Green and efficient one-pot three-component synthesis of novel drug-like furo[2,3-d]pyrimidines as potential active site inhibitors and putative allosteric hotspots modulators of both SARS-CoV-2 M^Pro and PL^Pro

Graphical abstract

Abstract

In this paper, an environmentally benign, convenient, and efficient one-pot three-component reaction has been developed for the regioselective synthesis of novel 5-aroyl(or heteroaroyl)-6-(alkylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-diones (4a‒n) through the sequential condensation of aryl(or heteroaryl)glyoxal monohydrates (1a‒g), 1,3-dimethylbarbituric acid (2), and alkyl(viz. cyclohexyl or tert-butyl)isocyanides (3a or 3b) catalyzed by ultra-low loading ZrOCl₂•8H₂O (just 2 mol%) in water at 50 ˚C. After synthesis and characterization of the mentioned furo[2,3-d]pyrimidines (4a‒n), their multi-targeting inhibitory properties were investigated against the active site and putative allosteric hotspots of both SARS-CoV-2 main protease (M^Pro) and papain-like protease (PL^Pro) based on molecular docking studies and compare the attained results with various medicinal compounds which approximately in three past years were used, introduced, and or repurposed to fight against COVID-19. Furthermore, drug-likeness properties of the mentioned small heterocyclic frameworks (4a‒n) have been explored using in silico ADMET analyses. Interestingly, the molecular docking studies and ADMET-related data revealed that the novel series of furo[2,3-d]pyrimidines (4a‒n), especially 5-(3,4-methylendioxybenzoyl)-6-(cyclohexylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4g) as hit one is potential COVID-19 drug candidate, can subject to further in vitro and in vivo studies. It is worthwhile to note that the protein–ligand-type molecular docking studies on the human body temperature-dependent M^Pro protein that surprisingly contains zinc^II (Zn^II) ion between His41/Cys145 catalytic dyad in the active site, which undoubtedly can make new plans for designing novel SARS-CoV-2 M^Pro inhibitors, is performed for the first time in this paper, to the best of our knowledge.

1. Introduction

The coronavirus disease 2019 (COVID-19), which was caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a life-threatening infectious disease that has posed significant global hazard concerning, including high mortality rate, economic breakdown, life distress, etc. [1]. By end of January 2023, based on World Health Organization (WHO) coronavirus disease situation dashboard and COVID-19 Map-Johns Hopkins Coronavirus Resource Center, over 754 million people had been infected with SARS-CoV-2 worldwide, and more than 6,800,000 reported deaths globally, unfortunately. The SARS-CoV-2 main protease (M^Pro) [2] and papain-like protease (PL^Pro) [3] are the most validated antiviral drug targets for combating COVID-19 because the SARS-CoV-2 M^Pro and PL^Pro are essential for viral replication, transcription, maintenance, and its life cycle. Therefore, the design and synthesis of small organic molecules that operate simultaneously as inhibitors of both SARS-CoV-2 M^Pro and PL^Pro targets are definitely worthwhile in this era. As a matter of fact, despite remarkable efforts in the nearly past three years, the scientific community's understanding about the SARS-CoV-2 phenomenon is still so limited that caused many serious restrictions on the design and preparation of new drugs or vaccines for the combating against the mentioned viral disease, regrettably [4]. Computer-aided drug design (CADD) approaches [5], which in recent years have become an indispensable constituent in medicinal chemistry, can be unquestionably practicable in a full-scale war with COVID-19 [6]. Notably, in the drug design field, computational studies are free from safety and ethical constraints and can increase the speed of a pharmaceutically relevant project and intensely reduce extravagant costs.

Heterocyclic compounds are valuable organic frameworks in myriad aspects of our life, especially in medicinal chemistry [7]. Furo[2,3-d]pyrimidine heterocyclic fused ring systems are structural analogs of purines and have diverse biological activities. As shown in Fig. 1 , some heterocyclic frameworks with the furo[2,3-d]pyrimidine core reported as inhibitors of epidermal growth factor receptor (EGFR) [8], activated Cdc42-associated kinase 1 (ACK-1) [9], lymphocyte-specific protein tyrosine kinase (LCK) [10], glycogen synthase kinase-3 beta (GSK-3β) [11], receptor-interacting serine/threonine-protein kinase 1 (RIPK-1) [12], and aurora kinase A (AK-A) [13]. Besides, Miyazaki and co-workers reported 1-(4-(4-amino-6-(4-methoxyphenyl)furo[2,3-d]pyrimidin-5-yl)phenyl)-3-(2-fluoro-5-(trifluoromethyl)phenyl)urea compound as dual inhibitors of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) [14]. Also, Gangjee et al. reported (4-((2,4-diamino-5-methylfuro[2,3-d]pyrimidin-6-yl)thio)benzoyl)-l-glutamic acid scaffold as dual inhibitors of Tie-2 and vascular endothelial growth factor 2 (VEGFR-2) [15]. In addition, some of the mentioned compounds were reported as antifolate [16] and potent anti-breast cancer [17] agents. Because of the mentioned significant biological features and many others, which are existed in scientific papers [18], furo[2,3-d]pyrimidines have become an attractive synthetic target for organic and medicinal synthetic groups.

Fig. 1.

Representative examples of bioactive molecules containing the furo[2,3-d]pyrimidine core.

Today, it is imperative to follow green chemistry protocols (GCPs) in designing or modifying a synthetic approach for the preparation of organic compounds (a), [19]. Nowadays, concerning the GCPs in the drug (or drug-like compounds) discovery process is undeniably significant and essential [20]. It is also worth mentioning that pharmaceutical manufacturing, with pioneering works, was one of the first industries to recognize the importance of the GCPs and applied all of (or most of) them as far as possible. In this regard, selecting the reaction solvent, catalyst, and procedure based on GCPs are significant. Among well-known environmentally benign chemical reaction mediums [21], water is the best because it is non-toxic, inexpensive, abundant, sustainable, and in the green chemistry solvent ranking list, holds a top and valuable place among others [22]. Furthermore, in aqueous media, it is unessential to dry co-solvents, substrates, and reagents before use, which cause saving costs and time. Catalysis is another main factor in the GCPs for the design of an environmentally benign organic reaction. Zirconium^IV oxychloride octahydrate (ZrOCl₂•8H₂O) as an available, low-cost, easy-handling, and moisture-stable catalyst with highly coordinating ability, has attracted the attention in the organic synthetic community [23]. In the past two decades, the mentioned green catalyst has been used frequently in various organic transformations, especially heterocyclic ones, including syntheses of 4H-chromenes [24], pyrimido[4,5-d]pyrimidinones [25], dibenzo[b,i]xanthene-tetraones [26], [1,3]oxazino[5,6-c]quinolin-5-ones [27], 1,8-dioxo-octahydroxanthenes [28], benzopyranopyrimidines [29], 2,4,6-triarylpyridines [30], tetrahydropyrimidine [31], isobenzofuran-1(3H)-ones [32], 1H-imidazoles [33], 2-aryloxazolines [34], dihydroquinolinones [35], 3,4-dihydropyrimidin-2(1H)-ones [36], hexahydroquinolines [37], pyrano[2,3-d:6,5-dʹ]dipyrimidinones [38], pyrimido[4,5-c]pyridazines [39], 5-amino-1-aryl-1H-tetrazoles,[40] and many others. From the green chemistry point of view, one-pot multi-component reactions (MCRs) are amiable, advanced, and innovative strategies in organic synthesis [41]. Rather than the classical sequential pathway approaches, these reactions have attracted expanded attention in combinatorial, synthetic, and pharmaceutical chemistry for their distinct advantages, such as straightforward reaction design, high atom-economy, time-effectiveness, simplified work-up procedures, high overall yields of desired products, and molecular diversity. A literature survey shows that arylglyoxal monohydrate-based [42] and isocyanide-based [43] one-pot multi-component reactions have a unique place in the synthesis of the heterocyclic compounds, each independently. Therefore, the combination of mentioned starting materials in specific one-pot MCRs assuredly leads to the creation of a new avenue in novel heterocyclic scaffolds synthesis.

In continuation of our research programs on the synthesis of pharmaceutically interesting heterocyclic frameworks [44], and also due to the importance of introducing new anti-SARS-CoV-2 agents, we wish to report an environmentally benign and efficient one-pot three-component regioselective synthetic strategy for the preparation of novel 5-aroyl(or heteroaroyl)-6-(alkylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-diones (4a‒n) through the sequential condensation of aryl(or heteroaryl)glyoxal monohydrates (1a‒g), 1,3-dimethylbarbituric acid (2), and alkyl(viz. cyclohexyl or tert-butyl)isocyanides (3a or 3b), which catalyzed by a tremendously small amount of ZrOCl₂•8H₂O (just 2 mol%), in water at 50 ˚C (Fig. 2 ). Besides, inhibitory activities of the newly synthesized fused heterocyclic frameworks (4a‒n) against the active site and putative allosteric hotspots of both SARS-CoV-2 M^Pro and PL^Pro investigated using molecular docking, and the obtained results compared with various medicinal compounds, which used, introduced, and or repurposed to fight against COVID-19 viral disease in nearly past three years. Drug-likeness properties of the mentioned furo[2,3-d]pyrimidines (4a‒n) were also explored by employing in silico ADMET analyses. It is worthwhile to note that 5-(3,4-methylendioxybenzoyl)-6-(cyclohexylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4g) as hit compound is potential COVID-19 drug candidate and can subject to further in vitro and in vivo studies.

Fig. 2.

Green one-pot three-component synthesis of novel drug-like furo[2,3-d]pyrimidines as potential active site inhibitors and putative allosteric hotspots modulators of both SARS-CoV-2 M^Pro and PL^Pro. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2. Results and discussion

2.1. Synthesis, characterization, and plausible mechanism of 5-aroyl(or heteroaroyl)-6-(alkylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-diones (4a‒n)

Initially, we commenced our studies with the optimization reaction conditions for the green one-pot regioselective synthesis of 5-benzoyl-6-(cyclohexylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4a) through the tandem three-component condensation of phenylglyoxal monohydrate (1a), 1,3-dimethylbarbituric acid (2), and cyclohexyl isocyanide (also known as isocyanocyclohexane) (3a) as a model reaction (Table 1 ). It is worthwhile to note that in all stages of optimization, we used water as an entirely environmentally benign reaction solvent because one of our main goals was to design a reaction based on green chemistry protocols. Under the catalyst-free conditions at room temperature, just 30% yield of the desired product (4a) was obtained, even after 48 h (Table 1, entry 1). Increasing reaction temperature from room temperature to 50 ˚C and Reflux caused preparation 4a in 56% and 43%, respectively, after 24 h (Table 1, entries 2 and 3). The gained poor results led us to use a catalyst to achieve better and more efficient outcomes. To this purpose, we used 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and 1,4-diazabicyclo[2.2.2]octane (DABCO) as base organocatalysts, and also zirconium^IV oxychloride octahydrate (ZrOCl₂•8H₂O) as a green metallic catalyst. When the one-pot three-component reaction was carried out in 2 mol% presence of the mentioned easily accessible catalysts, we observed that the catalytic performance of ZrOCl₂•8H₂O is better than others at 50 ˚C (Table 1, entry 7). Further investigations showed that the decreasing and even increasing amount of ZrOCl₂•8H₂O not only did the conditions of the one-pot reaction not improve, but it made the situation worse (Table 1, entries 8 and 9). Once we had the optimized reaction conditions in hand (Table 1, entry 7), we evaluated the scope and limitations of the mentioned green one-pot three-component regioselective synthetic protocol, as shown in Fig. 3 . The one-pot three-component reaction tolerated several aryl(or heteroaryl)glyoxal monohydrates (1a‒g) and alkyl(cyclohexyl or tert-butyl)isocyanides (3a or 3b), affording the corresponding 5-aroyl(or heteroaroyl)-6-(alkylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-diones (4a‒n) with good-to-excellent yields. Notably, the yields of the desired furo[2,3-d]pyrimidine heterocyclic products containing cyclohexyl isocyanide (3a) were slightly better rather than tert-butyl isocyanide (also known as 2-isocyano-2-methylpropane) (3b), as shown in Fig. 3.

Table 1.

Optimization reaction conditions for the one-pot three-component synthesis of 4a.

Entry	Catalyst (mol%)	Temperature conditions	Time (h or min)	Yield (%)
1	Catalyst-free	Room temperature	48 h	30
2	Catalyst-free	50 ˚C	24 h	56
3	Catalyst-free	Reflux	24 h	43
4	DBN (2 mol%)	50 ˚C	1 h	71
5	DBU (2 mol%)	50 ˚C	1 h	75
6	DABCO (2 mol%)	50 ˚C	1 h	80
7	ZrOCl2•8H2O (2 mol%)	50 ˚C	45 min	90
8	ZrOCl2•8H2O (1 mol%)	50 ˚C	70 min	81
9	ZrOCl2•8H2O (10 mol%)	50 ˚C	45 min	82

Fig. 3.

Green one-pot three-component regioselective synthesis of 5-aroyl(or heteroaroyl)-6-(alkylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-diones catalyzed by ZrOCl₂•8H₂O. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The structure of all mentioned fused heterocyclic products (4a‒n) was confirmed by Fourier transform infrared spectroscopy (FT-IR), ¹H and ¹³C nuclear magnetic resonance (¹H NMR and ¹³C NMR), and carbon–hydrogen–nitrogen (CHN) analyses. In this regard, the ¹H NMR spectrum of 4a, as a simple instance (Figure S1, section A), revealed a doublet peak at δ _H 8.61 ppm (J = 8.3 Hz) for the ─NH─ proton. The five aromatic protons of the phenyl ring system appeared at δ _H 7.62 ppm (d, J = 6.9 Hz, 2H, Ph─H), 7.51 ppm (t, J = 7.3 Hz, 1H, Ph─H), and 7.40 ppm (t, J = 7.5 Hz, 2H, Ph─H). The proton of the N─CH_cyclohexyl has also appeared as a multiplet peak at δ _H 3.81–3.70 ppm. The two sharp singlet peaks at δ _H 3.58 ppm and 3.29 ppm are related to the two methyl groups of the pyrimidine ring (N─CH₃). Also, the four multiplet peaks at δ _H 2.11–1.41 ppm showed ten hydrogens related to the five methylene (─CH₂─) groups of the cyclohexyl homocyclic ring system. On the other hand, the ¹H-decoupled ¹³C NMR (¹³C{¹H} NMR) spectrum of 4a (Figure S1, section B) showed seventeen peaks in agreement with the represented structure. In this regard, the peak of the benzoyl carbonyl group is revealed at δ _C 189.20 ppm, and the peaks of the two amide carbonyl groups of the pyrimidine ring are exposed at δ _C 161.09 ppm and 156.56 ppm. It is worth noting that the characteristic peaks at δ _C 149.97 ppm and 148.90 ppm are related to the two carbon atoms of the furan ring, which are nearby the oxygen heteroatom (namely C₆ and C_7a). Also, the other two carbon atoms of the mentioned furan ring, which are far from the furan’s oxygen heteroatom (namely C₅ and C_4a), are visible at δ _C 94.40 ppm and 92.55 ppm. The carbons of the phenyl ring appeared at δ _C 140.39 ppm, 131.15 ppm, 128.46 ppm, and 127.33 ppm. Furthermore, the ¹³C{¹H} NMR spectrum of 4a represented one peak at δ _C 51.91 ppm for the methine group of the cyclohexyl moiety (N─CH_cyclohexyl), and two distinct peaks at δ _C 33.43 ppm and 29.69 ppm for the two N─CH₃ groups of the pyrimidine ring, and also three peaks at δ _C 28.69 ppm, 25.24 ppm, and 24.34 ppm for the cyclohexyl ring methylene groups (─CH₂─).

A plausible mechanism for this valuable green one-pot three-component regioselective transformation using ZrOCl₂•8H₂O catalyst in water at 50 ˚C is also depicted in Fig. 3. In the first step, selected aryl(or heteroaryl)glyoxal monohydrate (1a‒g) was activated by ZrOCl₂•8H₂O and gave intermediate I. In the second step, a regioselective Knoevenagel condensation reaction between enol form (6) of 1,3-dimethylbarbituric acid (2) with the formyl group of the activated aryl(or heteroaryl)glyoxal (I) led to the formation of 1,3-dimethyl-5-(2-oxo-2-aryl(or heteroaryl)ethylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (intermediate II) along with the elimination of one water molecule. In the third step, a heteroannulation reaction between the activated intermediate II and related alkyl(cyclohexyl or tert-butyl)isocyanide (3a or 3b) caused the formation of (Z)-5-aroyl(or heteroaroyl)-6-(alkylimino)-1,3-dimethyl-5,6-dihydrofuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (intermediate III). It is worth noting that the mentioned step probably proceeds from a simple Michael-type addition and subsequently intramolecular heteroannulation reaction (path A) and or through a formal [4 + 1] heteroannulation process (path B). Finally, the spurred iminolactone (intermediate III) generated desired furo[2,3-d]pyrimidine product (4a‒n) by a [1,3]-hydrogen transfer.

2.2. Molecular docking studies

To explore the protein–ligand interactions between the newly synthesized 5-aroyl(or heteroaroyl)-6-(alkylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-diones (4a‒n) and various pockets of the SARS-CoV-2 M^Pro and PL^Pro, in silico molecular docking studies performed using AutoDock Vina as an open-source program for doing molecular docking along with UCSF Chimera as a graphical user interface. First of all, we carried out a molecular docking process for our synthesized furo[2,3-d]pyrimidines (4a‒n) on the active site of M^Pro protein (PDB ID: 7AEH). It should be noted that M^Pro of the SARS-CoV-2 contains a highly conserved catalytic dyad comprising amino acid residues His41 and Cys145 that are buried in the active site cavity of the mentioned protein, in which His41 acts as a general acid or base, and on the other site, Cys145 acts as a nucleophile. The obtained binding energies (also known as binding free energies, binding affinities, and or binding scores) of the molecular docking investigations were ranging from −7.2 Kcal/mol to −8.5 Kcal/mol (Table S1). From the binding energy point of view, the attained results demonstrated that 5-(3,4-methylendioxybenzoyl)-6-(cyclohexylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4g) with a binding energy of −8.5 Kcal/mol is slightly better than other synthesized furo[2,3-d]pyrimidine heterocyclic frameworks (4a‒n). As shown in Fig. 4 (sections A and B) and Table S1, the compound 4g was able to form two conventional hydrogen bonds with residues Ser144 (2.625 Å) and His163 (2.415 Å) of the SARS-CoV-2 M^Pro active site. On the other hand, 4g has some hydrophobic interactions with His41/Cys145 catalytic dyad. In this regard, the compound 4g exhibited π–alkyl interaction with His41 and revealed alkyl and π–alkyl interactions with Cys145. Furthermore, the mentioned fused heterocyclic compound (4g) interacts with residues Met49 (alkyl), His163 (π–alkyl), His164 (carbon hydrogen bond), Pro168 (π–alkyl), Gln189 (carbon hydrogen bond and π–σ), and Thr190 (carbon hydrogen bond), as shown in Fig. 4 (sections A and B) and Table S1. Interestingly, as shown in Table S1, the compound 5-(4-hydroxy-3-methoxybenzoyl)-6-(cyclohexylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4f), with a binding energy of −7.9 Kcal/mol, capable to creation eight hydrogen bonds with the SARS-CoV-2 M^Pro active site residues, especially Cys145 (2.341 Å). In 2022, Ebrahim and co-workers published a valuable and attention-grabbing paper about the temperature-dependent conformational ensemble of the SARS-CoV-2 M^Pro [45]. Their studies revealed that in human body temperature (310 K), the mobile zinc^II (Zn^II) ion interleaved between the His41/Cys145 catalytic dyad (Figure S2). The mentioned occurrence can inspire the scientific community to make new and different plans for designing new covalent and non-covalent SARS-CoV-2 M^Pro inhibitors. To this purpose, we shifted our investigations to the Zn^II-containing active site of the SARS-CoV-2 M^Pro protein (PDB ID: 7MHK). For this stage, the obtained binding energies were ranging from −6.5 Kcal/mol to −7.7 Kcal/mol (Table S2). Gratifyingly, in terms of binding energy we saw that the best heterocyclic compound was 4g (−7.7 Kcal/mol). As shown in Fig. 4 (sections C and D) and Table S2, the compound 4g can form five conventional hydrogen bonds with His41 (3.165 Å), Cys145 (3.099 Å), Glu166 (2.809 Å), and Gln189 (2.532 Å and 2.890 Å). In addition, we observed a π–cation interaction between 4g and Zn^II ion (Zn503). Furthermore, the compound 4g interacts with Met49 (alkyl and π–alkyl), Phe140 (carbon hydrogen bond), Leu141 (carbon hydrogen bond), Cys145 (π–alkyl), His163 (π–alkyl), and Met165 (carbon hydrogen bond) (Fig. 4 (sections C and D) and Table S2). It is worth noting that, as shown in Table S2, the compound 4f, with a binding energy of −7.5 Kcal/mol, is capable to create eleven hydrogen bonds with the SARS-CoV-2 M^Pro active site residues, especially three hydrogen bonds with Cys145 (2.470 Å, 2.830 Å, and 3.128 Å), and also can form a metal–acceptor interaction with Zn503.

Fig. 4.

Close-up views (3D and 2D) of 4g in the metal-free active site (A and B) and Zn^II-containing active site (C and D) of the SARS-CoV-2 M^Pro, respectively.

Inhibition of allosteric hotspots is an emerging paradigm in modern pharmacology to the extent that recent years have seen an unprecedented and astonishing level of innovation in the discovery and development of allosteric drugs [46]. Because of the importance of this issue, we investigated the molecular docking process for two putative allosteric pockets of the SARS-CoV-2 M^Pro, including allosteric site I (known as dimerization site, which could interrupt the dimerization conformation and inactive the M^Pro) and allosteric site II (Known as cryptic site) [47]. The molecular docking results for the allosteric site I (PDB ID: 7VLP) showed that the binding energies of the investigations were ranging from −5.3 Kcal/mol to −6.4 Kcal/mol (Table S3), which the compound 4g with a binding energy of −6.4 Kcal/mol is somewhat better than others, and as shown in Fig. 5 (sections A and B) and Table S3, it was able to form two conventional hydrogen bonds with residue Gly302A (2.065 Å and 2.922 Å) and three conventional hydrogen bonds with residue Asn142B (2.179 Å, 2.398 Å, and 3.379 Å). Furthermore, we observed a carbon hydrogen bond interaction with Gly302A and a π–σ hydrophobic interaction with Asn142B, as shown in Fig. 5 (sections A and B) and Table S3. On the other hand, the molecular docking outcomes for allosteric site II (PDB: 7MHK) exhibited that the binding energies were ranging from −6.6 Kcal/mol to −8.1 Kcal/mol (Table S4), which again the compound 4g with a binding energy of −8.1 Kcal/mol is slightly better than others. As shown in Fig. 5 (sections C and D) and Table S4, 4g can able to create twelve conventional hydrogen bonds with residues Lys102 (2.317 Å and 3.117 Å), Gln110 (2.380 Å, 3.343 Å, and 3.460 Å), Asn151 (2.397 Å, 2.623 Å, 3.382 Å, and 3.445 Å), Ser158 (2.895 Å), and Arg298 (2.157 Å and 2.486 Å). Besides, 4g has alkyl interaction with Lys102, and alkyl and π–alkyl interactions with Val104 along with carbon hydrogen bond interactions with Gln110 and Ser158, as shown in Fig. 5 (sections C and D) and Table S4.

Fig. 5.

Close-up views (3D and 2D) of 4g in the allosteric site I (A and B) and allosteric site II (C and D) of the SARS-CoV-2 M^Pro, respectively.

The SARS-CoV-2 PL^Pro is another essential factor for the COVID-19 proliferation cycle. The mentioned SARS-CoV-2 PL^Pro active site contains the catalytic triad that is formed by Cys111, His272, and Asp286. In continuation of in silico studies, we investigated another molecular docking process for our synthesized furo[2,3-d]pyrimidines (4a‒n) on the active site of SARS-CoV-2 PL^Pro protein (PDB ID: 6WX4). The molecular docking investigations showed that obtained binding energies were ranging from −7.5 Kcal/mol to −8.4 Kcal/mol (Table S5). Among the mentioned furo[2,3-d]pyrimidines (4a‒n), the compound 4g with a binding energy of −8.4 Kcal/mol is again slightly better than others. As shown in Fig. 6 (sections A and B) and Table S5, this ligand (4g) formed three conventional hydrogen bonds with the amino acids residues Arg166 (3.027 Å), Tyr273 (2.977 Å), and Thr301 (3.376 Å). In addition, other interactions were observed (Fig. 6 (sections A and B) and Table S5) for this case, including Leu162 (alkyl), Asp164 (π–anion), Arg166 (carbon hydrogen bond and π–alkyl), Met208 (π–sulfur), Ala246 (amide–π stacked), Pro247 (van der Waals), Tyr264 (π–alkyl), Tyr268 (carbon hydrogen bond, π–π T-shaped, and π–alkyl), and Asp302 (carbon hydrogen bond). Remarkably, the SARS-CoV-2 PL^Pro (PDB ID: 6WX4) allosteric [48] inhibitory properties of our synthesized heterocyclic compounds were also investigated. The binding energies for this case were from −5.8 Kcal/mol to −7.8 Kcal/mol (Table S6). The compound 4g, which is better than others in terms of binding energy (−7.8 Kcal/mol), can able to form two conventional hydrogen bonds with residues Arg65 (2.355 Å) and Val66 (3.164 Å), as shown in Fig. 6 (sections C and D) and Table S6. On the other hand, 4g have hydrophobic interactions with residues Pro59 (π–alkyl), Arg65 (π–alkyl), Val66 (alkyl), Ala68 (π–alkyl), and Phe69 (π–σ), along with an electrostatic interaction with residue Asp62 (π–anion) (Fig. 6 (sections C and D) and Table S6).

Fig. 6.

Close-up views (3D and 2D) of 4g in the active site (A and B) and allosteric site (C and D) of the SARS-CoV-2 PL^Pro, respectively.

2.3. In silico ADMET prediction

Most of the designed and prepared chemical compounds for medicinal purposes fail and flop in the drug development process due to their poor pharmacokinetics and toxicity problems, which is an undeniable fact worth pondering. Such drawbacks that arise during drug development should be addressed at the early stage in the pipeline of this process to prevent the loss of material and intellectual capital and to achieve the desired goal in a shorter period of time and with low costs. In silico ADMET (absorption, distribution, metabolism, excretion, and toxicity) prediction is a significant analysis to rolling out undesired effects of a proposed drug candidate at the initial step of the drug discovery process [49]. In this regard, in silico ADME analysis of the prepared 5-aroyl(or heteroaroyl)-6-(alkylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-diones (4a‒n) investigated using free web tool SwissADME, from Swiss Institute of Bioinformatics (http://www.swissadme.ch) [50]. The data related to physicochemical properties and lipophilicity (Table S7), water solubility (Table S8), pharmacokinetics (Table S9), and drug-likeness and medicinal chemistry (Table S10) of the synthesized heterocyclic compounds (4a‒n) have been collected in Supporting Information (SI). Interestingly, the obtained results demonstrated the mentioned furo[2,3-d]pyrimidines (4a‒n) generally possess drug-like behavior because they could successfully be passed fundamental drug-likeness filters, including Lipinski (Pfizer), Ghose (Amgen), Veber (GSK), Egan (Pharmacia), and Muegge (Bayer). Also, the Abbot bioavailability score [51] value for all synthesized compounds (4a‒n) was 0.55 (55%), which indicates the probability of their bioavailability, and it is based on the total charge of compound, topological polar surface area (TPSA), and violation of Lipinski filter. Furthermore, pan assay interference structures (PAINS) and Brenk filters are applied to provide information concerning potentially problematic fragments (putatively toxic, metabolically unstable, or possessing properties responsible for poor pharmacokinetics). Gratifyingly, our heterocyclic frameworks (4a‒n) have no alert for PAINS and Brenk. On the other hand, the BOILED-Egg plot between WLOGP and TPSA was used to predict gastrointestinal absorption and brain penetration of the furo[2,3-d]pyrimidines (4a‒n). As can be seen from the BOILED-Egg plot (Fig. 7 ), all of the mentioned one-pot synthesized compounds (4a‒n) show satisfactory gastrointestinal (GI) absorption and have no blood–brain barrier (BBB) permeability, and the red dots as P-glycoprotein non-substrates (PGP−) demonstrate predictions that our fused heterocyclic compounds (4a‒n) cannot be effluxes from the central nervous system (CNS) by PGP. Overall, from the related BOILED-Egg plot data, especially from the positions of the red dots, we can conclude that the BBB penetration property and PGP effect for our compounds are negative, and the GI absorption property is positive. Besides, the in silico toxicity evaluation was carried out using an online server, pkCSM (https://biosig.lab.uq.edu.au/pkcsm/prediction) [52]. As shown in Table S11, the in silico Ames toxicity test investigation shows that all the newly synthesized fused heterocyclic frameworks (4a‒n) successfully passed the mentioned imperative test and predicted them as non-mutagenic compounds. Also, LD₅₀ (oral rat acute toxicity) amounts for these heterocyclic structures (4a‒n) were from 2.37 mol/kg to 2.793 mol/kg, which highest one related to 4g (Table S11).

Fig. 7.

BOILED-Egg plot of 5-aroyl(or heteroaroyl)-6-(alkylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-diones.

2.4. Comparative study

To demonstrate the 5-aroyl(or heteroaroyl)-6-(alkylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-diones (4a‒n) value and power in fighting against COVID-19 disease, we compared hit one (4g) with seventy-three medicinal compounds (Fig. 8 ) that used, repurposed, investigated, and introduced against SARS-CoV-2 in closely three past years. From the binding energy point of view, the mentioned comparison revealed that 4g could have a unique place in this viral war. As shown in Table 2 , binding energies of the medicinal compounds from the SARS-CoV-2 M^Pro metal-free active site (PDB ID: 7AEH) showed that only three of them (viz. Elbasvir, Ensitrelvir, and Lumacaftor) are better than 4g, and one of them, namely Idarubicin, has equal binding energy with 4g. Also, the molecular docking results of 4g from the M^Pro Zn^II-containing active site (PDB ID: 7MHK), the allosteric site I (PDB ID: 7VLP), and the allosteric site II (PDB ID: 7MHK) of the SARS-CoV-2 were satisfied and acceptable rather than others. It should be noted that Ensitrelvir (known as S-217622), which is a better inhibitor than 4g in both SARS-CoV-2 M^Pro metal-free and Zn^II-containing active sites and allosteric sites (Table 2, entry 21), was reported in 2022 as the first oral non-covalent and non-peptidic SARS-CoV-2 M^Pro inhibitor clinical candidate with IC₅₀ value of 13 nM and have EC₅₀ values for wild type (WT) and other strains of SARS-CoV-2 including alpha, betta, gamma, delta, and omicron equal 0.37 μM, 0.33 μM, 0.40 μM, 0.50 μM, 0.41 μM, and 0.29 μM, respectively [53]. Furthermore, a comparison between entries 1, 49, and 57 of Table 2 revealed that 4g has more suitable binding energy than both Nirmatrelvir and Ritonavir as two parts of the oral Pfizer’s Paxlovid SARS-CoV-2 M^Pro inhibitor drug for COVID-19 [54]. As shown in Table 2 (entries 1 and 44), the compound 4g is also better SARS-CoV-2 M^Pro inhibitor than ML-188 (with a IC₅₀ of 2.5 ± 0.3 μM) as a non-covalent M^Pro inhibitor [55]. In the SARS-CoV-2 PL^Pro active site (PDB ID: 6WX4), Lumacaftor, MI-09, and Tropifexor (Table 2, entries 37, 40, and 70) showed slightly better binding energy than 4g, and also in the SARS-CoV-2 PL^Pro allosteric site (PDB ID: 6WX4), Lumacaftor and Paritaprevir (Table 2, entries 37 and 51) were somewhat better than 4g, and Simeprevir (Table 2, entry 61) had equal with 4g. Notably, 4g has better binding energy in the SARS-CoV-2 PL^Pro active and allosteric sites than GRL-0617 (Table 2, entry 27) as a SARS-CoV-2 PL^Pro inhibitor with IC₅₀ of 1.50 ± 0.08 μM [56]. Also, a comparison between binding energies of 4g and Tropifexor (Table 2, entry 70), which is an efficient repurposed drug for inhibition of SARS-CoV-2 PL^Pro (with a IC₅₀ of 5.11 ± 1.14 μM and EC₅₀ of 4.3 ± 0.5 μM) [57], the results related to the PL^Pro active site were so closed and for the PL^Pro allosteric site, the compound 4g was better one.

Fig. 8.

Examples of the reported medicinal compounds as anti-SARS-CoV-2 agents.

Table 2.

Comparative molecular docking studies between 4g and reported medicinal compounds as anti-SARS-CoV-2 agents.

Entry	Compound name	Binding energy (Kcal/mol)
		Main protease (MPro)				Papain-like protease (PLPro)
		Metal-free active site (PDB ID: 7AEH)	ZnII-Containing active site (PDB ID: 7MHK)	Allosteric site I (PDB ID: 7VLP)	Allosteric site II (PDB ID: 7MHK)	Active site (PDB ID: 6WX4)	Allosteric site (PDB ID: 6WX4)
1	4g	−8.5	−7.7	−6.4	−8.1	−8.4	−7.8
2	Atazanavir	−7.1	−6.9	−5.5	−7.3	−6.4	−6.6
3	Azelastine	−7.5	−7.2	−6.1	−7.7	−7	−7.1
4	Baicalein	−7.3	−7.1	−6.3	−7.3	−7	−7.1
5	Baricitinib	−7.6	−7.4	−5.5	−7.3	−7.3	−6
6	Bedaquiline	−7.3	−7.6	−5.4	−7.4	−7.2	−7.5
7	Bemnifosbuvir	−7.5	−7.2	−5.8	−7.4	−7.1	−6.4
8	Boceprevir	−7.1	−7.1	−5.5	−7.2	−7.2	−5.7
9	Calpeptin	−6.1	−6.3	−4.8	−6.5	−5.8	−5.5
10	Carfilzomib	−6.5	−7	−6.2	−6.3	−6.3	−5.8
11	Carmofur	−6.2	−6.2	−4.9	−6.4	−6.4	−5.7
12	Cinnoxicam	−7.9	−7.8	−6.5	−8.4	−7.2	−6.7
13	Cobicistat	−6.2	−7.3	−5.9	−7.4	−7.3	−5.8
14	Darunavir	−7.6	−7	−5.8	−7.9	−7.1	−6.1
15	Dexamethasone	−7.8	−7.5	−5.4	−7.4	−7.6	−5.8
16	Ebselen	−6.2	−6.7	−5.3	−6.5	−6.1	−6.1
17	Edoxudine	−6.4	−6.4	−5.2	−6	−6.1	−5.6
18	Efonidipine	−7.3	−8.5	−6.1	−8.6	−7.8	−6.5
19	Elbasvir	−8.7	−7.5	−6.3	−8.7	−7.9	−7
20	Elexacaftor	−7.8	−7.8	−6.5	−8.7	−7.6	−6.7
21	Ensitrelvir	−9.9	−8.2	−6.6	−8.6	−7.8	−7.2
22	Famotidine	−6.2	−6.1	−4.8	−5.3	−6.8	−5
23	Favipiravir	−5.7	−5.5	−5.3	−5.5	−5.8	−5.1
24	Galidesivir	−6.6	−6.2	−6.1	−6	−6.5	−5.8
25	GC-376	−6.9	−7.5	−5.6	−7.8	−7.2	−6
26	Grazoprevir	−7.4	−7.2	−6.4	−7.8	−6.6	−6
27	GRL-0617	−7	−7.1	−6.5	−7.7	−7.7	−6.9
28	GS-441524	−6.6	−6.7	−5.1	−6.5	−6.4	−6
29	Idarubicin	−8.5	−7.4	−6.2	−7.8	−7.3	−6.8
30	Ifenprodil	−7.6	−6.9	−7	−7.2	−7	−6.6
31	Indinavir	−7.6	−7.3	−6.6	−7.7	−7.6	−7
32	Ivacaftor	−7.2	−7	−5.9	−8.4	−7.1	−6.6
33	Lapatinib	−8.4	−7.9	−7	−8.1	−7.8	−7.4
34	Lercanidipine	−7.9	−7.7	−5.1	−7.7	−7.5	−6.4
35	Lopinavir	−7.3	−7.3	−5.8	−7.1	−7.3	−6.4
36	Lufotrelvir	−7.6	−7.4	−5.6	−7.6	−7.8	−5.7
37	Lumacaftor	−8.9	−9.1	−7.9	−9.3	−8.9	−8.1
38	Manidipine	−8.3	−7.1	−5.4	−8.3	−7.9	−6.8
39	Masitinib	−7.9	−8.2	−6.6	−9.3	−8.1	−7.5
40	MI-09	−8.3	−7.8	−5.8	−8.2	−8.5	−7.3
41	MI-23	−8.2	−8.3	−6.4	−8.1	−7.4	−6.8
42	MI-30	−8.1	−8	−6.4	−8.1	−7.9	−6.7
43	Mizoribine	−6.7	−6.7	−4.5	−6.4	−6	−5.6
44	ML-188	−7.3	−7	−5.6	−6.9	−7.1	−5.5
45	Molnupiravir	−7.3	−7	−5.7	−7	−7.4	−5.9
46	MPI3	−6.7	−7.1	−4.5	−6.7	−7.3	−5.4
47	Nelfinavir	−8.2	−7.2	−6.3	−8.1	−6.8	−6.4
48	Niclosamide	−7.3	−7.1	−5.4	−7	−7	−6.4
49	Nirmatrelvir	−8	−7.6	−5.8	−7	−8.2	−6.2
50	Oseltamivir	−5.9	−5.8	−4.2	−5.8	−6.3	−5.2
51	Paritaprevir	−7.7	−7.9	−7.6	−7.8	−8	−8.3
52	Pelitinib	−7.9	−7.3	−6.1	−7.7	−7.2	−6.6
53	Perampanel	−8.1	−7.5	−5.5	−7.6	−8	−7.3
54	Periciazine	−7.7	−7.8	−5.7	−6.8	−7.4	−6.4
55	Remdesivir	−7.9	−7.3	−5.1	−6.7	−7.3	−5.7
56	Ribavirin	−6.4	−6.3	−5.4	−6.3	−6.5	−5.3
57	Ritonavir	−6.9	−6.7	−6	−7.9	−6.4	−6.1
58	Rupintrivir	−7.6	−7.3	−5.6	−6.9	−7	−6
59	Saquinavir	−7.8	−7.9	−6.5	−9.9	−7.9	−7.1
60	Shikonin	−7.3	−6.8	−5.8	−7.2	−6.9	−7.6
61	Simeprevir	−8	−7.9	−6.8	−9.5	−7.6	−7.8
62	Sofosbuvir	−7.6	−7.5	−5.6	−7	−7.3	−6.4
63	Talampicillin	−8.4	−8.1	−6.8	−8.2	−8.3	−7.1
64	Telaprevir	−7.8	−7.1	−6	−7.8	−7	−5.7
65	Tezacaftor	−7.7	−7.6	−6.7	−8.5	−7.4	−6.9
66	Thapsigargin	−6.1	−5.9	−4.6	−6.6	−6	−4.3
67	Tideglusib	−7.1	−7.4	−6.3	−7.7	−7.6	−7.3
68	Tipranavir	−7.8	−6.6	−6.7	−8.1	−7.8	−7.5
69	Tolperisone	−5.8	−6.1	−5.2	−6.7	−6.3	−6.1
70	Tropifexor	−8.1	−8.6	−6.3	−8.6	−8.6	−7.1
71	UAWJ9-36-1	−7.6	−7.9	−5	−7.3	−6.5	−6
72	UAWJ9-36-3	−7.5	−7.7	−5.9	−7.5	−7.1	−6.3
73	Umifenovir	−6.2	−6.5	−4.5	−6	−6.4	−5.5
74	Vaniprevir	−8.3	−8.4	−7.3	−7	−7.1	−6

3. Experimental section

3.1. Reagents, samples, and apparatus in the novel furo[2,3-d]pyrimidines (4a‒n) synthesis

1,3-Dimethylbarbituric acid (2), and alkyl(viz. cyclohexyl or tert-butyl)isocyanides (3a or 3b), and catalysts were commercially available (purchased from Merck, Sigma-Aldrich, and Fluka companies) and used directly without further purification. Aryl(or heteroaryl)glyoxal monohydrates (1a‒g) were prepared through the Riley oxidation of related aryl(or heteroaryl) methyl ketones using SeO₂ in dioxane:water mixture solvent, in which the amount of water is genuinely trivial compared to dioxane [42b]. Melting points were determined on an Electrothermal 9200 apparatus. Fourier transform infrared (FT-IR) spectra were recorded on a PerkinElmer Spectrum Two FT-IR spectrophotometer, measured as potassium bromide (KBr) disks. ¹H and ¹³C{H} NMR spectra were recorded on a Bruker Avance 300 MHz spectrometer at 300 MHz and 75 MHz, respectively. Chemical shifts were measured in CDCl₃ as solvent relative to tetrametylsilane (TMS) as the internal standard. Elemental analyses were performed using a Leco Analyzer 932.

3.2. General procedure for the one-pot three-component synthesis of 5-aroyl(or heteroaroyl)-6-(alkylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-diones (4a‒n)

In a round-bottom flask (10 mL) equipped with a magnetic stirrer, a mixture of related aryl(or heteroaryl)glyoxal monohydrate (1 mmol), 1,3-dimethylbarbituric acid (1 mmol), and ZrOCl₂•8H₂O (2 mol%, 6.4 mg) in the water solvent (5 mL) was prepared and stirred at 50 ˚C for an appropriate time. After the formation of 1,3-dimethyl-5-(2-oxo-2-aryl(or heteroaryl)ethylidene)pyrimidine-2,4,6(1H,3H,5H)-trione intermediate, intended alkylisocyanide (1 mmol) was added to the reaction environment. After compilation of the reaction, the obtained solid product was filtered and washed with hot ethanol (5 mL) to afford the desired pure furo[2,3-d]pyrimidine heterocyclic product.

3.2.1. Physicochemical properties and spectroscopic data of the novel furo[2,3-d]pyrimidines (4a‒n)

3.2.1.1. 5-Benzoyl-6-(cyclohexylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4a)

Yellow solid; mp 191–192 ˚C; FT-IR (KBr) ʋ _max 3412, 3351, 3285, 3089, 3060, 3027, 2937, 2867, 1712, 1624, 1601, 1504, 1492, 1457, 1352, 1305, 1185, 1058, 963, 808, 736, 591, 517, 456 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ _H 8.61 (d, J = 8.3 Hz, 1H, ─NH─), 7.62 (d, J = 6.9 Hz, 2H, Ph─H), 7.51 (t, J = 7.3 Hz, 1H, Ph─H), 7.40 (t, J = 7.5 Hz, 2H, Ph─H), 3.81–3.70 (m, 1H, N─CH_cyclohexyl), 3.58 (s, 3H, N─CH₃), 3.29 (s, 3H, N─CH₃), 2.11–2.00 (m, 2H, ─CH₂─), 1.87–1.77 (m, 2H, ─CH₂─), 1.71–1.58 (m, 2H, ─CH₂─), 1.51–1.41 (m, 4H, 2 × ─CH₂─); ¹³C{¹H} NMR (75 MHz, CDCl₃) δ _C 189.20, 161.09, 156.56, 149.97, 148.90, 140.39, 131.15, 128.46, 127.33, 94.40, 92.55, 51.91, 33.43, 29.64, 28.69, 25.24, 24.34; Anal. Calcd for C₂₁H₂₃N₃O₄: C, 66.13; H, 6.08; N, 11.02. Found: C, 66.18, H, 6.10; N, 11.12.

3.2.1.2. 5-(4-Bromobenzoyl)-6-(cyclohexylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4b)

Yellow solid; mp 180–181 ˚C; FT-IR (KBr) ʋ _max 3518, 3269, 2934, 2859, 1707, 1674, 1650, 1624, 1589, 1573, 1509, 1487, 1455, 1300, 1173, 965, 747, 519, 455 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ _H 8.65 (d, J = 8.3 Hz, 1H, ─NH─), 7.53 (d, J = 8.3 Hz, 2H, Ph─H), 7.49 (d, J = 8.7 Hz, 2H, Ph─H), 3.79–3.71 (m, 1H, N─CH_cyclohexyl), 3.58 (s, 3H, N─CH₃), 3.31 (s, 3H, N─CH₃), 2.11–1.99 (m, 2H, ─CH₂─), 1.89–1.77 (m, 2H, ─CH₂─), 1.61–1.27 (m, 6H, 3 × ─CH₂─); ¹³C{¹H} NMR (75 MHz, CDCl₃) δ _C 187.64, 161.31, 156.64, 149.89, 149.01, 139.10, 130.56, 130.17, 125.77, 94.14, 92.28, 51.96, 33.40, 29.69, 28.70, 25.20, 24.33; Anal. Calcd for C₂₁H₂₂BrN₃O₄: C, 54.79; H, 4.82; N, 9.13. Found: C, 54.83, H, 4.76; N, 9.18.

3.2.1.3. 5-(4-Chlorobenzoyl)-6-(cyclohexylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4c)

Yellow solid; mp 187–188 ˚C; FT-IR (KBr) ʋ _max 3686, 3523, 3277, 2930, 2856, 1714, 1671, 1625, 1542, 1506, 1490, 1454, 1308, 1180, 1090, 964, 845, 747, 513, 459 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ _H 8.65 (d, J = 8.3 Hz, 1H, ─NH─), 7.57 (d, J = 8.2 Hz, 2H, Ph─H), 7.37 (d, J = 7.8 Hz, 2H, Ph─H), 3.79–3.70 (m, 1H, N─CH_cyclohexyl), 3.59 (s, 3H, N─CH₃), 3.31 (s, 3H, N─CH₃), 2.10–2.01 (m, 2H, ─CH₂─), 1.89–1.79 (m, 2H, ─CH₂─), 1.57–1.30 (m, 6H, 3 × ─CH₂─); ¹³C{¹H} NMR (75 MHz, CDCl₃) δ _C 187.59, 161.29, 156.65, 149.89, 149.00, 138.66, 137.18, 129.99, 127.64, 94.17, 92.31, 51.96, 33.41, 29.68, 28.69, 25.20, 24.33; Anal. Calcd for C₂₁H₂₂ClN₃O₄: C, 60.65; H, 5.33; N, 10.10. Found: C, 60.67, H, 5.30; N, 10.14.

3.2.1.4. 5-(4-Fluorobenzoyl)-6-(cyclohexylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4d)

Yellow solid; mp 174–175 ˚C; FT-IR (KBr) ʋ _max 3682, 3510, 3293, 3080, 2938, 2867, 1712, 1671, 1626, 1600, 1547, 1507, 1460, 1222, 1152, 967, 845, 747, 619, 517, 464 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ _H 8.59 (d, J = 8.3 Hz, 1H, ─NH─), 7.69–7.58 (m, 2H, Ph─H), 7.14–7.00 (m, 2H, Ph─H), 3.77–3.69 (m, 1H, N─CH_cyclohexyl), 3.59 (s, 3H, N─CH₃), 3.31 (s, 3H, N─CH₃), 2.11–1.99 (m, 2H, ─CH₂─), 1.87–1.77 (m, 2H, ─CH₂─), 1.60–1.28 (m, 6H, 3 × ─CH₂─); ¹³C{¹H} NMR (75 MHz, CDCl₃) δ _C 187.60, 166.22, 161.19, 156.70, 149.90, 149.01, 136.47, 130.87, 114.56, 114.26, 94.25, 92.28, 51.94, 33.42, 29.68, 28.69, 25.22, 24.33; Anal. Calcd for C₂₁H₂₂FN₃O₄: C, 63.15; H, 5.55; N, 10.52. Found: C, 63.14, H, 5.51; N, 10.55.

3.2.1.5. 5-(3-Bromobenzoyl)-6-(cyclohexylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4e)

Yellow solid; mp 179–180 ˚C; FT-IR (KBr) ʋ _max 3421, 3355 3285, 3166, 3064, 2932, 2856, 1717, 1677, 1625, 1569, 1546, 1505, 1477, 1451, 1307, 1259, 1176, 1149, 1047, 964, 894, 754, 716, 605, 521 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ _H 8.66 (d, J = 8.3 Hz, 1H, ─NH─), 7.73 (s, 1H, Ph─H), 7.61 (d, J = 7.8 Hz, 1H, Ph─H), 7.53 (d, J = 7.8 Hz, 1H, Ph─H), 7.29 (t, J = 7.5 Hz, 1H, Ph─H), 3.83–3.73 (m, 1H, N─CH_cyclohexyl), 3.58 (s, 3H, N─CH₃), 3.31 (s, 3H, N─CH₃), 2.10–2.02 (m, 2H, ─CH₂─), 1.84–1.78 (m, 2H, ─CH₂─), 1.72–1.63 (m, 2H, ─CH₂─), 1.51–1.42 (m, 4H, 2 × ─CH₂─); ¹³C{¹H} NMR (75 MHz, CDCl₃) δ _C 187.14, 161.30, 156.57, 149.90, 149.02, 142.13, 133.85, 131.14, 128.88, 127.02, 121.49, 94.07, 92.36, 51.97, 33.37, 29.69, 28.70, 25.20, 24.31; Anal. Calcd for C₂₁H₂₂BrN₃O₄: C, 54.79; H, 4.82; N, 9.13. Found: C, 54.73, H, 4.80; N, 9.10.

3.2.1.6. 5-(4-Hydroxy-3-methoxybenzoyl)-6-(cyclohexylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4f)

Yellow solid; mp 187–189 ˚C; FT-IR (KBr) ʋ _max 3392, 3162, 3072, 2928, 2855, 1712, 1669, 1605, 1509, 1488, 1467, 1429, 1386, 1355, 1307, 1299, 1283, 1181, 907, 829, 778, 755, 654, 603, 517, 504 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ _H 8.40 (d, J = 8.3 Hz, 1H, ─NH─), 7.28 (s, 1H, Ph─H), 7.21 (d, J = 8.1 Hz, 1H, Ph─H), 6.88 (d, J = 8.2 Hz, 1H, Ph─H), 6.23 (bs, 1H, Ph─OH), 3.89 (s, 3H, Ph─O─CH₃), 3.77–3.70 (m, 1H, N─CH_cyclohexyl), 3.58 (s, 3H, N─CH₃), 3.31 (s, 3H, N─CH₃), 2.04–1.99 (m, 2H, ─CH₂─), 1.83–1.79 (m, 2H, ─CH₂─), 1.48–1.40 (m, 6H, 3 × ─CH₂─); ¹³C{¹H} NMR (75 MHz, CDCl₃) δ _C 187.74, 162.34, 160.97, 156.70, 149.95, 148.96, 145.73, 132.27, 124.09, 113.07, 111.22, 92.20, 90.41, 58.85, 51.88, 33.44, 29.65, 28.67, 25.24, 24.31; Anal. Calcd for C₂₂H₂₅N₃O₆: C, 61.82; H, 5.90; N, 9.83. Found: C, 61.85, H, 5.87; N, 9.80.

3.2.1.7. 5-(3,4-Methylendioxybenzoyl)-6-(cyclohexylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4g)

Yellow solid; mp 179–180 ˚C; FT-IR (KBr) ʋ _max 3678, 3523, 3273, 3166, 3084, 2934, 2855, 2781, 1709, 1675, 1627, 1505, 1491, 1452, 1350, 1307, 1281, 1258, 1246, 1166, 1094, 1040, 967, 931, 856, 821, 767, 748, 669, 521, 494 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ _H 8.66 (d, J = 8.3 Hz, 1H, ─NH─), 7.73 (s, 1H, Ph─H), 7.61 (d, J = 7.9 Hz, 1H, Ph─H), 7.29 (d, J = 7.9 Hz, 1H, Ph─H), 6.03 (s, 2H, O─CH₂─O), 3.81–3.74 (m, 1H, N─CH_cyclohexyl), 3.59 (s, 3H, N─CH₃), 3.31 (s, 3H, N─CH₃), 2.08–2.02 (m, 2H, ─CH₂─), 1.85–1.80 (m, 2H, ─CH₂─), 1.51–1.39 (m, 6H, 3 × ─CH₂─); ¹³C{¹H} NMR (75 MHz, CDCl₃) δ _C 187.15, 162.33, 161.30, 156.57, 149.90, 149.01, 142.13, 131.51, 128.89, 127.01, 121.49, 103.00, 94.07, 92.36, 51.98, 33.37, 29.69, 28.70, 25.21, 24.31; Anal. Calcd for C₂₂H₂₃N₃O₆: C, 62.11; H, 5.45; N, 9.88. Found: C, 62.15, H, 5.49; N, 9.90.

3.2.1.8. 5-Benzoyl-6-(tert-butylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4h)

Yellow solid; mp 179–180 ˚C; FT-IR (KBr) ʋ _max 3428, 3363, 3162, 3060, 2962, 2933, 2872, 1718, 1705, 1676, 1660, 1618, 1601, 1490, 1454, 1287, 1226, 1186, 1107, 967, 902, 878, 784, 765, 744, 735, 611, 525, 512, 470 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ _H 8.85 (s, 1H, ─NH─), 7.68–7.60 (m, 2H, Ph─H), 7.18–6.95 (m, 3H, Ph─H), 3.61 (s, 3H, N─CH₃), 3.31 (s, 3H, N─CH₃), 1.53 (s, 9H, 3 × CH_{3 ter t-butyl}); ¹³C{¹H} NMR (75 MHz, CDCl₃) δ _C 187.70, 161.38, 156.67, 149.86, 149.26, 136.45, 132.14, 131.00, 129.83, 114.58, 114.29, 93.85, 93.21, 53.58, 30.04, 29.72, 28.69; Anal. Calcd for C₁₉H₂₁N₃O₄: C, 64.21; H, 5.96; N, 11.82. Found: C, 64.20, H, 5.93; N, 11.85.

3.2.1.9. 5-(4-Bromobenzoyl)-6-(tert-butylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4i)

Yellow solid; mp 176–178 ˚C; FT-IR (KBr) ʋ _max 3539, 3363, 3105, 2978, 2933, 2872, 1715, 1664, 1622, 1586, 1574, 1502, 1485, 1454, 1401, 1370, 1227, 1185, 1012, 960, 933, 908, 857, 774, 755, 743, 669, 588, 532, 513, 483 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ _H 8.91 (s, 1H, ─NH─), 7.52 (d, J = 8.5 Hz, 2H, Ph─H), 7.48 (d, J = 7.8 Hz, 2H, Ph─H), 3.59 (s, 3H, N─CH₃), 3.30 (s, 3H, N─CH₃), 1.53 (s, 9H, 3 × CH_{3 ter t-butyl}); ¹³C{¹H} NMR (75 MHz, CDCl₃) δ _C 187.73, 161.50, 156.61, 149.86, 149.25, 139.08, 130.59, 130.15, 125.82, 93.74, 93.16, 53.63, 30.01, 29.73, 28.69; Anal. Calcd for C₁₉H₂₀BrN₃O₄: C, 52.55; H, 4.64; N, 9.68. Found: C, 52.53, H, 4.60; N, 9.65.

3.2.1.10. 5-(4-Chlorobenzoyl)-6-(tert-butylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4j)

Yellow solid; mp 183–184 ˚C; FT-IR (KBr) ʋ _max 3539, 3371, 3105, 3080, 2982, 2941, 2870, 1716, 1662, 1622, 1587, 1577, 1502, 1488, 1455, 1402, 1369, 1227, 1184, 1032, 1010, 960, 935, 910, 862, 755, 743, 632, 619, 560, 509, 473 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ _H 8.91 (s, 1H, ─NH─), 7.63–7.45 (m, 2H, Ph─H), 7.42–7.24 (m, 2H, Ph─H), 3.60 (s, 3H, N─CH₃), 3.31 (s, 3H, N─CH₃), 1.53 (s, 9H, 3 × CH_{3 ter t-butyl}); ¹³C{¹H} NMR (75 MHz, CDCl₃) δ _C 187.67, 161.49, 156.63, 149.86, 149.26, 138.65, 137.23, 130.59, 129.98, 127.66, 93.77, 93.20, 53.63, 30.02, 29.73, 28.70; Anal. Calcd for C₁₉H₂₀ClN₃O₄: C, 58.54; H, 5.17; N, 10.78. Found: C, 58.51, H, 5.15; N, 10.75.

3.2.1.11. 5-(4-Fluorobenzoyl)-6-(tert-butylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4k)

Yellow solid; mp 178–179 ˚C; FT-IR (KBr) ʋ _max 3419, 3351, 3072, 2978, 2965, 2943, 2880, 1718, 1704, 1675, 1656, 1618, 1602, 1547, 1503, 1461, 1448, 1225, 1186, 1161, 1083, 1054, 973, 921, 849, 778, 758, 745, 619, 517, 478 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ _H 8.85 (s, 1H, ─NH─), 7.70–7.59 (m, 2H, Ph─H), 7.13–7.01 (m, 2H, Ph─H), 3.61 (s, 3H, N─CH₃), 3.31 (s, 3H, N─CH₃), 1.52 (s, 9H, 3 × CH_{3 ter t-butyl}); ¹³C{¹H} NMR (75 MHz, CDCl₃) δ _C 187.71, 166.26, 161.38, 156.68, 149.87, 149.26, 136.49, 130.96, 114.60, 114.29, 93.86, 93.22, 53.58, 30.04, 29.73, 28.70; Anal. Calcd for C₁₉H₂₀FN₃O₄: C, 61.12; H, 5.40; N, 11.25. Found: C, 61.14, H, 5.43; N, 11.24.

3.2.1.12. 5-(3-Bromobenzoyl)-6-(tert-butylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4l)

Yellow solid; mp 179–180 ˚C; FT-IR (KBr) ʋ _max 3413, 3351, 3155, 3072, 2982, 2953, 2884, 1718, 1702, 1676, 1659, 1615, 1572, 1546, 1504, 1477, 1451, 1296, 1224, 1185, 1097, 1035, 1001, 986, 941, 849, 753, 740, 700, 651, 632, 608, 525, 512, 472 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ _H 8.93 (s, 1H, ─NH─), 7.73 (s, 1H, Ph─H), 7.62 (d, J = 7.9 Hz, 1H, Ph─H), 7.53 (d, J = 7.8 Hz, 1H, Ph─H), 7.27 (t, J = 8.1 Hz, 1H, Ph─H), 3.61 (s, 3H, N─CH₃), 3.31 (s, 3H, N─CH₃), 1.54 (s, 9H, 3 × CH_{3 ter t-butyl}); ¹³C{¹H} NMR (75 MHz, CDCl₃) δ _C 187.23, 161.51, 156.56, 149.87, 149.28, 142.11, 133.90, 131.60, 131.53, 127.01, 121.50, 93.68, 93.22, 53.69, 30.01, 29.74, 28.70; Anal. Calcd for C₁₉H₂₀BrN₃O₄: C, 52.55; H, 4.64; N, 9.68. Found: C, 52.57, H, 4.63; N, 9.70.

3.2.1.13. 5-(4-Hydroxy-3-methoxybenzoyl)-6-(tert-butylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4m)

Yellow solid; mp 182–183 ˚C; FT-IR (KBr) ʋ _max 3191, 3027, 2986, 2953, 2839, 2789, 1718, 1703, 1661, 1615, 1598, 1492, 1400, 1285, 1249, 1215, 1179, 1124, 1099, 1051, 980, 865, 851, 838, 788, 759, 746, 652, 628, 603, 517, 471 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ _H 8.62 (s, 1H, ─NH─), 7.29 (s, 1H, Ph─H), 7.22 (d, J = 6.2 Hz, 1H, Ph─H), 6.88 (d, J = 8.1 Hz, 1H, Ph─H), 5.84 (bs, 1H, Ph─OH), 3.90 (s, 3H, Ph─O─CH₃), 3.61 (s, 3H, N─CH₃), 3.33 (s, 3H, N─CH₃), 1.51 (s, 9H, 3 × CH_{3 ter t-butyl}); ¹³C{¹H} NMR (75 MHz, CDCl₃) δ _C 187.87, 162.33, 156.69, 149.94, 149.26, 149.01, 145.75, 132.26, 124.16, 113.06, 111.19, 94.25, 93.36, 77.45, 76.92, 76.61, 55.84, 53.46, 30.10, 29.71, 28.67; Anal. Calcd for C₂₀H₂₃N₃O₆: C, 59.84; H, 5.78; N, 10.47. Found: C, 59.86, H, 5.80; N, 10.48.

3.2.1.14. 5-(3,4-Methylendioxybenzoyl)-6-(tert-butylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4n)

Yellow solid; mp 177–178 ˚C; FT-IR (KBr) ʋ _max 3424, 3359, 3093, 2978, 2958, 2912, 2888,2802, 1720, 1660, 1609, 1547, 1503, 1453, 1288, 1258, 1239, 1185, 1038, 923, 891, 865, 781, 756, 743, 663, 610, 589, 569, 517, 490 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ _H 8.62 (s, 1H, ─NH─), 7.20 (d, J = 8.0 Hz, 1H, Ph─H), 7.16 (2, 1H, Ph─H), 6.80 (d, J = 7.9 Hz, 1H, Ph─H), 6.02 (s, 2H, O─CH₂─O), 3.60 (s, 3H, N─CH₃), 3.32 (s, 3H, N─CH₃), 1.30 (s, 9H, 3 × N─CH_{3 ter t-butyl}); ¹³C{¹H} NMR (75 MHz, CDCl₃) δ _C 187.65, 162.34, 161.12, 156.68, 150.31, 149.92, 149.22, 147.03, 134.43, 124.57, 107.12, 101.42, 94.12, 93.36, 53.49, 30.08, 29.70, 28.70; Anal. Calcd for C₂₀H₂₁N₃O₆: C, 60.14; H, 5.30; N, 10.52. Found: C, 60.15, H, 5.33; N, 10.54.

3.3. In silico molecular docking and ADMET

The molecular docking simulation was carried out using AutoDock Vina (version 1.1.2) as an open-source program incorporating UCSF Chimera (version 1.15) as a graphical user interface on Apple MacBook Pro (Retina, 13-inch, Mid-2014, is equipped with a 2.8 GHz dual-core Intel core i5 processor, 8 GB 1600 MHz DDR3 memory, Intel Iris 1536 MB graphics, and 500 GB Apple SSD SM0512F storage.). All 3D desired crystal structures were downloaded from the Protein Databank (https://www.rcsb.org). Before the molecular docking process, the protein structures were cleaned from the non-standard residues and then prepared and minimized in UCSF Chimera. The 2D structures of the newly synthesized furo[2,3-d]pyrimidines were generated in ChemBioDraw Ultra (version 14.0.0.117). Conversion of the 2D skeletons to the related 3D structures of the ligands and their energy minimization processes (by the MM2 force field calculation method) was carried out by ChemBio3D Ultra (version 14.0.0.117). Also, structure editing steps (including dock prep and minimize structure) for the 3D ligands were repeated in UCSF Chimera. The 3D structures of medicinal compounds, which are existed in Fig. 8, were downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov) and ChemSpider (http://www.chemspider.com), and for some cases 2D version was generated from ChemBioDraw Ultra and then converted to the desired 3D format using ChemBio3D Ultra. All the 3D structures of the mentioned medicinal compounds underwent the aforementioned structure editing steps in UCSF Chimera. All the protein–ligand interactions were analyzed in UCSF Chimera and BIOVIA Discovery Studio (version v21.1.0.20298). The 3D figures of the protein–ligand interactions were visualized by UCSF Chimera, and the related 2D projects were drawn by ChemBioDraw Ultra. The in silico ADME and toxicity analyses were investigated using SwissADME (http://www.swissadme.ch) and pkCSM (https://biosig.lab.uq.edu.au/pkcsm/prediction), respectively.

4. Conclusions

Here, we described a green and efficient one-pot three-component regioselective synthetic strategy for the preparation of novel 5-aroyl(or heteroaroyl)-6-(alkylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-diones (4a‒n) in good-to-excellent yields, and then demonstrated their satisfactory multi-targeting inhibitory properties against the active site and putative allosteric hotspots of both SARS-CoV-2 M^Pro and PL^Pro based on molecular docking studies, especially in comparison with various medicinal compounds which used or investigated to fight against COVID-19 to yet. Besides, the drug-likeness properties of the synthesized heterocyclic frameworks (4a‒n) were predicted using in silico ADMET analyses. Furthermore, our studies in this paper showed that the novel series of furo[2,3-d]pyrimidines (4a‒n), especially 4g as hit one, can be a potential COVID-19 drug candidate. Notably, research to find and develop new and green synthetic strategies for the pharmaceutically interesting heterocyclic frameworks, especially anti-SARS-CoV-2 agents, is currently underway in our research group.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Data availability

Data will be made available on request.