Design, synthesis, molecular modelling and antitumor evaluation of S-glucosylated rhodanines through topo II inhibition and DNA intercalation

Abstract In the present study, 5-arylidene rhodanine derivatives 3a–f, N-glucosylation rhodanine 6, S-glucosylation rhodanine 7, N-glucoside rhodanine 8 and S-glucosylation 5-arylidene rhodanines 13a–c were synthesised and screened for cytotoxicity against a panel of cancer cells with investigating the effective molecular target and mechanistic cell death. The anomers were separated by flash column chromatography and their configurations were assigned by NMR spectroscopy. The stable structures of the compounds under study were modelled on a molecular level, and DFT calculations were carried out at the B3LYP/6-31 + G (d,p) level to examine their electronic and geometric features. A good correlation between the quantum chemical descriptors and experimental observations was found. Interestingly, compound 6 induced potent cytotoxicity against MCF-7, HepG2 and A549 cells, with IC50 values of 11.7, 0.21, and 1.7 µM, compared to Dox 7.67, 8.28, and 6.62 µM, respectively. For the molecular target, compound 6 exhibited topoisomerase II inhibition and DNA intercalation with IC50 values of 6.9 and 19.6 µM, respectively compared to Dox (IC50 = 9.65 and 31.27 µM). Additionally, compound 6 treatmnet significantly activated apoptotic cell death in HepG2 cells by 80.7-fold, it induced total apoptosis by 34.73% (23.07% for early apoptosis, 11.66% for late apoptosis) compared to the untreated control group (0.43%) arresting the cell population at the S-phase by 49.6% compared to control 39.15%. Finally, compound 6 upregulated the apoptosis-related genes, while it inhibted the Bcl-2 expression. Hence, glucosylated rhodanines may serve as a promising drug candidates against cancer with promising topoisomerase II and DNA intercalation.


Introduction
Worldwide, cancer is a major contributor to mortality rates and a major bottleneck to extending the human lifespan.Estimates for 2020 indicate that there would be 19.3 million new cases of cancer and about 10.0 million deaths from cancer worldwide.With an expected 1.8 million fatalities (18%), lung cancer continued to be the largest cause of cancer-related mortality.This was followed by colorectal (9.4%), liver (8.3%), stomach (7.5%), and female breast (6.9%) cancers 1 .With the continuous failure of current medicines on one side and the development of drug resistance on the other, cancer offers one of the greatest challenges to medical science 2 .Topoisomerases are nuclear enzymes that temporarily break DNA strands to facilitate DNA topological manipulation 3,4 .Topoisomerase I intentionally tear off individual molecules.In order to unwind tightly wound DNA, topoisomerase II creates double-strand breaks and shuttles the strands through a nick in the strand 5 .Topoisomerases are enzymes that are present in all eukaryotic cells and are crucial to their survival 6 .One of the most effective ways to combat cancer is by inhibiting human DNA topoisomerase II.Drugs that inhibit topoisomerase II have shown great promise in the clinic, however, drug resistance in cancer cells can reduce their usefulness 7 .Combination therapy and multitarget medications have been proposed in numerous research to increase the efficacy of anticancer medication 8 .Inhibitors of topoisomerase have been used extensively in chemotherapy.Etoposide, doxorubicin, daunorubicin, mitoxantrone, teniposide, and amsacrine are some of the most widely utilised topoisomerase II inhibitors 9 .Topo II inhibitors were initially quite effective in treating the disease, but further treatments proved unsuccessful 10 .As a result, new medications targeting topoisomerase II are needed to eliminate these limitations 11 .
Computational chemistry has come a long way in the past few decades, and it is now commonly used alongside experimental methods to study organic and biological structures and reactions.Structures, characteristics of molecules, processes, and selectivity of reactions can all be better understood with the use of computations 59 .Density functional theory (DFT) is widely used to calculate many different types of molecular properties, including but not limited to molecular structures, vibrational frequencies, chemical shifts, non-linear optical (NLO) effects, natural bond orbital (NBO) analysis, molecular electrostatic potential, frontier molecular orbitals, and thermodynamic properties [60][61][62][63][64][65][66][67][68] .Herein, we detail the design, synthesis, anticancer screening, and spectroscopic analysis of a series of nitrogen glucosylated carrying 2-thioxo-4-thiazolidinone bases.The purpose of this work is to use density functional theory to analyse how alterations to molecular and electronic structure affect the biological activity of the substances under research, and to try to locate a strong correlation between theoretical data and actual observations.

Results and discussion
Chemistry 2-Thioxo-4-thiazolidinone (1) was reacted with aromatic aldehydes (2a-f) in ethanol and morpholine as secondary amine at room temperature to give 5-((Z)-arylidene)-2-thioxo-4-thiazolidinones (3a-f) (Scheme 1) [69][70][71] .The compositions of 5-((Z)-arylidene)-2-thioxo-4-thiazolidinones (3a-f) were confirmed on the basis of elemental analysis and spectroscopic data (IR, 1 H NMR, 13 C NMR and MS).The infra-red absorption spectrum of compound 3a was characterised by the presence of signals for the NH and CO groups at 3190 and 1726 cm À1 , respectively.The The silylation of the nucleoside base 1 was accomplished with bis(trimethylsilyl)acetamide (BSA) in anhydrous MeCN at 70-80 C, and furnished the trimethylsilylated derivative 4.This derivative 4 was condensed, devised by Vorbr€ uggen et al. 73 , with 1,2,3,4,6penta-O-acetyl-a-D-glucopyranose (5) in the presence of trimethylsilyl trifluoromethyanesulfonate (TMSOTf) as catalyst at 70-80 C for 60 min.The nucleosides 6 and 7 were isolated by silica gel column chromatography in 6% and 30% yields, respectively.The structures of 6 and 7 were established and confirmed by elemental analyses and spectral data (IR, 1 H NMR, 13 C NMR and MS).The absence of signal for NH and the presence of signal for the thiocarbonyl group at v max 1225 cm À1 characterised the IR absorption spectrum of compound 6.While the IR absorption spectrum of compound 7 was characterised by the absence of signal for thiocarbonyl group.The 1 H NMR spectrum of compound 6 showed the anomeric proton as a doublet at d H 6.82 ppm (J ¼ 9.30 Hz) indicating the presence of the b-D-glucopyranose moiety [35][36][37][38][39] .The 13 C NMR (CDCl 3 ) spectrum of compound 6 showed a singlet at d C 172.1 and 201.5 ppm assigned to the carbonyl at C-4 and the thiocarbonyl group at C-2, respectively.These data are also in agreement with the 13 [35][36][37][38][39] .The 13 C NMR (CDCl 3 ) spectrum of compound 7 showed a singlet at d c 186.5 and 198.83 ppm assigned to the carbonyl at C-4 and the thiocarbonyl group at C-2, respectively.These data are also in agreement with the 13 C NMR (CDCl 3 ) spectrum of hitherto known 2-methylmercapto-4-thiazolidinone (11) 70 (Scheme 2) since the methylmercapto at C-2 appears at d C 15.9 ppm, the carbonyl at C-4 appears at d C 187.1 ppm and the thiocarbonyl group at C-2 appears at d C 202.5 ppm indicating the presence of S-glycosylation.Treatment of the protected nucleoside 6 with conc.HCl/MeOH (3.5%) at 50 C for 2 h afforded in both cases the corresponding deprotected nucleoside 8 indicating N-glycosylation.In the same manner, treatment of 7 with conc.HCl/MeOH (3.5%) at 50 C for 2 h furnished hitherto known 2,4-thazolidinedione (9) 74 indicating S-glycosylation.This type of cleavage explains why we did not successful in the preparation of the corresponding deprotected nucleoside of 7 (Scheme 3).

Computational method
DFT (density functional theory) was utilised to optimise the molecule structures of the substances under study by utilising the Beck's three parameter exchange functional and the Lee-Yang-Parr non local correlation functional (B3LYP) [75][76][77] with 6-31 þ G(d,p) basis set which is implemented in Gaussian 09 program package 78 .The DFT/B3LYP combination was used to determine molecular characteristics relevant to molecular reactivity 79 .Highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO), global hardness and softness, electronegativity, electron affinity, and ionisation potential are examples of molecular characteristics [80][81][82] .
computations, the geometrical structures of the organic molecules under study are not planar.
Our calculations began with a comparison of the Z-and E-form stabilities of the compounds under study; we found that, for all molecules under study, the Z-form is more stable than the E-form by a factor of 0.003-0.062au, which is in good agreement with experimental results.Thus, we used density functional theory (DFT) computations to determine the Z-form stable structures of all molecules.
We started our calculations to make a comparison between the stability of the investigated compounds in Zand E-forms and the calculations showed that they are more stable in the Z-form than E-form by 0.003-0.062au, for all investigated molecules which is in a good agreement with the experimental observations.So, we performed DFT calculations on the stable structures of Z-form for all molecules.

Compounds 3a-f, 10 and 14
The optimised molecular structures with minimum energies obtained from the calculations of the investigated compounds are shown in Figure 1.The calculations were done to investigate the effect of substituents on the biological activity of the thioxothiazoline-4-one 3a compound.In general, it was shown from the quantum chemical parameters obtained from the calculations that the presence of substituents on thioxothiazoline-4-one increase the reactivity of the inhibitors 3b-f.
The interaction between the HOMO and LUMO levels of the reacting species determines chemical reactivity, according to the frontier molecular orbital theory, or FMO.The EHOMO and ELUMO symbols on a molecule represent its capacity to give electrons to an applicable acceptor with vacant molecular orbitals and its capacity to accept electrons, respectively.The ability of the molecule to receive electrons increases with the value of ELUMO.Comparison the reactivity between compound 3a and 3d, the insertion of methoxy group substituent increase the energy of the HOMO of 3d inhibitor by 0.017 au, which means that this compound could react as nucleophile (electron donor), Table 1.Also, many theoretical models for describing the structure and conformation barriers in molecular systems make use of the HOMO-LUMO energy gap, E, which is an important stability index.For a given molecule, the lower its value of E, the greater the likelihood that it possesses inhibitory efficiency.The calculations showed that inhibitor 3d has a smaller DE (0.132 au) than that of 3a (0.146 au), Figure 2, which means increasing the biological activity for 3d compound which agree well with experimental observations, Table 1.The dipole moment, D, the first derivative of the energy with respect to an applied electric field, was used to discuss and rationalise the structure.There is a good correlation between D and inhibition efficiency.This was shown from increasing the dipole moment.The molecule with higher efficiency, compound 3d, has higher dipole moment (5.229 D) than that of compound 3a (3.738 D) (Table 1).
Absolute hardness, g, and softness, r are important properties that measure both the stability and reactivity of a molecule.The energy gap between a hard molecule and a soft molecule is considerable for the former and small for the latter.Because they are more likely to donate electrons to an acceptor, soft molecules are more reactive than their rigid counterparts.A biological system's inhibitor serves as a Lewis base, whereas the enzyme plays the role of a Lewis acid.The increasing the softness and chemical potential of 3d inhibitor (15.383 au À1 , and À0.153 au) respectively could be responsible for increasing its biological activity more than that 3a (13.158 au À1 and À0.163 au) respectively, Table 1, which agrees well with the experimental observations.It could be observed from the calculations that the insertion of hydroxyl and methoxy groups on the oxothizlidine-4-one moiety, increases highly the reactivity of 3e molecule with respect to 3a inhibitor.This was shown by increasing the energy of HOMO, dipole, softness and decreasing the DE, and electronegativity (À0.219 au, 0.130 au, 4.616 D, 15.384 au À1 and 0.154 au) respectively, Table 1.
To compare the effect of dioxolane, 3f, and dioxane, 3c, substituents on the biological activity of inhibitor 3a, the calculations showed that inhibitors 3f and 3c have higher activity than that of unsubstituted 3a inhibitor.Moreover, the calculations showed that the dioxolane substituent has higher reactivity than that of dioxane substituent.This was shown from the decreasing the energy of the LUMO (À0.087 au) which means that 3f inhibitor has more electron accepting ability from enzyme than that of 3c compound (-0.084au), Table 1.Also, 3f has a lower energy gap, DE (0.131 au), than that of 3c (0.134) which could be responsible for increasing the reactivity of 3f more that of 3c.Meanwhile, increasing the softness and chemical potential of compound 3f (15.152 au À1 and À0.153 au, respectively) could increase its reactivity with respect to 3c with dioxane substituent.This is in a good agreement with the experimental data.
Meanwhile, one can see that the presence of dichloro-substituent on thiazlolidine-4-one moiety is slightly increased the reactivity of compound 3b with respect to compound 3a.This could be attributed to increasing the electron affinity, softness and electronegativity of 3b inhibitor (0.099 au, 13.889 au À1 and 0.172 au, respectively) more than that of 3a inhibitor (0.090 au, 13.158 au À1 and 0.163 au, respectively).Also, the lowering of DE value, which is a function of reactivity, for 3b (0.144 au) more than that of 3a (0.146 au) could be responsible for increasing the its reactivity towards the enzyme, Table 1, which is in agreement with experimental observation.
Experimental observations showed that the replacement of sulphur atom in thiazolidine-2,4-dione moiety by oxygen atom is highly decreased the biological activity of inhibitor 14 with respect to inhibitor 3a.This was confirmed from the calculations which showed the effect of substituent on the calculated quantum chemical parameters.The calculations showed the highly increasing the energy gap between HOMO-LUMO of inhibitor 14 (0.161 au) which means increasing its stability (less reactive) more than that of 3a (0.146 au), Figure 2. Also, the decreasing the energy of HOMO, dipole moment, electron affinity and softness (À0.248 au, 1.350 D, 0.087 au, and 12.500 au À1 , respectively) and increasing the electronegativity (0.167) could be responsible for the decreasing the reactivity of inhibitor 14 and accordingly decreasing its biological activity which is in a good agreement with the experimental observation.
Surprisingly, the morpholinomethyl-2-thioxothiazolidin-4-one, inhibitor 10, was found experimentally to be biological inactive which was confirmed from the calculated quantum parameters.The calculations showed that compound 10 has high energy gap, DE (0.142 au), low electron affinity (0.065 au) which probably responsible for biological inactive which agrees well with experimental data.
From the above, we could conclude the importance of computational chemistry to explain the reactivity of the investigated molecules and accordingly the biological activity.

Compounds 6 and 7
Quantum chemical calculations were performed to investigate the effect of structural parameters on the biological activity of the investigated compounds 6 and 7.It was shown from  the calculations that the geometrical structures of the investigated organic compounds are not planner.The optimised molecular structures with minimum energies obtained from the calculations of the investigated compounds are shown in Figure 3.We started our calculations to make a comparison between the stability of the investigated compounds 6 and 7 and the calculations showed that compound 6 is more stable than compound 7 by about 0.035 au. which is in a good agreement with the experimental observations.Also, the experimental data showed that the presence of sugar moiety at the nitrogen atom of thioxothiazoline-4-one moiety, compound 6, showed a higher biological activity than that at sulphur atom of the same moiety, compound 7.
The calculations showed that the efficient inhibitor 6 has a lower energy for LUMO (À0.069 au) which increase its ability to accept charges from the enzyme and accordingly increase its biological activity, which is in a good agreement with the experimental observations.It was shown from the calculations that the inhibitor 6 has the smaller HOMO-LUMO gap (0.166 au) compared with the that of molecule 7 (0.181 au), Table 1.Accordingly, it could be expected that the molecule 6 has more inclination to interact with reactivity higher than that of molecule 7 which agrees well with the experimental data.
The dipole moment, D, was used to discuss and rationalise the structure.There is a good correlation between D and inhibition efficiency.The molecule with higher efficiency, compound 6, has higher dipole moment (11.057D) than that of compound 7 (5.089D) (Table 1).Absolute hardness, g, and softness, r are important indices which measure both the stability and reactivity of a molecule.Accordingly, it was concluded from the calculations that inhibitor 6 with higher r value (12.048 au À1 ), has higher ability inhibition efficiency compared to inhibitor 7 (11.050au À1 ), Table 1, which is in a good agreement with the experimental data.Also, the calculations showed that inhibitor 6 has higher v (0.152 au), Table 1, which leads to increase its donation ability to the enzyme and accordingly enhance its inhibition efficiency.It is concluded from the above discussion that the quantum chemical parameters confirm that the inhibitor with sugar substituents on nitrogen atom of thioxothiazoline-4-one moiety has higher inhibition efficiency than that on sulphur atom which agrees well with the experimental observations.
Additionally, the HOMO and LUMO levels-two widely used quantum chemical parameters-have an impact on how a molecule interacts with other molecules.Figure 3 displays the charge density distribution for the investigated compounds at the HOMO and LUMO level.The studied compounds revealed that the HOMO levels, which could react with the biological target as a nucleophile (hydrogen bond acceptor), are primarily localised on the thioxothiazolidine-4-one moiety with the lone pairs of sulphur, oxygen, and nitrogen atoms, indicating that these moieties are the preferred sites for the attacking electrophile at the enzyme.The LUMO, which could be reacted as an electrophile (hydrogen bond donor) with the biological target is also completely localised on localised on the thioxothiazoline-4-one moiety as antibonding n-p Ã character of C-S, C-N and C-O groups which means that these moieties could be reacted as electrophile (electron acceptor).Surprisingly, the calculations showed that there is no contribution at all for sugar moiety which means that the insertion of sugar moiety will not effect on the biological activity.

Compounds 13a-c
The DFT molecular modelling calculations were performed to study the effect of inserion the sugar moiety on the sulphur atom of the thiooxothiazolin-4-one on the inhibition efficiency of the investigated compounds for enzyme.The molecular structures of the investigated compounds 13a, 13b and 13c obtained from the calculations are shown in Figure 4.By comparing between the inhibition of 3a, 3b and 3c compounds with that of 13a, 13b and 13c compounds, the calculations showed that the presence of sugar moiety decreases the biological activity which is in a good agreement with the experimental observations.Surprisingly, the calculations showed that inhibitors 3a, 3 b, and 3c have the lower energies of LUMO (À0.090,À0.099, and À0.094 au, respectively) than that of 13a, 13b and 13c (À0.077,À0.096, and À0.071 au, respectively), which means that these compounds could react as electrophiles (electron acceptor) with enzyme, Table 1.Also, the decreasing the energy gap, DE, between HOMO-LUMO for compounds 3a, 3b, and 3c (0.146, 0.144, and 0.134 au, respectively), means that those inhibitors are probably more favourable for the reactivity towards the enzyme.The increasing of the chemical potential, electronegativity and mean increasing the reactivity of the molecule and accordingly increase the biological activity, which agrees well with the experimental observations (Table 1).
Absolute hardness, g, and softness, r are important indices that measure both the stability and reactivity of a molecule.Accordingly, it was concluded that inhibitors (3a, 3b, and 3c) with higher r value (13.158, 13.884, and 14.925 au À1 , respectively) have higher ability inhibition efficiency compared to inhibitors 13a, 13b, and 13c (13.158, 13.333, and 14.885 au À1 , respectively), Table 1, which is in a good agreement with the experimental data.Also, the calculations showed that inhibitors 3a, 3b, and 3c have higher v (0.153, 0.172 and 0.151 au, respectively), which leads to increase its donation ability to the enzyme and accordingly enhance its inhibition efficiency.It was concluded from the above discussion that the quantum chemical parameters confirm that the inhibitor with sugar substituents on nitrogen atom of thioxothiazoline-4-one moiety has higher inhibition efficiency than that on sulphur atom which agrees well with the experimental observations.

Biological activities
The synthesised compounds were screened for their cytotoxicity against breast, liver and lung cancer cells using the MTT assay.As seen in Table 2, most exhibited good cytotoxicity against MCF-7, hepG2 and A549 cells with promising IC 50 values.Interestingly, compounds 6, 7 and 13a showed potent cytotoxicity with IC 50 values of (11.7, 0.21, 1.7 mM), (12.4,0.76, 0.31) and (3.1, 17.2, 6.1), respectively compared to Dox (7.67, 8.28, 6.62 mM).While some compounds were found to be not active with IC 50 values higher than 50.Hence, these compounds 6, 7 and 13a were investigated for molecular target and apoptosis activity.

Topo II and DNA intercalation assay
Compounds 6, 7 and 13a with the highest cytotoxic activity against HepG2 cells were tested against the Topo II and DNA intercalation activities to highlight their mechanistic study.As seen in Table 3, the tested compounds exhibited promising dual Topo II and DNA intercalation activities, interestingly, compound 6 had IC 50 values of 6.9 and 19.6 mM, respectively compared to Dox (IC 50 ¼ 9.65 and 31.27mM).Additionally, compounds 13a and 7 exhibited promising inhibition activity with IC 50 values of 8.3 and 9.1 mM against Topo II and 22.6 and 29.6 mM against DNA intercalation.Hence, compound 6 was further investigated for apoptotic cell death in HepG2 cells.

Apoptotic investigation of compound 6 against HepG2
Flow cytometric examination of Annexin V/PI staining was used to examine apoptotic cell death in untreated and treated HepG2 cells to determine the apoptotic activity of compound 6 (IC 50 ¼ 0.21 M, 48 h). Figure 5(A) showed compound 6 significantly activated apoptotic cell death in HepG2 cells by 80.7-fold, it induced total apoptosis by 34.73% (23.07%for early apoptosis, 11.66% for late apoptosis) compared to the untreated control group (0.43%).
Afterward, DNA flow cytometry was used to determine the cell population in each cell phase following treatment with a cytotoxic agent.As seen in Figure 5(B), compound 6 treatment significantly increased the cell population at the S-phase by 49.6% compared to control 39.15%, while cells in G1 weren't significantly increased, and cells in G2/M were decreased.Consequently, compound 6 induced apoptsis in HepG2 cells arresting the cell proliferation at S-phase.

RT-PCR of apoptosis-inducing agents against HepG2
For validating the apoptotic cell death in HepG2 cells upon treatment with compound 6, gene expression level using RT-PCR was investigated for the apoptosis-mediaged genes of P53, Bax, caspase-3,8,9 and Bcl-2 in both untreated and treated HepG2 cells.As seen in Table 4, compound 6, upregulated the apoptosis-related genes by 6.16, 5.7, 8.3, 3.29, 6.14-fold change, while it inhibted the Bcl-2 expression by 0.36-fold.Hence, these findings indicated the apoptosis-mediated cell death of compound 6 treatment through intrinsic and extrinsic pathways.

Molecular docking
Molecular docking study was conducted to highlight the mechanism of binding of the promising compound 6 against topoisomerase II enzymes, as they are important molecular drug targets and inhibitors of these enzymes are widely used as effective anticancer agents.Molecular docking using AutoDock vina software was validated by calculating the RMSD value to be 1.87 Å, and this was seen by overlying the co-crystallised ligand structuers in the selfdocking.As seen in Figure 6, binding disposition and ligand receptor interacion of compound 6 inside the protein active site with active amino acids of Arg 503, Gln 778, and Ala 779.Compound 6 was properly docked with binding energy of À11.31 Kcal/mol, and it formed a good binding interactions with Arg 503 as key amino acid.

Conclusion
We have carried out the successful synthesis of hitherto unreported 3-(2 0 ,3 0 ,4 0 ,6 0 -tetra-O-acetyl-b-D-glucopyranosyl)-2-thioxo-4thiazolidinone ( 6), 2-(2 0 ,3 0 ,4 0 ,6 0 -tetra-O-acetyl-b-D-glucopyranosyl)-2-thioxo-4-thiazolidinone (7), 3-(b-D-glucopyranosyl)-2-thioxo-4thiazolidinones (8) and their corresponding 5-(Z)-arylidene derivatives (13a-c).The conformational analyses of their most stable configurations were established by NMR spectroscopy.The antiviral and other antitumor activities of the new prepared compounds are being investigated and will be reported in due course.The nucleoside base 1 can be used as a starting material for the synthesis of other carbohydrate derivatives such as deoxy, amino and azido nucleosides.To examine the stable structure of the compounds, DFT calculations using the B3LYP/6-31 þ G (d,p) level were used to infer the electronic and geometric structures.The calculated quantum chemical parameters and the experimental findings exhibited a strong correlation.Interestingly, compounds 6 exhibited promising cytotoxicity against MCF-7, HepG2 and A549 cells with IC 50 values of 11.7, 0.21, and 1.7 mM, compared to Dox 7.67, 8.28, and 6.62 mM, respectively.For the molecular target, compound 6 exhibited topoisomerase II inhibition and DNA intercalation with IC 50 values of 6.9 and 19.6 mM, respectively compared to Dox.Additionally, compound 6 treatmnet significantly activated apoptotic cell death in HepG2 cells by 80.7-fold, arresting the cell cycle at the S-phase by 49.6% Hence, glucosylated Rhodanines may serve as a promising drug candidates against cancer through apoptosis with promising topoisomerase II and DNA interchelation.

Chemistry and analysis
Melting points have been identified on the B€ uchi apparatus and have not been corrected.TLC was performed on a silica gel of aluminium 60 F 254 (Merck) sheet, and detected by short UV light.Infra-red spectra of potassium bromide pellets were obtained using a Pye Unicam 1000 spectrometer. 1H NMR and 13 C NMR spectra were measured on a 300 MHz Bruker Advance DPX spectrophotometer in DMSO-d 6 or CDCl 3 using TMS as an internal standard.Chemical shifts are given in ppm and J in Hz.Analytical data were acquired with a C, H, N Elemental Carlo Erba 1106 analyser.Mass spectra were recorded by EI on a Varian MAT 112 spectrometer and FAB on a Kratos MS spectrometer.

General procedures for the synthesizing 3a-3f
The nucleoside bases 3a-f, were prepared form the direct condensation of 1 with the appropriate aromatic aldehydes (5a-f) according to our previous reported procedure with using morpholine instead of piperidine in the previous method 70 as the following: To a mixture of 2-thioxo-4-thiazolidinone (1) (1.33 g, 10 mmol), anhydrous morpholine (0.87 g, 10 mmol) and anhydrous EtOH (30 ml), appropriate aromatic aldehydes (2a-f) were added (11 mmol).The mixture was stirred until the starting material was consumed (12 h; TLC).The reaction mixture was diluted with water and neutralised with dilute hydrochloric acid.The yellow solid that was separated by filtration was collected and recrystallized from ethanol to give products 3a-f in quantitative yields.

General procedures for the synthesizing 13a-13c
To a mixture of the protected nucleoside 7 (0.46 g, 1 mmol), anhydrous morpholine (0.09 g, 1 mmol) and anhydrous ethanol (10 ml) was added the appropriate aromatic aldehydes (12a-c) (1 mmol).The mixture was stirred until the starting material was consumed (12 h; TLC).The reaction mixture was neutralised with HCl/MeOH.After stirring for 5 min, the solution was filtered and evaporated in vacuo and the residue was purified by flash chromatography (eluent 10-50%, ether/petroleum ether, 40-60 C) to give the products 13a-c as yellow solids.General procedures for the synthesizing 5-((Z)-2,4dichlorobenzylidene)-2,4-thiazolidindione (14)   Method A To a stirred suspension of protected nucleoside 13b (620 mg, 1 mmol) in anhydrous MeOH (15 ml) a portion of NaOMe (0.06 g, 1.1 mmol) in anhydrous MeOH (15 ml) was added at 0 C. Then the reaction mixture was stirred for 2 h at room temperature.An ion exchange resin (Amberlite IR-120, H þ -form) was added to the resulting solution, which was pre-washed with MeOH.After stirring for 5 min, the solution was filtered and evaporated in vacuo and the residue was purified by flash chromatography (0-5% elution, CHCl 3 /MeOH) to provide 214 mg (78%) of 14 as a pale yellow solid.

Method B
Protected nucleoside 13b (620 mg, 1 mmol) was suspended in MeOH (15 ml) and concentrated HCl (0.5 ml) was added.The reaction mixture was heated for 2 h at 50 C.An ion exchange resin (Amberlite IR-120, OH --form) was added to the resulting solution, which was pre-washed with MeOH.After stirring for 5 min, the solution was filtered and evaporated in vacuo and the residue was purified by flash chromatography (0-5% elution, CHCl 3 /MeOH) to provide 255 mg (93%) of 14 as a pale yellow solid. This

Cytotoxicity against cancer cells
Using RPMI-1640 medium L-Glutamine (Lonza Verviers SPRL, Belgium, cat#12-604F), we successfully cultured liver (HepG2), breast (MCF-7), and lung (A549) cells bought from the National Research Institute, Egypt.Each culture was supplemented with 10% foetal bovine serum (FBS, Sigma-Aldrich, MO, USA) and 1% penicillin-streptomycin (Lonza, Belgium).Using a 96-well plate, cells were seeded at a density of 5,000 cells per well in triplicate.The chemicals were added to the cells on day 2 at doses of (0.01, 0.1, 1, 10, and 100 M).To determine cell viability, MTT solution was used after 48 h (Promega, USA) 84 .Using GraphPad Prism 7, as was previously reported in, we computed the survival rate in comparison to the control and obtained the IC 50 values 85 .

Topo II and DNA intercalation assay
Topoisomerase II (TopoGEN, Inc., Columbus) and DNA intercalator (methyl green (20 mg) and Calfthymus DNA (10 mg) were tested for their capacity to block the enzyme's activity.Following equation was used to determine compound inhibition percentages: 100 À A control A treated À ControlÞ h i , IC 50 was determined using GraphPad prism7 using inhibition curves at five different concentrations of each compound 86 .

Investigation of apoptosis
Annexin V/PI staining and cell cycle analysis After incubation overnight, 6-well culture plates were populated with HepG2 cells (3-5 10 5 cells/well).After that, compound 6 was used to treat the cells for 48 h at concentrations that were found to be IC50 for them.Cells and medium supernatants were then collected and washed in ice-cold PBS.The next step was suspending the cells in 100 mL of annexin binding buffer solution "25 mM CaCl2, 1.4 M NaCl, and 0.1 M Hepes/NaOH, pH 7.4" and incubation with "Annexin V-FITC solution (1:100) and propidium iodide (PI)" at a concentration equals 10 mg/mL in the dark for 30 min.The Cytoflex FACS system was then used to acquire the stained cells.cytExpert was used for the statistical analysis [87][88][89] .
Real time-polymerase chain reaction for the selected genes The gene expression of P53, Bax, Caspasses-3,8,9 as proapoptotic genes and Bcl-2 as an anti-apoptotic gene were evaluated to investigate the apoptotic pathway.The IC50 values for compound 6 were then used to treat HepG2 cells for 48 h.Afterwards, the standard operating procedure called for an RT-PCR reaction to be performed.The Ct values were then used to determine the relative expression of each gene in each sample, normalised to the b-actin as house-keeping gene [90][91][92] .

In silico studies
Chimaera-UCSF and AutoDock Vina were used for molecular modelling research on a Linux-based system.Binding sites within proteins were identified by measuring the dimensions of grid boxes surrounding the co-crystallised ligands; this entire process was performed utilising Maestro to build and optimise the structures of both proteins and compounds.After standard procedures, the Topo II (PDB¼ 3qx3) protein structures were docked against the chemicals under study using AutoDock Vina 84,[93][94][95][96] .Protein and ligand structures were optimised with Vina to make them more energetically favourable.The results of the molecular docking were interpreted by the binding activities in terms of the binding energy and the interactions between the ligand and the receptor.Chimaera was then used to complete the visualisation.The Supporting Information is available free of charge at 13 C NMR, 1 H NMR, and IR spectra of the new synthesised compounds (PDF).
C NMR (CDCl 3 ) spectrum of 3-(4-morpholinomethyl)-2-thioxo-4-thiazolidinone (10) (Scheme 2).The later was prepared from the reaction of 1 with morpholine and formaldehyde in EtOH at room temperature, since the carbonyl at C-4 appears at d C 175.1 ppm and the thiocarbonyl group at C-2 appears at d C 203.2 ppm indicating the presence of N-glycosylation.The 1 H NMR spectrum of compound 7 showed the anomeric proton as a doublet at d H 5.82 ppm (J ¼ 10.4 Hz) indicating the presence of the b-D-glucopyranose moiety

Figure 1 .
Figure 1.The optimised molecular structures of the investigated inhibitors obtained from the calculations.

Figure 3 .
Figure 3.The optimised molecular structures, charge density distributions (HOMO and LUMO) for the investigated compounds 6 and 7.
Figure2.The calculated electronic transition between HOMO-LUMO for the highest and lowest biologically active 3a and 14 inhibitors.

Table 2 .
Cytotoxic IC 50 values of the tested compounds against MCF-7, HepG2 and A549 cell lines using the MTT assay.