Synthesis , Characterization and Antibacterial Evaluation With Computational Study of New Schiff Bases Derived from 7-Hydroxy-4-Methyl Coumarin

New derivatives of 7-hydroxy-4-methylcoumarin were synthesized via oxidation of methyl group at C4 of parent coumarin nucleus using SeO2 as an oxidizing agent. Then the condensation with different aromatic amines and amino acids under conventional method. The structures of the title Schiff bases were elucidated by spectral analysis: FT-IR,13CNMR, and MS.All the compounds were tested in vitro for their preliminary antibacterial activity against two Gram-positive and two Gram –negative bacteria, using serial dilution method, and determining their minimum inhibitory concentrations for the title compounds. Most of the derivatives showed moderate to high antibacterial activity.Compound 7 showed a good antibacterial activity against Grampositive Staphylococcus aureus and Micrococcus luteus also very potent antibacterial activity against Gram-negative E.coli. Density functional theory (DFT)calculations for the synthesized coumarins were performed using a molecular structure with optimized geometry. Molecular orbital calculations supported a full description of the orbitals, including spatial characteristics, nodal patterns, and the contributions of individual atoms.Highest occupied molecular orbital/lowest unoccupied molecular orbital energies and structures are demonstrated. keywords: Antibacterial activity, broth dilution method, density functional theory(DFT), 7-hydroxy-4-methylcoumarin, Schiff base,


INTRODUCTION
Coumar ins are impor tant class of compounds, they demonstrate useful and different activities, often depending on the substituents they include, benzopyran moiety 1,2 .Coumarins are natural compounds produced by various plant sources.Their structures contain fused benzene and pyrone rings.Recently, they have fascinated and attractive research work, due to their remarkable biological and pharmacological activities, which include antibacterial, antifungal, anticoagulant, antioxidant, anticancer, and anti-inflammatory properties 3,4 .Coumarins showed strong antibacterial activity against both Gram-positive and Gram-negative bacteria, especially against Gram-negative bacteria, by damaging cell membrane.
The advantages of coumarins as potential antibacterial compounds, due to their broad range of antibacterial activity; also they are secreted by plants as phytoalexins and have defensive mechanism against attacks from pathogens, and they are environmentally friendly and not vulnerable to develop bacteria resistance [4][5][6] .
Azomethine group (-C=N-) containing compounds usually known as Schiff bases have been synthesized by the treatment of primary amines with active carbonyls.Schiff bases form a significant class of compounds in medicinal chemistry, with diverse biological applications that include antioxidant, antibacterial [7][8][9][10] .Coumarins containing a Schiff base moiety are predictable to have improved antitumor and other distinctive activities 11 .Some literatures show that the reaction of 4-methyl benzo coumarin with selenium dioxide reveals the corresponding 4-formyl derivative, which eventually has been reacted with some primary aromatic amines to get Schiff bases with significant antibacterial activities 12 Due to the importance and established pharmacological activity of these compounds, the aim of the present work is to synthesize new Schiff bases at C4 of the parent coumarin nucleus, as promising antibacterial agents, against both Gram positive and Gram negative bacteria.Also, a computational study was planned to calculate some thermodynamic parameters.Thus, the synthesis of new series of Schiff bases derived from 7-hydroxyl-4-methyl coumarin has been achieved.

MATERIALS AND METHODS
Chemicals used during the synthesis, supplied by Sigma-Aldrich.Solvents were dried and distilled before use.Completion of reactions and the purity of compounds were ascertained by thin-layer chromatography (TLC) , using Silica gel GF 254 (type 60) pre-coated Aluminium sheets, Merck (Germany).Melting points were determined using Stuart/SMP3 melting point apparatus version 5.0 in open capillary tubes, and were uncorrected.IR(KBr) spectra υcm -1 were recorded using Thermo Scientific™ Nicolet™ iS™10 FT-IR Spectrometer in Pioneer company for pharmaceutical industry-Sulaimani-Kurdistan-Iraq.

Synthesis of amino acid Schiff bases (7-8) 17-20
A solution of compound 1 (0.01 mol, 1.9 g) in absolute ethanol(20 ml) was added dropwise to a homogenous solution of each amino acid (0.01mol); glycine (0.75 g and alanine 0.89 g) and sodium hydroxide (0.01mole, 0.4 g) in absolute ethanol (20 ml) with continuous stirring.The solution's color changed immediately and after 2-3 minutes.(1 ml) acetic acid was added.Stirring continued for 24 h and 12 h for glycine and alanine respectively, the obtained precipitate was collected by filtration, washed with cold ethanol, dried and recrystallized from methanol.

Antibacterial Activity
The new title compounds 2-8 were screened for their preliminary antibacterial activity by using macro broth dilution method against two Grampositive (G +ve ) bacteria Staphylococcus aureus and Micrococcus luteus, and two Gram-negative G -ve ) bacteria Escherichia coli and Pseudomonas aeruginosa to determine the MIC in vitro.A series of 13 tubes containing 1 ml of Muller-Hinton media autoclaved and cooled then inoculated with 50

Chemistry
The new title coumarin derivatives, (Scheme 1) have been synthesized from 7-hydroxy-4-methyl coumarin by oxidation of the methyl group (CH 3 ) at the carbon 4, using SeO 2 as an oxidizing agent and hot xylene, to afford 7-hydroxy-4-formy coumarin 1, which is characterized by an IR band of aldehyde (C=O) stretching displayed at 1698cm -  cm -1 , in addition, the 13 CNMR-exhibited characteristic peak due to aldehydic (C=O) at 194.54ppm.
The aromatic amine Schiff bases 2-6 produced in a satisfactory yield, and by the condensation of compound 1 with different aromatic amines using absolute ethanol as a solvent.The(C=N) band showed in the IR spectra at the range 1640-1607 cm -1 and in the 13 CNMR-displayed characteristic peak at 160.90-159.88ppm.
The amino acid Schiff bases 7-8 were prepared by the reaction of the compound 1 with each of glycine and alanine in a basic medium (NaOH), using absolute ethanol as a solvent.The (C=N) stretching of the amino acid Schiff base, has been characterized in the IR at 1617 and 1600 cm -1 and in 13 CNMR-displayed peaks at161.18 and 161.01ppm for alanine and glycine, respectively.
It must be noted, for the mass spectral data of each title compound 2-8, showed a characteristic peak (m/z) assigned the molecular ion peak[M] +. , also, prominent peaks assigned for the fragment of each derivative,(experimental part).

Antibactrial Activity
From the results illustrated in table 2, showed that all title coumarin derivatives have antibacterial activity in vitro against both Gram positive and Gram negative bacteria with MICs range between 31-300 µg/ml in which they vary according to the derivatives and bacteria.
The lowest MIC is 31µg/ml for compound 7, ( most potent), against Escherichia coli, and also MIC is 40 µg/ml observed for the compounds 6 and 7 against Gram-positive Staphylococcus aureus and Micrococcus luteus, respectively.While the highest MIC is 300µg/ml for compound 8 against Staphylococcus aureus.

Computational Studies
The quantum chemical calculations were validated on the new coumarin derivatives using Density Functional Theory (DFT) methodology with 3-21G* basis,while the molecular atomic charges were approved using the Mulliken population analysis.
The molecular representation sketch of the new coumarin compounds was plotted using Chem.Bio-Office 2010 software.
The following chemical parameters calculated using DFT are: total energy (E), chemical hardness (η), electronic chemical potential (m) and electrophilicity (ω)

Structural and electronic properties
Compounds 1, 3, 4, 6,7 and 8 with optimized geometries and 3D geometrical structures are demonstrated in figure 1, and the data listed in table 2 showed the highest atomic charge of all coumarin compounds 1-8 and confirmed the atoms are the most reactive toward the addition substitution reactions.The calculated bond angle and twist angle, stretch., bend, stretch-bend, torsion and stereochemistry, indicated the title coumarin derivatives are non-planar molecules.
The quantum mechanical method have been approved for compounds 1, 3, 4, 6,7 and 8.The molecular structure (optimized form) of the most stable one is shown in figure 1  It is remarkable to view that both orbitals are considerably circulated over the conjugation plane.
In addition, it can also be seen from the figures 2-7 that the HOMO orbitals are located on the substituted molecule, while LUMO orbitals look like those obtained for the unsubstituted molecule and therefore, the substitution has contributed a control on the electron donation ability while influencing only a small effect on electron acceptance ability.Table 4. Showed the HOMO and LUMO gap .The lower values in the HOMO and LUMO energy gap would give details the eventual electron-donor-acceptorcomplex interaction occurring within the molecules.The values of HOMO for compounds 1, 3, 4, 6,7 and 8 are low.Indicate these molecules have low ionization energies inferring that, they can lose the electrons easily 20 .Chemical hardness is related with the stability and reactivity of a chemical compounds , it measures the resistance to change in the electron distribution or charge transfer.The molecule with the higher HOMO-LUMO energy gap, is harder and more stable/less reactive molecule.. Electronic chemical potential (m) is known as the negative of electronegativity of a molecule 21 and describes the escaping tendency of electrons from an equilibrium system.Global electrophilicity index (ω), is calculated using the electronic chemical potential and chemical hardness.This index measures capacity of a species, to accept electrons 22,23 .It is a total of the stabilization in energy after a system accepts an additional amount of electronic charge from the environment 24 .
The more reactive or less stable compound, is the higher chemical potential,.Therefore, compound 4 is the more reactive than other compounds.While compound 1 is the least reactive.The electrophilicity values (ω) (Table3, row 6) for the compound 1,3,4,6,7 and 8 are 20.174eV, 31.38 eV , 0.0274 eV, 0.847 eV, 15.464eV and 6.013 eV, respectively.Among the compounds, compound 3 is considered the strongest nucleophile while compound 1 is considered as a strongest electrophile.

Electrostatic potential charges and related quantum chemical properties
The distribution of the electronic density (electrostatic potential charges), related quantum chemical parameters [dipole moment (Table 3, row 2), and the partition coefficients of the compounds (log P; Table 3, row 3) were calculated for observed compound 1,3,4,6,7 and 8.These values and properties are very helpful and can be used in order to estimate chemical properties and possibilities for the interaction of compounds 1,3,4,6,7 and 8 with target sites (receptors, enzymes) as well as their transporting system through the cell membranes and brain-blood barrier.All these structural, electronic parameters and log P can be used for a construction of quantitative structure-activity relationship (QSAR) model because all of them are closely correlated to pharmacokinetics and pharmacodynamics properties 21 .
Redox potential can be measured from HOMO/LUMO gap; it is significant for the oxidativemetabolism pathway involving cytochrome P450 enzymes.The observed HOMO/LUMO gap ,(∆E) (Table 4), is used to calculate redox potential.The lower negative values of both HOMO and LUMO energies designate that these compounds have the general properties of the reductors.This information may be significant for drug metabolism, as the oxidation (and sometimes the reduction) is the main metabolic pathway for many drug molecules.
All title coumarin derivatives are polar molecules, depending on their electric dipole moments (Table 3), The most polar molecule is compound 7, while compound 6 is found to be the most lipophilic according to the log P values (Table 3row 3).
The UV-VIS absorption spectra of the compounds: 1, 3and 4 were recorded in (ethanol).The absorption peaks are observed at 248, 287 and 222 nm, and 267 and 295 nm for the compounds 6, 8.It can be conclude that these peaks imply to the n→p* and p→p* transitions.The 3D plots of the, HOMO-1, HOMO, LUMO, LUMO+1, and the corresponding energy levels of the compounds 1, 3, 4,6,7 and 8 are shown in the figures 2, 3,4,5 and 6, respectively.The theoretical electronic transfer (ET) for compound 1, 3,4,6 were corresponding to the UV-VIS spectral absorption peaks and the electronic transfer of HOMO plus LUMO and HOMO plus LUMO+1, respectively.The increased theoretical absorption wavelengths of the title compounds have slight blue-shifts.

CONCLUSION
In the present work a series of new coumarin derivatives have been synthesized from 7-hydroxy-4-methylcoumarin by the oxidation of methyl group at C4 of the parent coumarin nucleus, using SeO 2 , to produce formyl group, then the formation of different derivatives under conventional method.The title coumarin derivatives were screened for their preliminary antibacterial activity using serial dilution method, by determining their minimum inhibitory concentrations (MIC), and showed moderate to highest antibacterial activity against Gram-positive and Gram-negative bacteria.

C o m p o u n d 7 s h owe d t h e h i g h e s t antibacterial activity against Gram-positive
Staphylococcus aureus and Micrococcus luteus, and potent antibacterial activity against Gramnegative E.coli.The synthesized derivatives were studied theoretically by using DFT calculations., and the stereochemistry was estimated and found that compounds 1, 3, 4, 6, 7 and 8. are not-planar molecules.

13 C-
NMR spectra were recorded on Bruker FT-NMR spectrophotometer 400, in the College of Pharmacy-Hamedan University of Medical Sciences-Hamedan-Iran, using deuterated dimethyl sulfoxide (DMSO-d 6 ) and deuterated chloroform (CDCl 3 ) as internal standards.Chemical shifts were expressed in d ppm.MS was run at College of Pharmacy-Hamedan University of Medical Sciences-Hamedan-Iran, using Agilent Technology (HP), GC/MS model 5973 network mass selective detector.The values were expressed as m/z.The synthetic method is depicted in Scheme 1.

table 3 .
The contour plots of the frontier orbitals for the ground state of compounds 1, 3, 4, 6,7 and 8 are shown in the figures: 2, 3 4,5,6 and 7 together with the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO).