Preliminary structure-activity relationship studies on some novel s-substituted aliphatic analogues of 5-{1-[(4-chlorophenyl) sulfonyl]-3-piperidinyl}-1, 3, 4-oxadiazol-2-yl sulfide

Purpose: To study the structure-activity relationships of synthetic multifunctional sulfides through evaluation of lipoxygenase and anti-bacterial activities. Methods: S-substituted derivatives of the parent compound 5-(1-(4-chlorophenylsulfonyl) piperidin-3-yl)-1, 3, 4-oxadiazole-2-thiol were synthesized through reaction with different saturated and unsaturated alkyl halides in DMF medium, with NaH catalyst. Spectral characterization of each derivative was carried out with respect to IR, 1 H - NMR, 13 C - NMR and EI - MS. The lipoxygenase inhibitory and antibacterial activities of the derivatives were determined using standard procedures. Results: Compound 5e exhibited higher lipoxygenase inhibitory potential than the standard (Baicalein®), with % inhibition of 94.71 ± 0.45 and IC 50 of 20.72 ± 0.34 µmoles/L. Compound 5b showed significant antibacterial potential against all the bacterial strains with % inhibition ranging from 62.04 ± 2.78, 69.49 ± 0.41, 63.38 ± 1.97 and 59.70 ± 3.70 to 78.32 ± 0.41, while MIC ranged from 8.18 ± 2.00, 10.60 ± 1.83, 10.84 ± 3.00, 9.81 ± 1.86 and 11.73 ± 5.00 µmoles/L for S. typhi, E. coli, P. aeruginosa, B. subtilis and S. aureus, respectively. Compounds 5d , 5e and 5g showed good antibacterial activity against S. typhi and B. subtilis bacterial strains. Conclusion: The results suggest that compound 5e bearing n-pentyl group is a potent lipoxygenase inhibitor, while compound 5b with n-propyl substitution is a strong antibacterial agent. In addition, compounds 5d , 5e and 5g bearing n-butyl, n-pentyl and n-octyl groups, respectively, are good antibacterial agents against S. typhi and B. subtilis.


INTRODUCTION
Millions of people suffer from bacterial infectious diseases which are among the leading causes of morbidity and mortality worldwide [1,2]. Thus infectious diseases of bacterial aetiology are of serious public health significance. However, the treatment and management of bacterial infectious diseases are hampered by numerous factors, including the emergence of multi-drug resistant strains of bacteria [3]. The phenomenon of multi-drug resistance has led to increasing attention on the search for new, synthetic compounds with potential anti-microbial properties. Indeed a large number of compounds have been synthesized and evaluated for their anti-microbial potential [4,5].
Oxadiazole compounds have potent pharmacological properties. They are 5membered heteroaromatic rings which exist in different isomeric forms. Oxadiazoles have been characterized as frequently occurring motifs in drug-like compounds [6]. The 2,5-di-substituted -1, 3 4-oxadiazoles associated with a wide range of hetero-atom rings are important parts of a variety of clinical drugs used for management of different diseases.

EXPERIMENTAL Materials
All chemicals were products of either Merck (Darmstadt) or Sigma Aldrich (St Louis). For column chromatography (CC), silica gel (70 -230 mesh) was used. Silica plates (0.25 mm) coated on alumina were used for thin layer chromatography (TLC) to check the purity of synthesized compounds. Ethyl acetate:n-hexane (30:70) was used as mobile phase. To visualize the fluorescent spots UV lamp was utilized at 254 nm. Using KBr pellet method Jasco FTIR spectrometer recorded the IR spectra. Bruker spectrometers working at 300 and 400 MHz were employed in recording the 1 H-NMR and 13 C-NMR spectra. CDCl 3 was used as solvent and TMS was the reference standard. Chemical shifts (δ) were given in ppm, while coupling constants (J) were recorded in Hz. EIMS spectra were recorded on JMS-HX 110 spectrometer. Melting points were determined on Griffin and George melting point apparatus using open capillary tube method.

Synthesis of Ethyl-1-[(4-chlorophenyl) sulfonyl] piperidine-3-carboxylate (1)
Ethyl piperidine-3-carboxylate (a) (30 mmol) (Scheme 1) was mixed with 4-chlorobenzene sulfonyl chloride (30 mmol) in 30 mL distilled water contained in 100 mL round bottom flask. During the reaction, the pH of reaction medium was maintained at 10 -11 by addition of a small amount of 15 % Na 2 CO 3 solution and the reaction mixture was stirred at room temperature. TLC was utilized for monitoring reaction progress, with n-hexane and EtOAc as mobile phase. At the end of the reaction, the flask contents were neutralized and precipitates were filtered, washed with distilled water and recrystallized in ethanol. The resultant crystalline product was designated as compound 1.

Synthesis of 1-[(4-chlorophenyl) sulfonyl] piperidine-3-carbohydrazide (2)
Compound 1 (25 mmol) was refluxed with 80 % hydrazine hydrate (40 mmol) in 50 mL methanol contained in 100 mL round bottom flask for 2 -3 h. Reaction was monitored by TLC and on completion of the reaction, excess methanol was distilled off. Cold distilled water was introduced to the reaction mixture and the precipitates were filtered, washed with water and recrystallized from ethanol. The product was tagged compound 2.

Synthesis of 5-(1-(4-chlorophenylsulfonyl)-3piperidinyl)-1, 3, 4-oxadiazole-2-thiol (3)
Compound 2 (65 mmol) was dissolved in 60 mL ethanol along with CS 2 (65 mmol) and KOH (0.13 mol) in 100 mL round bottom flask. KOH was used to provide an alkaline environment to enhance the electrophilicity of CS 2 . The reaction mixture was refluxed for 5 h and reaction progress was monitored with TLC. Excess ethanol was distilled off at the completion of reaction. The reaction contents were dissolved in distilled water and acidified to obtain the oxadiazole precipitates. The precipitates were filtered and washed with distilled water to yield compound 3.
Different saturated/unsaturated alkyl halides (1 mmol) were added separately to the reaction mixture in the round bottom flask and the mixture was stirred for 4 h. At the end of reaction, cold distilled water was added and the precipitates were washed thoroughly with water. Seven different S-substituted derivatives resulted from the different alkyl halides ( Table 1).

Antibacterial assay
The antibacterial activity of each S-substituted alkyl derivative was evaluated using the methods of Kaspady et al [15] and Yang et al [16]. Two gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), and three gramnegative bacteria (Escherichia coli,

Pseudomonas aeruginosa and Salmonella typhi)
were clinically isolated and stored on appropriate agar media to facilitate bacterial growth. All the strains were obtained from a local hospital. Test samples (20 µg), after suitable dilution was added to 180 µL of diluted fresh bacterial cultures in nutrient broth in a microplate. The initial absorbance at 540 nm was taken and kept at values between 0.12 -0.19. Ciprofloxacin R was used as standard drug. The microplates with lids were incubated at 37 o C for 16 -24 h. Absorbance was read at 540 nm in a microplate reader, before and after incubation and the difference was used as an index of bacterial growth. Percent inhibition was calculated as in Eq 1. (1) where C (i.e., control) = total enzyme activity without inhibitor, and T (i.e., test sample) = activity in the presence of test compound. The results are expressed as mean ± SEM (n = 3). Minimum inhibitory concentration (MIC) was measured with suitable dilutions (5 -30 µg/well) and the results were analyzed using EZ-Fit (Perrella Scientific Inc. Amherst USA) software.

Lipoxygenase assay
Lipoxygenase activity was assayed using the methods as described previously [17,18] ) was used as a positive control. Percentage inhibition and IC 50 values were calculated as in Eq 2. (2) where C (i.e., control) = total enzyme activity without inhibitor, and T (i.e., test sample) = activity in the presence of test compound. IC 50 values (concentration at which enzyme inhibition is 50 %) were calculated using EZ-Fit Enzyme Kinetics Software (Perrella Scientific Inc. Amherst, USA).

Statistical analysis
All the measurements were done in triplicate and statistical analysis was performed using Microsoft Excel 2010. Results are presented as mean ± SEM with 85 % confidence limit.

RESULTS
Target compounds were synthesized (Table 1) by following a series of reactions as described in experimental section and shown in Scheme-1. All the molecules were characterized by spectral data of IR, 1 H-NMR, 13 C-NMR and EIMS.

Biological activities
The lipoxygenase inhibitory and antimicrobial activities of the compounds are shown in Tables  2 and 3, respectively, while their MIC values are shown in Table 4. Compounds 5a, 5e, 5f and 5g showed very strong lipoxygenase inhibitory potential with 5e having % inhibition comparable to that of the standard, Baicalein R .
Results for antimicrobial activities revealed that although none of the compounds exhibited % inhibition comparable to the standard drug Ciprofloxacin® most of them had good % inhibition values with some (5a, 5b 5d and 5e) having 70 % and above with respect to S. typhi, E. coli and B. subtilis.
Compound 5b consistently exhibited the lowest MIC values (relative to the other compounds) for all the bacterial strains used. The MIC values of 5b were closest to those of the standard drug, Ciprofloxacin® when compared with MIC values for 5a, 5c 5d, 5e 5f and 5g (Table 4).   sulfonyl]-3-piperidine cyanide cationic fragments respectively. Base peak appeared at m/z 175 corresponding to 4chlorophenyl sulfonylcation and the molecular ion peak was at m/z 401. The structures of all the other compounds were arrived at on the basis of these spectral information.
Compounds 5e, 5g and 5f exhibited% inhibition values 94.71 ± 0.45, 81.41 ± 0.98 and 78.09 ± 0.56, respectively, compared to standard Baicalein with % inhibition of 93.79 ± 1.27. Compound 5e exhibited excellent IC 50 value 20.72 ± 0.34 probably due to n-pentyl group substitution at 2-thiol position of oxadiazole ring which provides the molecule a more favorable geometry that probably facilitates its binding to active site of the enzyme, as compared to Baicalein R with IC 50 of 22.41 ± 1.30.
The different compounds showed different activities against the different bacterial strains. Some showed good % inhibition but their MIC values were not so appreciable. Compounds 5a, 5b, 5d,5e and 5g gave good % inhibition values against S. typhi bacterial strain but only compounds 5b,5d, 5e and 5g gave good MIC values (8.18 ± 2.00, 9.50 ± 1.03, 8.24 ± 3.36 and 9.06 ± 1.26, respectively). This is most probably due to aliphatic straight chain substitutions, as compared to standard Ciprofloxacin R 7.15 ± 1.29.
The compounds 5a -5g are S-substituted alkyl derivatives of 1, 3, 4 oxadiazole. The antimicrobial and anti-inflammatory properties of these compounds seen in this study are in agreement with results reported by other studies involving anti-microbial and anti-inflammatory potential of other synthetic 1, 3, 4-oxadiazole derivatives. Some novel 1, 3, 4-oxadiazole derivatives have been shown to exhibit antimicrobial activities against S. aureus, B. subtilis, E. coli and Pseudomonas [19]. In addition, potent antimicrobial and antiinflammatory derivatives of 1, 3, 4 oxadiazole have been chemically synthesized by condensation of 4-methoxybenzo hydride with different aromatic acids [20]. New antimicrobial derivative of 1 3, 4-oxodiazole with 5 -chloro -2 -methoxyphenyl moiety have also been reported by other investigators [21].

CONCLUSION
Our results indicate that, of the seven derivatives synthesized and studied, compound 5e shows the best lipoxygenase inhibitory activity, probably due to the effect of the n-pentyl chain on its orientation, while compound 5b was the most active antibacterial agent against all the bacterial strains tested.