INTRODUCTION:

Most of the benzofuran compounds [1,2] frequently occur in natural products and are good chelating agents. The compound amiodarone hydrochloride used as an ideal antiarrhythmic drug [3] contains a 2, 3-substituted benzofuran moiety. The synthesis of ester of 1-benzofuran-2-carboxylate derivatives was reported by number of scientists. They can be synthesized by direct condensation of 2-hydroxybenzophenones with ethyl 2-bromoacetate in dry toluene in the presence of sodium hydride, sodium ethoxide in refluxing absolute ethanol [4].

4-Hydroxy benzofuran-6-carboxhydrazide has been synthesized from furfuraldehyde and dimethyl succinate via series of reaction [5]. 5‐Chlorobenzofuran‐2‐carbohydrazide [6] were synthesized from ethyl 5‐Chlorobenzofuran‐ 2‐caboxylate and condensed with various substituted aromatic aldehyde to give Schiff base. 5-Bromosalicylaldehyde was treated with hydroxylamine hydrochloride in N,N-dimethyl formamide under reflux conditions forming 5-bromo salicylonitrile which is further treated with ethyl chloroacetate in anhydrous acetone in presence of anhydrous potassium carbonate forming its ethyl ester. The crude ester was treated with potassium carbonate in DMF under reflux condition forming ethyl 5-bromo-3-amino-1-benzofuran-2-carboxylate [7]. Molecular modeling can accelerate and guide to the chemist or scientist for drug design and contribute to the understanding of the biochemical functions of gene products. These molecular modeling techniques used for the study of organic/inorganic/bio molecules use theoretical and computationally based methods to model or mimic the behavior of molecule/s and have been widely applied for understanding and predicting the behavior of molecular systems [8]. The approaches and methodologies used in drug design have changed over time, exploiting and driving new technological advances to solve the varied bottlenecks found along the way. There are several programs used for docking [8], including DOCK-6, FlexX, GLIDE, GOLD, FRED, Cresset, and SURFLEX has been assessed and these programs proved to generate reliable poses in numerous docking studies. Until 1990, the major issues were lead discovery and chemical synthesis of drug-like molecules; the emergence of combinatorial chemistry [9], gene technology, and high-throughput tests [10,11] has shifted the focus, and poor absorption, distribution, metabolism, and excretion (ADME) properties of new drugs captured more attention [12]. Protein docking is a computational problem to predict the binding of a protein with potential interacting partners. The docking problem can be defined as: Given the atomic coordinates of two molecules, predict their correct bound association [13], which is the relative orientation and position after interaction.

Computational Molecular properties:

ChemBio3D is used to build, visualize, and analyze 3D models of chemical structures. ChemBio3D comes with GAMESS, an ab initio quantum chemistry package. ChemBio3D supports Gaussian 03 and Gaussian 09. Using the ab initio and semi-empirical quantum mechanics, Gaussian predicts the minimum energies, molecular structures, vibrational frequencies, optimum properties, IR/Raman spectra, NMR spectra and chemical properties of molecules and reactions in a variety of chemical environments. Gaussian can be applied to both stable compounds and compounds that are difficult or impossible to observe experimentally such as short-lived intermediates and transition structures. CS MOPAC performs semi-empirical calculations on atoms and molecules to determine details of molecular structures and properties. Auto Dock is an automated docking tool that helps you predict how small molecules bind to a receptor of known 3D structure. ChemBio3D maintains an interface to Auto Dock to perform the docking calculation. CONFLEX is a conformational analysis package. CONFLEX can be used to search for chemically significant conformers in flexible molecules and displays the conformers as fragments in your model. pKa, logS and logP for predicting acid dissociation constants, aqueous solubility and octanol/water distribution coefficients of chemical compounds are computational calculator modules based on Molecular Networks' chemo informatics platform MOSES. Using the ChemDraw panel, you can draw 2D structure drawings and convert them to 3D models. Alternatively, you can build a 3D model and convert it to a 2D drawing that displays in the panel. The various properties listed below of the molecule can be studied by using ChemBio3D:

LogP	= 0.828083 log units	LogS	= -1.96355 Log Units
Balaban Index	= 39365	pKa	= 6.74145 Log Units
Cluster Count	= 14	Molecular Topological Index	= 2029
Num Rotatable Bonds	= 2 Bond(s)	Polar Surface Area	= 84.58 A²
Radius	= 4 Atom(s)	Shape Attribute	= 12.0714285714286
Shape Coefficient	= 0	Sum of Degrees	= 30
Sum of Valence Degrees	= 56	Topological Diameter	= 7 Bond(s)
Total Connectivity	= 0.0080187537387448	Total Valence Connectivity	= 7.47103 x E -05
Wiener Index	= 284	Formal Charge	= 0
Connolly Accessible Area	= 359.679 A²	Connolly Molecular Area	= 167.577 A²
Connolly Solvent Excluded Volume	= 132.051 A³	Exact Mass	= 192.0534921335 g/Mol
Number of H-Bond Acceptors	= 4	Number of H-Bond Donors	= 3
Ovality	= 1.33629545278138	Mol Refractivity	= 4.98049974441528
Partition Coefficient	= 1.44719982147217	Critical Volume	= 469.5 cm³/mol
Henry's Law Constant	= 9.47	Gibbs Free Energy	= -16.9 kJ/mol
Stereochemistry	= C(8)-C(7): (E	Total Energy	= -0.3415 kcal/mol
Energy of HOMO (N = 36)	= -6.525 eV	Energy of LUMO (N = 37)	= -1.512 eV
Parachor	= 382.4 ± 4.0 cm³	Index of refraction	= 1.694 ± 0.02
Surface tension	= 69.8 ± 3.0 dyne/cm

SAR study:

Forge (software) is a molecular design and SAR interpretation tool. It will generate detailed 3D models of binding and pharmacophores that will help to define the requirements of the protein of interest, aiding the synthetic chemist in the designing of new actives. It also gives rationale for the polarization of the molecules for synthesis. Fore describe the molecules based on their molecular fields not on their structure. The interaction between a ligand and a protein involves electrostatic fields and the surface properties (e.g. H-bonding, hydrophobic surface, etc). If any two molecules binds to a common active sites tends to make a similar interactions with protein and hence have highly similar field properties. Accordingly, using these properties to describe molecules is a powerful tool for the medicinal chemist as it concentrates on the aspects of the molecules that are important for biological activity. Forge condense the molecular field down to a set of points around the molecule termed field points. The field points are local extrema of the electrostatic, van der Waals and hydrophobic potential of the molecule. They have size/strength information associated with them i.e. all H-bond donor are not treated the same; some make stronger bonds than the others. The bigger fields points are generated by charged groups such as ammonium or carbonyl group or highly polar group. The colours of the field points indicates – Blue – Negative field points (like to interact with positives or H-bond donor present on protein); Red – Positive field points (like to interact with negatives or H-bond acceptors present on protein); Gold or Orange – Hydrophobic field points (describe the regions with high polarizability or hydrophobicity); Yellow – van der Waal field points (describe possible surface or rdW interactions). In general, ionic groups including those forming hydrogen bonding; give rise to the strongest electrostatic field. Aromatic groups encode both electrostatic and hydrophobic fields. Aliphatic groups give rise to hydrophobic and surface points but are essential electrostatically neutral.

Activity atlas of Forge actually performs three types of analysis: useful for quantitative information can be gained from 3D model. This model shows that what the average active molecule looks like by making an analysis of what have in common the active molecules in the data set. The average electrostatic of actives (red or blue) shows the region where the active molecule in general shows either a positive or a negative field. As this field is associated with a high biological activity, new molecules that show either positive or negative fields in the same field should also be active. The average hydrophobic of the actives (yellow) contributions shows the regions where the active molecules is general make hydrophobic interactions with the target of interest.

The force field points of the 3-hydroxy-1-benzofuran-2-carbohydrazide was compared with ciprofloxacin (anti-TB; considered as reference molecule). The molecular field similarity between them was found to be 0.658 which was quite good. The reference molecule containing highly polarized group such as carboxylic acid and amino group which are exist in zwitter ion form at neutral conditions, increases it molecular electrostatic potential. Such highly ionized groups are not present in the hydrazide but containing hydrazide and hydroxyl group; therefore molecular field similarity score is comparatively good.

Density functional study of 3-hydroxy-1-Benzofuran-2-carbohydrazide:

Minimum Binding Energy of 3-hydroxy-1-Benzofuran-2-carbohydrazide:

It is the structure of 3-hydroxy-1-benzofuran-2-carbohydrazide obtained at minimum binding energy. The white colour ball indicates presence of hydrogen, red ball indicates presence of oxygen, blue ball indicates presence of nitrogen and gray colour ball is of carbon. The minimum binding energy of the molecule is calculated from the HOMO-LUMO energy gap (from density of states) i.e. band gap or given directly

Density of states of 3-hydroxxy-1-Benzofuran-2-carbohydrazide (DOS):

The DFT calculation has been performed for the determination of electronic DOS of molecule in terms of electron density in k space. It gives energy in terms of Fermi Energy, not in term of absolute energy. The density of state (DOS) of 3-hydroxy-1-benzofuran-2-carbohydrazide shows conduction band indicates the molecule has electrical conductivity property. The DOS is also used to calculate minimum energy required for the excitation of electrons from HOMO to LUMO (for electronic transition). The DOS of the molecule also shows HOMO of the molecule is completely filled as shown in following figure.

Molecular docking:

The three dimensional structures of all proteins were taken from the PDB database. The native autoinducer and all water molecules were removed from basic protein structures. Hydrogen were added using the templates for the protein residues. The three-dimensional structure of the ligand [3-hydroxy-1-benzofuran-2-carbohydrazide] was constructed. The ligand was then energy-minimized in the in-built ChemSketch module of the software. The active site of each protein were first identified and defined using an eraser size of 5.0 Å. The ligand was docked into the active site separately using the ‘Flexible Fit’ option. The ligand-receptor site complex was subjected to ‘in situ’ ligand minimization which was performed using the in-built CHARMm force field calculation. The nonbond cutoff and the distance dependence was set to 11 Å and (ε = 1R) respectively. The determination of the ligand binding affinity was calculated using the shape-based interaction energies of the ligand with the protein. Consensus scoring with the top tier of s=10% using docking score used to estimate the ligand-binding energies.

The binding sites for the docking are generated by using Glide software. The site of the protein having more site score is considered for the docking of ligand. The site which having maximum site points, locate on the site in different colors as hydrophobic and hydrophilic maps. The hydrophilic maps are further divided into donor, acceptor, and metal-binding regions. Other properties characterize the binding site in terms of the size of the site, degrees of enclosure by the protein and exposure to solvent, tightness with which the site points interact with the receptor, hydrophobic and hydrophilic character of the site and the balance between them, and degree to which a ligand might donate or accept hydrogen bonds. The docking site score of 1VOM (1.074) and 1RJB (1.073) receptor/protein is higher while that of 2BOU (0.464) is lowest is indicates that the 1VOM and 1RJB proteins PDB are more favorable for docking than the others. The size of 4BBG (223) and 1VOM (222) are higher while volume of 3FDN (760.77) and 1VOM (618.77) available for docking is higher but exposure to the ligand as compared to 3LAU and 3V3M is lower.

The exposure to the ligand is maximum in 2BOU and 3V3M and minimum in 1RJB while reverse is the case for the enclosure area, it is higher in 1RJB and 1TE6 while minimum in 3MK2. The overall contact area to the ligand is higher in 1RJB (1.124) and 1TE6 (0.993). The hydrophobic nature or character and balance between hydrophobic and hydrophilic nature of the active site is higher in 4BBG and 3LAU respectively while that of lower in 1TE6 (0.008). The hydrophilic nature or character of the active site is higher in 1TE6 (1.703) and lower in 3MK2 (0.717). The ligands having more hydrophilic nature are more tightly binds with 3MK2 and weakly binded to 1TE6. The order of protein in the decreasing order of hydrophilic character and increasing order of hydrophobic character is - 1TE6 > 2BOU > 3V3M > 1RJB > 3FDN > 3MK2 > 4BBG > 1VOM > 3LAU. This indicates that the ligands having more hydrophobic nature are binds easily 3LAU. The hydrogen bond donor/acceptor character ratio is higher in 2BOU (1.433) and 3FDN (0.880) while lower in 3V3M (0.510) therefore the ligand contains more hydrogen bond acceptor atoms/groups are more tightly binds to 2BOU and 3FDN while those containing hydrogen bond donor atoms/groups are bind to 3V3M. The order protein in the decreasing order of H-bond donor to H-bond acceptor ratio is –2BOU > 3FDN > 3LAU > 4BBG > 1VOM > 1RJB > 3MK2 > 1TE6 > 3V3M. The estimation of binding affinity of the ligand-receptor/protein complex is still a challenging task. Scoring functions (docking score) in docking programs take the ligand-receptor/protein poses as input and provides ranking or estimation of the binding affinity of the pose. These scoring functions require the availability of receptor/protein-ligand complexes with known binding affinity and use the sum of several energy terms such as van der Waals potential, electrostatic potential, hydrophobicity and hydrogen bonds in binding energy estimation. The second class consists of force field-based scoring functions, which use atomic force fields used to calculate free energies of binding of ligand-receptor/protein complex. The docking score and other different docking properties of 3-hydroxy-1-benzofuran-2-carbohydrazide are shown in following table.

Table 1: Docking score and other different docking properties of 3-hydroxy-1-benzofuran-2-carbohydrazide

Description	Proteins
Description	1RJB		3FDN	3LAU	4BBG	3V3M	2BOU			1TE6	1VOM	3MK2
Potential Energy OPLS 2005 = 45.703
RMS Derivative OPLS 2005 = 0.002
Glide lignum	1	3		11	4	3		3	3		3	3
Docking Score	-6.089	-6.950		-6.693	-5.003	-4.702		-5.010	-4.969		-6.353	-5.065
Glide Ligand efficiency	-0.435	-0.496		-0.395	-0.357	-0.336		-0.358	-0.355		-0.454	-0.362
Glide Ligand efficiency sa	-1.048	-1.190		-0.580	-0.861	-0.809		-0.862	-0.855		-1.094	-0.872
Glide Ligand efficiency In	-1.673	-1.197		-1.521	-1.375	-1.292		-1.377	-1.365		-1.746	-1.392
Glide gscore	-6.089	-6.950		-.5.535	-5.003	-4.702		-5.010	-4.969		-6.353	-5.065
glide lipo	-0.996	-1.114		-1.533	-1.125	-1.008		-0.916	0.0		-1.924	-1.481
glide hbond	-0.608	-1.222		-0.441	-0.254	-1.569		-0.304	-0.494		-0.219	-0.155
glide metal	0.0	0.0		0.0	0.0	0.0		0.0	0.0		0.0	0.0
glide rewards	-2.549	-4.740		-2.939	-1.995	2.009		-2.184	-2.099		-2.522	-2.099
Glide evdw	-27.913	-25.134		-24.204	-24.577	-22.419		-19.933	18.490		-24.374	-18.070
Glide ecoul	-6.513	-5.254		-3.791	-4.129	-6.227		-6.966	-11.251		-6.036	-5.325
glide erotb	0.436	0.436		0.436	0.436	0.436		0.436	0.436		0.436	0.436
glide esite	0.0	0.0		0.0	0.0	0.040		0.0	-0.200		0.0	-0.064
Glide emodel	-46.409	-40.911		-38.377	-42.210	-34.598		-33.598	-40.097		-41.430	-31.373
Glide energy	-34.425	-30.388		-27.993	-28.706	-28.646		-26.899	-29.741		-30.410	-23.395
Glide einternal	0.198	0.001		0.256	0.260	3.979		2.341	0.581		3.413	0.499
glide confnum	1	3		1	1	1		2	1		2	1
Glide posenum	248	13		147	8	6		338	205		6	148
XP GScore	-6.089	-6.95		-6.693	-5.003	-4.702		-5.010	-4.969		-6.353	-5.065
H-Bond	02	01		03	02	03		02	05		02	02
pi-pi /pi-cation interactions	00	00		00	00	00		00	03		00	00

1RJB

3FDN

3LAU

4BBG

1TE6

1VOM

3V3M

3MK2

2BOU

Fig 2: Docking images of 3-hydroxy-1-benzofuran-2-carbohydrazide with different PDBs.

The docking score table indicate that 3-hydroxy-1-benzofuran-2-carbohydrazide is more active against 3LAU (docking score -6.693) and 3FDN (docking score -6.905) while is less active against 3V3M (docking score -4.702). Glide esite explains the polar interaction in the active site between ligand and amino acid residue at the docking site after recombination. The polar interactions between the aldehyde and amino acid residues of the protein are only observed in 1TE6 (-0.200) and 3MK2 (-0.064) but these are totally absent in 3LAU, 3FDN, 1RJB, 3V3M, 2BOU, 1VOM and 4BBG. The aldehyde shows higher polar interactions with 1TE6 PDB. The amino acids of backbone of PDBs such as MET, ARG, LEU, TYR and GLY and side chain of the amino acids such as ARG, GLN and LYS are forming hydrogen bonding with 3-hydroxy-1-benzofuran-2-carbohydrazide. Glide evdw explains the van der Waal energy of the complex of ligand and amino acid residue at the docking site after recombination. The comparison between glide evdw and glide energy shows that van der Waal energy shows major contribution than coulombic energy for the stabilization of complex. The van der Waal interaction is depends on surface area (polar and non-polar) of the ligand, as surface area increases, van der Waal energy increases and vice versa. The Glide evdw of the interaction in decreasing order is as 1RJB > 3FDN > 4BBG > 1VOM > 3LAU > 3V3M > 2BOU > 1TE6 > 3MK2. Glide energy is summation of coulomb and van der Waal energy of interaction. The glide energy table indicates that, the comparatively coulombic force and van der Waal interactions (energies) are higher for the Hydrazide-1RJB complex. The 3-hydroxy-1-benzofuran-2-carbohydrazide has higher glide energy during the interaction with PBDs in the decreasing order as 1RJB > 1VOM > 3FDN > 1TE6 > 3V3M > 4BBG > 3LAU > 2BOU > 3MK2. The above docking images [Electrostatic interactions (blue)] shows that, two amino acids in all proteins as ARG and LYS shows positive interactions (hydrogen bonding between proton of protein and O/N of ligand or electrostatic interaction between positive centre of protein and negative / electron density of ligand). 3-hydroxy-1-benzofuran-2-carbohydrazide shows stronger such interaction with same amino acids of 3FDN, 4BBG, 3V3M, 1TE6, 3MK2 and 1VOM indicates that orientation of the molecule does not change during docking in major extend by the changing of skeleton or functional group. But such type of interaction is weaker in 3LAU and 2BBOU whereas is absent with 1RJB. The above docking images [Electrostatic interactions (pink)] shows that, two amino acids in all proteins as ASP and GLU shows negative interactions (hydrogen bonding between proton of ligand and oxygen of protein or electrostatic interaction between positive centre of ligand and negative / electron density of protein). This type interaction depends on the number of positive charge centre present in the ligand molecules and number of donor amino acids present in the docking site. 1RJB, 4BBG, 1TE6 and 3MK2 PDBs shows maximum number of such type of interactions with 3-hydroxy-1-benzofuran-2-carbohydrazide while these interactions are weaker with 3FDN, 3LAU, 2BOU, 1VOM, and 3V3M shows minimum number of such interactions. Glide lipo explains the lipophilic and lipophobic attraction between ligand and amino acid residue at the docking site after recombination. The molecule is undissociated and thus available for penetration through various lipid barriers. The rate of penetration is strongly depends on the lipophilicity of the drug molecule in its unionized form. The lipophilic-hydrophilic balance plays very important role in passive transport and active transport along with drug metabolism. As length of hydrophobic chain increases, both partion coefficient and anaesthetic potency increases. Lipophilic and phobic attraction between 3-hydroxy-1-benzofuran-2-carbohydrazide and amino acid residue at the docking site in the order of 1VOM > 3LAU > 3MK2 > 4BBG > 3FDN > 3V3M > 1RJB > 2BOU PDBs at the neutral pH = 7. At lower pH, amine get protonated and its lipophilicity character goes on decreasing. The aldehyde shows weaker lipophilic and hydrophobic attraction in 1TE6.

The electron rich pi-system (containing electron donating group) are generally interact with other electron deficient pi-system having electron withdrawing group. These are denoted by green colour and are called as hydrophobic interactions. Also, electron rich pi-centre interacts with cation (denoted by dark blue colour) and electron deficient centre interact with anion (denoted by pink colour). The 3-hydroxy-1-benzofuran-2-carbohydrazide shows the pi-pi interactions with the amino acid residue containing aromatic ring or pi electrons, the amino acids such as ARG (C=N bond) and PHE, HIE and HID (aromatic ring) shows such interactions with it. The pi-cation interaction are shown by those amino acid residue containing free cation or partial positive charge centre in their side chain such as LYS and ARG, both containing amino groups which get protonated and forming quaternary ammonium cation which get interact with pi-electrons of aromatic rings. The polar hydroxyl group (hydrogen having partial positive charge/oxygen having partial negative charge/lone pair of electrons of oxygen) interact with aromatic ring. These type of interactions are depends on the orientation of the molecule in the docking site and amino acid arrangement in the same. The 2BOU, 1TE6 and 3MK2 PDBs are shows weak interaction with 3-hydroxy-1-benzofuran-2-carbohydrazide which can be explained by their low docking score.

Synthesis of 3-hydroxy-1-benzofuran-2-carbohydrazide:

Dissolve 15.2 g (0.1 mol) of methyl salicylate in 80 ml of acetone; add 20.7 g (0.15 mol) of potassium carbonate slowly with constant string by using mechanical stirrer. Stir the solution for further 30 minutes at room temperature. Add 0.1 mole of ethyl chloroacetate slowly with constant stirring at room temperature and reflux the reaction mixture in water bath for about 4 hours. Check completion of reaction by monitoring TLC. After completion of reaction, distilled out excess acetone under reduced pressure. Dissolve the residue in methanol and add 0.1 mol of sodium methoxide with constant stirring, and reflux the reaction mixture in water bath at about 3 hours. Distilled out excess methanol under reduced pressure and acidify the reaction mixture with 1 N hydrochloric acid at cold condition. Add 80 ml cold water and small amount of ether, white solid is separated at the junction of two liquids (very small amount of product goes into ether), filter the white colour ethyl 3-hydroxy-1-benzofuran-2-carboxylate which is directly used further for acyl hydrazide synthesis.

Dissolve 0.1 mol of ethyl 3-hydroxy-1-benzofuran-2-carboxylate in methanol add 0.12 mol of hydrazine hydrate with constant stirring and catalytic amount of acetic acid. Reflux the reaction mixture for 6 hours, cool the reaction mixture to room temperature forming red colour solid which is washed with small amount of toluene and n-hexane obtain faint yellow colour solid. The formation of product was confirmed by monitoring TLC. The yield of the product formed 59 %. m.p. 190°C. Characterized the product by using FT-IR, NMR and mass spectroscopy.

Molecular formula: C₉H₈N₂O₃;

Colour: faint yellow solid;

Yield: 59%;

m.p.: 190 ⁰C.

FT-IR (in KBr): 3411, 3320, 3270, 3010, 1650, 1589, 1533, 1490, 1359, 1253, 1135, 966, 825, 759, 738 cm^-1.

NMR spectra (δ in ppm): 10.030 (s, 1H, NH-CO); 7.77 (d, 1H, Ar-H); 7.34 (d, 1H, Ar-H); 6.89-6.81 (m, 2H, Ar-H); 4.61 (bs, 2H, NH2); 3.82 (s, 1H, Ar-OH).

Mass spectra: 193 (M + 1).

Estimation of Toxic Hazard:

Toxtree is a full-featured and flexible user-friendly open source application, which is able to estimate toxic hazard by applying a decision tree approach. Toxtree has been designed with flexible capabilities for future extensions in mind (e.g. other classification schemes that could be developed at a future date). It predicts the toxicological hazard (when administered orally) from the molecular structure. This study explain - Carcinogenicity (genotox and nongenotox) and mutagenicity rulebase by ISS, in vitro mutagenicity (Ames test) alerts by ISS, Skin irritation / skin corrosion, Eye irritation and corrosion, Skin sensitization reactivity domains, START Biodegradability, Cytochrome P450-Mediated Drug Metabolism, Structure Alerts for the in vivo micronucleus assay in rodents, Structural Alerts for Functional Group Identification (ISSFUNC), Protein binding Alerts, DNA binding Alerts. By applying various decision tree approaches to the three dimensional structure of 3-hydroxy-1-benzofuran-2-carbohydrazide to estimate their toxic hazards, it shows class III toxicity for oral administration, low probability of a life time cancer risk greater than 1 to 10⁶, narcosis or baseline toxicity, negative for genotoxic and nongenotoxic carcinogenicity, structural alert for S. typhimurium mutagenicity, non-irritating or corrosive to skin and eyes (predicted lipid solubility is 10% and water solubility is 1%), capability to form Schiff bases with skin, persistent chemical (not easily biodegradable), three sites for metabolism, one positive structural alert for the micronucleus assay.

Anti-Mycobacterium Activity:

Tuberculosis (TB) is a lung infection caused mainly by Mycobacterium tuberculosis (M. tuberculosis [MTB]). It is considered to be one of the most contagious and deadly diseases and is a major threat for public health. Antibiotics are most effective against actively growing M. tuberculosis, as these persistent organisms exhibit a phenotypic drug resistance; i.e., their resistance is not associated with genetic changes but with their extant metabolic state. The structures of the developing tuberculosis lesions may effectively define the metabolic status of their bacterial inhabitants, and it has been speculated that at least four significant subpopulations of bacteria exist for which different drugs could be efficacious. These might include active growers that may be killed by isoniazid (INH), those with sporadic metabolic bursts that could be killed by rifampicin (RIF), a population with low metabolic activity that is considered likely to experience acidic surroundings and hypoxia that may be susceptible to pyrazinamide (PZA), and finally dormant bacilli that are not killed by any current agents. These complex phenomena are poorly understood and add a further barrier to the already formidable challenges associated with drug development and treatment of the disease. Despite its superlative early bactericidal activity (EBA), INH is no more effective than other drugs after this period and RIF becomes the most significant bactericidal drug. Its activity against sporadically active M. tuberculosis is crucial for preventing relapses, and INH then serves to limit the emergence of RIF resistance. Because of its apparent ability to kill a subset of bacteria not killed by the other drugs, supposed sporadically active organisms subject to an hypoxic and possibly acidic environment, PZA represents an important component of combination therapy. Phenazinamine derivatives closely related to the anti-leprosy drug clofazimine, CFM 42 [(E)-N,5-bis(4-chlorophenyl)-3-(isopropylimino)-3,5-dihydrophenazin-2-amine, etc are active against a range clinical M. tbc. isolated including MDR strains. Clofazimine and its derivatives stimulate intracellular synthesis of hydrogen peroxide which inhibits the multiplication of the cells because of binding to the guanine in DNA. The anti Mycobacterial activity of hydrazide should be assessed against M. tuberculosis using micro plate Alamar Blue assay (MABA). Finally we conclude the activity of the hydrazide as they were active or not. This methodology is non-toxic, uses a thermally stable reagent and shows good correlation with proportional and BACTEC radiometric method. Briefly, 200μl of sterile de-ionized water was added to all outer perimeter wells of sterile 96 wells plate to minimized evaporation of medium in the test wells during incubation. The 96 wells plate received 100 μl of the Middle-brook 7H9 broth and serial dilution of compounds was made directly on plate. The final drug concentrations tested were 100 to 0.2 μg/ml. Plates were covered and sealed with parafilm and incubated at 37ºC for five days. After this time, 25μl of freshly prepared 1:1 mixture of Almar Blue reagent and 10% between 80% was added to the plate and incubated for 24 hrs. A blue color in the well was interpreted as no bacterial growth, and pink color was scored as growth. The MIC was defined as lowest drug concentration which prevented the color change from blue to pink. Strain used for anti-TB study [14] is M. Tuberculosis (H37 RV strain): ATCC No – 27294. The standard or reference used for the anti-TB study are pyrazinamide, streptomycin and ciprofloxacin and their standard values for the anti-TB test which was performed her are - 3.125 μg/ml, 6.25 μg/ml and 3.125 μg/ml respectively while that of target compound is 1.6 μg/ml.

RESULT AND DISCUSSION:

The electron density present on the heteroatoms of the molecule is carbonyl group oxygen atom of –CONHNH₂ group is -0.44, hydroxy group oxygen is -0.37, furan oxygen is -0.11, nitrogen of –CONH- group (-0.24) and –NH₂ nitrogen is -0.50. These electron distribution indicates the strength of hydrogen bonding is N (NH₂) > O (-CONH-) > O (-OH) > N (-CONH-) > O (furan ring). The hydrogen attached to the –OH group is more electropositive (0.33) than the hydrogen attached to the nitrogen atom (NH₂ hydrogen - 0.28; NH hydrogen – 0.21) which are able to form hydrogen bonding. This can be confirm from NMR spectra and IR-spectra signal intensity. The force field points of the 3-hydroxy-1-benzofuran-2-carbohydrazide was compared with ciprofloxacin (anti-TB; considered as reference molecule) is 0.658 which quite good but with respect to ciprofloxacin, target molecule shows better results. The docking score table indicate that 3-hydroxy-1-benzofuran-2-carbohydrazide is more active against 3LAU (docking score -6.693) and 3FDN (docking score -6.905) while is less active against 3V3M (docking score -4.702).

In IR spectra, the carbonyl group (carbon-oxygen double bond) appears in many interacting compounds, and this bond acts like a well behaved localized vibration. The carbonyl group of the hydrazide shows absorption in the region at 1650 cm^-1. The H – O stretching vibrations of the phenolic group is also appearing in the 3420 cm^-1 while H – N bonds of the hydrazide group shows vibration in the region 3320 – 3270 cm^-1. The region below 1000 cm^-1 often reveals strong bands that are useful for the characterizing aromatic compounds. The benzofuran ring shows strong absorption band in the region of 1050 – 980 cm^-1 whereas C=C bonds of the aromatic ring shows absorption band in the region of 1600 – 1500 cm^-1. In NMR spectra, the NH-CO proton shows singlet at 10.03 ppm while that of –NH₂ protons at 4.61 ppm. The –OH proton shows singlet at 3.82 ppm. Finally the formation of the molecule was confirm by the mass spectra, it shows (m + 1) peak at 193. The anti Mycobacterium activity of hydrazide should be assessed against M. tuberculosis using micro plate Alamar Blue assay (MABA). The standard or reference used for the anti-TB study are pyrazinamide, streptomycin and ciprofloxacin and their standard values for the anti-TB test which was performed her are - 3.125 μg/ml, 6.25 μg/ml and 3.125 μg/ml respectively while that of target compound is 1.6 μg/ml.

ACKNOWLEDGEMENT:

The authors are grateful to the Schrödinger software organization and Cresset software organization for their support.

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Received on 13.01.2016 Modified on 25.01.2016

Asian J. Research Chem. 9(3): Mar., 2016; Page 116-126

DOI: 10.5958/0974-4150.2016.00021.3