Iraqi J Pharm Sci, Vol.29(2) 2020 New vanillic acid derivatives as antibacterials DOI: https://doi.org/10.31351/vol29iss2pp129-138 129 Synthesis, Characterization, and Antibacterial Evaluation of New Vanillic Acid Derivatives Mostafa F. Tawfeeq*,1 and Ahlam J. Qassir** * Department of Pharmacy, College of Pharmacy, University of Tikrit, Salahuddin, Iraq ** Department of Pharmaceutical Chemistry, College of Pharmacy, University of Baghdad, Baghdad, Iraq Abstract 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 (1HNMR). 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. Keywords: Hydrazide, Schiff Base, Oxadiazole, Antibacterial. وتقييم الفعالية المضادة للبكتريا لمشتقات جديدة لحامض الفانلين ، تشخيص ، تصنيع *و احالم جميل قصير 1*,مصطفى فايز توفيق . العراق ،صالح الدين ،جامعة تكريت ،كلية الصيدلة ،فرع الكيمياء الصيدالنية . العراق ،بغداد، جامعة بغداد ،كلية الصيدلة ،فرع الكيمياء الصيدالنية الخالصة لهما العديد من الفعاليات الحيوية كأن 5و2اوكسادايزول المعوضة في الموقعين -1,3,4قواعد شف و الحلقة األروماتية الغير متجانسة أو أن تكون مضادات لأللتهابات أو تعمل كمسكنات لأللم. لذلك الخاليا السرطانيةو الفايروساتو بكتريا التدرنو الفطرياتو تكون مضادات للبكتيريا اوكسادايزول كوحدة بناء رئيسية-1,3,4باألعتماد على الحلقة األروماتية الغير متجانسة (8-5) المركباتن حامض الفانليلجديدة شتقاتتم تصنيع م عن طريق مطياف األشعة تحت الحمراء, الرنين و عن طريق الكروماتوغرافيا )استشراب الطبقة الرقيقة( تم تشخيصها ه المركباتطوات وهذخ بعدة وسطة أظهرت فعالية مت (8-5) اتالمركب. تم تقييم فعالياتها المضادة للبكتريا باستخدام طريقة االنتشار (8-5) اتالمركبالنووي المغناطيسي للبروتون. ولكنها كانت أقل من فعالية السفيكزيم والنيتروفيورانتوين حيث البكتريا االشريكية القولونيةيمكن مقارنتها بفعالية االموكسسلين وااليزونيازايد ضد البكتريا الكلبسيلة الرئويةيمكن مقارنتها بفعالية االموكسسلين والسفيكزيم ضد أظهرا فعالية متوسطة ( 7)و( 6) انالمركبكانت فعاليتهما عالية. يمكن مقارنتها بفعالية أظهرا فعالية متوسطة ( 7)و( 6) انالمركبولكنها كانت أقل من فعالية النيتروفيورانتوين حيث كانت له فعالية عالية. كانت له فعالية (6)المركب لية عالية. بينما السفيكزيم والنيتروفيورانتوين كانت لهما فعا البكتيريا الكروية العنقودية الذهبيةاالموكسسلين ضد بينما فعالية االموكسسلين والسفيكزيم والنيتروفيورانتوين كانت فعالية عالية. البكتريا العصوية الرقيقةمتوسطة ضد الكلمات المفتاحية: هيدرازايد, قواعد شف, اوكسادايزول, مضادات بكتيرية. 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, hybridization- combination 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), anti- inflammatory(17), and analgesia.(18) 1corresponding author E-mail: m8mostafa@gmail.com Received:27 /1 /2020 Accepted:20 / 5/2020 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol29iss2pp129-138 Iraqi J Pharm Sci, Vol.29(2) 2020 New vanillic acid derivatives as antibacterials 130 Material and Methods Chemicals supplied by hyper-chem (China) were used. Aluminum sheets pre-coated with Silica gel GF254 (type 60), exposed to UV-254 nm, were used for thin-layer chromatography (TLC) to monitor the completion of reactions and to test the purity of compounds. Two solvent systems (S1 and S2) were used: S1(toluene:ethylacetate:ethanol (3:2:1)) and S2(ethylacetate:methanol:ammonia (5:3.5:1.5)). Melting points were incorrected and detected by using Stuart SMP3 melting point apparatus in open capillary tubes. All synthesized derivatives were characterized by spectroscopic analysis (Fourier-transform Infrared (FTIR) which was performed at Baghdad university-college of pharmacy) and (Proton nuclear magnetic resonance (1HNMR) which was performed by Chemistry Analysis Center (CAC)) at Iraq/ Baghdad (19-22) . Chemical synthesis The target compounds were synthesized by multistep(s) reactions as shown in the scheme in Figure (1). Figure 1. Multistep(s) scheme for synthesis of targeted compounds. Synthesis of Methyl 4-hydroxy-3- methoxybenzoate; 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). Iraqi J Pharm Sci, Vol.29(2) 2020 New vanillic acid derivatives as antibacterials 131 Synthesis of 4-Hydroxy-3- methoxybenzohydrazide; compound (2)(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). Synthesis of 4-(5-Mercapto-1,3,4-oxadiazol-2-yl)- 2-methoxyphenol; compound (3)(25): 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). Synthesis of Ethyl 2-((5-(4-hydroxy-3- methoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)acetate; compound (4)(26): Compound (3), (0.5g, 2.23mmole,) was dissolved in 2ml of absolute ethanol (99.9%), (0.155g, 1.115mmole) of anhydrous K2CO3 was added with stirring, after few minutes a white precipitate was formed, (0.37241g, 2.23mmole) of ethyl 2-bromoacetate was added dropwise. The reaction mixture refluxed for 2 hours (monitored by TLC) and left for stirring gently overnight. Then distilled water (D.W) was added gradually until precipitate was formed; then filtered, dried and crystallized from minimum amount (the amount that was just covered the powder) of boiled absolute ethanol giving 0.5g of pure product; compound (4). Synthesis of 2-((5-(4-Hydroxy-3-methoxyphenyl)- 1,3,4-oxadiazol-2-yl) thio) acetohydrazide; compound (5)(27): Compound (4), (0.5g, 1.6167mmole) was dissolved in a 8ml of hot absolute ethanol (99.9%), (0.16167g, 3.233mmole) of 80% hydrazine hydrate was then added, after 2 hours of vigorous mixing at room temperature, precipitate was formed, and left to stir gently overnight. Then, washed with (10ml) absolute ethanol, filtered, dried, and used without further purification. Synthesis of N'-(4-hydroxy-3- methoxybenzylidene)-2-((5-(4-hydroxy-3- methoxyphenyl)-1,3,4-oxadiazol-2-yl) thio)acetohydrazide; compound (6), N'- ((1H-pyrrol-2-yl)methylene)-2-((5-(4- hydroxy-3-methoxyphenyl)-1,3,4-oxadiazol- 2-yl)thio)acetohydrazide; compound (7), and 2-((5-(4-hydroxy-3-methoxyphenyl)- 1,3,4-oxadiazol-2-yl)thio)-N'-(4- methoxybenzylidene)acetohydrazide; compound (8)(28): An appropriate aldehydes; 4-hydroxy-3- methoxybenzaldehyde (Vanillin) (0.039g, 0.257mmole) for compound (6), 1H-pyrrole-2- carbaldehyde (0.0257g, 0.257mmole) for compound (7), and 4-methoxybenzaldehyde (p- Anisaldehyde) (0.035g, 0.257mmole) for compound (8), were dissolved in 5ml of absolute methanol (99.8%) and stirred for 15-30 minutes in the presence of 2-3 drops of glacial acetic acid; then (0.075g, 0.25mmole) of compound (5) was added and the suspension that formed had been refluxed for 1-2 hours. The reaction mixture was left to stir overnight, then the formed precipitate was filtered, dried and crystallized from boiled methanol. Antibacterial essay Well diffusion assay was carried out through using bacterial suspension of nearly (1.5×108CFU/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) Results Chemistry Compound (1) (C9H10O4): off-white powder, yield 91%, m.p: 60-62˚C (reported m.p: 64˚C)(30). The characteristic bands in FTIR spectrum in cm-1: (1686) C=O stretching vibration band, (1277) C-O stretching vibration band of aromatic ester. 1HNMR (500MHz, DMSO-d6) in ppm: 3.80, 3H, s, set (a) protons; 3.83, 3H, s, set (b) protons; 6.87- 6.89, 1H, d, set (c) proton; 7.44-7.49, 2H, m, set (d) protons; 9.97, 1H, s, set (e) proton. Compound (2) (C8H10N2O3): white powder, yield 70%, m.p: 208-210˚C (reported m.p: 210-211˚C)(31). The characteristic bands in FTIR spectrum in cm-1: (3310) phenolic OH stretching vibration band overlapped with NH2 asymmetric stretching vibration band, (3256) NH amide stretching Iraqi J Pharm Sci, Vol.29(2) 2020 New vanillic acid derivatives as antibacterials 132 vibration band, (3209) NH2 symmetric stretching vibration band, (1628) stretching vibration band of carbonyl amide, (1601) NH bending vibration band of hydrazide amine, (1585) NH bending vibration band of hydrazide amide. 1HNMR(400MHz, DMSO-d6) in ppm: 3.82, 3H, s, set (a) protons; 4.42, 2H, s, set (b) protons; 6.81- 6.83, 1H, d, set (c) proton; 7.33-7.44, 2H, m , set (d) protons; 9.56, 2H, s, set (e) protons. Compound (3) (C9H8N2O3S): faint yellow powder, yield 59%, M.P: 186-189˚C. The characteristic bands in FTIR spectrum in cm-1: (3452) phenolic OH stretching vibration band, (3155) NH broad (br) stretching vibration band, 2669 weak (w) SH stretching vibration band. 1HNMR(400MHz, DMSO-d6) in ppm: 3.84, 3H, s, set (a) protons; 6.92-6.94, 1H, d, set (b) protons; 7.30-7.37, 2H, m, set (c) protons; 10.04, 1H, s, set (d) proton; 14.58, 1H, s, set (e) proton. Compound (4) (C13H14N2O5S): white crystalline powder, yield 65.5%, m.p: 118-120˚C. The characteristic bands in FTIR spectrum in cm-1: (3400-2800) broad phenolic OH stretching vibration band, (1744) carbonyl stretching vibration band of aliphatic saturated ester, (1173) stretching vibration band of C-O of aliphatic saturated ester. 1HNMR(500MHz, DMSO-d6) in ppm: 1.18-1.21, 3H, t, set (a) protons; 3.86, 3H, s, set (b) protons; 4.14-4.18, 2H, q, set (c) protons; 4.26, 2H, s, set (d) protons; 6.95-6.96, 1H, d, set (e) proton; 7.41-7.43, 2H, m, set (f) protons; 9.97, 1H, s, set (g) proton. Compound (5) (C11H12N4O4S): white powder, yield 65%, m.p: 196-199˚C. The characteristic bands in FTIR spectrum in cm-1: (3506) stretching vibration band of phenolic OH, (3341) asymmetric NH2 stretching vibration band, (3256) NH amide stretching vibration band, (3217) symmetric NH2 stretching vibration band, (1682) C=O carbonyl amide stretching vibration band, (1655) NH2 bending vibration band. 1HNMR(500MHz, DMSO-d6) in ppm: 3.86, 3H, s, set (a) protons; 4.00, 2H, s, set (b) protons; 4.36, 2H, s, set (c) protons; 6.93-6.95, 1H, d, set (d) proton; 7.41-7.44, 2H, m, set (e) protons; 9.41, 1H, s, set (f) proton; 9.93, 1H, s, set (g) proton. Compound (6) (C19H18N4O6S): white powder, yield 40%, m.p: 196-200˚C. The characteristic bands in FTIR spectrum in cm-1: (3449) two Phenolic OHs stretching vibration bands, (3182) NH secondary amide stretching vibration band, (1678) C=O amide carbonyl stretching vibration band, (1601) C=N imine stretching vibration, C=N stretching vibration of oxadiazole and C=C aromatic skeletal stretching vibration (overlapped) bands. 1HNMR(500MHz, DMSO-d6) in ppm: 3.81, 3.80, 3H, 2s , set (a) protons [syn/anti-syn]; 3.83, 3H, s, set (b) protons; 4.16, 4.61, 2H, 2s, set (c) protons [syn/anti-syn]; 6.80-6.83, 1H, m, set (d) proton; 6.90-6.93, 1H, m, set (e) proton; 7.07-7.09, 1H, m, set (f) proton; 7.26-7.27, 1H, m , set (g) proton; 7.39-7.42, 2H, m, set (h) protons; 7.91, 8.08, 1H, 2s, set (i) proton [syn/anti-syn]; 9.52, 1H, s, set (j) proton; 9.94, 1H, s, set (k) proton; 11.60, 11.63, 1H, 2s, set (l) proton [cis/trans]. Compound (7) (C16H15N5O4S): white powder, yield 53.4%, m.p: 226-228˚C. The characteristic bands in FTIR spectrum in cm-1: (3302) phenolic OH and NH pyrrole stretching vibration (overlapped) bands, (3209) NH amide stretching vibration band, (1663) amide carbonyl stretching vibration band, (1620) NH pyrrole bending vibration and imine stretching vibration (overlapped) bands. 1HNMR(500MHz, DMSO-d6) in ppm: 3.83, 3H, s, set (a) protons; 4.17, 4.60, 2H, 2s, set (b) protons [syn/anti-syn]; 6.11-6.14, 1H, m, set (c) proton; 6.44-6.49, 1H, m, set (d) proton; 6.90-6.95, 2H, m, set (e) protons; 7.40-7.43, 2H, m, set (f) protons; 7.86, 8.04, 1H, 2s, set (g) proton [syn/anti-syn]; 9.94, 1H, s, set (h) proton; 11.40, 1H, s, set (i) proton; 11.45, 11.48, 1H, 2s, set (j) proton [cis/trans]. Compound (8) (C19H18N4O5S): White powder, yield 72%, m.p: 190-193˚C. The characteristic bands in FTIR spectrum in cm-1: (3495) Phenolic OH stretching vibration band, (3182) NH secondary amide stretching vibration band, (1666) C=O amide carbonyl stretching vibration band, (1609) C=N imine stretching vibration, C=N stretching vibration of oxadiazole and C=C aromatic skeletal vibration (overlapped) bands. 1HNMR(500MHz, DMSO-d6) in ppm: 3.79, 3.80, 3H, 2s , set (a) protons [syn/anti-syn]; 3.83, 3H, s, set (b) protons; 4.18, 4.59, 2H, 2s, set (c) protons [syn/anti-syn]; 6.90-7.01, 3H, m, set (d) protons; 7.39-7.42, 2H, m, set (e) protons; 7.61-7.65, 2H, m, set (f) protons; 7.97, 8.14, 1H, 2s, set (g) proton [syn/anti-syn]; 9.94, 1H, s, set (h) proton; 11.64, 11.69, 1H, 2s, set (i) proton [cis/trans]. 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 table (1), compound (5) showed moderate antibacterial activity against Escherichia coli (E. coli). Compound (6) showed moderate activity towards Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Escherichia coli (E. coli), and Klebsiella pneumoniae (K. pneumoniae). Compound (7) showed moderate activity towards Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Klebsiella pneumoniae (K. pneumoniae). Compound (8) was slightly active against Staphylococcus aureus (S. aureus) and moderately active against Escherichia coli (E. coli). Iraqi J Pharm Sci, Vol.29(2) 2020 New vanillic acid derivatives as antibacterials 133 No one of the synthesized compounds had shown activity against Streptococcus pyogenes (S. pyogenes) and Pseudomonas aeruginosa (P. aeruginosa). Table 1.The antibacterial activities of synthesized compounds. Comp. no. Conc. μg/ml Gram (+)ve Gram (-)ve S. aureus S. pyogenes B. subtilis E. coli K. pneumoniae P. aeruginosa Zone of inhibition(ZI) in (mm) Comp.5 103 - - - 12 - - Comp.6 103 11 - 13 11 11 - Comp.7 103 10 - - 11 10.5 - Comp.8 103 9.5 - - 11 - - Amoxicillin 103 13 5 40 11 11.5 28 Cefixime 103 16 6 20 22 13 6 Nitrofurantoin 103 21 9.5 31 16 20 17 Isoniazid 103 8 - - 12 - - DMSO Solvent control - - - - - - (-)= No activity, slightly active (ZI =5-10 mm), moderately active (ZI= 10-15 mm), highly active (ZI= more than 15 mm).(32,33) Discussion 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 1HNMR 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 Iraqi J Pharm Sci, Vol.29(2) 2020 New vanillic acid derivatives as antibacterials 134 normal basic condition in which the rate- determining 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). Figure 3. Hydrazinolysis of ester (35). 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-1C=O stretching vibration band of amide, and 1601cm-1NH2 bending vibration band. 1HNMR 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. 1HNMR was revealed the presence of new signals at 9.41 ppm and 4.36 ppm related to CONH and CONHNH2, respectively. Synthesis of compound (3) had been carried out by refluxing an ethanolic suspension of compound (2) with CS2, in the presence of (KOH) and the cyclization which involved formation of potassium salt of dithiocarbamate as an intermediate could be done through keto form of hydrazide carbonyl or enol form. Enol form is the preferred explanation because of the stability of enol form by intramolecular hydrogen bonding.(36) Figure 4. enol form of dithiocarbamate stabilized by intramolecular hydrogen bonding.(36) FTIR spectrum of compound (3) was characterized by NH stretching vibration band at 3155cm-1, weak SH stretching vibration band at 2669cm-1, and in addition to the absence of amide band at 1628 cm-1. 1HNMR was characterized by the appearance of new signal related to SH proton at 14.58 ppm. Compound (4) was obtained by a nucleophilic substitution (SN2) reaction between compound (3) and ethyl 2-bromoacetate in absolute ethanol and in the presence of anhydrous potassium carbonate as a catalyst. Figure 5. synthesis of compound (4) by nucleophilic substitution (SN2) reaction (37). FTIR spectrum was characterized by 1744cm-1 stretching vibration band of saturated ester carbonyl. 1HNMR 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). Iraqi J Pharm Sci, Vol.29(2) 2020 New vanillic acid derivatives as antibacterials 135 Figure 6. steps of Schiff base (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. 1HNMR 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- 42) Antibacterial activities Four antibacterial standards were used, amoxicillin to compare anti-gram (+)ve activities of the derivatives with it; cefixime to compare anti- gram(-)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. 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