Modeling of Quinoacridinium Derivatives as Antitumor Agents using a QSAR analysis

Email: ruslinhadanu@gmail.com A QSAR analysis has been performed on a compound series of 111quinoacridinium derivatives as internal test compounds, and compounds of 12-15quinoacridinium derivatives as external test compounds. The electronic descriptors used in this study were atomic net charge (q), dipole moment (μ), ELUMO, EHOMO, polarizability (α), and Log P. They were calculated through Hyper Chem for Windows 8.0 software using semi-empirical PM3 method. The antitumor activity (IC50) of quinoacridinium derivative compounds was obtained from literature. Furthermore, the model of QSAR equation was analyzed through RML method which produced the best QSAR equation model: Log IC50 = -13.010+15.338(qC3) 4.31(qC4) 155.308(qC9) + 33.626(qC11) + 26.626(qC12) + 24.631(qC14) 0.228(μ) 0.621(ELUMO) 0.066(α) + 0.233(Log P). The model of QSAR equation has a correlation coefficient n =11, (r) = 1.00, (r2) =1.00, SE =0, and PRESS = 0.003. Among 28 compounds of quinoacridinium derivative which were designed, only 15 compounds, namely 16, 19-20, 22-28, 30-32, 39, and 40 compounds, have been recommended to be synthesized in the laboratory.


INTRODUCTION
Tumors and cancers still become dangerous health problem in the world because of their high morbidity and mortality.In 2012, there were 14.1 million new cancer cases, with 8.2 million cancer deaths (Torre et al., 2015).In 2000, productivity costs which was lost due to cancer deaths were 115.8 billion US dollars, and by 2020, these costs are predicted to reach 147.8 billion US dollars (Bradley et al., 2008).The main problems in a cancer therapy are low selectivity and high toxicity.Therefore, it is necessary to develop antitumor drugs with high selectivity as well a slow toxicity, so that the therapy will be more effective.One of the antitumor compounds that has been widely studied and continuously developed by previous researchers is the derivatives of acridinium compounds.
A recent study on acridinium derivatives was conducted in 2008 (Cheng et al., 2008).It was a synthesis on a series of salt-derivative compound with 8, 13-dimethylquino[4,3,2-kl]acridinium as an antitumor.Derivatives of the acridinium compounds used as an antitumor have been studied (Cooksonet et al., 2005).Research on the derivatives of acridine compounds has been done by various researchers since1984.They isolated acridine compounds from plant species, Lactarius necator (Agaricales) (Fugmann et al., 1984).Furthermore, after 13 years later, the ring of acridine compounds was successfully synthesized and antitumor activity was tested (Hagan et al., 1997).After that in 1998, several researchers conducted a study to convert 9-azidoacridine to 7H-pyrido [4,3,2-kl]acridine compound and succeeded in changing the pyridine ring to 7Hpyrido[4,3,acridine compound to increase the antitumor derivative activity of acridine compounds (Hagan et al., 1998;Julino and Stevens, 1998).In 2000, the ring of acridine compound was developed into tetra-, penta-, and hexacyclic rings in a hetero aromatic system through a cyclical process of9-anilinoacridineto enhance antitumor activity of derivatives of acridine compounds which were synthesized in the previous years (Stanslaset al., 2000;Elliset al., 2001).

G a l l e y P r o o f
Research on the derivatives of acridine compounds continues being developed by researchers.Some of them are the synthesis of acridine polycyclic compounds, i.e. 8, 13-diethyl-6-methylquino[4,3,2-kl]acridiniumiodide and 3, 11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl] acridiniummethosulfate (Missailidiset al., 2002;Leonetti, 2004).The synthesis of the acrylic polycyclic derivative was intended to increase the antitumor activity and the solubility of the compound (Missailidis et al., 2002;Leonetti, 2004).Efforts to obtain a more active antitumor through the derivatives of acridine compounds were continued by researchers through the synthesis of more polar and complex compounds using the alkylation method of polycyclic acridine compounds (Cookson et al., 2005).
The development of antitumor compounds from polycyclic acridine has been carried out (Cheng et al., 2008).It is a salt compound of 8, 13dimethylquino[4,3,2-kl]acridinium and has an antitumor activity (IC50) around 0.21 to 2.00μM.The salt compound which has the highest activity is derivatives of 8,13-dimethylquino[4,3,2kl]acridinium which is bound up both with anionic iodine and an ester functional group on C2 atom of such cyclic compound ring.Antitumor activity of salt derivatives 8, 13-dimethylquino[4,3,2kl]acridinium compound is still low when it is compared with antitumor compounds that have been circulating in the market (Cheng et al., 2008).
Based on the results of the research above, the efforts to develop antitumor compounds from other compounds have also been developed recently, such as derivatives of Au(III)dithiocarbamate, cisplatin, benzoisoselenazolon, and gallium-pyridine complexes known as metal-based drugswith a very high anticancer activity (Schmitt and Dou, 2013;Florea and Büsselberg, 2011;Luo et al., 2012).However, the development of an anticancer compound from heavy metals causes many metabolic risks to the human body.Other studies on anticancer and antitumor have also been done by many researchers (Miladiyah et al., 2016;Hosny et al., 2012;Alamet al., 2016;Reddy et al., 2012;Bladt et al., 2013;Heliawati et al., 2015;Noolvi and Patel, 2013;Deep et al., 2016;Shelton et al., 2016;Tripodiet al., 2012;Su et al., 2011) (Miladiyah et al., 2016;Hosny et al., 2012;Alam et al., 2016;Reddy at al., 2012;Bladt et al., 2013;Heliawati et al., 2015;Noolvi and Patel, 2013;Deep et al., 2016;Shelton et al., 2016;Tripodi et al., 2012;Su et al., 2011).The antitumor activities of all compounds afore mentioned before are still low when they are compared with antitumor compounds that have been circulating in the market.All compounds which were isolated and modified generally have a functional group of primary amine (-NH2).Based on the results of the research above, to design the structure of antitumor or anticancer molecules, it is necessary to make a model of anticancer molecules whose structures are cyclic compounds and have functional groups of amine .In this study it was attempted to synthesize quinoacridinium derivative compounds that have an amine functional group, so that they are antitumor compounds that meet this criterion.
Based on the explanation above, the effort to find an antitumor compound from the 8, 13dimethylquino[4,3,2-kl]acridinium which has a functional group -NH2 is still very potential to be a candidate for antitumor drugs which are expected to have activity high and friendly to the body.Efforts to develop an antitumor drug from 8, 13dimethylquino[4,3,2-kl]acridinium derivatives need to be done through a computational chemistry approach with a Quantitative Structure-Activity Relationship (QSAR) analysis to obtain more active and body-friendly compounds.
The QSAR analysis is one of the latest methods in the phases of developing new drugs.The results of QSAR analysis are generally used as a guide to design new drugs theoretically.In this study, the results of QSAR analysis are used as guidance in designing compounds of 8, 13dimethylquino[4,3,2-kl]acridinium derivatives that are potentially to be antitumor and have never been previously synthesized.The QSAR analysis approach as an effort to design drug compounds is very important because it minimizes the use of chemicals and energy.

G a l l e y P r o o f
It also saves time because it can avoid trial and error experiments in laboratory.However, it can still provide a relatively high level of confidence (Hadanu and Syamsudin, 2013).The QSAR analysis aims to find an empirically consistent relationship between the molecular properties and the biological activity of a series of homologous structural compounds of the drug.The QSAR study began to grow rapidly after1960.It was pioneered by Corwin Hansch who connected chemical structures with drug biology activities through common chemical-physical properties such as fat solubility, ionization degree, and molecular size.Later on in the last few decades, it was developed more intensively into a quantitative relationship between biological activity and various chemicalphysical parameters such as net atomic charge, ELUMO-EHOMO, solubility in fat (Polaris abilities), solubility in water and alcohol (Log P), molecular size, hydrophobic parameters, electronics and sterics in a series of molecules (Hadanu and Syamsudin, 2013).The relationship between physical as well as chemical properties and biological activity is proposed by (Ferguson et al., 1997) with an equation that can be used to relate some activity data with the following parameters: Log BR = f(P1, P2, P3)……………………………………….( 1) BR (Biological Response) is a biological activity or biological response as an algebraic function of 3 parameters P1, P2, and P3 which is the nature of the structure under investigation.The development of QSAR analysis in the next period uses the net charge of the atom as an estimator and expressed by: Pi is the fitting parameter for i th atom, and q i is the net charge of the atoms and other parameters for the i th atom and C is constant.In the development of subsequent research, QSAR study is very helpful in the search for new drugs with greater activity, higher selectivity, toxicity or minor side effects, and greater comfort.In addition, using the QSAR equation model can save more money because to get a new drug with high activity, the experimental factor can be minimized as much as possible (Hadanu et al., 2015).Some attempts at cancer treatment have been performed in various ways such as surgery, radiation, anticancer drug treatment or chemotherapy.However, these efforts have not achieved satisfying results; even the effects of surgical failure can cause cancer to spread to other parts of the body with severe conditions (Nugraha et al., 2018).This encourages the development of new medicine from8, 13-dimethylquino[4,3,2kl]acridinium compound which is more active, friendly to the body, and expectedly to have a good therapeutic effect.

MATERIAL AND METHODS
The materials used in this study were quinoacridinium derivative compounds that have been synthesized by Cheng et al., (2008).Inhibition Concentration (IC50) was used as the dependent variable (Table I and II) (Cheng et al., 2008).

Instrumentation
In this study, the tools used for QSAR test were computer hardware devices namely a Sony Vaio Laptop with Intel® Dual Core Processor 2.20 GHz; 1 GHz RAM, and HDD 250 GB.Meanwhile, the software used in this study is HyperChem 8.0 for Windowsforoptimizationpurposesof3Dstructurea ndgeometryoptimizationofthechemical structure of tested compounds (compounds 1-11), external test compounds (compounds 12-15) and the structure of quinoacridinium derivative compounds (compound 16-43).For statistical analysis to obtain the QSAR equation, SPSS 19.0 for Windows was used.

Calculating the descriptors
Internal fitting compounds (Table I),external fitting compounds (Table II), and modeling compounds (Table V) were constructed respectively in three-dimensional (3D) structures with the HyperChem 8.0 for Windows programming package.Furthermore, the geometric structures of all fitting compounds and model compounds were optimized to obtain more stable structural conformation using the semiempirical PM3 method.This method is chosen because semi-empirical methods can be performed descript tor calculations quickly and accurately.The semi-empirical method is faster than the ab-initio method although it is less accurate and the semi-empirical method is more accurate than the AM1 method even though it is slower than the AM1 method.In addition, semi-empirical methods are methods that use experimental and theoretical data sets.When viewed with the molecular structure used, semi-empirical methods are suitable, because the quinoacridinium molecule have a medium molecular weight and functional groups -NH2, -NHR, and -NR2.

G a l l e y P r o o f
The structural optimization was performed by using Polak-Ribiere algorithm optimization method with RMS value = 0.001 kcal/(Å.mol),which was recorded through the single point menu.Via the data fromthe single point menu, the electronic parameters such as the net atomic charge, the dipole moment (μ), the polarization (α), and the Log P were obtained.Meanwhile, the ELUMO and the EHOMO were obtained from compute and orbital menus which were presented (Table III and  IV).The selection of descriptor type was adjusted to the type of descriptor used by Motta et al., (2006) in conducting QSAR analysis.The structure of the quinoacridinium derivative compounds as the study material was calculated by involving an ion counter of salt.
Table I.Chemical structure and activity data of antitumor compounds of quinoacridinium derivatives obtained from Cheng et al., (2008 Cheng et al., (2008).

QSAR equation analysis by using a linear regression method
The dependent variable in this study was the antitumor activity with IC50value based on experiment (Table I), while the independent variables used were net atomic charges, dipole moment (μ), ELUMO, EHOMO, polarizability (α), and Log P (Tables III and IV).All variables were analyzed using enter method through MLR to find out which sequence of independent variables influenced the antitumor activity value of quinoacridinium derivative compounds.The result generated QSAR equations as well as statistical parameters such as r, r 2 , SE, and PRESS values.In addition to the statistical parameters, the calculation result also obtained the constant value and the coefficient value of each independent variable involved in the equation result.The obtained coefficient value was used to calculate the theoretical antitumor activity (IC50 theoretical value) toward quinoacridinium derivative compounds.Furthermore, after finding out the square difference betweenthe IC50 experimental value andthe IC50theoretical value, the PRESS value can be calculated to know the quality and prediction ability of the best equation of the QSAR model.

Design of new antitumor molecules
The design of new antitumor drug molecules in this study was aimed to discover novel compounds of quinoacridinium derivatives which have higher antitumor activity than previously synthesized compounds.The molecular design was performed by varying the type and position of substituent or functional group positions in the main framework structure of quinoacridinium derivative compounds.The substituent position was focused on the active center area by considering the feasibility of synthesis in the laboratory.The substituent or functional group is the dominantly responsible atoms of the antitumor activity of the quinoacridinium derivatives.After finding out the quinoacridinium molecular sequence series, the next step is calculating the descriptor of the new compound of modeling design (Table VI and VII) by using the semi-empirical method of PM3 using HyperChem 8.0 for Windows.Based on the best QSAR equations, we can calculate the theoretical antitumor activity of the design compounds (Table VI and VII).

RESULT AND DISCUSSION
The stages of quantitative relationship analysis of structures carried out in this study were (a) determining series of quinoacridinium compounds which had IC50 values based on experiment in the laboratory Cheng et al., (2008); (b) optimizing the basic structural framework of the most stable quinoacridinium derivative as the initial compound in the process of optimizing the test compound for further analysis; (c) determining descriptor (independent variable); (d) calculating descriptor through optimization structure of tested compounds (optimization of derivative structure of tested compound) (Table I and II); (e) performing correlation analysis between variables through bivariate correlation method; (f) performing multi linear regression analysis to obtain the model of QSAR equation; (g) determining the best QSAR equation model (Hadanu and Syamsudin, 2013;Hadanu et al., 2015) and (h) designing a new compound of quinoacridinium derivatives based on the best QSAR equation model.

G a l l e y P r o o f
The quinoacridinium derivative compounds as the materials in this study has the following requirements: (1) the basic framework structure of the homologous compound should be possessed by all fittings compounds, external test compounds, and the modeled compound; (2) the fitting compound; and (3) the external test compound should have IC50value which is experimented in a laboratory.The optimization of the structures of all fitting compounds, external test compounds, and the modeled compounds was carried out by the same method to obtain the most stable structural compound with the lowest energy profile.When all of compound structures are at the lowest profile energy, they are in the most stable condition.In that condition, the descriptor (independent variable) required in the next stage of QSAR analysis can be obtained.
To test the accuracy of the model of the obtained QSAR equation, it is necessary to calculate the IC50 prediction value of the internal test compound (Table I).The proof of the accuracy of the model of QSAR equation can be seen on the graph of Log IC50value of prediction with an IC50 value of the experimental internal test (compounds 1-11) (Figure 1).The graph shows the relationship between the Log IC50 prediction value of the internal test compound and Log IC50 experiment with obtained valuer 2 = 0.993.The value of r 2 is close to 1, which indicates that the internal test model of QSAR equation has a very high level of trust.
To test the validity of the model of QSAR equation, a validity test has been performed by using external test compounds (compounds 12-15).The external test compound is a quinoacridinium compound derivative which has been known as IC50 experimental value, but it is not included in the calculation process in determining QSAR equation model.This is intended to validate the QSAR model more accurately since it is validated with internal test compounds and external test compounds.The IC50 value of external compound test (Table III).
The graphic of correlation between Log IC50 calculated and Log IC50 experiments value has r 2 =0.9701 (Figure 2).It shows that the QSAR equation model obtained in this research is the best QSAR equation for determining the IC50 value of compound derivative quinoacridinium.The high value of r 2 (close to 1) displayed in the correlation graph between Log IC50 calculated from the external test compound and the Log IC50 value of the experiments reinforces the QSAR model as an equation with high validity.
Based on the value of r 2 = 0.9701 obtained from the correlation between Log IC50of calculated from external test compound and Log IC50 experiment, it proves that the relationship between calculated IC50 value of external test compound and Log IC50 value of experiment is very strong.Thus, it can be concluded that the model of QSAR equation obtained by using MLR analysis is very significant to determine the value of Log IC50 compounds models of quinoacridinium derivatives.

Design of new antitumor quinoacridinium derivative compounds
The molecular design of the quinoacridinium derivative remains concerned with the active side of the homologous and atomic frameworks or the functional groups bound to the basic framework structure of the compound.The differences of atoms or functional groups bound to the basic frameworks of homologous compounds of quinoacridinium derivatives may cause different charges of atoms and differences in physical and chemical properties.In addition, it also causes differences in antitumor activity of such compounds (Hadanu et al., 2015).The obvious that compounds which have different structures can produce different antitumor activity (Table I Based on this fact, it can be concluded that different structures have electronic properties and molecular properties (different net charge of atoms and other descriptors).Compounds that have different electronic and molecular properties absolutely produce different antitumor activity.
Based on the above explanation and QSAR model equations (3), atomic charge values of qC3, qC4, qC9, qC11, qC12, qC14, μ, ELUMO, α, and Log P G a l l e y P r o o f for respective designed compounds are variables influencing the value of antitumor activity (IC50) of quinoacridinium derivatives.In designing the molecule, it is necessary to consider the attachment of the atom or functional groups to the main framework of quinoacridinium derivatives which can cause the change of atomic charge value of qC3, qC4, qC9, qC11, qC12, qC14, μ, ELUMO, α, and Log P, so that it can cause different predictions ofIC50value (Table V).The calculation of atomic charges and other descriptors obtained from each of the new compounds is incorporated into the QSAR model of equation (3) in order to obtain theoretical IC50 values of the novel compound derived from the modeling design.Compounds that have a small IC50 value are antitumor compounds that have the highest antitumor activity.The smaller IC50value of a quinoacridinium derivative compound is, the higher the chances of the compound as an antitumor drug is and it may be proposed for synthesis in the laboratory.
The calculated IC50value of new molecules from the quinoacridinium derivatives design (Table VI and VII).Some compounds from quinoacridinium (Table VI and VII) derivative design are proposed for synthesis, in which they have an IC50 value smaller than IC50 of fifteen quinoacridinium derivatives (Tables I and II).Thus the quinoacridinium derivatives compounds derived from the recommended modeling design are: compounds 16 (IC50=0.000002μM),19 (IC50=0.125555μM),20 (IC50=0.173970μM),22 (IC50=0.127722μM),23 (IC50=0.091970μM)24 (IC50=0.000118μM),25 (IC50=0.000398μM),26 (IC50=0.018086μM),27 (IC50=0.055728μM)28 (IC50=0.122898μM),30 (IC50=0.034668μM) 31 (IC50=0.000004μM),32 (IC50=0.017990μM),39 (IC50=0.040617μM),and 40 (IC50=0.156537μM).Theoretically, they can be proposed for synthesis in the laboratory.Certainly, in the synthesis process in the laboratory, it is prioritized to synthesize compounds that have smaller IC50 values and compounds that have the easy synthesis pathways in the laboratory, as well as the availability of chemicals (Hadanu and Syamsudin, 2013;Hadanu et al., 2015).The theoretical IC50 value of the quinoacridinium derivatives is less than the IC50 value determined by the National Cancer Institute (NCI) for the compounds extracted from the natural material.According to the National Cancer Institute (NCI), an extract is considered to have an active anticancer activity if its IC50 value is <30 μg/mL, moderate active anticancer activity ifits IC50value is ≥ 30 μg/mL, IC50<100 μg/mL, and inactive anticancer activity if its IC50 value is >100 μg/mL. 2  Based on the structure model of the QSAR analysis recommended for synthesis in the laboratory, it can be concluded that the quinoacridinium derivatives which have high and potential antitumor activities are quinoacridinium derivatives which have functional groups as follows: -Cl is bound toC3 and C11 atoms; the -CH3 function group is bound to C6, C8, and C13 atoms, anion H3COSO3 -;-OCH3 functional group is bound to C6 atom, anion iodide (I -); functional group of-CH=CHCON(CH2CH2)2Ois bound to C6 and C10 atoms; the functional group of-(CH2)3OCOCH3is bound to C3, C6, and C10;-C2H5 functional group is bound to C13 atom, anion H5C2OSO3 -.Those all are bound to the main framework of quinoacridinium compounds.Nearly all compounds modeled according to Lipinski's Rule are quinoacridinium derivative compounds having fewer than 5

Figure 1 .
Figure 1.Correlation between Log IC50 calculated from the internal test compound and Log IC50 experiment

Figure 2 .
Figure 2. Correlation between Log IC50 of the external test compound and Log IC50 experiment G a l l e y P r o o f

Table II .
The chemical structure of external standard compounds of quinoacridinium derivatives from

Table III .
Log IC50 experiments and Log IC50 calculated of external test compounds

Table IV .
Descriptors/independent variables used for QSAR analysis of antitumor compounds of quinoacridinium derivatives calculated by the semi-empirical PM3 method

Table VII .
Predicted Log IC50 calculated using the best QSAR model

Table V .
Descriptors/independent of external standard for QSAR analysis of antitumor compounds of quinoacridinium derivatives calculated by the semi-empirical PM3 method hydrogen bond donors (nitrogen or oxygen atoms with one or more hydrogen atoms), less than 10 hydrogen bond acceptors (nitrogen or oxygen atoms), and having an octanol-water partition coefficient (Log P) is less than 5. 40.