Synthesis and Molecular Docking Analysis of New Thiazo-isoindolinedione Hybrids as Potential Inhibitors of the SARS-CoV-2 Main Protease

Herein, we report the synthesis of novel thiazo-isoindolinedione derivatives in excellent yields (up to 92%) from the reaction of thiazolidinedione and isoindoline-dione. The structures of the novel compounds were elucidated by 1 H, 13 C-NMR, and MS analyses. Furthermore, molecular docking analysis was performed to study the potential inhibition of the SARS-CoV-2 main protease (M pro ) by the new thiazo-isoindolinediones. The present study revealed that the new thiazo-isoindolinediones could inhibit the M pro and represent a promising platform for the experimental development of new antiviral drugs based on thiazo-isoindolinedione scaffolds.


INTRODUCTION
In December 2019, the World Health Organization (WHO) declared COVID-19, a disease caused by the coronavirus 2 (SARS-CoV-2), a global health emergency 1,2 .According to the WHO statistics, SARS-CoV-2 resulted in over 33 million infections and caused more than 1 million deaths 3 .There is currently no approved specific treatment for COVID-19; however, immunization can reduce the risk of severe illness and death.The essential chymotrypsin-like cysteine protease (M pro ) is among the potential targets proposed for SARS-CoV-2 inhibition, which plays a crucial role in viral transcription and replication [4][5][6] .Within this context, heterocyclic compounds have been extensively studied as potential lead inhibitors of the SARS-CoV-2 M pro owing to their diverse biological properties, including antiviral, antiparasitic, and antimicrobial activities, making them among the most investigated pharmacologically active scaffolds 7 .
Fur ther more, nitrogen-and sulfurbased heterocycles demonstrated favorable binding affinities to various biological targets due to their ability to form exceptionally high intermolecular interactions via the nitrogen and sulfur heteroatoms 8 .In this regard, isoindolinediones serve as a core structure for several medically essential agents.Furthermore, they are commonly utilized as starting building blocks for synthesizing alkaloids, pesticides, and polymers 9 .In addition, isoindoline-diones derivatives also manifested potent pharmaceutical properties, such as antiviral, anti-inflammatory, anticancer, and anti-HIV properties 10 .Within this context, isoindolinedione scaffolds were used to synthesize the a-glucosidase inhibitor I 11 .Furthermore, isoindoline II is a potent papainlike cysteine protease (P Lpro ) 11 inhibitor.Moreover, norcantharimide III is a bioactive isoindoledione with potential antitumor activity against breast and lung cancers 12 .The 1,3-isoindolinedione tethered triazole IV possessed a promising antituberculosis mycobacterium activity 13 .
Interestingly, combining bioactive pharmacophores targeting different pathways into a single compound is a major challenge for developing and discovering novel drugs acting simultaneously on multiple targets 18 .This strategy has shown considerable success and is currently employed to develop new therapies for diseases such as tuberculosis, malaria, anticancer, and Alzheimer's diseases 19,20 .
Within this context, we envisage the synthesis of novel thiazo-isoindolinedione hybrids.The synthetic strategy involves a nucleophilic substitution reaction key step between thiazolidine-2,4-dione and bromo-substituted N-alkyl phthalimides.The target compounds are designed to comprise thiazolidine-2,4-dione and 1,3-isoindolinedione linked together by three carbon atoms, as illustrated in Fig. 2. Additionally, a molecular docking tool will be used to explore the chemical and electrical properties of the new compounds to inhibit the M pro required for SARS-CoV-2 replication.

The synthesis of compound 8
Compound 7 (1.2 mmol) and compound 4 (1 mmol) (1 mmol) were dissolved in DMF (10 ml) and heated at 80 o C for 4 hours.TLC was used to monitor the process, and after completion, the reaction was poured onto ice to give a white powder.Compound 8 was obtained from the reaction of compound 4 (1 mmol, 253 mg) with thiazolidine-2,4-dione potassium salt 7 (1.2 mmol, 186 mg) in DMF (10 mL) at 80 o C for 4 hours.The reaction was followed by TLC (EtOAc/heptane 1:3; Rf=0.32), isolated as a white solid with 88% yield (and its m.p. =158-159 o C. 1

The synthesis of compound 9
Compound 9 was obtained from the reaction of compound 7 (1.2 mmol) with compound 5 (1 mmol) in DMF (10 mL) at 80 o C for 4 hours.The mixture was cooled to room temperature and then poured over ice to give a white powder.

In silico studies Molecular docking
The novel two thiazo-isoindolinedione hybrids 8 and 9 were subjected to a molecular docking study using the MOE software 25,26 to investigate their potential inhibitory effect on the SARS-CoV-2 M pro .Besides, the co-crystal (O6K) was inserted as a reference standard in the docking process.
Each examined compound was sketched in the ChemDraw and then transferred to the MOE window, subjected to partial charge corrections and energy minimization, as mentioned before 27 .Next, the target Mpro protein receptor of SARS-CoV-2 was extracted from the Protein Data Bank (PDB ID: 6Y2G, https://www.rcsb.org/structure/6Y2G)and opened in the MOE window.The M pro protein was corrected and 3D hydrogenated before energy minimization as a final step of protein preparation 28 .Finally, a general docking process was performed by inserting a database of compounds 8 and 9 with the co-crystal (O6K) of SARSCoV-2 M pro .The default setting options were adjusted to match the selected docking methodology 29 .
Notably, a validation process by redocking O6K of SARSCoV-2 Mpro within its receptor pocket was carried out, and the validly applied forcefield was confirmed by obtaining low Root Mean Square Deviation (RMSD) values<2 Å 30.

Synthesis and characterization
Thiazo-isoindolinedione hybrids 8 and 9 were synthesized according to the synthetic Scheme 1.The condensation of phthalic anhydride with 2-aminoethyl bromide hydrobromide (2) and 3-aminopropyl bromide hydrobromide (3) under neat conditions afforded compound 4 and compound 5. Furthermore, the reaction of chloroacetic acid and thiourea in water afforded the corresponding 2,4-thiazolidinedione 6.The latter is converted to the corresponding potassium salt via reaction with KOH at room temperature and in ethanol.The nucleophilic substitution reaction of the potassium salt 7 with the bromo derivatives 4 and 5 afforded the corresponding compound 8 and 9 in 88% and 92% yields, respectively.
docking study to investigate their potential inhibitory effect on the SARS-CoV-2 M pro .Besides, the co-crystal (O6K) was inserted as a reference standard in the docking process.
Observing the O6K binding mode, it was clear that Glu166 and Cys145 are the most crucial amino acids to produce their inhibitory potential towards the SARS-CoV-2 M pro .The docked O6K achieved a binding score of -8.41 kcal/mol (RMSD=1.58Å) and could bind Glu166, Asn142, and Gly143 with three hydrogen bonds.On the one hand, compound 8 showed a binding score of -5.83 kcal/mol (RMSD=1.16Å).It bound crucial amino acids (Glu166 and Cys145) with two pi-hydrogen interactions and one hydrogen bond, respectively.On the other hand, compound 9 interacted with Glu166 (two hydrogen bonds) and Met165 (one pi-hydrogen bond), Table 1.Its binding score was recorded at -6.05 kcal/mol (RMSD=1.50Å), superior to compound 8. Based on the above, compound 9 with the three carbons bridge (propylene) between the 1,3-dioxoisoindoline and thiazolidine-2,4-dione moieties was superior to compound 8 with the two carbons bridge (ethylene) as SARS-CoV-2 M pro inhibitor.This may be attributed to the flexibility of compound 9, which produced more and deeper fitting within the SARS-CoV-2 M pro target receptor.In silico studies

Molecular docking
The novel two thiazo-isoindolinedione hybrids 8 and 9 were subjected to a molecular CONCLUSION I n t h i s s t u d y, w e d e s i g n e d a n d synthesized new thiazo-isoindolinedione hybrids from readily available starting materials and in good yields (up to 92%).The chemical structures of the new compounds were characterized by IR, 1 H-and 13 C-NMR, and MS techniques.In addition, a molecular docking study clarified that compound 9 with the three carbons bridge (propylene) between the 1,3-dioxoisoindoline and thiazolidine-2,4-dione moieties was superior to compound 8 with the two carbons bridge (ethylene) as SARS-CoV-2 M pro inhibitor.This may be attributed to the flexibility of compound 9, which produced more and deeper fitting within the SARS-CoV-2 Mpro target receptor.