Synthesis and in vitro antimycobacterial potential of novel hydrazones of eugenol

2.1 General

All chemicals, reagents and solvents were procured from Sigma-Aldrich and Merck Pvt Ltd. and were used without further purification. The reactions were carried out in oven-dried glassware (120 °C) under atmospheric condition. Chemicals and related solvents were procured from Merck Chemicals. From fifty-one in silico docked compounds, the selective ten compounds were subjected to synthesis. The reactions and purity of compounds were monitored by thin layer chromatography (TLC) over percolated plates coated with 0.2 mm Merck 60 F254 silica gel using butanol, acetic acid and methanol (4:3:1, v/v) eluent mixture, and were visualized by UV irradiation (254 nm). The synthesized compounds were purified using column chromatography. The melting points were determined using B-540 melting point apparatus using open capillaries and are uncorrected. The infrared spectra were recorded on a Shimadzu, MIRacle-10, IR Affinity-1 in the range of 400–4000 cm⁻¹. The ¹H NMR spectra were recorded in CDCl₃ using Agilent VNMRS 400 instrument at 300 MHz with chemical shift 0–10. The chemical shifts, δ are reported in ppm from 0 to 10 using tetramethylsilane (TMS) as internal standard. The mass spectra were performed using LCMS6103 at m/z values: 0–500.

2.2 Molecular docking

Fifty one compounds (Table 1) were docked in Small-Molecule Drug Discovery Suite of Schrodinger. All compounds were targeted on two enzymes such as 2NSD and 2X22 involved in tuberculosis activity. InhA, the enoyl-ACP reductase in Mycobacterium tuberculosis is an attractive target for the development of novel drugs against tuberculosis. The generated lower energy conformers of all ligands were docked into generated grid of active site of enzymes by XP precision of docking inside Glide-v7.4 (Joshi et al., 2016; Rohane and Makwana, 2019).

Table 1 . List of compounds screened for molecular docking.

2.3 Synthesis of ethyl aryloxy acetate 2

A mixture of compound 1 (eugenol: 0.1 mol), ethyl chloro acetate (0.1 mol) and anhydrous potassium carbonate (0.15 mol) in dried acetone was refluxed for 12 h. Resultant mixture was distilled off and poured on to ice-cold water and stirred. Residue was extracted with ether and the extract was dried over anhydrous sodium sulphate and was purified under reduced pressure to yield compound 2.

2.4 Synthesis of ethylaryloxyacetyl hydrazine 3

A mixture of compound 2 (0.05 mol) and hydrazine hydrate (0.075 mol) in ethanol was refluxed for 4 h and after distilling off the solvent the residue was recrystallized from methanol to yield compound 3.

2.5 Synthesis of hydrazones 4, 5, 11, 18, 30, 34, 35, 37, 42, and 45

A mixture of compound 3 (0.01 mol) and 2, 4-dihydroxy benzaldehyde (0.01 mol) was refluxed for 2 h using acetic acid. The crystals formed were washed with ice-cold water, dried and recrystallized from methanol to yield compound 4. Following the same procedure using respective aldehydes / ketones, other compounds 5, 11, 18, 30, 34, 35, 37, 42, and 45 were synthesized Scheme 1.

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Scheme 1

Synthesis of novel hydrazones.

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2.6 Anti-tubercular activity

The synthesized compounds (4, 5, 11, 18, 30, 34, 35, 37, 42, and 45) were evaluated for their anti-tubercular potential against standard strain of H37Rv. The method used was microplate Alamar Blue assay (MABA). The final drug concentrations tested were 100–0.2 µg/ml. Plates were covered and sealed with parafilm and incubated at 37 °C for five days. After addition of Alamar Blue reagent and incubating for 24 h, the results were observed. A blue color in the well was interpreted as no bacterial growth, and pink color was scored as growth.

Being a non-toxic method, it has several advantages such as thermal stability of the reagent, and good correlation with BACTEC radiometric method.

3.1 Molecular docking

The Insilco study of all fifty-one compounds was performed using Small-Molecule Drug Discovery Suite of Schrödinger. The compound 11exhibited good docking score and predicted interaction with enzymes. The docking result of novel hydrazone revealed that the binding energies were in the range of −6.097 kcal/mol to −10.393 kcal/mol, with the minimum binding energy of −10.393 kcal/mol (Table 2). The molecules were tested for structure analysis by the visualization tool. The entire compounds protein-ligand complex showed H - bond with the active site residue TYR 158 and PHE 149of 2NSD (Fig. 1) and GLY96 and TYR 158 of X22 (Fig. 2).

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Fig. 1

The Interaction between the compound 4 with the active site of PDB 2NSD.Abbreviation: PDB, protein data bank.

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Fig. 2

The Interaction between the compound 4 with the active site of PDB 2X22.

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3.2 Synthesis and characterization of synthesized compounds

The ten hydrazone compounds 4, 5, 11, 18, 30, 34, 35, 37, 42 and 45 were selected for synthesis based on their in-silico docking results. The derivatives were synthesized by condensation of arylhydrazide with various aromatic aldehydes or ketones using ethanol. Physical data of all synthesized compounds are given in Table 3 and characterization data mentioned in supplementary file. In the IR spectra, all hydrazone derivatives displayed characteristic band from 1700 to 1650 cm⁻¹ attributed to C⚌O stretching vibration. The N—H stretching vibration of the compounds exhibited a band at near 3150 cm⁻¹. The stretching bands for C⚌C and C⚌N groups were observed at 1610–1490 cm⁻¹. In general, the IR stretching frequencies for –OH groups varied for the compounds 4, 18, 34 and 42 in the region 3200–3650 cm⁻¹. In the ¹H NMR spectra of all the compounds, the aromatic and aliphatic protons were observed at the specified ppm scale. Aromatic protons were observed at about δ 6.15–7.78 ppm. Synthesized hydrazones (4, 5, 11, 18, 30, 34, 35, 37, 42 and 45) displayed characteristic NMR signals for —OH, —NH, —CH⚌N— protons as coupled peaks at δ value of 4.90–5.10 ppm, 7.10–6.90, and 3.35–2.53 respectively.

Table 3 Physical characteristics of synthesized hydrazones.

Compound- No	Ar’	Mol. Formula	Mol. Weight	MP oC	Yield %
4		C21H24N2O5	384.19	220–221	73.08
5		C19H18I2N2O3	576.18	235–236	62.00
11		C21H25N3O3	367.44	191–192	72.03
18		C19H20N2O4	340.37	229–230	75.00
30		C21H25N3O3	367.44	190–191	59.15
34		C21H24N2O4	368.43	217–218	74.08
35		C21H23N3O5	397.42	235–236	60.08
37		C21H25N3O3	367.44	190–191	72.03
42		C21H24N2O4	368.43	219–220	76.90
45		C21H24N2O3	352.43	184–185	65.55

3.3 Antitubercular activity

The compounds 4, 5, 11, 18, 30, 34, 35, 37, 42 and 45 were evaluated for in vitro antimycobacterial activity against M. tuberculosis H37Rv by microplate alamar blue assay (MABA) method, using isoniazid as standard. The results expressed in minimum inhibitory concentration (MIC) are given in Table 4 and the colour change observed during assay method is shown in Fig. 3. The obtained results indicate the biological potential and varying activity depending on the type of substituent on hydrazide nucleus. The results of anti-tubercular activity revealed all newer hydrazones possess inhibitory potential against Mycobacterium tuberculosis. Whereas compound 11 possess highest sensitivity against M. tuberculosis H37Rv at 25 µg/ml level. The antibacterial activity is strongly connected with the position of the electronegative substituent on phenyl ring of aldehydes in relation to the hydrazone skeleton (Fuloria et al., 2017).

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Fig. 3

Assessment of Microplate Alamar blue assay result.

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