Biological evaluation of a series of benzothiazole derivatives as mosquitocidal agents

1 Introduction

Mosquitoes are one of the most dangerous insect vectors and infectious disease carriers in developing countries [1]. Among mosquito species, Aedes aegypti L. transmits yellow fever, dengue fever, chikungunya, and more recently, Zika virus [2]. Dengue is an endemic viral disease found mainly in the tropical and subtropical regions across the globe [3,4]. Dengue is characterized by fever, headache, muscle, and joint pain together with nausea and vomiting [4, 5, 6]. Zika virus also causes severe brain defects and threatens the lives and health of adults and newborns from infected mothers [7].

The battle against mosquitoes has become a crucial environmental, economic and social health issue. Generally, chemical insecticides are considered as the first option for reducing vector-borne disease but evolving resistance caused by cytochrome P450 monooxygenases (P450s), which are capable of metabolizing many insecticides, as well as decreases in target site sensitivity can limit the success of insecticide treatment [8, 9, 10]. In particular, overexpression of P450s such as the CYP9J32 gene in Ae. aegypti is associated with pyrethroid resistance [11,12].

Sterol carrier protein-2 (SCP-2), a nonspecific intracellular lipid carrier, is expressed throughout the animal kingdom including insects. Moreover, single SCP-2 domain genes have also been proven to be expanded in mosquitoes [13]. Cholesterol is crucial for insects in order to grow, develop and reproduce, but they are not capable of synthesizing cholesterol de novo [14,15]. The mosquito SCP-2, Ae. aegypti SCP-2 (AeSCP2), has been reported to be involved in cholesterol and fatty acid uptake in the midgut in both larval and adult mosquitoes [16]. A small number of studies have focused on developing new insecticides that prevent cholesterol biosynthesis targeting AeSCP2 [17, 18, 19]. Fifty seven compounds were identified in silico as possible inhibitors of the cholesterol-binding capacity of SCP-2 from the library of 16000 compounds [20]. Therefore, targeting this cholesterol transport pathway associated with AeSCP2 could be an alternative target for the development of specific mosquitocidal agents.

Benzothiazole (BT) is a privileged bicyclic ring system present in a wide variety of synthetic and natural products. BT and its derivatives play a distinctive role in medicinal chemistry due to their diverse biological activities such as antiprotozoal, antimicrobial, anticancer, antischizophrenia, antihypertensive, anti-inflammatory, and antiviral activities [21, 22, 23, 24, 25]. Venugopala et al. 2013 [26], also screened benzothiazole analogs for their mosquitocidal and repellent properties against Anopheles arabiensis by mosquito feeding-probing assay, cone bio-assay and standard World Health Organization (WHO) larvicidal assay. Similarly, piperidine is a strong base mainly found in several natural alkaloid skeletons [27]. Diversely substituted piperidines are the leading heterocycles in the structure of several important pharmaceuticals such as bupivacaine, troxipide, tofacitinib, fexofenadine, astemizole, fentanyl, haloperidol, loperamide, trimeperidine, etc. [28].

On the basis of these findings, benzothiazole-piperidine derivatives, which had been reported previously for their antimicrobial effects on pathogenic bacteria and Candida species by our research group [29], were evaluated for their insecticidal activities against Ae. aegypti. In addition, molecular docking studies were performed for the most effective compounds within the active site of AeSCP2 (PDB code: 1PZ4) to provide a mechanistic approach for further studies [30].

2 Experimental

2.1 Chemistry

2-Chloro-N-(benzothiazol-2-yl)acetamide derivatives were synthesized via the reaction of 2-aminobenzothiazoles with chloroacetyl chloride in the presence of triethylamine. N-(Benzothiazol-2-yl)-2-(piperidin-1-yl)acetamide derivatives (1-24) (Table 1) were obtained by the nucleophilic substitution reaction of 2-chloro-N-(benzothiazol-2-yl) acetamides with piperidine derivatives in the presence of potassium carbonate. The synthetic protocol and spectral data of the compounds were reported previously by our research group [29].

Table 1

Structures of compounds 1-24 [29].

	Compound	R	R’
	1	H	4-methyl
	2	H	3-methyl
	3	H	2,6-dimethyl
	4	H	3,5-dimethyl
	5	Cl	4-methyl
	6	Cl	3-methyl
	7	Cl	2,6-dimethyl
	8	Cl	3,5-dimethyl
	9	CH3	4-methyl
	10	CH3	3-methyl
	11	CH3	2,6-dimethyl
	12	CH3	3,5-dimethyl
	13	OCH3	4-methyl
	14	OCH3	3-methyl
	15	OCH3	2,6-dimethyl
	16	OCH3	3,5-dimethyl
	17	OC2H5	4-methyl
	18	OC2H5	3-methyl
	19	OC2H5	2,6-dimethyl
	20	OC2H5	3,5-dimethyl
	21	NO2	4-methyl
	22	NO2	3-methyl
	23	NO2	2,6-dimethyl
	24	NO2	3,5-dimethyl

3 Results and Discussion

Compounds 1-24 were investigated for their larvicidal and adulticidal activities against Ae. aegypti Initial screening of the first instar larvae was completed at four concentrations (1.0, 0.5, 0.25, 0.1 mg/μL) with seven compounds producing more than 80% mortality at the 0.1 mg/μL dose (Table 2). Compounds 6 and 10 produced more than 80% mortality in initial screening of adult Ae. aegypti females (Table 2). LC₅₀ or LD₅₀ values were subsequently determined for compounds that produced

>80% mortality at the discriminating dose in either or both bioassays by further subdilutions and additional assays (Table 2). Compounds 11, 6, 4, 2, 13, 8, and 7 showed the highest larvicidal activity with LC₅₀ values of 0.034 (107.3 μM), 0.053 (163.8 μM), 0.065 (214.5 μM), 0.096 (332.2 μM), 0.098 (307.2 μM), 0.123 (364.4 μM), and 0.128 (379.2 μM) μg/μL, respectively. Compounds 6 and 10 exhibited the highest adulticidal activity with LD₅₀ values of 2.084 (6442.0 μM) and 2.962 (9775.6 μM) μg/mosquito, respectively. Compounds 1, 3, 5, 9, 12, 14-24 did not show strong larvicidal or adulticidal activity.

Generally, 4-methyl substitution on the piperidine ring and 6-methoxy substitution on the benzothiazole ring, apart from compound 13, caused the loss of larvicidal activity. On the other hand, 6-chloro substituted benzothiazoles (6, 7 and 8) displayed reasonable larvicidal activity; only compound 5 did not possess any antimosquito properties related to 4-methyl substitution on the piperidine ring. This outcome indicated the importance of the chloro substitution at the sixth position of the benzothiazole scaffold. Among nonsubstituted benzothiazoles (1-4), only 3-methyl substituted piperidine-based compound 2 and 3,5-dimethyl substituted piperidine-based compound 4 showed significant larvicidal activity, indicating that the methyl substitution at the third position of the piperidine ring enhanced the larvicidal activity. The presence of 3-methyl moiety on piperidine ring also influenced adulticidal activity positively as observed in compounds 6 and 10. In general, the loss of larvicidal activity was detected with 6-ethoxy and 6-nitro substitutions on the benzothiazole ring as observed in compounds 17-24.

According to docking results, compounds 2, 4, 6, 7, 8, 10, 11, and 13 showed high affinity and substrate-specific interactions in the active site of AeSCP2 [30,34] (Figure: 1). Benzothiazoles of compounds 2, 6, 7, 10, 11, and 13 presented π-π interactions with Arg15 and Phe105 residues of the binding site of AeSCP2. The methoxy substitution on the benzothiazole ring of compound 13 could account for all interactions of this compound. Piperidines of compounds 4, 6, 7, 8, 10 and 11 displayed cation-π interaction with the Arg15 residue and only 4-methyl substitution on the piperidine ring did not have a positive influence. All interactions of compounds 4 and 8 were related to 3,5-dimethyl substitution on the piperidine ring. Moreover, the acetamido moieties of compounds 2 and 10 served as H-bond donors for the in pocket residue Leu102. The docking score, glide gscore and glide emodel results of compounds 2, 4, 6, 7, 8, 10, 11, and 13 are also given in Table 3. The emodel score is appropriate for the comparison of different conformations of the same ligand, but the docking score is generally used for the comparison of different ligands [35]. The docking scores of the compounds were determined to range between -8.19 and -8.95 kcal/mol as similar to each other.

Figure: 1

Docked poses of compounds 2, 4, 6, 7, 8, 10, 11 and 13 (Yellow dashes: Hydrogen bonding; Blue and green dashes: π-π interactions) (A) and interactions of compound 6 in the active site of AeSCP2 (B).

4 Conclusions

In the present work, compounds carrying benzothiazole and piperidine rings were investigated for first instar larvicidal activity and adulticidal activity against Ae. aegypti. The most potent larvicidal and adulticidal compounds in this series were also analyzed for molecular docking interactions in the active site of the AeSCP2 to provide mechanistic insight for further in vitro enzyme studies. According to mosquitocidal assays, compounds 2, 4, 6, 7, 8, 11 and 13 were found to be the most promising larvicidal agents, whereas compounds 6 and 10 were identified as the most promising adulticidal agents. Based on the results, 3-methyl substitution on the piperidine ring enhanced the larvicidal and adulticidal activity. Besides, 6-chloro substitution on the benzothiazole ring also increased the larvicidal activity. The docking results of compounds 2, 4, 6, 7, 8, 10, 11 and 13 suggested that π-π interactions and hydrogen bonds were responsible for the observed affinity in the active site of the AeSCP2. This outcome supported the hypothesis that the larvicidal and adulticidal activities of these compounds could result from the inhibition of AeSCP2.