Design and synthesis of new spirooxindole candidates and their selenium nanoparticles as potential dual Topo I/II inhibitors, DNA intercalators, and apoptotic inducers

Abstract A new wave of dual Topo I/II inhibitors was designed and synthesised via the hybridisation of spirooxindoles and pyrimidines. In situ selenium nanoparticles (SeNPs) for some derivatives were synthesised. The targets and the SeNP derivatives were examined for their cytotoxicity towards five cancer cell lines. The inhibitory potencies of the best members against Topo I and Topo II were also assayed besides their DNA intercalation abilities. Compound 7d NPs exhibited the best inhibition against Topo I and Topo II enzymes with IC50 of 0.042 and 1.172 μM, respectively. The ability of compound 7d NPs to arrest the cell cycle and induce apoptosis was investigated. It arrested the cell cycle in the A549 cell at the S phase and prompted apoptosis by 41.02% vs. 23.81% in the control. In silico studies were then performed to study the possible binding interactions between the designed members and the target proteins.


Introduction
Multitarget therapies have, recently, attracted the medicinal chemists' interest, especially in the scope of complicated diseases including inflammation, thrombotic disorders as well as cancer 1,2 . The progress of such diseases can be regulated by several factors 3 . The primary or acquired resistance to almost all single-target therapies is the main challenge facing the treatment of these diseases due to the activation of compensatory mechanisms [4][5][6] . With respect to cancer, the currently available chemotherapeutic drugs achieved remarkable success in either curing the patients or, at least, extending their lives 7 . However, targeting two or more of the diverse pathways that participate in cancer development gives a synergistic effect to the designed drug and weakens the resistance against it 4,5 . The construction of a multitarget drug is quite more difficult than the single-target one as it requires the overlapping of the pharmacophoric features needed by the multiple targets 8,9 . Apoptosis, the process of programmed cell death, has received the researchers' attention in the last decades and became the target for almost all anticancer agents since the progress of tumours is associated with the failure of the apoptotic security pathways in deregulated cells 10 . Therefore, apoptosis induction in cancer cells is considered an effective pathway to eradicate uncontrolled proliferating cells. Thus, targeting DNA or, even, the enzymes involved in its metabolism is nowadays among the most promising strategies for cancer treatment 11,12 . Topoisomerases are a group of enzymes that resolve DNA topological problems by untangling the DNA duplexes 13 . The later fact potentiates the topoisomerases' role in DNA transcription and replication 13 . These enzymes can relax the DNA supercoiling upon the formation of transient single-stranded (topoisomerase I or Topo I) or double-stranded (topoisomerase II or Topo II) splits in DNA molecules 14 . Following, the DNA segment can then pass through the formed splits releasing the DNA molecule tension. However, the high levels of Topo I and Topo II enzymes in malignant cells compared to that of normal cells make the enzymes promising objectives for cancer chemotherapy 15 . Thus, DNA Topo I and Topo II inhibitors are classified as antitumor agents that form the basis of different chemotherapy combinations and are used widely in a broad spectrum of tumours 16 .
However, targeting Topo II by Topo II inhibitors motivates a cell resistance mechanism via a decrease in the cellular Topo II levels. This leads, subsequently, to an elevation in the Topo I cellular levels with an increase in the cells' sensitivity to Topo I inhibitors. The opposite phenomenon also occurs regarding resistance towards Topo I inhibitors 17 . Owing to this reason, targeting both Topo I and Topo II not only gives a synergistic effect but also weakens the probability of the development of resistance against such inhibitors 17 . Unfortunately, severe neutropenia and anaemia have been clinically reported after the co-application of Topo I and Topo II inhibitors 18 . An alternative approach to such a combination was the use of a dual inhibitor of both targets. Thus far, several dual Topo I/II inhibitors have been recognised 19 .
Moreover, dual Topo I/II inhibitors are classified into either DNA intercalating or DNA non-intercalating molecules according to their mechanism of action. The DNA intercalating molecules represent the majority of well-known dual Topo I/II inhibitors. Also, DNA intercalators are molecules that possess a planer (flat) aromatic system that can be sandwiched between two adjacent DNA nucleobases causing structural modifications to the DNA molecule 11 . Structural modification for the DNA intercalating molecules by linking their flat aromatic system to an amino group enables them to interact with Topo I/II enzymes. Figure 1 illustrates some reported Topo I/II inhibitors with potent in vitro anticancer effects against different types of cancers 17,[20][21][22][23][24][25][26] .
The spirooxindole-based structures are among the most vital nitrogen-containing compounds, particularly in the treatment of cancer due to the structural rigidity attributed to the spirocarbon present in these heterocycles [27][28][29] . However, spirooxindoles were reported to inhibit Topo I as well as Topo II enzymes 30,31 . In accordance with this, high interest was directed towards either discovering novel anticancer spirooxindole-based members or finding a simple and novel way to synthesise spirooxindoles. On the other hand, pyrimidine-based scaffolds also have a clear role in building several biologically active compounds, particularly in the field of cancer treatment 32 . The biological significance of pyrimidines may be ascribed to the ability of the nitrogen atom to bind with DNA by hydrogen bonding 33 . However, several pyrimidine-based structures were reported to inhibit topoisomerases including, benzo [4,5] Based on the importance of spirooxindoles and pyrimidines, we decided to design a novel hybrid of DNA intercalating Topo I/II inhibitors based on the spiro[indoline-3,5 0 -pyrido[2,3-d:6,5d 0 ]dipyrimidine] scaffold hoping to complete our efforts in searching for potent anticancer agents ( Figure 2). Selenium (Se) is a crucial trace element required for different cellular functions through incorporation into selenoproteins 40 . The significance of selenium nanoparticles (SeNPs) stems from their interaction with proteins, bioavailability, low toxicity, and biocompatibility related to organic and inorganic selenium. At high doses, SeNPs have been shown to have potent anticancer activity. SeNPs have excellent optoelectronic and semiconducting properties and are used in various applications ranging from photovoltaic cells, semiconductors, rectifiers, and copiers to biology and medicine 41 . SeNPs have been used in biomedical applications such as drug and targeted gene delivery, anti-inflammatory, antibacterial, and anticancer 42 . Many reports described the synthesis of SeNPs using different methods such as microwave-assisted synthesis, laser ablation, chemosynthetic reduction, electrodeposition, solvothermal, and green synthesis, all as examples of green synthesis. However, acidic pH, harsh chemical conditions, and high temperatures limit their use in biomedical applications 43 .

The molecular design rationale
The design of a new wave of compounds that dual-target Topo I and II enzymes and intercalate onto DNA depends basically upon the overlap between the reported pharmacophoric features of both Topo inhibitors and DNA intercalators. With this respect, we designed a new series of spiro[indoline-3,5 0 -pyrido[2,3-d:6,5d 0 ]dipyrimidine] candidates that fulfilled the requirements needed for a considerable binding with both receptors. Camptothecin, a Topo I inhibitor, and doxorubicin (Dox), a potent Topo II inhibitor and DNA intercalator, were our guides in the recent study. As so, the newly designed members achieved the following three features: (A) a planer heteroaromatic pyridodipyrimidine system to intercalate in between the DNA base pairs; (B) an NH basic centre to bind the negatively charged phosphate group of the sugar moiety; (C) a groove binder side chain to fit the DNA minor groove; and (D) a hydrogen bonding side chain to interact with both enzymes (Figure 3).

Chemistry
General SMP 30 apparatus was applied to record the melting point of the obtained congeners and the results were uncorrected. The progress of the synthetic steps was checked by the pre-coated silica gel plates (Merck 60 F 254 , Darmstadt, Germany) followed by visualising the applied spots with a UV lamp (254 nm). 1 H NMR and 13 C NMR (400 and 100 MHz, respectively) spectra were carried out by a Bruker NMR spectrometer (Billerica, MA). DMSO-d 6 was the solvent used in the NMR analysis. While mass spectra were obtained using Varian MAT 311-A (70 eV).

Materials
The starting compounds, 6-aminouracil derivatives (1a-e) were synthesised following the reported protocol [44][45][46] . Reagents and chemicals were supplied either from the USA or from Aldrich Chemicals Co. (St. Louis, MO). However, some reagents were obtained from commercial sources.  few traces of glacial acetic acid (6 mL). Next, the developed precipitate was filtered off. The collected product was then washed with methanol followed by a recrystallisation from DMF.

5-
In a conical flask, a solution of selenious acid (H 2 SeO 3 , 0.013 g, 0.01 mmol) in deionised water (80 mL) was prepared. The later solution was then heated and added to a DMSO (10 mL) solution of members 5b or 7d (0.01 mmol). The solutions were stirred continuously for 1 h at 60 C. Ascorbic acid (200 mL, 40 mM) was introduced to the solutions as a catalyst. The ruby red SeNPs were then suspended. Different methods were used to confirm and characterise the formation of SeNPs as described in the following subsections.

Ultraviolet-visible (UV-vis) spectra
The 1st method to confirm the SeNPs formation is to characterise their UV-vis spectra. The Shimadzu spectrophotometer is used for the current experiment. These spectra were taken in the range of 400-700 nm.

Transmission electron microscopy (TEM)
Another confirmation method is to determine the SeNPs' size as well as shape. High-resolution transmission electron microscopy (HRTEM), JEOL (JEM-2100 TEM; Tokyo, Japan) was applied in the current work. Particle size distribution A particle size analyser was used to determine the average diameter and size distribution (Nano-ZS, Malvern Instruments Ltd., Worcestershire, UK). Where the sample was sonicated before the assessment to measure size distribution for 30-60 min.

Cytotoxicity of different samples on five cancer cell lines
The cytotoxic activities of the constructed members were assessed via the reported MTT method 49-52 (Supporting information).
The mode of cell death of the promising cytotoxic samples (5b NPs and 7d NPs) Herein, the acridine orange/ethidium bromide (AO/EtBr) fluorescing stain and the different modes of cell death were investigated under the fluorescent microscope using the reported methodology 53 .

Dual topoisomerase I/II inhibitory assay
The tested members were subjected to Topo I inhibitory activity evaluation using the previously reported method 54 . While the Topo II inhibitory assay was done according to the method described by Patra et al. 55 (Supporting information).

DNA intercalation assay
The reported procedure by Atwal et al. was our guide to test the DNA intercalating ability of our candidates 56 (Supporting information).

Cell cycle analysis
The propidium iodide (PI) staining strategy explained in the Supporting information was adopted in the cell cycle analysis 57,58 .

Cell apoptosis analysis
The applied procedure 59 used for monitoring the apoptotic effect was mentioned in detail in the Supporting information.

Molecular docking studies
Two molecular docking studies of the novel candidates (3, 5a-e, 7a-e, and 9a,b) were performed using the Schrodinger suite 2021 60 to investigate their potential activities against the human DNA in complex with either Topo I or Topo II targets, respectively. Collectively, each chemical structure was built in ChemDraw and transferred to the working window, and corrected. The structures were then energy minimised as discussed previously 61 . Each prepared series was inserted in one database together with the native ligand in each receptor. The human DNA in complex with Topo I (1T8I) and Topo II (3QX3) target receptors were extracted. Each target protein was prepared for docking as reported previously 62 . Then, two docking processes were accomplished using the corresponding database and following the default setting options and methodology as before 9 . Also, PyMOL software 63 was applied for the visualisation of the selected poses. Moreover, two validation processes were performed by redocking each native ligand and the obtained RMSD values were <2 Å indicating a valid performance for the two docking processes 64 .

Results and discussion
Chemistry The spirooxindoles were synthesised by refluxing isatin with two equivalents of 6-aminouracil derivatives 44 dipyrimidine]-triones 3, 5a-e, 7a-e, and 9a,b by refluxing in acidic medium (Scheme 1). The investigation was extended to the uses of initiated isatin by electron acceptor group to yield more amounts of spiro derivatives and reduce the reaction time. All the resulting candidates were confirmed by spectral data 1 H, 13 C NMR, mass spectra, and elemental analysis. The 1 H NMR of compounds 3, 5a, 5b, 5d, 5e, 7a, 7b, 7d, 7e, and 9a,b showed singlet signals characteristic for NH-3 0 , NH-7 0 protons at d 11.93-11.06 ppm, furthermore, and singlet signals at the range d 11. The plausible mechanistic pathway for the formation of spiroindoles may be preceded as reported that the 3-position of the isatin is more reactive towards nucleophiles [68][69][70]  In situ synthesis of SeNPs using synthetic heterocyclic compounds 5b and 7d The team's work aimed to create new SeNPs using the synthesised compounds 5b and 7d having appropriate low reducing but high stabilising properties during SeNPs preparation. Accordingly, the organic heterocycle derivatives have reduced the Se þ cation to Se0 using an ascorbic acid catalyst, which acts as an aldehyde to form SeNPs and stabilise the nano-structure of SeNPs 71 . The organic compounds with reductive groups, such as -OH, -NH 2 , -NH, etc., can reduce Se þ cation. Figure 4 depicts the structure of the synthesised candidates.
The first indication for the formation of nanoparticles is the colour of the solution that turned from pale yellow to red after adding the dissolved compounds (5b and 7d) in DMSO to the solution of H 2 SeO 3 and stirring for one hour at 60 C ( Figure 5(A)). To easily monitor the formation of SeNPs, UV-vis spectroscopy was used ( Figure 5(B)). The absorption peak of the selenium colloidal solution is at 460 nm, according to surface plasmon resonance peaks in absorption spectra.
Transmission electron microscopy. It is indicating that NPs were formed and dispersed without the formation of aggregations in an aqueous solution 72 . Figure 6 shows how TEM confirmed the characterisation of selenium colloidal NPs. TEM was used to examine the prepared SeNPs, either alone or loaded onto the synthesised heterocyclic compounds. Figure 6 shows the presence of formed SeNPs in the tested specimens. SeNPs in spherical shapes are depicted in Figure 6(A). Minor aggregates can be found. The size of these nanoparticles ranges from 24.98 to 60.78 nm as shown in Figure 6. When SeNPs are loaded onto the investigated compounds 1, 2, 3, 4, 5, and 6, they spread uniformly through both samples (Figure 6(A,B)). The synthesised compounds helped to the prevention of such agglomerates in SeNPs. It appears that the nitrogen-based compounds' stabilising effect 73 contributed to the existence of SeNPs separated from each other with a convenient distribution.
Dynamic light scattering (DLS). The SeNPs' particle size was estimated by DLS measurements as demonstrated in Figure 7. Results revealed the compounds' particle size fell in the range of 30.43-65.90 nm (Table 1).

Biological testing
Cytotoxicity of different samples on five cancer cell lines The cytotoxic screening was performed using the MTT assay 74 . The assayed samples showed diverse effects on each cell line. The most sensitive tested cell lines were HepG2 and Caco-2 cells as nine out of the tested samples exhibited considerable cytotoxicity for them. However, only five tested compounds showed cytotoxic effects on the A549 cell line. Besides, the HCT-116 one was sensitive to only two samples, while the MCF7 was sensitive to one sample only. Two standard cytotoxic drugs: Dox and 5-fluorouracil (5-FU) were also applied on the same used cancer cell lines as references. The dose-response curves for each cell line are shown in Figure 4, and the respective IC 50 s are shown in Table 2.
After the cytotoxicity evaluation, some compounds were selected for the synthesis of in-situ SeNPs. The synthesised SeNPs were confirmed using the UV-vis (spectrophotometer), TEM, and particle size distribution. On the nanoscale, the toxicity was much enhanced for samples 5b NPs and 7d NPs. All the IC 50 values were around 10 lg/mL and less, except for 7d NPs on the Caco-2 cell line, which reached 18.9 lg/mL.
The dose-response curves for the nanoform on each cell line are shown in the Supporting information, and the respective IC 50 s are shown in Table 3. The most promising cytotoxic samples were further investigated for their cytotoxic effects on normal mice bone marrow cells. The overall trending results were increasing the proliferation of the bone marrow cells at high concentrations of the samples, especially the 5b NPs nanoform sample, which          On the macro scale, the most sensitive samples on the respective cell lines are illustrated in Figure 9. The photos showed the prevailed apoptotic mode of cell death with less necrotic changes.
Dual topoisomerase I/II inhibitory assay Since our main target for the recent study is to discover potent dual Topo I/II inhibitors, the highly active cytotoxic members were subjected to further assessment to measure their Topo I and Topo II inhibitory effects. Camptothecin, the potent Topo I inhibitor, and Dox, the potent Topo II inhibitor, were used as references in this study. Thus, compounds 5b and 7d in addition to their nano complexes 5b NPs and 7d NPs were evaluated for their enzyme inhibitory effects.
With regard to the Topo I enzyme, members 5b and 7d exhibited excellent inhibitory activities with similar and better IC 50 Figure 10. The inhibitory effects of the assessed compounds (5b, 7d, 5b NPs, and 7d NPs) against Topo I and Topo II compared to the reference drugs. values (0.651 and 0.419 mM), respectively, compared to camptothecin (0.694 mM). However, a drastic increase in the inhibitory activity was noticed regarding the nano complexes 5b NPs and 7d NPs. Compounds 5b NPs and 7d NPs were 5.8-fold and 16.5-fold more active than camptothecin with IC 50 values of 0.119 and 0.042 mM, respectively ( Figure 10).
Concerning the Topo II enzyme, results also confirmed the potentiality of the tested members as congener 7d was more active than Dox with IC 50 of 2.66 mM while its nano complex 7d NPs showed a potential inhibitory effect with IC 50 value of 1.172 mM. In contrast, compound 5b was slightly less potent than Dox with an IC 50 of 4.469 mM. Nevertheless, member 5b NPs possessed a higher inhibitory effect than that of Dox with an IC 50 value of 1.464 mM (Figure 10).

DNA intercalation assay
The ability of the designed members to intercalate onto the DNA molecules and increase the levels of Topo I-DNA as well as Topo II-DNA cleavage complexes by stabilising the enzymes-DNA covalent complexes were also assayed. Thus, the amount of linear DNA formed by compounds 5b and 7d, in addition to their nanoforms 5b NPs and 7d NPs, was measured. Even at low concentrations, the tested members caused considerable formation of linear DNA. As so, the designed congeners were then considered promising DNA intercalating members ( Figure 11).

Cell cycle analysis
The potent cytotoxic activity of congener 7d NPs, besides its superior dual Topo I/Topo II inhibitory effects, motivated us to perform a cell cycle analysis to explore the specific phase at which 7d NPs can halt the cell cycle. With this respect, the most sensitive A549 cells were treated with compound 7d NPs at a concentration equivalent to that produced the cytotoxic effect (1.9 ± 0.1 mg/mL).
The obtained results were compatible with the cytotoxicity study. Compound 7d NPs arrested the cell cycle progression at the S phase as it caused a dramatic increase to 41.02% vs. 23.81%, compared to the untreated cells. Moreover, the percentages of cells at G0-G1 and G2/M phases were relatively reduced from 61.33% and 14.86% in the control cells to 52.39% and 6.59% after incubation with compound 7d NPs, respectively ( Figure 12).

Cell apoptosis analysis
The ability of congener 7d NPs to prompt apoptosis in A549 cells was examined through the Annexin-V/PI staining assay 75 . The present strategy depends upon the incubation of A549 cells with the tested member, 7d NPs, at the compound's IC 50 concentration (1.9 ± 0.1 mg/mL) for 24 h. Based on the given results, 7d NPs caused a marked induction of apoptosis in the treated cells by a percentage exceeding 56%. In particular, a significant increase in both early and late apoptotic populations from 0.51% and 0.18% in the control cells to 28.77% and 22.03% in the 7d NPs treated cells confirmed that its cytotoxic activity is associated with a potent apoptotic effect ( Figure 13).

Structure-activity relationship (SAR)
Observation of the obtained biological findings clarified different notes about the SAR of the designed candidates ( Figure 14): a. HepG2, Caco-2, and A-549 cell lines were the most sensitive cells to the examined members among the tested cell lines.
As nine members out of 13 were active against HepG2 and Caco-2 cells while seven members were potent against A-549 cells. b. Two compounds were active regarding HCT-116 cells and only one congener of the tested members was active against MCF7 cells. c. The SeNPs complexes 5b NPs and 7d NPs exhibited a dramatic increase in the cytotoxic activity against almost all the examined cell lines. d. The fluorinated member 9b possessed a superior cytotoxic activity regarding HepG2 cells followed by the chlorinated members 7d and 7e, which were more potent than the nitro compound 5b.
e. In contrast, the nitro derivatives 5b and 5d showed better cytotoxic activities than that of the chlorinated member 7a against Caco-2 cells. f. The same observation was also reported for HCT-116 cells, as the nitro derivative 5c was more cytotoxic than the chlorinated member 7e. g. Concerning A-549 cells, the descending order of activity of the tested members was 7a > 5d > 3 > 9b. h. However, comparing the Topo I/Topo II inhibitory activities as well as the DNA intercalating potential of compounds 5b and 7d to the 5b NPs and 7d NPs revealed a tremendous increase in the activity of the nano complexes over the native members.

Molecular docking studies
Two molecular docking studies of the novel designed members (3, 5a-e, 7a-e, and 9a,b) were performed to investigate their potential activities against the human DNA in complex with both Topo I (https://www.rcsb.org/structure/1T8I) and Topo II (https:// www.rcsb.org/structure/3QX3) targets, respectively. First, the receptor pockets of the aforementioned targets were studied to clarify the binding mode and accordingly the most important nucleotides and amino acids within each. The co-crystallised poison of the human DNA in complex with Topo I enzyme (camptothecin) was able to bind Asp533, Arg488, DT10, DA113, DC112, and TGP11. Besides, the co-crystallised poison of the human DNA in complex with Topo II enzyme (etoposide) interacted with Gln778, Met782, Asp479, DA12, and DG13. Moreover, each docked co-crystallised ligand was inserted as a reference and showed a similar binding mode as the native one indicating a valid molecular docking software 76 . Herein, the superior anticancer members (5b and 7d) were selected for further investigation. Both 5b and 7d candidates recorded very promising binding scores towards the human DNA in complex with both Topo I and Topo II targets, which were comparable to the co-crystallised ligand in each process. They showed interaction scores of (-9.08 and À7.50 kcal/mol) and (-9.73 and À8.99 kcal/mol), respectively, with respect to camptothecin (-10.24 kcal/mol) and etoposide (-10.84 kcal/mol). All root mean square deviation values were in the range of (1.09-1.56 Å) indicating valid selected poses 77 .
Compound 5b within the Topo I binding site was able to interact with DA113 with a hydrogen bond and DC112 with a pi-pi interaction. However, compound 7d described three interactions with DA113 (hydrogen bond), DA113 (pi-pi bond), and TGP11 (pipi bond) as depicted in Table 4. Moreover, compound 5b within the Topo II binding site was found to bind both DA12 (hydrogen bond) and DG13 (hydrogen-pi bond). Furthermore, compound 7d bound DG13 and His775 with two hydrogen bonds as represented in Table 4.
According to the above results, we can observe the very promising scores and binding modes of both compounds (especially 7d) to be recommended as a potential DNA intercalator with dual Topo I/Topo II inhibitory activities.
It is worth mentioning that the C═S functional group of both 5c and 7c compounds contributed largely to the interactions with the key amino acids of Topo I and Topo II receptors indicating their great importance (Supporting information).

Conclusions
To find potent multitarget Topo I/II inhibitors, a new wave of compounds was constructed and synthesised based on the hybridisation of spirooxindoles and pyrimidines. The designed compounds were also constructed to act as DNA intercalators. Thirteen congeners were synthesised and in vitro assessed for their cytotoxic effects against five human cancer cell lines, HepG2, HCT-116, Caco-2, A-549, and MCF7. Considerable cytotoxic effects were observed regarding the tested cells. However, better cytotoxic compounds were selected for the synthesis of in situ SeNPs to enhance their activity. The nanoform compounds 5b NPs and 7d NPs exhibited excellent antitumor activities towards the tested cells (IC 50 values ranging from 1.9 ± 0.1 and 18.9 ± 2.4 mg/mL). Meanwhile, the former two nanoform members were examined for their Topo I and Topo II inhibition effects. Compounds 5b NPs and 7d NPs exhibited superior activities against Topo I (IC 50 ¼ 0.119 and 0.042 mM, respectively). Also, they inhibited Topo II (IC 50 values ¼ 4.469 and 1.172 mM, respectively). Furthermore, compound 7d NPs halted the cell cycle in the S phase. Candidate 7d NPs, moreover, induced apoptosis in A549 cells. Molecular docking also showed very promising binding scores and binding modes (especially for 7d) to be recommended Figure 14. SAR of the tested compounds (3, 5a-e, 7a-e, and 9a,b). Table 4. 2D interactions, 3D interactions, and 3D positioning of 5b and 7d within the binding pockets of the human DNA in complex with Topo I (1T8I) and Topo II (3QX3) target receptors. as a potential DNA intercalator with dual Topo I/Topo II inhibitory potentials.