Anticancer Activities and QSAR Study of Novel Agents with a Chemical Profile of Benzimidazolyl-Retrochalcone

The present pharmacochemical and modelling work focused on a benzimidazolyl-chalcone series. These previously synthesized compounds were evaluated in vitro for their anticancer activities against a panel of seven human cancer cell lines and normal fibroblasts. Among the new benzimidazole-supported chalcones, nine (9) compounds (compounds 1 4, 6 8 and compounds 10 and 11) showed promising anticancer activities with IC50s ranging from 0.83 to 2.58 μM. Compounds 2 and 6 with IC50s of 0.83 and 0.86 μM, respectively, were shown to be potent inhibitors of HCT-116 colon cancer cell proliferation. It was therefore necessary, for a development of this new series of chalcones, to establish through a QSAR study, their quantum descriptors according to the DFT calculation method and following the B3LYP/6-31+G (d,p) theory. These descriptive and predictive studies focused on the colon HCT 116 cell line which was found to be more sensitive to the anticancer action of our benzimidazolyl-retrochalcones. QSAR study showed that the electronic energy (Eelec), lipophilicity (logP), chemical softness (S) and chemical hardness (η) of benzimidazolyl-retrochalcones play an important role in inhibiting cancer cell proliferation.


Introduction
Cancer is characterized by a wide range of conditions involving abnormally large and unregulated cell proliferation in normal organism tissue. This proliferation can affect any part of the organism [1]. Cancer is currently a major cause of death worldwide. Indeed, it is the second leading cause of death, with 8.8 million deaths in 2015 according to the WHO [1] [2] [3]. Moreover, the growing economic impact of cancer is considerable. In 2010, the total annual cost of the disease was estimated to be approximately $1.16 trillion (US $) [4]. Among the main types of cancer that are fatal are lung, liver and colorectal cancer [1]. Chemotherapy treatment of cancer remains one of the preferred strategies for cancer management. Unfortunately, current cancer drugs are either ineffective for some people or too expensive for many populations.
It is in this perspective of contributing to the accessibility of new high-performance anti-cancer drugs that we have initiated this pharmacochemical and particularly a molecular modelling research [5] [6]. To this end, we have focused on 1,3-diphenylpropenones (chalcones) and the benzimidazole nucleus as pharmacophore and heterocyclic carrier, respectively. Indeed, taking into account their multiple intrinsic biological properties, it was normal to imagine that the combination of these chemical entities according to the concept of hybrid molecule, could generate new molecules likely to present new pharmacological activities, especially interesting anticancer ones.
Also, our benzimidazolyl-chalcones, which are actually retrochalcones, have been obtained in one of our previous work [24], by combining 1H-benzimidazo-2-yl-carbaldehyde derivatives with various acetophenones and analogues. These novel retrochalcones were evaluated in vitro for their antitumor activity only on the NCI-H727 lung cancer cell line. In addition, their cytotoxicity was established in normal human skin fibroblast cells [24]. Taking into account our previous results on benzimidazolyl-chalcone series, in particular their anthelmintic and antifungal activities [25]- [30], as well as the first promising anticancer results in this retrochalcone series [24].
We propose in the present work, to extend their screening to six (06) other human cancer cell lines. The other objective of this work was to establish, by a QSAR study, the quantum descriptors according to the DFT calculation method and the B3LYP/6-31+G (d, p) theory and from the cancer line whose proliferation was most inhibited by our compounds. The aim of such a study was to lay the foundations for the development of new anticancer agents with a chemical profile of benzimidazolyl-retrochalcone.

Chemistry
We have recently published the synthesis and physico-chemical characteristics of the new series of benzimidazolyl-retrochalcone derivatives and their anticancer activity against human lung cancer cell line NCI-H727 [24]. The chemicalstructures of 13 of the benzimidazolyl-retrochalcone derivatives are reported in All culture media were added with 10% of FBS, 1% of penicillin-streptomycin and 2 mM glutamine and were incubated at 37˚C in a 5% CO 2 atmosphere. Twenty-four hours after seeding, cells were exposed to chemicals. After 48 h of treatment, cells were washed in PBS and fixed in cooled 90% ethanol/5% acetic acid for 20 minutes. Then, the nuclei were stained with Hoechst 33,342 (B2261 Sigma).

Cytotoxic Assay
Image acquisition and analysis were performed using a Cellomics Array Scan VTI/HCS Reader (Thermo Scientific). The survival percentages were calculated as the percentage of cell number after compound treatment over cell number after DMSO treatment.

Materials and Methods of Calculation
The nine (09)

Level of Calculation
The calculation to establish the relation between the biological activities values of the studied molecules and their molecular structures were obtained by quantum chemistry calculations using Gaussian software 09 [33]. DFT [43]. The B3LYP/6-31+G (d, p) theory level was used to determine the molecular descriptors. The modelling was done using the multilinear regression method implemented in Excel [44] and XLSTAT [45].  [48]. Lipophilicity is an important measure for the identification of the similarity with a drug [49]. All these descriptors are determined from optimized molecules follow by frequency calculation. The calculation of the partial correlation coefficient between the descriptors studied is less than 0.70 (a ij < 0.70); which means that these different descriptors are independent of each other [50].

Estimation of the Predictive Ability of a QSAR Model
The quality of a model is determined according to certain criteria such as the coefficient of determination R 2 , the standard deviation S, the correlation coeffi- The Fischer coefficient F is also used to express the level of statistical significance of the model, that is to say the quality of the choice of the descriptors constituting the model.

Pharmacochemical Section
All retrochalcone derivatives (compound 1 -13) were evaluated for their anti-   For the retrochalcones that showed promising anticancer activities, we can note that they carry on their benzene ring (Ar) either a hydroxyl group or a methoxyl group in isomeric position 2 or 3 (compounds 2, 3 and 4). When this benzene ring is unsubstituted (compound 1), the activity obtained is moderate against all tumour cell lines with IC 50 values ranging from 1.48 to 7.77 µM. Also, the replacement of benzene ring by furanyl (compound 6) and benzimidazolyl (compound 7) heterocycles type showed better activities than retrochalcone 1.
However, compound 5 substituted with nitro group, led to a significant decline in anticancer activities compared to compound 1.

Training and Validation Set
The results of training set of nine (09) benzimidazolyl-retrochalcones and the four (04) molecules of the validation set are reported in Table 3. As for Table 4, it presents the values of partial correlation coefficients a ij of the descriptors. The partial correlation a ij between the descriptors is less than 0.70. This shows the independence of the descriptors used in the model.

QSAR Model Validation
The   The statistical indicators of multilinear regression are shown in Table 5.
The electronic energy (E elec ) and the lipophilicity (logP) have a positive sign in the model, which suggest that increased activity can be achieved by increasing the electronic energy and the lipophilicity.
The chemical hardness (η) and the chemical softness (S) have a negative sign in the model, which suggest that increased activity can be achieved by decreasing the chemical hardness and softness. A good model is qualified by its high value of the coefficient of Fischer F = 170.803, the cross-validation coefficient 2 CV Q which is 0.954. The values of log(1/C) theo /log(1/C) exp ratio of external validation set are shown in Table 6.
All values of log(1/C) theo /log(1/C) exp tend to 1. This indicates the good correlation between the theoretical and experimental anticancer activity of the molecules studied. The regression line between the experimental and theoretical anticancer activity of training set (blue dots) and the test set (red dots) is illustrated in Figure 1.

Analysis of the Contribution of Descriptors in the Model
The relative contribution of the descriptors in the prediction of anticancer activity of the molecules is shown in Figure 2.
The chemical hardness (η) and the chemical softness (S) have a wide contribution. The lipophilicity presents a weak contribution.

Conclusion
In this work, we evaluated a series of benzimidazolyl-retrochalcone derivatives for their in vitro cytotoxic activity against a panel of seven (7)