Synthesis of 3,3′-methylenebis(4-hydroxyquinolin-2(1H)-ones) of prospective anti-COVID-19 drugs

Abstract

Abstract

During formylation of 2-quinolones by DMF/Et₃N mixture, the unexpected 3,3′-methylenebis(4-hydroxyquinolin-2(1H)-ones) were formed. The discussed mechanism was proved as due to the formation of 4-formyl-2-quinolone as intermediate. Reaction of the latter compound with the parent quinolone under the same reaction condition gave also the same product. The structure of the obtained products was elucidated via NMR, IR and mass spectra. X-ray structure analysis proved the anti-form of the obtained compounds, which were stabilized by the formation hydrogen bond. Molecular docking calculations showed that most of the synthesized compounds possessed good binding affinity to the SARS-CoV-2 main protease (M^pro) in comparable to Darunavir.

Graphic abstract

Electronic supplementary material

The online version of this article (10.1007/s11030-020-10140-z) contains supplementary material, which is available to authorized users.

Introduction

Dimethylformamide (DMF) can react as either an electrophilic and/or a nucleophilic agent. Therefore, DMF can be considered as the source of various key intermediates mediating a plethora of important reactions [1]. More significantly, DMF can participate in many reactions by serving as a multipurpose building block for various units, such as CH₃, N(CH₃)₂, HCO₂, CHO, O, H⁻, H., (CH₃)₂CO, etc. (Fig. 1).

Fig. 1.

DMF as a precursor of various functional groups

Alkyl-quinolones AQ analogs (Fig. 2) act synergistically to inhibit bacterial growth [2, 3] (i.e., two examples assigned as HHQ and HQNP).

Fig. 2.

The structures of 2-heptylquinolin-4(1H)-one (HHQ) and 2-heptyl-4-hydroxyquinoline 1-oxide (HQNO) as alkyl-quinolone (AQ) analogues

Quinolones show a significant similarity to some anticancer [4], anticonvulsant [5–7], anti-dermatities [8], antibacterial [9], antimicrobial [10], anti-Alzheimer [11] and pain relief [12] in addition to their medical, agricultural and industrial uses [13–15]. In previous work with quinolones, Aly et al., synthesized various classes of 2-quinolones such as 2′-amino-2,5′-dioxo-5′,6′-dihydro-spiro(indoline-3,4′-pyrano[3,2-c]quinoline)-3′-carbonitriles [16], 3-(methyl-thio)-4-oxo-4,5-dihydrofuro[3,2-c]quinolone-2-carbonitriles [17], 3-(methylthio)-4-oxo-4,5-dihydro-furo[3,2-c]quinolone-2-carboxamides [17], naphtho[2′,3′:4,5]furo[3,2-c]quinoline-6,7,12(5H)-trione derivatives (as ERK inhibitors with efficacy in BRAF-mutant Melanoma) [18], 2,3-bis-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)succinates, arylmethylene-bis-3,3′-quinoline-2-ones [19], N-2,3-bis(6-substituted-4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)naphthalene-1,4-diones and substituted N-(methyl/ethyl)bisquinolinone triethylammonium salts [20].

Han and Zhou [21] reported that the reaction of two equivalents of quinolone derivatives with one equivalent of aromatic aldehydes and potassium phtalamide under reflux at water–ethanol solution, gave the corresponding 3,3′-arylmethylene-bis(4-hydroxyquinolin-2(1H)-ones. Aly et al. [19] also reported another method of preparing arylmethylene-bis-3,3′-quinoline-2-ones via the reaction of equal equivalents of aromatic amines and diethyl malonate together with half equivalent of the corresponding aromatic aldehydes. 3,3′-Arylmethylene-bis(4-hydroxyquinolin-2(1H)-ones have a great biological activity especially in the composition of vitamin K [22, 23] and anticoagulation [24]. Choudhary et al. [25] synthesized some 3,3′-methylenebis(substituted-4-hydroxyquinolin-2(1H)-ones from the condensation between two molecules of quinolones and one molecule formaldehyde but also neither mechanism nor NMR spectra were discussed for the products. Previously, irradiation of only N-ethyl(methyl)-4-hydroxyquinol-2-ones, was tested in ethanol and afforded their corresponding 3,3′-methylenebis(substituted-4-hydroxyquinolin-2(1H)-ones, virtually eliminating the solvent as a source of formaldehyde [26]. The method suffered from low yields of the obtained products besides to its hazard condition. Moreover the stereochemistry of the obtained products was not discussed.

Utilizing by the expected formylation process during the reaction of 2-quinolones with dimethylformamide/triethylamine (DMF/Et₃N) mixture [27, 28], we explain the abnormal formation of 3,3′-methylenebis(4-hydroxyquinolin-2(1H)-ones). Also, the previous two aforementioned methods could not afford a general preparation method and suffered from low yields, hazardous conditions and no spectroscopic detailed data compared to our announced method of preparation.

Coronavirus disease (COVID-19) is a respiratory infectious disease caused by a novel virus strain, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) [29–32]. Molecular docking is utilized as a substantial tool in the drug discovery process to predict the binding mode and affinity of a drug candidate with a target. To combat COVID-19, the main protease of SARS-CoV-2 (M^pro) would be targeted due to its significant role in the viral replication process. Therefore, the binding modes and affinities of 3,3′-methylenebis(4-hydroxyquinolin-2(1H)-ones) as prospective SARS-CoV-2 inhibitors were predicted against M^pro using Darunavir as a drug reference. Darunavir (DrugBank code: DB01264) is a human immunodeficiency virus (HIV) protease inhibitor and has been recently clinical investigated as anti-COVID-19 drug [33, 34]. The aforementioned encouraged us to synthesize various derivatives of 3,3′-methylenebis(4-hydroxyquinolin-2(1H)-ones) and established a general method of preparing the former compounds. In addition, we investigate the molecular docking of 3,3′-methylenebis(4-hydroxyquinolin-2(1H)-ones) as anti-COVID-19 using Darunavir as a prospective drug reference.

Results and discussion

Upon addition of equimolar amounts of 4-hydroxy-2(1H)-quinolones 1a–g and Et₃N and gently heating in an oil path at 70–80 °C using DMF for 10–12 h, the resulting yellowish orange coloration of the solution was converted gradually to brown color and the 3,3′-methylenebis(substituted-4-hydroxyquinolin-2(1H)-ones 3a–g were precipitated in 70–87% yields (Scheme 1).

Scheme 1.

Formation of 3,3′-methylenebis(substituted-4-hydroxyquinolin-2(1H)-ones from the reaction of 4-hydroxy-2-quinolones 1a–g with DMF 2 and Et₃N

The structural assignment of all the obtained products 3a–g were based on IR, NMR (¹H NMR and ¹³C NMR,) and mass spectra were performed; these and elemental analyses were in good agreement with the assigned structures. As an example, 3,3′-methylene-bis(1-ethyl-4-hydroxyquinolin-2(1H)-one (3g). The elemental analysis and mass spectrometry of compound 3g have the gross formula C₂₃H₂₂N₂O₄. The IR spectrum of 3g indicated the presence of OH at ν = 3500 (OH), 3030 (Ar–CH), 2867 (Alpihatic –CH) and 1643 cm⁻¹ (C=O), whereas CH₂ group at ν = 1458 cm⁻¹. The ¹H NMR spectrum of 3g exhibited a triplet at δ_H = 1.24 and a quartet at 4.38 ppm with the coupling constant J = 7.50 Hz arising from ethyl group. The ¹H NMR spectrum of 3g also showed the methylene protons at δ_H = 3.89. Eight aromatic protons give rise to characteristic signals in the aromatic region of the spectrum, whereas the hydroxyl protons resonated at δ_H = 12.65. The presence of methylene (CH₂) group is evident from ¹³C-DEPT-NMR spectra; exhibiting positive signal at δ_H = 12.95 ppm and negative signal at δ_H = 37.59 ppm (CH₂). The ¹³C NMR spectrum of 3g showed signals at δ_C = 131.50, 122.67, 123.30 and 116.74 ppm due to Ar–CH (C-7), (CH-6), (CH-5) and (CH-8), respectively (Fig. 3). The ¹³C NMR spectrum of 3g supported the ¹³C NMR spectroscopic data by the distinctive appearance of carbon signals representing quinolone C-4a and C-8a (Fig. 3) and resonated at δ_C = 115.15 and 136.70 ppm, respectively. Also, the observed δ_C values for carbon atoms in C-2 at δ_C = 164.84, C-4 at 159.63 and C-3 at 108.53 ppm.

Fig. 3.

Structure and numbering of compound 3g

The structure of 3g was unambiguously determined by a single crystal structure determination showing the bismethylene system (Fig. 4 and see CIF file, note that the crystallographic numbering does not correspond to the systematic IUPAC numbering rules). The bond lengths C(3)–C(21) and C(13)–C(21) are 1.5085 (15) Å and 1.5104 (14) Å, respectively, and have single bond character, while C=O of 1.2536 (13) Å and 1.2605 (13) Å, has double bond character. Whereas, bond lengths C(3)–C(4) 1.3637 (15) Å, C2–C3 1.4384 (15) Å and N1–C2 1.3796 (14) Å indicate the presence of hydrogen bond between O2–H14–O14 and O12-H4-O4.

Fig. 4.

X-ray structure analysis of 3g (displacement parameters drawn at 50% probability level)

The anti-form of the formed compound is established and stabilized by the formed hydrogen bonding. On the basis of the previous reports [1, 27], the formation of 3,3′-methylenebis(substituted-4-hydroxyquinolin-2(1H)-ones 3a–g can be rationalized as depicted in Scheme 2. It would be proposed that Et₃N would abstract a hydrogen proton from the active methylene in C-3 of 1a–g and therefore increasing the nucleophilicty of CH-3 of the quinolone moiety. Thereafter, a nucleophilic addition of the anion CH-3 of 1a–g to the carbonyl carbon of DMF would give the intermediate 4 accompanied by elimination of a molecule of dimethylamine, (CH₃)₂NH to give 4-formyl-2-quinolones (5). Reaction of 5 with 2 via the nucleophilic attack of the oxygen lone pair to the carbonyl in 2 would form intermediate 6 (Scheme 2). Subsequently, elimination of another molecule of dimethylamine (CH₃)₂NH would give the intermediate 7. Further nucleophilic attack of a molecule of 1 to vinylic-carbon in 7 would form the intermediate 8. Finally, decarboxylation of 8 would form compound 3 (Scheme 3). The reaction pathway was also supported via isolation of (CH₃)₂NH, which was identified by TLC analysis.

Scheme 2.

The proposed mechanism describes the formation of compounds 3a–g

Scheme 3.

Formation of compounds 3a–g from the reaction of 3-formyl-4-hydroxy-2-quinolones 5a–g with 1, 2 and Et₃N

Having established reaction conditions in hand, we investigated the formation 3a–g from the reaction of 3-formyl-4-hydroxy-2-quinolone derivatives 5a–g with 1a–g under the condition illustrated in Scheme 3. We reacted 5a–g with their resemble derivatives in 1a–g to obtain symmetric compounds like those in Scheme 1. Fortunately, the target symmetric products of 3,3′-methylenebis(substituted-4-hydroxyquinolin-2(1H)-ones) 3a–g were formed in 60–77% yields (Scheme 3).

Molecular docking calculations

Utilizing molecular docking technique, the binding modes and affinities of compounds 3a–g as prospective SARS-CoV-2 inhibitors were predicted against the main protease (M^pro). The geometrical structures of 3a–g were prepared and docked into the active site of SARS-CoV-2 M^pro using AutoDock 4.2.6 software with docking parameters of GA= 250 and eval= 25,000,000. The predicted binding scores and features are summarized in Table 1. The 2D representations of binding modes of the investigated compounds inside the active site of SARS-CoV-2 M^pro are depicted in Fig. 5.

Table 1.

Molecular docking scores and binding features for compound 3a–g and Darunavir with SARS-CoV-2 main protease (M^pro)

No.	Compound	Docking score (kcal/mol)	Binding features (hydrogen bond length in Å
1	3a	− 8.28	ARG188 (2.18 Å), MET165 (2.63 Å), HIS164 (2.14 Å), GLU166 (2.17 Å, 2.79 Å)
2	3b	− 8.14	ARG188 (2.81 Å), GLN192 (2.37 Å), THR190 (2.09 Å), GLU166 (2.03 Å)
3	3c	− 7.05	ARG188 (1.82 Å), THR190 (2.60 Å) GLN192 (1.93 Å), GLU166 (1.82 Å, 1.96 Å)
4	3d	− 8.30	GLU166 (2.05 Å), ARG 188 (1.80 Å), THR190 (2.08 Å), GLN192 (2.38 Å)
5	3e	− 8.63	THR190 (2.1 Å), GLN192 (2.38 Å), ARG188 (1.79 Å), GLU166 (2.08 Å)
6	3f	− 7.72	GLN189 (1.94 Å), GLU166 (2.01 Å, 2.33 Å)
7	3g	− 7.38	GLU166 (2.82 Å)
8	Darunavir	− 8.19	GLU166 (1.94 Å, 2.88 Å), LEU167 (1.96 Å)

Fig. 5.

2D representation of predicted binding mode of 3a–g inside the active site of COVID-19 main protease (M^pro)

What is interesting about the data in Table 1 is that compounds 3a–g demonstrated good binding affinities toward SARS-CoV-2 M^pro with docking scores ranged from −8.63 to −7.05 kcal/mol. Besides, compounds 3a-g exhibited the same binding modes inside the active site of M^pro, forming an essential hydrogen bond with key amino acid GLU166 residue (Fig. 5). Further interactions including van der Waals, hydrophobic and pi-based interactions were also observed between the compound and the key amino acids inside the SARS-CoV-2 M^pro active site (Fig. 5).

Among the examined compounds, 3e showed the highest binding affinity with docking score of −8.6 kcal/mol against SARS-CoV-2 M^pro. The high potentiality of 3e as SARS-CoV-2 M^pro inhibitor would be returned to its capability to form four hydrogen bonds with THR190, GLN192, ARG188 and GLU166 amino acid with bond lengths of 2.10, 2.38, 1.79 and 2.08 Å, respectively (Figs. 5, 6).

Fig. 6.

3D representations of interactions of 3e and Darunavir with important amino acid residues of COVID-19 main protease (M^pro)

The binding affinity and features of Darunavir were investigated and compared to compound 3e as SARS-CoV-2 M^pro inhibitors. According to molecular docking calculations, Darunavir showed a good binding affinity of −8.19 kcal/mol, forming three hydrogen bonds with GLU166, and LEU167 with bond lengths of 1.94, 2.88 and 1.96 Å, respectively (Figs. 5, 6). A comparison of the molecular docking results revealed the competing binding affinity of 3e with regard to Darunavir as prospective SARS-CoV-2 M^pro inhibitor.

Conclusion

Formylation of 2-quinolones by DMF/Et₃N mixture caused an insertion of a methylene group between two symmetrically quninolones. DMF/Et₃N mixture was proved as a formylatig agent of the parent 2-quinolones. Reaction of 4-formyl-2-quinolone with the parent 2-quinolone under the same reaction condition gave the same product. The aforesaid 3-formyl-2-quinolones would prospectively be used to prepare various symm and/or asymm substitutents of the desired compounds. Molecular docking calculations demonstrated the competing binding affinity of 3e with regard to Darunavir as a prospective SARS-CoV-2 M^pro inhibitor.

Experimental

The IR spectra were recorded by ATR technique (ATR = Attenuated Total Reflection) with a FT device (FT-IR Bruker IFS 88), Institute of Organic Chemistry, Karlsruhe University, Karlsruhe, Germany. The NMR spectra were measured in DMSO-d₆ on a Bruker AV-400 spectrometer, 400 MHz for ¹H, and 100 MHz for ¹³C; and the chemical shifts are expressed in δ (ppm), versus internal tetramethylsilane (TMS) = 0 for ¹H and ¹³C, and external liquid ammonia = 0. The description of signals includes: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublet and m = multiplet. Mass spectra were recorded on a FAB (fast atom bombardment) Thermo Finnigan Mat 95 (70 eV). Elemental analyses were carried out at the Microanalytical Center, Cairo University, Egypt. TLC was performed on analytical Merck 9385 silica aluminum sheets (Kieselgel 60) with Pf₂₅₄ indicator; TLC’s were viewed at λ_max = 254 nm.

Starting materials

1,6-Disubstituted-quinoline-2,4-(1H,3H)-diones 1a–g were prepared according to the literature [35, 36] whereas carbaldehydes 5a, 5b, 5c–f and 5g were synthesized according to the literature [37–40].

General procedure

Method a: A mixture of 1a–g (1 mmol), 15 ml of DMF (2), 0.100 g (1 mmol) Et₃N was gently heated with stirring in an oil path at 70–80 °C for 10–12 h. The time period until the reactants had disappeared, as mentioned in Scheme 1, was monitored by TLC. The formed precipitate was then washed with ethanol (50 mL) and recrystallized from the stated solvents to give pure crystals of 3a–g. The filtrate was concentrated on vacuum and (CH₃)₂NH was obtained and was identified by TLC analysis.

Method b: A a mixture of 1a–g (1 mmol), 5a–g (1 mmol) and 0.100 g (1 mmol) of Et₃N in 2 15 ml of 2 was gently heated with stirring for 8–10 h in an oil path at 70–80 °C. Compounds 3a–g were obtained (i.e., Scheme 3) in pure state as above mentioned.

3,3′-Methylenebis(4-hydroxyquinolin-2(1H)-one) (3a). Orange crystals (DMF/H₂O), yield (method a): 0.233 g (70%) or yield (method b) 0.200 g (60%); ¹H NMR (400 MHz, DMSO-d₆): δ = 3.59 ppm (s, 2H, CH₂), 7.05–7.07 (m, 2H, Ar–H), 7.17–7.19 (m, 2H, Ar–H), 7.30–7.35 (m, 2H, Ar–H), 7.65–7.71 (m, 2H, Ar–H), 11.99 (s, 2H, NH), 12.54 ppm (s, 2H, OH); ¹³C NMR (100 MHz, DMSO-d₆): δ = 19.16 (CH₂), 109.12 (C-3), 115.85 (C-4a), 115.96 (C-8), 122.52 (C-6), 122.78 (C-5), 130.87 (C-7), 136.80 (C-8a), 160.77 (C-4), 165.94 ppm (C-2); MS (Fab, 70 eV, %): m/z = 334 (M⁺, 15), 227 (15), 136 (62), 120 (23), 107 (27), 89 (15). Anal. Calcd. for C₁₉H₁₄N₂O₄ (334.33): C, 68.26; H, 4.22; N, 8.38. Found: C, 68.38; H, 4.35; N, 8.42.

3,3′-Methylenebis(4-hydroxy-6-methylquinolin-2(1H)-one) (3b) [25]. Orange crystals (DMF/EtOH), yield (method a): 0.267 g (74%) or yield (method b): 0.231 g (64%); ¹H NMR (400 MHz, DMSO-d₆): δ = 2.25 (s, 6H, CH₃), 3.78 (s, 2H, CH₂), 7.20–7.29 (m, 2H, Ar–H), 7.30–7.40 (m, 2H, Ar–H), 7.65–7.71 (m, 2H, Ar–H), 12.21 (s, 2H, NH), 12.78 ppm (s, 2H, OH); ¹³C NMR (100 MHz, DMSO-d₆): δ = 19.23 (CH₂), 20.59 (CH₃), 109.12 (C-3), 115.77 (C-4a), 115.88 (C-8), 122.14 (C-5), 131.66 (C-7), 132.15 (C-6), 134.82 (C-8a), 160.62 (C-4), 165.71 ppm (C-2); MS (Fab, 70 eV, %): m/z = 362 (M⁺, 25), 226 (25), 136 (63), 120 (22), 107 (28), 89 (13). Anal. Calcd. for C₂₁H₁₈N₂O₄ (362.38): C, 69.60; H, 5.01; N, 7.73. Found: C, 69.74; H, 4.89; N, 7.83.

3,3′-Methylenebis(4-hydroxy-6-methoxyquinolin-2(1H)-one) (3c). Orange crystals (DMF/CH₃OH), yield (method a): 0.230 g (76%) or yield (method b): 0.260 g (66%); mp = 330–332 °C; IR (KBr): ν = 3450 (OH), 2910 (NH), 3008 (Ar–CH), 1660 (CO), 1453 cm⁻¹ (CH₂); ¹H NMR (400 MHz, DMSO-d₆): δ = 3.81 (s, 6H, OCH₃), 3.78 (s, 2H, CH₂), 7.24–7.30 (m, 2H, Ar–H), 7.32–7.38 (m, 2H, Ar–H), 7.60–7.72 (m, 2H, Ar–H), 12.13 (s, 2H, NH), 12.96 ppm (s, 2H, OH); ¹³C NMR (100 MHz, DMSO-d₆): δ = 19.23 (CH₂), 55.37 (OCH₃), 109.11 (C-3), 115.76 (C-4a), 115.87 (C-8), 122.13 (C-5), 131.62 (C-7), 132.13 (C-6), 134.82 (C-8a), 160.61 (C-4), 165.69 ppm (C-2); MS (Fab, 70 eV, %): m/z = 394 (M⁺, 20), 136 (63), 120 (9), 107 (18), 89 (13). Anal. Calcd. for C₂₁H₁₈N₂O₆ (394.38): C, 63.96; H, 4.60; N, 7.10. Found: C, 63.84; H, 4.72; N, 7.19.

3,3′-Methylenebis(7-chloro-4-hydroxyquinolin-2(1H)-one) (3d) [25]. Orange crystals (DMF/CH₃OH), yield (method a): 0.322 g (80%) or yield (method b): 0.274 g (68%); ¹H NMR (400 MHz, DMSO-d₆): δ = 3.78 (s, 2H, CH₂); 7.22–7.28 (m, 2H, Ar–H), 7.30–7.39 (m, 2H, Ar–H), 7.62–7.70 (m, 2H, Ar–H), 12.15 (s, 2H, NH), 12.86 ppm (s, 2H, OH); ¹³C NMR (100 MHz, DMSO-d₆): δ = 20.01 (CH-₂), 109.00 (C-3), 115.70 (C-4a), 115.02 (C-8), 122.13 (C-6), 130.00 (C-5), 132.13 (C-7), 136.82 (C-8a), 160.62 (C-4), 164.69 ppm (C-2); MS (Fab, 70 eV, %): m/z = 403/402 (/20/18), 136 (63), 120 (9), 107 (18), 89 (13). Anal. Calcd. for C₁₉H₁₂Cl₂N₂O₄ (402.02): C, 56.60; H, 3.00; N, 6.95. Found: C, 56.49; H, 3.12; N, 7.14.

3,3′-Methylenebis(7-bromo-4-hydroxyquinolin-2(1H)-one) (3e) [25]. Orange crystals (DMF/EtOH), yield (method a): 0.406 g (83%) or yield (method b): 0.357 g (73%); ¹H NMR (400 MHz, DMSO-d₆): δ = 3.77 (s, 2H, CH₂), 7.22–7.25 (m, 2H, Ar–H), 7.26–7.30 (m, 2H, Ar–H), 7.70–7.82 (m, 2H, Ar–H), 12.14 (s, 2H, NH), 12.90 ppm (s, 2H, OH); ¹³C NMR (100 MHz, DMSO-d₆): δ = 19.80 (CH₂), 109.10 (C-3), 115.76 (C-4a), 115.10 (C-8), 122.10 (C-6), 128.90 (C-5), 132.98 (C-7), 136.82 (C-8a), 160.58 (C-4), 165.12 ppm (C-2); MS (Fab, 70 eV, %): m/z = 490/489 (20/18), 136 (63), 120 (10), 107 (20), 89 (10). Anal. Calcd. for C₁₉H₁₂Br₂N₂O₄ (489.12): C, 46.37; H, 2.46; N, 5.69. Found: C, 46.48; H, 2.36; 5, 7.73.

3,3′-Methylenebis(4-hydroxy-1-methylquinolin-2(1H)-one) (3f) [26]. Orange crystals (DMF/EtOH), yield (method a): 0.308 g (85%) or yield (method b): 0.272 g (75%); IR (KBr): ν = 3450 (OH), 3040 (Ar–CH), 2820 (CH-Aliphatic), 1641 (CO), 1417 cm⁻¹ (CH-₂); ¹H NMR (400 MHz, DMSO-d₆): δ = 3.72 (s, 6H, CH₃), 3.60 (s, 2H, CH₂), 7.10–7.14 (m, 2H, Ar–H), 7.25–7.31 (m, 4H, Ar–H), 7.72–7.78 (m, 2H, Ar–H), 12.87 ppm (s, 2H, OH); ¹³C NMR (100 MHz, DMSO-d₆): δ = 19.15 (CH₂), 38.01 (CH₃), 109.13 (C-3), 115.80 (C-4a), 115.97 (C-8), 122.53 (C-6), 122.77 (C-5), 130.98 (C-7), 136.82 (C-8a), 160.66 (C-4), 165.80 ppm (C-2); MS (Fab, 70 eV, %): m/z = 362 (M⁺, 33), 136 (63), 120 (10), 107 (20), 89 (10). Anal. Calcd. for C₂₁H₁₈N₂O₄ (362.38): C, 69.60; H, 5.01; N, 7.73. Found: C, 69.72; H, 5.12; N, 7.65.

3,3′-Methylenebis(1-ethyl-4-hydroxyquinolin-2(1H)-one) (3g) [26]. Orange crystals (DMF/EtOH), yield (method a): g 0.340 (87%) or yield (method b): 0.300 g (77%); IR (KBr): ν = 3500 (OH), 3030 (Ar–CH), 2867 (CH-Aliphatic), 1643 (CO), 1458 cm⁻¹ (CH₂); ¹H NMR (400 MHz, DMSO-d₆): δ = 1.24 (t, 6H, CH_3-), 3.89 (s, 2H, CH₂), 4.38 (q, 4H, CH₂), 7.00–7.05 (m, 2H, Ar–H), 7.29–7.35 (m, 2H, Ar–H), 7.59–7.70 (m, 4H, Ar–H), 7.90–8.07 (m, 2H, Ar–H), 12.65 ppm (s, 2H, OH); ¹³C NMR (100 MHz, DMSO-d₆): δ = 12.95 (CH-₂-Et), 21.11 (CH₂), 37.59 (CH₃-Et), 108.52 (C-3), 115.15 (C-4a), 116.74 (C-8), 122.67 (C-6), 123.30 (C-5), 131.50 (C-7), 136.70 (C-8a), 159.63 (C-4), 164.83 ppm (C-2); MS (Fab, 70 eV,  %): m/z = 390 (M⁺, 18), 202 (12), 136 (62), 120 (12), 107 (20), 89 (20). Anal. Calcd. for C₂₃H₂₂N₂O₄ (390.42): C, 70.75; H, 5.68; N, 7.17. Found: C, 70.82; H, 5.77; N, 7.29.

Crystal structure determination

The single-crystal X-ray diffraction study of 3g was carried out on a Bruker D8 Venture diffractometer with Photon II detector at 123(2) K using Cu-Kα radiation (λ = 1.54178 Å). Dual space/intrinsic methods (SHELXT) [41] were used for structure solution and refinement was carried out using SHELXL-2014 (full-matrix least-squares on F²) [42]. Hydrogen atoms were localized by difference electron density determination and refined using a riding model (H(O) free). A semi-empirical absorption correction was applied.

3g: Orange crystals, C₂₃H₂₂N₂O₄, M_r = 390.42, crystal size 0.36 × 0.24 × 0.12 mm, monoclinic, space group P2₁/c (No. 14), a = 13.2293 (3) Å, b = 17.0327 (4) Å, c = 8.5503 (2) Å, β = 101.919 (1)°, V = 1885.11 (8) ų, Z = 4, ρ = 1.376 Mg/m⁻³, µ(Cu-K_α) = 0.77 mm⁻¹, F(000) = 824, 2θ_max = 144.4°, 16886 reflections, of which 3689 were independent (R_int = 0.024), 268 parameters, 2 restraints, R₁ = 0.034 (for 3587 I > 2σ(I)), wR₂ = 0.089 (all data), S = 1.06, largest diff. peak/hole = 0.27/− 0.19 e Å⁻³.

Molecular docking calculations

All molecular docking calculations were carried out using Autodock 4.2.6 software [43]. The crystal structure of SARS-CoV-2 main protease (M^pro; PDB code: 6LU7 [44]) was taken as a template for all molecular docking calculations. Water molecules, ions and the ligand were deleted. The protonation state of M^pro was evaluated using H⁺⁺ server, and all missing hydrogen atoms were added [45]. All docking parameters were kept to default values, except the number of genetic algorithm (GA) run and the maximum number of energy evaluation (eval) which were set to 250 and 25,000,000, respectively. The docking grid was set to 60 Å × 60 Å ×  60 Å with a grid spacing value of 0.375 Å, and the grid center was placed at the center of the active site of M^pro. The geometrical structures of all examined synthesized compounds were minimized with MMFF94s force field using SZYBKI software [46] and the partial atomic charges were assigned using Gasteiger method [47].

Supporting Information

CCDC 2011538 (3g) contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Funding

No funds were applicable.