Synthesis, characterization and biological evaluation of novel hydrazone compounds

Abstract

Hydrazones are important class of organic compounds with the structure –C=NNH2 and are related to aldehydes and ketones by the replacement of the oxygen with the NNH2 functional group. Literature studies on these hydrazones compounds have shown that these organic compounds possess various biological activities, such as antibacterial, antifungal, antitumor, anticonvulsant, anti-inflammatory, antiplatelet and antimicrobial. Due to its significant biological activities, these compounds arouse considerable interest. However, the study of hydrazones derivatives of carbonyl compounds on the intestinal smooth muscle contraction was rarely found. Thus, the present study was designed to synthesize the novel hydrazones compounds, characterize its structure and to investigate in-vitro antibacterial activity, ex-vivo intestinal smooth muscle contraction and in-vivo gastrointestinal motility. Methods: The novel hydrazones were synthesized by the reaction of 3,5 dihydroxy benzoic acid hydrazide with corresponding aromatic aldehydes in methanolic solution. The structure of the compound was characterized by using IR spectra, thermo gravimetric analysis and single-crystal X-ray diffraction. In-vitro antibacterial activity of the compounds was studied by using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) method. The colorimetric assay was carried out on Caco2 cell culture to evaluate the toxic effects of these new synthesized compounds. The contractility of jejunal smooth muscle (CJSM) of rats was chosen as a major index to investigate the effects of these synthesized compounds on smooth muscle contraction. The acute oral toxicity of the active compounds was determined by up- and -down experimental design to estimate LD50.The diarrhea predominant rat model was established to explore the role of compound on gastrointestinal motility. Western blot analysis was used to detect the 5HT serotonin transporter (SERT) protein content on intestinal fragments of diarrhea afflicted rats. Results: The light yellow crystalline compounds were obtained from the synthesis. The novel synthesized compounds are 3,5 dihydro-N′-(3-Nitrobenzylidene)benzohydrazide(compound 1),3,5dihydro-N′-(4-Nitrobenzylidene)benzohydrazide(compound 2), 3,5dihydro-N′-(2-Nitro benzylidene)benzohydrazide(compound 3), 3,5dihydro -N′- (2-bromo, 4-methoxy, 5-hydroxy benzylidene)benzohydrazide(compound 4),3,5dihydro-N′-(2-hydroxy,3-methoxybenzylidene) benzohydrazide(compound5),3,5dihydro-N′-(2-hydroxy,5-chlorobenzylidene)benzohydrazide (compound6),3,5dihydro-N′-(3,5-ditertiarybutyl,2-hydroxybenzylidene)benzohydrazide (compound 7). These novel synthesized compounds exhibited mild to moderate antibacterial activities against S. aureus and K. pneumonia. The compounds exhibited the low toxic effects on Caco-2 cell culture. In ex-vivo assay, these new synthesized compounds exhibited the inhibitory effects on CJSM. Among them, 3,5 dihydro-N′-(3-nitrobenzylidene)benzohydrazide(DNBB) showed strong inhibitory effects on contractility of jejunum smooth muscle. They inhibited CJSM in a dose dependent manner, in normal contractile state. They decreased the stimulatory effects on CJSM induced by acetylcholine, high ionic concentration of calcium and potassium respectively. Alpha adrenergic receptor phentolamine and beta adrenergic receptor propanolol partially abolished the inhibitory effects of novel compounds. The oral Lethal Dose 50 (LD50) of the active novel compound DNBB in mice using up-and-down procedure was over 2.0g/kg. In vivo assay indicated that DNBB reduced the feces quantity and its water content of Diarrhea predominant (DP) rat model. SERT protein expression (detected as bands at 70kDa) was faintly detected in jejunum and colon segments of diarrhea afflicted rats. Discussion and Conclusion: The structural analysis of these novel hydrazones compounds elucidated that the compounds with nitro, hydroxyl, methoxy, halogen substituent groups on benzene ring were effective on intestinal contractility and in-vitro antibacterial activity. This suggests that the biological activity of the compounds is positively related with different functional groups and its arrangement and position on the benzene ring. The innovations of the present study are: 1. The novel hydrazones compounds were synthesized by applying simple, reliable and green or sustainable chemical procedure. 2. The compounds (1-7) possessed low toxic effects. 3. Compound (4, 5 and 6) showed mild to moderate antibacterial activities against S. aureus and K. pneumonia. 4. Compound (1-7) exhibited the inhibitory effects on CJSM in a dose dependent manner in normal contractile states. Among them, compound 1(DNBB) containing nitro group at meta position showed the strong inhibitory effects on CJSM. 5. DNBB alleviated the symptoms of diarrhea predominant (DP) rat model. Finally, this is the first attempt to study the effects of novel hydrazones compounds on intestinal contractility. The inhibitory effects of novel hydrazones on CJSM and its possible mechanism suggest its clinical implication on the treatment of intestinal hyper contractility.