Synthesis of new series of quinoline derivatives with insecticidal effects on larval vectors of malaria and dengue diseases

Mosquito borne diseases are on the rise because of their fast spread worldwide and the lack of effective treatments. Here we are focusing on the development of a novel anti-malarial and virucidal agent with biocidal effects also on its vectors. We have synthesized a new quinoline (4,7-dichloroquinoline) derivative which showed significant larvicidal and pupicidal properties against a malarial and a dengue vector and a lethal toxicity ranging from 4.408 µM/mL (first instar larvae) to 7.958 µM/mL (pupal populations) for Anopheles stephensi and 5.016 µM/mL (larva 1) to 10.669 µM/mL (pupae) for Aedes aegypti. In-vitro antiplasmodial efficacy of 4,7-dichloroquinoline revealed a significant growth inhibition of both sensitive strains of Plasmodium falciparum with IC50 values of 6.7 nM (CQ-s) and 8.5 nM (CQ-r). Chloroquine IC50 values, as control, were 23 nM (CQ-s), and 27.5 nM (CQ-r). In vivo antiplasmodial studies with P. falciparum infected mice showed an effect of 4,7-dichloroquinoline compared to chloroquine. The quinoline compound showed significant activity against the viral pathogen serotype 2 (DENV-2). In vitro conditions and the purified quinoline exhibited insignificant toxicity on the host system up to 100 µM/mL. Overall, 4,7-dichloroquinoline could provide a good anti-vectorial and anti-malarial agent.

www.nature.com/scientificreports/ Nyberg et al. 27 ). As the plasmodium parasite becomes more resistant to quinoline based anti-malarial drugs, it becomes even more important to design a potent anti-malarial molecule 28,29 . Hence, finding new compounds to treat malaria is urgently needed for the treatment of dangerous mosquito borne diseases 30,31 . This work provides a general overview of quinoline advantages for the discovery of more efficient compounds 32,33 . In continuation of the study for the preparation of a 4-diamine substituted-7-dichloroquinoline compounds against vector borne diseases 34 we report herein the anti-malarial and anti-dengue potential of a novel quinoline compound.
The quinoline skeleton is utilized for some important engineered agrochemicals and to plan manufactured mixtures providing several pharmacological effects. Quinoline and its related compounds belongs to a significant class of antimalarial sedates that affect the parasite's hemoglobin breakdown pathway. Earlier studies reported that for some time this compound was utilizing quinoline to battle malaria 35 . Along these lines, it is significant to re-look into the antimalarial movement of existing quinoline libraries or blend some unique quinoline subsidiaries with improved action. A methodical and broad investigation is needed to find a compelling antimalarial compound structure 4-aminoquinoline based framework 36 . In the present research, we have orchestrated several analogs of 4,7-dichloroquinoline and screened against jungle fever parasites, dengue (DENV-2) and their respective mosquito vectors. Also, we reported the synthesis of N2-2-((7-chloroquinolin-4-yl) amino) ethyl)-N4, N6-bis(4-nitrophenyl)-1,3,5-triazine-2,4,6-triamine. Whose synthesis have been planned for the bi-substituted cyanuric chloride using p-nitroaniline incorporated N1-(7-chloroquinoline-4-yl) ethane-1,2-diamine. Synthesized molecules can be analyzed by IR, 1 HNMR, 13 C, mass and elemental analysis to characterize their molecular structure. This is a new compound that is easily synthesized by substituting cyanuric chloride to provide s-triazine derivatives. Substituted quinolines are historically among the most important antimalarial drugs and are expected to achieve a substantial reduction of malaria infections.

Toxicity effects on developmental instars of Aedes aegypti and A. stephensi. The mosquitoes
A. aegypti and A. stephensi were cultured and maintained following Murugan et al. 37 . For toxicology studies, 25 individuals of both A. stephensi and A. aegypti larva (1st, 2nd, 3rd, and 4th) and pupae were placed for a 24 h treatment in a tank filled with 500 mL of distilled water at concentrations of 4,7-dichloroquinoline (2, 4, 6, 8 and 10 ppm) 38 . In each treatment, 3 replications were carried out, in addition to negative controls. Mortality rate in percentage was studied applying the following formula: Antiplasmodial cell culture assays on P. falciparum. CQ-sensitive strain 3D7 and CQ-resistant strain INDO of Plasmodium falciparum were used to test the antimalarial activity of 4,7-dichloroquinoline. They were maintained according to the method described by Murugan et al. 39 . Formulations of 4,7-dichloroquinoline in DMSO were evaluated by the procedure of Murugan et al. 40 , modified after Smilkstein et al. 41 . Microscopic examination of Giemsa stained smear samples of normal Plasmodium falciparum exposed to 4,7-dichloroquinoline was following Bagavan et al. 42 .
In vivo antiplasmodial assays on P. falciparum. Following the method of Murugan et al. 43 , male albino mice (weight 27-30 g) were tested. They were maintained as reported by Murugan et al. 43 . For each experiment, three albino mice were used to test the antimalarial potential of the synthesized compound, 4,7-dichloroquinoline following a four-day inhibition technique by Murugan et al. 43 . Chloroquine (Sigma-Aldrich, Germany) was used as a positive control drug with normal saline (0.9%) at 5 mg/kg, while the negative control group was treated with 1 mL deionized water. The parasites inoculated in mice were noticed after 4 days of infection through microscopic observations of the blood 44 . Chemosuppression (%) was analyzed for every concentration of the parasitemia following the method of Argotte et al. 45 .
Infection and toxicity towards cells. We  www.nature.com/scientificreports/ was incubated at 37 °C in a 5% CO 2 atmosphere. We decreased the serum concentration to 2% when viral cultures were used. As described by Murugan et al. 43 Dengue virus type-2 (DEN-2) New Guinea C strain was raised through adopting the cell line and were retrieved after the expression of cytopathic effects (CPE), commonly seven days after infection. Infected viral cells were stored at -70 °C. Cytotoxicity assays and viral quantification assays were following Sujitha et al. 46 with minor modification.
Statistical analysis. Data from Probit analysis allowed the analysis of the effective lethal concentrations of the mosquito larvicidal and pupicidal experiments 47 . From the drug concentration-response curves the IC 50 s of Plasmodium were calculated. In vivo antimalarial data were checked for normality and analysed using ANOVA with two factors (i.e. dose and treatment). DEN-2 PFU and cytotoxicity data were determined by ANOVA followed by the HSD test of Tukey with the following probabilities (P = 0.05). All analyses were commonly carried out with the SPSS software package version 16.0.

Results and discussion
N1-(7-chloroquinoline-4-yl) ethane-1, 2-diamine effects analyzed by in-silico approaches. The synthesized compound showed no tumorigenic, irritative, nor reproductively significant effects in silico. Besides, LogP and LogS values (Table 1) indicated that the synthesized compound was hydrophilic with a high probability of being distributed along with hydrophilic environments such as insect lymph or cellular cytosol. Molinspiration analysis indicated that the values regarding, GPCR ligand, kinase inhibitor, nuclear receptor ligand, ion channel modulator, protease inhibitor and enzyme inhibitor scores were high. Molinspiration analysis generally indicated that the larger the value of the score was, the more the compound would have biological effects. Therefore, according to in silico analysis, N1-(7-chloroquinoline-4-yl) ethane-1,2-diamine is likely to affect ion channels, kinases, and some important enzymes. The above results could be related to acute toxicity on young instars of A. aegypti and A. stephensi and highly increased the growth inhibition of Plasmodium falciparum 48  Antiplasmodial activities. As a result of antiplasmodial assays, when compared to chloroquine, the synthesized 4,7-dichloroquinoline expressed significant growth inhibition against both CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of P. falciparum (Fig. 2). Similarly, Kumawat et al. 57 investigated 7-Chloro-4-aminoquinoline derivatives causing moderate growth inhibition on CQ-sensitive P. falciparum (RKL-2). Also, Faruk Khan 58 noticed that the cyclen 4-Aminoquinoline anlog, bisquinoline, exhibited in vitro and in vivo antiplasmodial properties on D6 W2 chloroquine-sensitive and chloroquine-resistant strains of P. falciparum with IC 50 values of 7.5 nM (D6 CQ-sensitive) and 19.2 nM (W2 CQ-resistance). Very recently, Pinheiro et al. 59 showed that quinoline and non-quinoline derivatives were highly effective against both P. falciparum W2 chloroquine-resistant strains of P. falciparum in infected mice. Quinoline drugs exhibited potential inhibitory effect of proteolysis, DNA replication, RNA synthesis and heme polymerization in Plasmodium spp 60,61 . Additionally, Aboelnaga and El-Sayed 62 reported that 7-chloroquinoline derivatives showed significant anticancer activity on cervical (Hela) cancer cell lines, human breast cancer (MCF-7) and colon carcinoma (HCT-116). Protein kinase inhibitors, topo isomerase inhibitors, carbonic anhydrase inhibitors, Hsp90 inhibitors are the anticancer mechanisms of quinoline derivatives 63 . Aderibigbe et al. 64 found that polymer loaded aminoquinoline were highly potent against the strain of P. falciparum which was chloroquine-sensitive. A new quinoline derivative, thiazolyl hydrazone were synthesized as effective antifungal and anticancer agents by Erguc et al. 65 . Dose-dependent chemosuppression against P. falciparum was demonstrated by Peters' 4-day chemo-suppressive activity assay (Fig. 3). After 4 days of 4,7-dichloroquinoline treated groups exhibited the percentage of parasitemia 10.6 ± 0.8% at 300 mg/kg/day than that of the control drug chloroquine (CQ) 1.0 ± 0.0% 37,66 . Tang   Table 2. Acute toxicity of synthesized 4,7-dichloroquinoline on young instars of Anopheles stephensi and Aedes aegypti. Control no mortality, LC 50 lethal concentration that kills 50% of the exposed organisms, LC 90 lethal concentration that kills 90% of the exposed organisms, χ 2 chi-square value, d.f. degrees of freedom, χ 2 0.05 level of significance indicates homogeneity of results.  www.nature.com/scientificreports/ et al. 67 showed antimalarial activities against the P. falciparum strain K173 with EC 50 values ranging from 0.38 to 0.43 mg/kg. Manohar et al. 68 found that 4-Aminoquinoline-pyrimidine hybrids exhibited 80% parasitemia suppression as compared to CQ (20%). Finally, Sahu et al. 69 found that low doses of tigecycline (3.7 mg/kg) showed 77-91% of parasitaemia suppression. Inhibition of parasitaemia of 77-91% was provided by 3.7 mg/kg dose of tigecycline for 4 consecutive days. Furthermore, the authors reported that in vivo treatment with tigecycline in combination with sub-curative doses of CQ provided 100% mortality of P. falciparum in infected mice.

Cytotoxicity effect of 4,7-dichloroquinoline on Vero cells.
In the present study, the viability of Vero cells was incorporated in various concentrations of 4,7-dichloroquinoline 70 . We observed that there were no adverse morphological differences in the treated groups when compared to control Vero cells (Figs. 4,5). For example, Tseng et al. 71 studied that the new derivatives of synthesized 2-aroyl-3-arylquinoline compounds provided substantial cytotoxicity against Huh-7 cells with less than 20% viability at doses of 100 μM of 4,7-dichloroquinoline. Cell death above a concentration of 60 μM of 4-methyl pyrimido (5,4-c) quinoline-2,5(1H, 6H)-dione on MDCK cells were shown by Paulpandi et al. 34 . Recently, Beesetti et al. 72 highlighted that quinoline derivatives, BT24 effectively inhibit DENV-2 protease with IC 50 of 0.5 μM.   Fig. 6). Furthermore, a plaque assay displayed after an individual exposure and with a minimum dosis that 4,7-dichloroquinoline effectively inhibited the production of dengue viruses. Post 48 h treatment duration of the viral production was 91 PFU/mL in the control, whereas it was 19 PFU/mL, after the treatment of in 4,7-dichloroquinoline at a concentration of 40 μL/mL (Fig. 7). Similarly, Guardia et al. 74 discovered that quinoline derivatives highly inhibited DENV-2 with IC 50 values ranging from 3.03 to 0.49 μM, respectively. Very recently, Devaux et al. 75 found that chloroquine/hydroxychloroquine significantly inhibited pandemic SARS-CoV-2. Furthermore, chloroquine highly inhibited HCoV-229E replication in epithelial lung cell cultures 76 . It became apparent that the Zika virus provided a regional threat for Latin America and the Caribbean 77,78 .

Conclusion
It is clear from previous reports that resistance to the malaria vector continues to grow. This is increasingly limiting our ability to control malaria worldwide. Our present study demonstrated the mosquitocidal potential of 4,7-dichloroquinoline derivatives against the key mosquito vectors, An. stephensi and Ae. aegypti. This would be a promising advance in the development of clean, non-toxic, and environmentally acceptable quinoline  www.nature.com/scientificreports/ compounds for their effect against mosquito vectors. Furthermore, 4,7-dichloroquinoline had a significant and promising anti-malarial potential to reduce the global threat malaria. Quinoline decreased virus proliferation and replication during protein synthesis at mRNA levels. No cell cytotoxicity was identified. A compound was recognized as a unique kind of structure different for additional improvement against DENV specialists. We have presented here novel quinoline subordinates that are fundamentally dynamic against dengue infection in a partially subordinate way. The discoveries presented here are significant as a starting point for additional clarification of the particular components of the antiviral action and to pick up the necessary information to additionally grow new, compelling, strong, and safe medications to lessen the risks from viral diseases.  www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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