Antioxidant activity, molecular docking, and modeling pharmacokinetics study of some benzo[f]quinoline candidates (2025)

Benzoquinoline and its derivatives play a crucial role in pharmaceutical chemistry, and they are considered as a precursor for the construction of heterocyclic compounds that possess a wide spectrum of biological activities including antioxidant^1,2, antibacterial, antifungal, anti-inflammatory, anticancer, anti-Alzheimer’s, and insecticidal properties^{3,4,5,6,7,8,9,10,11,12,13}. Quinoline and its candidates are extensively employed as devoted compounds to prepare a diversity of heterocycles with a broad array of pharmacological actions^14,15,16,17. Also, benzoquinolines were found in many pharmaceuticals and natural products and were utilized as templates for the synthesis of many drugs^4,8. These compounds might act as an antioxidant by reacting with various free radicals through hydrogen atom or single electron transfer. Oxidative stress is one the primary contributors to significant severe diseases, which arises from the over-production of reactive oxygen species (ROS) in the human body.

ROS describes several reactive molecules and free radicals derived from molecular oxygen such as hydrogen peroxide (H₂O₂), hydroxyl radical (^•OH), peroxyl radical (ROO^•), and superoxide anion radical (O₂^•−). The high levels of such species can damage biological molecules such as proteins, carbohydrates, lipids, and DNA, leading ultimately to cellular dysfunction. Therefore, there is a significant demand for the development of new potent antioxidants that can delay or prevent oxidative damage in the human body via scavenging or regulating the formation and elimination of ROS.

Otherwise, benzoxazinone derivatives are an essential class of benzo-fused heterocycles due to their facile ring opening with nitrogen nucleophiles producing acyclic and heterocyclic candidates with diverse biological activity^{18,19,20,21,22,23}. In continuation to our program for the synthesis of various biologically active candidates^{24,25,26,27,28}, these findings directed our attention to a design and synthesize new heterocyclic system that incorporated both benzoquinoline and benzoxazine frameworks and studied its reactivity towards some mono- and bi-dentate nitrogen nucleophiles aiming to obtain new heterocycles with potential antioxidant activity (to protect cellular components from free radical damage) and supported by in silico studies.

Rationale and design

One of the most important targets for the design of new antioxidant drugs is polyaromatic ligands of larger surface area and applicable steric properties¹. Some of these planar polyaromatic candidates were biologically active agents, which were shown in Fig.1. The nature, tautomerism, and size of chromophores are important parameters that govern the radical scavenging activity^26,28. Recently, much consideration has been promoted to design and synthesize novel and efficient targeted antioxidant agents encompassing benzo[f]quinoline scaffold^29,30.

The candidates with proper functional substituents (e.g., OH or NH) can exhibit great efficacy in antioxidant activity due to their potent abilities to scavenge ROS directly through hydrogen or electron transfer. Thus, this work reports the molecular design and synthesis of a series of new benzo[f]quinolines (planar polyaromatic ligands) encompassing either side chain (hydrophilic and hydrophobic groups as compounds 3 and 4), phenyl, or heterocyclic scaffolds (enhancing lipophilic interactions) at position-3 of quinoline core. The rationale for the design of these substrates can be shown in Fig.2 starting from a benzoquinoline scaffold through an insertion of side chains and heterocycles (like benzoxazinone, quinazolinone, pyrimidine, oxadiazole, imidazole, and benzimidazole) with or without linkers (amide groups). Compound 19, a benzo[f]quinoline bearing a benzimidazole scaffold, displayed the best antioxidant properties among the synthesized candidates, through possible tautomerism and extended conjugation increasing its scavenging activity via increasing hydrogen bonding interactions and enhancing the lipophilicity properties.

Chemistry

Owing to their diverse biological activities, our research focused on developing a framework of benzoxazinone skeleton based on a benzo[f]quinoline core. Thus, 3-chlorobenzo[f]-quinoline-2-carbaldehyde (1)³¹ condensed with ethyl cyanoacetate to afford the arylidene derivative 2 which was easily transformed into the corresponding acid 3 via hydrolysis using alcoholic NaOH followed by an acidification with dilute hydrochloric acid (10%). The IR spectrum of acid 3 lacked the ester carbonyl group and exhibited absorption bands for OH at ν 3440cm^-1 and acid C = O at ν 1706cm^-1. Treating the acid 3 with thionyl chloride yielded the corresponding acid chloride 4. The IR spectrum of acid chloride 4 revealed that the absence of acid carbonyl and OH groups and the presence of an absorption band for C = O of acid chloride group at ν 1761cm⁻¹.

The benzoic acid derivative 5 was obtained from the treatment of the acid chloride derivative 4 with anthranilic acid in dry dioxane and triethyl amine (Et₃N) at room temperature. The IR spectrum of compound 5 exhibited an absorption band for acid C = O at ν 1695cm^-1 and amide C = O at ν 1667cm^-1. Its ¹H NMR spectrum exhibited an exchangeable OH proton in the downfield region δ 12.85ppm, in addition to a singlet signal for the NH proton at δ 9.00ppm. Cyclization of the benzoic acid derivative in the presence of acetic anhydride furnished the benzoxazinone derivative 6 (Fig.3). The IR spectrum of benzoxazinone 6 showed an absorption band for lactone C = O at ν 1774cm^-1. Another evidence for its structure was achieved from its ¹H NMR spectrum which lacked the labile protons of OH and NH. The mass spectra of compounds 3–6 provided the correct molecular ion peaks.

The benzoxazinone 6 was submitted to react with some mono- and bi-nitrogen nucleophiles to construct other heterocyclic systems. Initially, the ammonolysis of benzoxazinone 6 with ammonium acetate under fusion condition for 6h afforded the quinazoline derivative 7. The IR spectrum lacked the lactone absorption band and the ¹H NMR spectrum showed one proton of NH group at δ 10.42ppm. Treating the benzoxazine derivative 6 with benzylamine, isobutyl amine or cyclohexylamine in refluxing n-butanol or ethanol/glacial acetic acid yielded the corresponding quinazoline derivatives 8–10 (Fig.4).

The IR spectra of compounds 8–10 exposed the absence of the lactone C = O and the appearance of an absorption band for amide C = O. The ¹H NMR of compound 8 revealed a singlet signal integrated into two protons at the up-field region δ 3.40ppm corresponding to CH₂ protons. The ¹H NMR of compound 9 exposed a doublet signal for CH₂ at δ 3.29ppm integrated for two protons, multiplet signal for CH at δ 2.62ppm, and doublet signal for two CH₃ at δ 0.90ppm integrated for six protons. The ¹H NMR of compound 10 was quite consistent with the proposed structure.

Quinazoline bearing a pyrazolone ring 11 was achieved via the reaction of benzoxazinone derivative 6 with 4-amino-1,5-dimethyl-2-phenyl-1,2-dihydropyrazol-3-one in 1,4-dioxane. The IR spectrum lacked lactone carbonyl absorption and displayed the amide carbonyl absorption. Heating benzoxazinone 6 with aromatic amines as 2-aminopyrimidine or 4-aminoacetophenone furnished the benzamide derivatives 12 and 13, respectively. Their IR spectra showed an absorption band for amide instead of lactone carbonyl absorption. The ¹H NMR spectra of 12 disclosed two singlet signals at δ 12.69 and δ 9.02ppm for 2 NH protons. While for compound 13, it displayed two singlet peaks at δ 12.71ppm and δ 8.90ppm for 2 NH and one singlet peak integrated for three protons for CH₃ group at δ 2.47ppm which indicate the formation of compound 13 (Fig.5).

On the other hand, to investigate the behavior of benzoxazine derivative 6 towards different 1,2-and 1,4-bidentate nucleophiles, it was submitted to react with ethyl carbazate, phenylhydrazine, ethanolamine, ethylenediamine, thiosemicarbazide and 2-aminoaniline (Figs. 6, 7). Indeed, treating benzoxazinone 6 with ethyl hydrazine carboxylates or phenylhydrazine in refluxing dioxane afforded the 5-hydroxy-1,3,4-oxadiazole derivative 14 and the quinazoline derivative 15, respectively. The IR spectra of compounds 14 and 15 lacked lactone carbonyl and showed an absorption band for C = O amide. The ¹H NMR spectrum of compound 14 disclosed two broad singlet peaks at δ 13.04 and 8.92ppm for OH and NH, respectively. Meanwhile, ¹H NMR spectrum of compound 15 exposed a broad singlet signal in the downfield region at δ 13.38ppm for NH. Conducting the benzoxazinone 6 with 1,4-binucleophile as 2-aminoethanol in dioxane yielded the quinazoline derivatives 16. Its ¹H NMR spectrum demonstrated a singlet signal at δ 3.73ppm for OH and two triplet signals for CH₂-CH₂ groups at δ 3.02 and 2.61ppm each integrated for 2 protons which confirmed the assigned structure (Fig.6).

On the other hand, the imidazoline derivative 17 was obtained from the condensation of benzoxazine derivative 6 with ethylenediamine in ethanol (Fig.7). Its IR spectrum lacked lactone carbonyl absorption, and displayed the amide carbonyl absorption, The ¹H NMR displayed two singlet signals at δ 13.20ppm and δ 8.97ppm for 2 NH and two triplet signals at δ 3.99ppm and δ 3.22ppm for two CH₂ groups. A plausible pathway for this reaction was represented in Fig. S1. Initially, a lactone ring opening of substrate 6 had occurred by nucleophilic attack of primary amino group of ethylenediamine followed by 5-exo-trig cyclization to remove a water molecule to form an imidazoline candidate 17.

The reaction of benzoxazinone 6 with thiosemicarbazide in ethanol and acetic acid underwent lactone ring opening to obtain the thiosemicarbazide derivative 18. The IR spectrum showed absorption bands for NH, NH₂, CN, and C = O groups. Its ¹H NMR spectrum showed three singlet signals at δ 12.10, 10.72, and 9.43ppm for 3 NH, and a singlet signal integrated for 2 protons at δ 7.83ppm corresponding for NH₂. Like the behavior with ethylenediamine, the benzimidazole derivative 19 was formed from the reaction of the benzoxazinone 6 with 2-aminoaniline in refluxing ethanol containing acetic acid. Its ¹H NMR spectrum exposed two singlet signals for 2 NH at δ 12.46ppm and 8.99ppm (cf. Figure7). The suggested pathways for the formation of compounds 18 and 19 can be visualized via Figs. S2 and S3, respectively.

Most of the prepared compounds were tested for their in vitro antioxidant activity according to phosphomolybdenum method using ascorbic acid as a reference^32,33. The total antioxidant capacity of a sample was measured in mg ascorbic acid equivalents (AAE)/g sample. The results in Table 1 showed that the benzimidazole derivative 19 exhibited very strong activity. In addition, compounds 3, 9, 13, and 17 showed strong activity. Otherwise, compounds 4, 7, 10, 16, and 18 displayed moderate activity while the other compounds showed weak activity. The higher potency of compound 19 may be attributed to the aromaticity and extended conjugation^34,35 integrated with the benzimidazole skeleton.

Molecular docking study

A molecular docking simulation was performed to disclose the modes of interactions of the most potent benzimidazole 19 toward HCV NS5B polymerase (PDB ID: 3SKA, Resolution: 1.73Å)³⁶. The binding affinity was measured by the binding energies (S-score, kcal/mol) and hydrogen bonds. The synthesized complexes were docked in the same groove of binding site of native co-crystallized ligand (053) (cf. Figure8).

The results of docking analysis of benzimidazole 19 with respect to the co-crystallized ligand (053) were depicted in 2D and 3D graphical representations (cf. Table S1). According to the data presented (cf. Table 2), ligand 19 exhibited binding energy of − 8.4812kcal/mol, which was greater than that of co-crystallized ligand, referring to strong binding to certain key nucleobases and amino acids (CYS 366 and ASN 411) of HCV NS5B polymerase through hydrogen bonding and pi-hydrogen interactions, revealing its potential usage as an antioxidant agent.

DFT study

DFT parameters and global descriptors were calculated for the most potent compounds to correlate the relation between chemical structure and activity (cf. Table S2 and Fig.9)^37,38,39. The structures of these compounds were drawn using ChemBio3D 14.0. The ∆E (E_LUMO—E_HOMO) values decrease in the order of ascorbic acid, 9, 17, 19, 13, 3, respectively. Among them, the lowest ∆E value (1.351kcal/mol) was discovered by compound 3, making it more reactive towards radical surface interactions, being efficient of donating electrons easily to hole surface^40,41 and therefore might display strong antioxidant efficiency. According to ionization potential (Ip, eV) values, compound 9 exhibited the lowest value (7.497eV) which means that it is more feasible to remove electrons from its last orbitals, HOMO. Regarding the 1,4-VDW (van-der Waal) interactions, the values increase in the order of ascorbic acid, 3, 17, 19, 13, and 9, respectively. Compound 9 exhibited the highest 1,4-VDW value.

Modeling pharmacokinetics study

The ADME profiles of the potent compounds including their physicochemical properties, lipophilicity, and drug-likeness were calculated in order to decrease the time required to select substrates from a huge collection of compounds in the early stages of drug discovery, biological activities, and development for potent drugs (cf. Table S3)^42,43,44,45. The antioxidant activity revealed a range of TAC values which can be also attributed to compound’s ADME profile. TPSA (topological polar surface area) is a key property linked to drug bioavailability, which should be less than 140 Ų. The potent compounds 3, 9, 13, 17, and 19 exhibited TPSA of 73.98, 71.57, 111.95, 90.17, and 94.46 Ų, respectively, which are predicted to exhibit good passive oral absorption. Also, their consensus Log P_o/w values were 3.25, 5.52, 5.24, 4.26, and 5.62, respectively, which offered good lipophilicity and found to comply with Lipinski’s rule of five. Compounds 3, 17, and 19 showed no violations to Lipinski’s rule. Based on the pink area on the radar chart for substances (cf. Figures S4-S20), the bioavailability of those substances was also calculated.

According to calculations, compounds 3, 9, and 17 exhibited a high GI absorption and an excellent bioavailability score of 0.85, 0.55, and 0.55, respectively, as shown in Table S3. The variance in GI absorption might depict the differences in observed antioxidant efficacy of compounds. High GI absorption enables effective internal distribution and interaction with receptors, enhancing antioxidant activity. These compounds were fully included in the pink area, and this supported their well-predicted oral bioavailability. Their skin permeation (Log K_P) parameters were -5.14, -4.38, -4.90, -5.44, and -4.12cm/s, which made the bioactive compounds easier to access through the skin, protecting the body from free radical cell damage, supporting the immune system and assisting the body in the absorption process.

Also, their cytochrome P450 isoenzymes (CYP1A2, CYP2D6, CYP2C9, CYP2C19, and CYP3A4), which play a substantial role in the biotransformation of medicines through O-type oxidation processes, were also estimated. All compounds are predicted as non-inhibitors of CYP2D6 and hence side effects (i.e. liver dysfunction) are not expected upon administration of them. Regarding the absorption property, compounds 5, 6, 7, 9, 16, and 17 showed gastrointestinal tract (GIT) absorption due to their being in the BOILED-EGG chart white area (cf. Figure10). Also, compounds 3 and 4 are expected to penetrate the blood–brain barrier (BBB), existed inside chart yellow area, which selectively regulates the permeability of drugs to the brain. Furthermore, they are not potential substances for permeability glycoprotein (PGP) which is indicated by red.

Regarding SAR of the antioxidant properties, it was noted that the active compounds satisfy the Lipinsk, Veber, and Ghose rules, with no violations. They exhibited a higher number of H-bond donors and acceptors (cf. Figure11 and Table S3). Also, they exhibited higher values of bioavailability score, Log K_P (skin permeation), and consensus Log P_o/w (lipophilicity). The benzimidazole 19 had the highest consensus Log P_o/w value (5.62), which strongly enhanced lipophilicity and the binding interactions with receptors (cf. Table 2). Also, the existence of tautomerism in compounds increases antioxidant activity via increasing hydrogen bonding interactions. Also, the extended conjugation increases the scavenging activity by enhancing the lipophilicity properties.

“Melting points are uncorrected and were measured on a MEL-TEMP II electric melting point apparatus. The IR spectra (ν, cm^-1) were recorded by KBr disks on Fourier Transform Infrared Thermo Electron Nicolet iS10 Spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) at Faculty of Science, Ain Shams University. The ¹H and ¹³C NMR spectra (δ, ppm) were run at a BRUKER 400 and 100 MHz using tetramethyl silane as an internal standard in deuterated dimethyl sulfoxide (DMSO-d₆) at Faculty of Pharmacy, Ain Shams University. Electron impact mass spectra were carried out on direct probe controller inlet part to single quadrupole mass analyzer in Thermo Scientific GCMS MODEL (ISQ LT) using Thermo X-CALIBUR software at the regional center for mycology and biotechnology (RCMB), Azhar University, Cairo, Egypt. Elemental analyses were measured using Perkin-Elmer 2400 CHN elemental analyzer at Faculty of Science, Ain Shams University, and satisfactory analytical data (±0.4) were obtained. The reactions were monitored by thin-layer chromatography (TLC) using Merck Kiesel gel 60F₂₅₄ obtained from Fluka, Switzerland.”

Ethyl 3-(3-chlorobenzo[f]quinolin-2-yl)-2-cyanoacrylate (2)

A mixture of 3-chlorobenzo[f]quinoline-2-carbaldehyde (1) (0.01mol, 2.41g) and ethyl cyanoacetate (0.01mol, 1.13ml) in ethanol (20ml) and piperidine (0.1mL) was refluxed for 30min. The formed product was filtered, washed with ethanol, dried, and recrystallized from ethanol to afford beige crystals, mp 206–208°C, Yield (90%). Anal. Calcd. for C₁₉H₁₃ClN₂O₂ (336.8): C, 67.76; H, 3.89; N, 8.32; Found: C, 67.74; H, 3.92; N, 8.35%. FTIR (ν, cm^-1): 2224 (CN), 1712 (C = O ester), 1621 (C = N). ¹H NMR (DMSO-d₆, δ, ppm): 9.10 (s, 1H, CH olefinic), 9.02 (d, 1H, C5-Ar, J = 7.4Hz), 8.62 (s, 1H, C4-H quinoline), 8.11–7.86 (m, 5H, Ar–H), 4.39 (q, 2H, CH₂, J = 6.7Hz), 1.35 (t, 3H, CH₃, J = 6.7Hz). ¹³C NMR (DMSO-d₆, δ, ppm): 191.1, 161.4, 150.1, 148.9, 147.1, 139.5, 130.7, 129.8 (2), 128.9 (2), 128.5 (2), 125.5 (2), 124.7, 108.4, 63.4, 14.5.

3-(3-Chlorobenzo[f]quinolin-2-yl)-2-cyanoacrylic acid (3)

To a solution of ethyl-3-(3-chlorobenzo[f]quinolin-2-yl)-2-cyanoacrylate (2) (0.01mol, 3.37g) in ethyl alcohol (20ml), sodium hydroxide (0.01mol, 0.4g) in ethyl alcohol (3ml) was added and then the reaction mixture was stirred for 6h at room temperature. The reaction mixture was poured onto crushed ice and diluted solution of HCl. The formed precipitate was filtered, dried, and recrystallized from dioxane to form a yellow precipitate, mp. > 300oC, Yield (90%). Anal. Calcd. for C₁₇H₉ClN₂O₂ (308.7): C, 66.14; H, 2.94; N, 9.07; Found: C, 66.18; H, 2.90; N, 9.11%. FTIR (ν, cm^-1): 3440 (br. OH), 2225 (CN), 1706 (C = O acid). ¹H NMR (DMSO-d₆, δ, ppm): 9.05 (s, 1H, CH olefinic), 8.54 (s, 1H, C4-H quinoline), 8.97 (d, 1H, C₅-Ar, J = 7.6Hz), 8.10–7.80 (m, 6H, Ar–H). MS, m/z (%): 308 (12), 265 (61), 116 (28), 77 (100).

3-(3-Chlorobenzo[f]quinolin-2-yl)-2-cyanoacryloyl chloride (4)

A mixture of acid derivative 3 (0.01mol, 3.08g) and thionyl chloride (15ml) was heated on water bath for 8h. The reaction mixture was left until all thionyl chloride evaporated. The formed precipitate was washed with hot petroleum ether (60–80) to furnish a yellow solid, mp. 252–254oC, Yield (85%). Anal. Calcd. for C₁₇H₈Cl₂N₂O (327.2): C, 62.41; H, 2.46; N, 8.56; Found: C, 62.45; H, 2.50; N,8.52%. FTIR (ν, cm^-1): 2224 (CN), 1761, 1715 (C = O). ¹H NMR (DMSO-d₆, δ, ppm): 9.01 (s, 1H, CH olefinic), 8.93 (d, 1H, CH, C₅-Ar, J = 7.8Hz), 8.50 (s, 1H, C4-H quinoline), 8.07–7.97 (m, 5H, Ar–H). ¹³C NMR (DMSO-d₆, δ, ppm): 162.7, 152.7, 149.2, 148.8, 147.1, 146.9, 139.1, 130.5, 129.6, 128.7, 128.4, 125.3, 125.2, 124.6, 115.4, 114.8, 113.1, 109.5, 103.5, 99.9. MS, m/z (%): 327 (22), 283 (100), 257 (40), 78 (92).

2-(3-(3-Chlorobenzo[f]quinolin-2-yl)-2-cyanoacrylamido)benzoic acid (5)

A mixture of acid chloride derivative 4 (0.01mol, 3.7g) and anthranilic acid (0.01mol, 1.37g) in dry dioxane (20ml) containing triethylamine (0.1mL) was stirred at room temperature for 10h. The solid obtained was filtered, dried, and recrystallized from dioxane to provide yellow crystals, mp.180–182oC, Yield (82%). Anal. Calcd. for C₂₄H₁₄ClN₃O₃ (427.8): C, 67.38; H, 3.30; N, 9.82; Found: C, 67.34; H, 3.33; N, 9.86%. FTIR (ν, cm^-1): 3422 (br. OH), 3212, 3172 (NH), 2207 (CN), 1695 (C = O acid), 1667 (C = O amide). ¹H NMR (DMSO-d₆, δ, ppm): 12.85 (br.s, 1H, OH, exchangeable), 9.20 (br.s, 1H, NH, exchangeable), 9.15 (s, 1H, CH olefinic), 8.66 (s, 1H, C4-H quinoline), 8.64–723 (m, 10H, Ar–H). ¹³C NMR (DMSO-d₆, δ, ppm): 179.9, 169.5 (C = O), 158.1 (C = N), 147.1 (C = C), 140.1, 138.9, 134.4, 133.7, 131.2, 130.0 (2), 129.2 (2), 128.3 (2), 127.8, 125.4 (2), 124.9 (2), 124.2, 123.9, 120.3, 117.0 (CN).

3-(3-Chlorobenzo[f]quinolin-2-yl)-2-(4-oxo-4H-benzo[d][1,3]oxazin-2-yl)acrylonitrile (6)

A suspension of benzoic acid derivative 5 (0.01mol, 4.27g) in acetic anhydride (25ml) was heated on water steam for 6h. The formed precipitate was filtered, dried, and recrystallized from benzene to yield a pale-yellow precipitate, mp. 280–282oC, Yield (78%). Anal. Calcd. for C₂₄H₁₂ClN₃O₂ (409.8): C, 70.34; H, 2.95; N, 10.25; Found: C, 70.38; H, 2.90; N, 10.19%. The FTIR (ν, cm^-1): 2226 (CN), 1774 (C = O), 1620 (C = N). ¹H NMR (DMSO-d₆, δ, ppm): 9.2 (s, 1H, CH olefinic), 8.69 (s, 1H, C4-H quinoline), 9.05 (d, 1H, C5-Ar, J = 8.3Hz), 8.22–7.37 (m, 9H, Ar–H). ¹³C NMR (DMSO-d₆, δ, ppm): 170.4, 158.4, 158.1, 148.9, 147.8, 147.0, 145.9, 139.4, 134.9, 130.6 (2), 130.2, 129.8 (2), 129.5 (2), 128.8 (2), 128.5 (2), 125.5 (2), 125.1, 124.6. MS, m/z (%): 411 (M^+. + 1, 19), 367 (44), 225 (10), 137 (18), 90 (29), 60 (100).

3-(3-Chlorobenzo[f]quinolin-2-yl)-2-(4-oxo-3,4-dihydroquinazolin-2-yl)acrylonitrile (7)

A mixture of benzoxazine derivative 6 (0.002mol, 1g) and ammonium acetate (2g) was fused for 8h. The formed precipitate was filtered, washed with ethanol, dried, and recrystallized from dioxane to form yellow crystals, mp. > 300°C, Yield (80%). Anal. Calcd. for C₂₄H₁₃ClN₄O (408.8): C, 70.51; H, 3.21; N,13.70; Found: C, 70.55; H, 3.18; N, 13.72%. FTIR (cm^-1): 3338 (NH), 2213 (CN), 1689 (C = O amide), 1624 (C = N). ¹H NMR (DMSO-d₆, δ, ppm): 10.42 (br.s, 1H, NH, exchangeable), 9.10 (s, 1H, CH olefinic), 8.59 (s, 1H, C4-H quinoline), 8.53 (d, 1H, Ar–H, J = 7.9Hz), 8.13–7.07 (m, 9H, Ar–H). MS, m/z (%): 408 (50), 354 (95), 313 (56), 298 (100), 220 (63), 164 (85), 91 (47).

2-(3-Benzyl-4-oxo-3,4-dihydroquinazolin-2-yl)-3-(3-chlorobenzo[f]quinolin-2-yl)acrylonitrile (8)

To a solution of benzoxazine derivative 6 (0.002mol, 1g) in n-butanol (20ml), benzylamine (0.002mol, 0.21ml) was added and then the reaction mixture was refluxed for 6h. The precipitated solid was filtered and washed with n-butanol, dried, and recrystallized from dioxane to produce pale-yellow crystals, mp. 242–245°C, Yield (85%). Anal. Calcd. for C₃₁H₁₉ClN₄O (499): C, 74.62; H, 3.84; N, 11.23; Found: C, 74.68; H, 3.79; N, 11.27%. FTIR (ν, cm^-1): 2207 (CN), 1687 (C = O amide), 1621 (C = N). ¹H NMR (DMSO-d₆, δ, ppm): 9.03 (s, 1H, CH olefinic), 8.96 (d, 1H, C₅-H), 8.56 (s, 1H, C4-H quinoline), 8.03–7.36 (m, 12H, Ar–H), 7.09 (t, 2H, Ph-H, J = 7.6Hz), 3.40 (s, 2H, CH₂). ¹³C NMR (DMSO-d₆, δ, ppm): 169.9 (C = O), 157.8, 148.23 (C = N), 146.15 (C = C), 145.6, 140.4, 138.7, 134.2, 131.1, 130.3, 129.8 (2), 129.1 (2), 129.0, 128.3 (2), 127.9 (2), 125.6, 125.1 (2), 124.9, 124.6 (2), 124.0 (2), 122.67, 119.04, 114.9, 113.4 (Ar–C).

3-(3-Chlorobenzo[f]quinolin-2-yl)-2-(3-isobutyl-4-oxo-3,4-dihydroquinazolin-2-yl)acrylonitrile (9)

A mixture of benzoxazine 6 (0.002mol, 1g) and isobutylamine (0.002mol, 0.15ml) in ethanol (20ml) containing glacial acetic acid (0.1mL) was refluxed for 10h. The solid obtained was filtered off and recrystallized from dioxane to produce yellow crystals, mp. 268–270°C, Yield (90%). Anal. Calcd. for C₂₈H₂₁ClN₄O (465): C, 72.33; H, 4.55; N, 12.05; Found: C, 72.36; H, 4.50; N, 12.09%. FTIR (ν, cm^-1): 2210 (CN), 1689 (C = O imide), 1611 (C = N). ¹H NMR (DMSO-d₆, δ, ppm): 9.13 (s, 1H, CH olefinic), 8.61 (s, 1H, C4-H quinoline), 9.03 (d, 1H, C₅-H, J = 7.4Hz), 8.56–7.12 (m, 9H, Ar–H), 3.29 (d, 2H, CH₂, J = 6.4Hz), 2.62 (m, 1H, CH), 0.90 (d, 6H, 2CH₃, J = 6.8Hz). MS, m/z (%): 465 (M^+., 7.6), 417 (75), 371 (100), 126 (66), 105 (43).

3-(3-Chlorobenzo[f]quinolin-2-yl)-2-(3-cyclohexyl-4-oxo-3,4-dihydroquinazolin-2-yl)acrylonitrile (10)

A mixture of benzoxazine 6 (0.002mol, 1g) and cyclohexylamine (0.002mol, 0.2g) in ethanol (20ml) containing glacial acetic acid (0.1mL) was refluxed for 10h. The solid obtained was filtered, dried, and recrystallized from dioxane to produce yellow crystals, mp. 272–274oC, Yield (88%). Anal. Calcd. for C₃₀H₂₃ClN₄O (491): C, 73.39; H, 4.72; N, 11.41; Found: C, 73.44; H, 4.75; N, 11.37%. FTIR (ν, cm^-1): 2207 (CN), 1693 (C = O amide), 1629 (C = N). ¹H NMR (DMSO-d₆, δ, ppm): 9.15 (s, 1H, CH olefinic), 8.63 (s, 1H, C4-H quinoline), 9.03 (d, 1H, C₅-H, J = 7.4Hz), 8.50–7.27 (m, 9H, Ar–H), 3.85 (m, 1H, CH–N), 1.86–1.35 (m, 10H, 5CH₂ cyclohexyl ring). ¹³C NMR (DMSO-d₆, δ, ppm): 194.0, 187.8, 167.6, 158.4, 147.9, 147.1, 139.6, 138.5, 129.8 (2), 128.9 (2), 128.6 (2), 125.6 (2), 121.6, 114.7, 103.1, 99.9, 40.6, 40.4, 40.3, 40.1, 39.9, 39.7 (2), 25.7, 25.4 (2). MS, m/z (%): 490 (18), 330 (24), 283 (100), 230 (50), 135 (36).

3-(3-Chlorobenzo[f]quinolin-2-yl)-2-(3-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-4-oxo-3,4-dihydroquinazolin-2-yl)acrylonitrile (11)

A solution of benzoxazine 6 (0.002mol, 1g) and 4-amino-1,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one (0.002mol, 0.4g) in dioxane (15ml) was refluxed for 8h. The obtained solid was filtered, dried, and recrystallized from ethanol to furnish orange crystals, mp. 263–265°C, Yield (55%). Anal. Calcd. for C₃₅H₂₃ClN₆O₂ (595.1): C, 70.65; H, 3.90; N, 14.12; Found: C, 70.61; H, 3.93; N, 14.15%. FTIR (ν, cm^-1): 2210 (CN), 1697 (C = O pyrazalone), 1650 (C = O imide), 1615 (C = N). ¹H NMR (DMSO-d₆, δ, ppm): 9.13 (s, 1H, CH olefinic), 9.02 (d, 1H, C5-H, J = 7.3Hz), 8.63 (s, 1H, C4-H quinoline), 8.58–7.83 (m, 14H, Ar–H), 3.19 (s, 3H, CH₃N), 1.20 (s, 3H, CH₃C = C).

2-(3-(3-Chlorobenzo[f]quinolin-2-yl)-2-cyanoacrylamido)-N-(pyrimidin-2-yl)benzamide (12)

A mixture of benzoxazine 6 (0.002mol, 1g) and 2-aminopyrimidine (0.002mol, 0.19g) in dioxane (15ml) was refluxed for 6h. The precipitated solid was filtered off, dried, and recrystallized from dioxane to produce orange precipitate, mp. > 300°C, Yield (85%). Anal. Calcd. for C₂₈H₁₇ClN₆O₂ (504.9): C, 66.60; H, 3.39; N, 16.64; Found: C, 66.63; H, 3.42; N, 16.61%. FTIR (ν, cm^-1): 3333, 3186 (NH), 2212 (CN), 1678 (C = O). ¹H NMR (DMSO-d₆, δ, ppm): 12.69 (br.s, 1H, NH, exchangeable), 9.13 (s, 1H, CH olefinic), 9.02 (s, 1H, NHCO, exchangeable), 8.67 (s, 1H, C4-H quinoline), 8.62–6.51 (m, 13H, Ar–H). MS, m/z (%): 504 (21), 395 (36), 368 (100), 342 (68), 236 (66), 125 (42).

N-(4-Acetylphenyl)-2-(3-(3-chlorobenzo[f]quinolin-2-yl)-2-cyanoacrylamido)benzamide (13)

Benzoxazine derivative 6 (0.002mol, 1g) was added to a solution of 4-aminoacetophenone (0.002mol, 0.27g) in dioxane (15ml) containing glacial acetic acid (0.1mL) was heated under reflux for 6h. The deposited solid was collected and recrystallized from dioxane to form orange crystals, mp. 282–284°C, Yield (75%). Anal. Calcd. for C₃₂H₂₁ClN₄O₃ (545): C, 70.52; H, 3.88; N, 10.28; Found: C, 70.48; H, 3.91; N, 10.23%. FTIR (ν, cm^-1): 3325, 3165 (NH), 2222 (CN), 1727 (C = O acetyl), 1648 (C = O amide), 1626 (C = N). ¹H NMR (DMSO-d₆, δ, ppm): 12.71 (br.s, 1H, ArNHCO, exchangeable), 9.13 (s, 1H, CH olefinic), 8.90 (br.s, 1H, NHCO, exchangeable), 8.50 (s, 1H, C4-H quinoline), 7.98–7.64 (m, 14H, Ar–H), 2.47 (s, 3H, CH₃). MS, m/z (%): 545 (14), 485 (72), 323 (52), 292 (64), 262 (100), 180 (96), 108 (48).

3-(3-Chlorobenzo[f]quinolin-2-yl)-2-cyano-N-(2-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl)acrylamide (14)

A mixture of benzoxazine 6 (0.002mol, 1g) and ethyl hydrazinecarboxylate (0.002mol, 0.2g) in dioxane (15ml) was refluxed for 6h. The formed solid was filtered, dried, and recrystallized from dioxane to produce yellow precipitate, mp. > 300°C, Yield (60%). Anal. Calcd. for C₂₅H₁₄ClN₅O₃ (467.9): C, 64.18; H, 3.02; N, 14.97; Found: C, 64.21; H, 3.06; N, 14.92%. FTIR (ν, cm^-1):3233 (NH), 2214 (CN),1694 (C = O oxadiazolone), 1670 (C = O amide). ¹H NMR (DMSO-d₆, δ, ppm): 13.04 (br.s, 1H, NH, exchangeable), 9.01 (s, 1H, CH olefinic), 8.92 (br.s, 1H, NHCO, exchangeable), 8.59 (s, 1H, C4-H quinoline), 8.57 (d, 1H, C₅-H, J = 8.6Hz), 8.04–7.76 (m, 9H, Ar–H).

3-(3-Chlorobenzo[f]quinoline-2-yl)-2-(4-oxo-3-(phenylamino)-3,4-dihydroquinazolin-2-yl) acrylonitrile (15)

Phenylhydrazine (0.002mol, 0.26ml) was added to a solution of benzoxazine 6 (0.002mol, 1g) in dioxane (15ml) and then, the reaction mixture was refluxed for 8h. The deposited solid was collected and recrystallized from dioxane to produce yellow crystals, mp. > 300°C, Yield (75%). Anal. Calcd. for C₃₀H₁₈ClN₅O (500): C, 72.07; H, 3.63; N,14.01. Found: C, 72.02; H, 3.65; N, 13.97%. FTIR (ν, cm^-1): 3341 (NH), 2216 (CN), 1691 (C = O amide), 1605 (C = N). ¹H NMR (DMSO-d₆, δ, ppm): 13.38 (br.s, 1H, NHPh, exchangeable), 9.1 (s, 1H, CH olefinic), 8.99 (d, 1H, C₅-H, J = 7.8Hz), 8.64 (s, 1H, C4-H quinoline), 8.12–7.21 (m, 14H, Ar–H). MS, m/z (%): 500 (M^+., 16), 480 (32), 452 (90), 340 (39), 263 (100), 162 (52), 79 (56).

3-(3-Chlorobenzo[f]quinolin-2-yl)-2-(3-(2-hydroxyethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)acrylonitrile (16)

A mixture of benzoxazine 6 (0.002mol, 1g) and ethanolamine (0.002mol, 0.12ml) in dioxane (15ml) was refluxed for 6h. The precipitated solid was filtered and recrystallized from dioxane to furnish yellow crystals, mp. > 300°C, Yield (55%). Anal. Calcd. for C₂₆H₁₇ClN₄O₂ (452.9): C, 68.95; H, 3.78; N, 12.37; Found: C, 68.90; H, 3.73; N, 12.41%. FTIR (ν, cm^-1): 2210 (CN), 1679 (C = O amide), 1645 (C = N). ¹H NMR (DMSO-d₆, δ, ppm): 9.19 (s, 1H, CH olefinic), 8.70 (s, 1H, C4-H quinoline), 9.11–7.20 (m, 10H, Ar–H), 3.73 (br.s, 1H, OH, exchangeable), 3.02 (t, 2H, CH₂CH₂OH, J = 6.7Hz), 2.61 (t, 2H, CH₂CH₂OH, J = 6.8Hz). MS, m/z (%): 452 (M^+., 30), 381 (43), 334 (100), 264 (89), 199 (66), 117 (40).

3-(3-Chlorobenzo[f]quinolin-2-yl)-2-cyano-N-(2-(4,5-dihydro-1H-imidazol-2-yl)phenyl)acrylamide (17)

Ethylenediamine (0.002mol, 0.12ml) was added to a solution of benzoxazine 6 (0.002mol, 1g) in ethanol (20ml) and then, the reaction mixture was refluxed for 8h. The precipitated solid was filtered and recrystallized from dioxane to produce brown crystals, mp. 260–262°C, Yield (65%). Anal. Calcd. for C₂₆H₁₈ClN₅O (451.9): C, 69.10; H, 4.01; N, 15.50; Found: C, 69.15; H, 4.04; N, 15.46%. FTIR (ν, cm^-1): 3365 (NH), 2211 (CN), 1681 (C = O amide). ¹H NMR (DMSO-d₆, δ, ppm): 13.20 (br.s, 1H, NH imidazole, exchangeable), 9.03 (s, 1H, CH olefinic), 8.97 (br.s, 1H, NHCO, exchangeable), 8.57 (s, 1H, C4-H quinoline), 8.07–7.76 (m, 10H, Ar–H), 3.99 (t, 2H, CH₂N = C, J = 7.2Hz), 3.22 (t, 2H, CH₂NH, J = 6.9Hz). MS, m/z (%): 451 (M^+., 100), 436 (54), 322 (47), 163 (21), 128 (30).

N-(2-(2-Carbamothioylhydrazine-1-carbonyl)phenyl)-3-(3-chlorobenzo[f]quinolin-2-yl)-2-cyanoacrylamide (18)

A mixture of benzoxazine 6 (0.002mol, 1g) and thiosemicarbazide (0.002mol, 0.18g) in ethanol (20ml) containing glacial acetic acid (0.1mL) was refluxed for 10h. The precipitated solid was filtered, dried, and recrystallized from ethanol to furnish pale-brown crystals, mp. > 300oC, Yield (69%). Anal. Calcd. for C₂₅H₁₇ClN₆O₂S (501): C, 59.94; H, 3.42; N, 16.78; Found: C, 59.90; H, 3.45; N, 16.74%. FTIR (cm^-1): 3448 (NH), 3314, 3273 (NH₂) 2204 (CN), 1688, 1650 (C = O amide), 1613 (C = N). ¹H NMR (DMSO-d₆, δ, ppm): 12.10 (s, 1H, NHCO, exchangeable), 10.72 (s, 1H, CONHNH, exchangeable), 9.43 (s, 1H, CONHNH, exchangeable), 9.12 (s, 1H, CH olefinic), 8.63 (s, 1H, C4-H quinoline), 8.38–7.30 (m, 10H, Ar–H), 7.83 (s, 2H, NH₂, exchangeable). MS, m/z (%): 500 (M^+., 42), 340 (44), 300 (100), 281 (64), 209 (70), 147 (34), 99 (36).

N-(2-(1H-Benzo[d]imidazol-2-yl)phenyl)-3-(3-chlorobenzo[f]quinolin-2-yl)-2-cyanoacrylamide (19)

A mixture of benzoxazine derivative 6 (0.002mol, 1g) and o-phenylenediamine (0.002mol, 0.22g) in ethanol (10ml) containing acetic acid (0.1mL) was refluxed for 6h. The precipitated solid was filtered and recrystallized from dioxane to produce pale-yellow crystals, mp. > 300°C, Yield (73%). Anal. Calcd. for C₃₀H₁₈ClN₅O (500): C, 72.07; H, 3.63; N,14.01; Found: C, 72.02; H, 3.65; N,13.97%. FTIR (ν, cm^-1): 2209 (CN), 1688 (C = O amide), 1606 (C = N). ¹H NMR (DMSO-d₆, δ, ppm): 12.46 (br.s, 1H, NH benzimidazole, exchangeable), 9.1 (s, 1H, CH olefinic),8.99 (br.s, 1H, NHCO, exchangeable), 8.66 (s, 1H, C4-H quinoline), 8.06–7.25 (m, 14H, Ar–H). MS, m/z (%): 500 (M^+., 52), 447 (60), 396 (68), 354 (95), 232 (88), 150 (82), 129 (100), 90 (80).

Determination of total antioxidant capacity (TAC)

“Antioxidant activity of each extract was investigated via phosphomolybdenum method using ascorbic acid as a standard. This assay is based on the reduction of Mo (VI) to Mo (V) by the sample analyte and subsequent formation of a green-colored [phosphate=Mo (V)] complex at pH< 7 with maximal absorption at 695 nm. In this method, 0.5mL of each extract (200 µg/mL) in methanol was combined in dried vials with 5mL of reagent solution (0.6 M sulfuric acid, 28mM sodium phosphate, and 4mM ammonium molybdate). The vials of the reaction mixture were capped and incubated in a thermal block at 95 °C for 90min⁴⁶. After the samples had cooled at room temperature, the absorbance was measured at 695 nm against a blank experiment. All experiments were executed in triplicate. The antioxidant activity of the sample was formulated as the number of ascorbic acid equivalent (AAE)^30,31. All data were recorded as mean ± S.D. using Graph Pad Prism software version 7 (https://www.graphpad.com). Differences between groups were considered statistically significant at p values < 0.05”.

Molecular docking

In the docking study, the crystal structure of the binding protein, HCV NS5B polymerase was retrieved from the Protein Data Bank (https://www.rcsb.org, PDB ID: 3SKA, Resolution: 1.73Å). AutoDock Vina (ADT, Version 1.5.7) available from Scripps Research Institute (http://autodock.scripps.edu/resources/adt) was employed to perform molecular docking study, focusing on the interaction between ascorbic acid, and the potent compound with HCV NS5B polymerase. To prepare the protein receptor, necessary steps such as 3D hydrogenation and energy minimization were carried out. Docking results were analyzed using Biovita Discovery Studio Visualizer (https://biovia-discovery-studio-64-bit-client.software.informer.com/4.5/).

Modeling pharmacokinetics

The ADME profiles of the synthesized compounds including their physicochemical properties, lipophilicity, and drug-likeness were obtained from the SwissADME free web tool (http://www.swissadme.ch/).