In Vitro Larvicidal and Antioxidant Activity of Dihydrophenanthroline-3-carbonitriles

Many naturally occurring and synthetic compounds containing dihydrocyanopyridine and cyanopyran moiety show pharmacological properties. The aim of this study is to investigate the larvicidal and antioxidant potential of dihydrophenanthroline-3-carbonitrile derivatives 4a–f. A novel series of 2-amino-10-chloro-4,12-diphenyl-1,4,5,6-tetrahydrobenzo[j][1,7]phenanthroline-3-carbonitrile derivatives were synthesized by reacting different substituted acridine chalcones through Michel addition. The compounds were synthesized in excellent yields and the structures were corroborated on the basis of FT-IR, 1H NMR, 13C NMR, and ESI Mass analysis data. All the synthesized compounds were evaluated for larvicidal activity against Aedes aegypti and Culex quinquefasciatus larvae. Furthermore, the antioxidant activity was studied by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay method. From the antioxidant assay, the compound 4c was reported with profound antioxidant potential.


Introduction
The special scopes of natural products are discovery of potential drugs with novel structures and varying biological activity. Compounds containing heterocyclic ring systems are of much importance in medicinal chemistry [1]. Because of their excellent chemotherapeutic characters, natural occurrence in living system, varied structure, and chemical properties researchers focused towards the synthesis of heterocyclic rings [2]. Among these, acridone is one of the scaffolds known to associate with several biological activities [3]. And also these acridine analogues are found in plant sources which possess various biological activities. Many plants, particularly plants pertaining to Rutaceae species, possess maximum number of acridine derivatives [4]. Dihydropyridine is an area of interest owing to the key role played in synthesis of intermediate for natural products [5] and other heterocycles. These hydropyridines are valuable synthetic intermediates and can be elaborated to the pharmacologically interesting polysubstituted piperidines and polycyclic alkaloid [6]. The reactivity of dihydropyridine is mainly involved in selective reductions [7] and electrophilic additions [8,9] and has allowed the completion of total synthesis of alkaloids [10,11]. Biological importance of dihydropyridines structures was elaborated in Figure 1 [12].
Vector control is facing a threat due to the emergence of resistance to synthetic insecticides. Nowadays researchers are focusing their research on a synthetic compound that kills the larvae at initial stage itself [13]. Plant extracts are acting as a potential larvicidal and antioxidant activity [14]. One of the present research interests is the synthesis of nanoparticles by biosynthetic methods. These nanoparticles are studied for larvicidal activity against various larvae [15]. All these kinds of applications are regarding the presence of various phytochemical compositions in plants. The growing interest is to synthesize heterocyclic compounds, and studying their potential uses in medicinal applications is well proved by the growing number of publications.  Taking these facts into account, our research group have been involved actively to synthesis a drug against larvae. Due to effect of synthetic compounds on larvicidal activity, our research group mainly focused on killing the larvae at initial stage itself. Earlier we reported the 7-chloro-3,4-dihydro-9phenylacridin-1(2H)-one shows an effective larvicidal activity against the early fourth instar larvae of filariasis vector, Culex quinquefasciatus, and Japanese encephalitis vector, Culex gelidus (Diptera: Culicidae). The compound exhibited high larvicidal effects at 50 mg/L against both the larvae with LC 50 values of 25.02 mg/L ( 2 = 0.998) and 26.40 mg/L ( 2 = 0.988) against C. quinquefasciatus and C. gelidus, respectively [16].
Nowadays, antioxidants that exhibit DPPH radical scavenging activity are increasingly receiving attention ( Figure 3) [17]. The generation of free radicals during the metabolic process is now observed to be responsible for wide range of human diseases such as aging, cancer, atherosclerosis, arthritis, viral infection stroke, myocardial infarction, pulmonary condition, inflammatory bowel disease, and neurogenerative disease and others may be produced by reactive oxygen species [18,19]. Antioxidants act as a major defence against radical mediated toxicity by protecting the damage caused by free radicals. Antioxidative agents are effective in the prevention and treatment of complex diseases like atherosclerosis, stroke, diabetes, Alzheimer's disease, and cancer [20,21].
In the present study, the efforts have been laid down to synthesis of 2-amino-10-chloro-4,12-diphenyl-1,4,5,6-tetrahydrobenzo[j] [1,7]phenanthroline-3-carbonitrile analogues, 4a-f, to evaluate the inherent larvicidal activity which was carried out against larva of Aedes aegypti and Culex quinquefasciatus. The model of scavenging of the stable DPPH radical is extensively used to evaluate radical scavenging activities in a very short span of time in comparison to other methods. Compound under investigation reacts with DPPH (1,1-diphenyl-picrylhydra-zine) due to its hydrogen donating ability at a very rapid rate [22]. When this electron becomes paired off, the absorption decreases stoichiometrically with respect to the number of electrons taken up. Such a change in the absorbance produced in this reaction has been widely applied to test the capacity of numerous molecules to act as free radical scavengers [23]. In this way, the scavenging effects of all the synthesized compounds on the DPPH free radical were evaluated.

General Synthesis of 2-Amino-10-choloro-4,12diphenyl-1,4,5,6-tetrahydrobenzo[j]
1a-f and malononitrile (0.66 g, 0.01 mol) was dissolved in absolute ethanol (15 mL). The reaction mixture was refluxed for 5 h. After completion of the reaction, the reaction mixture was cooled and poured into crushed ice. The crude products were separated by column chromatography, ethyl acetate, and petroleum ether as a solvent. All synthetic pathways were elaborated in Scheme 1 and synthesized derivatives and physical data of all synthesized compounds, 4a-f, were summarized in Table 1. and dechlorinated tap water. The numbers of dead larvae were counted after 24 h of exposure, and the percentage of mortality was reported from the average of three replicates. The experimental media in which 100% mortality of larvae occurs alone were selected for dose-response bioassay.

Dose-Response Bioassay.
From the stock solution, different concentrations ranging from 3.12 to 50 ppm were prepared for larvicidal activity. Based on the preliminary screening results, synthetic compounds, 4a-f, were subjected to dose-response bioassay for larvicidal activity against the larvae of A. aegypti and C. quinquefasciatus. The numbers of dead larvae were counted after 24 h of exposure, and the percentage of mortality was reported from the average of three replicates. However, at the end of 24 h, the selected test samples turned out to be equal in their toxic potential.

Statistical Analysis.
The average larval mortality data were subjected to probit analysis for calculating LC 50 , LC 90 , and other statistics at 95% fiducial limits of upper confidence limit and lower confidence limit, and chi-square values were calculated using the software [26]. Results with < 0.05 were considered to be statistically significant.

Antioxidant Activity.
All the synthesized compounds, 4a-f, were to be examined for antioxidant activity. Antioxidant assay [27] is based on the measurements of the scavenging ability of compounds towards the stable 2,2-diphenyl-1-picrylhydrazyl radical (DPPH). The disappearance of this commercially available radical is measured spectrophotometrically at 517 nm in an ethanolic solution. The antioxidant activity was expressed as the 50% inhibitory concentration (IC 50 ) based on the amount of compound required for a 50% decrease of the initial DPPH radical concentration. DPPH antiradical scavenging activity was also time dependent. The radical scavenging activity of the tested samples, expressed as percentage inhibition of DPPH, was calculated according to the following formula: where is the absorbance value of the tested sample and 0 is the absorbance value of blank sample at a particular time.
The data for antioxidation is presented as means ± SD of three determinations. The synthesized compounds used for antioxidant assay are of 1 mM concentration. Absorbance taken     [1,7]

Result and Discussion
Results on the larvicidal activities of 4a-f obtained in this study that were summarized in Table 2   The entire synthesized compounds scavenged DPPH radical significantly in a concentration-dependent manner. Their comparable scavenging activities were expressed in IC 50 (concentration required for 50% inhibition of l M DPPH concentration) value. As presented in Table 4, compound 4c with IC 50 values in the range of 41 M showed good radical scavenging activities in comparison with ascorbic acid which was attributed to the presence of two methoxy aryl group of 4c (that can donate hydrogen atoms) and governs the main factor behind their ability to be scavenged by DPPH. After donating a hydrogen atom, compounds exist in their radical form, and the electron conjugation effect in the structure stabilizes the radical of DPPH.
The difference in activity amongst compounds 4a-f was due to the difference in the substitution of these compounds. Amongst them, compounds 4b and 4c with two methoxy substituents showed higher hydrogen donor ability to DPPH radical. Compounds 4b and 4c IC 50 values were 54 and 41, respectively, since the corresponding IC 50 values for all synthesized compounds were higher than compounds 4b and 4c. Compound 4c has two methoxy groups which are para to each other. Therefore, radical ion resulting from the abstraction of -H atom by DPPH would stabilize by other hydroxyl groups. While in compound 4b both the methoxy groups are in meta and para positions, the radical ion resulted from the abstraction of H atom not that much stabilized from methoxy group compared with 4c. Thus, compound 4d shows better scavenging effect compared to compounds 4e and 4f due to the presence of single methoxy group. Compounds 4a, 4e, and 4f are less active compared with other derivativesbecause of the absence of any electron releasing group. The results suggest that our compound possesses less activity when compared to 9-aminoacridine propranolol (IC 50 = 13.6) [28].