Stereoselective Synthesis of the Di-Spirooxindole Analogs Based Oxindole and Cyclohexanone Moieties as Potential Anticancer Agents

A new series of di-spirooxindole analogs, engrafted with oxindole and cyclohexanone moieties, were synthesized. Initially, azomethine ylides were generated via reaction of the substituted isatins 3a–f (isatin, 3a, 6-chloroisatin, 3b, 5-fluoroisatin, 3c, 5-nitroisatin, 3d, 5-methoxyisatin, 3e, and 5-methylisatin, 3f, and (2S)-octahydro-1H-indole-2-carboxylic acid 2, in situ azomethine ylides reacted with the cyclohexanone based-chalcone 1a–f to afford the target di-spirooxindole compounds 4a–n. This one-pot method provided diverse structurally complex molecules, with biologically relevant spirocycles in a good yields. All synthesized di-spirooxindole analogs, engrafted with oxindole and cyclohexanone moieties, were evaluated for their anticancer activity against four cancer cell lines, including prostate PC3, cervical HeLa, and breast (MCF-7, and MDA-MB231) cancer cell lines. The cytotoxicity of these di-spirooxindole analogs was also examined against human fibroblast BJ cell lines, and they appeared to be non-cytotoxic. Compound 4b was identified as the most active member of this series against prostate cancer cell line PC3 (IC50 = 3.7 ± 1.0 µM). The cyclohexanone engrafted di-spirooxindole analogs 4a and 4l (IC50 = 7.1 ± 0.2, and 7.2 ± 0.5 µM, respectively) were active against HeLa cancer cells, whereas NO2 substituted isatin ring and meta-fluoro-substituted (2E,6E)-2,6-dibenzylidenecyclohexanone containing 4i (IC50 = 7.63 ± 0.08 µM) appeared to be a promising agent against the triple negative breast cancer MDA-MB231 cell line. To explore the plausible mechanism of anticancer activity of di-spirooxindole analogs, molecular docking studies were investigated which suggested that spirooxindole analogs potentially inhibit the activity of MDM2.


Introduction
According to GLOBOCAN report in 2018, there were 9.6 million mortalities and 18 million new cases of cancer. In addition, the report describes cancer as the second leading cause of death worldwide. Therefore, development of anticancer agents with low toxicity, high efficacy, low drug resistance, and acceptable bioavailability is an urgent need to meet this global health challenge [1]. Drug discovery based on medicinal chemistry of natural and synthetic products have gained much attention [2,3]. In the past century, there has been an enormous success in drug innovation in the area of oral availability and biological compatibility, but drug resistance has emerged as a challenge for the medicinal chemists. Therefore, pharmacologists and chemists are focussing on functional diversity of drug leads [4], nano-formulation, and drug delivery development [5][6][7], with an aim of overcoming the existing problems. Specifically, the alkaloids spirooxindole scaffold, as a member of the oxindole class of natural products [8] has received much attention. The first member of this series was isolated from Apocynaceae and Rubiaceae plants. The spirooxindole scaffold is a privileged structure consisting of two basic sub-units: the first is oxindole with multiple functionalities, which can interact as acceptors or donors with the biological targets via hydrogen bonding. The second unit is a carbocyclic or heterocyclic moiety fused with oxindole ring at the C-3 position. It provides an opportunity to regulate many physicochemical properties and the liposolubility of spirooxindoles [9]. Accordingly, the significant biological activities (e.g., anti-inflammatory, anticancer, analgesic, antimicrobial, antimalarial, antioxidant, antiviral, antidiabetic, antiatherosclerotic, and insecticidal properties) and unique spatial architecture of spirooxindoles have attracted a remarkable attention of pharmacologists and chemists [10,11]. In the last few decades, several approaches towards new of spirooxindole analogs with structural diversity have been explored [12]. Based on the literature survey, the spirooxindole scaffold has shown to be a promising candidate for anti-cancer drug discovery.
In 2014, Santos's group reported the synthesis of some novel spiropyrazoline based oxindole scaffold, and subsequently examined the cytotoxicity in vitro toward MCF-7 breast cancer cell line ( Figure 1). The hit with high efficacy towards the breast cancer line was checked against MDA-MB-231 cell line. The results demonstrated that compound I exhibited a high activity with higher selectivity between the two cell lines with GI 50 < 7.4 µM and >10-fold than the MDA-MB-231 cell line. Interestingly, the promising compound I behaved safely, and was noncytotoxic to HEK293T normal cell line [13].
Zhou's group synthesized a family of spirooxindoles II, grafted with five-membered carbocyclic, with substituted oxindoles via set of chemical transformations, including Knoevenagel condensation/Michael cyclization ( Figure 1). The synthesized compounds were evaluated for anticancer activity against three cancer cell lines, lung A549, human leukemia K562, and prostate-cancer cell lines PC-3. From this study, some representative compounds showed a comparable or stronger inhibitory effect against human leukemia cells K562 (IC 50 = 7.4 to 32.8 µM) as compared to cisplatin (up to 3.4-fold). Moreover, other hits have either shown equivalent inhibition toward A549 cell line or slightly increased inhibitory activity against prostate cancer PC-3 cell line as compared to cisplatin [14].
In 2019, Tumskiy et al. synthesized five spirooxindolepyrrolidines and examined the cytotoxic activity against some cell lines (Vero normal and HeLa cancer cells). The results demonstrated that hit III having a pyridine moiety with the chlorine atom in the ortho position exhibited a moderate selectivity (3-fold) between HeLa cancer cells and Vero healthy cells [18].
In continuation of research work, Barakat et al. reported the synthesis of spirooxindolepyrrolothiazoles having a 3-cinnamoyl moiety. The results of cytotoxicity activities assay disclosed that compound V was the most active member of the series towards HCT-116, HepG2, and PC-3 cancer cells (IC 50 < 4 µM). The selectivity index for the cancer cells versus the normal cells was superior to 2. Additionally, the research group carried out a set of biological assays which indicated that compound V could inhibit cell migration, colony formation, arrest cancer cell growth at the G2/M phase and induce apoptosis through extrinsic and intrinsic pathways [19] (Figure 1).
In 2019, Barakat et al. reported the synthesis and cytotoxicity activities (HeLa) of the hit depicted in Figure 1 (i.e., VI). The antiproliferative assay showed that the compound can inhibit the proliferation of HeLa cancer cell line (IC 50 = 11.2 µM), but less than the anticancer drug, doxorubicin (IC 50 = 1.2 µM) [20]. The large library of the spirooxindole scaffold was generated with diverse pharmaceutical activities including low toxicity, acceptable bioavailability, and high efficiency [21][22][23][24][25][26][27][28][29]. In this paper, we describe in detail the synthesis of the spirooxindole analogs with significant bioactivities against the cancer cell in vitro. Molecular docking studies were also carried out to explore the plausible mechanism of anticancer activity of di-spirooxindole analogs.

HMQC (Supplementary Materials
Next, COSY analysis mapping helped us to find the neighboring protons association. H 1 and H 2 coupled with each other while H 2 also coupled with two other protons (H 9,10 ). Proton H 3 had three strong coupling signals with H 5 and H 18 , 20 . Similarly, proton H 5 had five adjacent protons since it had five coupling signals with H 3 , H 9,10 , and H 11,13 (Figure S18, Supplementary Materials). HMQC Spectra explain the 1-3 and 1-4 interaction of H 1 with C 2 , C 3 , C 7 , C 11 , and CO which further confirm the position of H 1 proton (Supplementary Materials Figure S19).

Molecular Docking Study
It has been reported that most of the human cancer cells overexpressed p-53 protein [4,11,36,37]. Therefore, docking studies were performed to rationalize the plausible mechanism of inhibition of p53-MDM2 protein-protein interactions.
A tumor suppressor protein p53 plays a pivotal role in preventing tumor progression and development. Cellular stress in response to DNA damage and hypoxia triggers the stimulation of p53. Up-regulated p53 stimulates the transcription of many important genes involved in apoptosis, senescence, DNA repair, and apoptosis. Consequently, suppression of p53 may be a requisite step in tumor formation. Murine double minute 2 (MDM2) is a central negative regulator of p53. Due to the vital role of MDM2 in inhibiting the tumor suppressor function of p53, blockade of protein-protein interaction of MDM2-p53 is an attractive anticancer therapeutic target. Furthermore, it has been extensively reported that spirooxindole analogs potentially inhibit the activity of MDM2 [19,[38][39][40]. Thus, in this study, molecular docking studies of the potential anticancer di-spirooxindole analogs were carried out using MDM2 crystal structure to explore the observed anticancer activity. The docking studies suggested that 4a, 4b, 4i, and 4l accommodated well in the binding site of MDM2 with a binding affinity of −7.20, −7.37, −7.83, and −7.90 kcal/mol, respectively ( Figure 3). Compound 4a showed a slightly different binding mode in comparison to 4b, 4i, and 4l. The unsubstituted oxindole ring of 4a interacts with the side chain of Leu54 and Gly58 while the substituted oxindole ring of 4b, 4i, and 4l is buried deeply in the binding cavity of MDM2 by interacting with Val93, His96, and Ile99. Substitution on the oxindole ring may account for the different binding mode of the di-spirooxindole analogs within the Compound 4a showed a slightly different binding mode in comparison to 4b, 4i, and 4l. The unsubstituted oxindole ring of 4a interacts with the side chain of Leu54 and Gly58 while the substituted oxindole ring of 4b, 4i, and 4l is buried deeply in the binding cavity of MDM2 by interacting with Val93, His96, and Ile99. Substitution on the oxindole ring may account for the different binding mode of the di-spirooxindole analogs within the binding cavity of MDM2. Similarly, compound 4a confers two hydrogen bond contacts with the carboxyl group of Leu54 and Gly58. Six-membered aromatic rings in the 4a mediate π-π and π-alkyl interactions with the binding site residues of MDM2, including Leu54, Ile61, Val75, Phe91, Val93, and Ile99, which projected 4a firmly in the binding pocket. The compound 4b resides comfortably in the binding site extending hydrophobic interactions with Leu54, Ile61, Tyr67, Gln72, Val93, and Ile99, while no hydrogen bond was observed. The docked pose of compound 4i suggested a number of significant hydrophobic interactions with binding site residues. Fluorinated phenyl rings stacked between Ile61, Tyr67, and Val93 produced hydrophobic effects. Similarly, Phe55 was also observed to mediate hydrophobic interactions. In addition, 4i confers two hydrogen bonds with the side chain hydroxyl group of Ser17 and a halogen bond with Gln72, whereas fluorinated phenyl rings of compound 4l establish hydrophobic interactions with Leu57, Phe55, Ile61, Tyr67, Val93, and Ile99. Moreover, two hydrogen bonds were observed with the side chain hydroxyl group and imidazol ring of Ser17 and His96, respectively.        3262, 2926, 2853, 1715, 1681, 1612, 1592,  1558, 1484, 1473, 1447, 1365, 1317, 1260, 1200, 1155, 1102, 1073 147. 1, 143.4, 138.1, 137.1, 136.1, 135.4 6, 129.5, 128.2, 127.9, 126.1, 126.1, 125.6, 121.6, 116.6, 116.5, 116.4, 116.3, 115.5, 115.3, 113.9, 113.7, 110.6, 79.7, 69.5, 64.4, 58.1, 51.6, 42.5, 36.2, 28.5, 28.3, 28.1, 26.8, 24.7, 21.5, 20.3

Molecular Docking Methodology
To rationalize the observed anticancer activity, molecular docking studies of the potential compounds were carried out using MDM2 crystal structure. The selected dispirooxindole analogs (4a, 4b, 4i, and 4l) were sketched in MOE v.2019 by using Builder module and subsequently subjected to geometry correction and protonation followed by minimization using MMFF94x force field. The 3D structure of MDM2 in complex with a benzodiazepine inhibitor (PDB ID 1T4E) was retrieved from ProteinData Bank [34]. Since there were no conserved water molecules in the utilized pdb, all water molecules were deleted and the structure was corrected and protonated using GB/VI as the electrostatics function with a dielectric value of 80 (for solvent). Consequently, the protein structure was minimized to remove the bad clashes using the Amber99 force field. A grid of 6 Å was generated centered on the co-crystalized ligands and the selected compounds. The triangular method was used as a placement method with an induce fit protocol. The resulting poses of the ligands were scored by London dG scoring function and the top ranked poses were visually analyzed. All the graphics were rendered using MOE software.

Conclusions
In conclusion, a new series of hybrid di-spirooxindole analogs, engrafted with substituted oxindole and cyclohexanone moieties, were synthesized successfully by a one-pot multicomponent reaction. The anticancer assay showed promising results, which makes these di-spirooxindole analogs suitable for further research. Synthesized di-spirooxindole analog 4b (IC 50 = 3.7 ± 1.0 µM) appeared to be a more potent candidate against PC3 cell line, whereas, di-spirooxindole analogs 4a (IC 50 = 7.1 ± 0.2 µM) and 4l (IC 50 = 7.2 ± 0.5 µM) possessed promising anticancer activity against cervical cancer HeLa cell line and triple negative breast cancer MDA-MB231 cell line. Compound 4i (IC 50 = 7.63 ± 0.08 µM) appeared to be more active among these di-spirooxindole analogs. The docking studies suggested that 4a, 4b, 4i, and 4l accommodated well in the binding site of MDM2. However, further mechanistic studies via in vivo animal models are required to validate the results of these in vitro assays.