Synthesis, Antimicrobial Evaluation and Docking Studies of Novel 4-acetamido-3-aminobenzoic acid derivatives As Microbial Neuraminidase Inhibitors

 

Mukesh Kumar Gupta

Institute of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (A Central University),

Bilaspur - 495009, Chhattisgarh, India

 

ABSTRACT:

Background: Neuraminidase has been considered as vital target for neuraminidase inhibitors (NAI) and designing drug against influenza like new virus strains. The objectives of research work is to synthesized novel 4-acetamido-3-(benzylideneamino)benzoic acid derivatives against microbial  neuraminidase (NA) enzyme. Methods: The series of Imine/Schiff base compound were synthesized by reaction of various aldehydes with 4-acetamido-3-amino benzoic acid. Compounds were characterized by IR, ¹H NMR and, ¹³C NMR. Evaluation of synthesized compounds was done against neuraminidase contained bacterial as well as fungal strains. Results: The Compounds 5k-5q, 5x and 5y of novel 4-acetamido-3-aminobenzoic acid derivatives were characterized as potent inhibitory action against the NA contained microbes. The zone of inhibition of these compounds at 125 µg/ml was found (16 ± 2.5), which is more than the standard compounds. Molecular docking of synthesized compounds 5k to 5q showed the highest docking score (> -9 Kj/mol). Conclusion: The compounds 5k-5q, 5x and 5y of novel 4-acetamido-3-aminobenzoic acid derivatives displayed potential neuraminidase inhibition against NA containing microbes. The molecular docking studies predict their binding mode interaction of NA inhibitor. These studies may be further employed, against the neuraminidase contained flu.

 

 

 

1. INTRODUCTION:

Neuraminidase (NA) is an attractive molecular target for the development of drugs that help to combat influenza (H1N1) infection. NA and Hemagglutinin (HA) are two vital glycoproteins present on the surface of influenza virus which plays an important role in transferring the virus from one cell to another cell by cleaving of neurominic acid (sialic acid) of host respiratory cell surface that causes severe respiratory infection [1, 2]. Neuraminidase sequences along with hemagglutinin make various subtypes of flu strains like H1N1, H5N1, H3N2, H7N7, H5N7, H7N9 etc., and cause pandemic as well as endemic diseases worldwide.

 

The NA inhibitors oseltamivir and zanamivir, are sialic acid analogs approved for the treatment of influenza [3, 4, 5].  Due to the mutation in neuraminidase enzyme, the current NA inhibitors seem to be ineffective because of drug resistance against the NA. So presently, there is no any effective drug or vaccine is available for the treatment of influenza caused by these new strains [5]. Therefore, scientists are trying to discover and develop new therapeutics which can act effectively against these resistant strains. In the present study, our aim is to design, synthesis and antimicrobial evaluation for the search of potential neuraminidase inhibitors with the hope to act as neuraminidase inhibitors for the treatment of flu.

 

The neuraminidase (NA) is also called sialidase present on the surface of bacterial, fungal, protozoan, trypnosomal and, mammalian cells, which have the similar structural and function of that of flu virus. All neuraminidase/sialidase has the potency to bind and cleaves sialic acid from various glycoconjugates in human respiratory epithelial cells surface and causes various pathological problems [6, 7, 8]. NA contains Streptococcus pneumonia prone to synergetic effect in influenza infection by cleaving the sialic acid bond during respiratory infection. In view of the presence of NA in microbes, it may take as target for the NA inhibitors and serve as the best and economic tool to screen neuraminidase inhibitors and may fight against the influenza neuraminidase [9]. Therefore in this work, we selected NA containing bacterial (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Vibrio cholera, Streptococcus pneumonia, Staphylococcus aureus) and fungal strains (Aspergillus nigar Candida. albicans, Aspergillus fumigatus) for screening NA inhibiting potential of newly synthesized compounds.

 

Various NA inhibitors and its derivatives have been developed for NA but resistance against these drugs in many viral strains makes it problems and interesting task to discover novel compounds as NA inhibitors. Zanamivir and oseltamivir were approved drug for the treatment of influenza. The mutation of amino acid (H274Y) of NA causes drug resistance against NA, thus it caused new challenged for design drug against NA. The structure activity relationship (SAR) studies of NA inhibitors showed that carboxylic acid and acetamido group with aromatic or hetrocyclic ring are the essential groups for the NA activity (Fig. 1). The hetero ring like pyrene ring flavones, biflavone, thiozolidine, pyrimidine, pyridine, pyrrolidine, dihydropyrane, caffiec acid, creteniside replaced by aromatic which increased the lipophilicity of molecules which important for NA inhibition [1, 4, 11, 12, 13, 14].

 

The aromatic ring like salicylic acid, cyclohexene, and other benzene ring with carboxylic acid and acetamido group found potent NA inhibitors (Fig. 1) [15, 16, 17]. The aromatic ring derivatives increased the lipophilicity of molecules and make the inhibitors more stable and easy synthetically accessible. The designed compound 4-acetammido 3-amino benzoic acid analogs have same binding pose as potent known inhibitors (Fig. 1). However 4-acetamodo-3-amino benzoic acid linked with Schiff base which itself acts as antimicrobial activity and synergetic effect in NA active site. SAR exposed the good platform for the design and easily synthesized new NA inhibitor from abounded commercial available PABA [18, 19, 20, 21, 22].

 

 

Figure 1: Structure of some potent NA inhibitor and designed compound

 

2. MATERIALS AND METHODS:

2.1 Chemistry:

All chemicals and solvents for the synthesis of compounds were used as commercial grade and used further without purification. Characterization of synthesized compounds was done by spectral analysis which recorded on FT-IR Spectrophotometer (SHIMADZU), using KBr powder. ¹H NMR and ¹³C NMR spectra of synthetic compounds were recorded on NMR Spectrometer (Bruker AV 400, at 400. 130 MHz and 100 MHz) using tetramethylsilane as an internal standard. Melting point apparatus was used for the measurement of melting points of all compounds by using open glass capillaries and are uncorrected. Reactions process was monitored by using TLC plates (silica gel G), by using solvents systems hexane: ethyl acetate (4:6 v/v) and benzene: acetone (9:1, v/v). The spots were visualized under UV-light. The synthetic pathway of series compounds (5a-y) is presented in Scheme 1.

 

2.1.1 Acetylation of para amino benzoic acid (starting material) (2):

Para-amino benzoic acid (PABA) 6.86 gm (0.05 mol.) was starting material, mixed with 4% (50ml) sodium hydroxide solution. Mixture was stirred well for 1 hour at room temperature meanwhile added acetyl chloride (2 ml/0.028 mol) followed by acetone (3 ml) drop-wise successively. The resultant solution kept on ultra-sonication for 1 hour. The saturated NaHCO₃ solution was added till the effervescence ceased. The progress of reaction was monitored by TLC at appropriate time interval. The solution was then acidified with conc. HCl. The sparingly soluble acetyl derivative was separated as white solid (2). It was filtered under suction and recrystallized through ethanol. The compounds were characterized by chromatographic under TLC- Hexane: Methanol (6:4). The yield was 72%, m.p. 260-263°C, IR (KBr): υ = 3337, 1708, 1608, 1592 cm^-1, ¹H NMR (300 MHz, DMSO-d6), 11.42 (s, 1H), 8.32, (d, J=8.2 Hz, 2H), 7.45 (d, J=7.78 Hz, 2H), 7.12 (s, 1H), 2.04 (s, 3H).

 

2.1.2 Nitration of 4-acetamido benzoic acid (3):

 The 4-acetamidobenzoic acid (0.05mol) dissolved in nitric acid (7.2 mole, 83.6%). In this solution, a mixture of two parts of sulfuric acid and one part of nitric acid was added limited interval of time till 30 minutes. The temperature was maintained at 4-8°C in the ice bath. A reddish-brown solution was formed, which stirred for one hour at same temperature, the mass was formed, then whole solution mixed with 100 parts of ice water and filter it, the filtrate product was again it washed with 200 parts of distilled water and dried it. After hydrolysis and recrystallization with ethanol, dried the final product which was 4-acetamido-3-nitrobenzoic acid (3) has pale yellow powder (Du Pont, nitration of 4-acetamidobenzoic acid, US3428673, 1989). Chromatography studies- TLC was done in ethyl acetate: methanol (6:4). Yield was 68% , M.P. 209° -212° C, IR (KBr): 3376, 3480, 1710, 1650, 1602, 1344 cm^-1, ¹H-NMR (300 MHz, DMSO-d6), 11.20 (s,1H), 8.69 (m, 1H) 8.54, (d, J=8.2 Hz,1H), 8.17 (d, J= 7.78 Hz, IH), 7.81 (s, 1H),  2.54 (s, 3H). ¹³C-NMR (300MHz, CDCl₃), 164.13, 154.56, 144.44, 143.14, 140.17, 137.36, 122.30, 120.40, 40.02, 39.23 ppm.

 

2.1.3 Reduction of 4-acetamido-3-nitrobenzoic acid (4):

 4-Acetamido-3-nitrobenzoic acid (1.5g, 3.4 mmol) mixed in 10 g  SnCl₂ (61.9 mmol) with ethanol (40 ml). Add the concentrated HCl in mixture to make clear solution and mixture was stirred for 1hour at room temperature. Further mixture was refluxed for 2-3 hours at 80-90 ⁰C. During this refluxed 4-5 drops of conc. HCl was added for acidification. A pale yellow clear solution of 4-acetamido-3-aminobenzoic acid (4) was formed. The mixture was separated by the brine solution and washed with ethyl acetate. TLC was done in ethyl acetate: methanol (6:4). The yield was 58%, M.P. was 215° -218° C, IR (KBr)- 3466, 3156, 3309, 1710, 1667, 1588 cm^-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =10.53 (s, 1H), 8.54 (d, J = 8.1 Hz, 2H), 8.17 (s, 1H), 7.84 (d, J = 8.6 Hz, 1H), 7.10 (s, 1H), 3.46 (s, 2H), 2.03 (s, 3 H). ¹³C NMR ((300 MHz) (DMSO-d6) δ ppm =, 168.76, 165.27, 144.44, 148.64, 141.11, 135.02, 129.56, 125.90, 117.40, 40.04, 38.78, 23.60 ppm.

 

4.1.4 Schiff base formation with aldehydes (General procedures) (5):

A requested of amount of aldehydes (0.25 mmol) dissolved in 200 ml of ethanol was mixed with 0.25 mmol (equimolar) of amine compound (4-acetamido-3-amino benzoic acid) in a 100 ml round bottle flask. The mixture was well stirred at room temprature. Mixture was acetylated with glacial acetic acid as drop wise. Subsequently whole mixture was refluxed for 4-5 hours at 80-90 ⁰C. The reaction mixture was cooled in bath then transferred in separating conical flask and wash with aqueous and ethyl acetate. Organic phase was treated with anhydrous sod.sulphate (Na₂SO₄) then filter it, and re-crystallized the filtrate with ethanol and dried as a brownish white color of Schiff compound formed as yield 60-70%.  All reactions was monitored by TLC in one hour interval of time using ethyl acetate:methanol (7:3), as mobile phase. Acetamido-3-aminobenzaimino derivatives (Schiff base) 5a-5d and 5e-5y were synthesized (Table 2) and its structure was elucidate by IR, and ¹H NMR spectroscopy.

 

Spectral characteristics of synthesized Imine base compounds.:

5a. 4-acetamido-3-(methyleneamino)benzoic acid:

IR v max (KBr) (cm-1): 3446, 3360, 3109, 1680, 1667, 1588 cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =10.73 (s, 1H), 8.01-7.80 (m, 3H), 7.82 (s, IH), 7.23 (s 1H,), 2.72 (s, 3H), M.P.-210-212

 

5b. 4-acetamido-3-(ethylideneamino)benzoic acid:

IR v max (KBr) (cm-1): 3396, 3300, 3112 1680, 1558 cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =10.73 (s, 1H), 8.04-7.88 (m, 3H), 7.80 (s, 1H), 7.43 (t 1H,), 7.23 (s, IH), 2.02 (s, 3H), 1.09 (d, J= 3.54, 2H), M.P. 205-207.

 

5c. 4-acetamido-3-(5-oxohexylideneamino)benzoic acid:

IR v max (KBr) (cm-1): 3396, 3300, 3133 1680, 1558 cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =10.73 (s, 1H), 8.04-7.83 (m, 3H), 7.55 (m, 3H,), 7.22 (s, 1H), 2.45 (m, 3H), 2.04 (s, 3H) , 1.8 (m, 5H), 1.09 (m, 3H), M.P.-218-222.

 

5d. 4-acetamido-3-(but-2-enylideneamino)benzoic acid: IR v max (KBr) (cm-1): 3410, 3330, 3118, 1707, 1638, 1509, 1250 cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm = 10.63 (s, 1H), 8.01-7.8 (m, 3H), 7.78 (s, 1H,), 7.40 (d, J= 21 Hz, IH), 5.77 (m, 1H), 5.03 (m, 2H), 2.09 (s, 3H), 1.85 (s, 1H), M.P.- 248-250.

 

5e. 4-acetamido-3-(benzylideneamino)benzoic acid: IR v max (KBr) (cm-1): 3377, 3350, 3280, 1700, 1645, 1638 cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm = 10.83 (s, 1H), 8.22 (s, IH ), 7.48-8.04 (m, 3H), 7.52-7.60 (m, 5H,),  7.33 (s, 1H), 2.03 (s, 3H), M.P.-200-205.

 

5f. 3-(4-chlorobenzylideneamino)-4-acetamidobenzoic acid: IR v max (KBr) (cm-1): 3455, 3340, 1710, 1607, 737 cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =10.73 (s, 1H), 8.62 (s, 1H), 8.04- 7.77 (m, 3H), 7.73 ( m, 2H), 7.61-7.54 (m, 2H), 7.44 (s, 1H), 2.72 (s, 3H), M.P.- 210-212.

 

5g. 3-(2-dichlorobenzylideneamino)-4-acetamidobenzoic acid: IR v max (KBr) (cm-1): 3415, 3365, 1708, 1607, 737 cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =10.73 (s, 1H), 8.61 (s, IH), 8.02-7.89 (m 1H), 7.79 (s, 1H), 7.44 (d, 6.6 Hz, 1H), 7.30 (t, 1H), 2.02 (s, 3H), M.P.- 215-220.

 

5h. 3-(3-chlorobenzylideneamino)-4-acetamidobenzoic acid: IR v max (KBr) (cm-1): 3385, 3315, 1708, 1617, 727 cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =10.73 (s, 1H), 8.39 (s, 1H), 8.03-7.87 (s, 3H), 8.0 ( m, 1H), 7.9- 7.73 (m 4H,), 2.06 (s, 3H,), M.P.- 208-212.

 

5i. 3-(4-bromobenzylideneamino)-4-acetamidobenzoic acid: IR v max (KBr) (cm-1): 3415, 3365, 1700, 1607, 657 cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =10.77 (s, 1H), 8.54 (s, 1H), 7.84- 8.04 (m, 3H), 7.67 (s 1H,), 7.75 (s, 2H), 7.42 (m, 1H), 2.02 (s, 3H), M.P.- 200-202.

 

5j. 3-(4-methylbenzylideneamino)-4-acetamidobenzoic acid: IR v max (KBr) (cm-1): 3415, 3365, 3010, 1700, 1635 1600, cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =11.01 (s, 1H), 8.34 (s, 1H), 8.24- 7.81 (m, 3H), 8.12 (s 1H,), 7.53 (m, 2H), 7.02 (m, 2H), 2.78 (s, 3H), 2.02 (s, 3H), M.P.- 206-207.

 

5k. 3-(4-methoxybenzylideneamino)-4-acetamidobenzoic acid: IR v max (KBr) (cm-1): 3445, 3265, 2980, 1700, 1730, 1628 1592, cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =10.78 (s, 1H), 8.46 (s, 1H), 8.0 (s, 1H), 8.03-7.8 (m, 3H,), 7.84 (d, j= 6.78Hz 3H,), 7,44 (s, 1H), 3.77 (s, 3H), 2.72 (s, 3H), M.P.-190-194⁰C.

 

5l. (E)-3-(3,4-dimethoxybenzylideneamino)-4-acetamidobenzoicacid: IR v max (KBr) (cm-1): 3415, 3365, 2950, 1710, 1720 (OCH3), 1635 1619, cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =10.78 (s, 1H), 8.46 (s, 1H), 8.0 (s, 1H), 7.8-6.69 (m, 5H,), 7.84 (s, 3H), 3.77 (s, 6H), 2.72 (s, 3H), M.P.-209-211⁰C.

 

5m. Z)-3-(2,3,4-trimethoxybenzylideneamino)-4-acetamidobenzoic acid: IR v max (KBr) (cm-1): 3400, 3355, 2950, 1730, 1686, 1630 1619, cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =10.78 (s, 1H), 8.46 (s, 1H), 8.0 (s, 1H), 7.84 (m, 3H,), 5.61 (s, 1H) 3.47 (s, 9H), 2.72 (s, 3H), M.P.-180-182⁰C.

 

5n. 3-(3,4, 5-trimethoxybenzylideneamino)-4-acetamidobenzoic acid: IR v max (KBr) (cm-1): 3393, 3275, 3050, 1710, 1680, 1635 1619, cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =11.78 (s, 1H), 8.36 (s, 1H), 8.06 (s, 1H), 8.09-7.69 (m, 5H,), 6.84 (m, 2H,), 3.75 (s, 9H), 2.06 (s, 3H), M.P.-200-204⁰C.

 

5o. Z)-3-(2,4, 6-trimethoxybenzylideneamino)-4-acetamidobenzoic acid: IR v max (KBr) (cm-1): 3435, 3265, 3010, 1720, 1708, 1635 1619, cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =10.78 (s, 1H), 8.46 (s, 1H), 8.0 (s, 1H), 7.8-6.69 (m, 5H,), 7.84 (d, j= 6.78Hz 2H), 3.95 (s, 9H), 2.72 (s, 3H), M.P.-188-191⁰C.

 

5p. 4-Aetamido-3-((3-bromo-4-methoxybenzylidene)aminobenzoic acid: IR v max (KBr) (cm-1): 3408, 3265, 3000, 1720, 1628 1592, cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm = 10.78 (s, 1H), 8.46 (s, 1H), 8.06-8.0 (m, 2H,), 8.0 (s, 1H), 7.80-7.02 (m, 3H ),  3.75 (s, 3H), 2.72 (s, 3H), M.P.-210-212⁰C.

 

5q. (E)-4-acetamido-3-((4-hydroxy-3-methoxybenzylidene amino)benzoic acid:  IR v max (KBr) (cm-1): 3505, 3245, 2980, 1730, 1678, 1605 cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =10.88 (s, 1H), 8.33 (s, 1H), 8.01-7.79 (m, 3H,), 7.68 (s, 1H), 7.46- 7.05 (m, 3H), 5.63 (s,) 1H), 3.68 (s, 3H), 2.72 (s, 3H), M.P.-175-178⁰C.

 

5r. 3-(2-hydoxy-4-methyoxlbenzylideneamino)-4-acetamidobenzoic acid: IR v max (KBr) (cm-1): 3385, 3265, 3010, 1705, 1636, 1625 cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =10.78 (s, 1H), 8.33 (s, 1H), 8.01 (s, 1H), 7.8-6.69 (m, 3H,), 6.26 (m, 1H), 5.03 (s,) 1H), 3.68 (s, 3H), 2.72 (s, 3H), M.P.-190-194⁰C.

 

5s. 4-acetamido-3-((4-hydroxybenzylidene)amino)benzoic acid: IR v max (KBr) (cm-1): 3435, 3175, 3010, 1730, 1636 cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm = 10.98 (s, 1H), 8.63 (s, 1H), 8.06-7.79 (m, 3H,), 7.66 (s, 1H), 7.26- 6,84 (m, 3H), 5.23 (s, 1H), 2.70 (s, 3H), M.P.-180-184⁰C.

 

5t.(Z)-3-(2-hydroxybenzylideneamino)-4-acetamidobenzoic acid:

IR v max (KBr) (cm-1): 3365, 3175, 1730, 1665, 1625 cm-^{1, 1}H-NMR (300 MHz) (DMSO-d6) δ ppm =11.08 (s, 1H), 9.33 (s, 1H), 8.03- 8.32(m, 2H), 7.80 (m, 1H,), 6.26- 7.55 (m, 4H), 5.53 (s,) 1H), 2.72 (s, 3H), M.P.-185-188⁰C.

 

5u. 4-acetamido-3-((E)-((E)-3-phenylallylidene)amino) benzoic acid:

IR v max (KBr) (cm-1): 3344, 3095, 1732, 1645, 1625 cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =11.08 (s, 1H), 8.03- 7.89 (m, 2H), 7.30 (m, 2H,), 7.26- 7.45 (m, 5H), 7.23 (m, IH), 6.65 (m, 1H), 2.72 (s, 3H), M.P.-240-242⁰C.

 

5v. (Z)-3-(4-nitrobenzylideneamino)-4-acetamidobenzoic acid:

IR v max (KBr) (cm-1): 3320, 3175, 1732, 1665, 1602, 1455 ( Ar-NO2) cm-1, ¹H-NMR (300 MHz) (DMSO) δ ppm =10.88 (s, 1H), 8.2 (m, 2H,), 7.78 (s, 3H), 8.03 (m, 2H), 2.72 (s, 3H), M.P.-220-224⁰C.

 

5w. (E)-3-(2,4-dinitrobenzylideneamino)-4-acetamidobenzoic acid:

IR v max (KBr) (cm-1): 3370, 3275, 1722, 1680, 1582, 1430 ( Ar-NO2) cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =11.08 (s, 1H), 9.55 (S, 1H,), 8.66-8.41 (m, 2H,), 8.06 (s, 1H), 2.72 (s, 3H), M.P.-228-230⁰C.

 

5x. (E)-4-acetamido-3-((4-hydroxy-2-nitrobenzylidene)amino)benzoic acid:

IR v max (KBr) (cm-1): 3440, 3175, 1732, 1680, 1602, 1540 ( Ar-NO2) cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm =11.98 (s, 1H), 8.69 (s, 1H), 8.06-7.89 (m, 3H,), 7.99 (d,J=12.5 Hz, 1H), 7.88(s, 1H), 5.43 (s, 1H), 2.70 (s, 3H), M.P.-218-220⁰C.

 

5y. (E)-3-(4-(dimethylamino) benzylideneamino)-4-acetamidobenzoic acid:

IR v max (KBr) (cm-1): 3420, 3334, 3177, 2970, 1722, 1680, 1582, 1530 cm-1, ¹H-NMR (300 MHz) (DMSO-d6) δ ppm = 12.01 (s, 1H), 8.34 (s, 1H), 8.11 (s 1H), 8.24- 7.81 (m, 3H), 7.43 (m, 2H), 6.82 (m, 2H), 2.98 (s, 3H), 2.07 (s, 3H).M.P.-200-202⁰C.

 

2.2. Experimental procedure for antimicrobial activity:

2.2.1. Well diffusion method:

Well diffusion method applied for inhibition of microbial growth around well. Antimicrobial activities of compounds were done by evaluation as per the guideline of National committee for Clinical Laboratory Standards (NCCLS, 1997) using the agar diffusion method. The area of inhibition around well formed in petri-dis known as zone that measured in diameter (mm). For antibacterial activity, a 24 h old culture of selected pathogenic bacteria viz. E. coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 10145), Salmonella typhi (ATCC 19430), Vibrio cholera (39541), Streptococcus pneumonia (ATCC 6301), Staphylococcus aureus (ATCC 25923) and a 48 h old culture of selected fungi viz. Aspergillus nigar (ATCC 16404), Candida. albicans (ATCC 10231) and Aspergillus fumigatus (NICM No.902) was prepared in sterile physiological saline solution. Out of the nine microbes Pseudomonas aeruginosa and Aspergillus nigar has no neuraminidase in cell. Further bacteria were cultured in Muellar-Hinton Agar and fungal strains were a subculture in Sabauraud dextrose agar (Himedia) culture media at 37⁰C. The well was formed in solidified inoculated media in petri dish, in which test compounds and standard compounds (oseltamivir, cefixim and fluconazole) were placed in different concentration. All the antimicrobial screening was performed in triplicate and the average was taken as final reading. The lowest concentration at which no microbial growth on agar media, consider as MIC of that compound [23-25]. The zone of inhibition at (3±1mm) considered as invisible growth or no growth in this study.

 

2.2.2. Determination of minimum inhibitory concentration:

Minimum inhibitory concentration (MIC) is defined as the lowest concentration of the compound which completely inhibits visible growth (turbidity on liquid media) of the microorganisms. The solutions of test compounds, oseltamivir, cefixime and fluconazole were prepared in dimethylsulfoxide (DMSO) at a concentration of 500 µg/ml. From this solution (stock), serial dilutions of 250, 125, 62.5 µg/ml were prepared to determine the MIC. All experiments were performed in triplicates and the average was taken as final reading. The standard drugs, oseltamivir, cefixime (100 µg/ml) for bacteria and fluconazole (100 µg/ml) for fungi were used as positive controls. The 100 µl of DMSO was used as a negative control. The lowest concentration, at which microbial growth was visualized, consider as MIC of that compounds. The zone of inhibition at 2 ± 1 µg/ml, consider as no growth (invisible growth) [19-26].

 

2.3. Docking study:

Molecular docking studies were carried our using AutoDock 4.2 and Glide v5.8 (Schrodinger, LLC, New York, NY). The docking study employing Glide starts with protein preparation followed by receptor grid generation and lastly ligand docking is performed [27-29].  The crystal structure of protein of NAs of bacterial as well as fungal was obtained from protein data bank. The four microbial NAs protein were selected from PDB database (www.rcsb.org) which ID is -4B7Q (influenza), 4YW5 (bacterial), 1KIT (bacterial) and 4M4N (fungal). The selection on basis of recent mutant protein and approved NAI as ligands bind to the all NA sites. Selected protein has also same grooves (pose) structure which taken from PDB database. The proteins were modeled and prepared by removing hydrogen bonds and water also by minimizing the energy with the help of ‘‘protein preparation wizard’’ in Maestro wizard v9.3 (Schrodinger, LLC, New York) [29]. The receptor grid was defined around on prepared protein centroid of the ligands using the grooves of NAs receptor. The validation of molecular docking studies have done using  comparison of docking pose (minimum energy of binding pose selected), RSMD, re-dock score with same pose score and minimum errors, coverage of contacts docked ligand with the co-crystallized structure [30, 31]. There final evaluation of ligands binding with common residues (Table 1) was participate in respective NA sites defined by Glide of Schrodinger and compared with standard compounds oseltamivir and zanamivir (Fig. 2 and 3).

 

 

 

 

 

 

Figure 2: Views of the favorable binding modes of the bacterial NA PDB 4B7Q (A), IKIT (B), and 4M4N (C) binding sites residues with the ligands 5k, 5n, 5q, 5x and 5y with residues are represented by the surface visualization. Ligands are represented by stick models and they colored in, green, purple, blue, red white, and yellow respectively. The polar H-bonds are represented in dotted yellow.

 

 

 

 

 

 

Figure 3. Interaction of amino acids with functional groups of potent compounds, A. 5k, B.5q, C.5n, D.5w, with microbial PDB 4B7Q.

Table 1. The groups of novel designed compounds involved in common interaction in microbial NA.

Docking interaction of Synthesized molecule	Common residues involved in interaction with all PDBs
-COOH	Arg118, Arg292, and Arg371
NHCOCH3	Ile222 and Trp178, Tyr402
-C=N-	Arg78, Arg158, Glu254
Aliphatic	Tyr356, Arg, 611,
Aromatic (OCH3, OH, NO2)	Arg78, Arg172, Tyr360, Ile234, Tyr405

 

3. RESULT AND DISCUSSION:

3.1 Chemistry:

The novel series of Schiff based 4- acetamido-3-(benzylideneamino) benzoic acid derivatives were synthesized as per scheme 1. The starting compound PABA was acetylated in presence of sodium acetate, sod. hydroxide and acetic acid, resultant formed 4-acetamido benzoic acid. Then nitration of 4-acetamido benzoic acid were done with different reaction conditions of HNO₃ (68%) and H₂SO₄ (32%) added at 4-8 ⁰C 30 min interval of time. They formed mono nitro 4-acetamido benzoic acid followed by reduction of nitro compound, formed 4-acetamido-3-aminobenzoic acid as lead compound. Further series of Schiff base compounds 5a-5d (aliphatic) and 5e-5y (aromatic) were synthesized on vigorously refluxed at 80- 90 ⁰C in presence of glacial acetic acid with respective aliphatic (a-d) and aromatic aldehydes (e-y). The all compounds 5a-5y were characterized by FT-IR, ¹HNMR and ¹³C NMR. Their spectral analysis gave confirmation of identification of synthesized compounds and its functional groups.

 

 

Scheme 1. Synthesis of Schiff base compounds

 

 

Compound code	R1	R2	R3	R4	R5
5e	H	H	H	H	H
5f	Cl	H	H	H	H
5g	H	Cl	H	H	H
5h	H	H	Cl	H	H
5i	H	H	Br	H	H
5j	H	H	CH3	H	H
5K	H	H	OCH3	H	H
5l	H	H	OCH3	OCH3	H
5m	OCH3	OCH3	OCH3	H	H
5n	H	OCH3	OCH3	OCH3	H
5o	OCH3	H	OCH3	H	OCH3
5p	H	H	OCH3	Br	H
5q	H	H	OCH3	OH	H
5r	OH	H	OCH3	H	H
5s	H	H	OH	H	H
5t	H	OH	H	H	H
5u	H	C6H5CH=CH-CH2CHO	H	H	H
5v	H	H	NO2	H	H
5w	NO2	H	NO2	H	H
5x	OH	H	NO2	H	H
5y	H	H	N (CH3)2	H	H

 

The FT-IR spectrum of Schiff base as stretching bands in the consistent region υ= of Schiff base (-N=CH-) stretching was υ = 1645 cm^-1, OH lies at 3467 and C=O- stretching came at region 1685 cm^-1.  The ¹H NMR of all novel compounds were recorded chemical shift (δ) of N=C lies in range 7.81- 8.30 ppm as singlet peak. The aromatic proton showed δ = 6.68 – 8.22, proton of Schiff bond lies at δ = 3.02 - 3.44, and carboxylic acid proton showed the peak at 10.45 – 11 ppm.

 

¹³C NMR spectra of compound 4-acetamido-3-aminobenzoic acid showed aromatic peaks displayed in region of δ= 129.56-117.39, but aromatic carbon with NH₂ group showed absorption at 138.64 ppm. The carbon of acetamido was displayed at 165.27 and C=O lies δ= 174.23 ppm confirmed the amine compound. ¹³C NMR spectra compound 4-acetamido-3-(ethylideneamino) benzoic acid (5b) was analyzed and it showed acetamido occurred at 163.33 ppm,- N=CH- (Schiff base) displayed at 166.65 ppm and aromatic carbon attached with Schiff base lies at 148.21 [17-19].

 

 

3.2. Antimicrobial effect  

The novel series of Schiff based 4- acetamido-3-(benzylideneamino) benzoic acid derivatives were synthesized and evaluated using in-vitro methods for their anti-microbial which contain NA and non-NA. The activity and potency of compounds was measured by of zone of inhibition and minimum inhibitory concentration (MIC) using anti-microbial assay of NA and non-NA containg microbes. The value (in mm) measured for all compounds three times for each strain (mm ± SD) was represented in graphs (Fig. 4, 5, 6). The synthesized compounds are structure analogues of oseltamivir, binds with NA of microbes as a result produce NA inhibition.  Schiff base also showed synergetic antimicrobial effects on microbes [18-22, 27].

The antimicrobial activity showed that concentration at 62.5μg/ml of compounds 5a-5h has insignificant effect (no visible growth). But at concentration 125 μg/ml, almost all compounds showed significant inhibition activity. Further the increasing concentration at 250 and 500μg/ml were showed doubled the previous value (zone of inhibition). MIC of fifteen (5k-5y) compounds including standard drugs was also revealed that the concentration 125μg/ml was act as significant effect concentration for all synthesized compounds against all nine strains as shown in table 2. Oseltamivir was the standard drug which significant affects against both microbes which contain neuraminidase while Cefixime and Fluconazole were showed significant effect to respective strains only (Fig. 4).

 

 

Table 2: Antimicrobial activity of synthesized compounds against both non-neuraminidase and neuraminidase contained strains.

			Gram –ve				Fungus
Test compounds Code (100μg/ml)	E. Coli	S. typhi	Pseudomonas Aresonasa	V. cholera	S. aureus	S. pneumoniae	A. nigar	Candida Albicans	Aspergillums fumigates
5a	1	2	2	2	1	1	1	2	2
5b	2	2	1	2	1	2	1	2	2
5c	2	2	1	3	2	1	2	1	1
5d	5	3	2	4	2	2	1	2	2
5e	5	4	4	4	5	4	2	3	2
5f	4	4	3	3	3	3	3	4	4
5g	4	3	3	3	3	3	3	4	3
5h	4	5	4	4	4	3	4	5	5
5i	6	6	7	7	6	6	7	6	5
5j	4	3	4	4	5	5	3	3	4
5k	10	10	11	12	10	12	4	4	5
5l	14	12	16	16	13	14	5	5	8
5m	14	14	14	14	16	18	4	7	8
5n	14	16	16	16	16	18	5	7	8
5o	14	16	14	16	16	18	4	7	7
5p	8	8	12	14	12	14	4	5	5
5q	14	16	16	18	17	18	8	10	10
5r	14	14	16	14	16	16	3	7	4
5s	12	14	12	12	16	16	4	6	4
5t	12	15	14	15	13	16	4	7	8
5u	10	12	10	12	14	14	2	4	6
5v	8	10	10	12	10	12	7	8	10
4w	10	10	10	11	12	12	7	8	11
5x	10	10	9	12	15	13	5	8	8
5y	8	10	9	14	14	16	8	10	12
DMSO (-ve control)	0	0	0	0	0	0	0	0	0
ETHANOL	6	6	3	8	7	7	2	0	0
CEFIXIME (+ve control)	24	17	25	24	27	20	5	1	1
OSELTAMIVIR	6	10	8	14	12	12	0	0	0
Fluconazole (+ve control)	0	0	0	0	0	2	12	10	14

 

 

Zone of inhibition of compounds at 5k, 5l, 5m, 5n, 5o, 5q, 5r, 5x, 5y were shown activity at certain extend  (10-18 ± 2.5 mm) rather than  rest of compounds which have  little activity for both bacterial and fungus microbes at the same MIC (Table 2). Graphical representation of standard compounds and synthesized compounds 5k, 5m, 5n, 5q, 5r, 5x, and 5y  against nine strains showed better zone of inhibition (14-18 ± 1.5 mm) (Fig.5, 6) especially against the strains S. aureus, V. cholera and S. pneumonia.

 

 

Figure 4. Zone of inhibition of standard compounds at conc. 125 μg/ml against nine strains

 

Figure 5. Zone of inhibition of at conc. 125 μg/ml against nine strain for potent compounds 5o, 5k, and 5q.

 

Figure 6. Zone of inhibition of at conc. 125 μg/ml against nine strain for potent compounds 5u, 5x and 5y.     

 

Result exposed  that compounds code 5k, 5l, 5m, 5q, 5x and 5y characterized effective even at 62.5 μg/ml against like S. aureus (+ve), S. typhi (-ve), S. pneumonia (+ve), V. cholera (-ve). So compounds 5k, 5l, 5m, 5q, 5x and 5y showed sensitivity at MIC 62.5 μg/ml against NA contained microbes. But double the concentration, zone of inhibition also doubled as previous zone of inhibition. As compared to standard drug Cefixime (25 ± 2-35 ± 5 mm), these  compounds has less sensitivity (Fig. 5) but a higher zone of inhibition then fluconazole (7-9 ± 1.5 mm) (Fig. 6) and a certain degree equal to oseltamivir (8 ± 2.5-18 ± 0.5 mm), against bacteria as well as fungus. The polarity of compounds was also accountable for inhibition of the bacteria and fungus under investigation. Compounds 5k, 5m-5l, 5o, 5q, 5t, 5x, 5y showed the high polar interaction with NAs in In-Silico as well as In-Vitro and have highest affinity and potency (Table. 1) than other compounds. The result of both studies revealed that these nine compounds were showed the effective zone of inhibition against the neuraminidase containing microbes. The five compounds 5l, 5m, 5q, 5x and 5y were broad-spectrum activity towards microbes (gram +ve, gram -ve) and provided inhibitory active in NA contains bacteria as well as fungus strains. The compound 5k, 5p and 5y were dissolved in ethanol as solvent, changed in polarity and shown more activity.

 

3.2. Molecular docking:

Ligand-receptor interaction study of synthesized compounds confirmed that acetamido and carboxylic acid as essential functional groups for the inhibitory activity (Fig. 1E). The functional group R= hydroxyl/methoxy a polar group at ring B was shown better binding affinity to neuraminidase active sites (Fig. 2) of protein with PDB id (4B7Q,  1KIT, 4M4N). The 4B7Q has same grooves structure as in 4YW5 and 4M4N and it linked with oseltamivir. When the imine (-N=C-) bond with aromatic aldehyde, binds with all model then it showed the some common residues interact as same as oseltamivir and imine bond act as potent hydrophobic groups that helped in interaction with active site residues.

 

Interactions showed 2-4 more hydrogen bonds by substitution of OCH₃, OC₂H₅, NO₂, NH₂, with ring B (Fig. 3). The hydrogen bonds interaction of amino acids, binding affinity (> -7.0 Kj/mol) and root mean square deviation (RMSD) [31, 32] was less than 3A⁰ revealed excellent receptor-ligand interaction towards the neuraminidase templates. RMSD represents the overall movement of the ligands relative to the active site protein during the simulation [32, 33]. The compounds code 5k, 5m, 5p, 5q, 5r, 5x and 5y had shown the (9.0 ± 2 Kj/mol) more binding affinity than all rest compounds and standard compounds. From these results it confirms that 4-acetamidobenzoic acid group (ring A) link to the benzene ring (Fig. 1D, ring B) assigning with methoxy (–OCH₃), nitro (NO₂) and hydroxyl (–OH) had more suitable for the active site of NA. The binding energy (-Kj/mole) of compounds 5k, 5m, 5q, 5w, 5x, and 5y had hydrophobic group oriented towards Arg78, Arg172, Tyr360, 405, Arg611 amino acids, hydrophilic group ARG224, Asn241, Ser294, Glu276 and carboxylic acid formed tried arginine bonding (Fig. 3) as strong interaction in all PDBs which showed promising inhibition. They give better interaction with the active site of all PDB templates and their H-bond interaction with additional group result from potent compounds (Table 1).

 

The present study highlighted that the imine group rather than amine and methoxy, ethoxy and hydroxyl aldehydes rather than methyl, ethyl, bromine, chloride showed synergetic action and better occupancy towards active site than oseltamivir, zanamivir, and other derivatives were recorded. Thus 4-acetamido-3-benzylidimino benzoic acid derivative was taken as lead compound and they evaluated against NA contains microbial strains. Increasing the OCH₃ and OH groups on ring B, showed better activity In-Vitro as well as In-Silico. It also provides the co-relation between the result of In-Silico and In-Vitro [34-36].

 

On basis of molecular docking and antimicrobial study of synthesized compounds it showed that carboxylic acid and acetamido group need for activity and  –N=CH- base at 4-acetamido provides multiple folds of the potency of analogous towards the NAs active site. The synthesized compounds having methoxy and hydroxyl at same ring showed the better and potent than standard compounds against NA contained microbes.

 

CONCLUSIONS:

We synthesized a series of twenty-five 4-acetamido-3-(benzylideneamino) benzoic acid derivatives compounds and screened for their in-vitro antimicrobial activities (Table 2) as oseltamivir analogues. Docking studies immensely helped in the different binding modes of designed compounds with different microbial NAs active site that confirmed better neuraminidase inhibitors than oseltamivir. The binding mode of compounds 5k, 5n, 5q, 5x and 5y against the PDB of NAs confirmed hydrogen bond interaction (Table 1) and also provided the inhibition property (16 ± 2.5) against neuraminidase contained microbes (Table 2). Compounds 5k, 5l, 5m, 5n, 5p, 5q, 5w and 5y exhibited strong potency against tested microbes which have neuraminidase proteins. Out of these 5k, 5q and 5y were highly active against all strains. This confirmed that synthesized compounds which had hydroxyl (-OH), methoxy (-OCH₃) groups at the adjacent site at ring B (Fig. 1) showed potent against the NA containing microbes. Thus novel 4- acetamido-3-aminobenzaimino (oseltamivir analogues) may be further employed for in-vitro evaluation of such compounds against influenza virus to explore their clinical potential in future.

 

 

 

 

 

ACKNOWLEDGMENTS:

One of authors, Mukesh kumar Gupta is grateful to Central of Scientific Industrial and Research (CSIR), New Delhi for providing financial assistance in the form of senior research fellowship.

 

CONFLICT OF INTEREST:

The authors confirm that this article content has no conflicts of interest.

 

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Accepted on 21.10.2018       © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(1): 303-313.