Synthesis and molecular docking of new hydrazones derived from ethyl isonipecotate and their biological activities

Purpose: To investigate the antibacterial and α-glucosidase inhibitory activities of hydrazone derivatives (8a-h) of ethyl isonipecotate. Methods: The reaction of ethyl isonipecotate (2) with 3,5-dichloro-2-hydroxybenzenesulfonyl chloride (1) in an aqueous basic medium yielded ethyl 1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]piperidin-4carboxylate (3). Compound 3 was subsequently converted to ethyl 1-[(3,5-dichloro-2ethoxyphenyl)sulfonyl]piperidin-4-carboxylate (5) via O-alkylation. Compound 5 on reaction with hydrated hydrazine yielded 1-[(3,5-dichloro-2-ethoxyphenyl)sulfonyl]piperidin-4-carbohyrazide (6) in MeOH. Target compounds 8a-h were synthesized by stirring 6 with different aromatic aldehydes (7a-h) in MeOH. All the synthesized compounds were structurally elucidated by proton nuclear magnetic resonance (H-NMR), electron impact mass spectrometry (EI-MS) and infrared (IR) spectroscopy. For antibacterial activity, solutions of the synthesized compounds were mixed with bacterial strains, and the change in absorbance before and after incubation was determined. For enzyme inhibitory activity, change in the absorbance of mixtures of synthesized compounds and enzyme before and after incubation with substrate was determined. Results: The target compounds were synthesized in appreciable yields and well characterized by spectral data analysis. Salmonella typhi was inhibited by 8e (MIC 8.00 ± 0.54 μM), Escherichia coli by 8f (8.21 ± 0.83 μM), Bacillus subtilis by 8c (8.56 ± 0.63 μM) and Staphylococcus aureus by 8c (8.86 ± 0.29 μM). Two compounds, 8e and 8d, were very effective inhibitors of α-glucosidase with IC50 values of 40.62 ± 0.07 and 48.64 ± 0.08 μM, respectively. Conclusion: Low IC50 values of the synthesized compounds against α-glucosidase demonstrates their potential in type-2 diabetes treatment. Furthermore, these compounds exhibit substantial antibacterial activity against the bacterial strains tested.


INTRODUCTION
The labile lone pair of nitrogens in hydrazones is known to play an active role in the bioactivity and chemical reactivity of this class of compounds [1,2].These compounds have been shown to have anti-tuberculosis [3], antimicrobial [4], antimycotic [5] and antihypertensive activities [6].
The cleavage of carbohydrates is delayed by αglucosidase inhibitors (AGI) in the small intestine [12,13].In search of bioactive compounds, an attempt was made to synthesize some new hydrazones bearing a sulfamoyl moiety.

EXPERIMENTAL
All chemical reagents were from Merck, Sigma Aldrich and Alfa Aesar and purchased through local suppliers, and they were used without further purification.Purity of the synthesized compounds was assured by thin layer chromatography (TLC); the plates were developed with n-hexane and ethyl acetate solvent systems and visualized under UV at 254 nm and also by spraying with ceric sulfate solution.
The melting points of all synthesized compounds were determined using open capillary tubes on a Griffin and George apparatus.IR spectra were recorded using the potassium bromide pellet method on a Jasco-320-A spectrophotometer with wave number in cm -1 . 1 H-NMR spectra were recorded in CDCl 3 on a Bruker spectrometer operating at 400 MHz.Chemical shift values are reported in ppm (δ) units taking TMS as reference and the coupling constants (J) are in Hz.Mass spectra (EIMS) were recorded on a JMS-HX-110 spectrometer.

Procedure for synthesis of sulfonamide (3) in aqueous medium
Ethyl isonipecotate (6.49mmol; 1 mL, 2) was added to a 100-mL round-bottom flask containing 15 mL basic aqueous medium prepared by addition of aqueous Na 2 CO 3 with pH adjusted to 8-9.Next, 3,5-dichloro-2-hydroxybenzenesulfonylchloride (6.49mmol; 1.698 g, 1) was gradually added to the reaction flask over 5-10 min.The reaction mixture was stirred for 6-8 h with pH maintained at 8-9 at ambient temperature.The completion of the reaction was checked by TLC until confirmed by a single spot.Product 3 was collected by acidifying the reaction mixture with dilute HCl to bring the pH to 5-7.The synthesized product was collected by filtration, washed with distilled water and dried for next use.

Procedure for synthesis of O-alkyl derivative (5)
Compound 3 (0.5249 mmol, 0.2 g) was dissolved in DMF (5 mL) in a 100-mL round-bottom flask.Solid KOH (0.5249 mmol, 0.0293 g) was added to activate the O-substitution.Ethyl iodide (0.5249 mmol; 42.2 µL, 4) was then added, and the mixture was stirred for 2-3 h at 60 ºC.Completion of the reaction was confirmed by TLC showing a single spot.Chilled distilled water was used to collect the precipitates of pure product 5. Precipitates were filtered, washed with distilled water and dried.

Procedure for synthesis of carbohydrazide (6)
Compound 5 (1.467 mmol, 0.6 g) was dissolved in MeOH (5-10 mL) in a 100-mL round-bottom flask and stirred for 2-5 min.Hydrazine hydrate (1.467 mmol) was then added and the reaction mixture was refluxed for 6 h.There was a color change in the reaction mixture from orangeyellow to grey as the reaction proceeded toward completion.The progress of the reaction was followed by TLC.After reaction completion, the mixture was quenched with cold distilled water and the precipitates of 6 were filtered, washed with distilled water and dried for further use.

General procedure for synthesis of various hydrazone derivatives (8a-h)
A methanolic solution of compound 6 (0.2523 mmol, 0.1 g) was placed in a 50-mL roundbottom flask and stirred at room temperature.Aromatic aldehydes (7a-h) were introduced in equimolar ratios to produce hydrazone derivatives.The reaction duration for different aldehydes varied 2-3 h.Reaction completion was confirmed by TLC, and distilled water was added to the mixture to precipitate the compounds synthesized, 8a-h, which were filtered, washed with distilled water and dried.

Evaluation of antibacterial activity
The broth microdilution method was employed to test for antibacterial activity [14].The solutions of synthesized compounds were mixed with bacterial strains and change in absorbance before and after incubation was determined.

α-Glucosidase assay
α-Glucosidase inhibitory activity was determined as previously described, with slight modification [15].Enzyme activity was based on the difference in absorbance of the mixture of synthesized compounds and enzyme before and after incubation with substrate.

Molecular docking study
The crystallographic structure of Saccharomyces cerevisiae isomaltase (PDB code 3AJ7; resolution 1.30 Å) showing 72.4 % sequence identity with the target was selected as a template.The 3D structure of α-glucosidase for Saccharomyces cerevisiae was predicted using the Molecular Operating Environment (MOE 2010.11)software package.MOE docking program was used to analyze the binding modes of the ligands with the protein molecule.The best conformations were analyzed for hydrogen bonding and π-π interactions [16][17][18].

Statistical analysis
The results are presented as mean ± SEM (n = 3) and were analyzed by Microsoft Excel 2010.

Biological studies
The results for in vitro antibacterial activity against Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus are presented in Table 1 and those for in vitro α-glucosidase inhibitory activity are presented in Table 2.

Molecular docking
With regard to hydroxyl-containing compounds (8b and 8e), the docking conformation of 8e (ortho analogue) showed good interaction network as well as good docking score compared  to 8b (para analogue).The binding mode of 8e with a docking score of -10.4311 displayed two hydrogen bonds with Asn 241 and Asp 408, an arene-arene interaction with Phe 157 and arenearene interaction with Arg 312 residue of the enzyme (Figure 2a).Compound 8b with a docking score of -7.2386 established a hydrogen bond with Asn 241 and an arene-arene interaction with Phe 157 residue of the enzyme (Figure 2b).In case of dimethoxy substituted compound 8g, the docking conformation showed moderate interaction pattern.The two methoxy groups present at the meta and para position at adjacent carbon atoms displayed poor interaction, which might have been due to higher steric strain.The docking conformation of compound 8g showed two arene-arene interactions with Phe 157 and His 239 residues of the enzyme (Figure 2c).

DISCUSSION
The synthesis of compound 8a yielded a white powder.Its molecular formula, ).In the aromatic region of the The screening of all these synthesized compounds against Gram-positive and -negative bacterial strains showed potent antibacterial activity in all but a few.Compounds 8e and 8c exhibited good inhibition percentage and MIC values against all bacterial strains, possibly due to the presence of a 2-hydroxyphenyl and 3nitrophenyl group, respectively, in comparison to ciprofloxacin, taken as reference standard.Similarly, evaluation of α-glucosidase inhibitory activity of all synthesized compounds showed moderate activity except 8a, 8c and 8h, the inactive ones.Good activity was shown by 8e and 8d with respective IC 50 values of 40.62 ± 0.07 and 48.64 ± 0.08 µM in comparison to 38.25 ± 0.12 µM for acarbose, the positive control.The activity of 8e and 8d was probably due to the presence of a hydroxyl and chloro group in these compounds.Compounds 8b, 5 and 8f were less active, and 3, 6 and 8g had very low poorly active.However, 8a and 8b showed outstanding activity and could be further evaluated for the treatment of type-2 diabetes.

CONCLUSION
The biological activity data obtained demonstrate that the target compounds are significant inhibitors of bacterial growth and α-glucosidase activity.On the basis of the aforementioned results, these newly synthesized compounds may be further developed for the treatment of type-2 diabetes and bacterial infections.

Figure 2 :
Figure 2: Binding models of compounds; (a): Compound 8e nicely binds to α-glucosidase through Asn 241 and Asp and two π-π interactions with Phe 157 and Arg 312.(b): Compound 8b binds well to α-glucosidase through Asn 241and one π-π interaction with Phe 157.(c): Compound 8g binds to α-glucosidase and shows two π-π interactions with Phe 157 and His 239 Compounds 8a, 8b, 8d, 8f, 8g and 8h demonstrated good to moderate activity against both Gram-positive and -negative bacterial strains.S. typhi was best inhibited by molecule 3 (ethyl ester) with a MIC of 7.99 ± 0.86 µM and 8e (bearing 2-hydroxyphenyl group) with a MIC of 8.00 ± 0.54 µM relative to 7.12 ± 0.21 µM for ciprofloxacin.Against E. coli, molecule 6 (carbohydrazide) with a MIC of 8.05 ± 0.52 µM and 8f (bearing a 4-(dimethylamino)phenyl group) with a MIC of 8.21 ± 0.83 µM were the most effective in comparison to 7.05 ± 0.28 µM for the reference.The synthesized compounds showed relatively moderate activity against B. subtilis with the lowest MIC for 8c (bearing a 3nitrophenyl group) at 8.56 ± 0.63 µM as compared to 7.65 ± 0.48 µM for the standard.The moderate to excellent activity against Staphylococcus aureus rendered 3 (ethyl ester) and 8c (bearing a 3-nitrophenyl group) the best ones with MIC values of 8.43 ± 0.79 and 8.86 ± 0.29 µM in comparison to 7.89 ± 0.27 µM.

Table 1 :
Antibacterial activity of synthesized compounds MIC (minimum inhibitory concentration) values of compounds were calculated using EZ-Fit EnzymeKinetics software (Perella Scientific Inc. Amherst, USA).Results are expressed as mean ± SEM.