Synthesis of New bis -furanyl-pyrrolo[3,4-b ]pyridin-5-ones via the Ugi-Zhu Reaction and Docking Studies on the Main Protease (M Pro ) from SARS-CoV-2

: The synthesis of ﬁve new bis -furanyl-pyrrolo[3,4-b ]pyridin-5-ones in 30 to 40% yields through a domino sequence based on the Ugi-Zhu three-component reaction is described. Then, on the main protease M Pro (PDB: 6lu7) from the SARS-CoV-2, the synthesized products and co-crystallized ligands of M Pro were in silico evaluated using the docking technique, ﬁnding moderate to good binding energies and some interesting interactions, demonstrating that the ligand 8c can be considered as a drug candidate against the SARS-CoV-2-M Pro due to its LE value ( − 5.96 kcal/mol), which is better than other synthesized and reported molecules in the literature. At the same time, hydrophobic interactions play a crucial role in the ligand target molecular couplings, demonstrated through a hydrophobicity surfaces analysis. Finally, 8a and 8b can also be considered as drug candidates. Thus, some synthesized bis -furanyl-pyrrolo[3,4-b ]pyridin-5-ones may be used for further in vitro assays against the virus.


Introduction
With the current pandemic COVID-19, it has become increasingly necessary to develop design-synthesis strategies based on robust computational methods behind understanding the response of the SARS-CoV-2 towards new candidate polyheterocyclic compounds for further in vitro antiviral activity studies.The rational drug design is a very valuable tool that minimizes the relationship between trial and error, providing some de novo design of antiviral compounds, including the use of nanocarriers [1][2][3].Thanks to advances in areas such as organic physical chemistry, theoretical chemistry, computational chemistry, and pharmacochemistry, amazing algorithms can be implemented behind predict the interactions between an organic ligand and its biological receptor.A widely used methodology is the molecular docking, a simulation method that is complemented with results from in vitro assays to evaluate new compounds with potential biological activity.In February 2020, the main protease from SARS-CoV-2 M pro (PDB code: 6lu7) was published [4], which has an important role in both replication and transcription of virus [5].
Moreover, heterocyclic chemistry is a branch of organic chemistry of special interest.Heterocycles are found in a wide variety of products such as drugs, natural products, agrochemicals, copolymers, materials, among others [6].It is known that 90% of new drugs contain one or more heterocycles in their structure, so their importance in medicinal chemistry is crucial [7].Isoindolin-1-one is a synthetic platform with biological properties incorporated into several products of pharmaceutical interest [8] (Figure 1a).An azaanalogue of this heterocyclic moiety is the pyrrolo [3,4-b]pyridin-5-one, found in many biological active compounds [9], analogues of natural products [10], and in some anticancer agents [11] (Figure 1b).Also, furan ring is contained in antibacterial, enzymatic inhibitors, antibiotics, antifungals, and so on [12,13] (Figure 1c).
Chem.Proc.2022, 8, 84 2 of 10 complemented with results from in vitro assays to evaluate new compounds with potential biological activity.In February 2020, the main protease from SARS-CoV-2 M pro (PDB code: 6lu7) was published [4], which has an important role in both replication and transcription of virus [5].Moreover, heterocyclic chemistry is a branch of organic chemistry of special interest.Heterocycles are found in a wide variety of products such as drugs, natural products, agrochemicals, copolymers, materials, among others [6].It is known that 90% of new drugs contain one or more heterocycles in their structure, so their importance in medicinal chemistry is crucial [7].Isoindolin-1-one is a synthetic platform with biological properties incorporated into several products of pharmaceutical interest [8] (Figure 1a).An aza-analogue of this heterocyclic moiety is the pyrrolo [3,4-b]pyridin-5-one, found in many biological active compounds [9], analogues of natural products [10], and in some anticancer agents [11] (Figure 1b).Also, furan ring is contained in antibacterial, enzymatic inhibitors, antibiotics, antifungals, and so on [12,13] (Figure 1c).Multicomponent reactions (MCRs) are powerful tools to synthesize quickly and efficiently novel polyheterocyclic compounds.MCRs belong to one-pot process and can be based on the use of isocyanides as principal reagents due to their duality as nucleophiles and electrophiles.MCRs are carried out directly or coupled to further transformations, for instance, ionic, pericyclic, radical-mediated, or metal-catalyzed [14].Isocyanide-based multicomponent reactions let afford a wide variety of complexes polyheterocyclic whom applied in drug discovery [15].A classic example that incorporates isocyanides in MCRs is three and four component-Ugi reaction [16].Within the Ugi-type reactions, it has been recently published a variant of the Ugi reaction that has been studied in our research group called the Ugi-Zhu three-component reaction (UZ-3CR), useful mainly to Multicomponent reactions (MCRs) are powerful tools to synthesize quickly and efficiently novel polyheterocyclic compounds.MCRs belong to one-pot process and can be based on the use of isocyanides as principal reagents due to their duality as nucleophiles and electrophiles.MCRs are carried out directly or coupled to further transformations, for instance, ionic, pericyclic, radical-mediated, or metal-catalyzed [14].Isocyanide-based multicomponent reactions let afford a wide variety of complexes polyheterocyclic whom applied in drug discovery [15].A classic example that incorporates isocyanides in MCRs is three and four component-Ugi reaction [16].Within the Ugi-type reactions, it has been recently published a variant of the Ugi reaction that has been studied in our research group called the Ugi-Zhu three-component reaction (UZ-3CR), useful mainly to synthesize 5-aminooxazoles that can act as synthetic platforms toward more complex polyheterocycles.The UZ-3CR involves the combination of aldehydes 1, amines 2, and aminoacid-derived isocyanides 3 in protic or aprotic solvents to assemble just the oxazole core 4.This MCR are eventually promoted by Lewis or BrØnsted acids [17] (Scheme 1).
synthesize 5-aminooxazoles that can act as synthetic platforms toward more complex polyheterocycles.The UZ-3CR involves the combination of aldehydes 1, amines 2, and aminoacid-derived isocyanides 3 in protic or aprotic solvents to assemble just the oxazole core 4.This MCR are eventually promoted by Lewis or BrØnsted acids [17] (Scheme 1).

Chemistry
To synthesize the desired bis-furanyl-pyrrolo [3,4-b]pyridin-5-ones 8a-e, the UZ-3CR was coupled to a cascade process: aza-Diels-Alder/N-acylation/aromatization (decarboxylation/dehydration) into a one pot process.The first attempt was carried out under the Zhu's classic conditions [18], and then optimized in our research group [19,20].It was found that initially dichloromethane as solvent and scandium triflate as Lewis acid catalyst worked well.However, the solvent used to synthesize all products was toluene due to better solubility observed for all reagents.It was necessary first to synthesize the isocyanides 3 from their corresponding aminoacids in three steps as reported by Zhu and coworkers [18].With the isocyanide in hands, according to the Ugi-Zhu mechanism, a condensation occurs between aldehydes 1a-b and amines 2a-b to access to imines 5a-e, which become in Lewis' iminium ions 6a-e after reacting with catalytic amount of scandium(III) triflate.The later intermediates are nucleophilically attacked by the isocyanides 3a-b to give the 5-aminooxazoles 4a-e via a non-prototropic chain-ring tautomerization.Subsequently, after adding maleic anhydride (7), an aza-Diels-Alder cycloaddition coupled into a cascade process N-acylation/aromatization (decarboxylation/dehydration) takes place to generate the corresponding pyrrolo [3,4-b]pyridin-5-ones 8a-e in 24 to 40% yields (Scheme 2).It is worth highlighting the structural complexity of each product, as well as the formation of several new C-C and C-N bonds while leaving only one molecule of water and one of carbon dioxide, which denotes a high atomic economy and a green approach of this synthetic methodology.Also, the use of furan-substituted starting materials for the structural decoration of the pyrrolo [3,4-b] pyridin-5-one nucleus had not been reported yet in the literature.

Chemistry
To synthesize the desired bis-furanyl-pyrrolo[3,4-b]pyridin-5-ones 8a-e, the UZ-3CR was coupled to a cascade process: aza-Diels-Alder/N-acylation/aromatization (decarboxylation/dehydration) into a one pot process.The first attempt was carried out under the Zhu's classic conditions [18], and then optimized in our research group [19,20].It was found that initially dichloromethane as solvent and scandium triflate as Lewis acid catalyst worked well.However, the solvent used to synthesize all products was toluene due to better solubility observed for all reagents.It was necessary first to synthesize the isocyanides 3 from their corresponding aminoacids in three steps as reported by Zhu and co-workers [18].With the isocyanide in hands, according to the Ugi-Zhu mechanism, a condensation occurs between aldehydes 1a-b and amines 2a-b to access to imines 5a-e, which become in Lewis' iminium ions 6a-e after reacting with catalytic amount of scandium(III) triflate.The later intermediates are nucleophilically attacked by the isocyanides 3a-b to give the 5-aminooxazoles 4a-e via a non-prototropic chain-ring tautomerization.Subsequently, after adding maleic anhydride (7), an aza-Diels-Alder cycloaddition coupled into a cascade process N-acylation/aromatization (decarboxylation/dehydration) takes place to generate the corresponding pyrrolo[3,4-b]pyridin-5-ones 8a-e in 24 to 40% yields (Scheme 2).It is worth highlighting the structural complexity of each product, as well as the formation of several new C-C and C-N bonds while leaving only one molecule of water and one of carbon dioxide, which denotes a high atomic economy and a green approach of this synthetic methodology.Also, the use of furan-substituted starting materials for the structural decoration of the pyrrolo [3,4-b] pyridin-5-one nucleus had not been reported yet in the literature.

In Silico Study
Once molecular docking was performed on the active site of the SARS-CoV-2-M Pro , which has been reported for our research group [1-3], the studied molecules were positioned as Figure 3 shows.

In Silico Study
Once molecular docking was performed on the active site of the SARS-CoV-2-M Pro , which has been reported for our research group [1][2][3], the studied molecules were positioned as Figure 3 shows.

In Silico Study
Once molecular docking was performed on the active site of the SARS-CoV-2-M Pro , which has been reported for our research group [1][2][3], the studied molecules were positioned as Figure 3 shows.The docking results show that all of the synthesized molecules in the current work present exergonic interactions with the SARS-CoV-2-M Pro (Table 1).Analyzing the results and considering that the more negative values indicate, the more stable ligand-target formed system, it is clear that 8c is the molecule with a higher interaction with the SARS-CoV-2-M Pro , with a ligand efficiency (LE) value of -5.96 kcal/mol.Note that P8 is a designed molecule by Díaz-Cervantes and co-workers, with a high ligand-target interaction against the SARS-CoV-2, and co-crystallized molecules are ligand experimental tested.Although the above molecules present high interactions, 8c shows a better LE than the studied molecules depicted in Table 1.The docking results show that all of the synthesized molecules in the current work present exergonic interactions with the SARS-CoV-2-M Pro (Table 1).Analyzing the results and considering that the more negative values indicate, the more stable ligand-target formed system, it is clear that 8c is the molecule with a higher interaction with the SARS-CoV-2-M Pro , with a ligand efficiency (LE) value of −5.96 kcal/mol.Note that P8 is a designed molecule by Díaz-Cervantes and co-workers, with a high ligand-target interaction against the SARS-CoV-2, and co-crystallized molecules are ligand experimental tested.Although the above molecules present high interactions, 8c shows a better LE than the studied molecules depicted in Table 1.8b and 8a show a LE of −5.90 and −5.89 kcal/mol, respectively, which are almost degenerate with 8c.This result denotes that the other synthesized molecules can also be considered potential drugs against the COVID-19.
The Table 1 shows that smallest size of 8c is a determinant factor to promotes a ligantarget interaction, due to H bond, electro and VdW energies are not the best for 8c compared with the other studied ligands.
In this order, the hydrophilic interactions have been analyzed; see the Figure 4, which demonstrates that the hydrophobicity then promotes one essential interaction in this kind of target.The hydrophobic side of the target confers a higher site in the center of the cavity, which promotes better interactions by 8c.
P8 [1] − The Table 1 shows that smallest size of 8c is a determinant factor to promotes a ligantarget interaction, due to H bond, electro and VdW energies are not the best for 8c compared with the other studied ligands.
In this order, the hydrophilic interactions have been analyzed; see the Figure 4, which demonstrates that the hydrophobicity then promotes one essential interaction in this kind of target.The hydrophobic side of the target confers a higher site in the center of the cavity, which promotes better interactions by 8c.

Conclusions
Considering the docking assay, the present studies demonstrate that 8c can be considered as a drug candidate against the SARS-CoV-2-M Pro due to its LE value (−5.96 kcal/mol), which is better than other synthesized and reported molecules.At the same time, the hydrophobic interaction plays a key role in the ligand targe molecular couplings, demonstrated through a hydrophobicity surfaces analysis.Finally, 8a and 8b can also be considered as drug candidates.

Conclusions
Considering the docking assay, the present studies demonstrate that 8c can be considered as a drug candidate against the SARS-CoV-2-M Pro due to its LE value (−5.96 kcal/mol), which is better than other synthesized and reported molecules.At the same time, the hydrophobic interaction plays a key role in the ligand targe molecular couplings, demonstrated through a hydrophobicity surfaces analysis.Finally, 8a and 8b can also be considered as drug candidates.

General Information, Instrumentation and Chemicals
1 H and 13 C NMR spectra were acquired on a Bruker Advance III (500 MHz) spectrometer.The solvent was deuterated chloroform (CDCl 3 ).Chemical shifts are reported in parts per million (δ/ppm).Internal reference for NMR spectra is in respect to tetramethyl silane (TMS) at 0.0 ppm.Coupling constants are reported in Hertz (J/Hz).Multiplicities of the signals are reported using the standard abbreviations: singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m).NMR data were treated using the MestReNova software (12.0.0-20080).Microwave-assisted reactions were performed in closed-vessel mode on a CEM Discover MW-reactor (Matthews, North Carolina, CA, USA).Reaction progress was monitored by thin-layer chromatography (TLC) and the spots were visualized under ultraviolet (UV) light (254 or 365 nm).Flash columns packed with silica-gel 60 and glass preparative plates (20 × 20 cm) coated with silica-gel 60 doped with UV indicator (F254) were used to purify the products.Mixtures of hexanes (Hex) and ethyl acetate (EtOAc) in 1:1 or 1:2 (v/v) proportion were used to run TLC, silica-gel columns, preparative plates, and to measure the retention factor (Rf) values (using the same mobile phase for all of the experiments).All starting reagents and solvents were used as received (without further purification, distillation, nor dehydration).Chemical structures were drawn using the ChemDraw Professional software (Ver.15.0.0.106,Perkin Elmer Informatics, Cambridge, MA, USA).The purity for all synthesized products (>95%) was assessed by NMR.

Figure 4 .
Figure 4. Hydrophobicity surfaces for 8c.Red and blue surfaces represent the hydrophilic and hydrophobic surfaces of the SARS-CoV-2-M Pro , respectively.Green and orange sticks are the hydrophilic and hydrophobic moieties of 8c.

Figure 4 .
Figure 4. Hydrophobicity surfaces for 8c.Red and blue surfaces represent the hydrophilic and hydrophobic surfaces of the SARS-CoV-2-M Pro , respectively.Green and orange sticks are the hydrophilic and hydrophobic moieties of 8c.

Table 1 .
Main interaction energies between the studied molecules and the SARS-CoV-2-M Pro *.