Synthesis of a series of pyrazoline derivatives carrying tosyl substitution and evaluation of their antimicrobial properties

Article history: Received on: 22/06/2016 Revised on: 30/07/2016 Accepted on: 22/08/2016 Available online: 29/11/2016 A series of pyrazoline derivatives with tosyloxy substitution were synthesized and characterized. They were evaluated for antimicrobial potential against Mycobacterium smegmatis, gram positive Staphylococcus aureus, gram negative Escherichia coli, and a fungi Candida albicans. The initial screening using zones of inhibition at 50μL concentrations revealed that they are moderately active against the tested strains, and therefore further studies have not been carried out.


INTRODUCTION
The antimicrobial agents with novel structural characteristics are increasingly in demand due to antimicrobial resistanceto the prescribed drugs (Bogatcheva et al., 2006).Various strategies are adopted to find newer drugs among them are synthesis of small chemical entities, complex molecular structures, and metal complexes of organic ligands or isolation of structural lead from natural plant products or from marine resources (Muller, 2003).
Other strategies are to synthesize novel molecules based on hybrid pharmacophores, where two or more therapeutically useful structural units are combined with appropriate chemical linkages (Kalaria et al., 2014).Pyrazoline compounds are having significant biological activities as they are structurally related to the pyrazolone family of drugs.Among isomeric pyrazoline derivatives, 1,2-pyrazolines are the most thermodynamically stable and therefore are explored widely for a range of biological applications, including antitubercular, antioxidant, anticancer and as antidepressant agents.Due to their biological significance, a variety of structural modifications have been achieved around pyrazoline ring (Ghorab et al., 2010;Rostom et al., 2003;Yeu et al., 2001;Amnerkar et al., 2010).We have recently explored the synthesis and antimicrobial activity of a series of compounds having aryloxy substituents attached to pyrazoline ring providing moderate antimicrobial action.
Introduction of tosyl group at an aromatic ring gives new molecules that can be utilized to probe their biological potential as well as further derivatization of such molecules are possible as tosyl group is easily replaceable with many nucleophiles.In one example, we have described the synthesis and antimicrobial properties of newer pyrimidinethione derivatives possessing tosyl substituents (Shubhalaxmi et al., 2016).In this paper, we report the synthesis, characterization and antimicrobial screening of a series of pyrazoline derivatives.

MATERIALS AND METHODS
All reagents were used as procured from commercial suppliers and used without further purification.The melting points were determined in open capillaries, using Thomas Hoover melting point apparatus and expressed in C.The reactions were monitored using TLC for completion and compounds were checked for purity by TLC on silica gel-G (Merck grade).Infrared spectra (IR) were recorded on Schimadzu 8400S Infrared Spectrophotometer using potassium bromide (KBr) pellets and the values are expressed in cm -1 .The 1 H NMR spectra of the compounds were recorded on Bruker Ascend 400 MHz NMR spectrophotometer using TMS as an internal standard and the values are expressed in δ ppm.

Procedure for the synthesis of chalcones
The required chalcones were synthesized by aldol condensation of the corresponding aldehydes and ketones.The detailed synthetic procedure and their characterization data were given in our earlier publications (Shubhalaxmi et al., 2016;Shubhalaxmi, Manjunatha et al., 2016) Procedure for the synthesis of pyrazoline derivatives (1a-f, 2 a-f) The various pyrazoline derivatives are synthesized by reacting chalcone derivatives with excess of hydrazine hydrate in presence of catalytic amount glacial acetic acid for 3-6 hours at 70 -80 0 C.The formation of the products were identified by thin layer chromatography and completion of the reaction is monitored.The products were recovered by pouring the reaction mixture into ice cold water or in few cases crystals were developed after keeping the mother liquor overnight.The products were isolated by filtration, dried and recrystallized from suitable solvent.The R f values were reported using mobile phase of hexane: ethylacetate (2:0.9).CH 3 ), 1504 (C=N str), 1157 and 1200 (asym, sym SO 2 str); 1 H NMR (DMSO, δ ppm): 2.472 (s, 3H, CH 3 ), 2.76-2.83(q, 1H, H of pyrazoline ring), 4.81-4.86(t,2H, H of pyrazoline ring), 7.00 -7.76 (m, 14H, aromatic H).

Antimicrobial Activity
The antimicrobial property of the synthesized compounds 1 a-f and 2a-f was evaluated by well diffusion method in nutrient agar media.Inhibition zones of the test compounds against microorganisms considered qualitatively suggest the antimicrobial potential (Sathish et al., 2012).12 h old bacterial culture of a tuberculosis variant bacteria Mycobacterium smegmatis (MTCC 944), gram positive bacteria Staphylococcus aureus (MTCC 3160), and gram negative bacteria Escherichia coli (MTCC 1687) were taken for the in vitro antibacterial evaluation of compounds (Palomino et al., 2002).Antifungal activity of the compounds were tested against pathogenic fungi Candida albicans (MTCC 7253).The bacterial and fungal cultures were obtained from the microbial type culture collection, IMTECH, Chandigarh, India and maintained the cultures as per the standard protocol.About 15-20 mL of nutrient agar media was poured into each petri plate and allowed to solidify for 15 minutes inside laminar air flow chamber.About 100 L of 0.5 McFarland standard of bacterial/fungal suspension was inoculated on the agar media and spread on the whole surface by swabbing with sterile cotton buds.Than 5 mm wells were dig on the seeded agar plates with a sterile cork borer.Working solutions of the test compounds is prepared in DMSO at 10 mg/mL as stock and were poured at different concentrations (25 and 50 g mL -1 ) in to the wells in triplicates.The test plates were incubated at 37 C for 12 h before observing for the zone of inhibition, which is measured in millimeter.DMSO was used as a negative control.Ciprofloxacin was used as antibacterial standard and fluconozole as antifungal standard (10 mcg discs).

Chemistry
The title compounds were synthesized starting from hydroxy acetophenones and hydroxy aldehydes by introducing tosyl substitution, as per the procedure described in detail in our previous publication (Shubhalaxmi et al., 2016).The structures of all the compounds were confirmed using FT-IR and 1 H NMR. The absence of the carbonyl peak of chalcones around 1650 cm -1 and the appearance of additional peaks due to NH stretch at around 3200 cm -1 along with C=N stretch at around 1550 cm -1 confirms the formation of pyrazoline ring.The presence of S=O asymmetric and symmetric stretching bands in FTIR spectra of the compounds also prove that tosyl substitution is unaffected under the experimental conditions.The PMR spectrum of the compound 1a show peaks at 2.4 for CH 3 group of tosyl moiety, 2.76-2.83due to H of pyrazoline ring, 4.81-4.86for 2H of pyrazoline ring, and the remaining aromatic protons appeared as multiplets in the range 7.00-7.76.In many cases, the NH protons were merged in the aromatic range and therefore not assigned separately.

Biological Evaluation
The zones of inhibition revealed that the compounds screened are moderately active towards all the tested gram positive bacteria.There were no inhibition zones exhibited against gram negative bacteria, E.coli, implying that these compounds are inactive against similar bacteria.Few of the compounds showed moderate zones of inhibition against the fungi considered for the study, C.albicans.It may be noted that the compounds without substitution at 4 of the aromatic ring, 2a, showed highest inhibition zone against M. smegmatis.The compound 2b, with4-chloro substitution in aromatic ring showed remarkably higher inhibition zone against S. aureus.The compounds 1a, 2a and 2e without substitution at aromatic rings and 3,4-dimethoxy substitution gave highest inhibition zones against C. albicans at 50 µL concentrations.

CONCLUSION
A series of 2-pyrazoline derivatives of different tosyloxy substituted chalcones were synthesized.Some of these compounds are positional isomers of one another.The synthesized compounds were characterized by spectral techniques and structures were assigned.The compounds were subjected to qualitative screening for their antimicrobial potential using well diffusion assay with drug Ciprofloxacin, and Fluconazole for antibacterial and antifungal action as standards respectively.Some of the compounds showed moderate zones of inhibition at 50 µL concentration.We intend to extend the scope of the present work by replacing tosyl group with better biologically active substituents by nucleophilic substitution.