Synthesis, Characterization, and Antibacterial Evaluation of New Vanillic Acid Derivatives

Hydrazide Schiff bases (hydrazones) and 2,5-disubstituted-1,3,4-oxadiazole derivatives exhibit diverse biological activities that include antibacterial, antifungal, antitubercular, antiviral, anticancer, anti-inflammatory, and analgesia; so that new derivatives, compounds (5-8) of vanillic acid based on 1,3,4-oxadiazole as scaffold unit were synthesized through multi-steps, and characterized by thin layer chromatography and spectroscopically by Fourier-transform Infrared (FTIR) and Proton nuclear magnetic resonance (HNMR). Compounds (5-8) were evaluated for their antibacterial activity by the disk diffusion method. Compounds (5-8) were showed moderate and comparable to the activities of amoxicillin and isoniazid against Escherichia coli (E. coli), but less than that of cefixime and nitrofurantoin which their activities were high. Compound (6) and (7) had shown moderate and comparable to the activities of amoxicillin and cefixime against Klebsiella pneumoniae (K. pneumoniae), but less than that of nitrofurantoin which its activity was high. Compound (6) and (7) had shown moderate and comparable to the activity of amoxicillin against Staphylococcus aureus (S. aureus), while the activities of cefixime and nitrofurantoin were high. Compound (6) was moderately active against Bacillus subtilis (B. subtilis), while the activities of amoxicillin, cefixime, and nitrofurantoin were high.


Introduction
Heterocyclic compounds are used in many biological fields, due to their different activities, and are considered as one of the principal classes of organic compounds, that are used in the development of several pharmaceutically essential compounds (1,2) . Oxadiazoles are important five-membered aromatic heterocyclic containing oxygen and two nitrogens in their structure. Because the oxadiazole ring is structurally rigid, various functional groups are easily introduced into the ring. Valuable biological activities are associated with oxadiazole derivatives (3) , such as antitumor (4) , anti-inflammatory (5) , antimicrobial (6) , antifungal (7) , and anticonvulsant (8) . In synthetic medicinal chemistry, to improve the biological activity of new drugs with respect to the corresponding lead compounds, hybridizationcombination of different pharmacophores in one structure-is one of the techniques being followed. (9,10) Hydrazide Schiff base derivatives (hydrazones) are good scaffolds for various pharmaceutical applications, and characterized by the presence of highly reactive azomethine group (-CO-NH-N=CH-). (11) Their biological activities include antibacterial (12) , antifungal (13) , antitubercular (14) , antiviral (15) , anticancer (16) , antiinflammatory (17) , and analgesia. (18)

Chemical synthesis
The target compounds were synthesized by multistep(s) reactions as shown in the scheme in Figure

Synthesis
of Methyl 4-hydroxy-3methoxybenzoate; compound (1) (23) : Vanillic acid (7.0g, 42mmole) was dissolved in 75ml of absolute methanol (99.8%), the temperature of this solution was lowered to 0˚C by ethanol-water ice bath, 5ml of concentrated H2SO4 was added dropwise, and the mixture was stirred at room temperature for 48 hours, then refluxed for 7 hours. The reaction mixture was poured in beaker containing crashed ice (100ml), the formed precipitate was filtered, collected, and washed with 5% NaHCO3 aqueous solution, filtered again, and dried by a warmed current of air, giving 7.0g of compound (1). (24) : Compound (1), (5.0g, 27mmole) was dissolved in a minimum amount (10ml) of absolute ethanol (99.9%), (13.5g, 270mmole) of 80% hydrazine hydrate was added gradually. The mixture was refluxed for 3-4 hours (monitored by TLC). Then the reaction mixture was cooled and precipitate begin to appear, which was filtered and dried in oven (adjusted at 60˚C), giving 3.5g of compound (2). (25) :

Synthesis of 4-(5-Mercapto-1,3,4-oxadiazol-2-yl)-2-methoxyphenol; compound (3)
To suspension of (1.25g, 6.86mmole) of compound (2) in 30ml of 50% aqueous ethanolic solution, (0.785g, 10.3mmole) of CS2 was added with stirring for few minutes, followed by the addition of (0.7g, 10.3mmole) potassium hydroxide (KOH), and the mixture was refluxed for 12 hours (monitored by TLC, and notation the evolution of H2S gas by strip soaked with lead acetate aqueous solution; which was turned black as indication of evolution of H2S gas). The mixture poured in a beaker containing crashed ice (30 ml) and then concentrated HCl was added dropwise until pH became (2-3). The formed precipitate was filtered, dried, and purified by base-acid precipitation method (i.e.; the product was dissolved in water by the aid of equimilimole of sodium hydroxide or triethylamine, filtered, and the clear filtrate was treated with concentrated HCl which was added dropwise until the precipitate formed again), giving 0.9g of compound (3).

Antibacterial essay
Well diffusion assay was carried out through using bacterial suspension of nearly (1.5×10 8 CFU/ml) obtained from McFarland turbidity standard (number 0.5). This was used to inoculate by swabbing the surface of Mueller Hinton Agar (MHA) plates. The excess liquid was dried by air under a sterile hood. In each agar plate of examined bacteria, four wells were made, and (80μl) of each concentration of the synthesized compound was poured to it. The plates were incubated at 37°C for 24 hours. The evaluation of antibacterial activity was based on the measurement of the diameter of the inhibition zone formed around the well. (29)

Anti-bacterial evaluation
The antibacterial activities of the synthesized compounds; compounds (5-8) were evaluated against six bacteria and compared with four standard antibiotics; amoxicillin, cefixime, nitrofurantoin, and isoniazid. Dimethyl sulfoxide (DMSO) was used as solvent and control. It's evident from the data displayed in the

Chemistry
Compound (1) was esterification product which resulted from reaction between carboxylic acid and an alcohol in the presence of an acid as catalyst. Two stages were involved: addition of a nucleophile followed by elimination of a leaving group. Protonation and deprotonation steps also occur during the ester formation which could explain the role of acid in the reaction. Under basic conditions, carboxylate anion will be formed which does not react with an electron-rich nucleophile, so the esterification will be happened in the presence of an acid. Formation of ester is necessary for the success of step 2 and 5 as explained later. (34) .

Figure 2. Steps of esterification. (34)
Compound (1) was characterized by carbonyl group of aromatic ester at 1686cm -1 in its FTIR spectrum and 1 HNMR signals confirmed the presence of COOCH3 at 3.80 ppm.

Synthesis of Compound (2) and (5)
is essentially a base catalyzed hydrolysis (hydrazinolysis of ester) which was run under normal basic condition in which the ratedetermining step involves two molecules of hydrazine in which a proton was being transferred between them. In the next step, one hydrazine molecule will be left slowly with one molecule of alcohol (35) . (35) .

Figure 3. Hydrazinolysis of ester
In case of compound (2); FTIR spectrum was characterized by two stretching vibration bands for the primary amine of hydrazide, at 3310 cm -1 and 3209 cm -1 , respectively, 3256 cm -1 NH amide stretching vibration band, 1628 cm -1 C=O stretching vibration band of amide, and 1601cm -1 NH2 bending vibration band. 1 HNMR signals confirmed the presence of CONH at 9.56 ppm, and NH2 at 4.42 ppm. For compound (5); FTIR spectrum was characterized by asymmetric and symmetric stretching vibration bands of NH2 at (3341 and 3217) cm -1 , respectively, 3256 cm -1 NH amide stretching vibration band, the amide carbonyl stretching vibration band at 1682 cm -1 , and NH2 bending stretching vibration band at 1655 cm -1 .     (37) . FTIR spectrum was characterized by 1744cm -1 stretching vibration band of saturated ester carbonyl. 1 HNMR was characterized by the presence of new signals at 4.14-4.18(2H, q, COCH2CH3), 1.18-1.21(3H, t, COCH2CH3), and the absence of SH signal at 14.58 and instead of the appearance of new signal at 4.26 ppm which was related to -SCH2.
Compounds (6-8) were Schiff base products (imines) which resulted from reaction between aldehydes with a primary amines in mildly acidic conditions and involves six steps; the first three steps produce an intermediate called a carbinolamine and the last three steps convert the carbinolamine into an imine (38) . FTIR spectra were characterized by absence of hydrazide NH2 asymmetric and symmetric stretching vibration bands; while N=CH imine stretching vibration bands were overlapped with other bands in FTIR spectra. 1 HNMR were characterized by the appearance of new signals related to CONHN which were due to cis and trans isomers showed 2signals between [11.60,11.63 for compound (6), 11.45,11.48 for compound (7), 11.64,11.69 for comp.8], and CONHN=CH which due to syn/anti-syn conformers showed 2signals between [7.91, 8.08 for compound (6), 7.86, 8.04 for compound (7), 7.97, 8.14 for compound (8)]. (39-

Antibacterial activities
Four antibacterial standards were used, amoxicillin to compare anti-gram (+)ve activities of the derivatives with it; cefixime to compare antigram(-)ve activities of the derivatives with it; nitrofurantoin because it is considered hydrazone and contains furan ring which is isoseter with oxadiazole ring, and isoniazid which is hydrazide compound resembles to compound (5). Because compound (6) and compound (7) are more polar than compound (8), they showed additional activities against K. pneumoniae [as polarity increased; there will be extended activity against gram(-)ve bacteria, while retained activities against gram(+)ve bacteria as in the case of penicillin G and aminopenicillins]. (43) Because Compound (5) is hydrazide; it is expected to show limited activity against test bacteria and showed agreement with isoniazid.

Conclusion:
New oxadiazole derivatives (hydrazide and its Schiff bases), derived from vanillic acid were successfully synthesized by conventional methods. They were characterized and evaluated for their antibacterial activities. Compound (6) had shown the broadest spectrum against tested bacteria showed activities against four out of six bacteria. Compound (7) had shown moderate activities against S. aureus, E. coli, and K. pneumoniae. Compound (5) was moderately and selectively active against E. coli. Compound (8) was slightly active against S. aureus and moderately active against E. coli.

Acknowledgments
We're grateful to the College of Pharmacy-Department of Pharmaceutical Chemistry-University of Baghdad, for conducting the research, Mr. Ahmed College Ibn -Al-Haitham, for Pure Science, for his polite style and his patience during evaluation of antimicrobial activity, and chemistry analysis center (CAC) stuff for their major role in 1 HNMR analysis and evaluation of antibacterial activities of the compounds.