Facile Synthesis, Characterization, and In Vitro Antimicrobial Screening of a New Series of 2,4,6-Trisubstituted-s-triazine Based Compounds

A series of new 2,4,6-trisubstituted-s-triazine was synthesized, assessed for antimicrobial activity, and characterized by FTIR, 1HNMR, 13CNMR, and elemental analysis. The tested compounds, 4d, 4g, 4h, 4k, and 4n, have shown considerable in vitro antibacterial efficacy with reference to the standard drug ciprofloxacin (MIC 3.125 μgmL−1 against B. subtilis, E. coli, and K. pneumoniae). It was observed that compounds 4d and 4h displayed equipotent antibacterial efficacy against B. subtilis (MIC 3.125 μgmL−1) and S. aureus (MIC 6.25 μgmL−1). The studies demonstrated that the para-fluorophenylpiperazine substituted s-triazine (4n) was potent and exhibited broad spectrum antibacterial activity against S. epidermidis, K. pneumoniae, and P. aeruginosa with MIC of 6.25 μgmL−1 and for E. coli, it showed an MIC of 3.125 μgmL−1 equipotent with reference to the standard drug. Among all the compounds under investigation, compound 4g also demonstrated significant antifungal activity (3.125 μgmL−1) against C. albicans.


Introduction
Over the past decades, the development of new antibiotics has plummeted while antimicrobial resistance (AMR) has increased. Only a limited number of new antimicrobials have been developed in the last decade [1] and overtreatment with the available antibiotics has led to the emergence of AMR [2]. Despite big advances in antimicrobial therapies and strategies in counteracting infections, the emergence of AMR represents an emergency situation [3]. A high percentage of hospital-acquired infections are caused by highly resistant bacteria such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci [4]. Further, infections caused by resistant microorganisms often fail to respond to the conventional treatment, resulting in prolonged illness and higher death risk. Considering the scenario, the World Health Organization has necessitated an urgent and consolidated effort to avoid regressing to the preantibiotic era [5].
Therefore, discovery of novel antimicrobial molecules and their rational use are crucial to combat microbial infections. In recent years, triazine derivatives have gained particular interest on account of their broad biological activities.
Recently, 4-benzyl piperazine derivatives of s-triazine were also found to be active against some Gram positive and Gram negative bacterial strains [16]. The importance of piperazine moiety has been substantiated by the fact that several N-alkyl and N-aryl piperazine derivatives showed potent antibacterial activity against resistant microbial strains [17,18]. Additionally, thiosemicarbazones of piperazine bearing arylmethylidene also displayed potent antimicrobial activity compared to ciprofloxacin [19]. Thus considering the above factors and with a goal to explore and improve the antimicrobial profile of s-triazines, we carried out the synthesis of a series of new 2,4,6-trisubstituted-s-triazines. All the compounds are sequentially substituted with aryl, diethylamino, and/or morpholinyl substituent at 2nd, 4th, and 6th position, respectively, of s-triazine. In addition, the compounds are invariably substituted with piperazine, Nmethylpiperazine or aryl substituted piperazine as one of its substituents of s-triazine scaffold. The precursor for the compounds is cyanuric chloride and the synthetic route involves temperature controlled nucleophilic substitution reaction. The reaction relies on differential reactivity of the substituted chlorine atoms and the easy displacement of the chloride with nucleophiles for nonsymmetrical trisubstituted functionalization [20]. The newly synthesized compounds were screened for their possible antimicrobial activity to identify new candidates as antibacterial and antifungal agents.

Materials and Methods
All the chemicals used in this experiment were of analytical grade and procured from Merck (India) and Hi-Media (India). Cyanuric chloride was obtained from Lonza Ltd., Switzerland, as a gift sample. Melting points were determined in open capillaries using Veego VMP1 melting point apparatus, Veego Instruments Corporation (Mumbai, India), and are uncorrected. The progress of the reactions was monitored by thin layer chromatography developed with nhexane/diethyl ether (1 : 1) and performed on Merck silica gel 60 F254 aluminium sheets and products were purified by recrystallization. IR spectra were recorded on a Perkin Elmer-Spectrum RX-1 spectrophotometer, PerkinElmer, Inc. (California, USA), on potassium bromide pellets and were recorded in cm −1 . 1 H NMR and 13 C NMR spectra were performed on a JEOL AL300 FT-NMR (300 MHz), Jeol Ltd., Japan, in DMSO-d 6 /CDCl 3 using tetramethylsilane as internal standard and the chemical shifts are reported in ppm ( ). Elemental analysis (C, H, and N) were performed on Exeter CE-440 elemental analyzer (Exeter Analytical Inc., USA).

2.1.
Chemistry. Scheme 1 outlines the general classic threestep temperature controlled method of synthesis of 2,4,6trisubstituted-s-triazine derivatives ( were added to it at 0-5 ∘ C and stirred for 2 h. Thereafter, sodium carbonate solution (10%) was added to neutralize the hydrochloric acid evolved during the reaction. The reaction mixture was then poured into crushed ice and the solid separated out was filtered, washed with water, and recrystallized from ethanol.

General Method of Synthesis of 2,4-Disubstituted-
6-chloro-s-triazine Derivatives (3a-3o). 2-Substituted-4,6dichloro-s-triazine derivatives (2a-2o) were added to 100 mL of acetone and different substituted nucleophiles (R 2 H) ( Table 1) (0.02 mol) were added to it and stirred at room temperature for 3 h. Sodium carbonate solution (10%) was added to neutralize the hydrochloric acidevolved during the reaction and the product was filtered and washed with cold water and recrystallized from ethanol.

Preparation of Seed
Organisms. 13.0 g of nutrient broth medium was suspended in 1000 mL distilled water. Then it was boiled to dissolve the medium completely and filtered from 5 sintered glass filter. A set of test tubes with nutrient broth (5 mL) was capped with cotton plugs and sterilized by autoclaving at 15 psig pressure (121 ∘ C) for 15 min. Then after cooling, a loop of organism was inoculated into liquid broth and incubated at 37±1 ∘ C and used within 12 h. The inoculum size for test strain was adjusted to 10 8 CFU/mL (colony forming unit per milliliter) by comparing the turbidity. Method). Petri plates containing 15 mL of Mueller Hinton agar media (Hi-Media) were used for all the bacterial strains and Sabouraud dextrose agar (SDA) media (Hi-Media) were used for the fungal strains. 0.1 mL of seed of various organisms was aseptically inoculated over the sterile solid agar medium. Whatman no. 1 filter paper discs (6 mm in diameter) impregnated with the synthesized compound (10 g/disc) dissolved in dimethyl sulfoxide (DMSO) were placed on the plates. All the sensitivity plates were then incubated at 37 ± 1 ∘ C for 24 h for bacterial strain and 72 h at 25 ∘ C for fungal strain. Ciprofloxacin (10 g/disc, Hi-Media) and fluconazole (10 g/disc, Hi-Media) were used as positive control for the assessment of antibacterial and antifungal activity, respectively. A paper disc impregnated with DMSO was used as negative control. The zone of inhibition on agar plate was measured in millimeters (mm) and the test was performed in triplicate and the average was taken as final reading (Table 2).

Determination of Minimum Inhibitory Concentration.
Compounds that displayed favorable zone of inhibition were considered for further assessment of minimum inhibitory concentration (MIC). MIC of a compound is defined as the lowest concentration at which it completely inhibits visible growth (turbidity on liquid media). Solutions of the test compounds, ciprofloxacin and fluconazole, were prepared in DMSO at a concentration of 100 g/mL. From this stock solution, serial broth dilutions of the compounds (3.125, 6.25, 12.5, 25, 50, and 100 g/mL) were prepared to determine the MIC. All the determinations were done in triplicate and the average was taken as the final reading. The standard antibiotics, ciprofloxacin and fluconazole (100 g/mL), were used as positive controls and 100 g/mL of DMSO was used as a negative control. At the end of the incubation period (37±1 ∘ C for 24 h), the MIC values were determined (Table 3).

Results and Discussion
3.1. Chemistry. Synthesis of the intermediates and target compounds was accomplished according to the steps illustrated in Scheme 1. The structure of the synthesized compounds was confirmed on the basis of IR, 1 H NMR, 13 C NMR, and elemental analysis and the spectral data are in accordance with the structures of the compounds (4a-4o). Only significant bands from IR are reported. The derivatives showed characteristic band in the range of 1570-1577 cm −1 for -C=N stretching of triazine nucleus. Further, strong absorption band in the range of 805-810 cm −1 indicates CN stretch of s-triazine moiety. A characteristic band appearing in the range of 1117-1121 cm −1 corresponds to the C-O-C stretching of morpholine in the IR spectra of compounds 4b, 4i, and 4o. The compounds also displayed -NH stretch for secondary amines that appears in the region 3315-3324 cm −1 . The 1 H NMR spectra of all the derivatives (4a-4o) displayed two triplets in the range 2.98-3.46 and 3.21-3.61 attributable to -CH 2 -protons of piperazine ring and each integrating for four protons except for compounds 4e