Synthesis, antimicrobial and antioxidant activities of pyridyl substituted thiazolyl triazole derivatives

In this present study, 63 different 5-[4-methyl-2-(pyridin-3/4-yl)thiazole-5-yl]-4-substituted-3-substituted benzylthio- 4H -1,2,4-triazole derivatives were synthesized, and evaluated for their in vitro antimicrobial activity against various human pathogenic microorganisms and antioxidant activity. The derivatives were synthesized in a multi-step synthesis procedure including triazole and thiazole ring closure reactions, respectively. The synthesized derivatives (A1-24; B1-39) were screened for their antibacterial, antifungal, and antioxidant activities compared to standard agents. The derivatives possessing 3-pyridyl moiety particularly exhibited relatively high antibacterial activity (MIC= < 3.09-500 µg/mL) against Gram-positive bacteria, and compounds possessing 4-pyridyl moiety showed remarkable antioxidant activity.


INTRODUCTION
Antimicrobial chemotherapy is a concern for many regions and centuries. The development of resistance against existing chemotherapeutics is still an important issue, although there are a large number and wide spectrum of antimicrobial drugs. In addition, disproportionate antibiotic use increases the associated problems, and prevents the treatment from reaching the desired outcome. Thus, there is an urgent need for new antimicrobial drugs (Tomasic et al., 2010;Zoumpoulakis et al., 2012;Perron et al., 2015;Blanco et al., 2016).
It is well-known that radicals such as superoxide anion and hydroxyl affect some pathological or physiological processes (Miliovsky et al., 2015). Organisms are suitable to provide the stability between the radicals and their antioxidant systems under normal conditions. However, in a pathological situation, endogenous antioxidants are not enough to cope with the raised levels of the radicals (Halliwell, 1996;Halliwell, 2001).
Pyridine compounds that bound to different azole rings are an important class of the heterocyclic system. The pyridine ring, which has many various biological effects, is present in more than 7000 drugs produced by the worldwide pharmaceutical industry (Hosseinzadeh et al., 2020;Elkanzi, Bakr, Ghoneim 2019;Zaki, Al-Gendey, Abdelhamid 2018;El-Naggar 2018;Chaubey, Pandeya, 2011).

General procedure for the synthesis of the compounds I-V 4-Methyl-2-(pyridin-3/4-yl)-5-carbethoxythiazole (I)
Pyridine-3/4-thiocarboxamide (15 g, 109 mmol) was dissolved in ethanol (200 mL), then ethyl 2-chloroacetoacetate (19.5 mL, 131 mmol) was added to this solution and the mixture was refluxed for 4 days. After TLC check, the solvent was evaporated and the residue was treated with water and neutralized with sodium acetate. The precipitate was filtered, washed with water, dried and recrystallized from ethanol. The physical properties and spectral data of the final compounds are given below;

Minimum inhibitory concentration (MIC):
The minimum inhibitory concentration (MIC) values were determined by broth microdilution methods (CLSI, 2006;CLSI, 2008). The results here were compared with standard antimicrobial agents. Test Compounds were diluted between [2000-3.9 μg/ mL for minimum inhibitory concentrations, and the antimicrobial standard agents comprised of ampicillin, chloramphenicol, ketoconazole, and oxiconazole (64-0.125 µg/mL) were prepared in dimethyl sulfoxide (DMSO) and water. The compounds (100 µL) were added to wells of row A, while the remaining wells in rows B to H received 50 µL of Mueller-Hinton Broth. Bacterial suspensions were grown overnight in double strength broth and standardized to 10 5 cfu/mL for bacteria. Each bacterial suspension (50 µL) was added to the appropriate well. Candida strain was inoculated on Potato Dextrose Agar (PDA) prior to the experiments at 35 °C. After incubation had grown, the microorganism was inoculated with sterile saline of 0.85%. Subsequently, it was standardized using a turbitometer (Biosan) (McFarland No: 0.5) to 5 x 10 3 cfu per well in RPMI medium inoculated under sterile conditions. Serial dilution series were prepared in 100 µl RPMI medium with an equal amount of the test compounds. After serial dilution, 100 µL microorganism suspension was pipetted into each well and then incubated at 35°C for 24 h. Positive growth controls (to assess the presence of turbidity) were performed in wells without standard antimicrobial agents. After incubation at 35°C for 24 h, the first well without turbidity was determined as the minimal inhibitory concentration (MIC, µg/mL). All experiments were repeated in triplicates, and mean values were reported.

1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity:
Serial dilutions were prepared from the stock solutions (4 mg/mL) of the test compounds to get the half concentrations of the previous one. To diluted solutions, DPPH • (equal amounts) was added.
After 30 min, UV absorbance was recorded at 517 nm. The experiment was performed in triplicate for extract and positive standard control, BHT (Butylated hydroxytoluene). The average of the absorptions was noted for each concentration. The percentage inhibition of triplicate experiments was calculated using Equation 1. The IC 50 value, which is the concentration of the test compound that inhibits 50% of the free radical concentration, was calculated as mg/mL using Sigma Plot statistical software (Kumarasamy et al., 2007). Table I were synthesized using the sequence of reactions depicted in Figure 1. In the first step, compound I derivatives were prepared via the reaction of pyridine-3/4-thiocarboxamide with ethyl 2-chloroacetoacetate. The resulting thiazole derivatives substituted with pyridyl moiety I, were treated with hydrazine hydrate to produce the hydrazide derivatives II, and then with appropriate aryl isothiocyanate derivative III, respectively. The obtained thiosemicarbazide derivatives were treated with potassium carbonate to form 1,2,4-triazole-3-thione compounds IV. Finally, compounds V (A1-A24 and B1-B39) were synthesized by the reaction of 1,2,4-triazole-3-thiones IV, and various benzyl bromide derivatives. The structures of the final compounds were elucidated by using spectral data, listed in the experiment. In the IR spectra of compounds A1-A24 and B1-B39, the aromatic C-H stretching vibrations gave rise to bands at 3086-3005 cm -1 and 3146-3004 cm -1 . C=N, C=C stretching bands were observed in the regions 1636-1573 cm -1 , and 1639-1586 cm -1 , respectively. The 1 H NMR spectra of compounds A1-A24 and B1-B39, exhibited singlet peaks owing to -S-CH 2 protons at 4.50-4.39 ppm, and 4.51-4.20 ppm upfield. Resonances of other aliphatic protons and aromatic protons were observed at the expected regions. Formation of different 1,2,4-triazoles linked with substituted benzyl groups through the thio linkage, derivatives were confirmed by their IR, 1 H NMR, and MS spectroscopic, and CHN analytical data, respectively. In 13 C NMR spectrum of the compound A2, carbon resonance of methylene carbon bridge between sulfur and phenyl appeared in the region 35.82 ppm. The thiazole and phenyl connected CH 3 carbons resonated at 17.22 and 19.01 ppm, respectively. The signals of thiazole, triazole, pyridine and phenyl ring systems were observed at 164. 64-155.70, 151.88-137.23, 148.59-117.15, 134.65-124.78 ppm, respectively (for more details evaluate the experimental section, and supplementary data).

Final compounds listed in
In the MS spectra of the synthesized compounds, the M+1 base peak was observed.

Antimicrobial activity
All compounds (A1-14, B1-39) were screened for their in vitro antimicrobial activity against E. coli (NRRLY B 3008), P. aeruginosa (ATCC 10145), S. aureus (ATCC BAA-1026), B. Cereus (NRRLY B 3711) and C. albicans (ATCC 24443) using the microbroth dilution method. Chloramphenicol, ampicillin, ketoconazole and oxiconazole were used as standard drugs, and positive control group was also studied for comparing the microbial growth. MIC (minimum inhibitory concentration) was determined for all compounds, and standard drugs, as the lowest inhibition concentration that killed all tested microorganisms after overnight incubation. Tested compounds were active mainly between 500-31.25 µg/ mL, as seen in Table II. Among the tested compounds, most of them showed high antimicrobial activity, particularly against Gram-positive bacteria and serial dilution was continued to 3.09 µg/mL as the second stage for antibacterial activity evaluation for the selected potent derivatives (see Table III). Compounds A2, A3, A8, A9, A10, A15, A16, A17, A18, A23, and A24, which exhibited relatively high inhibition against two Grampositive bacteria, and Candida albicans, were tested at lower three concentrations (      The structure-activity relationship analysis of the triazole derivatives revealed that compounds differ from each other with 3-pyridyl-, 4-pyridylmoieties, and with substituents on both phenyl rings. The substituents were varied as methyl-, methoxy-, and chloro-functions on ortho-, meta-and, parapositions of the phenyl rings. Compounds from series A possessing 3-pyridyl-residue -were remarkable due to their antimicrobial activity. Also, compounds bearing ortho-, and para-substituents on phenyl rings stand out with high antibacterial activity, especially against Gram-positive pathogens.

Antioxidant activity
All final test compounds (A1-24, B1-39) were screened for their antioxidant activity using in vitro DPPH radical scavenging assay, which is one of the assays that depend on measuring the consumption of stable free radicals (Bayomi et al., 2015;Li et al., 2015). The obtained results are presented in Table IV, reported in IC 50 values (the concentration of the tested compounds which inhibited half percentage of free radicals) for compounds B7, B8, B10, B11, B13, B28, B30, B32, B33, and B34. The IC 50 value could not be calculated for the other compounds at the highest tested concentration (> 4 mg/mL). Interestingly, the antioxidant property was not found for the compounds in A1-A24 series, including 4-pyridyl moiety, contrary to their relatively high antimicrobial activity.  lowest IC 50 , which were as low as 17.10 and 17.40 µg/ mL. Compounds B7, B8, B10, B11, and B13, including 2-methyl phenyl moiety at fourth position of triazole ring, have attracted attention with antioxidant activity potential. Additionally, other indicated compounds contain chloro substituted phenyl moiety at triazole ring. The remaining parts of the molecules differ from each other with the substituents on benzyl residue at the third position of triazole ring linked to the sulfur atom. Among them, 2-chloro, 2-methyl, 3-chloro and 3-methoxy substituents have come into prominence with the presence of repetitive antioxidant activity.

CONCLUSION
To the best of our knowledge, the new compounds were tested for the first time for their biological and pharmacological potential. Thus, in this present study, 63 new triazole derivatives were synthesized, and evaluated for their in vitro antimicrobial, and antioxidant activities, respectively. The antibacterial screening revealed that compounds from series A possessing 3-pyridyl-residue showed relatively high antibacterial activity against Gram-positive bacteria. In addition, compound; B7 and B8 exhibited the highest antioxidant activity, which can be further evaluated in vitro for potential drug development. According to the present findings, further detailed biological and pharmacological investigations are worthwhile for the synthesized heterocyclic compounds.