Xanthone as Antimalarial : QSAR Analysis , Synthesis , Molecular Docking and In-vitro Antimalarial Evaluation

The rational design of eighteen new antimalarial compounds from xanthone derivatives has been conducted based on Quantitative Structure-Activity Relationship (QSAR) calculation using semiempirical AM1 methods. The best equation model obtained from QSAR calculation was Log pIC50 = 2.997 29.256 (qO8) 138.234 (qC9) 6.882 (qC12) 107.836 (qC14) + 48.764 (qO15). Among the designed compounds, 3,6-dihydroxy-9H-xanthen-9-one (26) and 3,4,6-trihydroxy-9H-xanthen9-one (27) have been synthesized and investigated their in-vitro antimalarial activities against the chloroquine-sensitive 3D7 strain. An in-vitro antimalarial activity of compound 26 and 27 showed to be highly potential as antimalarial compounds with IC50 of 0.71 and 0.11 μM respectively. Molecular docking studies of compound 26 and 27 showed the formation of a binding interaction between the compounds with the amino acids Ala16, Ser108, Phe58, Asp54 and Leu46, which is the crucial amino acids for antimalarial activity based on the protein-ligand co-crystal structure of WR99210 (1,3,5-triazine, a pre-clinical molecule as P. falciparum DHFR-TS inhibitor).


INTRODUCTION
There are five types of malaria parasites that commonly infect humans, the first one is Plasmodium falciparum, which is the most fatal among other Plasmodium parasites.Furthermore, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae that cause mild diseases in human and not generally fatal.The fifth parasite, Plasmodium knowlesi, is known to cause malaria in monkeys, which is reported can also infect humans.3] In order to prevent more victims caused by malaria disease, searching and finding for new candidates of the antimalarial drug must be conducted continuously because the absence of new antimalarial drugs may cause malaria being an uncured disease in the next ten years.One of the strategies to find new antimalarial drugs is to explore natural products, particularly from plants, which have been traditionally utilized as an antimalarial drug.5][6] However, further research to find optimum antimalarial activities of xanthone compounds is still needed.Hence, the research to discover xanthone compounds with the best antimalarial activity should be conducted.
Xanthone compound was also reported for having some activities as anticancer, [7][8][9] antitumor, 10- 11 antioxidant, [12][13] cytotoxic, [14][15][16] leukemia, 17 antidiabetic. 18These facts show that xanthone has tremendous potential activities.In general, the reported compounds were the result of the isolation process, which means it is more difficult to conduct further development in enhancing the activity because of the low yield of compounds afforded.Therefore, the research that can truly maximize the potential of the xanthone compound is required, such as by making design and synthesis of xanthone compounds that have optimum activity.This kind of research could generate higher yield of the compounds obtained and further modification and their biological activity assay can be conducted effectively.QSAR analysis of xanthone as an antimalarial has been repor ted by Amanatie et al (2010) 19 .They have used PM3 method and concluded the descriptors which influence the antimalarial activity were atom charges of O7, C12, and C13.However, their research did not report the design of xanthone which had the best antimalarial activity.
In this research, QSAR studies were carried out to determine the descriptors which were responsible for the antimalarial activity.QSAR equation from the resulted descriptor was used to design new xanthone compounds which have the best-predicted antimalarial activity.Actually, QSAR studies could help to determine what the functional group should be attached to the compounds in order to get the best activity.Herein, molecular docking with the crystal structure (1J3I.pdb) of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (DHFR-TS) was conducted to observe the binding interaction of the xanthone with the amino acid.The designed compound was further synthesized and their in vitro antimalarial activity was tested to prove the accuracy of QSAR calculation.

Data Set
1] .This data set was divided into two parts i.e. internal evaluation (training set) and external evaluation (test set).The test set compounds were selected manually in order to consider the structural diversity and a wide range of antimalarial activity.The in vitro antimalarial activities expressed as the inhibition concentration [IC 50 (µg/mL)] values were converted to the logarithmic pIC 50 = log IC 50 and used as dependent variables in QSAR analysis.

Instrumentation
In this study a PC with Intel® (TM)2 Quad CPU Q8200 @2.33GHz was running under a Windows XP Professional operating system.All quantum mechanical calculation was executed using Gaussian 09 software.Correlation of QSAR models was evaluated by multiple linear regression analysis using SPSS statistics 23.0.Docking studies were performed with the cDOCKER protocol under the receptor-ligand interaction section in Discovery Studio 3.1 (Accelrys, Inc., San Diego, CA, USA).Other molecular modeling software was used throughout this study including CHIMERA 1.9 and ChemOffice®.
The melting point of the synthesized compounds was determined using Electrothermal 9100 with temperature gradient 10°C/min.ESI-HRMS spectra were recorded on a Bruker micrOTOF Mass Spectrometer.Meanwhile, 1 H and 13 C-NMR spectra were recorded on a JEOL 500 MHz spectrometer with TMS as an internal standard.All reagents were

QSAR Analysis
In order to find the best calculation method, one of the xanthone compounds (24) was calculated for 1 H-NMR chemical shift using the semi-empirical method of Austin Model 1 (AM1), Parameterized Model (PM3 and PM6), and Gaussian 09 software.The chemical shift data were then compared to the data of 1 H-NMR from the experiment.AM1, PM3 or PM6 calculation methods which gave the closest values to the experimental data was selected as a method of study throughout the investigation.

Statistical analysis
The predominant descriptors which affecting the antimalarial activity of the prenylated xanthone analogues were selected according to the correlation analysis by SPSS 23.0 statistical software.In this process, each electronic descriptor was appointed as independent variables and pIC 50 as the dependent variable.Moreover, this regression method estimates the values of the regression coefficients by applying least square curve fitting method.The model for QSAR calculation was chosen based on some statistical parameters such as r 2 , standard estimation of error (SEE), F-ratio between the variance of prediction and observation activity, and PRESS value (predictive residual sum of square), where: PRESS = S (predicted value-observed value) 2,22 in criteria r 2 > 0.6, 23 ; SEE < 0.3, 24 ; Fcal/Ftab ≥ 1. 25 The best-selected model obtained from the previous step was used to predict the log IC 50 of the test set and the model was validated by criteria r 2 prediction > 0.5. 26neral procedure for the synthesis of compounds 3,6-dihydroxy-9H-xanthen-9-one (26) and 3,4,6-trihydroxy-9H-xanthen-9-one (27)   A mixture of 2,4-dihydroxybenzoic acid (5 mmol) and resorcinol or pyrogallol (5 mmol) was stirred in Eaton's acid (5 ml) and heated to 80 °C for 3 h.After the completion of reaction (monitored by TLC), the product was poured into an ice water and the precipitate formed was filtered, washed with water and 5% NaHCO 3 .The dried product was purified by silica gel column chromatography with n-hexane/ethyl acetate (gradient 0-30% ethyl acetate) to afford compound 26 and 27.

Molecular Docking Studies
The protein crystal structure of the inhibitorbound DHFR was retrieved from Brookhaven Protein Data Bank (PDB codes: 1J3I).The protein was pretreated before the docking process.Hydrogen atoms were added to the protein structure, and all ionizable residues were set at their default protonation of pH 7.2 while the ligands were prepared and minimized.During the docking process, the receptor was held rigid while the ligands were allowed to flex during the refinement.A number of polar or nonpolar receptor hotspots for conformer matching starting were set at 500 with the docking tolerance of 0.25 Å.The conformations ligands generated from the process was set to 500 within the threshold of relative energy of 20 kcal/mol.

In-vitro Antimalarial activity Assay
An in-vitro antimalarial assay was conducted against Plasmodium falciparum 3D7 strain which  27 For this assay, the compounds were dissolved in DMSO and prepared in a series of concentration, i.e. 100, 10, 1, 0.1 and 0.01 µg/mL in RPMI-1640 media.Into the sample, Parasitemia and hematocrit, ±1% and 5% were added respectively.The culture was incubated for 48 hours at 37 °C, treated with 20% Giemsa dyes and made it as thin blood layer.After that, the percentage of parasitemia and also the inhibition percentage of P. falciparum growth were determined by calculating the number of the infected erythrocytes for every 1000 erythrocytes.
Based on the inhibition percentage data, analysis of the correlation between concentrations of the compound with the inhibition percentage was conducted using probit log analysis to determine the IC 50 .

Validation method
Calculation of 1 H-NMR chemical shift of compound 24 using AM1, PM3, and PM6 method showed that AM1 method was the best method, which gives the closest result to the experimental data.This result is clearly shown by the PRESS value of AM1 method (2.13) which is smaller than PM3 (3.53) and PM6 (3.57) as presented in Table 2. Therefore, AM1 method was used to calculate the descriptors of each compound.

Selection of the Best Model
Statistical multi-linear regression calculation of the descriptors using SPPS give 5 QSAR models as shown in Table 3.It shows that all models depict the linear correlation between biological activity and descriptors as can be seen by the r-value of each model.Basically, the best model is determined by the number of variable and statistical parameters which belong to that model.Validation Model QSAR equation [1] and [2] were used to predict antimalarial activity (log pIC 50 ) of 5 test compounds in order to find the best model in predicting the antimalarial activity of xanthone derivatives (Table 4).The predicted antimalarial activity of 5 test set generated from model 4 and 5 was compared and then plotted with the experimental data by linear regression calculation to see the correlation for each equation, as shown in Fig. 1.
Table 4 shows that PRESS value of model 5 (0.821) is smaller than model 4 (0.962), which mean model 5 give a closer result to experimental data compared to model 4.This result is supported by Fig. 1 where model 4 has r 2 = 0.887 while model 5 has r 2 = 0.963.These numbers mean that there is a significant correlation between an independent variable (in equation 2) and antimalarial activity of xanthone compound.As for model 5, the r 2 value of 0.963 means the changing of independent variable (atomic charges of qO8, qC9, qC12, qC14 and qO15) could influence 96.3% of antimalarial activity (log IC 50 ) of xanthone derivate.Therefore, it can be concluded that model 5 is the best model to design new xanthone compounds with better antimalarial activity.
Furthermore, by introducing the descriptor of qC9, qC14, and qO15 as an active center of antimalarial in xanthone derivatives, it revealed that conjugated double bond with carbonyl group was an important feature on the antimalarial activity.This result showed the similarity with the reported QSAR study conducted et al for chalcone derivatives. 28

Design of New Antimalarial
Model 5 as the best QSAR model was used as a guidance to predict the antimalarial activity in the rational design of the new antimalarial compound from xanthone derivatives.R substituents were introduced to the new molecule as a descriptor which will influence the antimalarial activity.From equation 2, it can be shown that in order to get the The designed new antimalarial compounds of xanthone derivatives based on the best QSAR model were listed in Table 5.Indeed, this study shows that R substituents variation gives a difference antimalarial activity.It can be seen in Table 5 that there is a very significant change of the predicted antimalarial activity occurred when substituent was changed to SO 3 H and NO 2 at C10 and C13.In addition, removing of SO 3 H from the compound gives significant influence in decreasing the antimalarial activity.This result is similar to the previously reported study, 28 that substitution of SO 3 H functional group is able to provide the best antimalarial activity towards chalcone compound.Meanwhile, electron donating group OH is a substituent that also gives the significant influence which leads to the better antimalarial activity.
Synthesis and an in-vitro antimalarial activity assay of compound 26 and 27 against chloroquinesensitive 3D7 strain have been successfully conducted.The experimental antimalarial activity of 26 and 27 are close to the results from the QSAR analysis (Table 6).This result proves the accuracy of the generated QSAR equation to predict the antimalarial activity of xanthone derivatives.Also, the antimalarial activity of compound 26 and 27 could be categorized as a good antimalarial although their IC 50 are slightly higher than the chloroquine as a control.
The active synthesized compound 26 and 27 as antimalarial was docked to the active site of the P. falciparum DHFR crystal structure to confirm their mechanism as an antimalarial drug.Thus, the protein crystal structure of DHFR (1J3I: 2.33Å) was retrieved from Brookhaven Protein Data Bank.P. falciparum dihydrofolate reductases-thymidylate synthase (PfDHFR-TS) is an important target of antimalarial drugs. 29The inhibition of this enzyme could prevent the dTMP production and DNA synthesis since it involved in the catalysis sequential reactions in the thymidylate cycle. 30The docking studies of compounds 26 and 27 displayed favorable binding affinity towards DHFR with -cDOCKER energy shown in Table 7 and the docking interaction showed in Figure 2.
Docking studies revealed the binding site of 26 and 27 form interactions with Ala16, Ser108, Asp54, Met55, and Phe58 amino acid which is the

Fig. 1 :
Fig. 1: Plot of experimental result versus predicted antimalarial IC 50 value of 5 test set compounds by model 4 and 5

Fig. 2 :
Fig. 2: Predicted binding mode from docking simulation of 26 and 27 into the active site of P. falciparum DHFR-TS (PDB ID: 1J3I).The coloring atom for the compound is in order as follows: carbons in gray, oxygen in red, nitrogen in blue, and hydrogen in white.The green line indicates hydrogen-bonding interaction with distance ascribed in angstroms, Å.

Table 1 : Xanthone derivatives compound used as data set for QSAR analysis
a) Test set purchased from Aldrich, Acros, and Merck and were used without further purification.All the solvents used in the synthesis were analar and synthesis grade.The solvents used in spectroscopic measurements were spectroscopic grade.