Study of 1,2,4-triazole-3(5)-thiol Behavior in Reactions with 1-phenyl-1H-pyrrole-2,5-dione Derivatives and 3-bromodihydrofuran-2(3H)-one and Antimicrobial Activity of Products

In present work we report our studies of the interaction of 1-phenyl-1H-pyrrole-2,5-dione derivatives (N-arylmaleimides) and 3-bromodihydrofuran-2(3H)-one (α-bromo-γ-butyrolactone) as possible [C2]2+ synthons with 1,2,4-triazole-3(5)-thiole targeting on synthesis of novel 5-substituted thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones. According to obtained results was establish that in above-mentioned interactions (conditions: convenient heating, range of solvents/reaction time) the thiol-ene click (for N-arylmaleimides) and SN (for α-bromo-γ-butyrolactone) processes take place, but not [2+3]-cyclocondensation reaction. The structure of compounds was studied using 1H, 13C NMR spectroscopy, LC-MS spectrometry, and X-ray analysis. The prescreening of antimicrobial activity for synthesized compounds was performed against Gram-positive and Gram-negative bacteria, as well as yeasts.

As a part of our ongoing interest to chemistry and pharmacology of azoles [19][20][21][22], in present paper we tried to study this issue more deeply using wide range of solvents/reaction conditions and clarify the structure of products in interactions of 1,2,4-triazole-3(5)-thiol with N-arylmaleimides using X-ray technique. Moreover, we report about early undescribed interaction between α-bromo-γ-butyrolactone and 1,2,4-triazole-3(5)-thiol and characterization of product. The paper also presents the screening results of antimicrobial activity for synthesized compounds against Gram-positive and Gram-negative bacteria, as well as yeasts.

General Experimental Methods
Melting points were measured in open capillary tubes on a BÜCHI B-545 melting point apparatus (BÜCHI Labortechnik AG, Flawil, Switzerland) and are uncorrected. The elemental analyses (C, H, N) were performed using the Perkin-Elmer 2400 CHN analyzer (PerkinElmer, Waltham, MA, USA) and were within ±0.4% of the theoretical values. The 500 MHz-1 H and 126 MHz-13 C spectra were recorded on Bruker AVANCE-500 spectrometer (Bruker, Bremen, Germany). All spectra were recorded at room temperature except where indicated otherwise and were referenced internally to solvent reference frequencies. Chemical shifts (δ) are quoted in ppm and coupling constants (J) are reported in Hz. LC-MS spectra were obtained on a Finnigan MAT INCOS-50 (Thermo Finnigan LLC, San Jose, CA, USA). Solvents and reagents that are commercially available were used without further purification. The synthesis of compound 1 was performed as described in [23], the synthesis and spectral properties of compound 2a-f were early described in [5].

Antimicrobial Activity
The antimicrobial activity of the compounds 2a, 2d, and 2e was determined by the agar diffusion method [24,25]. Nutrient agar (0.5% peptone, 0.3% beef extract, 1.5% agar, 0.5% sodium chloride, distilled water, pH ~ 6.8) was used as a nutrient medium. The test cultures suspensions (in concentration 1 × 10 7 CFU/mL), standardized previously by the optical standard of turbidity, were uniformly sown in Petri dishes with the nutrient agar. Aliquots (20 μL) of 0.1% tested derivatives (concentration 1000 μg/mL) in EtOH/DMSO/water (2:1:1) were placed into wells (diameter of 4.0 ± 0.1 mm) in agar in Petri plates with test microorganisms. The antimicrobial activity was evaluated by measuring the diameter of zone inhibition of microbial growth. The plates were incubated for 24 h at 37 °C. The inhibition zone appeared after 24 h and was measured in mm around the well in each plate. Digital images of culture growth on dishes were obtained and processed with a computer program UTHSCSA ImageTool 2.0 (The University of Texas Health Science Center in San Antonio, San Antonio, TX, USA, © 1995-1996) for calculation of growth inhibition zone diameters. Each experiment was performed in triplicate. Results were expressed as the means ± S.D. The experiments were carried out on microorganism strains, which were isolated in the laboratory of the microbiology research of the Department of Microbiology, Virology and Immunology of the Ivano-Frankivsk National Medical University from ambulatory patients. The following bacterial strains were used: methicillin-sensitive Staphylococcus aureus ATCC 25,923 (MSSA); methicillin-resistant Staphylococcus aureus (MRSA), clinical isolate; methicillin-resistant Staphylococcus epidermidis (MRSE), clinical isolate; Escherichia coli ATCC 25922; yeasts Candida albicans (clinical isolate). Staphylococci of clinic origin were identified using chemical micro-tests "STAPHYtest 16" (Lachema, Czech Republic). Fungi culture was identified on the basis of 40 biochemical tests using the VITEK 2 system with the VITEK 2 YST ID card (bioMerieux, Craponne, France). The sensitivity of strains to antibiotics was determined by disc diffusion and serial dilutions methods.
Since the application of spectral methods to analyze the structure of products is not completely convincing the X-ray crystallographic analysis was used for structure determination of compounds 2b and 2e. The ORTEP drawing and atomic numbering are shown in Figure 1. The reaction between 1,2,4-triazole-3(5)-thiol (1.0 eq.) and α-bromo-γ-butyrolactone (1.0 eq.) were studied in glacial acetic acid and ethanol medium. Such bases as anhydrous sodium acetate (1.0 eq.) and triethylamine (1.0 eq.) were used for reaction. The process was monitored by TLC. It should be noted that nucleophilic substitution is very fast process in mentioned conditions and SN product's (compound 3, Scheme 1) spot had been observed in TLC already after 10-15 min of reflux. The extending of reflux even to 24 h did not lead to the formation of new products accordingly to TLC and finally LC-MS, NMR data. In the 1 H NMR spectrum of compound 3 the broad signal at 14.20 ppm belongs to the NH proton of 1,2,4-triazole ring and indicates the absence of bicyclic annelated product (Figure 2).

Conclusions
In present report the interaction of 1,2,4-triazole-3(5)-thiol with such electrophilic synthons [C2] 2+ equivalents as N-arylmaleimides and α-bromo-γ-butyrolactone have been studied. The X-ray analysis and spectral methods were used for products structure determination. According to dates was establish that in mentioned interactions the thiol-ene click process and nucleophilic substitution take place, but not [2+3]-cyclocondensation reaction as could be expected. The obtained results of pharmacological screening suggest to coming design and synthesis of new 1,2,4-triazol/pyrrolidine-2,5-dione hybrids as potential molecules with promising antimicrobial properties. Funding: This work was partially supported by COST Action NutRedOx-CA16112 "Personalized Nutrition in aging society: redox control of major age-related diseases" (for S.H.).