeruginosa and Staphylococcus aureus bacterial strains.


Introduction
Earlier, by Quantitative Structure Activity Relationship (QSAR) studies, most of the rifamycin derivatives were found to be biologically active compared to other compounds [1].For example, the hydrazones obtained from 3-formyl rifamycin and N-amino-N-methyl piperazine derivatives were found to be biologically active and tested for oral treatment of infections in animals [2].Recently, a lot of biologically important hydrazone derivatives with a number of functional groups have been synthesized from aromatic and aliphatic compounds [3].Hydrazone derivatives are molecules containing highly reactive azomethine group (CO-NH-N=CH) and thus useful in new drug development [4].Also, these are found to possess anti-microbial [5][6][7], anti-mycobacterial [8], anti-convulsant [9], analgesic [10], anti-inflammatory [11,12], anti-platelet [13], anti-tubercular [14][15][16] and anti-tumoral [17][18][19] activities.Diflunisal hydrazones were also prepared as possible dual acting antimicrobial and anti-tuberculosis agents with anti-inflammatory properties [20].Moreover, hydrazones has been recently established as a good precursor for one-pot synthesis of C-4 functionalized 1,2,3,4-tetrahydro quinolones containing a quaternary stereo center [21].Due to the growth of population and changes in climatic conditions several new diseases are likely to affect the human beings.So, there is a continuous need for the synthesis of new biologically active organic compounds by using a fast and efficient approach which may act as potential antimicrobial agents.Based on the higher bio-reactivity of hydrazones, we have synthesized novel hydrazones (10-13) from Piperdine-4-carboxylic acid methyl ester (1) coupled with 2-chloro pyrimidine (2) along with other vanillin derivatives (6)(7)(8)(9).The anti-bacterial studies were effectively done for newly synthesized hydrazones by standard disc diffusion method [22] with different concentrations.

Synthesis
Earlier studies on pyrimidine shows, that heterocyclic compounds containing pyrimidine moiety shows various biological activities.Therefore, we were tempted to synthesize vanillin related hydrazones with a pyrimidine moiety.A series of vanillin related hydrazones are synthesized and their purity is checked by thin layer chromatography (TLC) and HPLC techniques.All the synthesized hydrazones structures are characterized by 1 HNMR along with 13 CNMR; LC-MS and FT-IR spectral techniques.There are three different types of coupling reactions taking place in syntheses of the hydrazone derivatives.In step-1, compound 1 is coupled with 2 to form 3 by "chloro-amine" coupling.In step-2 the product 3 was reacted with 4 to form 5 by "ester-amine" condensation.In step-3, vanillin derivatives (6-9) react with 5 to form hydrazone derivatives (10-13) by "aldehyde-amine" coupling.From the 1 H NMR spectra, the structures of the synthesized compounds (10-13) were confirmed on the basis of the fact that the aldehydic proton (which was visible at δ 10.55) in the starting compound 6 disappeared, and a new singlet due to the azomethine (CH=N) group appeared at δ values between 8.06 -8.11 ppm in all the compounds.The CONH protons appearing as singlets resonated at δ values between 11.20 and 11.25 ppm.Furthermore, the protons of CONH and CH=N exhibited two separate signals in 1HNMR spectra in between 11.20 -11.25 ppm and 8.06 -8.11 ppm respectively due to the nitrogen inversion, which is shown in The three -CH protons of the pyrimidine (pm) were centered at δ value 8.35 ppm as doublets by integrating in two proton and at δ value 6.60 ppm as triplets by integrating in one proton.
In the 13 CNMR spectra of 6 the carbon signal due to (-CHO), was observed at δ 188.97 ppm.However in products 10-13 this signal was found to be absent and a new signal at δ values between 150.04 and 151.04 ppm arose due to the presence of CH=N in compounds (10)(11)(12)(13).
The carbon signal of C=O group appeared at δ values between 175.8 and 176.2 ppm.The molecular mass of the synthesized compounds were recorded by LC-MS techniques, which was registered in positive ion (+M) mode.The FT-IR spectra of compounds (10)(11)(12)(13) showed absorption bands at 1652 -1655 cm -1 due to the presence of C=O functional group, while the bands observed at 1582 -1586 cm -1 corresponded with C=N linkage and 3280 -3413 cm -1 observed due to the -NH group.The absorption peak at 2845 -2867 cm -1 was due to the CH linkage and the band appearing at 3845.4 cm -1 in the IR spectrum of the compound (10) represented OH group.The synthetic conditions and melting points of the newly synthesized compounds are summarized in Table 1.

Antibacterial Activity
The anti-bacterial results showed that some of the compounds were active against both Gram-positive S. aureus and Gram-negative P. aeruginosa bacteria.Among the tested solutions (10-13), the compounds ( 12) and ( 13) showed good antibacterial activity against the test organisms and 11 had moderate effective against S. aureus and less effective against P. aeruginosa.The compound 10 had no anti-bacterial activity against P. aeruginosa and lowest activity against S. aureus.It was observed that maximum antibacterial activity was shown by compounds containing the butoxy, methoxy and methylethoxy group with highly reactive azomethine (-NH-N= CH-) group.On the other hand, compared to the standard antibacterial drugs namely, Ciprofloxacin and Cefaclor our synthesized hydrazones were having moderate activeity against test organisms.The obtained results of antibacterial activity have been summarized in Table 2.

Experimental
All synthetic manipulations were conducted in the dry and nitrogen atmosphere.Solvents for synthesis were reagent grade and dried by standard procedures [23].The starting materials are such as (1), (2), Hydrazine hydrate (4), Vanillin (6) and Veratraldehyde (7) were obtained from Sigma-Aldrich chemicals and acetone, methanol, ethanol, acetonitrile and dichloromethane, which were obtained from SRL Chemical Limited, India.The intermediate vanillin derivatives such as 4-butoxy-3-methoxy benzaldehyde (8) and 3-methoxy-4-(2-methoxy-ethoxy) benzaldehyde ( 9) were prepared by typical procedures [24,25].Melting points of as synthesized compounds were determined with open capillary tube on a Gallenkamp melting point apparatus.The 1 H and 13 CNMR were recorded on a Bruker Avance-III, 300 MHz and 400 MHz.Liquid chromatography mass spectra (LCMS) were run on "LCMS-Agilent Technologies-1200 Series" and purity was checked by "HPLC-Agilent Technogies-1200 Series".IR spectra were recorded by "FT-IR Nicolet 6700" spectrometer.All compounds were routinely checked by TLC on silica gel G plates using petroleum ether/ethyl acetate (7:3; 6:4; 5:5 by V/V) as solvent system and the developed plates were visualized by UV light, iodine vapour and KMnO 4 solution.The detailed scheme of synthesis has been shown in Scheme 1.The anti-bacterial studies performed in Center for Biotechnology, University of Allahabad, India.
The reaction mixture cooled to room temperature, concentrate under reduced pressure.The crude was purified by crystallization, washed with petroleum ether and filtered.White solid, 0.96 g Yield: 96%.

General Procedure for Synthesis of (10-13)
Vanillin derivatives (6-9) (1.5eq.) were added to compound (5) (1eq.)separately in dry ethanol/acetic acid (5:1 mL) under nitrogen atmosphere and the reaction mixtures were refluxed at 85 o C in sealed tube for 8 hrs.These were slowly brought to room temperature and concentrated under reduced pressure.The crude products purified by fractional crystallization method by using ethyl acetate or dichloromethane along with petroleum ether or diethyl ether were filtered and dried under vacuum to give the corresponding products (10-13) as white solids.

Anti-Bacterial Assay
All the synthesized hydrazones were tested for their anti-bacterial activity against a set of bacterial strains, namely, Staphylococcus aureus, and Pseudomonas aeruginosa by paper disc diffusion method with different concentrations of the solutions prepared in Dimethyl sulfoxide (DMSO).The reason of choosing DMSO for antibacterial studies was that it has no effect on the above mentioned bacterial strains.Nutrient agar was used as the culture medium for the growth of bacterial colony that was prepared by using peptone (3.0 g), NaCl (3.0 g), Yeast (1.5 g), Agar (6.0 g) in 300 mL of distilled water with pH at 7.0 The as prepared medium is autoclaved at 15 pa for 20 minutes and kept at 85 º C for 30 minutes to sterilize the media.This media was then poured into petridishes slowly in laminar flow environment, allowed to solidify and kept at 30˚C for 24 hrs.The bacterial strains were inoculated by spreading in peptidases and its temperature is maintained at 30˚C for 24 hr.Using paper disc (8 mm) in nutrient agar culture medium, different concentrations (50, 100, 150, 200, 250 µg/mL) of the newly synthesized hydrazones (10-13) were loaded through bacteria free micro pipettes.The anti-bacterial activity was determined by measuring the zone of inhibition in millimeters and compared with standard drug Ciprofloxacin and Cefaclor.

Conclusions
We have developed the simple and crucial synthetic technique of vanillin related hydrazone derivatives and the reactions occurred very fast, under mild condition using reasonable reagents and solvents, yield is also higher.The anti-bacterial activity of synthesized novel hydrazones were effectively screened against Gram positive S. aureus and Gram-negative P. aeruginosa bacterial strains.Most of these compounds show moderate antibacterial activity comparable with to marketable compounds.The zone of inhibition of tested compounds shows, the vanillin coupled hydrazone derivatives encompass potent bio-activities against bacterial strains.Due to the strong bio-activity of our synthesized hydrazones can be further allowed to attempt other bio-activities against a number of diseases and this work will be precious for further studies in terms of toxicity effect and Quantity Structural Activity Relationship (QSAR) to improve their biological and pharmacological properties.

Figure 1 .
Figure 1.The three -CH protons of the pyrimidine (pm) were centered at δ value 8.35 ppm as doublets by integrating in two proton and at δ value 6.60 ppm as triplets by integrating in one proton.In the13 CNMR spectra of 6 the carbon signal due to (-CHO), was observed at δ 188.97 ppm.However in products 10-13 this signal was found to be absent and a new signal at δ values between 150.04 and 151.04 ppm arose due to the presence of CH=N in compounds(10)(11)(12)(13).The carbon signal of C=O group appeared at δ values between 175.8 and 176.2 ppm.The molecular mass of the synthesized compounds were recorded by LC-MS techniques, which was registered in positive ion (+M) mode.The FT-IR spectra of compounds(10)(11)(12)(13) showed absorption bands at 1652 -1655 cm -1 due to the presence of C=O functional group, while the bands observed at 1582 -1586 cm -1 corresponded with C=N linkage and 3280 -3413 cm -1 observed due to the -NH group.The absorption peak at 2845 -2867 cm -1 was due to the CH linkage and the band appearing at 3845.4 cm -1 in the IR spectrum of the compound (10) represented OH group.The synthetic conditions and melting points of the newly synthesized compounds are summarized in Table1.