Synthesis, Antibacterial and Antifungal Properties of Cyclohexane Tosyloxyimine Derivative

Introduction

Antimicrobial resistance is a global health problem and major obstacle in the eradication of infectious diseases. This leads to exploration of synthetic organic compounds as new antimicrobial agents with unique mode of action [1]. Functionally substituted derivatives of cyclohexane possess diverse biological properties. Anticancer activity, antioxidant activity, cytotoxic activity, anti-inflammatory activity and antimicrobial activity of different derivatives of cyclohexane has been reported in literature [2, 3, 4]. Keeping this in mind, wide range of functionally substituted cyclohexane derivatives are being explored as potential antimicrobial agents.

Diacetyl (diethoxycarbonyl) hydroxycyclohexanones act as valuable construction blocks of organic synthesis. This is due to existence of an extensive source of raw materials in the form of available 1,3- dioxocompounds (acetylacetone, ethyl acetoacetate), aliphatic and aromatic aldehydes. Furthermore, these substances have high chemical potential due to presence of oxogroups of various types [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15]. The interaction of diethoxycarbonyl hydroxycyclohexanones with hydroxylamine corresponding oximes is studied in literature [16].

However, transformations of these oximes with use of hydroxyl group as reactionary center, has yet not been investigated. Here we report the synthesis and antimicrobial characterization of diethyl -4- hydroxy -4- methyl -2- phenyl -6- ((tosyloxy)imino) cyclohexane -1, 3- dicarboxylate which is formed by reaction of diethyl 4- hydroxy-6- (hyd-roxyimino)- 4-methyl- 2- phenylcyclohexane-1,3 -dicarboxylate with 4- toluenesulfonyl chloride in boiling acetone in the presence of equimolar triethylamine.

Material and Methods

Synthesis of Diethyl -4-hydroxy-4-methyl-2-phenyl- 6-((tosyloxy) imino) cyclohexane-1,3-dicarboxylate A solution of 5 mmol of diethyl 4-hydroxy-6- (hydroxyimino)-4-methyl-2-phenylcyclohexane-1,3- dicarboxylate, 5 mmol of 4-toluene-sulfonyl chloride and 5 mmol of triethylamine in 20 ml of acetone is boiled for 6 hours. To the resultant solution, 50 ml of cold water is added. After 24 hours, the precipitated powder is filtered, recrystallized from ethanol.

1H and 13C NMR spectra recorded on a Bruker AC-300 instrument (300 MHz on 1H and 75 MHz nuclei at 13C cores) in a (CDCl3 solution, residual signals of the solvent used as the standard. The melting points were determined on a Kofler's table. TLC monitored the purity of the resulting compounds on Silufol UV-254 plates, eluent acetone-hexane 1:2, developer-iodine vapor, UV detector.

Agar Well Diffusion Assay

Standard agar well diffusion method [17] was used to evaluate antimicrobial properties of newly synthesized derivative. Mueller-Hinton agar was used to determine in vitro antibacterial properties against four Gram-positive bacteria (Staphylococcus aureus BDU-23, Bacillus Subtilis BDU-50, Bacillus mesentericus BDU-51 and Bacillus megaterium BDU-N20) and four Gram-negative bacteria (Escherichia coli BDU-12, Klebsiella pneumoniae BDU-44, Acinetobacter baumannii BDU-32 and Pseudomonas aeruginosa BDU-49). Test compound was screened for antifungal properties against Candida tropicalis BDU LK30, Candida pelliculosa BDU KT55 and Candida pseudotropicalis BDU MA88 using sabouraud dextrose agar (SDA). All the test cultures were obtained from collection of Department of Microbiology, Baku State University. Test compound was dissolved in dimethyl sulphoxide (DMSO) and three different concentrations of test compound (0.3%, 0.1% and 0.05%) were evaluated for antimicrobial activity. All the experiments were repeated three times and DMSO was used as control due to its inert nature.

Results and Discussion

Synthesis of Diethyl - 4-hydroxy-4-methyl-2-phenyl- 6-((tosyloxy) imino) cyclohexane-1,3-dicarboxylate As shown in scheme 1, the reaction of diethyl 4- hydroxy-6-(hyd-roxyimino)-4-methyl-2- phenylcyclohexane-1,3-dicarboxylate (I) with 4- toluenesulfonyl chloride (II) in boiling acetone in the presence of equimolecular triethylamine resulted in formation of diethyl - 4-hydroxy-4-methyl-2-phenyl-6- ((tosyloxy)imino) cyclohexane-1,3-dicarboxylate (III):

Scheme 1: Synthesis of test compound.

The catalytic role of Triethylamine (Scheme 2) act as catalyst and as a base, it chips off a proton of hydroxyl group of an oxime fragment forming an anion intermediate (A):

Scheme 2: Catalytic role of triethylamine.

Further this anion reacts with 4-toluenesulfonyl chloride leading to formation of a product (III) (Scheme 3):

Scheme 3

Diethyl - 4-hydroxy-4-methyl-2-phenyl-6-((tosyloxy) imino) cyclohexane-1,3-dicarboxylate: The compound was colorless crystals and yield was 63%. Melting point was found to be 190°C. 1Н NMR spectrum (300 MHz, CDCl3, δ, ppm: 0.77 (t, 3Н, OСН2СН3); 0.99 (t, 3Н, OCH2СН3); 1.31 (3H, s, CH3); 2,0 (1H, d, CH2); 2,43 (3H, s, CH3); 2.78 (1H, d, CH2); 3.51 (1H, s, OH); 3.56 (1H, d, CH); 3.58 ( 1Н, d, CH); 3.73-4.00 (5H, m, OCH2 , CH); 7.15-7.32 (7H, m, CHar), 7.80 (2H, d, CHar). 13С NMR spectrum (75 MHz, CDCl3 , δC, ppm: 13.56 (OCH2СН3); 13.83 (OCH2СН3); 28.48 (CH2); 37.95(CH); 44.93 (CH); 53.69 (CH); 57.08 (OCH2); 60.99(OCH2); 70.92 (C); 46.27 (CH); 50.32 (CH); 63.86 (OСН2); 127.84 (CHar); 128.11 (CHar); 128.61 (CHar); 129.08 (CHar); 129.46 (CHar); 132.46 ( CHar); 137.98 (CHar); 145.00(CHar); 163.01 (C=N); 167.50 (CO2C2H5) ; 173.64 (CO2C2H5). Found, %: C-60.51; H-6.14; N-2.88; C26H31NO8 S, Calculated, %: C-60.33; H-6.04; N- 2.71 (Figure 1).

Table 1 show that test compound showed variable antimicrobial properties against different test cultures. Test compound was found to be more active against gram-negative bacteria as compared to gram-positive bacteria. At 0.3% concentration, cyclohexane derivative showed remarkable antimicrobial activity against Escherichia coli, Acinetobacter baumannii, Bacillus subtilis and Candida pseudotropicalis. Weak to moderate antibacterial activity was observed against Escherichia coli, Acinetobacter baumannii and Candida pseudotropicalis at 0.1% concentration. At 0.05% concentration, test compound was inactive against all the test cultures.

Acinetobacter baumannii was found to be most sensitive Gram-negative bacteria (20 mm zone of inhibition), while Bacillus subtilis was most sensitive Gram-positive bacteria (16.7 mm zone of inhibition). Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacillus megaterium and Bacillus mesentericus were found to be resistant against test compounds. Among the fungal cultures, Candida tropicalis and Candida pelliculosa were found to be resistant while Candida pseudotropicalis was highly sensitive. Thus, cyclohexane tosyloxyimine derivatives can act as potential antimicrobial agents in future.

(-): Inactivity Table 1: Average diameter of inhibition zone in mm.