Synthesis, antimicrobial activities and GAPDH docking of novel 1, 2, 3-triazole derivatives

Purpose: To synthesize new triazole derivatives in order to overcome the problem of side effects of antimicrobial agents and microbial resistance, while broadening the spectrum of antimicrobial activity. Methods: The starting triazole, compound 1, was prepared through click chemistry and reacted with chloroacetyl chloride to yield compound II. Triazole 1 was reacted with acids and aldehydes to produce oxadiazole (III) and azomethine (IV) which cyclized in acetic anhydride to give a new acetylated oxadiazole (V). Minimum inhibitory concentration (MIC) and resorufin assays were used for antibacterial and anti-parasitic screening, respectively. Compounds II and IVb were subjected to molecular docking studies using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Molecular Operating Environment (MOE) program. Results: Novel oxazole-triazole derivative (III) showed high activity against Pseudomonas aeruginosa and moderate activity against Staphylococcus epidermidis, whereas compound IVc showed moderate activity against Staphylococcus epidermidis. Chloro-acetyl-triazole II and 2-hydroxyphenyl-triazole Schiff base (Ivb) showed pronounced activity against the kinetoplastid parasites, Leishmania major, Leishmania mexicana and Trypanosoma brucei. Conclusion: The new synthesized triazoles represent a new antimicrobial scaffold and identifies potential new lead compounds for follow-up and for further mechanistic studies.


INTRODUCTION
Compounds containing triazole cores have numerous pharmacological activities and applications in the pharmaceutical field [1 -4] .
Combination of a triazole with triazines (3), pyridines (2), pyridazines (3), pyrazines (1), and pyrimidines (4) form new scaffolds with pronounced activities. The incorporation of the N-C-S skeleton into triazole and thiadiazine produces compounds with significant antimicrobial activities [5]. The nucleophilic amino group is the center for the synthesis of heterocyclic rings [6] and for obtaining known Schiff base antimicrobial derivatives [7]. On the other hand, derivatives of oxadiazole are reported to display significant antimicrobial activities, and the merging of two or more different pharmacophores is considered a promising avenue for the design of potent antimicrobials against which resistance does not easily develop [8].
The main kinetoplastid pathogens are classified in the genera Trypanosoma and Leishmania. Leishmaniasis is the result of a Leishmania infection, whereas an infection of Trypanosoma brucei rhodesiense or Trypanosoma brucei gambiense results in sleeping sickness (human African trypanosomiasis, HAT). Moreover, the parasite Trypanosoma cruzi is responsible for Chagas disease, an often-fatal infection particularly prevalent in South and Central America. Worldwide, some 20 million people are infected with these kinetoplastid pathogens, resulting in estimated 95,000 deaths per year [9]. The main challenges of kinetoplastid disease therapies are extensive toxicity, lack of efficacy and potential resistance of the Leishmania species [10], T. brucei [11] and T. cruzi [12]. In the present study, the main target was the synthesis and evaluation of antimicrobial and anti-kinetoplastid parasite activities in new scaffolds of phenyl-triazole rings merged with azomethine, oxadiazole and acetylated oxadiazole rings which are expected to confer high antimicrobial activities with reduced toxicities. The target compounds were obtained through the synthetic pathways shown in Scheme 1.

EXPERIMENTAL Chemistry
The identities, purity and structures of the novel compounds were confirmed according to reported methods [13][14][15]
The precipitated product was filtered and washed with cold water. Then, compound III was crystallized from ethanol.

Preparation of compounds IVa -IVd
Appropriate amounts of aldehyde (0.01 mole) and hydrazide compound I (0.01 mole) in ethanol (50 mL) with two drops of glacial acetic acid, were refluxed for 10 h. The reaction solution was cooled in ice bath, filtered, and crystallized from ethanol.

Synthesis of compounds Va -Vc
Azomethine compound IV (0.01 mole) and acetic anhydride (30 mL) were refluxed for 10 h. The reaction solution was cooled in ice bath and continuously stirred with distilled water (100 mL) for 6 h. Thereafter, the new oxadiazoles were filtered and crystallized from ethanol.

Assessment of antibacterial activity
The microorganisms used were ATCC or LMG standard isolates kindly provided by Beni-Suef University, Faculty of Pharmacy Microbiology and Immunology Department. The minimum inhibitory concentrations (MICs) of the synthesized compounds were determined using the agar dilution method according to the Clinical Laboratory Standards Institute (CLSI) [17]. For each sample, different concentrations were diluted with Muller Hinton agar to give final concentrations ranging from 200 to 6.25 µg/mL. Dimethyl sulphoxide (DMSO) was used as a negative control plate. All bacterial isolates were sub-cultured on Brain Heart Infusion Agar (BHIA) and incubated at 37 °C for 24 h [17]. Each experiment was performed in duplicate.

Determination of anti-kinetoplastid activity
All parasite strains were kept and cultured at the University of Glasgow, and were standard strains used in many published papers [28].

Culturing of Trypanosoma brucei bloodstream forms (BSF) in-vitro
Two strains of the bloodstream forms of Trypanosoma brucei were utilized. The first strain was the wild type of Trypanosoma brucei (s427-WT) and the other was clone B48, which was acquired from a TbAT1-KO strain created by the genetic deletion of the TbAT1 gene from s427-WT, followed by in vitro exposure to incremental concentrations of pentamidine, creating a high level of resistance to pentamidine, diminazene and melaminophenyl arsenicals [19]. The culturing of Trypanosoma brucei bloodstream forms (BSF) in vitro was done according to the method reported earlier [20].

Culturing of Leishmania major and Leishmania mexicana promastigotes
Leishmania major strain Friedlin (LmjF) and a strain of Leishmania mexicana (MNYC/BZ/62/ M379) were propagated in essential medium (HOMEM) at a pH of 7.4 and were supplemented with a 10 % heat-inactivation FCS at 25 ºC, as described earlier [21]. The resultant cultures were then passed through fresh medium three times per week.

Resorufin assay
Resorufin sodium salt (Alamar blue) is usually used as an indicator of cell metabolic functions. It is a non-fluorescent blue dye that is metabolized by live cells to a red fluorescent compound resorufin. The assay was done according to the method reported in [22].

Alamar blue assay
This assay was essentially performed as described previously [23]. Drug solutions (20 mM) for testing in the resazurin assay were prepared in DMSO, ensuring a final concentration of exactly 0.5 % DMSO in the cell suspension (this level of DMSO does not influence cell growth). Plates (96-well) were set up with test concentrations starting from 100 µM and 23 double dilutions over two rows of the plate, with the final well used as a drug-free control. Each well of the plate received 100 µL of medium; the first well in the dilution series also received 100 µL of the 200 µM test compound; 100 µL went to the next well and so on, until 23 dilutions were made. To each well was then added 100 µL of cell suspension containing 2 × 10 5 cells/mL, and the plates were incubated for 48 h at 37 ºC in an incubator containing 5 % CO 2, after which 20 µL of the resazurin solution was added to each well, followed by a further 24 h incubation. Fluorescence was then read using a FLUOstar Optima fluorimeter (BMG Labtech; λ excitation = 530 nm and λ emission = 590 nm). The 50 % effective concentrations (EC 50 ) were calculated from a plot of the data using the equation for a sigmoidal curve with a variable slope, with Prism 5.0 software (GraphPad Software Inc., California, USA).

Resazurin assay
The resazurin assay for L. major promastigotes and L. mexicana was performed exactly as described earlier [24]. It was very similar to the procedure for Trypanosoma brucei, except for the use of HOMEM medium (Section 3.3.2) and 1 × 10 5 cells per well. The incubation time was 72 h prior to the addition of the resazurin solution, with additional incubation for 24 h after the addition.

Molecular modelling studies
In this study, Molecular Operating Environment (MOE) version 2010.08 (Chemical Computing Group Inc., Montreal, Quebec, Canada) was used in the docking experiments. The crystal structure of nicotinamide adenine dinucleotide (NAD) bound to glyceralde-3-phosphate dehydrogenase (GADPH, PDB: ID 1GYP) was obtained from the RCSB Protein Data Bank. Docking of the co-crystallized ligands was used to study the scoring energies, root mean values, and interactions of amino acids [18].

Trypanosoma brucei bloodstream forms (BSF) in-vitro
In this research, two strains of the bloodstream forms of Trypanosoma brucei were utilized. The first was a standard drug-sensitive laboratory strain of Trypanosoma brucei (s427-WT). The other was TbAT1-B48, which was derived from s427-WT through a genetic knockout of the TbAT1/P2 drug transporter, followed by exposure to a gradual increase in the concentration of pentamidine [19]. It is usually highly resistant to pentamidine and diminazene as well as melaminophenyl arsenicals, lacking both the TbAT1/P2 transporter and the high-affinity pentamidine transporter [18]. The results are shown in Table 2.

Promastigotes of Leishmania major and Leishmania mexicana
The antileishmanial activity closely mirrored the activity against the related kinetoplastid parasite T. brucei (Table 2), with compounds II and IVb displaying EC 50 values of 10.1 ± 2.1 µM and 18.0 ± 0.8 µM, respectively, against L. major promastigotes.
Compounds II and IVb had moderate inhibitory effects against kinetoplastid parasite, while the other compounds showed no activity.

Molecular docking results
In an attempt to explore the mechanisms of action of the newly synthesized target compounds as anti-kinetoplastid parasite agents, the compounds were subjected to molecular docking studies using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which catalyzes the sixth reaction in the glycolytic sequence [20]) In kinetoplastids such as Trypanosoma and Leishmania, the glycolytic pathway is localized mainly in

DISCUSSION
The structure of chloroacetyl-triazole II was confirmed by NMR in which the new NMR peaks that appeared at δ 4.46 (HNMR) and 44.47 (CNMR) were equivalent to CH 2 Cl, and the presence of chloride in the mass spectra confirmed the structure of chloro-acetyl-triazole II. The disappearance of the carbonyl group peaks in the IR spectrum and NH peaks in the 1 H-NMR spectra indicated the structure of oxadiazole-triazole III. The molecular structure of each azomethine derivative was confirmed by the appearance of new and characteristic de-shielded azomethine peaks (CH=N) and the disappearance of NH 2 peaks. The azomethine CH peak appeared at δ 8.51-8.61 ppm, while the methoxy groups of azomethine derivatives appeared in the aliphatic region in the 1 H-NMR or 13 C-NMR spectra. The methoxy group peaks appeared at δ 3.37-3.85 ( 1 H-NMR) and 50-56 ( 13 C-NMR) ppm. The new phenolic (OH) peak of compound IVb appeared at δ 11.82 ppm ( 1 H-NMR). New NMR peaks (of methyl group) indicating the acetyl moiety appeared at δ 1.92-2.14 ( 1 H-NMR) and 11-20 ( 13 C-NMR) ppm, and confirm the structure of oxadiazole derivatives Va-Vc.
Only compound oxadiazole III displayed activity against Pseudomonas aeruginosa, with an MIC of 12.5 µg/mL, which may be attributed to the presence oxadiazole ring.
The chloroacetyl compound II and azomethine containing hydroxyl group IVb, displayed moderate activity against T. brucei, with EC 50 values close to 10 µM, and importantly, no loss of activity against the multi-drug resistant strain B48. The moderate activities of compounds II and IVb may be due to their polarity.
It was observed that the EC 50 values against L. mexicana were very similar to those found against L. major, confirming that the scaffolds have broad activity against kinetoplastid parasites, and that the anti-kinetoplastid activity could be further improved by exploring more of the structure-activity relationships and identifying the targets to enable structure-aided design.
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is an attractive drug target in trypanosomes since their bloodstream forms lack the citric acid cycle and the classical electron transport chain in their minimalized mitochondrion, making them depend solely on glycolysis for their energy requirements. Glyceraldehyde 3-phosphate dehydrogenase from the pathogenic trypanosomatids T. brucei is quite similar to GAPDH from Leishmania Mexicana. However, the GAPDH in these pathogens is structurally different from the human GAPDH. This difference is exploited in the structure-based design of compounds that selectively inhibit the trypanosomatid enzymes but not the human homologue. Moreover, GAPDH was used in the present study as a target hunt. The novel synthesized triazoleoxazole scaffold has promising antimicrobial activity as a result of its multi-targets and mutual combination of two active structures.

CONCLUSION
The oxadiazole nucleus of compound Vc or nonacetylated III has important antibacterial activity, especially against Pseudomonas aeruginosa. However, the 2-hydroxy group in compound IVb and the chloroacetyl group in compound II strongly improve the anti-parasitic activities of these compounds; they may affect the conformation of the structure with respect to formation of H-bonds with GAPDH receptors, if GAPDH is confirmed to be the cellular target. Thus, the antimicrobial activity of the novel triazoles may be beneficial for the discovery of new antimicrobial drugs.