Novel Functionalized Spiro [Indoline-3,5'-pyrroline]-2,2'dione Derivatives: Synthesis, Characterization, Drug-Likeness, ADME, and Anticancer Potential

Abstract

A highly stereo-selective, one-pot, multicomponent method was chosen to synthesize the novel functionalized 1, 3-cycloaddition spirooxindoles (SOXs) (4a-4h). Synthesized SOXs were analyzed for their drug-likeness and ADME parameters and screened for their anticancer activity. Our molecular docking analysis revealed that among all derivatives of SOXs (4a-4h), 4a has a substantial binding affinity (∆G) -6.65, -6.55, -8.73, and -7.27 Kcal/mol with CD-44, EGFR, AKR1D1, and HER-2, respectively. A functional study demonstrated that SOX 4a has a substantial impact on human cancer cell phenotypes exhibiting abnormality in cytoplasmic and nuclear architecture as well as granule formation leading to cell death. SOX 4a treatment robustly induced reactive oxygen species (ROS) generation in cancer cells as observed by enhanced DCFH-DA signals. Overall, our results suggest that SOX (4a) targets CD-44, EGFR, AKR1D1, and HER-2 and induces ROS generation in cancer cells. We conclude that SOX (4a) could be explored as a potential chemotherapeutic molecule against various cancers in appropriate pre-clinical in vitro and in vivo model systems.

Keywords: ADMET studies; ROS generation; and anticancer effects; cancer cells; drug-likeness; isatin-derived spirooxindoles (SOXs).

Figure 1

Representation of naturally occurring (1–6) and synthetic and biologically active SOXs (7–12).

Scheme 1

Proposed reaction for the synthesis of the substituted spirooxindole-pyrroline (4a).

Scheme 2

Proposed mechanism for the synthesis of substituted SOXs (4a–h).

Scheme 3

Plausible approach of 1,3-dipolar cycloaddition for the formation of regio- and diastereoselective spirooxindole-pyrrolines (4a–h).

Figure 2

Substituted SOX (4a) is a potent inhibitor of human CD44 (PDB ID: 1UUH). (A) Binding pattern of the substituted SOX (4a) against human CD44 (∆G: −6.65 Kcal/mol); (B) Binding pattern of doxorubicin against human CD44 (∆G: −5.83 Kcal/mol). Subpanels (a) represent the interaction of 4a and doxorubicin inside the active pocket of the target protein (represented as a red surface). Subpanels (b) show the 2D image of the interacting residues of the CD44 with our drugs.

Figure 3

Substituted SOX (4a) is a potent inhibitor of the tyrosine kinase domain of the EGFR (PDB ID:1M17). (A) Binding pattern of the substituted SOX (4a) against human EGFR (∆G: −6.55 Kcal/mol); (B) Binding pattern of doxorubicin against human CD44 (∆G: −10.11 Kcal/mol). Subpanels (a) represent the interaction of 4a and doxorubicin inside the active pocket of the target protein (represented as a red surface). Subpanels (b) show the 2D image of the interacting residues of the EGFR with our drugs.

Figure 4

Substituted SOX (4a) inhibits the activity of 5-β-reductase/AKR1D1 (PDB ID: 3CAQ). (A) Binding pattern of the substituted SOX (4a) against human AKR1D1 (∆G: −8.73 Kcal/mol); (B) Binding pattern of doxorubicin against human CD44 (∆G: −10.01 Kcal/mol). Subpanels (a) represent the interaction of 4a and doxorubicin inside the active pocket of the target protein (represented as a red surface). Subpanels (b) show the 2D image of the interacting residues of the AKR1D1 with our drugs.

Figure 5

Substituted SOX (4a) occupies the binding pocket of HER-2 (PDB ID: 3PP0). (A) Binding pattern of the substituted SOX (4a) against human AKR1D1 (∆G: −7.27 Kcal/mol); (B) Binding pattern of doxorubicin against human CD44 (∆G: −8.94 Kcal/mol). Subpanels (a) represent the interaction of 4a and doxorubicin inside the active pocket of the target protein (represented as a red surface). Subpanels (b) show the 2D image of the interacting residues of the HER-2 with our drugs.

Figure 6

Cytotoxicity of 4a and doxorubicin against PC-3 cells. Values (percent cell viability) are mean ± SEM from triplicate measurements. Significantly different from untreated PC-3 cells at *** p < 0.001; ** p < 0.01; and * p < 0.05. The statistical analysis (ANOVA) was performed using GraphPad Prism Software version 9.5.0., San Diego, CA, USA.

Figure 7

Phase contrast micrographs showing the impact of substituted SOX (4a) on the morphological features of PC-3 cells after 24 h. For the assessment of morphological features, an equal population of the PC-3 cells was seeded in the 96-well plate and incubated with varying doses of 4a (0, 6.25, 12.5, 25, 50, and 100 μM). Following the incubation period, photomicrographs were taken using a FLoid^TM Imaging Station, Thermo Fisher Scientific, Waltham, MA, USA. Scale bar: 100 μM.

Figure 8

The substituted SOX (4a) triggers ROS generation in PC-3 cells. For the detection of ROS-specific fluorescence, PC-3 cells seeded in each well were treated with 0.0 to 100 μM substituted SOX (4a) for 24 h, and the cells were then stained with 10 µM DCFH-DA and incubated for half an hour and imaging was done using a FLoid^TM imaging station, Thermo-Scientific, Waltham, MA, USA. Scale bar: 100 μM.

Figure 9

The substituted SOX (4a) stimulates nuclear condensation/apoptosis in PC-3 cells. The PC-3 cells were challenged with varying doses of 4a (0.0–100 μM) and incubated for 24 h and then stained with DAPI, and the DAPI-specific fluorescence was captured using a blue filter in a FLoid^TM Imaging station (Thermo-Scientific, Waltham, MA, USA). Scale bar: 100 μM.