Multi-drug resistant microbial infection, a major problem in the treatment of infectious disease, is increasing considerably from the last decades (Ahmad et al., 2012; Makki et al., 2015) and to overcome this problem, development of new antimicrobial agents is the need of the hour (Lindahl, 2015; Venkatesh et al., 2016). Hydrazones, belonging to Schiff base family with chemical formula R2C=NNR2, are of incredible interest because of their various biological activities reported in literature, viz., anti-bacterial, anti-fungal, anti-convulsant, anti-inflammatory, anti-cancer, anti-viral, anti-malarial & anti-tuberculosis, anti-oxidant, and antidepressant (Alam et al., 2012; Kumar and Chauhan, 2014; Marwa and Ahmed, 2018; Neda et al., 2018; Nurkenov et al., 2017; Popiolek, 2017; Rayam et al., 2015; Verma et al., 2014). Along with the diverse activities, their ease of synthesis, increased stability, and tendency toward crystallinity make hydrazones a perfect choice to synthesize more effective and novel antimicrobial agents (Bala et al., 2011; 2013; Singh et al., 2016).
The antimicrobial (Chaudhary et al., 2008; Kapoor and Dahiya, 2016; Khatkar et al., 2017) and anti-oxidant (Velika and Kron, 2012; Pontiki and Litina, 2019) perspective of organic acids can be well recognized from the literature. Phenolic compounds (Abouzeed et al., 2018; Benslama et al., 2017; Fu et al., 2016; Sahloul et al., 2014) are also proved as successful antibacterial as well as anti-oxidant activity which might be due to the fact that anti-oxidants act by creating scavenging environment which may inhibit bacterial growth. p-hydroxy benzoic acid, a phenolic derivative, is also found to possess various pharmacological activities, viz., antimicrobial, antialgal, antisickling, antimutagenic, antiviral, anti-inflammatory, anti-oxidant, etc., (Manuja et al., 2013). Antimicrobial potential of p-hydroxy benzoic acid derivatives, viz., NÊ¹-(4-methoxybenzylidene)-4-hydroxybenzohydrazide, NÊ¹-benzylidene-4-hydroxybenzohydrazide have also been reported (Sapra et al., 2014; Suzana et al., 2017). Therefore, the present study was designed with the aim of synthesizing novel hydrazone derivatives of p-hydroxy benzoic acid for exploring their antimicrobial and anti-oxidant potential.
MATERIALS AND METHODS
Ester of 4-hydroxy benzoic acid was synthesized using Fischer esterification which was further treated with hydrazine hydrate in ethanol to yield corresponding 4-hydroxybenzoic acid hydrazides. The synthesized hydrazides were further reacted with substituted aldehydes (aromatic) in presence of glacial acetic acid (2–3 drops) to yield 4-hydroxy-NÊ¹-[(1E)-substituted phenylmethylidene] benzohydrazide (AR1–AR10) (Scheme 1) which were then characterized by spectral and analytical means (Harer et al., 2010; Narang et al., 2012; Rajput and Rajput, 2009).
Evaluation of antibacterial activity
Antibacterial activity of synthesized products was evaluated against Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), and Enterococcus faecalis (ATCC 29212) using Norfloxacin standard as it is the most widely used antibiotic in bacterial infections (Table 2) (Cappucino and Sherman, 1999) by tube dilution method using double strength nutrient broth IP followed by incubation at 37°C ± 1°C.
Hydrogen peroxide radical scavenging (H2O2) assay
Hydrogen peroxide plays an important role as bactericidal agent (Halliwell, 1995) by acting directly or indirectly via its reduction product, OH- (second messenger in synthesis and activation of several inflammatory mediators) (Sprong et al., 1998).
Anti-oxidant activity by the H2O2 method was determined as per the method reported (Ruch et al., 1989). Different dilutions of synthesized (20–60 μg/ml), as well as standard compound (Ascorbic acid) in distilled water, were added to 40 mM H2O2 solution prepared in phosphate buffer and absorbance was measured at 230 nm after 10 minutes against a blank solution.
DPPH radical scavenging activity
DPPH radical scavenging, a standard rapid assay for antioxidant studies (Soares et al., 1977), was done by adding ethanolic DPPH solution (0.1 Mm) to test solutions as well as standard compound to make different dilutions (20–60 μg/ml) and then by measuring their absorbance at 517 nm after 30 minutes. Scavenging activity in both methods was expressed as inhibition percentage and calculated using the following formula:
% inhibition = (Ao−At)/ Ao × 100
where Ao is the absorbance control (blank); At is the absorbance of the test (Shukla et al., 2009).
|Scheme 1. Synthesis of 4-hydroxy-N'-[(1E)-substituted-phenylmethylidene] benzohydrazide analogs.|
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RESULTS AND DISCUSSION
Melting points of synthesized compounds were determined (Elico melting point apparatus, India) and purity was ascertained using single spot TLC (Table 1). The structures were confirmed by spectral studies (IR spectra on FTIR-Shimadzu spectrometer and 1H NMR spectra on BRUKER AVANCE II 400 NMR spectrometer using DMSO). The data obtained from spectral studies were in agreement with assigned molecular structures.
Analytical data for compound AR1
M.P.(°C): 243–247; Yield: 77.4%; IR (cm−1): 1,738.90 (C=O stretch), 1,627.99 (C=N stretch), 3,731.45 (OH stretch), 1,547.47 (C=C stretch), 3,227.35 (NH stretch), 2,995.45 (C-H stretch) ; 1H NMR: (δ) 9.9 (s, 1H, N=CH), 8.4 (s, 1H, NH-N=), 7.8 (d, 2H, Ar-H), 7.5 (m, 2H, Ar-H), 7.4 (m, 3H, Ar-H), 6.9 (m, 2H, Ar-H), 5.2 (s, 1H, OH).
Analytical data for compound AR2
M.P.(°C): 211–214; Yield: 72%; IR (cm−1): 1,720.13 (C=O stretch), 1,625.98 (C=N stretch), 3,636.48 (OH stretch), 3,279.74 (NH stretch), 3,035.96 (Aromatic CH stretch), 1,521.84 (Aromatic C=C stretch), 671.23 (C-Cl stretch), 2,961.45 (C-H stretch), 630.42 (CH Rocking), 1,148.46 (C-C stretch); 1H NMR: (δ) 9.1 (s, 1H, -NH-N=), 8.1 (s, 1H, -N=CH-), 7.8 (d, 2H, Ar-H), 7.6 (d, 2H, Ar-H), 7.3 (d, 2H, Ar-H), 6.9 (d, 2H, Ar-H), 5.4 (s, 1H, OH).
Analytical data for compound AR3
M.P. (°C): 225–227; Yield: 56.5%; IR (cm−1): 1,706.11 (C=O stretch), 1,665.45 (C=N stretch), 3,715.31 (OH stretch), 3,269.13 (NH stretch), 3,056.34 (Aromatic CH stretch), 1,504.54 (Aromatic C=C stretch), 2,921.32 (C-H stretch), 640.39 (CH Rocking), 1,148.46 (C-C stretch); 1H NMR: (δ) 8.0 (s, 1H, -NH-N=), 8.1 (s, 1H, -N=CH), 7.8 (d, 2H, Ar-H), 7.5 (d, 2H, Ar-H), 7.1 (d, 2H, Ar-H), 6.9 (d, 2H, Ar-H), 5.1 (s, 1H, OH).
Analytical data for compound AR4
M.P. (°C): 170–173; Yield: 55.3%; IR (cm−1): 1,717.68 (C=O stretch), 1,627.99 (C=N stretch), 3,558.27 (OH stretch), 3,237.65 (NH stretch), 1,573.98 (NH band), 3,024.51 (Aromatic CH stretch), 1,544.08 (Aromatic C=C stretch), 1,302.01 (C-O stretch), 3,042.44 (C-H stretch), 659.68 (CH Rocking). 1,023.28 (C-C stretch); 1H NMR: δ 9.2 (s, 1H,-NH-N=), 8.1 (s, 1H,-N=CH-), 7.8 (d, 2H, Ar-H), 7.0 (m, 2H, Ar-H), 6.9 (d, 2H, Ar-H), 6.7 (d, 2H, Ar-H), 5.6 (s, 1H, OH), 3.7 (s, 3H, -OCH3).
Analytical data for compound AR5
M.P. (°C): 247–249; Yield: 67.9%; IR (cm−1): 1,710.70 (C=O stretch), 1,638.00 (C=N stretch), 3,562.60 (OH stretch), 1,560.92 (NH band), 3,081.60 (Aromatic CH-stretch), 1,590.92 (NH band), 1,545.65 (C=C stretch), 636.51 (CH rocking); 1H NMR: δ 9.8 (s,1H,NH), 8.4 (s,1H,OH), 7.8 (d, 2H, Ar-H), 7.7 (d, 2H, Ar-H), 7.6 (d, 2H, Ar-H), 7.3 (d, 2H, Ar-H), 7.1 (d, 2H, Ar-H), 7.0 (s, 1H, OCH3), 6.8 (d, 2H, Ar-H).
Analytical data for compound AR6
M.P. (°C): 177–177; Yield: 87.8%; IR (cm−1): 1,690.68 (C=O stretch), 1,636.67 (C=N stretch), 3,554.41 (OH stretch), 3,277.20 (NH stretch), 3,037.05 (Aromatic CH stretch), 1,540.23 (Aromatic C=C stretch), 1,092.72 (C-O stretch), 2,772.79 (C-H stretch), 659.68 (CH Rocking), 1,033.89 (C-C stretch); 1H NMR: δ 8.0 (s, 1H, -NH-N=), 8.1 (s, 1H, -N=CH-), 7.7 (d, 2H, Ar-H), 7.1 (m, 2H, Ar-H), 7.2 (m, 2H, Ar-H), 6.9 (d, 2H, Ar-H), 5.4 (s, 1H, OH), 3.7 (s, 3H, -OCH3).
|Table 1. Physical data of synthesized derivatives. |
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|Table 2. Antibacterial activity of synthesized compounds. |
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Analytical data for compound AR7
M.P. (°C): 164–168 (°C); Yield: 74.9%; IR (cm−1): 1,730.11(C=O stretch), 1,690.25(C=N stretch), 3,675.15(OH stretch), 3,234.45 (NH stretch), 3,024.11 (Aromatic CH stretch), 1,560.45(Aromatic C=C stretch), 1,290.25 (C-O stretch), 2,755.31(C-H stretch); 1H NMR: δ 10.5 (s, 1H, CH), 9.9 (s, 1H, NH), 7.9 (s, 1H, OH), 6.8 (d, 2H, Ar-H), 3.1 (s, 6H, 2OCH3), 2.4 (d, 2H, Ar-H), 2.2 (s, 3H, CH3).
Analytical data for compound AR8
M.P. (°C): 212–215; Yield: 59.8%; IR (cm−1): 1,690.68 (C=O stretch), 1,691.64 (C=N stretch), 3,758.27 (OH stretch), 3,282.99 (NH stretch), 1,576.87 (NH band), 3,026.44 (Aromatic CH stretch), 1,544.68 (Aromatic C=C stretch), 1,105.26 (C-O stretch), 2,779.54 (C-H stretch), 660.65 (CH Rocking), 1,026.17 (C-C stretch); 1H NMR: δ 9.6(s, 1H, NH), 8.4(s, 1H, OH), 7.8(d, 2H, Ar-H), 7.7(d, 3H, Ar-H), 7.6(d, 2H, Ar-H), 7.5 (d, 2H, Ar-H), 3.2(s, 3H, CH3).
Analytical data for compound AR9
M.P. (°C): 125–128; Yield: 48.1%; IR (cm−1): 1,705.10 (C=O stretch), 1,645.74 (C=N stretch), 3,575.45 (OH stretch), 3,258.90 (NH stretch), 3,021.45 (Aromatic CH stretch), 2,650.45 (C-H stretch), 1,510.64 (Aromatic C=C stretch), 675.71 (CH Rocking). 1,019.21 (C-C stretch); 1H NMR: δ 7.8 (d, 2H, Ar-H), 7.7 (d, 2H, Ar-H), 7.4 (m, 2H, Ar-H), 6.9 (d, 2H, Ar-H), 7.0 (s, 1H, -NH-N=), 5.1 (s, 1H, OH).
Analytical data for compound AR10
M.P. (°C): 112–115; Yield: 97.7%; IR (cm−1): 1,720.18 (C=O stretch), 1,685.71 (C=N stretch), 3,658.27 (OH stretch), 3,368.85 (NH stretch), 3,156.44 (Aromatic CH stretch), 1,642.55 (Aromatic C=C stretch), 1,576.87 (NH band), 3,079.54 (C-H stretch), 742.32 (C-Cl stretch), 670.55 (CH Rocking). 1,026.17 (C-C stretch); 1H NMR: δ 7.8 (d, 2H, Ar-H), 7.3 (m, 2H, Ar-H), 7.2 (m, 2H, Ar-H), 6.9 (d, 2H, Ar-H), 7.0 (s, 1H, -NH-N=), 5.1 (s, H, OH), 1.3 (s, 3H, -CH3)
In E. coli, compounds AR7 (pMIC = 1.420) and AR10 (pMIC = 1.412); in P. aeruginosa, compounds AR1 (pMIC = 1.62) and AR6 (pMIC = 1.284); in S. aureus, compounds AR7 and AR8 and in E. faecilis, compounds AR4, AR7, and AR8 were emerged as the most active one.
From antibacterial studies, compound AR7, NÊ¹-(3,4,5-trimethoxybenzylidene)-4-hydroxybenzohydrazide in case of E. coli, S. aureus, and E. faecilis and compound AR6, NÊ¹-(3-methoxybenzylidene)-4-hydroxybenzohydrazide in case of P. aeruginosa were emerged as the most active compound indicating that methoxy group (e-donating group) on benzene ring is essential for antibacterial activity (Emami et al., 2008). All compounds show good antibacterial property which may be due to the presence of benzylidene/1-phenyl-ethylidene which imparts lipophillicity, a parameter responsible for penetration of molecules across the microbial membrane. In case of E. coli, the compound AR10 containing electron withdrawing chlorine group was also found effective which confirmed the fact that different structural requirements are requisite for activity against different microorganisms (Sortino et al., 2011). The above facts are summarized in Figure 1.
To verify the fact that antibacterial compounds also possess anti-oxidant activity, the synthesized compounds were evaluated and it was observed that compound AR10 possesses maximum anti-oxidant activity followed by compounds AR3 and AR7 as governed by hydrogen peroxide scavenging activity method, whereas in DPPH scavenging activity, compound AR8 possesses maximum antioxidant activity followed by compound AR9 > AR10. All these compounds show even better antioxidant activity than reference compound Ascorbic acid as depicted by their IC50 (Table 3; Fig. 2a and b)
The high activity of compounds AR2 and AR10 can be explained by the fact that halogens like chloro enhance antioxidant activity due to its redox activity through one electron transfer mechanism (Bala et al., 2012; Dudhe et al., 2013; Hossain et al., 2009). The high activity of compounds AR3, AR7, AR6, AR8, and AR9 can be explained by the fact that methoxy and alkyl/aryl enhanced the stabilization of the generated radical during oxidation (Hadi et al., 2013). There is no correlation between results obtained by both methods, which governs that completely different mechanisms are involved in these two anti-oxidant determination methods.
|Figure 1. SAR studies.|
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|Table 3. IC50 of synthesized derivatives. |
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|Figure 2. (a and b) IC50 of synthesized compounds (H2O2 and DPPH method).|
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A series of 4-hydroxy-NÊ¹-[(1E)-substituted-phenylmethylidene] benzohydrazide derivatives were synthesized and evaluated for its antibacterial and anti-oxidant activity. Compound AR7 was found most potent antimicrobial against E. coli, S. aureus, E. faecilis, and and compound AR6 against P. aeruginosa which might be due to the presence of methoxy group (electron donating group) on benzene ring indicating the importance of electronic parameters in governing potency. Compounds AR10 and AR8 show maximum anti-oxidant activity when governed by hydrogen peroxide and DPPH scavenging activity, respectively, signifying different mechanisms are involved in antioxidant determination.
The authors would like to thank the M. M. College of Pharmacy, M. M. (Deemed to be University), Mullana, Ambala for providing support and facility to carry out research.
CONFLICT OF INTEREST
The authors declared no conflict of interest.
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