Synthesis and Evaluation of Antimicrobial Activity of New Imides and Schiff Bases Derived From Ethyl-4-Amino Benzoate

A series of disubstituted 1,3,4-oxadiazole derivatives, including: imides and Schiff bases, was achieved from the starting material, ethyl-4-aminobenzoate, which was converted to the corresponding 4-aminobenzohydrazide (1), by its reaction with hydrazine hydrate in absolute ethanol. Two oxadiazole parent nuclei had been synthesized from (1), the first nucleus 5-(4-aminophenyl)-1,3,4-oxadiazol-2amine(2), and the second is 5-(4-aminophenyl)-1,3,4-oxadiazole-2-thione (3). Compound (2) obtained from stirring methanolic solution of (1) with cyanogen bromide(CNBr) and sodium bicarbonate (NaHCO3) at RT. While compound (3) was synthesized by refluxing of (1) with CS2 in the presence of (KOH), the produced potassium salt of hydrazide underwent cyclization by acidification with 10% HCl. Meanwhile, the cyclic imides derivatives (4-6) and (10-12) were synthesized by thermal fusion of (2) or (3) with acid anhydrides, while Schiffs bases derivatives (7-9) and (13-15) were synthesized by a conventional method involved refluxing of (2) or (3) with different aromatic aldehydes, in acidic medium (using glacial acetic acid). The new derivatives had been tested against three Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus, and Bacillus pumilus) and two Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) and two fungal species: (Saccharomyces cerevisiae and Candida albicans). Among the synthesized derivatives, compound (15) displayed a moderate to potent antibacterial activity, against different (Gram positive and Gram-negative) bacteria, and also showed a slight to moderate antifungal activity. keywords: Antimicrobial, Imides, Schiff base, Synthesis, 1,3,4-Oxadiazole derivatives.

It has been documented that Gram negative bacteria are much more resistant to different antimicrobial agents, as compared to Gram positive bacteria 19 .The differences may be due to, the constituent of the cell wall in Gram positive bacteria is of a simple and single layer, while the cell wall in Gram negative bacteria possesses an "outer membrane" with high lipid and lipoprotein content, which is not found in Gram positive bacteria 20 .Therefore the lipophilic nature of the cell membrane of Gram negative bacteria is more resistant towards antibacterial agents, which functions as a strong barrier for a variety of antimicrobial agents.Therefore, compounds possessing hydrophilic properties, will not be able to penetrate the cell membranes of Gram negative bacteria.Meanwhile, the cell wall of Gram positive bacteria is not complex structure like Gram negative bacteria.Antibacterial agents can quickly collapse the bacterial cell wall of Gram positive bacteria, which leads to disruption of the cytoplasm 21 .This work targets to synthesize of new derivatives of cyclic imides and Schiff bases derived from two parent nuclei: 5-(4-aminophenyl)-1,3,4oxadiazol-2-amine (2), and 5-(4-aminophenyl)-1,3,4-oxadiazole-2-thione (3), respectively.Imides are essential to lead components for natural products and drugs 22 .While, Schiff bases have been widely studied and the focus of different researches, due to their extensive use and distinct biological activities 23 .
Fur thermore, 1 HNMR spectra were recorded on Bruker model Ultra shield 300 MHz, vance II-at (Al-Bayt University-Jordan) using tetramethylsilane (TMS) as an internal standard, the chemical shift was expressed as (δ=ppm), DMSO-d 6 and acetone-d 6 were used as solvents.

General method for synthesis of imide derivatives (4-6).
In a pyrex test tube, added compound (2) (0.0011 mol, 0.19 g), and phthalic anhydride (0.0022 mol, 0.325 g) or of maleic anhydride(0.0022mol, 0.215 g), or succinic anhydride (0.0022 mol, 0.22 g), mixed, and heated using oil bath 290 O C. The mixture stirred continuously by using pyrex glass road for 30 min.then washed with diethyl ether and left to dry.

Antibacterial Activity
The new title compounds 4-15 were tested for their preliminary antibacterial activity and measured using well diffusion technique 26 in vitro, against three types of tested microorganisms: Gram-positive (G+ve) bacteria (S. aureus, Micrococcus luteus, and Bacillus pumilus), and two Gramnegative (G-ve) bacteria (Pseud.aeruginosa, and E. Coli), were clinically activated and maintained on nutrient agar medium for testing antibacterial activity, and potato dextrose agar medium for antifungal activity 27 .Cefotaxime was used as a standard drug for antibacterial activity, while miconazole was used as a standard drug for antifungal activity, using a minimum inhibitory concentration, (MIC) of 1000& 100μg/ml of synthesized derivative in DMSO.

RESULTS AND DISCUSSION
The hydrazide (1) obtained by refluxing of the starting material ethyl-4-aminobenzoate with hydrazine hydrate in absolute ethanol.(1)  characterized by FTIR, due to formation of two doublet peaks for the primary amine of hydrazide, at 3271 and 3234 cm -1 ,in addition, a prominent peak for the(C=O) amide stretching, at 1626 cm -1 .Two parent nuclei (2 and 3), had been synthesized from (1), where (2) obtained from stirring a methanolic solution of (1) with cyanogen bromide (CNBr), and sodium hydrogen carbonate, (NaHCO 3 ) at ambient temperature, while (3) obtained from refluxing an ethanolic suspension of (1) with CS 2 in the presence of KOH, the cyclization involved formation of potassium salt of hydrazide, which underwent cyclization by acidification with 10% HCl.Each parent nucleus (2 ) and (3), displayed two peaks, at 3327and 3224 cm -1 and 3315 and 3220 cm -1 , respectively, that confirmed the presence of (NH 2 ) group.In addition, (3) recorded a peak at 2257 cm -1 due to thiol group (SH), and a peak at 1290 and 1271 cm -1 for (2)and (3), respectively, due to asym.(C-O-C) stretching, also, other peak at 1068 and 1065 cm -1 , respectively, owing to sym (C-O-C) stretching, (oxadiazole ring formation).The 1 HNMR spectrum for the parent nucleus (2) exhibited a singlet peak at δ=7.40 ppm, attributed to primary aromatic-NH 2 group, as well as, a peak appeared at δ=6.60 ppm, due to (NH 2 )-oxadiazole.
The four aromatic protons of (2), displayed at δ=6.85-6.50ppm, as a multiplet, and , the second parent nucleus (3), displayed a peak at δ =7.78 ppm corresponds to the presence of primary -NH 2 -group, while the four aromatic protons, for each (2) and ( 3), appeared at their expected region.See (exp.Part).
The IR spectrum of (4),displayed a distinct band at 3307 cm -1 owing to (OH) stretching of the carboxylic group.This indicate the opening of one imide ring, at one side of a ring fusion , besides that, a peak displayed at 1749 cm -1 , due to (C=O) stretching of the carboxylic acid.Other peaks assigned for (C=O) amide stretching, at 1681 and 1651 cm -1 , belong to the second fused imide ring.The 1 HNMR spectrum for (4) ), exhibited a prominent peak, at δ =10.54 ppm, attributed to (OH) signal of the carboxylic acid, this confirm the opening of one of phthalic imide ring , while the aromatic rings integrating for twelve protons, appeared at their expected aromatic region.(5) showed a prominent IR band at 1664 cm -1 , due to conjugated (C=O) amide stretching, and 1 HNMR displayed two signals, each as a doublet , appeared at δ=7.12 and 6.36 ppm, respectively, owing to the two protons of each (maleimide ring).
The IR spectrum for (6) showed a characteristic carboxylic acid absorption, due to (OH) stretching at 3315 cm -1 =, and a peak displayed at 1725 cm -1 , assigned for (C=O) acid stretching.Other peaks displayed at 1701 and 1666 cm -1 belong to (C=O) stretching of amide, while 1 HNMR analysis for ( 6) is similar to (4), exhibited a peak at δ= 12.10, as a singlet, due to (OH) signal of the carboxylic acid, (this will confirm the opening of one of succinimide ring), besides that, a peak integrating for two protons, belonging to (CH 2 ) group, displayed at δ =2.76 ppm, as a triplet.The second aliphatic (CH 2 ) group, and the two (CH 2 )groups of succinimide ring, were masked with DMSO-d6 peak.(10) and (11) represent the product fusion, of each of phthalic anhydride, and maleic anhydride with (3), respectively, The NH-stretching of thioamide, for each, (10) and (11), displayed at 3225 and 3168 cm -1 respectively, also IR spectra recorded two peaks assigned for the (C=O) amide stretching, for (10) at 1739 and 1712 cm -1 , while (11) displayed a (C=O) amide stretching, at 1687 cm -1 due to (conjugation).The 1 HNMR (10) showed peaks, as multiplet, at the range of δ =8.06-7.69ppm, due to eight aromatic protons, While (11) showed a peak at δ =10.68 ppm, due to NH-thioamide, also another two distinct signals, each attributed for one proton, appeared as a doublet, at δ=6.51 and δ=6.33 ppm , respectively, (maleimide ring).
The IR spectrum for ( 12) is similar to (6), in which succinimide ring is opened during thermal fusion.A recorded band at 3450 cm -1 , assigned for (OH) stretching of a carboxylic acid, (ring opening), a new peak at 1697 cm -1 , due to (C=O) of the carboxylic acid stretching, While The 1HNMR showed a signal for (OH) of the carboxylic acid appeared at δ=14.60 ppm, in addition, two other signals, one displayed at δ=12.10 and 10.28 ppm, attributed to NH-thioamide and NH-amide.
The characteristic imine group displayed in IR spectra at 1606, 1576 cm -1 for ( 7), and at A ll the aromatic protons for (7-9) and (13-15), are well characterized and displayed at their expected aromatic regions.For more details, for the rest of the functional groups, see (exp.part).

CONCLUSION
A new oxadiazoles derivatives ( imides and Schiff bases), derived from ethyl -4-amino benzoate, were successfully synthesized, by thermal fusion and conventional methods, respectively, they were characterized and evaluated for their antimicrobial activities.Compound (11) showed a slight activity against some of Gram positive bacteria, while (13)   showed a moderate activity against all Gram positive bacteria used in this evaluation.Furthermore, (15)   displayed a moderate to potent antibacterial activity, against different (Gram positive and Gram negative) bacteria, and also showed a slight to moderate antifungal activity.

ACkNOWLEDGMENT
We're grateful to the College of Pharmacy-Department of Pharmaceutical Chemistry-University of Baghdad, for conducting the research, and Dr. Raeid M. Al-Sayab/College Ibn -Al-Haitham, for Pure Science, for running 1 HNMR analysis.