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

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.


Introduction
Cancer stands amongst the foremost public health problems worldwide and is the second leading cause of death in the United States. As per the Cancer statistics report of the American Cancer Society, around 2 million new cancer cases and 610,000 cancerassociated deaths have been anticipated to occur in the year 2023 [1]. Prostate cancer is the most prevalent malignancy among males. WHO (2020) reported that 1.41 million new instances of prostate cancer and around 10 million deaths worldwide would be attributed to cancer [2]. Indeed, androgens and androgen receptors play a significant role in the growth and development of prostate cancer. The androgen receptor has been the target of several therapeutic drugs during the past ten years. Moreover, one of the most widely explored membrane-bound receptors involved in cancer cell adhesion, invasion, and metastasis is CD44, which regulates the glycolytic pathway in different carcinomas, hence cell proliferation. The expression of CD44 has been reported to be expressed in prostate-specific PC-3 cells but was absent in LNCaP cells [3,4]. Considering its association with the establishment of prostate cancer, different strategies, i.e., miRNA (miR-383), have been tested for their ability to restrict tumor initiation in CD44-positive prostate cancer cells [5].  (1)(2)(3)(4)(5)(6) and synthetic and biologically active SOXs (7)(8)(9)(10)(11)(12).

SOX Synthesis
The synthesis of the target SOXs was optimized using a one-pot, three-component strategy (Isatin derivatives/dioxindoles, arylamines, and dimethyl acetylenedicarboxylates, or DMAD) through a 1, 3-dipolar cyclo-addition reaction in the presence of an acid solution of p-toluene sulphonic acid and ethanol (p-TSA-EtOH) and purified through column chromatography using n-hexane and ethyl acetate. The proposed reaction (general) for the synthesis of newly formed SOXs is represented in Scheme 1. Optimization reactions were performed in ethanol at room temperature using 0.0 to 0.8 mM p-TSA (p-toluenesulphonic acid) as a sole catalyst, wherein 0.5 mM p-TSA resulted in the maximum yield (Table 1). Reaction condition: The reaction (10 mL) was carried out with isatin (1 mM), dimethyl acetylenedicarboxylate (1 mM), 4-chloro 3-(trifluoromethyl) aniline (1 mM), and (0.5 mM) p-TSA acid catalyst. The isolated yield was calculated. Reaction completion was monitored by TLC. RT = room temperature. ND = not detected.
Further, to predict all the possible interactions between two intermediates (i.e., 3 and Reaction mechanisms for the synthesis of SOXs have been schematically explained in Scheme 2. To summarize, compounds 1 and 2 reacted at their active sites to form 3-(phenylimino)indolin-2-one (Schiff base) 3. Simultaneously, H2O was nucleophilically added to by the alkyne system of dimethyl acetylenedicarboxylate 6, followed by a proton shift to generate a 1,3-dipolar intermediate (enamino ester or dimethyl 2-hydroxyfumarate). The Huisgen reaction then occurred between intermediates 3 and 7, resulting in a 1,3dipolar cycloaddition product (8). To produce stable SOXs, the product (8) went through cyclization (4a-h).
Further, to predict all the possible interactions between two intermediates (i.e., 3 and Reaction mechanisms for the synthesis of SOXs have been schematically explained in Scheme 2. To summarize, compounds 1 and 2 reacted at their active sites to form 3-(phenylimino)indolin-2-one (Schiff base) 3. Simultaneously, H2O was nucleophilically added to by the alkyne system of dimethyl acetylenedicarboxylate 6, followed by a proton shift to generate a 1,3-dipolar intermediate (enamino ester or dimethyl 2-hydroxyfumarate). The Huisgen reaction then occurred between intermediates 3 and 7, resulting in a 1,3dipolar cycloaddition product (8). To produce stable SOXs, the product (8) went through cyclization (4a-h).
Further, to predict all the possible interactions between two intermediates (i.e., 3 and Reaction mechanisms for the synthesis of SOXs have been schematically explained in Scheme 2. To summarize, compounds 1 and 2 reacted at their active sites to form 3-(phenylimino)indolin-2-one (Schiff base) 3. Simultaneously, H2O was nucleophilically added to by the alkyne system of dimethyl acetylenedicarboxylate 6, followed by a proton shift to generate a 1,3-dipolar intermediate (enamino ester or dimethyl 2-hydroxyfumarate). The Huisgen reaction then occurred between intermediates 3 and 7, resulting in a 1,3dipolar cycloaddition product (8). To produce stable SOXs, the product (8) went through cyclization (4a-h). Reaction mechanisms for the synthesis of SOXs have been schematically explained in Scheme 2. To summarize, compounds 1 and 2 reacted at their active sites to form 3-(phenylimino)indolin-2-one (Schiff base) 3. Simultaneously, H2O was nucleophilically added to by the alkyne system of dimethyl acetylenedicarboxylate 6, followed by a proton shift to generate a 1,3-dipolar intermediate (enamino ester or dimethyl 2-hydroxyfumarate). The Huisgen reaction then occurred between intermediates 3 and 7, resulting in a 1,3dipolar cycloaddition product (8). To produce stable SOXs, the product (8) went through cyclization (4a-h). Reaction mechanisms for the synthesis of SOXs have been schematically explained in Scheme 2. To summarize, compounds 1 and 2 reacted at their active sites to form 3-(phenylimino)indolin-2-one (Schiff base) 3. Simultaneously, H2O was nucleophilically added to by the alkyne system of dimethyl acetylenedicarboxylate 6, followed by a proton shift to generate a 1,3-dipolar intermediate (enamino ester or dimethyl 2-hydroxyfumarate). The Huisgen reaction then occurred between intermediates 3 and 7, resulting in a 1,3dipolar cycloaddition product (8). To produce stable SOXs, the product (8) went through cyclization (4a-h). Reaction mechanisms for the synthesis of SOXs have been schematically explained in Scheme 2. To summarize, compounds 1 and 2 reacted at their active sites to form 3-(phenylimino)indolin-2-one (Schiff base) 3. Simultaneously, H 2 O was nucleophilically added to by the alkyne system of dimethyl acetylenedicarboxylate 6, followed by a proton shift to generate a 1,3-dipolar intermediate (enamino ester or dimethyl 2-hydroxyfumarate). The Huisgen reaction then occurred between intermediates 3 and 7, resulting in a 1,3dipolar cycloaddition product (8). To produce stable SOXs, the product (8) went through cyclization (4a-h). Further, to predict all the possible interactions between two intermediates (i.e., 3 and 7) and the possibility of getting desired SOXs, we analyzed the more specific regioselectivity and diastereoselectivity phenomenon (Scheme 3). Taking the regioselectivity into account, the carbonyl group at C-3 of isatin residue of 1 is more active than C-2 of the same due to the possibility of resonance between N-1 and C-2 to form lactim and lactam forms of isatin, making further nucleophilic attacks at C-2 less feasible, whereas, the same phenomenon is not possible at C-3 of intermediate 3.
On the other hand, the H atom of C-3 of intermediate 7 is active and prone to attack the C-3 of intermediate 3 to form SOXs 4a-h, whereas the C-2 of intermediate 7 contains a -OH group providing it with more electron density and making it unable to generate a carbanion at C-2 required to attack the C-3 of intermediate 3. Therefore, the reaction proceeds in a Path A-dependent manner, while Path B is aborted. On the other hand, as far as diastereoselectivity is concerned, the newly synthesized SOXs (4a-h) will attain trans configuration (Path C) rather than cis conformers (Path D; 4a-h ) in order to get more stability as the cis conformers will have -OH and -COOR at same plane making them overcrowded and unstable.  13 13 13 13 13 13

HPLC Analysis of SOX 4a
The purity of the synthesized compound, used for in vitro cell culture studies, was assessed through HPLC at Integral University, Lucknow. Briefly, 20 µL SOX 4a was analyzed using Shimadzu 20AD Gradient LC System with PDA Detector system. A C-18 column (4.6 mm × 250 mm, 5 µm) was used to achieve the chromatographic separation. The mobile phase comprising methanol: H 2 O (70:30, v/v) was applied at a flow rate of 0.5 mL/min. The SOX 4a was detected at 254 nm with a retention time of 7.928 min in the HPLC system thermo-stated at 25 • C.

Computational Chemistry
The drug discovery program (DDP) involves the assessment of a set of distinct factors to identify the drug-likeness of a chemical entity. To date, various artificial intelligence (AI)-based strategies, i.e., absorption, distribution, metabolism, excretion, and toxicology (ADMET) and drug-likeness are currently being exploited as a part of the DDP to avoid unnecessary wasting of time, budget, and manpower [46][47][48]. In the same context, we also implied AI-based approaches, i.e., analysis of Lipinski's rule of five and ADME to assess the drug-like properties of our newly synthesized spirooxindole-pyrrolines (4a-h). Lipinski's rule of five includes some physicochemical descriptors that each and every chemical entity should follow in order to be qualified as an appropriate therapeutic agent. These descriptors include the molecular weight (M.W. < 500 Da), H-bond donors (HBDs: <5), H-bond acceptors (HBAs: <10), rotatable bonds (<10), topological polar surface area (TPSA) not exceeding the thresholds of 140 Å 2 and octanol-water partition coefficient (Log P) not exceeding the value of 5 [48,49]. The initial drug-likeness analysis depicted that all the newly synthesized spirooxindole-pyrrolines (4a-h) fall under the acceptable scores of Lipinski's rules of five if only one violation was permitted (Supplementary Table S1). Briefly, the M.W. for all the substituted spirooxindole-pyrrolines was found to be less than 500 except 4f, which had a M.W. of 531.66, whereas the number of HBDs and HBAs for 4a-h were recorded in the desirable thresholds of less than 5 and 10, respectively. On the other hand, the number of rotatable bonds was also found to be either three or four for all the substituted spirooxindole-pyrrolines (4a-h), which falls within the permissible thresholds of less than five [48].
On the other hand, considering the potential of hydrophobicity in drug distribution patterns, analysis of Log P has widely been used to assess the permeability of drug candidates, whereas the TPSA is used as the measure of the polarity and trans-membrane transport of compounds [50,51]. The results of our Log P and TPSA analysis showed that all the synthesized spirooxindole-pyrrolines (4a-h) have desirable Log p values (Log p ≤ 5, ranging from 1.48 for 4g to 2.91 for 4f). Moreover, the values for TPSA were also within the permissible thresholds (<140 Å 2 ) except for the two spirooxindole-pyrrolines, i.e., 4g and 4h, where each of the here-mentioned spirooxindole-pyrroline exhibited TPSA > 140 Å 2 (141.76 Å 2 for each); however, these violations (slightly higher TPSA) were not significant enough to rule out these newly synthesized spirooxindole-pyrrolines from this study. These values for the TPSA for all the newly synthesized spirooxindole-pyrrolines (4a-h) were similar to that reported in previously published reports [48,52]. These properties signify the fact that the newly synthesized spirooxindole-pyrrolines qualify for all the tests for drug-likeness and can further be assessed for other pharmacological effects.

Substituted Spirooxindole-Pyrrolines (4a-h) Have Acceptable ADME Properties
The prediction of distinct pharmacological measures, i.e., ADME through AI-based in silico strategies, is the most fundamental and persuasive step in the screening of chemical libraries for further drug discovery [46,48]. Our ADME analysis depicted that the newly synthesized spirooxindole-pyrrolines exert varying aq. solubility ranging from 0.330 to 100.057 mg/L for 4f and 4c, respectively (Supplementary Table S2). However, the reference anticancer agent, doxorubicin, had the greatest aq. solubility (112.691 mg/L). On the other hand, blood-brain barrier (BBB) penetration scores for all the substituted spirooxindolepyrrolines (4a-h) were less than 2 (0.013 to 0.623 C. Brain/C. Blood). Most importantly, the substituted 4h showed the lowest affinity against the BBB (BBB score: 0.013 C. Brain/C. Blood) as chemical entities with a BBB score > 2.0, between 2.0~0.1, and <0.1 correspond to the highest, moderate, and the lowest absorption across the BBB, respectively [48,53,54]. To sum up, all the substituted spirooxindole-pyrrolines (4a-h) exhibited either moderate or low transport against the BBB.
On the other hand, the assessment of Caco-2 and MDCK cell permeability has been established as the crucial measure of the DDP [54][55][56]. The Caco-2 and MDCK cell permeability was predicted through PreADMET. Our results from PreADME analysis showed that the substituted spirooxindole-pyrrolines (4a-h) exhibited a Caco-2 permeability range of 19.275 for 4h to 21.090 for 4g nm/s, which was suggestive of the fact that all of these newly synthesized spirooxindole-pyrrolines (4a-h) exert moderate permeability against Caco-2 cells. Similarly, the MDCK cell permeability score ranged from 0.1555 for 4f to 9.255 for 4c nm/s, which signifies that all the substituted spirooxindole-pyrrolines synthesized in the current study exhibited lower permeability across MDCK cells (score < 4.0) except 4c which showed a moderate permeability across these cells (score 9.255 nm/s). The permeability scores ≤ 4, >4-70, and >70 (nm/s) are suggestive of low, middle, and high permeability, respectively, in various cellular models [48,57].
Besides the permeability across the BBB, Caco-2, and MDCK cells, evaluation of the permeability across the skin/epidermal linings is thought to be of great importance when it comes to the transdermal drug delivery as well as the unintended exposure to harmful chemical entities [48,58]. In this regard, the skin permeability score (Log Kp) for the newly synthesized spirooxindole-pyrrolines was reported to be negative (from −2.723 to −4.233 cm/h for 4f and 4b, respectively), which is considered the acceptable range of Log Kp for therapeutic agents [59]. The fate of the drugs beyond absorption into the blood to the target tissues is governed by a set of factors (i.e., high M.W., solubility, and HBAs/HBDs) but plasma protein binding (PPB), perhaps, has been established amongst the major detrimental factors [48]. Chemical species with a very high PPB score tend to have comparatively higher persistence in the circulation or an elongated plasma half-life (t 1/2 ). More importantly, high PPB efficiency also impacts drug efficacy as usually the circulating fraction of drugs is accredited to the potent pharmacological actions [60,61]. In the same vein, the PPB for substituted spirooxindole-pyrrolines (4a-h) ranged from 89.411 to 92.711% for 4b and 4h, respectively. In contrast, doxorubicin exerted comparatively lower PPB efficiency (only 32.789%), which could be associated with poor absorption/distribution as well as its limited efficiency in the management of different malignancies [62].
Human intestinal absorption (HIA), a key determinant of drug suitability in the current drug discovery program, is derived from the bioavailability, absorption, and excretion of the drugs [48,49,63]. The establishment of different cancers and associated complications are attributed to distinct factors, i.e., reduced apoptosis, the state of oxidative imbalance, inflammatory cascades, and the accumulation of distinct advanced glycation end products (AGEs) in the circulation as well as various tissues/organs [31,[64][65][66]. Therefore, the therapies advised against distinct malignancies should not only be focused on the regulation of the apoptosis or cell cycle arrest but also on ameliorating the here-mentioned multiple targets residing either in the circulation or various tissues/organs [67,68]. In this regard, a pleiotropic drug with ameliorative efficacy against all these ghosts must have enhanced absorption as well as bioavailability in the circulation and other target tissues.
In the same context, the HIA for all the substituted spirooxindole-pyrrolines (4a-h) in our study ranged from 81.85% for 4g to 95.58% for 4f. Most strikingly, HIA for all the spirooxindole-pyrrolines ranged from 90-95%, except 4g, which showed an HIA score of 81.85% only. The high HIA scores of substituted spirooxindole-pyrrolines synthesized in the current study are advocating the fact that these substituted derivatives (4a-h) may have potent pharmacological efficacies owing to their elevated HIA as well as bioavailability. In contrast, the standard antiproliferative drug, doxorubicin, recorded a very poor HIA score of 31.95%, and such a poor bioavailability and absorption alone has been linked with the compromised anticancer efficacy of this commercially available drug [62]. Even the previous pharmacokinetic evaluations reported that the maximum plasma concentration (C max ) for doxorubicin was found to be only 29.2, 75.0, and 142.0 ng/mL when orally administered in rats at the dosage of 20, 50, and 100 mg/kg, respectively [62].
Moreover, the cytochrome P450 2D6 (CYT.p450 2D6 or CYP2D6) has been associated with around 25% of the drug metabolism in various tissues, organs, and circulation, as well as excretion from the body [48,69]. The fate of CYP2D6-directed drug metabolism differs from person to person as they may behave either as ultra-metabolizers or poor metabolizers of drugs, where the former suffer from the poor availability of drugs while the latter are supposed to be challenged by the compromised clearance from the body [69,70], therefore, making CYP2D6-directed drug metabolism a detrimental factor in dose selection criteria [48,71]. The results from this study showed that all the substituted spirooxindolepyrrolines (4a-h) neither behaved as CYP2D6 inhibitors nor CYP2D6 substrates, which signified the fact that the metabolism of these substituted spirooxindole-pyrrolines (4a-h) undergoes more or less in a CYP2D6-independent manner.
In this case, their potent pharmacological effects may be attributed to their higher PPB ability (>90%), which makes them more persistent as well as more bioavailable and also delays their instant removal from the circulation. In addition, none of the newly synthesized spirooxindole-pyrrolines (4a-h) acted as CYP2D6 substrates, which also reflects their persistent nature as well as enhanced therapeutic efficacy once these are administered in the body. These corroborations are well supported by recent reports [48,69,72].

Substituted SOXs (4a-h) Are Potent Inhibitors of CD44
Previous studies have confirmed the association of CD44 with various malignancies, including prostate cancer [3,4]. In an attempt to screen the substituted SOXs for their ability to occupy the binding pocket of CD44, the PyRx, a web-based server for virtual screening of drug-like candidates, was implied. As a result, all the SOXs exhibited significant interaction against the active pocket of CD44, and their binding score ranged from −6.5 to −7.4, whereas SOX 4a showed the best binding affinity with the least negative binding score (−7.4) ( Table 3). On the other hand, the interaction of doxorubicin with CD44 was not as strong as reported in the case of substituted SOX 4a, as evident by its slightly higher binding score (binding score: −7.0). Moreover, the strong interaction of the best SOX (4a) against CD44 was further validated through Autodock 4.0, which revealed that 4a strongly interacted with the active pocket of CD44 with a binding energy (∆G) of -6.65 Kcal/mol ( Table 4). The binding of 4a was facilitated by the involvement of Arg29, Phe34, Phe56, Asn120, Thr130, Ser131, Val132, Thr133, Asp134, Pro136, and Ser158 residues of CD44 ( Figure 2A). In contrast, this interaction of 4a with CD44 was better than that reported in the case of doxorubicin (∆G: −5.83Kcal/mol). Moreover, the doxorubicin-CD44 complex was stabilized by the interaction of Arg29, Asn57, Asn120, Ala121, Ser122, Ala123, Pro124, Asn128, Thr130, Ser131, Val132, Thr133, Asp134, Leu135, and Pro136 residues residing in the active pocket of CD44 ( Figure 2B). Table 4. Deep analysis of the 4a binding affinities and interacting residues of human CD44, EGFR-Tyrosine kinase domain, 5-β-reductase (AKR1D1), and HER-2.

Complex
Binding

Substituted SOXs (4a-h) Are Potent Inhibitors of the Tyrosine Kinase Domain of the EGFR
Various researchers have reported overexpression as well as enhanced functionality of EGFR in tumor tissues [21,22]. Our initial virtual screening through PyRx identified SOX 4a as the best inhibitor of the tyrosine kinase domain of EGFR, with a binding score of −8.7 among other newly synthesized SOXs (4a-h). The reference standard doxorubicin also formed a stable doxorubicin-EGFR complex with a better binding score of −10.1 Kcal/mol ( Table 3). The inhibitory effect of the substituted SOX with the best binding score against EGFR (4a) was further validated through a deep interacting analysis using Autodock 4.0. The result from our docking studies showed that the substituted 4a formed a stable complex with EGFR with a ∆G score of −6.55 Kcal/mol (Table 4), which falls within the ideal interaction energy as ∆G anything less than −4.0 Kcal/mol is considered a substantial interaction [73,74]. This 4a-EGFR complex was stabilized by the interaction of Leu694, Val702, Ala720, Lys721, Leu768, Met769, Gly772, Cys773, Arg817, Asn818, Leu820, Thr830, and Asp831 residues of EGFR ( Figure 3A). In contrast, the doxorubicin-EGFR complex showed a ∆G of −10.11 Kcal/mol, and the formation of this complex became feasible due to the involvement of Leu694, Phe699, Ala719, Lys721, Glu738, Met742, Val762, Leu764, Thr766, Gln767, Leu768, Met769, Pro770, Gly772, Arg817, Asn818, Leu820, Thr830, Asp831, and Phe832 residues of the EGFR ( Figure 3B).

Substituted SOXs (4a-h) Occupy the Binding Pocket of HER-2
Blocking the enhanced activity of HER-2 has been proven as a promising anticancer strategy in the last few decades [7,8]. To further validate the antiproliferative effects, we screened our newly synthesized SOXs (4a-h) for their ability to inhibit the active pocket of HER-2 via implying the PyRx server. In this attempt, the SOXs (4a-h) occupied the binding pocket of HER-2 (binding score: −6.6 to −7.8) with the greatest affinity exhibited by 4a (binding score: −7.8), whereas doxorubicin also exhibited a comparable binding score (−7.7) ( Table 3). Subsequent detailed molecular docking analysis through Autodock 4.0 also confirmed the strong binding of substituted SOX (4a) against HER-2 ( Table 4). The 4a-HER-2-complex was stabilized by the involvement of Gly77, Ser78, Lue726, Gyl729, Val734, Ala751, Leu800, Met801, Gly804, Cys805, Asp808, Asp850, Leu852, Arg859, Thr862, and Asp863 amino acid residues and potent binding energy (∆G: −7.27 Kcal/mol) ( Figure 5A). On the other hand, doxorubicin exerted stronger binding with HER-2 (∆G: −8.94 Kcal/mol) when compared to the ∆G of substituted SOX (4a). The interaction of doxorubicin with HER-2 was supported by different amino acid residues of HER-2 including Lys724, Val725, Leu726, Val734, Ala751, Thr798, Leu800, Met801, Pro802, Tyr803, Gly804, Cys805, Asp808, Arg849, Asn850, Leu852, and Thr862 ( Figure 5B). To corroborate our findings from the in silico molecular modeling studies, we further opted for distinct strategies implying PC-3 cells as a prostate cancer model. The very first among those strategies was the assessment of the cytotoxicity effect of the abovescreened best in silico modulator (4a) of multiple enzymes/biomarkers specific to the cancer establishment, i.e., CD44, EGFR, AKR1D1, and HER-2. The in vitro cytotoxicity effect of substituted SOX 4a was determined using an MTT assay, the most widely used assay to observe the toxicity of the test drugs in cell culture settings [66]. In this attempt, the substituted SOX (4a) was found to have substantial cytotoxicity against PC-3 cells in a dose-dependent fashion (IC 50 : 72.51 ± 2.35 µM) ( Figure 6). Moreover, the cytotoxicity effect of substituted 4a was further compared to the effect that was reported in the case of the reference standard doxorubicin, which showed an IC 50 of 37.90 ± 2.10 µM. These findings are well supported by the previous report, which also showed similar cytotoxicity effects of doxorubicin on PC-3 cells [75].
Usually, all cells have their own characteristic cytological features and morphologies, and so do the cancerous cells. In most cases, the cancerous cells are distinguishable from the noncancerous ones due to their comparatively larger nucleus as well as irregular morphologies [76]. However, these characteristic features may be altered when these cells are exposed to chemotherapeutic agents. In the same context, we also assessed the possible aberrations in the cytomorphological characteristics of the androgen-independent PC-3 cells after they were exposed to the above-screened substituted SOX (4a). In this attempt, our phase contrast microscopic analysis revealed that exposure to different dosages of substituted SOX (4a) resulted in diverse morphological aberrations in the PC-3 cells that were easily characterized in terms of cellular size, cytoplasmic abnormalities, nuclear condensation, granule formation as well as dispersed cellular counterparts (Figure 7). These morphological abnormalities, together with the cytotoxic effects on PC-3 cells (evident through MTT assay), are indicative of the substantial antiproliferative potential of substituted SOX (4a).

The Substituted SOX (4a) Triggers ROS Generation in PC-3 Cells
An imbalanced redox state is well-reckoned to cause the establishment of distinct non-communicable ailments such as atherosclerosis [26,73,77], neuro-degeneration [29], hyperglycemia [30,78] and associated complications, particularly nephropathy [32]. Interestingly, the ability of robust ROS generation is thought to be the key mechanism behind the antiproliferative efficacy of the majority of the commercially available chemotherapeutic agents [79]. In the same context, we also assessed the impact of our substituted SOX, i.e., 4a, on ROS generation as a probable mechanism behind its ability to negatively affect the survival of PC-3 cells. The observations from the fluorescence microscopic analysis demonstrated that incubation with substituted 4a led to robust ROS generation in PC-3 cells (Figure 8). This phenomenon was amplified with an increase in the dosages of 4a (6.25 to 100 µM). Such robust ROS production by the exposure of 4a may be attributed to its potent chemotherapeutic effect on PC-3 cells as ROS are well known to trigger distinct injuries to the cells, including damage to the cellular macromolecules, membrane oxidation and inflammatory signaling [27,28,66,80,81]. These exciting findings are strongly advocating the chemotherapeutic potential of substituted SOX (4a).

The Substituted SOX (4a) Stimulate the Nuclear Condensation in PC-3 Cells
The rationale behind the success of most of the commercially implied chemotherapeutic drugs in the last few decades is thought to be their capability to induce apoptosis in cancerous cells. The phenomenon of apoptosis is characterized by a set of key events, including the shrinkage of the nucleus, condensation of chromatin, DNA cleavage/fragmentation, the appearance of membrane protrusions (or cytoplasmic blebs), and the formation of apoptotic bodies [82,83]. Therefore, the assessment of nuclear fragmentation/condensation has been established as one of the most preferred strategies to screen the efficacy of the test chemotherapeutic agents. In this order, we also determined the effect of the substituted SOX (4a) on the apoptosis-like phenomenon in PC-3 cells and observed that the treatment with 4a significantly triggered the nuclear condensation/fragmentation as evident by the enhanced DAPI-specific fluorescence in a dose-dependent fashion against an untreated control ( Figure 9). This enhancement in the DAPI-specific fluorescence after 4a treatment could be attributed to the ability of this fluorophore to combine within the A=T rich milieu of the minor groove of the fragmented/condensed DNA, and the same has already been established by previous reports [83,84]. These findings are clearly indicating the chemotherapeutic potential of the substituted 4a via induction of ROS generation as well as nuclear condensation, which ultimately led to cell death or apoptosis in PC-3 cells.  On the basis of the findings from these optimizations hits, we concluded that the optimal conditions for the synthesis of the substituted spirooxindole pyrroline (4a) were p-TSA: 0.5 mM and solvent: EtOH. The rest of the compounds (4b-h) were synthesized by using distinct isatin derivatives, aryl amine derivatives, and dimethyl acetylenedicarboxylate (remained constant) in the presence of 0.5 mM p-TSA and ethanol at RT.

Apparatus Used for the Characterization
The melting point of all the synthesized compounds was determined with the help of the open capillary tube method using IA 9100 MK-Digital uncorrected melting Point analyzer Griffin Apparatus at the Department of Chemistry, Integral University. Infrared Spectroscopic analysis was done on Agilent Cary 630 FT-IR Spectrometer (Range: 4000-450 cm −1 ) Perkin-Elmer Spectrum version 10.03.06 at CSIR-CDRI, Lucknow, and the spectra were expressed as wave number (cm −1 ) with KBr discs. 1 H NMR Spectra was determined using Advance-400/800 MHz Bruker, Switzerland, and Bruker AVLL-300/800 MHz (Bruker, Fällanden, Switzerland) using DMSO-d 6 as a solvent.
The chemical shifts were denoted by δ ppm units using trimethylsilane as the internal standard. 13 C NMR spectra were recorded using Bruker AVLL-100 MHz (Bruker, Switzerland) using DMSO-d 6 . High-Resolution Mass Spectra were taken on Acquisition SW-6200 series TOF, version Q-TOF B.05.00 at CBMR, SGPGIMS, Lucknow. The completion of all the reactions was monitored with the help of thin-layer chromatography (TLC) and using variable proportions of ethyl acetate and petroleum n-hexane.

Ligand Preparation
The two-dimensional structures of synthesized SOX derivatives were drawn with the help of ChemDraw Professional v15.1 and subjected to prepare a three-dimensional structure with the help of Chem3D v15.1. The structural modification, geometrical correction, and optimization were performed with the help of Merck Molecular Force Field (MMFF94). Protoss is an online tool that automatically predicts hydrogens for the interaction between protein-ligand complexes (https://proteins.plus/), accessed on 5 February 2022. The substituted SOXs were subjected to a single-step minimization by the steepest descent method for 500 steps and an RMS gradient of 0.01 [85]. To perform docking analysis, the eutectic state of protonation of the ligands was found at pH 7.4.

ADME and Drug-Likeness of Substituted SOXs
The pharmacokinetic details of the substituted SOXs were obtained from an online ADME tool, and the drug-likeness of the newly synthesized SOXs was analyzed through the SwissADME tool (http://www.swissadme.ch); accessed on 15 February 2022 [49].

Retrieval of the Human CD-44, EGFR, AKR1D1, and HER-2
The 3D structures of human CD-44, EGFR, AKR1D1, and HER-2 were taken from the PDB (http://www.rcsb.org/) (accessed on 21 February 2022) using the PDB IDs 1UUH, 1M17, 3CAQ, and 3PP0, respectively, and visualized through BIOVIA Discovery Studio Visualizer 2020. These proteins had the resolutions of 2.2 Å, 2.6 Å, 2.2 Å, and 2.25 Å, respectively, and were processed as described earlier to remove the co-crystalized chemical entities [27,74]. The steepest descent and conjugate gradient approaches were implied for the energy minimization of the above-mentioned targets involved in the cancer progression. The human CD-44, EGFR, AKR1D1, and HER-2 were subjected to the DEEPSITE (available at: https://www.playmolecule.com/), accessed on 5 March 2022, for the prediction of the binding pockets [86].
3.2.4. Molecular Docking of Substituted SOXs against CD-44, EGFR, AKR1D1, and HER-2 The newly synthesized SOXs (4a-h) were firstly subjected to PyRx to screen the best inhibitor of the hyaluronan binding domain of human CD-44, EGFR, AKR1D1, and HER-2.

Investigations on the Cytotoxic Effects of Substituted SOX (4a) on PC-3 Cells through MTT Assay
The above-screened substituted SOX derivative (4a) was evaluated for its anticancer potency together with reference standard doxorubicin against prostate-specific PC-3 cancer cells by the MTT method [66]. The PC-3 cells in RPMI-1640 medium were seeded in a 96-well plate at a seeding density of 5 × 10 3 cells/well and left overnight at 5% CO 2 for adherence at 37 • C. After 24 h, varying concentrations (0, 25, 50, 100, 200, and 300 µM) of substituted SOX (4a) were added to these adhered PC-3 cells and incubated for a further 24 h in similar atmospheric conditions. The SOX 4a was first dissolved in DMSO and then serially diluted in the cell culture media (RPMI-1640) to obtain different concentrations ensuring that the final concentration of the DMSO should not exceed 0.5%. After that, 0.2 mL of MTT (20% v/v, prepared in RPMI-1640 medium) was added to these cells, incubated for 4 h at 37 • C in the dark, and the generation of formazan crystals was assessed after solubilizing them into the 0.15 mL DMSO by putting the plate on a shaker, and the plate was read at 570 nm.

Assessment of the Impact of the Substituted SOX (4a) on the Morphological Features of PC-3 Cells
The morphological examination of the PC-cells, either in the presence or absence of 4a, was performed in order to assess the antiproliferative effects of the substituted SOX (4a). In brief, the PC-3 cells (10 4 cells/well) were seeded and incubated with varying doses of 4a (0, 6.25, 12.5, 25, 50, and 100 µM) at 5% CO 2 for adherence at 37 • C. Following the incubation period, a FLoidTM Imaging Station, Thermo Fisher Scientific, Waltham, MA, USA, was implied to spot any morphological alterations in the cells.

Detection of ROS Generation
The efficacy of the substituted SOX (4a) in prompting ROS production within PC-3 cells was evaluated using DCFH-DA as described recently [66]. In brief, PC-3 cells (10 4 cells/well) seeded in each well were treated with 0, 6.25, 12.5, 25, 50, and 100 µM substituted SOX (4a) for 24 h at 37 • C. The cells were then stained with 10 µM DCFH-DA and incubated for half an hour, and imaging was done using a FLoid imaging station (Thermo-Scientific, Waltham, MA, USA).

DAPI Staining
The ability of substituted SOX (4a) to prompt apoptosis in PC-3 cells was done through DAPI staining [83]. The PC-3 cells were challenged with varying doses of 4a (0, 6.25, 12.5, 25, 50, and 100 µM and incubated for 24 h at 37 • C and then stained with DAPI. DAPI-specific fluorescence in 4a-treated and untreated cells was captured using a blue filter in a FLoid Imaging station (Thermo-Scientific, Waltham, MA, USA).

Conclusions
These SOXs (4a-h) exhibit suitable drug-like features and ADME indices. Moreover, molecular modeling studies using PyRx revealed that 4a, among all SOXs, was found to be the best inhibitor of distinct biomarkers associated with cancer progression, i.e., human CD-44, EGFR, AKR1D1, and HER-2. The enzyme inhibitory activity assessed through AutoDock confirmed that 4a exhibits substantial binding affinity for CD-44, EGFR, AKR1D1, and HER-2 (∆G: −6.65, −6.55, −8.73, and −7.27 Kcal/mol, respectively), which were also comparable to the standard doxorubicin. Apart from the in-silico analysis, the 4a also greatly affected the morphological features of the PC-3 cells. Moreover, the DCFH-DA and DAPI staining also confirmed the antiproliferative potential of 4a via substantial ROS generation as well as nuclear condensation into the PC-3 cells. This initial in silico and in vitro report demonstrate substituted SOX (4a) as a potent chemotherapeutic agent via its ability to interfere with the human CD-44, EGFR, AKR1D1, and HER-2 as well as instigating the ROS generation and nuclear condensation in PC-3 cells.