INDOLE DERIVATIVES: DESIGN, SYNTHESIS, IN-VITRO BIOLOGICAL EVALUATION AND MOLECULAR DOCKING STUDY AS ANTICANCER AGENTS

New hetero annulated indoles were synthesized and structurally characterized by spectral means. In order to understand the nature of interactions of these molecules, we carried out molecular docking studies using the protein kinase CK2 inhibitors. The docking results provided some useful information for the futuredesign of more potent inhibitors. The in vitro cytotoxicity was evaluated for all the new compounds by MTT assay against HeLa and compared with the standard drug ellipticine. All the compounds showed moderate to potent activity against the cell lines. The preliminary structure–activity relationships were carried out.


INTRODUCTION
Cancer continues to be one of the major health problems worldwide and one of the leading causes of death despite the advances that have led to the development of new therapies. So, there is a continuing need for designing and developing new chemotherapeutic agents for cancer treatment.
The indole ring is the most ubiquitous heterocyclic substructure in nature. Owing to its great diversity in both structure and biological activity, it is not surprising that the indole ring is an important structural component in many pharmaceuticals (1)(2)(3)(4)(5). Particularly fused-polycyclic indole framework is potential candidates for drug discovery because this structural motif is present in a wide variety of biologically active alkaloids (6). It is known for its variety of pharmacological characteristics as e.g. anti-fungal, anti-bacterial, antitumor, anti-HIV and DNA interaction properties (7)(8)(9)(10)(11)(12).
The development of an efficient synthetic method of cyclopenta[b]indole derivatives has attracted broad attention in medicinal chemistry and synthetic organic chemistry. Extensive efforts are therefore focused on this topic (13)(14)(15).
Prompted by the findings from the literature studies, we set out to explore and synthesise new pyrazolo-and isooxazolo-cyclopenta[b]indole derivatives. Furthermore, the compounds were evaluated for their active site with Human Kinase CK2 Protein by molecular docking study and cytotoxicity against HeLa human cervical cancer cell line also was carried out.
The formation of compound 3a confirmed by its IR spectrum which showed sharp and strong bands at 3153 and 1671 cm -1 assigned to NH and C=O group respectively. 1 H NMR spectrum lacked C2methylene proton signals and the displayed signals for C1-methylene and thiophene-CH protons, suggested the structure of 3a to be a thiophen-2ylmethylene compound. The proton NMR spectrum of 4a exhibited two broad singlets at 11.94 ppm and 9.01 ppm attributed to indole-NH and pyrazole-NH protons respectively. The aromatic protons appeared as multiplet in the region 6.76-7.87 ppm. IR spectrum of 5a registered absorption band at 1626 cm -1 assigned to cyano functional group. The proton NMR spectrum of 5a showed one broad singlet at 11.96 ppm attributed to indole-NH. The aromatic protons appeared as a multiplet between the regions δ 7.20-7.96 ppm. Analytical data are in accordance with the proposed structure for compound 5a. The identities of the other compounds were established in the same way with all spectroscopic data readily assignable. To understand the interaction of Human Kinase CK2 protein receptor with (3a-3d, 4a-4d and 5a-5d), the crystal structure of CK2 protein was downloaded from protein data bank and studied with the glide program. All the glide and E model compounds. In addition, glide is primarily concerned with generating an accurate pose for each compound and enrichment (the separation of actives from inactive) Fig.1. In the binding mode, compounds were attractively bound to CK2 via hydrophobic interaction, and Pi-Pi stacked interaction. The scoring functions of the docking program ranked that the binding interactions of the intermediate were less than those of the cyclised products. The compounds 5a-5d displayed better binding interaction compared to the intermediate; this might be due to the presence of the isoxazole moiety. The next best interactions were found among the compounds 4a-4d which hold the pyrazole moiety. Among the cyclised products compound 5c exhibited the best lowest binding energy and ligand efficiency; this might be due to the presence of the isoxazole moiety which was further reinforced by favourable electrostatic interaction of the chloro group at the 7 th position of the indole moiety. In general it was found that the isoxazole moiety favours better binding interactions compared to pyrazole moieties and intermediates.

Cytotoxicity studies
The in vitro cytotoxicity assay proves that the compound 5c has potent anticancer effect on human cervical cancer cells. Morphological shift of HeLa cell line by the compound is shown in Fig.2, revealing that the morphological alteration occurs in all the concentrations. From Fig.3, it is evident that compound 5c showed a dose-dependent anticancer activity. The maximum anticancer activity was obtained at 100 μM. The percentage of cell growth inhibition is found to be 100 % for compound 5c. The estimated IC50 value is 15.24 µ. Therefore, it is evident that the compound 5c has cell growth inhibition value closer to the standard drug ellipticine (9.62µ). The results obtained from the MTT assays help us to understand that the compound 5c have higher efficiency due to the presence of chloro group in the exact position in benzene moiety to have a good interaction with receptor protein active site. This, again, may be reason for the notable difference in IC50 value.

STRUCTURE ACTIVITY RELATIONSHIP (SAR) STUDIES
For the structure activity relationship, heterocyclic indole motif derivatives (3a-5d) such as pyrazolo and isoxazolo groups with indole ring were synthesized and evaluated for anticancer activity. It was observed that compounds 5a-5d comprising the isoxazolo framework possessed excellent anticancer activity against the tested cell line, where the anticancer activity was found to be in the order (5c (15.24μM)> 5d (17.67 μM) > 5b (19.33 μM) > 5a (25.63 μM)). Such results suggested that the halogen substituents enhance the antitumor activity when compared to other substituent. Compounds 4a-4d comprising pyrazolo moiety displayed moderate anticancer activity. It was noted that the intermediates showed comparatively less cytotoxic activity than the cyclised derivatives Fig. 4.

CONCLUSION
In conclusion, a new series of fluorine substituted pyrazolo-and isoxazolo-derivatives were chosen through target based drug discovery and synthesized, and characterized by IR, 1 H NMR and 13 C NMR spectroscopy. Molecular docking studies establish that these molecules exhibit significant molecular interactions with Protein kinase CK2 target protein. In vitro cytotoxicity study of the synthesized molecules against cervical cancer cells (HeLa), revealed that compound 5c exhibited IC50 value 15.24 μ. Compound 5c inhibited the growth of HeLa cells, showing a cell growth inhibition of 100%. The structure activity relationship of cytotoxic studies discovered that the chloro substituted isoxazolo-cyclopenta[b]indole display high activities than their counterparts.