Larvicidal and histopathological efficacy of cinnamic acid analogues: a novel strategy to reduce the dengue vector competence

Background: A novel strategy such as conjugation of amino, Schiff's bases, and thiadiazole moieties to the cinnamic acid nucleus has been adopted in this study to discover new molecules that target the dengue envelope protein (DENVE). Aim: Among the different domains of dengue virus envelope protein (PDB ID 1OKE), we have selected a ligand-binding domain for our structure-based drug design. The designed compounds have also been docked against DENVE protein. Methodology: Based on the in silico results and synthetic feasibility, three different schemes were used to synthesize twenty-three novel cinnamic acid derivatives. Sci-finder ascertained their novelty. The synthesized derivatives were consistent with their assigned spectra. The compounds were further evaluated for their larvicidal activity and histopathological analysis. Multiple linear regression analysis was performed to derive the QSAR model, which was further evaluated internally and externally for the prediction of activity. Results and discussion: Four compounds, namely CA 2, CA 14, ACA 4, and CATD 2, effectively showed larvicidal activity after 24, 48, and 72 h exposure; particularly, compound CA2 showed potent larvicidal activity with LC50 of 82.15 μg ml−1, 65.34 μg ml−1, and 38.68 μg ml−1, respectively, whereas intermittent stages, causes of abscess in the gut, and siphon regions were observed through histopathological studies. Conclusion: Our study identified some novel chemical scaffolds as effective DENVE inhibitors with efficacious anticipated pharmacokinetic profiles, which can be modified further.


Introduction
The primary carrier of viruses that cause dengue fever, Aedes aegypti, is found in vast areas of the tropics and subtropics. There are currently no known therapies for dengue fever. However, standard fever treatment is available (nursing care, uid balance, electrolytes, and blood clotting parameters). As a result, the only way to reduce the prevalence of this disease is by mosquito management, which involves interrupting the disease propagation cycle by attacking mosquito larvae at breeding sites. Besides vector control, a recently authorized dengue vaccine immunizes against all four serotypes. Dengvaxia is Sano Pasteur's (Paris, France) chimeric yellow fever-dengue-tetravalent dengue vaccine (CYD-TDV), the rst and only of its type, and has been registered for use in 19 countries but is only available in 10.2 of them. In addition, several phase II and phase III trials have resulted in specic vaccine restrictions, such as CYD TDV being delivered exclusively to those who have previously been infected with dengue and live in endemic regions. [13][14][15][16] Presently around the world, dengue is endemic in 112 countries. 1,2 Mostly in tropical and subtropical areas, each year, 50-100 million individuals are infected with DENV, resulting in nearly 500 000 severe life-threatening illnesses and 25 000 deaths. 3,4 One attractive approach could be the interruption of the virus replication at an early stage of attachment. 5 DENV enters the cell by receptor-mediated endocytosis followed by viral E protein-mediated membrane fusion. Membrane fusion is a major molecular event during viral entry into the host cell. 6 E (envelope) protein is a signicant component of the virion surface, plays an essential role in binding to the host receptor, and assists virus fusion. 7 Among the three domains present in the E protein, the hinge region movement of domains I and II facilitates the fusion process. 8 Upon lowering the pH, the E protein undergoes signicant conformational changes in the hinge region, springing upwards to bring the fusion peptide closer to the host membrane for fusion to occur. Small molecules' rearrangement and/or conformational changes in the hinge region may interrupt the fusion process. 9 A small detergent molecule, n-octyl-b-D-glucoside (bOG), occupies the hydrophobic pocket, which is located in the hinge region and plays a signicant role in the rearrangement. It has been reported that mutations within this binding pocket lead to the alterations of the pH threshold for fusion. In addition to this, the protonation of histidine residues present in the hydrophobic domain would also impart the conformational change of the E protein. The previously reported fusion inhibitors such as peptidic antivirals suffer from poor absorption from the gastrointestinal tract, necessitating intravenous delivery and high manufacturing costs. 10 We, therefore, wanted to develop nonpeptidic small molecules to inhibit dengue virus entry.
While searching for small molecules, we came across an exciting scaffold, zosteric acid, isolated from marine eelgrass, Zostera marina. It has been reported that zosteric acid, chemically p-sulphoxycinnamic acid, and its analogs act as entry inhibitors by inhibiting the fusion process. Based on the reported antiviral potency of zosteric acid, we moved on to a similar readily available scaffold cinnamic acid, the naturally occurring precursor of avonoids. It has excellent potential for a therapeutic role, showing antimicrobial, antifungal, and antitumor activities. 11 A compound derived from trans-cinnamic acid, p-sulphoxy-cinnamic acid, showed antiviral activity against the dengue virus, suggesting a possible antiviral potential of trans-cinnamic acid. 12 Besides, the modication of the -COOH group of cinnamic acid analogs leads to decreased IC 50 values against DENV (Fig. 1). Keeping all facts in mind, the present work is aimed to identify novel bioactive molecules bearing cinnamic acid as a key signature of DENV entry.

Materials and methods
Sigma Aldrich, SD Fine, and Carbanio supplied all the chemicals used which were of analytical grade. The solvents were puried and dried by industry standards. The 1 H and 13 C NMR data were analyzed using a Bruker 400 MHZ spectrometer. The internal standard was tetramethyl silane (TMS), and the solvent was DMSO-d 6 . Chemical shis were reported downeld from TMS in parts per million (ppm). For analytical thin-layer chromatography, the plates used were MERCK aluminium back precoated silica gel 60-F 254 (0.5 mm) plates. An iodine chamber or UV light was used to identify the spots on TLC plates visually. Shimadzu recorded mass spectra.

In silico design
The ADME, molecular docking, and molecular dynamics studies were conducted using Discovery Studio (D.S.) 4.1 and PyRx 0.8. The MMGBSA assay was performed using Schrodinger 2017.2 and the 3D QSAR studies were performed using MOE 2009.10 soware. 17 The preparation of protein (PDB ID 1OKE) and a library of seventy-two novel ligands was carried out by the standard method. [18][19][20] The CDOCKER module was used for calculating ligand binding affinity and energies. 21 The protein binding site was identied using existing ligands in the PDB database, and the sphere concept identied the location. 22 All other remaining procedure was followed and maintained by the reported methods. [23][24][25] The protein-ligand interactions were visualized and evaluated with DS 4.1.
PyRx 0.8 was used to perform the docking research of designed cinnamic acid analogues against FK506-binding protein (FKBP) from Aedes aegypti (PDB ID 3UQI). PyRx is a Python-based programming language that runs on almost any modern machine, from personal computers to supercomputers. PyRx has been used to determine the binding affinity of a ligand for a protein to facilitate molecular docking. PyRx, a structurebased docking program, was used to screen cinnamic acid analogues for Aedes aegypti (PDB ID 3UQI) at a resolution of 1.30 A. Additionally, ligands for energy reduction interact in good ways. The MMFF94 force eld performed the minimization in 200 steps with an RMS gradient of 0.1. Following the devaluation, the ligands were transferred to the PDBQT format. First, we chose the macromolecule that will dene the produced protein's binding site. Next, the active docking site was constructed utilizing bound ligand binding locations. Then, molecular docking was performed on a molecular window, with all produced ligands interacting with the specied active site. 34,35 First, all ligands were categorized according to their binding affinity as determined by the PyRx score. Following that, the ligands were classied according to their binding energy levels.
2.1.1. ADME studies. The ADME module of DS 4.1 was used to evaluate the compound's ADMET properties. 26 2.1.2. Insecticide likeness studies. The designed cinnamic acid analogues were screened using the InsectiPAD database (Insecticide Physicochemical-properties Analysis Database), a unique agrochemical database to provide information of all approved insecticides, and qualitative and quantitative evaluation of insecticide likeness of small molecules.

Chemistry
Twenty-three acid analogues were selected from a library of seventy-two cinnamic acid derivatives that showed signicant in silico results and were synthetically feasible.
2.2.1. General procedure for the synthesis of amino cinnamic acid hydrazides (ACA 1-4). The 4-amino cinnamic acid (40 mmol, 5.2 g) in acetonitrile with a coupling reagent N,Ndicyclohexyl carbodiimide (48 mmol, 80 ml) has been used in the presence of hydroxy benzotriazole (48 mmol, 9.98 g) (HOBT). The reaction mixture was stirred at room temperature for ten h, and the reaction process was monitored by TLC to obtain the 4-amino cinnamic acid ester (ACE). To the dried ACE, different hydrazines (3.8 mmol) in acetonitrile were added consecutively with stirring at 0 C and 5 C for one h and at R.T. for ve h. Then, the obtained dicyclohexyl urea (DCU) was removed by ltration, and the ltrate was allowed to stand at 0 C overnight and ltered again (Scheme 1). A mixture of the carbon disulde (0.015 mmol, 14 ml), potassium hydroxide (0.015 mmol, 8.4 g) in 100 ml of 99% ethanol, and cinnamic acid hydrazide (0.01 mmol, 1.62 g) was stirred for 20 h. Dry ether (150 ml) was added to the resulting mixture, and the precipitate potassium dithiocarbazinate was collected by ltration (Scheme 2). The obtained potassium salt (0.015 mmol, 4.4 g), hydrazine hydrate (2 ml), and distilled water (10 ml) mixture were reuxed with stirring to obtain the white residue on cooling. Then the concentrated hydrochloric acid was used to acidify, followed by ltration to obtain the desired product, which was recrystallized with ethanol.
2.2.3. General procedure for the synthesis of Schiff bases (CA [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. Equimolar mixtures of 1,2,4-triazole obtained from Scheme 2 with various aromatic aldehydes and a catalytic amount of 3-5 drops of concentrated sulphuric acid were reuxed in an ethanol medium for 5-8 h. The resultant was cooled to room temperature; the sediment solid was ltered and recrystallized in hot ethanol.  CH 3 ), 6.20 (d, J ¼ 6.6 Hz, H, CH), 5.08 (d, J ¼ 6.6 Hz, H, CH), 1.53 (s, H, SH). 13    In vitro cytotoxicity assay. The in vitro cytotoxicity assay was performed by the reported method (27). The mitochondrial enzyme succinate dehydrogenase cleaves the tetrazolium salt MTT into a blue coloured substance (formazan). The number of cells used was discovered to be equal to the amount of formazan produced by the cells used. 27 2.3.2. Haemolytic assay. The synthesized compounds in fresh anti-coagulated human blood cells were centrifuged for 10 min at 2000 rpm, following which the cell pellets were resuspended in NaCl 0.9% to prepare a 2% v/v cell suspension. Next, the red blood cell suspension (100 ml) was added to a 96well plate and incubated for one h at 37 C in 5% CO 2 . As an index of red blood cell lysis, the release of haemoglobin was determined spectrophotometrically at 540 nm. The complete hemolysis (positive control) was achieved by adding 1% (v/v) NaOH, while cells in 0.9% (w/v) NaCl solution served as a negative control. 28 The percentage of hemolysis was calculated as 2.3.3. Larvicidal bioassay. The larvicidal bioassay was assessed in the third stage instar larvae of Aedes aegypti following the standard World Health Organization larval susceptibility test method with minor modications. 29 The synthesized compounds at concentrations ranging from 50 to 250 mg ml À1 were mixed with 50 ml of dechlorinated sterile water and 25 larvae were used in this assay. Aer 24, 48, and 72 h, the mortality of Aedes Aegypti larvae was determined, respectively. The infected larvae were examined under a stereo zoom microscope, and these were done in triplicate.
Aer larval exposure to test compounds aer 24 hours and 48 hours, mortality was noted. The LC 50 and LC 90 values were further determined with the help of probit regression analysis. In addition, the chi-square value, degree of freedom, and pvalue were documented. All the analysis was performed using SPSS 16. Any 'p'-value <0.05 was considered statistically significant. The mortality of the larvae did not exceed 10% in the control group. Hence, the present data did not require any additional statistical analysis.
2.3.4. Histopathological analysis. Following alcohol wash to harden the tissue, the larval samples were embedded in paraffin wax and subsequently sectioned (2 mm) using a microtome (Weswox Optik-1090A). The sections were mounted, and stained with eosin and hematoxylin, and different cells were visualized at 10Â and 40Â magnications under a stereomicroscope. 30

QSAR studies
The half-maximal lethal concentration (LC 50 ) measures the effectiveness of a substance in inhibiting a specic biological or biochemical function. The LC 50 values were manually converted into pLC 50 (predicted LC 50 ) using the formula pLC 50 ¼ Àlog LC 50 . For performing the QSAR study, a dataset of 24 synthesized compounds was chosen, from which 19 training sets and 5 test set compounds were assigned accordingly. The seven descriptors were chosen to describe the structure of the compounds comprising the series to study viz., dipole moment (AM1_dipole), molar refractivity (M.R.), lowest unoccupied molecular orbital (AM1_LUMO), partition coefficients (log P(o/ w)), highest unoccupied molecular orbital (AM1_HOMO), van der Waals energies (EvdW), and topological polar surface area (TPSA). 31 The Multiple Linear Regression (MLR) procedures were used to create the QSAR model, which was constructed from the QSAR descriptor panel of the MOE 2009.10 suite, 32 and the RMSE and R 2 values were derived from the QSAR t. 33 A QSAR correlation plot was generated by plotting the values of pLC 50 on the x-axis and the predicted values ($PRED) on the yaxis for all the nineteen compounds (Fig. 9).

Results and discussion
3.1. In silico study of cinnamic acid analogues against DENVE (PDB ID 1OKE) A compound library of seventy-two new substances substituted with various aldehydes and benzoic acids at the 2 nd and 4 th positions of cinnamic was designed ( Table 1). The proposed compounds were postulated as new molecules by Sci-nder soware.
The most effective ligand binding site was discovered in pocket number 26, which had a volume of 1178.9Å 3   Thiadiazole substitution and its position Play on a signicant role in the inhibition of viral fusion with the above amino acid residues, and the Ramachandran plot further validated the prepared protein (Fig. 3). In addition, the CDOCKER energy of the synthesized compounds was lower than that of the reference inhibitor P02 (À132.553), as shown in Table 2, indicating that these molecules may have a higher binding affinity for DENVE (Fig. 4).
The results of the ADME study showed that all the compounds' log P values were found to be within limits (i.e. less than ve). AlogP is a critical parameter that affects drug bioavailability,   membrane permeability and drug delivery and clearance paths. This parameter is also important in drug pharmacological and toxicological properties. Plasma protein binding is another parameter to assess the concentration of a medicinal compound. It may be restrictive or permissive and is known to affect drug distribution. A Bayesian score $2.226 was observed for the compounds, indicating their likelihood to bind to a carrier protein in the blood. All compounds had a >140 polar surface area (Table 3), indicating the potential for high intestinal absorption (Fig. 5). All of the compounds were found to have high blood barrier levels. All compounds were CYP2D6 non-inhibitors, with none associated with signicant drug interaction toxicity. The MM-GBSA approach for binding free energy calculation revealed that van der Waals and coulombic terms were the most relevant positive contributors. A preferential trend towards the van der Waals component was observed, as indicated by the van der Waals and Coulomb energy interactions between À17. 19 and À57.5 kcal mol À1 , and À1.36 and À41.97 kcal mol À1 , respectively (Table 4). CA2 bounds best with DG (À75.87 kcal mol À1 ), with a slightly higher van der Waals contribution (À43.1 kcal mol À1 ), favourable lipophilic energy (25.67 kcal mol À1 ), and a favourable coulomb energy term (À41.97 kcal mol À1 ). van der Waals was deemed the driving force behind ligand binding. a Solubility level -0 extremely low; 1 no very low but possible; 2 yes low; 3 yes good; 4 yes optimal; 5 no too soluble. b BBBblood-brain barrier level -0 very high; 1 high; 2 medium; 3 low; 4 undened. c CYP2D13cytochrome 450 inhibition. d Hepatotoxicity. e PPBplasma-protein binding. f Absorption -1 very poor, 2 poor; 3 medium; 4 good. g AlogP98partition coefficient of octanol/water system, h PSApolar surface area. In PyRx, binding affinity parameters were considered for selecting the best "HITS" and compared with the known Cocrystal. PyRx binding energy is the interaction energy between the protein and the ligand. This value strongly indicates the extent of the interaction of proteins and ligands. The synthesized molecules whose binding energy was above the co-crystal FKBP-type peptidyl-prolyl cis-trans isomerase (À4.7) are shown in Table 5, indicating that the compounds were effectively bound to the active site of 3UQI. The hydrogen bonds and Pi-Pi interactions of these compounds were analyzed. The CA 2 showed the highest binding affinity for 3UQI (À7.8) and the 3UQI-6 protein CA 2 complex made one conventional-hydrogen bonding (Arg 43), four hydrophobic interactions (Glu 55, Ile 57, Phe 100 and Trp 60), and two pi-alkyl interactions (Val 56, and Tyr 57).

Prediction of insecticide likeness
Insecticides are likely to have signicantly different bioavailability related property proles compared with drugs. Therefore, we evaluated the insecticide likeness of our cinnamic acid analogues by the radar (Fig. 6) (the pink area represents the optimal range for each property) and histograms at rst glance or according to the insecticide likeness scores (the higher the score, the better the pesticide likeness of our compounds). Our compounds have shown properties which are similar to those of the standard insecticide upyradifurone, which are given in Table 6.

Biological studies
3.5.1. In vitro cytotoxicity assay. The MTT assay was performed to screen the synthesized compounds on Vero cells, and the results are shown in Table 7. Even at higher concentrations   of cinnamic acid derivatives for treatment, no cytotoxicity was observed in normal cells. 3.5.2. Hemolytic assay. The hemolytic activities of synthesized compounds were tested at various concentrations (25, 50, and 100 mg ml À1 ). The least hemolysis was 50 mg ml À1 (Table 8). Because compounds affect blood cells explicitly or implicitly, blood compatibility research is critical at the nanotoxicity level. Erythrocyte circulation through various organs leads to cell membrane injury, DNA damage, and congenital malformation. In this case, biocompatibility analyses of ligands that release erythrocytes were more critical than compound toxicity analyses.
3.5.3. Larvicidal activity. The larvae's growth (mortality) was analyzed for the selected compounds to understand the relationship between compound growth rate and larvae mortality. During the 72 h exposure period, the LC 50 and LC 90 for the best larvicidal activity were determined. Among all the compounds tested, CA 2 strains demonstrated signicant larvicidal activity at minimum concentrations even aer 24, 48, and 72 h of subjection (Table 11).
Different concentrations (50, 100, 150, 200 and 250 mg ml À1 ) were used to determine the mortality aer 24, 48 and 72 h of subjection. The mortality rate is affected by concentration and subjection period. However, observing CA 2 treatment at a minimum concentration for 24 and 48 hours resulted in the highest mortality range. ACA 4 showed slow mortality in the 24, 48, and 72 h exposure times, but they inhibited larval  In both larvae, the drug samples CA 2, CA 14, ACA 4, and CATD 2 resulted in severe lesions that primarily affected the midgut epithelium and the caeca to a lesser extent (Fig. 8).  Histopathological variations were observed qualitatively based on location along the midgut and quantitatively based on incubation time. Aer 72 h of infection, anterior midgut cells did not differ morphologically from normal ones, but for mild apical lysis of transparent cells and no lateral junctional complex disturbances. Compared with monitors, transparent cell lysis was accelerated at the brush border, basal membranes, and cytoplasmic organelles before bursting into the gut lumen. A posterior midgut partial lysis was initiated following local detachment in dark cells, with basal membrane dilatation and loss of the peritrophic membrane, with enlargement of cells.

QSAR studies
The QSAR approach is used to identify the structural parameters for the enhanced bioactivity, which leads to the design of the new scaffold with improved biological effects. A QSAR correlation between larvicidal activities with the series of nineteen synthesized compounds was performed by multiple linear regression analysis. The LC 50 (mM) values of the training set were rst converted into a negative logarithmic scale (pLC 50 ) to achieve the normal distribution ( Table 9). The derived QSAR model shows a good correlation between biological activity and  parameters, further validated through test set compounds (Table 11). The descriptors showed a positive correlation among all parameters selected for the QSAR model. The positive coef-cients suggest that including such hybrid structures together leads to an increase in biological activity. Further, the regression graph was plotted for pLC 50 vs. $PRED. (Fig. 9). The crossvalidated correlation coefficient (r 2 ) value is 0.61664, and the root mean square error value is 0.06233.  The formula for the generated QSAR linear model is Thus, the developed QSAR model can be applied to develop potent modied molecules with nitrogen, sulfur, and oxygen atoms, as potential anti-dengue agents.

Conclusion
The present research study suggests that our synthesized compounds may be the "LEAD" molecules for early DENV inhibition, which prevents or lessens the chances of patients progressing to dengue hemorrhagic fever/dengue shock syndrome. Cost-effectiveness is a crucial issue for this still poverty-linked disease, and this issue is also gured out by selecting early DENV inhibitors. Overall, this study concludes CA-2 and ACA-4 as potent compounds for biologically controlling Aedes aegypti. The docking studies also support the insecticidal potency of the synthesized compounds. Interestingly, in silico docking ndings show that the molecule CA-2 also regulates the 1OKE protein through the b-OG bag. As a result, CA-2 may be used as a lead molecule in anti-viral entry inhibition. The QSAR model developed may be used to design candidates, with nitrogen, sulfur, and oxygen atom substituents, as promising anti-dengue compounds. The compounds CA 2 and ACA 4 may also be considered lead compounds for other mosquitoborne viral diseases.