Synthesis and Antimicrobial Activities of Some New 1,2,4-Triazole Derivatives

Some novel 4,5-disubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one (3, 6, 8, 9) derivatives and or 3-(4-methylphenyl)[1,2,4]triazolo[3,4-b][1,3]benzoxazole (5) were synthesized from the reaction of various ester ethoxycarbonylhydrazones (1a-e) with several primary amines. The synthesis of 4-amino-5-(4-chlorophenyl)-2-[(5-mercapto-1,3,4-oxadiazol-2-yl)methyl]-2,4-dihydro-3H-1,2,4-triazol-3-one (13) was performed starting from 4-Amino-5-(4-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (2) by four steps; then 13 was converted to the corresponding Schiff base (14) by using 4-methoxybenzaldehyde. Finally, two Mannich base derivatives of 14 were obtained by using morpholine or methyl piperazine as amine component. All newly synthesized compounds were screened for their antimicrobial activities and some of which were found to possess good or moderate activities against the test microorganisms.


Introduction
The synthesis of high nitrogen containing heterocyclic systems has been attracting increasing interest over the past decade because of their utility in various applications, such as propellants, explosives, pyrotechnics and especially chemotherapy [1]. In the medicinal chemistry, azoles are widely used and studied class of antimicrobial agents due to their safety profile and high therapeutic OPEN ACCESS index. Among these, Conazoles are a major class of azole-based drugs such as Itraconazole, Fluconazole, Voriconazole, Ravuconazole etc. [2][3][4][5]. Some of other major applications of conazoles are on crop protection. As pharmaceuticals, they are used for the treatment of local and systemic fungal infections, which are important problems in phytopathology and especially in medicine, and they are frequently observed in immune-compromised patients suffering from AIDS or subjected to invasive surgery, anti-cancer therapy or graft receivers.
Several lines of evidence suggest that the primary target of azoles is the heme protein, which cocatalyzes cytochrome P-450-dependent 14α-demethylation of lanosterol. Inhibition of 14demethylase leads to depletion of ergosterol and accumulation of sterol precursors, including 14methylated sterols (lanosterol, 4,14-dimethylzymosterol, and 24-methylenedihydrolanosterol), resulting in the formation of a plasma membrane with altered structure and function. The more recent triazole derivatives, such as fluconazole, itraconazole, and voriconazole owe their antifungal activity at least in part to inhibition of cytochrome P-450-dependent 14α-sterol demethylase [6].
The diseases caused by fungal species cause not only improve the cost of therapy but also may lead mortality. Due to the inadequacy of alone standard antibiotic therapy in certain circumstances, more efforts have been focused on addressing the problem of multidrug-resistant bacteria and the decreasing of costs and consequences the obtained results from this [7][8][9]. Tuberculosis (TB) that is another mortal infection, causes to death with approximately three million patients in the world every year. According to the World Health Organization (WHO), about 30 million people will be infected within next 20 years [10]. Thus, the treatment of infections has become an important and challenging problem because of the increasing number of multi-drug resistant microbial pathogens [11]. In spite of a large number of antibiotics and chemotherapeutics available for medical usage, the increasing resistance made it necessary to continue the search for new antimicrobial substances. Though various molecules designed and synthesized for this aim, the efforts have demonstrated that 1,2,4-triazoles and their derivatives could be considered as possible antimicrobial agents, some of them studied in our laboratories [12][13][14][15][16][17][18][19][20][21][22][23][24].
Moreover, synthesis of 1,2,4-triazoles fused to another heterocyclic ring has attracted wide spread attention due to their diverse applications as antibacterial-, antidepressant-, antiviral-, antitumorial-and anti-inflammatory agents, pesticides, herbicides dyes, lubricant and analytical reagents [25,26]. Among these, the commonly known systems are generally triazoles fused to pyridines, pyridazines, pyrimidines, pyrazines and triazines. Although there are not many triazoles fused to thiadiazines or thiadiazoles, a number of them are incorporated into a wide variety of therapeutically important compounds possessing a broad spectrum of biological activities [26][27][28][29].
In the present study, as a continuation of our studies on obtaining bioactive molecules, we have performed the synthesis of some new 1,2,4-triazole derivatives and investigation of antimicrobial activities of newly synthesized compounds (Scheme 1, Scheme 2).
The synthesis of ethyl [3-(4-chlorophenyl)-5-oxo-4-{[phenylmethylene]amino}-4,5-dihydro-1H-1,2,4-triazol-1-yl]acetate (11) was performed by the reaction of compound 10 [27] with ethyl bromoacetate in the presence of sodium ethoxide (Scheme 2). The reaction of compound 11 with hydrazine hydrate in water afforded compound 12 but not 16. It was reported that the treatment of Schiff bases derived from type 2 compounds might cause to the hydrolysis of alkylidenamino group to free amino group via the attack of hydrazine hydrate (or water) to imine bond at the same time with the attack to exocyclic ester group [23]. The 1 H NMR spectrum of compound 12 showed complete absence of signals relevant to structure 16. In addition, compound 12 gave M+1 ion peak in the mass spectrum and good elemental analysis results.
The treatment of 12 with carbon disulphide in basic media caused to the conversion of hydrazide side chain into 5-mercapto-1,3,4-thiadiazole ring, thus, compound 13 was obtained. It is known that 5-mercapto-1,3,4-oxadiazoles are exist as their mercapto-thioxo tautomeric forms [13][14][15]. As a result of this tautomerism, the IR spectrum of 13 displayed two stretching bands, one of which observed at 2750 cm -1 belongs to -SH group, the other recorded at 1164 cm -1 represents the existent of -C=S group. Moreover, compound 13 gave satisfactory NMR, mass and elemental analysis data.
The synthesis of compounds 15a,b was carried out by the Mannich reaction of compound 14 with methyl piperazine (for 15a) or morpholine (for 15b) in the presence of formaldehyde solution. Additional signals belonging to methyl piperazine or morpholine moiety and methylene linkage were recorded at the related chemical shif values in the 1 H and 13 C NMR spectra of 15a,b. It was reported that, the compounds having Schiff base structure may exist as E/Z geometrical isomers about the -N=CH-double bond [12,19,33,34]. The literature survey revealed that, the compounds containing imine bond are present in higher percentage in dimethyl-d 6 sulfoxide solution in the form of geometrical E isomer about -N=CH-double bond [33]. The Z isomer can be stabilized in less polar solvents by an intramolecular hydrogen bond. In the 1 H NMR spectra of compounds 14 and 15a,b, the -N=CH-signals were observed as two sets due to the existence of E and Z isomers.
Among the Mannich bases of compound 14, 15b displayed good antimicrobial activities against the test microorganisms that contain an additional morpholine moiety beside 1,2,4-triazole and 1,3,4-oxadiazole rings, while other Mannich base, 15a, that has a methyl piperazine nucleus instead of morpholine, exhibited good or moderate activities towards the test microorganisms except Escherichia coli (Ec) and Klebsiella pneumoniae (Kp).

General
Melting points were determined on a Büchi B-540 melting point apparatus and are uncorrected. 1 H NMR and 13 C NMR spectra were recorded on a Varian-Mercury 200 MHz spectrometer. The IR spectra were measured as potassium bromide pellets using a Perkin-Elmer 1600 series FTIR spectrometer. Mass spectra were obtained at a Quattro LC-MS (ESI, 70 eV) Instrument (except compounds 5, 9a, 9b and 15a). Combustion analysis was performed on a Costech Elementel Combution System CHNS-O elemental analyzer. All the chemicals were obtained from Fluka Chemie AG Buchs (Switzerland). Compounds 1a-e, 2 and 10 were prepared by the way reported earlier [30][31][32].

General Method for the synthesis of compounds 15a, 15b
To the solution of corresponding compound 14 (10 mmol) in dichloromethane, formaldehyde (37%, 1.55 mL) and methyl piperazine (for 15a) or morpholine (for 15b) (10 mmol) were added and the mixture was stirred at room temperature for 3 h. After removing the solvent under reduce pressure, a solid was obtained. This crude product was treated with water, filtered off and recrystallized from ethyl acetate/petroleum ether (1:2) to yield the title compounds.  The antimicrobial effects of the substances were tested quantitatively in respective broth media by using double dilution and the minimal inhibition concentration (MIC) values (µg/mL) were determined [35,36]. The antibacterial and antifungal assays were performed in Mueller-Hinton broth (MH) (Difco, Detroit, MI) at pH 7.3 and buffered Yeast Nitrogen Base (Difco, Detroit, MI) at pH 7.0, respectively. The MIC was defined as the lowest concentration that showed no growth. Ampicillin (10 μg) was used as standard antibacterial and antifungal drugs, respectively. Dimethylsulphoxide with dilution of 1:10 was used as solvent control. The results are shown in Table 1.

Conclusion
This study reports the successful synthesis of some new 1,2,4-triazol-3-one derivatives, one of them into the corresponding Shiff and Mannich bases. The antimicrobial screening studies were also performed in the study. 1,2,4-Triazole nucleus is one of the active components present in many standard drugs and it is known to increase the pharmacological activity of the molecules. The presence of N-methylpiperazine or morpholine moiety is also instrumental in contributing to the net biological activity of a system. Also we already reported antimicrobial activities of some biheterocyclic triazole derivatives incorporating indole, imidazole, 1,3,4-oxadiazole and piperazine moieties. Hence herein we combined all these potential units, namely 1,2,4-triazole and 1,3,4-oxadiazole, imidazole, indole, morpholine, piperazine or methyl piperazine ring.
The antimicrobial screening suggests that among the newly synthesized compounds, the compounds 3 and 8 exhibited moderate activities towards Escherichia coli (Ec) and Klebsiella pneumoniae (Kp), similarly, compounds 11 and 12 showed moderate activities against Enterobacter aerogenes (En), Staphylococcus aureus (Sa), Enterococcus faecalis (Ef) and Bacillus careus (Be); while good antimicrobial activities were found for compounds 13 and 14 against the test microorganisms. Also the Mannich bases 15a,b displayed good or moderate antimicrobial activities against the test microorganisms. On the other hand, none of the synthesized compounds showed antimicrobial activity against Candida tropicalis (Ct) and Candida albicans (Ca).