Synthesis and Antimicrobial Evaluation of Novel Hydrazones and 1 , 3 , 4-Oxadiazoles Incorporating Bumetanide Derivatives .

a Pharmaceutical Chemistry Department, Suez Canal University, Faculty of Pharmacy, Ismailia, Egypt. b Medicinal Chemistry Department, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt. c Pharmaceutical Chemistry Department, Faculty of Pharmacy, Taif University, Taif, Saudi Arabia. d Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig, 44519, Egypt. e Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah, 21589, Saudi Arabia. f Department of Pharmaceutical chemistry, Faculty of Pharmacy, Port Said University, Port Said, Egypt. g Department of Microbiology and Immunology, Faculty of Pharmacy, Port Said University, Port Said, Egypt.


Introduction
The antimicrobial resistance has turned into a menace that confronts the community over the last few decades.The use and misuse of antimicrobial agents incited the bacteria to develop resistance against them through different defensive mechanisms like enzymatic degradation, modulation of the drug targets or modifying bacterial cell membrane permeability ORIGINAL ARTICLE (Sefton;2002).Thus a great number of infectious diseases have become uncontrollable which contributes to high levels of morbidity and mortality (El-Sayed et al.;2017).According to the Centers for Disease Control and Prevention (CDC), the resistant bacteria could infect up to two million patients in the United States every year, while around 23000 person of them meet their fate due to the treatment failure (Srinivasan et al., 2012).On the other hand, the World Health Organization (WHO) stated that though the antimicrobial agents lose their activity, there are insufficient researches that stand up to this catastrophe which means that our competence to overcome the antimicrobial resistance declines dramatically (Gorton et al., 2017).
The researches in the field of medicinal chemistry evolved a limited number of substantial moieties, frequently present in several drug molecules and interestingly, sulfonamide moiety (SO 2 NH 2 ) is one of them (Scozzafava et al., 2012).Sulfonamides are a pharmacologically active class of drugs that exert different pharmacological activities like antibacterial (Kamal et al. 2013;Zhang et al., 2017), antifungal (Saha et al., 2000;Zaidi et al., 2007), anticarbonic anhydrase (Saluja et al., 2014;Supuran et al., 2018), diuretics (Fravolini et al., 1991;Al-Kahtani et al., 2016), antiinflammatory (Rodge et al., 2012;Brusco et al., 2015) and anticancer (Ahmed et al., 2015;Alafeefy et al., 2016).Among this class of drugs are sulfa drugs that are the first successfully synthesized antimicrobial agents that exert their action via competitive inhibition of folic acid synthesis, and consequently turn off the microbial nucleic acid replication.So far, a group of clinically tried sulfa drugs comprised of benzenesulfonamides bearing antibacterial active aromatic heterocycles are now widely used (Scozzafava et al., 2012).For instance, Sulfamethoxazole, Sulfathiazole, Sulfadiazine and Sulfachloropyridazine are composed of benzenesulfonamide conjugated with isoxazole, thiazole, pyrimidine and pyridazine moieties respectively.Thus, new researches were conducted to discover novel benzenesulfonamide derivatives of antimicrobial action to overcome the antimicrobial resistance problem.For example, addition of coumarin ring to benzenesulfonamide contributed to the antimicrobial activity of compounds (1, 2) as displayed in figure (1) (Patecl et al., 2010).(Pakkath et al., 2014).Recently, Ghorab et al. (Soliman et al., 2019) could prepare new antimicrobial agents compounds (5,6) through conjugating thioureidobenzensulfonamide moiety with substituted pyrimidine as shown in figure (1).
On a parallel approach, in the present investigation, we aimed at introducing new antimicrobial agents to face the antimicrobial resistance challenge.Our research is based on incorporation of well reputed antimicrobial moieties like hydrazones (Mazi et al., 2003) and 1,3,4-oxadiazoles (Othman et al., 2014) into Bumetanide as a precursor of benzenesulfonamide.
The structures of the novel compounds were confirmed from their spectral and micro analytical data.Structure of compound (2) was confirmed by the presence of one singlet signal in H 1 NMR spectrum at 3.87 ppm due to (OCH 3 ) protons.Furthermore, its 13 CNMR spectra revealed one additional signal of aliphatic carbon at 52.5 due to (OCH 3 ) carbon.Formation of compound (3) was indicated in 1 HNMR spectrum by disappearance of singlet signal of (OCH 3 ) protons and appearance of the signals of NHNH 2 protons at 2.49 ppm (NH-NH 2 ) and 9.88ppm (NHNH 2 ), while 13 CNMR spectrum elucidate disappearance of one signal of aliphatic carbon of (OCH 3 ) group which clarifies the nucleophilic substitution reaction.Moreover, the structures of compounds (4 a-i ) were confirmed by 1 HNMR spectra that showed disappearance of the signal of primary amino group protons (NHNH 2 ) in compound (3) spectrum which proved the condensation with different aldehydes.Moreover, an increase in the number of signals in the aromatic region with one signal corresponding to a methylidene proton at the region between 8.30-8.80ppm except compound (4 h ) it exhibited at 10.65 ppm.

Antimicrobial Activity
The antimicrobial activity of the newly synthesized compounds were screened against several pathogenic microbes representative Gram-positive bacteria: Staphyllococcus aureus (RCMB010010), Gram-negative bacteria: Pseudomonas aeruginosa (RCMB 010049) and Escherichia coli (RCMB 010058) and Fungi: Aspergillusfumigatus(RCMB 02568) and Candida albicans (RCMB 05036).The microbial suspensions equivalent to the turbidity of 0.5 McFarland (10 8 CFU/ml) standard were prepared from a fresh subculture of tested bacteria in Mueller Hinton Broth (MHB) and tested fungi in Sabouraud Dextrose Broth (SDB) then this suspension was diluted to 10 6 CFU/ml using MHB for bacteria and Sabourand dextrose Broth (SDB) for tested fungi.The adjusted microbial inoculums (100 µl) were added to each well of sterile 96-well flat-bottomed micro titer plate containing the tested concentration of tested samples (100 µl/well).As a result,last inoculum concentration of 5 X 10 5 CFU/ml was obtained in each well.Three wells containing microbial suspension with no sample using DMSO employed for dissolving the tested compound (Growth control) and two wells containing only media (background control) were included in this plate.Optical densities were measured after 24 hours at 37 ○ C for bacteria and after 48 hours at 28 ○ C for fungi using a multi-detection microplate reader at The Regional Center for Mycology and biotechnology (sun Rise -Tecan, USA) at 600 nm.Sulfamethoxzole and Amphotericin B were used as standards for Gram positive bacteria, Gram negative bacteria and fungi respectively.The inhibitory percentage of the tested compounds were illustrated in the Table (1) (Ankem et al., 2009).
The microbial suspensions equivalent to the turbidity of 0.5 McFarland (108 CFU/ml) standard were prepared from a fresh subculture of tested bacteria in Mueller Hinton Broth (MHB) and tested fungi in Sabouraud Dextrose Broth (SDB) then this suspension was diluted to 106 CFU/ml using MHB for bacteria and Sabourand dextrose Broth (SDB) for tested fungi.The adjusted microbial inoculums (100 µl) were added to each well of sterile 96-well flat-bottomed micro titer plate containing the tested concentration of tested samples (100µl/well).As a result, last inoculum concentration of 5 X 105 CFU/ml was obtained in each well.Three wells containing microbial suspension with no sample using DMSO (Sigma-Aldrich, Germany) employed for dissolving the tested compound (Growth control) and two wells containing only media (background control) were included in this plate.
Optical densities were measured after 24 hours at 37 ○C for bacteria and after 48 hours at 28 ○ C for fungi using a multi-detection microplate reader at 600 nm.Sulfamethoxazole and Amphotericin B were used as standards for Gram positive bacteria, Gram negative bacteria and Fungi respectively.The inhibitory percentage of the tested compounds was illustrated in the  The results clarified that all of tested compounds had no activity against the used fungal strains.Most of the newly synthesized compounds show significant antibacterial activity against Escherichia coli and Pseudomonas aeruginosa with MIC values ranging from 6.4 to 51.2μg/mL.However, compounds (4 c , 4 e , 4 g ) showed activity against all tested bacterial strains.Interestingly, compound (4 i ) showed an excellent antibacterial activity against Escherichia coli (6.4μg/mL) that exceed Sulfamethoxazole reference standard (12.8μg/mL).Moreover, compounds (4 a , 4 b , 4 c , 4 f , 4 g , 4 h ) showed similar activity to Sulfamethoxazole against Escherichia coli (12.8μg/mL).Furthermore, compounds (4 g , 5 d ) exert similar activity to Sulfamethoxazole against Pseudomonas aeruginosa (12.8μg/mL).Compounds (1, 2, 3, 5 a , 5 e ) did not exhibit any activity against all the tested strains.
The structure activity relationship (SAR) analysis revealed that benzenesulfonamides bearing hydrazone moieties carrying electronwithdrawing groups or atoms like pyridine (N), Nitro and Chloro at position 4 as compounds (4 c , 4 e , 4 g ) conferred a significant antibacterial activity against all the tested bacterial strains.While compounds possessing electron-donating groups like 3indolyl, 2-furyl, 2-thienyl, methoxy, benzyloxy or timethoxy as compounds (4 a , 4 b , 4 d , 4 f , 4 h , 4 i ) exhibited antibacterial activity against Escherichia coli only.On the other side, benzenesulfonamides bearing oxadiazoles having electron-withdrawing groups like Chloro and Nitro at position 4 of the aromatic ring as compounds (5 b , 5 c ) exhibit a good antibacterial effect against Escherichia coli only, while only compounds attached to electron-donating groups at position 4 of aromatic ring like phenoxy group as compound (5 d ) resulted in a good activity against Pseudomonas aeruginosa.

General techniques
All starting materials in this investigation from (Sigma-Aldrich, Germany).Melting points (°C) were determined with a Gallenkamp melting point apparatus (London, UK), and are uncorrected.Elemental analysis was performed in the Regional center for Mycology and Biotechnology, Faculty of Science, Al Azhar University, Nasr city, Cairo, Egypt. 1 HNMR and 13 CNMR were performed in NMR department, Faculty of Science, Al Mansora University. 1 HNMR spectra were recorded on Jeol resonance DELTA2-NMR (500 MHz) (Japan) using dimethyl sulfoxide (DMSO)-d 6 as a solvent and tetramethylsilane (TMS) as internal standard (chemical shift in δ, ppm).

Table (3). Table (1): Mean of inhibitory % ± standard derivation produced on a range of clinically pathogenic microorganisms using (125 µg) concentration of tested samples. Results are depicted in the following table:
Staphyllococcusaureus, Ps. aeruginosa: Pseudomonas aeruginosa,E.coli: Escherichia coli.