Novel Hydralazine Schiff Base Derivatives and Their Antimicrobial, Antioxidant and Antiplasmodial Properties

Two novel Schiff bases, 3-[1-(2-(phthalazin-1-yl)hydrazono)ethyl)-1,3-oxa- zinane (PHEO) and 2-[(2-(phthalazin-1-yl)hydrazono)methyl]phenol (PHMP), derived from hydralazine hydrochloride, an effective drug against hypertension, were synthesized and characterized by spectroscopic methods, Infrared (IR), Proton Nuclear Magnetic Resonance (1H NMR) and Carbon-13 Nuclear Magnetic Resonance (13C NMR). PHEO showed low antimicrobial activity against one bacterial strain with MIC value of 250 μg/ml while PHMP showed interesting activity against 4 bacterial strains with MIC of 31.25 - 250 μg/ml compared to the standard drug, amoxicillin. PHEO and PHMP showed higher reducing activity on ferric ions compared to Vitamin C. On lipid peroxidation, PHEO showed higher inhibition while PHMP showed lower inhibition compared to Vitamin C. Both compounds presented lower stimulating effect and lower catalase activity compared to the standard Vitamin C. PHEO and PHMP showed less than 80% inhibition in the preliminary antiplasmodial assay and so were not considered for the dose-response studies.


Introduction
Health problems have been a source of sadness and pain to humanity and research in the domain of health has afforded a great variety of therapeutics to fight against a wide range of diseases and ailments. Despite this effort and many others, some drugs have become ineffective as the micro organism develops resistance when the usage is recurrent, reason why illnesses like malaria, bacterial and yeast infections, gastrointestinal disorders, diarrhoea, dysentery, cholera, as well as chronic degenerative diseases like cancer, continue to inflict suffering in developing countries, Africa and the world. There is therefore urgent need to discover new drugs.
Apart from their pharmaceutical and medicinal importance, Schiff bases are widely used as catalysts, dyes, stabilizers in polymer formulations, pigments and corrosion inhibitory agents [12].
The growing resistance of infectious microorganisms to therapeutics is a serious concern and represents a health threat in both developed and developing world [13]. Introduction of new classes of antimicrobial drugs is desperately needed to hamper the tendency of drugs to quickly develop drug-resistant pathogens [13]. Therefore, drug discovery efforts will facilitate the identification of novel antimicrobial compounds and new drug combinations will facilitate the development of better dosing regimens and novel strategies to manage drug resistance and prevent the dissemination of resistant bacteria [14]. In Cameroon, infectious diseases such as malaria and bacterial infections are amongst the most commonly notified diseases and largest cause of mortality [15]. Malaria continues to be a major cause of childhood mortality and was responsible for an estimated 303,000 (165,000 -450,000) deaths in children under 5 years old in 2015.
The majority is caused by the Plasmodium falciparum infections. However, the 60% reduction observed in mortality since 2000 is heavily associated to the widespread deployment of effective control measures such as Artemisinin-based Combination Therapies (ACTs). The recent emergence and spread of Plasmodium falciparum parasites resistant to ACTs, worsened by the spread of insecticide resistance hindered the elimination of malaria [16] [17] [18].
An antioxidant is any molecule that delays, prevents or removes oxidative damage to target cells. Oxidative damage or oxidative stress produces free radicals which are dangerous to the body. Reactive oxygen species (ROS) is a term which encompasses all highly reactive molecules including free radicals like hy-A. F. Awantu et al. International Journal of Organic Chemistry droxyl radical, superoxide anion radical, hydrogen peroxides, singlet oxygen, nitric acid radical and various peroxides [19]. When oxidative forces exceed the antioxidant systems, the cell is subjected to oxidative stress [19]. Oxidative stress is among the major causative factors in induction of many chronic and degenerative diseases including atherosclerosis, ischemic heart disease, aging, diabetes mellitus, cancer, immunosuppression, neurodegenerative diseases, male infertility, infectious diseases and others [20]. Therefore, development of new antimalarial, antibacterial and antioxidant chemotypes is an urgent priority. Our objective in this study is to synthesize new hydralazine-derived Schiff bases and to evaluate them for their antiplasmodial, antibacterial and antioxidant activity in vitro.

Materials
Hydralazine hydrochloride, 4-acetylmorpholine, 2-hydroxybenzaldehyde and other solvents were purchased from commercial sources and used without further purification. All chemicals used were of reagent grade. Methanol was used as solvent all through the synthesis. Glass apparatus with standard interchangeable joint were used after being washed with concentrated sulphuric acid, distilled water and methanol. All the synthesis was carried out in a 250 mL round-bottomed flask fitted with a quick Liebig condenser where it involved refluxing. All reactions were performed on a hot plate equipped with a magnetic stirrer. Elemental analysis was performed on a VARIO EL (Heraeus) analyzer. IR spectra were obtained from a Perkin-Elmer spectrum 100-FT-IR spectrometer. 1 H-NMR spectra were obtained on a Varian unity plus 400 MHz instrument. 13 C-NMR spectra were recorded on a Bruker AV 100 MHz instrument.

Synthesis of 3-[1-(2-(Phthalazin-1-yl)Hydrazono)Ethyl)-1,3-Oxazinane (PHEO)
The ligand was prepared by the condensation reaction between hydralazine hydrochloride and 4-Acetylmorpholine. (3 mmol, 0.6 g) of hydralazine hydrochloride and sodium acetate (buffering agent) (3 mmol, 0.4 g) was dissolved in 20 mL of ethanol, and (3 mmol, 0.4 g) of 4-Acetylmorpholine was also measured. The two solutions were transferred into a 250 mL round bottom flask and the mixture was refluxed for three hours while stirring at a temperature of 70˚C and allowed to cool overnight. The yellow precipitate obtained (PHEO) was separated by filtration, washed with ethanol and allowed to air dry giving a yield of 50%. The filtrate was sealed using paraffin in a vial and kept for further crystalization by slow evaporation to improve on the yield. The synthetic route for PHEO is shown in Scheme 1.

Organisms and Growth Conditions
Microorganisms were obtained from the culture collections of the Antimicrobial and biocontrol Agents Unit at the Department of Biochemistry, University of

Evaluation of the Antioxidant Reducing Power
The antioxidant reducing power of the synthesized compounds PHEO and PHMP was evaluated using the method as described by Kamtekar and collaborators in 2014 [21]. The standard ascorbic acid or synthesized compounds (50, 100, 150, 200, 250 and 300 µg/mL) was introduced in the tube, then 0.4 mL of phosphate buffer (pH 6.6, 0.2 M) and 0.4 mL of 1% KFe(CN) 6 were successively added. After homogenization, the mixture was incubated at 50˚C for 20 min, then cooled, and centrifuged (3500 rpm, 10 min, 25˚C). To 1 mL of supernatant was added 1 mL of 10% TCA, 1mL of distilled water and 0.2 mL of 0.1% FeCl 3 .
After homogenization, the absorbance was measured at 593 nm using a UV-Vis spectrophotometer.

Statistical Analyses
For ferric reducing power and lipid peroxidation, the fifty percent efficient (EC 50 ) and inhibitory (IC 50 ) concentration of the tested compound were determined.
Differences between treatments were assessed by one factor ANOVA followed by the Student-Newman-Keulstest, and P values less than 0.05 were considered statistically significant. All analyses were performed using Graphpad Instat software Version 3.0.

Infrared Spectra
The IR spectrum of the PHMP (Figure 3) recorded in the 4000 -400 cm −1 region displayed strong bands at 3400 cm −1 and 3100.1 cm −1 corresponding to the O-H and the N-H stretching vibrations respectively [25]. A sharp band was seen at

1 H NMR Spectra
The proton NMR spectrum of PHMP (  Figure 6 shows the 1

Anti-Plasmodial Activity
PHEO and PHMP were tested for anti-plasmodial activity using the SYBR green I based fluorescence assay and the results are presented in Table 1. PHEO inhibited the growth of the malaria parasites with a percentage inhibition of 78.55%, lower than the 80.41% obtained with the standard treatment of malaria, Artemisinin. Likewise, PHMP inhibited the growth of the malaria parasites with a percentage inhibition of 78.06%, lower than the 80.41% obtained with the standard treatment of malaria, Artemisinin. Considering that their percentage inhibitions in the preliminary assay were less than 80%, PHEO and PHMP were not considered for the Dose-Response studies.

Antimicrobial Activity
Antibacterial activities of PHEO and PHMP are recorded in Table 2

Ferric Reducing Powder and Lipid Peroxidation
The effects of different compounds and vitamin C on reduction of ferric ions A. F. Awantu et al.
and peroxidation of lipids are presented in Table 3. In general PHEO and PHMP showed higher reducing activity on ferric ions than the reference compound vitamin C. On lipid peroxidation, PHEO showed higher inhibition (P < 0.05) while PHMP showed lower inhibition (P < 0.05) compared to vitamin C.

Catalase Activity
The catalase activity in the presence of PHEO, PHMP and Vitamin C is depicted in Figure 9. Catalase activity showed concentration dependent effect in the presence of all compound. In general Vit C presented higher stimulatory effect on the enzyme activity than PHEO and PHMP. PHMP showed a lower effect on catalase activity.

Conclusions
PHEO and PHMP were synthesized from hydralazine hydrochloride and characterized by spectroscopic methods. PHEO showed low anti-microbial activity only on one bacterial strain with   PHEO and PHMP inhibited the growth of the malaria parasites with percentage inhibitions of 78.55% and 78.06% respectively; lower than the 80.41% obtained with the standard treatment of malaria, Artemisinin. Considering that their percentage inhibitions in the preliminary assay were less than 80%, PHEO and PHMP were not considered for the Dose-Response studies.
The anti-microbial, anti-oxidant and anti-plasmodial activities of these compounds are reported for the first time and the results indicate that these novel ligands can be exploited as anti-microbial and anti-oxidant agents.