Development of β-carboline-benzothiazole hybrids via carboxamide formation as cytotoxic agents: DNA intercalative topoisomerase IIα inhibition and apoptosis induction
Graphical abstract
Introduction
“War on cancer,” officially, National cancer act which aims to eradicate cancer by finding a cure through effective treatments using targeted drug therapies was signed in the year 1971. Ever since then, continuous efforts are laid in research to discover a perfect therapy with minimal/no after-effects. Nevertheless, nearing about five decades cancer is still a global health problem with sky-scraping incidences and deaths as established in statistics reports of International Agency for Research on Cancer (GLOBOCAN) [1]. However, research using small molecules targeting different biological targets aiding cell proliferation is always in greater demand in the development of novel cancer therapeutic agents.
In this view, the therapeutic agents that inhibit the function of topoisomerase IIα trigger the activation of multiple pathways leading to cell death. As in rapidly dividing cells, expression of topo IIα is often observed as it holds the ability to impel topological changes in genetic material through several steps. However, to induce changes in DNA, topo II requires hydrolysis of ATP and metal ion Mg2+ [2]. Further, a considerable number of topo IIα acting neoplastic agents act by intercalative mode with DNA [3]. Intercalative topo IIα agents have the potential to be developed into efficient clinical agents towards cancer in near future.
Additionally, most of the agents used in the treatment of cancer have their origin either directly or indirectly linked to the natural sources. In such ways, β-carboline core was found in near about 50 natural products of different origin [4] (marine, plant, food products) that posses vivid biological potential [5] with magnificent excellence in cancer [6]. This bioactive heterocyclic entity is long known for its DNA binding efficiency [7] which might affirm the efficiency in several diseases. Of all the types available, fully saturated β-carboline was profoundly known for its intercalation properties with DNA [8]. Further, topoisomerase II inhibition of β-carboline was long known as the hybridized entities were reported as both catalytic inhibitory compounds [3c] and topo poisons [3](c), [9] indicating its efficiency as an anticancer agent.
Lately, Bathini et al., reported sulfonyl piperazines (secondary amine) amidated with β-carbolines (A) exhibiting superior efficacy in MG-63 cancer cells along with topo II inhibition [10]. Kamal and co-workers developed the β-carboline carboxamide based derivatives with podophyllotoxin (B) [11] and combretastatin (C) [3c] and explored them as potential anticancer agents and excellent in vitro cytotoxicity was exhibited by B and C in DU145 and A549 cancer cell lines respectively as well as the effectiveness towards inhibition of enzyme topoisomerase II (Fig. 1). On the other hand, scaffolds namely, benzothiazole has acquired the status of universal privileged heterocyclic scaffold owing to its vast pharmacological applications in multiple diseases, targeting several biological enzymes [12], receptors [13], pathways and other applications in polymer chemistry [14], dyes [15], imaging [16] and photographic sensitizers [17] and also, it has a special status in cancer biology [18], towards the design of diverse cytotoxic molecules. Anticancer molecules with benzothiazole ring include small molecule like D (NSC 710305) with an aryl carboxamide side chain [19] inspired the development of newer cytotoxic agents, where Choi et al., developed E by solid-phase combinatorial method using trityl resin and reported its strongest ability to inhibit topoisomerase II and cytotoxicity against myeloid leukemic cancer cells (HL-60) [20]. Kamal and co-workers reported benzothiazole carboxamides hybridized with naphthalimide piperazines [21] and phenyl pyrazoles [22] and best cytotoxicity was exhibited by compounds F and G on HT-29 and PC-3 cancer cell lines respectively. The effective performance of benzothiazole based small molecules as topoisomerase inhibitors along with exertion on other tumor targets pronounced them as successful candidates in cancer-based chemical research.
Carboxamide aka amide has been used as a linker in adjoining different pharmacophores to build novel cytotoxic agents. Amide bonds are prevalent in diverse biomolecules, and the hydrogen bonding ability, stability and ease of construction are few of the reasons for their success in the design and development of new biologically important agents [23]. By giving importance to the pharmacophoric characteristics and DNA intercalative topo II inhibition properties of β-carboline and benzothiazole and taking advantage of the structural framework, and our venture in the development of novel small heterocycles against neoplasms [24], we have designed and synthesized a new series of carboxamide bridged β-carboline-benzothiazole derivatives.
Section snippets
Chemistry
The pattern of carboxamide-linked β-carboline and benzothiazoles 8a–ad synthesis is depicted in Scheme 1. The β-carboline acid components 6a–f are synthesized based on an earlier approach [10], [11]. The two functionalities of amino acid tryptophan are utilized as starting material in the construction of 8a–ad. Commercially available l-tryptophan 1 is subjected to esterification results in the formation of methyl ester of tryptophan 2, and the amine functionality is condensed using diverse
In vitro biological activity
The new series of synthesized compounds 8a–ad were tested for their in vitro cytotoxic potential against two different sets of cancer cell lines by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [25] and their IC50 values were presented in Table 1. In solid tumors, live cells adhere to the surface whereas in liquid tumors like leukemia (blood cancers), cells are suspension type and no adherence is observed. Table 1 denotes the cytotoxicity of compounds on
Topoisomerase IIα inhibition studies
Based on the results obtained from in vitro cytotoxicity study (Table 1), molecular docking (Fig. 4, Fig. 5) and binding energy values, the compounds 8u and 8f were chosen for topoisomerase II enzymatic assay. Topo enzymes are known for imperative steps during the cell multiplication like DNA nicking and ligation. Specifically, topo II enzymes introduce a double-strand break unlike topo I, which causes a single-strand break. The catalytic reaction of topo II is roughly categorized into seven
DNA binding studies
To reveal the affinity of these hybrids with DNA, the best performers of in vitro cytotoxicity table (8u and 8f) were considered for DNA binding studies based on absorbance, fluorescence. Further, the circular dichroism spectroscopy and displacement studies along with viscosity experiments were also performed using CT-DNA to confirm the mode of interaction. Actinomycin D was used as positive control in all the DNA binding studies.
Annexin V PI dual staining assay
Quantification of apoptotic cell fatality is measured by Annexin V/PI dual staining assay and it aids in the detection of necrotic cells (Q1, UL; AV–/PI + ), live cells (Q2, LL; AV–/PI–), early apoptotic (Q3, LR; AV+/PI–) and late apoptotic (Q4, UR; AV+/PI+) cells [36]. To appraise the phase of apoptosis, annexin V assay was performed in A549 cells using different concentrations of compound 8u and analyzed using a flow cytometer. From Fig. 12A, it is inferred that there is a dose-dependent
In vitro cell migration assay
To evaluate the effect of compound 8u on cell migration, wound healing assay [38] was performed in A549 cells. This assay is a comparison of the migratory potential of cells towards the closure of an artificial wound with and without treatment by compound 8u. A scratch was made using sterile 200 μL pipette tip on the confluent monolayer of A549 cells, followed by treatment with compound 8u and the cells were allowed to migrate towards the scratch. Fig. 14 represents the phase-contrast
Lipinski’s and Jorgensen’s rules
Lipinski rule of five details about the druggability of a determinate molecule and is a “thumb rule” used to determine the potential of biologically active compounds regarding their oral bioavailability depending on the physical and chemical properties [39]. Compound 8u and 8f satisfy all the rules and fall in the recommended range proposed after analyzing 95% of the known drugs [40] and are tabulated in Table 2.
Jorgensen’s rule of three is extensively followed for the lead-like properties and
Conclusion
To sum up, a series of intercalative topoisomerase IIα inhibitors to target cancer was logically designed basing on the pharmacophoric features (in vitro cytotoxicity & topo II inhibition) of each structural element like β-carboline, benzothiazole and carboxamide as a bridge. A total of 30 new molecules 8a–ad were synthesized by relying on rational approach and amide coupling between the acid component of β-carboline and amine functionality bearing benzothiazole. Preliminary evaluation to
Chemistry
Materials and methods: The chemical reagents and solvents were obtained from commercial suppliers and were used without further purification. Analytical thin-layer chromatography (TLC) was performed on MERCK precoated silica gel 60-F254 (0.5 mm) aluminum plates. The spots on TLC plates were visualized under UV light. Wherever required, column chromatography was performed using silica gel (60–120). The anhydrous reactions were carried under nitrogen positive pressure and with freshly distilled
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
Authors are thankful to DoP, Ministry of Chemicals & Fertilizers, Govt. of India, New Delhi, for the award of NIPER fellowship. We acknowledge Dr. Chandraiah Godugu and his lab members, Department of Regulatory Toxicology, NIPER, Hyderabad for all time support to carry out biological studies. NIPER-H Research Communication No.: NIPER-H/2020/M058.
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