Anticancer, Antioxidant Activities and Molecular Docking Study of Thiazolidine -4-one and Thiadiazol Derivatives

Liver cancer accounts for a major portion of the global cancer burden. In many nations, the prevalence of this condition has risen in recent decades. New series of thaiazolidinones and thaiadiazolidine have been designed, synthesized, and evaluated for potential antioxidant and antihepatocarcinogenic activity. The antioxidant activity of synthesized compounds was evaluated using a DPPH assay. Furthermore, we examined the compounds against HepG2 cells using MTT assay, ow cytometry analysis through the cell cycle, reactive oxygen species, and apoptosis. The result showed that compound 6b has the highest antioxidant activity with IC 50 =60.614±0.739 µM. The anticancer activity showed that compounds 5 and 6b have signicant toxicity against liver cancer cells HepG2, IC50 values (9.082 and 4.712) µM, respectively. Flow cytometry experiments revealed that compound 5 arrested HepG2 cells in the S process, while compound 6b arrested HepG2 cells in the G1. Compound 6b had a greater reduction in reactive oxygen species and late apoptosis than compound 5. Substantially, compound 5 had anity energies of -7.6 and -8.5 for Akt and CDK4 proteins, respectively, but compound 6b had anity energies of -7.8 and -10.1 for Akt1 and CDK4 proteins, respectively. Consequently, compound 6b had lower binding energies than compound 5. In this work, we used multiple bioinformatics methods to shed light on the prospective therapeutic use of these series as novel candidates to target immune cells in the tumor microenvironment of hepatocellular carcinomas such as CD8+ T cells, endothelial cells, and hematopoietic stem cells. The results of antioxidant, anticancer, molecular docking studies, and bioinformatic analysis showed that compound 6b has a potential impact and could be developed for drug discovery with further research. Metastases in 1 to 3 axillary lymph nodes) using TCGA database.


Introduction
The incidence of cancer is increasing, according to the annual report of the Health Organization, as there are millions of new cases [1]. Since 1980, the incidence of liver cancer has more than quadrupled, while the mortality rate has more than doubled. In the United States, there are expected to be 42,230 new cases and 30,230 deaths from liver cancer in 2021( https://www.cancer.org). Liver hepatocellular carcinoma (LIHC) is fatal cancer with no viable therapy. The Cyclin-dependent kinases 4/6 (CDK4/6) and PI3K/AKT signaling pathways are important in carcinogenesis and offer prospective therapeutic options for LIHC [2]. Akt1 ablation has been shown in genetic research in mice to signi cantly reduce hepatic carcinogenesis.
In terms of Akt1, a 2019 study found that Akt1-mediated phosphorylation of mTORC2 is critical for starting hepatocarcinogenesis in humans and animals, as it activates c-Myc, thus promoting cellular growth [3]. Strong evidence suggests that CDK4 is a clinical prognostic marker for LIHC patients [4]. As a result, the development of novel potential medicines targeting AKT1 and CDK4 may represent an unmet medical need. Tumor microenvironment (TME) is a dynamic biological environment surrounded by tumors that include macrophages, stroma, broblasts, dendritic cells, stem cells, lymphocytes, pericytes, adipocytes, and blood vessels [5]. However, no studies on the connection between AKT1 and CDK4 and invading immune cells in the TME have been undertaken.
Given these ndings, the current study's goal is to synthesize thaiazolidinone and thaiadaiazoline derivatives. The synthesized heterocyclic compounds were characterized using spectroscopic methods such as IR, 13C, 1H NMR, and mass spectroscopy. They were then evaluated as antioxidants, anticancer (HepG-2), molecular docking studies of a target protein Akt1, and CDK4, and bioinformatic analysis of immune cell in ltration in the TME of LIHC patients.

Antioxidant potential
For many living things, oxidation is necessary for the production of energy [34]. However, the continually generated free radical reactive oxygen (ROS) species may destructively cause RNA, DNA, causing mutations, chromosomal damage, and the oxidation of unsaturated fatty acids. ROS promotes heart disease, neurological disease, cancer, and aging, and contributes to enhancing oxidative damage [35]. However, antioxidants help protect the body from free radical damage and are necessary for the human body to scavenge the radicals are produced from mitochondria leak, pollution, sunlight, ultraviolet ray, and smoking [36].
Several antioxidant scans are collected under the subject of HAT or single electron transfer (SET). The DPPH concentration reduce with time and change in color from purple to yellow and/or colorless owing to the transfer of the atomic hydrogen, was used in the measurements of scavenging activity of studied compounds (2,3a,3b, 3c, 3d, 3e, 4, 6a, and 6b) at concentrations of 50, 75, 100, and 200 µM. At a concentration of 200 µM, the inhibition activity was (9.02% -77.778%). The inhibition activity percentage of all studied compounds is shown in Figure 1 and data were collected in Table 1. Table 1 In-vitro antioxidant inhibition activities of compounds (2,3a,3b, 3c, 3d, 3e, 4, 6a, and 6b Several antioxidant scans are gathered under the topic of hydrogen atom transfer (HAT) or single electron transfer (SET). Their action can be clari ed through the oxidative mechanism of phenolic antioxidants. The values of the scavenging activity of the synthesized compounds (2,3a,3b, 3c, 3d, 3e, 4, 6a, and 6b) at concentrations 50, 75, 100, and 200 µM, were measured by the decrease of DPPH absorbance at 517 nm with time and the change of the DPPH color from purple to yellow or colorless due to the transfer of hydrogen atom. The inhibition activity at a concentration of 200µM within the range (9.02% -77.778% ), the inhibition percentage activity of the studied compounds shows in gure 1, and data was gathered in Table 1.
The inhibitions activity of compounds 6a, 6b, and 2 were quite high (200 µM) in DPPH. While the other compounds exhibited less inhibitory activity of DPPH, due to for absence of the phenolic OH group, which provided the 6a, 6b, and 2 compounds with a strong radical activity of scavenging by giving radicals of DPPH hydrogen atom and inhibiting radical activity with HAT [37]. Consequently, the radical scavenging activity of compounds 6a, 6b, and 2 were in the order: 6a> 6b>.2> The half-maximal inhibitory concentration (IC50) for the studied compounds was calculated by

Anticancer activity
Liver cancer is one of the most common malignant tumors in the world [38]. The fast growth in liver cancer incidence, lack of adequate treatment have led us to look for new and more e cient molecules [39].
Because of the action of the thaiazolidinone and thaiadaiazoline rings, as well as their ability to communicate with biological targets, they have a variety of biological applications, including antimicrobial and antitumor [40,41]. As a result, it may be a starting point for developing new anticancer agents. We tested ve new thaiazolidinone and thaiadaiazoline derivatives against liver cancer cells in this study (HepG2). The compounds had varying anticancer activity against the HepG2 cell line, gure (2 ) shows the IC 50 Figure 4 depicted the impact of compounds 5 and 6b on reactive oxygen species, DCFH+ for the control was 90.8%, which was increased to 92.8% by using the IC50 value of compound 5, and raised to 96.0% by using the IC50 value of compound 6b. This increase in DCFH ratio compared to the control revealed that compound 6b has a greater impact on HepG2 cells than compound 5, which was improved by the apoptosis experiment. Figure 3(A) shows that the IC50 value of compound 5 has a late apoptosis ratio (Q2) of about 15.1%, compared to the control late apoptosis ratio of 42.1%, and compound 6b increased the late apoptosis ratio to 19.6% when compared to live cells (Q4). Therefore, these results suggest that compounds 5 and 6b are potential cytotoxic agents. One reason for apoptosis might be the observed capacity of compounds to cause cell cycle arrest. The principal regulatory mechanisms for cell growth and proliferation are known as cell cycle and apoptosis. When speci ed control points of the cell cycle are detained, apoptotic cell death is triggered [42]. Accordingly, numerous anti-cancer drugs lead to cell cycle stoppage and clinically effective cancer treatment has been demonstrated [43]. This suggests that compound 6b has a stronger impact on HepG2 cells than compound 5. Compound 5 has an amino group bonded to a thaiadaiazoline ring, and free of the hydroxyl group. Whereas compound 6b has two aromatic rings bonded to a thaiadaiazoline ring and two hydroxyl groups. This range of functional groups boosts the compound's e cacy against liver cancer cells, paving the way for further research.

Molecular docking results
Molecular Bioinformatics Docking studies in the eld of drug development are currently of considerable use, reducing the money and efforts required to screen novel compounds by directing and restricting the research to possible targets/targets. A molecular docking simulation is, thus, an essential way of anticipating a substrate interacting with its receptor.
To determine whether compounds 5 and 6b inhibits Akt1 and CDK4 proteins, thus, affecting cellular migration of HepG2, molecular docking simulation was carried out to determine the binding mode of 5 and 6b into the Akt1 and CDK4 active site. The 5 and 6b molecules were precisely docked into the active pocket of Akt1 and CDK4 in ADT software [44].
The results of the docking study showed a good t into the binding of CDK4 protein active site with a nity energy -8.7 KJ/mol (6b ) and -7.9 (5), the a nity energy of Akt1 protein with compound 6b ( -10.0 KJ/mol). Moreover, the binding interactions between the amino acid residue and ligands (compounds 5 and 6b) are shown in Table 2 and Figures (5and 6 ). shown in gure 5 Compound 5 formed two hydrogen bonds, as a side-chain acceptor and backbone acceptor with amino acids (GLU A75 and LYS A180) residue of CDK4 protein. In addition, several hydrophobic interactions with amino acid residue include Lys A72, Gin B183, Glu A69, Lys B180and Thr B184. Compound 6b ( gure 5) formed four hydrogen bonds as a side-chain acceptor with amino acid (GLU A69, Thr A184, GLU A69, and GLU A75) and ionic bond with GLU A69 of CDK4 protein, in addition, hydrophobic interactions with amino acid residue including Leu B188, Gin B261, Ser B258, Leu A65, and Cys A68. The interaction of compound 6b with Akt1 target protein ( gure 6) shows two hydrogen bonds from binding with critical amino acid residues Trp A80, and Lys A268 as well as ionic interaction with Asp A292, Moreover, the hydrophobic interactions with amino acid residue including Tyr B205, Thr A82, Gin Arg B273, Val B270, Ser B205, Arg B273, and Leu B264.

Molecular Dynamics simulation
Molecular dynamics is an analytical approach that uses computer simulation to analyze the physical motions of a predetermined amount of time is provided for the interaction of the atoms and molecules to reveal the system's complicated development. The design and discovery of novel medicines, it has shown to be a helpful tool. As a result of molecular dynamics research, thermodynamics and kinetics related to the compound-protein recognition and binding may be more precisely estimated. Root mean square deviation (RMSD), Root mean square uctuation (RMSF), and Radius of gyration data as a function of time were used to analyze the molecular dynamic simulations [45]. In addition, Fig. (7) demonstrated that compound 6b-CDK4 complex has a higher ratio of water-binding with 17 amino acids for the active site of CDK4 as well as few hydrogen bonds. compound 5-CDK4 complex demonstrated a higher ratio of hydrogen bonds and water binding. As a result, compound 6b may have an active effect on CDK4, agreeing with the IC 50 value. RMSD and RMSF were used to analyze the molecular dynamic simulations, and assess internal motions conformation change and stability of compounds 5-CDK4, 6b-CDK4, 5-ART1, and 6b-ART1. The RMSD and RMSF values were shown in (Fig. 8, 9). In the compound 6b-CDK4 complex the scalar distance between atoms show an upward trend uctuation from 0-25 nanosecond and the downward trend uctuation from 25-100 nanosecond, the value of RMSD 1.7 Å, compound 5-CDK4 complex show an upward from 20-40, and 70-90 nanosecond, and downward from 40-70 nanosecond, the value of RMSD 1.6 Å. while compound 5-AKT1, and 6b-AKT1 (Fig. 8C, D)

Bioinformatic results and discussion:
In the current study, we utilized UALCAN as a bioinformatics tool to highlight the expression of AKT1 and CDK4 in LIHC based on sample type (normal/primary tumor) and nodal metastasis status (N0: No regional lymph node metastasis; N1: Metastases in 1 to 3 axillary lymph nodes) using TCGA database. Substantially, our ndings showed that AKT1 was expressed at much greater levels in primary tumors than in normal tissues( Figure 10A)and more abundant in N1 than in N0 and normal( Figure 10B).
Similarly, CDK6 was highly expressed in primary tumors than in normal tissues( Figure 11A)and more considerable in N1 than in N0 and normal ( Figure 11B). Our bioinformatic analysis showed that AKT1 and CDK4 are promising a druggable target to eradicate LIHC. As a consequence of the foregoing ndings, it is indicated that the proposed compounds can combat LIHC and additional research on compound 6b is advised. We anticipate that the study will give ample references towards the development of a suitable scaffold for the LIHC.

Chemistry
The synthesis of two heterocyclic series of ( thiazolidine-4-one and thiadiazole) derivatives are performed in multi-steps( Scheme 1 and 2), Initially, prepared thiosemicarbazone (1), then was treated with chloroacetic acid as a cyclized agent to prepare the rst series from thaiazolidin-4-one [46]. The derivatives of thiazolidine-4-one (3a-e) were prepared by the reaction of a corresponding aromatic aldehyde with thiazolidine-4-one and sodium acetate as a reagent. the next series of thiadiazole was prepared from the reaction thiosemicarbazone with acetic anhydride [28], the product was treated with hydrazine monohydrate to hydrolysis the acetyl groups and prepare 2-amino thiadiazoline (5), the product was reacted with the corresponding aldehyde to form thiadiazole derivatives (6a and 6b).

Spectral characterization
The IR spectra of the synthesized compounds exhibits show bands of expected functional groups, the IR spectrum of thiosemicarbazone (47,48), shows two bands at 3412 and 3346 cm -1 attributed to symmetric and asymmetric stretching of an amine group, strong band at 1087cm -1 attributed to stretching vibration of C=S, the thiosemicarbazone behavior has been reported in the literature (49)(50)(51).
Thiazolidine-4-one (2) shows a strong band within the range υ (1705-1770) cm -1 attributed to C=O, the absence of absorption bands of NH 2 and C=S group due to participate to form the thiazole ring with the synchronous appearance of new bands assigned to the carbonyl group within range υ (1705-1770) cm -1 .
The IR spectrum of thaiadiazoline (4)  Comparison of thiazolidine-4-one chemical shifts with thiosemicarbazide indicates that the NH2-proton signal in the spectrum is absent. The disappearance of NH2 proton signals from the 1 H NMR Thiazolidine-4-one spectrum supports cyclization of the thiosemicarbazone, which is con rmable by the appearance simultaneously of new signals of the ve-membered ring. the spectra of (3a-3e) Thiazolidine-4-one derivatives display new signals with range (6.89-7.20) ppm attributed to C=CH of coupled groups with an absence of -CH 2 -protons of cyclic rings. Also, the spectra display signals of aromatic protons and protons signals of coupled groups. Thaiadiazolin (4) is produced from cyclization of the thiosemicarbazone. the spectrum shown two signals at 2.03 and 2.20 ppm attributed to OCH 3 also display signal at 11.73 ppm attributed to NH proton, all protons of cyclic, aromatic, and methyl can be seen In the predicted region, the Comparison of Thaiadiazolin (4) chemical shifts with thiosemicarbazide indicates that the NH 2 proton signal in the spectrum is absent. This con rms the formation of the Thaiadiazolin ring. The spectra of compound 5 display a new signal at 6.23 ppm attributed to NH 2 protons with absent the signals of methyl protons, This supports the acetamide (HN-COCH 3 ) is hydrolysis and formed primary amine. The Thaiadiazolin derivative (6a-6b) spectra display the signal of azomethine within range (10.3-11.22) ppm and signals of phenolic protons within range (10.3-11.22) ppm as well as signals of aromatic protons.
The 13 C NMR spectra of synthesized compounds, the 13 C NMR spectrum of thiosemicarbazide (1) display signal at δ 178.3 ppm attributed to C=S carbon, also exhibits signal from C=N carbon at δ 149.5ppm. Furthermore, the 13 C NMR spectra give good evidence of cyclization of thiosemicarbazide through the absence of C=S from the thiazolidine-4-one spectrum, and a new signal appears at 33.2 ppm attributed to the C-S carbon signal from thiazolidine-4-one. Also, the spectrum of Thaiadiazolin (4) displays a signal at 85.6ppm attributed to C-S, it is from the cyclization of thiosemicarbazide through the absence of C=S from the spectrum. as well as the signals of C=O at 167.9, 170 ppm. the 6a and 6b spectra display azomethine (CH=N ) carbon signal 164.6 ppm and 166.5 ppm respectively. All other carbon assignments were as anticipated (54). Mass spectrometry was used to determine the molecular ion peaks of synthesized compounds. The peak intensity provides information about the stability of fragments, especially with the base peak; the molecular ion con rms the proposed structural elucidation.

Conclusions
A new class of thiazolidine-4-one and thiadiazol derivatives have been designed and synthesized (1,2, 3a-3e, 4, 5, and 6a-6a). Using the DPPH method, we assessed the scavenger activity of compounds and discovered that compound 6b has the highest antioxidant activity, followed by compound 5. Furthermore, the cytotoxic activity of compounds 5 and 6b against HepG2 liver cancer cells revealed that compounds 5 and 6b are more toxic. Compound 5 arrested HepG2 cells in the S phase, while compound 6b resulted in a cellular arrest in the G1 phase, and both compounds decreased ROS and apoptosis. Docking studies with proteins Akt1 and CDK4 improved the results of compounds 5 and 6b. Docking studies revealed that both compounds make good contacts with protein binding sites. As a result, these two compounds should be developed as lead compounds for new liver cancer agents that inhibit Akt1 and CDK4.

Instrumentation and spectral measurements
The IR spectra were collected by using FTIR-A nity -1 spectrophotometer in the region 4000-400 cm − An equimolar amount of thiazolidine-4-one (2) and the corresponding aldehyde (0.2 m.mol) Dissolved in glacial acetic acid (20ml), then added anhydrous sodium acetate (1.2g) the was heated at 120 o C in an oil bath, the reaction mixture was monitored by TLC using (chloroform: ethanol) (8:2 v/v), after cool down to room temperature, the mixture reaction was poured to (100ml) ice water and kept it overnight, the solid product was ltered, recrystallization from an appropriate solvent.

Dpph Radical Scavenging Assay
The antioxidant activity of all the synthesized compounds was an evaluation by determining the ability of radical scavenging according to the Blois method [55]. The inhibitory activity of DPPH was measured by The cell line was mainly grown in 96-well plates at 1x105 cells per well for 24 hours under optimum conditions (37°C, 5% CO2 in a humidi ed incubator). Next, the cells were washed twice with PBS after the removal of the growth medium (10% FBS). New culture media containing the studied compounds at a concentration (5,10,25,25 ) μM were added to the cells followed by incubation for24, 48, and 72 h.
Quintet wells were analyzed for each concentration. A 10μL solution of freshly prepared (5 mg/mL) MTT in PBS was added to each well and then incubated for an additional 4 hours. After successful incubation, the resulting medium was aspirated and the MTT formazan which has been generated in this step was dissolved in the 100 μL of DMSO. The solubilization of formazan crystals was obtained by gently shaking the plates. A microplate reader was then employed to measure the absorbance at 545 nm. The cellular toxicity percentage, as well as the half-maximal inhibitory concentration (IC 50

Apoptosis
Flow cytometry analysis, which was adapted from the Wang et al. method [60], was used to identify early and late apoptosis. Compounds 5 and 6b IC 50 values were applied to HepG2 cells for 48 hours. The cells were collected, rinsed twice with PBS, and labeled with 5 ul FITC-conjugated annexin V, as directed by the manufacturer. After being incubated in the dark for 10 minutes and then labeled with PI. Samples were then analyzed on a ow cytometer (Beckman Coulter, Epics XL).

Molecular Docking
As usual, the receptor input les were created. To begin, we removed any water molecules, ligand atoms, and ions that did not belong to the receptor's active site from the PDB le. After that, hydrogen atoms, protons, and partial charges from amino acid side chains were added (following the protonation state at the physiological). This was followed by a local minimization to relieve potential bad contacts.
Minimization was performed in the presence of restraints to maintain the protein conformation. The structure of ligands (compound 5 and 6b) was drawn using Chem Draw Ultra from the Chem O ce software package. Then, it was copied into Chem3D Ultra (same program package), before using ADT, it's a good idea to check that the le contains all hydrogen atoms. ADT now immediately computes Gasteiger charges (empirical atomic partial charges) and distinguishes between hybridization states and atom groups after opening the ligand. The software speci es the ligand's rotatable bonds as part of the preparation process so that different conformers for docking can be produced. Where it was subjected to a simpli ed energy minimization search to a minimum root mean standard deviation gradient of 0.100.
The obtained structure with local minimum was saved in convenient mol2 format.
Docking studies [61].was confuted using Auto Dock vena to obtain binding interaction between the ligand (compound 5 and 6b ) and binding pockets of two distinct proteins of liver cancer cell (HepG-2). From the protein data bank ( https://www.rcsb.org/ ). The crystal structure of Hepg-2 proteins was obtained: Akt1 (PDB: ID 5KCV) [62], and CDK4 (DPB: ID 2W96) [63]. Energy minimization and hydrogen bonding optimization were carried out after docking. For each ligand the docking simulation was run multiple times for further study, the highest binding scores were used in addition. Discovery studio visualizes 2019 was used to do a thorough examination of the docking effects.

Statistical Analysis
IC 50 values of the compounds were performed by plotting dose-response curves vs. the concentrations using GraphPad Prism version 8.1 for windows. The experiment was repeated thrice.

Bioinformatic Analysis
To highlight many discoveries linked to our targets AKT1and CDK4, we used a variety of bioinformatics approaches. First, UALCAN is a web-based resource for cancer research that is comprehensive, userfriendly, and interactive. UALCAN is meant to give users quick access to publically available TCGA data, allowing them to nd biomarkers and perform in silico validation of possible genes of interest, as well as display expression pro les and patient survival statistics in graphs and plots for various cancer types [64].
Second, TIMER is a comprehensive resource for studying immune in ltrates in malignancies of diverse types methodically. We used the latest version of the website to estimate the amount of immune in ltrates using multiple immune deconvolution algorithms, produce high-quality gures dynamically, and examine tumor immunological, clinical, and genetic features in-depth in the current study [5].
We utilized TIMER as a bioinformatic tool to show the Kaplan-Meier curves for the associated immune in ltrates (CD8+ T cells, CD4+ T  into low and high levels of in ltration. Furthermore, the gene module displays a scatter plot and the purity-adjusted spearman's rho to visualize the association of AKT1 and CDK4 expression with immune in ltration level in LIHC. Because most immune cell types are negatively related to tumor purity, tumor purity is a signi cant confounding factor in our investigation [65].
We also used the gene outcome modules to generate Kaplan-Meier curves to show the clinical signi cance of AKT1 and CDK4 expression in LIHC patients. We utilized the Cox proportional hazard model to assess the outcome signi cance of gene expression in this study [66].
Third, Cycle base 3.0 is a web-based database that incorporates new mRNA and protein expression data, as well as cell-cycle phenotypic information from high-content screens and model-organism databases used in genome-wide cell-cycle research [67]. We evaluated the role of AKT1 and CDK4 expression during distinct stages of the cell cycle in the current proposal.