New Isatin-Indole Conjugates: Synthesis, Characterization, and a Plausible Mechanism of Their in vitro Antiproliferative Activity

Abstract

Background: Cancer remains the leading cause of human morbidity universally. Hence, we sought to assess the in vitro antiproliferative activity of new isatin-based conjugates (5a-s) against three human cancer cell lines.

Methods: The antiproliferative activities of compounds 5a-s were evaluated in vitro and their ADME (absorption, distribution, metabolism and excretion) was carried out using standard protocols. Subsequently, Western blot analysis was conducted to elucidate the potential antiproliferative mechanism of compounds 5a-s.

Results: The in vitro antiproliferative activities of compounds 5a-s against the tested cancer cell lines ranged from 20.3 to 95.9%. Compound 5m had an IC₅₀ value of 1.17 µM; thus, its antiproliferative potency was approximately seven-fold greater than that of sunitinib (IC₅₀ = 8.11 µM). In-depth pharmacological testing was conducted with compound 5m to gain insight into the potential antiproliferative mechanism of this class of compounds. Compound 5m caused an increase in the number of cells in the G1 phase, with a concomitant reduction of those in the G2/M and S phases. Additionally, compound 5m significantly and dose-dependently reduced the amount of phosphorylated retinoblastoma protein detected. Compound 5m enhanced expression of B cell translocation gene 1, cell cycle-associated proteins (cyclin B1, cyclin D1, and phosphorylated cyclin-dependent kinase 1), and a pro-apoptotic protein (Bcl-2-associated X protein gene), and activated caspase-3. ADME predictions exposed the oral liability of compounds 5a-s.

Conclusion: Herein, we revealed the antiproliferative activity and ADME predictions of the newly-synthesized compounds 5a-s and provided a detailed insight into the pharmacological profile of compound 5m. Thus, compounds 5a-s can potentially be exploited as new antiproliferative lead compounds for cancer chemotherapeutic.

Keywords: antiproliferative; cancer cell line; indole; isatin; synthesis.

Figure 1

Chemical structures of compounds I, II, and 5a–s.

Scheme 1

Preparation of compounds 5a–s. Reagents and conditions: (i) Few drops of concentrated sulfuric acid, absolute methanol, reflux, 4 h; (ii) H₂N-NH₂•H₂O, absolute methanol, reflux, 2 h; (iii) Few drops of glacial acetic acid, absolute ethanol, reflux, 4 h.

Figure 2

Dose-dependent reductions in the numbers of adherent cells. A-549 NSCLC cells were treated for 24 or 48 h, as indicated, followed by fixation and addition of the fluorescent label. Values are presented as the average number of cells per 4 fields within each well.

Figure 3

Dose-dependent reductions in Rb phosphorylation. A-549 NSCLC cells were treated for 24 or 48 h (as indicated), fixed, and subjected to quantitative indirect immunofluorescence and image analysis.

Figure 4

Tumor selectivity. A-549 NSCLC cells and non-tumorigenic cells derived from intestine, breast, and fibroblasts were treated for 72 h, as indicated, for use in a luminescence-based, growth-inhibition assay.

Figure 5

Evaluation of susceptibility to efflux. A-549 NSCLC cells and NCI-H69AR cells expressing the ABCC1 (MRP1) multi-drug resistance efflux transporter were treated for 72 h, as indicated, for use in a luminescence-based, growth-inhibition assay.