In Silico Toxicological, Anti-Tubercular Effect Evaluation And In Vitro Marine Pathogenic Bacteria Inhibition of N-[(3-Chloro-4-Nitro-Phenyl) Methyleneamino]Pyridine-4-Carboxamidine

The hydrazone; N-[(3-chloro-4-nitro-phenyl) methyleneamino] pyridine-4-carboxamidine (H) was selected for in silico toxicological and in vitro bactericidal studies. Toxicological investigation was carried out using software program, such as eMolTox and Gusar, for the toxic substructure determination, and acute rat toxicity prediction respectively. In vitro bactericidal effect evaluation was investigated using tow marine pathogenic bacteria; Vibrio anguillarum and Photobacterium damselae. Computational results determinate toxicophores of (H), which are nitroaromatic part, hydrazine group, and quaternary carbon, were predicted as responsible for Idiosyncratic toxicity metabolic activation, covalent bond with DNA, and hepatotoxicity respectively. In addition, the predicted LD50 of (H) are 1086, 244, 1816, and 823.40 mg/kg in intraperitenial, intravenous, oral and subcutaneous administration respectively. For bactericidal results, H exhibited an excellent effect with inhibition percentages of 98.65 and 98.83% at the concentrations of 1000 and 500 μg/mL against Vibrio anguillarum respectively, the same effect was demonstrated against Photobacterium damselae with inhibition percentages of 97.74 and 97.98 % at the same concentrations. For anti-tubercular effect prediction, results revealed that H has an excellent effect with probability percentage of 84.6%. Keyword: Hydrazone, toxicophore, LD50, Anti-tubercular, Vibrio anguillarum, Photobacterium damselae. Article Info: Received 02 Sep 2019; Review Completed 11 Oct 2019; Accepted 16 Oct 2019; Available online 15 Nov 2019 Cite this article as: Mokhnache K, Soltani E, Madoui S, Khither H, Karbab A, Charef N, Arrar L, In Silico Toxicological, Anti-Tubercular Effect Evaluation And In Vitro Marine Pathogenic Bacteria Inhibition of N-[(3-Chloro-4-Nitro-Phenyl)Methyleneamino]Pyridine4-Carboxamidine, Journal of Drug Delivery and Therapeutics. 2019; 9(6):23-27 http://dx.doi.org/10.22270/jddt.v9i6.3653 *Address for Correspondence: Kamel Mokhnache, Laboratory of Applied Biochemistry, University Ferhat Abbas Setif 1, 19000, Algeria


INTRODUCTION
Heterocyclic hydrazones present in many structures, with biological and pharmacological interest. They are a particular class of the Schiff base family, play an important role in medicinal chemistry. This class of compounds has excellent antibacterial, antifungal, antimicrobial and anticonvulsant activity 1 . In this context, heterocyclic hydrazones derived from the anti-tubercular drug; isoniazid (IZD) showed antituberculosis activity against Mycobacterium tuberculosis, and some of these compounds are more active and less toxic than isoniazid 2 . For these reason, our objective is to evaluate the toxicity, anti-tubercular activity and the antibacterial effect of new hydrazone derived from isoniside; N-[(3-chloro-4nitro-phenyl) methyleneamino] pyridine-4-carboxamidine (H) (Scheme 1).

MATERIAL AND METHODS
All chemicals and reagents used throughout this study were obtained from commercial sources and used without further purification; Isoniazid (BHD chemicals Ltd Poole England).

Antibacterial activity evaluation
Two pathogenic marine bacteria Vibrio anguillarum and Photobacterium damselae, were employed to estimate the bactericidal effect of H. The bacteria were grown on tryptic soy agar (TSA) at 25 ° C. After 18h, colonies of 1 to 2 mm were diluted in 5 mL of liquid culture medium (TSB) and cultured for 16 h at 25 ° C. The antimicrobial activity of H was determined by evaluating their effects on bacterial growth curves; 10 μL aliquots of each of the bacterial dilutions (1/10) were placed in 96-well flat-bottomed plates (Nunc) and incubated with equal volumes of H (concentration of 1000 and 500 μg/mL) for two hours. Then the absorbance of the samples was measured at 620 nm (BMG, Star GalaxyFluoro) at 30 minute intervals for 24 h at 25 ° C. Samples without bacteria were used as blanks (negative control). Samples without H were used as positive control 3 .

Toxicity predictions
The online software programs used for toxicological studies are summarized in the following

Toxic substructure prediction
Drug undesirables and toxic effects were traduced by unfavorable reactions affecting human organs, inducing harmful effects, are the principle problems in drug discovery and development 8 . These toxic actions can be related to chemical structures, or a part of the structures which defined by substructures or toxicophores, which could be an atom or fragments of chemical structure 9 . In order to predict the toxicity of IZD and its derivative H, Table 2, represents toxic substructures (in red color) and their adverse effects. The obtained results defined the toxic fragment in common of all investigated compounds; the hydrazine group (-NH-N-), predicted as responsible in hepatotoxicity effect induction. In addition to the hepatotoxic effect, the hydrazone has other toxic substructures: the nitro-aromatic part of H, presents the covalent bond with DNA, in the quaternary carbon atom, the nitro group bond with quaternary carbon, presents toxicity metabolic activation.

Acute rat toxicity prediction
The major advantage of computational approaches for the acute toxicity and the toxicity classification of the investigated hydrazone, is to give important information before they are synthesized. Additionally, due to the ethical consideration, the time consumption and the high cost in animal models, the computational methods were used as a substitute in the in vivo toxicological studies.
LD50 values and the predicted toxicity class of the IZD and its derivative were predicted by GUSAR software in rodent with different administration routes (intraperitoneal, intravenous, oral and subcutaneous). Results in Table 3, showed that the intraperitoneal route was the low toxic way of H with LD50 value of 1086 mg/kg, classified in the class 5, the LD50 of the investigated hydrazone in oral administration route were 1816 mg/kg.

Mutagenicity prediction
Mutagenicity is defined as the induction of changes in genes and/or chromosomes that are permanent or transmissible in cells or organisms 10 . Probability of IZD and its derivatives to introduce genetic changes showed total mutagenic effect of INH with probability of 100%. From H, the predicted results depicted in Table4, indicate the reduction in the mutagenic effect from 100% to 71% in. In the literature, studies indicated that the hepatotoxic effect of IZD can lead to the genetic variations in enzymes in Asian populations (Caucasian population) 11 . These variations could be attributed to the interaction between IZD-reactive metabolites with DNA.  (Fig. 1).

Antibacterial effect evaluation
Fish infection caused the decrease in marine productivity, and heavy economic costs 12 . This problem led to search of new safe antibiotics for marine uses. For this reason, the hydrazone H was examined for their bactericidal effect towards tow marine pathogenic bacteria; Vibrio anguillarum and Photobacterium damselae. The results displayed in Fig.2.
indicate an excellent bactericidal effect of H against Vibrio anguillarum with inhibition percentages of 98.38% and 98.65% at 500 µg/mL and 1000 µg/mL respectively. Also, IH exhibited good effect against Photobacterium damselae with inhibition percentages of 97.79% and 97.74% at the same concentrations respectively.

In silico anti-tubercular activity evaluation
According to World Health Organization statistics, tuberculosis is the principle infectious disease responsible of mortality in the word 13 , which leads to search of potential anti-tubercular drugs 14 . In this context, the computational study is the essential step for the designing of new efficient drugs. The hydrazone H was investigated for these effects. The predicted results (Fig.3) were compared with the antitubercular drug IZD, and indicated excellent effects of this hydrazone with probabilities of anti-tubercular effects of 84.6, while the standard anti-tubercular drug IZD has the probability of 81.3 %. These results showed that H has higher effect than IZD. These results are in good agreement with the literature precedent investigations, which showed that hydrazones derived from IZD have a similar or improved effect than the drug itself 15 .

Molecular docking
Molecular docking study was performed to predict the possible binding modes and to rationalize the observed biological activity 16 . In order to comprehend the mechanism of action of the new hydrazones, the active molecules were subjected to molecular docking studies against InhA which is considered as a promising target for the discovery of novel anti-tubercular drugs 17 . The molecular docking studies of active compounds were accomplished against the active site of InhA enzyme structure (PDB code: 4DRE) using SwissDock server. FullFitness and Gibbs free energy (ΔG) were evaluated. Favorable binding modes were scored based on FullFitness and cluster formation. Ranking of the cluster was accomplished using the value of FullFitness. As shown in

CONCLUSION
In silico toxicological studies were undertaken using online software programs; from toxicophores, acute toxicity, and mutagenicity; these studies showed low toxic effect of H with predicted LD50values of 1816 mg/kg. Additionally, reduction in the mutagenic effect was observed with probability value of 29%. The anti-tubercular effect was predicted, results demonstrated the persistence of the effect in the investigated compound with predicted value of 84.6 %. Results of in vitro bactericidal effect evaluation using microorganism (Vibrio anguillarum and Photobacterium damselae) indicated an excellent effect of this compound at the doses of 1000 and 500 µg/mL. We conclude that the hydrazone can be a marine antibiotic.