Synthesis, antimalarial activity evaluation and molecular docking studies of some novel dispiro-1,2,4,5-tetraoxanes

Seven novel dispiro-1,2,4,5-tetraoxane derivatives were synthesized and characterized by a number of analytical and spectroscopic techniques. The molecules were subsequently screened for in vitro antimalarial activity against chloroquine resistant strain of  Plasmodium falciparum  (RKL-9). At antimalarial activity screening, two compounds, namely 5d (MIC = 15.6 µg/mL or 64.5 µM) and 5f (MIC = 15.6 µg/mL or 54.6 µM) were found to be about 1.5 times more potent against chloroquine resistant strain-RKL-9 compared to chloroquine (MIC = 25.0 µg/mL or 78.3 µM). Molecular docking studies of potent ligands were also performed in cysteine protease binding pocket residues of falcipain-2 as a target protein.

the synthesized compounds were recorded on UVvisible spectrophotometer (Shimadzu UV-1800). Infrared spectra were recorded on an FT-IR Perkin-Elmer spectrometer. The 1 H and 13 C NMR spectra were recorded at 400 MHz and 100 MHz, respectively, on a Bruker Avance-II 400 NMR spectrometer using either  or CDCl3 as solvent with tetramethylsilane (TMS) as an internal standard. Mass spectra were obtained on a Waters Q-TOF MICROMA SS LC mass spectrometer. Elemental analyses (CHN and O) were carried out on Eager Xperience Elemental analyzer (Coates, 2000;Pasto et al., 1992;Mathieson, 1965;Silverstein and Webster, 1963).

Synthesis of intermediate dihydroperoxides (Step I)
Cyclic aldehyde/ketone (1 mL, 10 mmol) was dissolved at room temperature in a CH2Cl2/CH3CN mixture (20 mL, 1:3 v/v) followed by 30% H2O2 (10.4 mL, 0.1 mol) and 0.5 mL of concentrated HCl. The reaction mixture was stirred for 2 hours at room temperature and quenched with saturated NaHCO3 and CH2Cl2. The organic layer was separated, and the water layer was filtered and dried Opsenica et al., 2008;Terent'ev et al., 2012).

Antimalarial activity
All the synthesized compounds were evaluated for in vitro antimalarial activity against chloroquine resistant strain-RKL-9 of P. falciparum (Pf) using 96 well-microtitre plates at the Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, Assam, India. The laboratory adapted strain of Pf was routinely cultured at 37ºC temperature and 5% CO2 environment in RPMI 1640 medium supplemented with 25 mM HEPES, 1% D-glucose, 0.23% sodium bicarbonate and 10% heat inactivated human serum. For antimalarial testing, the asynchronous parasites of Pf were synchronized to obtain only the ring stage parasitized cells by 5% D-sorbitol treatment. For carrying out the assay, the initial ring stage parasitemia of 0.8-1.5% at 3% hematocrit in a total volume of 100 mL of medium RPMI-1640 was uniformly maintained. A stock solution (1 mg/mL) of sample was prepared by dissolving the test compounds in DMSO and subsequent dilutions were made with the culture medium. Hundred microlitres of the test compounds at 100 µg/mL concentrations in triplicate was incubated with parasitized cell preparation at 37ºC and 5% CO2 in a CO2 incubator. After an incubation period of 36-40 hours, blood smears were prepared from each well and stained with 3% Giemsa stain. The slides were microscopically observed and the percent dead rings and schizonts were scored against 200 asexual parasites with respect to the control group. Chloroquine was used as the standard reference drug (Trager and Jensen, 1976).

Molecular docking studies
The three dimensional (3D) crystal structure of falcipain -2 (PDB code 3BPF) was retrieved from the protein data bank (PDB) (Source:www.rcsb.org/pdb). The native autoinducer and all water molecules were removed. The CHARMm force field (FF) was used to add atom types and hydrogens in the proteins. 3D structures of all synthesized compounds were constructed and energy minimized using the Discovery Studio 2.5/ Builder module. Docking studies were performed using the CDOCKER module of Discovery Studio 2.5. CDOCKER is a grid-based molecular docking method where the receptor is held rigid while the ligands are allowed to flex during the refinement. The CHARMm force field was used as an energy grid force field for docking and scoring function calculations. Random ligand conformations were generated from the initial structure through high temperature molecular dynamics, followed by random rotations which were further refined by grid-based (GRID 1) simulated annealing and a final grid-based minimization. Of the 10 best poses, one (conformation) having a highest docking score (-CDOCKER energy) was used for the binding energy calculations and further analysis. The higher negative value of CDOCKER energy represents more favorable binding of the complex. This means that ligands with high docking scores are able to fit snugly in the active site pocket with the minimal steric clashes. CDOCKER score (-CDOCKER Energy) includes inter-nal ligand strain energy and receptor-ligand interaction energy, and is used to sort the different conformations of each input ligand (Oliveira et al., 2013;Liu et al., 2012).
FT-IR spectra showed the stretching frequency range between region 2850-2950 cm -1 due to aliphatic cycloalkyl -C-H stretching, 1250-1000 cm -1 due to C-C-O stretching and 900-750 cm -1 due to peroxide, C-O-Ostretching. 1 H NMR spectra of the compounds showed a triplet or multiplet at δ (ppm) 1.00-2.50 due to cycloalkyl -C-H which further confirmed the formation of the desired compounds. The analytical and spectral data of the compounds were in conformity with the structure of the synthesized compounds.

Molecular docking studies
A molecular docking study was undertaken to gain insight into the key structural requirements and the basis of the distinct activity profile of the test compounds in P. falciparum parasite. The docking studies of the target compounds were performed into the binding pocket of falcipain-2 (PDB code 3BPF). The results and docked conformations of the ligands in the active site are illustrated in Table II and Figure 1, respectively.

Discussion
Seven novel dispiro-1,2,4,5-tetraoxane derivatives were synthesized and characterized by a number of analytical and spectroscopic techniques. Two compounds, namely 5d (MIC = 15.6 µg/mL or 64.5 µM) and 5f (MIC = 15.6 µg/mL or 54.6 µM) were found to be about 1.5 times more potent against chloroquine resistant strain-RKL-9 compared to chloroquine (MIC = 25.0 µg/ mL or 78.3 µM) on antimalarial activity screening. Molecular docking study results showed that the targeted molecules were snugly fitted into the active site with considerable and diverse CDOCKER energy (-1.6870 to -23.1300) with FP-2 along with the formation of numerous hydrogen bonds and hydrophobic interactions. Vennerstrom and co-workers (1992) reported that high  steric hindrance close to the peroxide ring is unfavorable for activity in dispirotetraoxanes. Amewu and co-workers  reported that dispirotetraoxane compounds was found to be equally potent as artemisinin.
Antimalarial activity results reflect that the dispirotetraoxanes are found to be potent compounds against chloroquine resistant Pf strain RKL-9. Substitution on dispirocycloalkane-tetraoxane with methyl and ethyl carboxylic acid groups make more active tetraoxanes than CQ against RKL-9 as observed with 5d and 5f, due to desirable lipophilicity to tetraoxane. Attachment of higher cycloalkanes e.g. six and seven member dispirocycloalkane ring (cyclohexane or cycloheptane) to tetraoxane ring enhanced effectiveness of tetraoxanes than that of lower cycloalkanes e.g. five member dispirocycloalkane ring (cyclopentane) towards their antimalarial activity. Chloro group substituted dispirotetraoxane become less active than corresponding unsubstituted dispirotetraoxanes.

Conclusion
A novel series of compounds with potent antimalarial activity has been developed. Designed molecules have the possibility to introduce chemical diversity around the core skeleton to generate newer and potent molecules.