Design, Microwave-Assisted Synthesis and In Silico Prediction Study of Novel Isoxazole Linked Pyranopyrimidinone Conjugates as New Targets for Searching Potential Anti-SARS-CoV-2 Agents

A series of novel naphthopyrano[2,3-d]pyrimidin-11(12H)-one containing isoxazole nucleus 4 was synthesized under microwave irradiation and classical conditions in moderate to excellent yields upon 1,3-dipolar cycloaddition reaction using various arylnitrile oxides under copper(I) catalyst. A one-pot, three-component reaction, N-propargylation and Dimroth rearrangement were used as the key steps for the preparation of the dipolarophiles3. The structures of the synthesized compounds were established by 1H NMR, 13C NMR and HRMS-ES means. The present study aims to also predict the theoretical assembly of the COVID-19 protease (SARS-CoV-2 Mpro) and to discover in advance whether this protein can be targeted by the compounds 4a–1 and thus be synthesized. The docking scores of these compounds were compared to those of the co-crystallized native ligand inhibitor (N3) which was used as a reference standard. The results showed that all the synthesized compounds (4a–l) gave interesting binding scores compared to those of N3 inhibitor. It was found that compounds 4a, 4e and 4i achieved greatly similar binding scores and modes of interaction than N3, indicating promising affinity towards SARS-CoV-2 Mpro. On the other hand, the derivatives 4k, 4h and 4j showed binding energy scores (−8.9, −8.5 and −8.4 kcal/mol, respectively) higher than the Mpro N3 inhibitor (−7.0 kcal/mol), revealing, in their turn, a strong interaction with the target protease, although their interactions were not entirely comparable to that of the reference N3.


Introduction
Coronaviruses (CoVs) tend to have a high zoonotic potential and according to the World Health Organization (WHO), these viral diseases have emerged as a serious health issue to the world [1]. Consequently, the WHO declared a state of global health emergency to coordinate scientific and medical efforts to rapidly develop a cure for patients [2]. The discovery of an antiviral drug is of immense importance in the current spread of the rapidly modifiable SARS-CoV-2. The aim of the present study was to develop an antiviral drug against the novel COVID-19 virus [3]. One of the ways that several research teams are thinking is to highlight promising molecules and drug compounds by making a virtual screening via molecular docking of drugs approved by the FDA, certain natural substances, and synthetic heterocyclic compounds such as pyranopyrimidinones linked the ways that several research teams are thinking is to highlight promising molecules and drug compounds by making a virtual screening via molecular docking of drugs approved by the FDA, certain natural substances, and synthetic heterocyclic compounds such as pyranopyrimidinones linked to isoxazoles for probable therapeutic outcome. In this context, antiviral compounds have seriously attracted the attention of organic medicinal chemists, who have thought of chemically modifying them in different ways in order to achieve much more active structural analogues.
On the other hand, heterocyclic scaffolds play a central role in drug discovery and development, thus constituting the key structural component of a majority of biologically active moieties. Conjugated pyranes are heterocyclic compounds arousing great interest due to their multiple biological activities, such as antimicrobial [9], anti-tumor [10], influenza inhibition [11] and antiviral (Figure 1, 1-C,1-D) [12]. On the other hand, pyrimidines are among the most important nitrogenous heterocyclic classes in medicinal chemistry due to their various extensive biological and therapeutic activities [13]. In this context, the condensed derivatives of pyrimidines are very attractive targets because of their various pharmacological effects, such as anti-tubercular [14], antitumor [15], antityrosinase [16], antiproliferative [17], anti-inflammatory [18] and antiviral ( Figure 2 2-A [19], 2-B [20]).
Furthermore, isoxazole derivatives possess various biological activities such as antiinflammatory [21], antileishmanial [22], trypanocidal [23], antimicrobial [24,25] and antiviral ( Figure 2, 2-C [26],D [27]). On the other hand, pyrimidines are among the most important nitrogenous heterocyclic classes in medicinal chemistry due to their various extensive biological and therapeutic activities [13]. In this context, the condensed derivatives of pyrimidines are very attractive targets because of their various pharmacological effects, such as anti-tubercular [14], antitumor [15], antityrosinase [16], antiproliferative [17], anti-inflammatory [18] and antiviral ( Figure 2, 2-A [19], 2-B [20]). Furthermore, isoxazole derivatives possess various biological activities such as antiinflammatory [21], antileishmanial [22], trypanocidal [23], antimicrobial [24,25] and antiviral ( Figure 2 In addition, numerous target-based virtual screenings were performed to discover promising protease inhibitors against SARS-CoV-2. In this context, a recent study showed that high throughput virtual screening of more than 10,000 molecules, based on the SARS-CoV-2 M pro structure, resulted in the identification of six small inhibitors molecules [28], one of which is a pyrimidine derivative (Figure 3, 3-A [28]) and the other contains the isoxazole nucleus ( Figure 3   In addition, numerous target-based virtual screenings were performed to discover promising protease inhibitors against SARS-CoV-2. In this context, a recent study showed that high throughput virtual screening of more than 10,000 molecules, based on the SARS-CoV-2 M pro structure, resulted in the identification of six small inhibitors molecules [28], one of which is a pyrimidine derivative (Figure 3, 3-A [28]) and the other contains the isoxazole nucleus ( Figure 3 In addition, numerous target-based virtual screenings were performed to discover promising protease inhibitors against SARS-CoV-2. In this context, a recent study showed that high throughput virtual screening of more than 10,000 molecules, based on the SARS-CoV-2 M pro structure, resulted in the identification of six small inhibitors molecules [28], one of which is a pyrimidine derivative (Figure 3, 3-A [28]) and the other contains the isoxazole nucleus ( Figure 3    In addition, the junction of the pyrimidine ring with different heterocyclic fragments in the same molecule could lead to a new category of hybrid molecules with pronounced biological power. In this context, and in the continuity of looking for new bioactive pyrimidine compounds [31][32][33], we report herein the synthesis of a new series of hybrid compounds 4, in three steps (Scheme 1), under conventional heating conditions and by microwave irradiation. We have chosen to integrate into the same molecule the naphthalene, pyran, pyrimidine and isoxazole fragments assigned as antiviral and anti-SARS-CoV-2 agents, as indicated above, with the aim to perform their virtual screening, using molecular docking studies, hoping to find out promising protease inhibitors against COVID-19. In addition, the junction of the pyrimidine ring with different heterocyclic fragments in the same molecule could lead to a new category of hybrid molecules with pronounced biological power. In this context, and in the continuity of looking for new bioactive pyrimidine compounds [31][32][33], we report herein the synthesis of a new series of hybrid compounds 4, in three steps (Scheme 1), under conventional heating conditions and by microwave irradiation. We have chosen to integrate into the same molecule the naphthalene, pyran, pyrimidine and isoxazole fragments assigned as antiviral and anti-SARS-CoV-2 agents, as indicated above, with the aim to perform their virtual screening, using molecular docking studies, hoping to find out promising protease inhibitors against COVID-19.

Chemistry
Our key starting materials, 2-amino-3-cyanonaphthopyranes 1a-c, were synthesized by adopting the method of Zayane et al. [34]. It is a multicomponent reaction of equimolar amounts of arylaldehyde, malononitrile and β-naphthol at the reflux of a hydroalcoholic solution in the presence of CuI as a catalyst. Their structures were confirmed on the basis oftheir 1 H and 13 C NMR spectra and by comparison with literature data [35].
Condensation reaction, of the last precursors, with acetic anhydride in the presence of polyphosphoric acid (PPA) under reflux for 2 h gives pyrimidinones 2a-c in yields ranging from 66% to 80%. From a mechanistic point of view, the reaction is carried out in two steps. The precursors 1a-c react with acetic anhydride to give the non-isolable intermediates 1 a-c which cyclize to 1"a-c after nucleophilic attack at the nitrile group. Dimroth-type intramolecular rearrangement [32,36] of the intermediate thus formed leads to pyranopyrimidinones 2a-c, which reacted with propargyl bromide in the presence of sodium hydride in anhydrous DMF at room temperature to give the corresponding N-propargylated dipolarophiles 3a-c in good yields (80-92%) (Scheme 2) [37]. The formed N-propargylatednaphthopyranopyrimidinones 3a-c were identified on the basis of their 1 H NMR and 13 C NMR spectra. The 1 H NMR spectra of these dipolarophiles allows to see, in addition to the signals of the protons introduced by the naphthopy-ranopyrimidinones2a-c, the disappearance of a singlet of the NH proton at δH12.60-12.61 and the appearance of three new signals, two doublets of doublets are observed at δH 4.58-4.61 and 5.08-5.12 (J=15.9, 2.4 Hz) which were attributed to methylenic protons (CH2) and a characteristic triplet of an acetylenic proton observed at δH 2.29-2.33 (J=2.4 Hz). The proximity of methylene to the nitrogen atom bearing four different substituents (hybridized sp3) including its free doulet perfectly conjugated with the carbonyl of pyrimidinone is at the origin of the non-isochrony, and thus the non-equivalence of the methylenic protons which logically appear at two different chemical shifts. Moreover, the 13 C NMR spectra of these derivatives exhibited the appearance of new signals at δC31.7-32.7, 71.8-72.4 and 75.3-76.1 relative to new carbons C4′, C5′ and C6′introduced by the propargyl moiety, The formed N-propargylatednaphthopyranopyrimidinones 3a-c were identified on the basis of their 1 H NMR and 13 C NMR spectra. The 1 H NMR spectra of these dipolarophiles allows to see, in addition to the signals of the protons introduced by the naphthopyranopyrimidinones 2a-c, the disappearance of a singlet of the NH proton at δ H 12.60-12.61 and the appearance of three new signals, two doublets of doublets are observed at δ H 4.58-4.61 and 5.08-5.12 (J = 15.9, 2.4 Hz) which were attributed to methylenic protons (CH 2 ) and a characteristic triplet of an acetylenic proton observed at δ H 2.29-2.33 (J = 2.4 Hz). The proximity of methylene to the nitrogen atom bearing four different substituents (hybridized sp3) including its free doulet perfectly conjugated with the carbonyl of pyrimidinone is at the origin of the non-isochrony, and thus the non-equivalence of the methylenic protons which logically appear at two different chemical shifts. Moreover, the 13 C NMR spectra of these derivatives exhibited the appearance of new signals at δ C 31.7-32.7, 71.8-72.4 and 75.3-76.1 relative to new carbons C 4 , C 5 and C 6 introduced by the propargyl moiety, respectively.
In order to access, via 1,3-dipolar cycloaddition reaction as shown in Scheme 3, to the new expected hybrid molecules 4a-l, the N-propargylated derivatives 3a-c were treated with various arylnitrile oxides, under conventional heating in refluxing DMF for 8-12 h (Method A) or under microwave irradiation in DMF for 3 to 5 min (Method B), both in the presence of copper(I) catalyst and Et 3 N as a base.
Until now, copper catalyzed nitrile oxide-alkyne click chemistry (CuAAC) is one of the methods of choice for regiospecific access to 3,5-disubstituted isoxazole derivatives [38].  This reaction sequence generates the new hybrid molecules 4a-l in moderate (method A/58-69%) to excellent yields (method B/91-97%). The absence of any other compound in the reaction mixture implies the strong regiospecificity of this reaction.
These resulting 3,5-regioisomers were confirmed from the NOE H-5 isoxazole /H methylene and H-5 isoxazole /H arom , and the non-observation of any NOE H methylene /H arom . Thus, this 3,5-regiospecificity was explained by the uses of copper(I) catalyst.
The comparison of the obtained results shown in Table 1 from the conventional heating method (method A) and those from the MW-assisted synthesis way (method B) showed in all cases much shorter reaction times and higher yields were achieved under MW irradiation compared to classical heating. This reaction sequence generates the new hybrid molecules 4a-l in moderate (method A/58-69%) to excellent yields (method B/91-97%). The absence of any other compound in the reaction mixture implies the strong regiospecificity of this reaction.
These resulting 3,5-regioisomers were confirmed from the NOE H-5isoxazole/Hmethylene and H-5isoxazole/Harom, and the non-observation of any NOE Hmethylene/Harom. Thus, this 3,5regiospecificity was explained by the uses of copper(I) catalyst.
The comparison of the obtained results shown in Table 1 from the conventional heating method (method A) and those from the MW-assisted synthesis way (method B) showed in all cases much shorter reaction times and higher yields were achieved under MW irradiation compared to classical heating.
The structures of the newly prepared compounds 4a-l were raised on the basis of their spectroscopic measurements. In fact, compared to compounds 3, the 1 H NMR spectra of isoxazoles derivatives 4a-l indicate the disappearance of the signal at δH 2.30-2.32 relative to the terminal acetylenic proton and the appearance of a new singlet at δH 6.50-6.51 (s, 1H) attributable to H2′of the isoxazole ring, in addition to signals at δH6.92-7.98 attributable to the protons of the aromatic moiety linked to the isoxazole system. Moreover, the 13 C NMR spectra reinforced the structures of 4a-l by showing the signals of the formed isoxazole moiety, as well as those from the dipolarophiles3a-c.
The ES-HRMS of compounds 3 and 4 showed the correct protonated molecular ion peaks [M+H] + that complied with the proposed structures. The structures of the newly prepared compounds 4a-l were raised on the basis of their spectroscopic measurements. In fact, compared to compounds 3, the 1 H NMR spectra of isoxazoles derivatives 4a-l indicate the disappearance of the signal at δ H 2.30-2.32 relative to the terminal acetylenic proton and the appearance of a new singlet at δ H 6.50-6.51 (s, 1H) attributable to H 2 of the isoxazole ring, in addition to signals at δ H 6.92-7.98 attributable to the protons of the aromatic moiety linked to the isoxazole system. Moreover, the 13 C NMR spectra reinforced the structures of 4a-l by showing the signals of the formed isoxazole moiety, as well as those from the dipolarophiles 3a-c.
The ES-HRMS of compounds 3 and 4 showed the correct protonated molecular ion peaks [M+H] + that complied with the proposed structures.

Molecular Docking Study
By studying the binding pocket of the SARS-CoV-2 M pro , it was found that the cocrystallized native inhibitor (N3) is located inside the pocket of the main protease receptor asymmetrically. The N3 ligand is a designed inhibitor for SARS-CoV-2 M pro that was built from amino acids based on the M pro pocket amino acids and cannot used medicinally. Molecular docking of the synthesized molecules (4a-l) and N3 inhibitor into the main protease binding site was performed. The binding energies and interaction details (number of interactions, number of interacting amino acids, interacting amino acids and hydrogen bonds) of ligands with the target enzyme are presented in Table 2.
All compounds were perfectly placed in the active site by variable scores and binding interactions with the amino acids of the receptor pocket. From the docking results ( Table 2), All the tested compounds (4a-l) achieved promising binding scores ranging from −7.6 to −8.9 kcal/mol, compared to the docked co-crystallized N3 inhibitor with a binding score of −7.0 kcal/mol. The synthesized compounds (4a-l) showed a much higher binding affinity towards M pro enzyme (PDB: 6LU7) than N3.
It is worth mentioning that, especially for compounds 4a, 4e and 4i, the obtained binding modes were quite similar to that of the docked N3 inhibitor, as depicted in Table 3. Moreover, these three selected compounds exhibited binding scores of −8.4, −8.5 and −8.5 kcal/mol, respectively, higher than of the docked co-crystallized N3 inhibitor (−7.0 kcal/mol) ( Table 2).
The Although compounds 4k, 4h and 4j did not exhibit very comparable interactions to those of N3 towards SARS-CoV-2 M pro , they, on the other hand, exhibited lower binding energy (−8.9, −8.5 and −8.4 kcal/mol, respectively) ( Table 2). This finding can be explained, in part, by the nature of interactions between these compounds and the amino acids that constitute SARS-CoV-2 M pro (Table 4) It is important to mention that the nature of the substituents allows the molecule to be placed into space in a well-defined conformation. Indeed, these substituents with different sizes promote or disadvantage possible free rotations, they also increase or decrease the electron density of certain groups essentially the aromatic ones by their indicative and/or mesomeric electronic effects. The spatial arrangement of the two benzene rings in compounds 4a-1 depends on the nature of their substituents (R and R 1 ). This arrangement can, in certain cases, inhibit the delocalization of the electronic doublets by mainly mesomeric effects towards the outside of the ring (case of the aromatic system carried by the isoxazole nucleus), which influences the interaction of these aromatic systems with certain amino acids. Moreover, the R and R 1 substituents do not need to engage in direct interactions with amino acids, but their sizes and electronic effects require special interactions of the corresponding aromatic rings with special amino acids. As mentioned in Table 3, the benzene ring at position C-6 of the pyran moiety and that carried by isoxazole both unsubstituted in 4a exhibit Pi-sulfur interactions with MET-49 (bond length: 5.38 Å) andMET-165 (bond length: 5.23 Å), respectively. The same observation was noted with compounds 4e and 4i where the aromatic ring carried by the pyran is para-methylated and para-chlorinated, respectively and the benzene ring carried by the isoxazole in both dervivatives is unsubstituted. This result can be explained by the effect of the size of the methyl group and the chlorine atom on the spatial arrangement of the aromatic ring throughout the molecule. In addition, the inductive donor effect (+ I) exerted by the methyl group and the two opposite electronic effects (-I) and (+ M) exerted by the chlorine atom appear to lead to practically the same result. It was observed that in the case of compound 4h, the benzenic nucleus attached to an isoxazole carrying in para position a chlorine atom (R 1 ) exerts two Pi-donor Hydrogen bonds interactions with ASN-142 (bond length: 3.65 Å). These types of interactions were not observed in the rest of the products with other R and R 1 which proves that the nature of these two substituents give the entire molecule a well-defined conformation which also guarantees well-defined interactions. With R = Cl and R 1 = OMe, the compound 4k, takes a new conformation which allows, among other things, the OMe group to engage in a hydrogen bond with THR-26 (bond length: 3.19 Å).

General Experimental Procedures
All synthesis reactions were checked by TLC using aluminium sheets of silica gel 60 F254, 0.2 mm. Melting points were measured on an electrothermal 9002 apparatus and were reported uncorrected. NMR spectra were run on a Bruker AC-300 spectrometer at 300 MHz ( 1 H) and 75 MHz ( 13 C). All chemical shifts were reported as δ values (ppm) relative to residual non-deuterated solvent. Mass spectra were recorded with HRMS-ES using the reflectron mode in the positive ion mode. Microwave-assisted synthesis was peformed in a Start Synth multimode microwave instrument producing controlled irradiation at 2.45 GHz (Milestone S.r.l., Sorisole, Italy). The device is equipped with an industrial magnetron and a microwave diffuser providing a continuous microwave output power from 0 to 1400 W. An open reaction vessel was employed in all reactions.
The starting materials 1 and 2 were prepared according to the literature [32].
3.1.2. General Procedure for the Preparation of Naphthopyranopyrimidinone N-propargylated Derivatives 3a-c 1 mmol of naphthopyranopyrimdinone 2a-c was dissolved in 15 mL of anhydrous DMF then 2 equivalents of sodium hydride (NaH) were added and the reaction mixture was stirred at room temperature under an argon atmosphere for 30 min; afterwards, 1.5 eq of propargyl bromide was added slowly, and the mixture was stirred for another 1.5 h 4h 4j

General Experimental Procedures
All synthesis reactions were checked by TLC using aluminium sheets of silica gel 60 F254, 0.2 mm. Melting points were measured on an electrothermal 9002 apparatus and were reported uncorrected. NMR spectra were run on a Bruker AC-300 spectrometer at 300 MHz ( 1 H) and 75 MHz ( 13 C). All chemical shifts were reported as δ values (ppm) relative to residual non-deuterated solvent. Mass spectra were recorded with HRMS-ES using the reflectron mode in the positive ion mode. Microwave-assisted synthesis was peformed in a Start Synth multimode microwave instrument producing controlled irradiation at 2.45 GHz (Milestone S.r.l., Sorisole, Italy). The device is equipped with an industrial magnetron and a microwave diffuser providing a continuous microwave output power from 0 to 1400 W. An open reaction vessel was employed in all reactions.
The starting materials 1 and 2 were prepared according to the literature [32].
3.1.2. General Procedure for the Preparation of Naphthopyranopyrimidinone N-propargylated Derivatives 3a-c 1 mmol of naphthopyranopyrimdinone 2a-c was dissolved in 15 mL of anhydrous DMF then 2 equivalents of sodium hydride (NaH) were added and the reaction mixture was stirred at room temperature under an argon atmosphere for 30 min; afterwards, 1.5 eq of propargyl bromide was added slowly, and the mixture was stirred for another 1.5 h

General Experimental Procedures
All synthesis reactions were checked by TLC using aluminium sheets of silica gel 60 F 254 , 0.2 mm. Melting points were measured on an electrothermal 9002 apparatus and were reported uncorrected. NMR spectra were run on a Bruker AC-300 spectrometer at 300 MHz ( 1 H) and 75 MHz ( 13 C). All chemical shifts were reported as δ values (ppm) relative to residual non-deuterated solvent. Mass spectra were recorded with HRMS-ES using the reflectron mode in the positive ion mode. Microwave-assisted synthesis was peformed in a Start Synth multimode microwave instrument producing controlled irradiation at 2.45 GHz (Milestone S.r.l., Sorisole, Italy). The device is equipped with an industrial magnetron and a microwave diffuser providing a continuous microwave output power from 0 to 1400 W. An open reaction vessel was employed in all reactions.
The starting materials 1 and 2 were prepared according to the literature [32].
3.1.2. General Procedure for the Preparation of Naphthopyranopyrimidinone N-Propargylated Derivatives 3a-c 1 mmol of naphthopyranopyrimdinone 2a-c was dissolved in 15 mL of anhydrous DMF then 2 equivalents of sodium hydride (NaH) were added and the reaction mixture was stirred at room temperature under an argon atmosphere for 30 min; afterwards, 1.5 eq of propargyl bromide was added slowly, and the mixture was stirred for another 1.5 h under positive atmosphere of argon. Upon completion of the reaction, monitored by TLC, we added distilled water and the formed precipitate was collected by filtration, washed with water and purified by flash column chromatography on silica gel (EtOAc/PE, 1:1, as an eluent). water molecules. Then, the polar hydrogens were added followed by appending Kollman charges. Hence, the grid box with dimensions of 40 × 40 × 40 points, spacing of 1.0 Å and centered with coordinates x: −11.993, y: 15.425, and z: 65.951, was generated based on N3 binding position in the target protein binding site. The molecular docking analysis of N3 and the synthesized compounds (4a-l) were performed using AutoDock Vina software (The Scripps Research Institute, La Jolla, CA, USA) [39]. Molecule-enzyme interactions were drawn and construed by employing the Biovia Discovery Studio Visualizer, BIOVIA, San Diego, CA, USA (2017).

Conclusions
In conclusion, we have successfully synthesized in this work a new series of 3,5isoxazole linked naphthopyranopyrimidinones 4a-l, in three steps, using the naphtopyranyl moiety as a building blocks support, via 1,3-dipolar cycloaddition reaction using arylnitrile oxides catalyzed by Cu(I) under classical and microwave irradiation conditions. The last condition offers several advantages including good-to-high yields, cleaner products, and a reduction in reaction time, making this method particularly attractive. In addition, to help fight COVID-19, molecular docking based on virtual screening was performed to identify among the newly synthesized compounds 4a-1 which ones has the potential to interact with COVID-19 M pro . Our results demonstrate that compounds 4a, 4e, 4h, 4i, 4j and 4k, exhibited good binding affinity towards the main protease of COVID-19 (M pro ) better than that made by the native co-ligand N3 inhibitor. These molecules could possibly be promising in terms of therapeutic effect against SARs-CoV-2. However, in vitro and then in vivo investigation are essential to validate our prediction results and pave the way for the discovery of anti-SARs-CoV-2 drugs.