Evaluation of Quinazolin-2,4-Dione Derivatives as Promising Antibacterial Agents: Synthesis, In Vitro, In Silico ADMET and Molecular Docking Approaches

Abstract

A series of new quinazolin-2,4-dione derivatives incorporating amide/eight-membered nitrogen-heterocycles 2a–c, in addition, acylthiourea/amide/dithiolan-4-one and/or phenylthiazolidin-4-one 3a–d and 4a–d. The starting compound 1 was prepared by reaction of 4-(2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-benzoyl chloride with ammonium thiocyanate and cyanoacetic acid hydrazide. The reaction of 1 with strong electrophiles, namely, o-aminophenol, o-amino thiophenol, and/or o-phenylene diamine, resulted in corresponding quinazolin-2,4-dione derivatives incorporating eight-membered nitrogen-heterocycles 2a–d. Compounds 3a–d and 4a–d were synthesized in good-to-excellent yield through a one-pot multi-component reaction (MCR) of 1 with carbon disulfide and/or phenyl isocyanate under mild alkaline conditions, followed by ethyl chloroacetate, ethyl iodide, methyl iodide, and/or concentrated HCl, respectively. The obtained products were physicochemically characterized by melting points, elemental analysis, and spectroscopic techniques, such as FT-IR, ¹H-NMR, ¹³C-NMR, and MS. The antibacterial efficacy of the obtained eleven molecules was examined in vitro against two Gram-positive bacterial strains (Staphylococcus aureus and Staphylococcus haemolyticus). Furthermore, Computer-Aided Drug Design (CADD) was performed on the synthesized derivatives, standard drug (Methotrexate), and reported antibacterial drug with the target enzymes of bacterial strains (S. aureus and S. haemolyticus) to explain their binding mode of actions. Notably, our findings highlight compounds 2b and 2c as showing both the best antibacterial activity and docking scores against the targets. Finally, according to ADMET predictions, compounds 2b and 2c possessed acceptable pharmacokinetics properties and drug-likeness properties.

1. Introduction

Nitrogen-heterocyclic compounds are of particular interest by virtue of their biological and pharmacological activity [1,2,3,4,5]. Quiet recently, compounds incorporating a quinazolin-2,4-dione moiety represented an inexhaustible inspiration for the design and development of novel semisynthetic or synthetic agents with a broad spectrum of bioactivities [6,7]. Quinazolin-2,4-diones stand out as promising candidates in pharmacology, having several biological activities, including anticancer [8], antibacterial [9], anti-malarial [10], and anti-inflammatory [8].

Eight-membered nitrogen-heterocycles such as azocine are considered privileged structures found in a variety of natural products and bioactive molecules [11,12,13,14]. They play a fundamental role in medicinal and pharmaceutical chemistry. They serve as a key scaffold for the design and development of various inhibitors, including broad-spectrum antibacterial drug candidates [15,16]. For example, AZOCIN-500^® (Azithromycin) is an antibiotic that is utilized in the treatment of bacterial infections and typhoid fever. AZOCIN-500^® stops bacterial growth and infection spread [12,17].

Recent studies on derivatives incorporating amide and acylthiourea moieties exhibited a broad spectrum of biological applications, e.g., antibacterial, antiviral, and antioxidant activity [18,19,20,21,22].

Thiazolidin-4-one is considered an essential heterocyclic scaffold in medicinal chemistry. Moreover, it has a broad range of biological activities, including antibacterial, anticancer, and anti-inflammatory [23,24,25], Figure 1.

Figure 1.

Reported antibacterial agents incorporating (A) quinazoline, (B) azocine, and (C) dithiolan-4-one and/or phenyl-thiazolidin-4-one moieties.

In addition, the molecular hybridization approach is responsible for good antibacterial activity [26].

Inspired by the data collected, as well as in continuation of our efforts to synthesize new and promising antibacterial inhibitors [19,27]. Herein, a new series of eleven compounds with various bioactive moieties such as quinazolin-2,4-dione, amide, eight-membered nitrogen-heterocycles, acylthiourea, dithiolan-4-one and/or phenyl-thiazolidin-4-one were synthesized. Virtual screening on diverse quinazolin-2,4-dione derivatives and standard drugs to unveil their inhibition potential against the target enzymes was performed. Furthermore, biological evaluations of the new quinazolin-2,4-dione derivatives were performed against two Gram-positive bacterial strains, namely, Staphylococcus aureus and Staphylococcus haemolyticus. Finally, the ADME/Tox and drug-likeness properties of the best-docked compounds and methotrexate were checked using AdmetSAR, Mol inspiration, and SwissADME web servers.

2. Results and Discussion

2.1. Chemistry

Compound 1 was reported earlier in our previous study [18]. Compounds 2a–c, quinazolin-2,4-diones attached to eight-membered nitrogen-heterocycles, such as oxa/thia/tri/tetr-azocine, were synthesized via the reaction of 1 with strong electrophiles, namely, o-aminophenol, o-aminothiophenol, and o-phenylene diamine, respectively (Scheme 1). The chemical structures of the new compounds 2a–c were elucidated based on spectroscopic data. For instance, the FT-IR spectrum of 2a showed bands at 3180, 1709, 1660, and 1609 cm⁻¹ attributed to NH, C=O, and C=N groups, respectively. A band at 2265 cm⁻¹ was assignable to a C≡N group, indicating that the nucleophilic attack did not occur at the C≡N group. The ¹H-NMR spectrum of 2a showed signals attributed to NH, CH₂N, and aromatic protons at 11.62, 4.02, and 7.74–8.04 ppm. Further evidence was gained from the mass spectrum; it showed the correct molecular ion peak at m/z 479 beside some other important peaks.

Scheme 1.

Synthetic routes of compounds 2a–c, 3a–d and 4a–d.

On the other hand, compounds with an activated methylene group react as carbanions in the presence of a base with the electrophilic carbon disulfide to yield dithiocarboxylates. This can be converted to ketene dithioacetals on treatment with an excess of the alkylating reagent [28]. Cyclization of the intermediate (A1) with ethyl chloroacetate afforded 3a. The reaction proceeded via nucleophilic addition of the carbanions on CS₂ to form the potassium salt intermediates (A1), followed by in situ cyclization through an SN² mechanism to yield the cyclic compound 3a (Scheme 2). Stirring of 1 with carbon disulfide in the presence of KOH in DMF followed by the addition in situ of ethyl iodide or methyl iodide or concentrated HCl afforded compounds 3b–d via intermediate (A1) (Scheme 2). The structures of compounds 3a–d were established by means of analytical and spectral data. The IR spectra of compounds 3a–d showed bands characteristic for NH, CN, C=O, and C=S groups in the range 3187–3110, 2205–2250, and 1722–1662 cm⁻¹, respectively. The ¹H-NMR spectra are in good agreement with the suggested structures. They are devoid of a signal corresponding to CH₂CN protons. However, they displayed signals related to two CH₃ and two CH₃S protons for compounds 3b and 3c at δ 1.36 ppm as triplet signals and 2.68 ppm as singlet signals, respectively. Two SCH₂ and SCH₂CO protons for compounds 3b and 3a appeared at δ 2.83 ppm as a quartet signal and δ 4.00 ppm as a singlet signal. The ¹³C-NMR spectra of compounds 3a–c exhibited signals at δ 34.55, 19.10, 24.35, and 14.44 ppm, respectively, indicating the presence of SCH₂CO, CH₃, CH₂S, and SCH₃. Further evidence was gained from the mass spectra, as they showed the correct molecular ion peaks for compounds 3a–d at m/z 538, 554, 526, and 498, respectively.

Scheme 2.

Synthetic pathway of compound 3a.

Furthermore, the reaction of compound 1 with phenyl isothiocyanate in the presence of KOH yielded the potassium salt intermediate (A2). Cyclization of (A2) in situ with ethyl chloroacetate furnished compound 4a. Also, followed by the addition in situ of ethyl iodide, methyl iodide, or concentrated HCl afforded compounds 4b–d via the molecule intermediate (A2) (Scheme 1). The structures of compounds 4a–d were elucidated on the basis of the elemental analysis and spectral data. The FT-IR spectra of compounds 4a–d displayed absorption bands corresponding to NH, CN, C=O, and C=S groups at 3182, 3333, 3211, 2203, 2202, 1673, and 1714 cm⁻¹, respectively. Furthermore, the ¹H-NMR spectrum of compound 4a showed characteristic signals for four NHs, aromatic protons, and CH₂ protons at 11.65, 11.61, 10.34, 9.61, 7.24–7.79, and 4.00 ppm, respectively. An extended analysis was performed using the mass spectrum, the recorded mass m/z at (597 [M]⁺) corresponding to the calculated molecular formula. The spectral data of all newly prepared quinazolin-2,4-dione derivatives are declared in Figures S1–S46 (Supplementary File).

2.2. Biological Studies

In the present study, all the synthesized compounds were screened for their in vitro antibacterial activity using MIC and MBC assays. Table 1 declares the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of compounds tested against two Gram-positive bacterial strains: Staphylococcus aureus and Staphylococcus haemolyticus. The MIC values ranged from 10 to 26 (mg/mL), indicating varying levels of antibacterial activity.

Table 1.

Antibacterial activity of compounds against S. aureus and S. haemolyticus.

No.	Compound	S. aureus (MIC/MBC)	S. haemolyticus (MIC/MBC)
1	2a	16 ± 2/18 ± 4.5	13 ± 1/18 ± 1.7
2	2b	13 ± 1/18 ± 1.7	10 ± 1/13 ± 2.6
3	2c	11 ± 1/20 ± 2.6	17 ± 1/22 ± 2.6
4	3a	10 ± 1/10 ± 1	20 ± 2/22 ± 2
5	3b	25.6 ± 1.6/27 ± 1.7	N.A/N.A
6	3c	12 ± 1.7/15 ± 1.7	N.A/N.A
7	4a	16 ± 1/18 ± 1.7	18 ± 1.7/22 ± 2
8	4b	21 ± 1.7/23 ± 1	18 ± 1.7/21 ± 3
9	4c	10 ± 1/13 ± 2.6	21 ± 1.7/22 ± 2.6
10	4d	19 ± 1/11 ± 1	22 ± 2/22 ± 2

MIC: Minimum Inhibitory Concentration (mg/mL); MBC: Minimum Bactericidal Concentration (mg/mL); N.A: Not Applicable.

Most of the tested compounds exhibited significant antibacterial properties, with marked differences observed between the MIC and MBC values. The lowest MIC values were recorded for compounds 2b and 2c, which both exhibited lower MICs, as well as 2b with 10 mg/mL against S. haemolyticus. Additionally, compound 2c showed MIC of 11 mg/mL against S. aureus, followed by 3c with 12 mg/mL, and 2a with 13 mg/mL against S. haemolyticus. Thus, both the 2b and 2c compounds have promising antibacterial activity against the two tested G+ve bacteria. Herein, a structure–activity relationship (SAR) study is reported, which focuses on the presence of –CH₂CN (cyanomethyl), amide, and/or thia/tri/tetr-azocine moieties, respectively. Gui Z et al. 2013 [29] reported that the function group of azocine in the antibiotic Azithromycin reduces the production of α-hemolysin and biofilm formation in S. aureus.

Regarding bactericidal activity, the lowest MBC value was found for 3a at 10 mg/mL against S. aureus, followed closely by 4d with an MBC of 11 mg/mL. Both compounds 2b and 4c demonstrated MBC values of 13 mg/mL against S. haemolyticus and S. aureus, respectively. Conversely, the highest MIC and MBC values were observed for 3b against both bacterial strains, indicating reduced efficacy. The main backbone of the tested compounds is a quinazoline-2,4-dione moiety, which was previously described as an inhibitor for enzymes of dihydrofolate reductase and purine synthesis in microorganisms [30].

Overall, compounds 2b and 2c exhibited the highest antibacterial activity (due to the molecular hybridization between quinazolin-2,4-dione scaffold and/or thia/tri/tetr-azocine moieties); all showed the lowest MIC values of 10 mg/mL, making them the most promising antibacterial agents in this study. The analysis of the MBC/MIC ratios is depicted in Figure 2, illustrating that most compounds had ratios ≤2, suggesting a strong bactericidal effect.

Figure 2.

Comparison of the MBC/MIC ratios of each compound tested against both bacterial strains. A ratio of ≤2 was considered indicative of strong bactericidal activity, and the majority of the compounds tested exhibited such effectiveness.

2.3. In Silico Studies and ADMET Analysis

In this study, a set of quinazolin-2,4-dione derivatives was screened by CADD to identify compounds showing potent enzyme activity and acceptable pharmacokinetic properties. Dihydrofolate reductase DHFR is considered an essential enzyme for thymidylate and purine synthesis in microorganisms [31]. In addition, the literature suggested that eukaryotic initiation factor 2 α (eIF2α) signaling may be active during bacterial infections [32]. Therefore, dihydrofolate reductase and eukaryotic initiation factor 2 α were selected as promising targets for the identification of new antibacterial inhibitors. Herein, in silico molecular docking studies were performed for a set of quinazolin-2,4-diones against the target enzymes of bacterial strains, dihydrofolate reductase (PDB ID: 2W9S), and eukaryotic initiation factor 2 α (eIF2α) (PDB ID: 1Q46) utilizing a PyRx-virtual screening tool [33]. For the standard drug (Methotrexate) and the reported antibacterial drug, the docking study was also performed in order to map important interactions with the active site of the targets. The results obtained from the docking study are depicted in Table 2. Figure 3 and Figure 4 exhibited 2D and 3D interactions between the best-docked compounds and standard drugs with the target enzymes.

Table 2.

The binding energy of the docked molecules, standard drug, and reported antibacterial drug against the target enzymes.

	Enzyme	Dihydrofolate Reductase Enzyme			Eukaryotic Initiation Factor 2 α Enzyme
No.		Binding Energy kcal/mol	Docked Complex (Amino Acid–Ligand) Interactions	Distance (Å)	Binding Energy kcal/mol	Docked Complex (Amino Acid–Ligand) Interactions	Distance (Å)
2a		−11.4	H-bonds ASN18:N—compound 2a arene-arene interactions PHE92—compound 2a PHE92—compound 2a arene-sigma interactions LEU20:CD1—compound 2a	2.95 4.92 5.61 3.99	−7.3	H-bonds LYS100:NZ—compound 2a	2.95
2b		−11.7	H-bonds ASN18:N—compound 2b arene–arene interactions PHE92—compound 2b PHE92—compound 2b arene–sigma interactions LEU20:CD1—compound 2b	2.91 5.01 5.92 3.82	−9.6	H-bonds TYR171:OH—compound 2b TYR141:OH—compound 2b arene–cation interactions ARG175:NH1--compound 2b ARG175:NH1--compound 2b	2.91 2.21 4.75 5.12
2c		−11.6	H-bonds ASN18:N—compound 2c arene–arene interactions PHE92—compound 2c PHE92—compound 2c	2.86 4.93 5.39	−9.5	H-bonds TYR141:OH—compound 2c arene–cation interactions ARG175:NH1—compound 2c ARG175:NH1—compound 2c	2.20 4.76 5.69
3a		−11.0	H-bonds ASN18:N—compound 3a THR46:OG1—compound 3a ILE14:O—compound 3a arene–cation interactions ARG57:NH1—compound 3a ARG57:NH1—compound 3a ARG57:NH2—compound 3a ARG57:NH2—compound 3a	2.99 2.95 2.38 5.26 5.06 5.50 5.64	−8.1	H-bonds LYS11:N—compound 3a ARG135:NH1—compound 3a ARG135:NH2—compound 3a arene–cation interactions ARG6:NH2—compound 3a ARG6:NH1—compound 3a LYS11:NZ—compound 3a LYS11:NZ—compound 3a	2.96 3.00 2.89 3.93 5.86 5.13 5.09
3b		−10.5	H-bonds ARG44:N—compound 3b LYS45:N—compound 3b GLN95:N—compound 3b TYR98:OH—compound 3b arene–cation interactions ARG57:NH1—compound 3b ARG57:NH1—compound 3b ARG57:NH2—compound 3b ARG57:NH2—compound 3b LYS45:NZ—compound 3b	3.00 2.98 2.97 2.83 5.25 4.63 5.36 5.23 4.81	−8.0	H-bonds TYR101:OH—compound 3b HIS108:ND1—compound 3b ARG112:NH1—compound 3b arene–cation interactions LYS117:NZ—compound 3b	2.97 3.00 2.81 5.60
3c		−11.1	H-bonds SER49:OG—compound 3c GLN95:N—compound 3c	2.99 2.95	−8.7	H-bonds SER109:OG—compound 3c TYR113:N—compound 3c TYR171:OH—compound 3c ARG175:NH1—compound 3c SER109:OG—compound 3c arene–cation interactions ARG175:NH1—compound 3c LYS145:NZ—compound 3c	3.00 2.98 2.95 2.86 2.17 5.54 5.69
3d		−10.4	H-bonds SER49:OG—compound 3d	2.95	−7.9	H-bonds TYR101:OH—compound 3d ARG112:NH1—compound 3d ARG112:NH1—compound 3d SER109:OG—compound 3d arene–cation interactions ARG112:NH2—compound 3d	2.97 2.87 2.99 1.92 5.22
4a		−10.3	H-bonds THR46:OG1—compound 4a arene–cation interactions ARG57:NH1—compound 4a LYS45:NZ—compound 4a	2.91 5.05 4.32	−8.0	H-bonds TYR171:OH—compound 4a ARG175:NH1—compound 4a TYR141:OH—compound 4a arene–cation interactions ARG175:NH1—compound 4a	2.94 2.99 1.96 4.72
4b		−9.7	H-bonds ARG44:NH2—compound 4b THR46:N—compound 4b THR46:OG1—compound 4b GLY94:N—compound 4b THR46:OG1—compound 4b arene–cation interactions ARG44:NH2—compound 4b	3.00 2.96 2.69 2.65 1.98 6.00	−8.3	H-bonds TYR101:OH—compound 4b LYS105:NZ—compound 4b THR106:OG1—compound 4b SER109:OG—compound 4b ARG112:NH1—compound 4b arene–cation interactions LYS105:NZ—compound 4b ARG112:NH1—compound 4b	3.00 2.85 2.98 2.93 2.82 3.61 5.03
4c		−10.2	H-bonds THR46:OG1—compound 4c THR121:OG1—compound 4c SER49:OG—compound 4c arene–cation interactions PHE92—compound 4c PHE92—compound 4c	2.83 3.00 2.46 5.88 5.33	−8.6	H-bonds SER109:OG—compound 4c SER109:OG—compound 4c ARG175:OXT—compound 4c arene–sigma interactions TYR113:CD1—compound 4c	2.75 2.41 2.37 3.63
4d		−9.5	H-bonds SER49:OG—compound 4d PHE92:O—compound 4d arene–cation interactions LYS52:NZ—compound 4d	1.90 2.31 5.44	−8.5	H-bonds SER109:OG—compound 4d SER109:OG—compound 4d ARG175:OXT—compound 4d arene–sigma interactions TYR113:CD1—compound 4d	2.70 2.45 2.38 3.57
Standard drug		−9.3	H-bonds ARG44:N—standard drug ARG44:NE—standard drug ARG44:NH2—standard drug LYS45:N—standard drug ASN64:N—standard drug LEU62:O—standard drug	2.81 2.94 2.99 2.98 2.18 2.07	−7.1	H-bonds ILE18:N—standard drug SER134:OG—standard drug TRP131:O—standard drug GLU9:OE2—standard drug ASP93:OD1—standard drug arene–cation interactions LYS100:NZ—standard drug	2.85 2.99 2.03 2.09 2.07 3.49
Reported antibacterial drug [34]		−9.3	H-bonds PHE92:O—reported drug	2.50	−7.0	H-bonds ARG175:O—reported drug	2.17

Figure 3.

2D (left side) and 3D (right side) interactions of best-docked compounds, standard drug, and reported antibacterial drug against 2W9S enzyme.

Figure 4.

2D (left side) and 3D (right side) interactions of best-docked compounds, standard drug, and reported antibacterial drug against 1Q46 enzyme.

In the case of dihydrofolate reductase, compound 2b (with thia/triazocine moiety) exhibited the best binding affinity, −11.7 kcal/mol, and docked to the target enzyme through one H-bond, two arene-arene, and one arene-sigma interaction with the residues ASN18, PHE92, and LEU20, while compound 2c (with tetrazocine moiety) showed binding energy of −11.6 kcal/mol and docked to the target through one H-bond and two arene-arene interactions with the residues ASN18 and PHE9.

In the case of eukaryotic initiation factor 2 α, compound 2b (−9.6 kcal/mol) docked to the target through two H-bonds and aren–cation interactions with the residues TYR171, TYR141, and ARG175. On the other hand, compound 2c (−9.5 kcal/mol) docked to the target through one H-bond and two arene–arene interactions with the residues TYR141 and ARG175, respectively.

For methotrexate (−9.3 kcal/mol), six H-bonds with dihydrofolate reductase through ARG44, LYS45, LEU62, and ASN64. In addition, it docked with eukaryotic initiation factor 2 α (−7.1 kcal/mol) through five H-bonds and one arene–cation interaction.

For reported antibacterial drug [34], it docked with dihydrofolate reductase (−9.3 kcal/mol) through one H-bond with PHE92 at 2.5 Å. Additionally, it docked with eukaryotic initiation factor 2 α (−7.0 kcal/mol) through one H-bond with the residue ARG175 at 2.17 Å.

The 3D interactions of the other docked compounds toward the target enzymes are represented in Figures S47 and S48 (Supplementary File).

By comparing the experimental antibacterial activity of the compounds reported in this study to their structures, the following structure–activity relationship (SAR) was postulated:

Compounds 2b and 2c exhibited the highest antibacterial activity, which may be due to the presence of –CH₂CN, amide, and/or thia/tri/tetr-azocine moieties, respectively. In addition, it was reported that the -C=N- bond is utilized in the design of antibacterial agents [35]. Further, the molecular hybridization between the quinazoline-2,4-dione scaffold and/or the thia/tri/tetr-azocine moieties is responsible for good antibacterial activity [26].

Table 3 declares the ADMET properties of the best-docked molecules, standard drugs, and reported antibacterial drugs. Their molecular weights are below 500 g/mol, indicating good absorption. Consequently, they have satisfied the Lipinski rule without any violation. They have rotatable bonds within the allowed range (<8 bond) that enhance their flexibility. In addition, they have acceptable HBA and HBD. In conclusion, compounds 2b and 2c are predicted to have acceptable bioavailability.

Table 3.

Physicochemical and pharmacokinetic properties of the best compounds, standard drug, and reported antibacterial drug.

#	Compound 2b	Compound 2c	Standard Drug	Reported Antibacterial Drug
MW (g/mol)	495.51	478.46	454.44	284.40
#Rotatable bonds	5	5	10	4
#HBA	6	6	9	2
#HBD	3	4	5	2
TPSA (Å2)	177.56	165.11	210.54	49.84
MLOGP	2.64	2.29	−0.46	3.37
%ABS	99.00	98.23	82.61	99.65
GI absorption	Low	Low	Low	High
BBB permeant	No	No	No	Yes
Lipinski #violations	0	0	1	0
Bioavailability Score	0.55	0.55	0.11	0.55

3. Experimental

3.1. Organic Synthesis

An electrothermal melting apparatus was used to measure the melting points, which were uncorrected. All chemical reactions were observed on a silica gel GF254 plate with thin-layer chromatography (TLC). FT-IR spectra υ/cm⁻¹ (KBr) were recorded on a Shimadzu 8101 PC spectrometer from South Valley University. The ¹H- and ¹³C-NMR spectra were run on a Varian Mercury spectrophotometer at 400 and 100 MHz, respectively, using tetramethylsilane TMS as an internal standard and DMSO-d₆ as a solvent. Electron impact mass spectra were obtained at 70 eV using a GCMS-QP 1000 EX spectrometer. Elemental analyses were carried out at the microanalytical center at Cairo University.

• Synthesis of N-[N′-(2-cyano-acetyl)-hydrazino-carbo-thioyl]-4-(2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-benzamide 1

The compound was described earlier by our group members [18].

• General procedures for synthesis of oxa/thia/triazocinyl/tetrazocinyl quinazolin-2,4-diones 2a–c

To a solution of compound 1 (0.003 mol, 1.5 g) in DMF (30 mL), o-aminophenol and/or o-aminothiophenol and/or o-phenylene diamine (0.003 mol) was added to the mixture. The reaction mixture was refluxed for 10 h. The separated solid was filtrated off, dried, and recrystallized to afford compounds 2a–c, respectively.

• Synthesis of N-2-(cyanomethyl)-4H-benzo-[g]-[1,3,4,6]-oxatriazocin-5-yl)-4-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-benzamide 2a

Dark brown crystals. Yield 62%; MP 226–228 °C. FT-IR (KBr, υ, cm⁻¹) = 3180 (NH’s), 2265 (CN), 1709, 1660 (C=O’s), 1608 (C=N). ¹H-NMR (DMSO-d₆, 400 MHz): δ (ppm) = 11.62 (s, 1H, NH), 7.74–8.04 (m, 14H, Ar-H+2NH), 4.02 (s, 2H, CH₂). ¹³C-NMR (DMSO-d₆, 100 MHz): δ (ppm)= 19.13, 114.68, 114.78, 114,79, 114.80, 115.78, 115.89, 116.13, 123.09, 128.07, 128.47, 129.82, 135.80, 140.34, 142.49, 143.51, 150.56, 156.15, 157.56, 159.09, 160.00, 160.49, 161.07, 162.47, 163.18. MS (EI): m/z (%) = 479 [M]⁺. Anal. Calcd for C₂₅H₁₇N₇O₄ (Mol. Wt.: 479): C, 62.63; H, 3.57; N, 20.45%. Found C, 62.75; H, 3.69; N, 20.33%.

• Synthesis of N-2-(cyanomethyl)-4H-benzo-[g]-[1,3,4,6]-thiatriazocin-5-yl)-4-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-benzamide 2b

Dark green crystals. Yield 65%; MP > 300 °C. FT-IR (KBr, υ, cm⁻¹) = 3195 (NH’s), 2053 (CN), 1710, 1671 (C=O’s), 1612 (C=N). ¹H-NMR (DMSO-d₆, 400 MHz): δ (ppm) = 11.61 (s, 1H, NH), 7.14–8.22 (m, 14H, Ar-H+2NH), 4.17 (s, 2H, CH₂). ¹³C-NMR (DMSO-d₆, 100 MHz): δ (ppm) = 20.20, 114.81, 115,26, 115.79, 116.52, 116.92, 122.95, 123.09, 123.51, 126.19, 127.26, 128.09, 128.14, 130.79, 131.61, 133.11, 135.18, 135.81, 135.89, 138.93, 140.34, 150.23, 150.52, 154.09, 162.56, 167.11. MS (EI): m/z (%) = 495 [M]⁺. Anal. Calcd for C₂₅H₁₇N₇O₃S (Mol. Wt.: 495): C, 60.60; H, 3.46; N, 19.79; S, 6.47%. Found C, 60.72; H, 3.58; N, 19.68; S, 6.58%.

• Synthesis of N-5-(cyanomethyl)-3,6-dihydrobenzo[e]-[1,2,4,7]-tetrazocin-2-yl)-4-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl) benzamide 2c

Pale brown crystals. Yield 70%; MP 280–282 °C. FT-IR (KBr, υ, cm⁻¹) = 3190 (NH’s), 2275 (CN), 1723, 1665 (C=O’s), 1610 (C=N). ¹H-NMR (DMSO d₆, 400 MHz): δ (ppm) = 11.57 (s, 1H, NH), 7.19–8.23 (m, 14H, Ar-H+2NH), 3.96 (s, 2H, CH₂). ¹³C-NMR (DMSO-d₆, 100 MHz): δ (ppm) = δ 21.51, 114.63, 115,78, 115.79, 122.13, 123.08, 128.02, 128.06, 128.07, 129.33, 129.66, 130.18, 135.78, 135.79, 140.33, 140.34, 150.51, 150.79, 151.51, 162.63, 162.64, 162.66, 172.33, 174.50. MS (EI): m/z (%) = 478 [M]⁺. Anal. Calcd for C₂₅H₁₈N₈O₃ (Mol. Wt.: 478): C, 62.76; H, 3.79; N, 23.42%. Found C, 62.87; H, 3.91; N, 23.32%.

• General procedures for the synthesis of compounds 3a–d

To a stirred suspension of finely powdered potassium hydroxide (0.002 mol, 1.12 g) in dry DMF (20 mL), compound 1 (0.002 mol, 1 g) was added. The resulting mixture was cooled at 10 °C in an ice bath, and then carbon disulfide (0.50 mL, 0.002 mol) was added slowly over the course of 10 min. After complete addition, stirring of the reaction mixture was continued for an additional 4 h. Then, ethyl chloroacetate, ethyl iodide, methyl iodide, or concentrated HCl (0.002 mol) was added to the mixture while cooling and stirring for 20 h. The mixture was then poured onto crushed ice; the resulting precipitate was filtrated off, dried, and recrystallized from the proper solvent to give compounds 3a–d, respectively.

• Synthesis of N-(2-(2-cyano-2-(4-oxo-1,3-dithiolan-2-ylidene)acetyl)hydrazine-1-carbono thioyl)-4-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)benzamide 3a

Orange crystals. Yield: 63%. MP > 300 °C. FT-IR (KBr, υ, cm⁻¹) = 3187, 2995 (NH’s), 2205 (CN), 1718, 1662 (C=O’s), 1271 (C=S). ¹H-NMR (DMSO-d₆, 400 MHz): δ (ppm) = 11.66 (s, 1H, NH), 11.64 (s, 1H, NH), 10.72 (s, 1H, NH), 7.26–8.06 (m, 8H, Ar-H), 4.00 (s, 2H, CH₂). ¹³C-NMR (DMSO-d₆, 400 MHz): δ (ppm) = 34.55, 94.94, 114.74, 115.80, 115.82, 116.17, 123.09, 128.03, 128.55, 129.51, 129.90, 129.95, 130.29, 132.43, 135.80, 139.47, 140.34, 150.43, 150.60, 162.33, 162.60, 165.51. MS (El): m/z (%) = 538 [M]⁺. Anal. Calcd for C₂₂H₁₄N₆O₅S₃ (Mol. Wt.: 538): C, 49.06; H, 2.62; N, 15.60; S, 17.86%, found: C, 49.21; H, 2.75; N, 15.49; S, 17.98%.

• Synthesis of N-(2-(2-cyano-3,3-bis-(ethylthio)-acryloyl)-hydrazine-1-carbonothioyl)-4-(2,4-dioxo-1,4-dihydroquinazol-in-3-(2H)-yl)benzamide 3 b

Yellowish brown crystals. Yield: 65%. MP 140–142 °C. FT-IR (KBr, υ, cm⁻¹) = 3135 (NH), 2235 (CN), 1722, 1671 (C=O’s), 1348 (C=S). ¹H-NMR (DMSO-d₆, 400 MHz): δ (ppm) = 11.69 (s, 1H, NH), 10.69 (s, 1H, NH), 10.55 (s, 1H, NH), 8.87 (s, 1H, NH), 7.23–8.10 (m, 8H, Ar-H), 2.81–2.85 (q, 4H, 2CH₂), 1.34–1.38 (t, 6H, 2CH₃). ¹³C-NMR (DMSO-d₆, 100 MHz): δ (ppm) = 19.10, 24.35, 102.01, 114.76, 115,79, 116.13, 123.10, 128.06, 128.54, 129.91, 132.42, 135.81, 139.46, 140.32, 150.46, 161.03, 162.33, 162.59, 165.36, 165.58, 171.88, 184.20. MS (El): m/z (%) = 554 [M]⁺. Anal. Calcd for C₂₄H₂₂N₆O₄S₃ (Mol. Wt.: 554): C, 51.97; H, 4.00; N, 15.15; S, 17.34%, found: C, 52.05; H, 4.13; N, 15.02; S, 17.43%.

• Synthesis of N-(2-(2-cyano-3,3-bis(methylthio)-acryloyl)-hydrazine-1-carbonothioyl)-4-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)benzamide 3 c

Pale yellow powder. Yield: 60%. MP 120–122 °C. FT-IR (KBr, υ, cm⁻¹) = 3110, 2925 (NH’s), 2250 (CN), 1719, 1670 (C=O’s), 1271 (C=S). ¹H-NMR (DMSO-d₆, 400 MHz): δ (ppm) = 11.64 (s, 1H, NH), 10.67 (s, 1H, NH), 10.60 (s, 1H, NH), 10.45 (s, 1H, NH), 7.24–7.99 (m, 8H, Ar-H), 2.68 (s, 6H, 2CH₃). ¹³C-NMR (DMSO-d₆, 100 MHz): δ (ppm) = 14.44, 114.76, 115,80, 123.09, 128.05, 128.33, 128.48, 129.52, 129.77, 129.93, 135.80, 138.55, 139.41, 140.34, 150.49, 162.60, 162.59, 165.67, 165.76, 168.80, 182.49. MS (El): m/z (%) = 526 [M]⁺. Anal. Calcd for C₂₄H₁₈N₆O₄S₃ (Mol. Wt.: 526): C, 50.18; H, 3.45; N, 15.96; S, 18.26%, found: C, 50.30; H, 3.59; N, 15.84; S, 18.39%.

• Synthesis of N-(2-(2-cyano-3,3-dimercaptoacryloyl)-hydrazine-1-carbono-thioyl)-4-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)benzamide 3d

Yellow crystals. Yield: 70%. MP 208–210 °C. FT-IR (KBr, υ, cm⁻¹) = 3489 (SH), 3200, 3135 (NH’s), 2230 (CN), 1718, 1668 (C=O’s), 1272 (C=S). ¹H-NMR (DMSO-d₆, 400 MHz): δ (ppm) = 11.68 (s, 1H, NH), 10.69 (s, 1H, NH), 10.53 (s, 1H, NH), 8.81 (s, 1H, NH), 7.23–7.98 (m, 8H, Ar-H), 1.24 (s, 2H, SH). ¹³C-NMR (DMSO-d₆, 100 MHz): δ (ppm) = 101.04, 114.73, 114.74, 115.82, 115.83, 123.09, 128.04, 128.55, 129.90, 129.91, 132.56, 135.81, 140.34, 150.43, 150.44, 162.60, 162.74, 162.75, 165.75, 181.04. MS (El): m/z (%) = 498 [M]⁺. Anal. Calcd for C₂₀H₁₄N₆O₄S₃ (Mol. Wt.: 498): C, 48.18; H, 2.83; N, 16.86; S, 19.29%, found: C, 48.30; H, 2.95; N, 16.74; S, 19.32%.

• General procedures for the synthesis of compounds 4a–d

To a dissolved compound 1 (0.003 mol, 1.5 g) in (DMF) (20 mL), potassium hydroxide (0.003 mol, 0.2 g) was added. The mixture was stirred at RT until the complete dissolution of potassium hydroxide, and then phenyl isothiocyanate (0.003 mol, 0.47 g) was added after completing the stirring for 5 h. After that, ethyl chloroacetate, ethyl iodide, methyl iodide, or concentrated HCl (0.003 mol) were added with stirring overnight. Then, it was quenched into water and acidified with 10% hydrochloric acid, and the obtained products 4a–d were collected by filtration and recrystallized, respectively.

• Synthesis of N-(2-(2-cyano-2-(4-oxo-3-phenylthiazolidin-2-ylidene)-acetyl)hydrazine-1-carbonothioyl)-4-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)benzamide 4a

Yellow crystals. Yield: 57%. MP 90–92 °C. FT-IR (KBr, υ, cm⁻¹) = 3205, 3058 (NH’s), 2088 (CN), 1719, 1667 (C=O’s), 1269 (C=S). ¹H-NMR (DMSO-d₆, 400 MHz): δ (ppm) = 11.65 (s, 1H, NH), 11.61 (s, 1H, NH), 10.34 (s, 1H, NH), 9.61 (s, 1H, NH), 7.24–7.79 (m, 13H, Ar-H), 4.00 (s, 2H, CH₂). ¹³C-NMR (DMSO-d₆, 100 MHz): δ (ppm) = 32.99, 68.59, 114.57, 114.77, 115.79, 115.80, 123.09, 126.50, 128.06, 128.21, 128.76, 128.90, 129.26, 129.86, 129.96, 135.81, 135.82, 140.33, 142.99, 147.80, 150.43, 150.46, 158.79, 162.59, 165.26, 175.57, 184.20. MS (El): m/z (%) = 597 [M]⁺. Anal. Calcd for C₂₈H₁₉N₇O₅S₂ (Mol. Wt.: 597): C, 56.27; H, 3.20; N, 16.41; S, 10.73%, found: C, 56.40; H, 3.33; N, 16.38; S, 10.85%.

• Synthesis of N-(2-(2-cyano-3-(ethylthio)-3-(phenylamino)-acryloyl)hydrazine-1-carbono thioyl)-4-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)benzamide 4b

Yellow crystals. Yield: 55%. MP 158–160 °C. FT-IR (KBr, υ, cm⁻¹) = 3262, 3070 (NH’s), 2213 (CN), 1717, 1693 (C=O’s), 1258 (C=S). ¹H-NMR (DMSO-d₆, 400 MHz): δ (ppm) = 12.17 (s, 1H, NH),11.64 (s, 1H, NH), 11.60 (s, 1H, NH), 10.63 (s, 1H, NH), 9.81 (s, 1H, NH), 7.22–7.98 (m, 13H, Ar-H), 3.15–3.21 (q, 2H, CH₂), 1.34–1.38 (t, 3H, CH₃). ¹³C-NMR (DMSO-d₆, 100 MHz): δ (ppm) = 14.74, 26.97, 115,78, 123.06, 124.12, 124.89, 126.44, 127.00 128.36, 128.49, 128.74, 128.81, 128.92, 129.10, 129.62, 129.80, 129.92, 130.40, 134.42, 135.78, 137.39, 140.26, 140.32, 150.43, 150.46, 152.64, 154.33, 162.55, 180.08. MS (El): m/z (%) = 585 [M]⁺. Anal. Calcd for C₂₈H₂₃N₇O₄S₂ (Mol. Wt.: 585): C, 57.42; H, 3.96; N, 16.74; S, 10.95%, found: C, 57.55; H, 4.06; N, 16.64; S, 11.05%.

• Synthesis of N-(2-(2-cyano-3-(methylthio)-3-(phenylamino)-acryloyl)hydrazine-1-carbono thioyl)-4-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)benzamide 4c

Orange crystals. Yield: 65%. MP > 300 °C. FT-IR (KBr, υ, cm⁻¹) = 3205, 3008 (NH’s), 2260 (CN), 1714, 1666 (C=O’s), 1269 (C=S). ¹H-NMR (DMSO-d₆, 400 MHz): δ (ppm) = 11.66 (s, 1H, NH), 11.64 (s, 1H, NH), 9.60 (s, 1H, NH), 9.59 (s, 1H, NH), 7.28–7.71 (m, 13H, Ar-H), 1.21 (s, 3H, CH₃). MS (El): m/z (%) = 571 [M]⁺. Anal. Calcd for C₂₇H₂₁N₇O₄S₂ (Mol. Wt.: 571): C, 56.73; H, 3.70; N, 17.15; S, 11.22%, found: C, 56.85; H, 3.82; N, 17.02; S, 11.34%.

• Synthesis of N-(2-(2-cyano-3-(phenylamino)-3-thioxopropanoyl)hydrazine-1-carbono thioyl)-4-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)benzamide 4d

Pale yellow crystals. Yield: 58%. MP > 300 °C. FT-IR (KBr, υ, cm⁻¹) = 3262, 3069, 3006 (NH’s), 2213 (CN), 1718, 1664 (C=O’s), 1258 (C=S). ¹H-NMR (DMSO-d₆, 400 MHz): δ (ppm) = 9.02 (s, 1H, NH), 7.10–7.31 (m, 14H, Ar-H+CH). MS (El): m/z (%) = 557 [M]⁺. Anal. Calcd for C₂₆H₁₉N₇O₄S₂ (Mol. Wt.: 557): C, 56.01; H, 3.43; N, 17.58; S, 11.50%, found: C, 56.23; H, 3.55; N, 17.46; S, 11.62%.

3.2. Antibacterial Susceptibility Testing

3.2.1. Bacterial Strains and Culture Conditions

The human pathogenic Gram-positive bacteria Staphylococcus aureus and Staphylococcus haemolyticus were used in this study. Bacterial strains were kindly obtained from the Faculty of Science—Botany and Microbiology Department—Bacteriology Laboratory. Bacterial strains were maintained on Tryptic Soy Agar (TSA) slants and incubated at 37 °C for 24–48 h. The inocula were spread over (TSA) plates prior to the antimicrobial activity tests.

3.2.2. Determination of Minimum Inhibitory Concentration (MIC) by INT Assay

The antibacterial activities of the compounds were assessed using MIC and MBC assays. The MIC, defined as the lowest concentration that inhibits visible bacterial growth after overnight incubation, was determined using the INT assay. Sterile 96-well microtiter plates were employed, with each well containing a 100 µL bacterial suspension adjusted to a 0.001 = OD₅₉₅ [36] and 10 µL serial dilutions of the chemical compounds.

The plates were incubated at 37 °C for 24 h, followed by the addition of INT (p-iodonitrotetrazolium violet) to assess bacterial growth. A total of 60 µL of INT (p-iodonitrotetrazolium violet, 0.2 mg mL⁻¹) was added to microplate wells and re-incubated at 37 °C for 2 h [37]. The MIC in the INT assay was defined as the lowest concentration of chemical substances that prevented color change, indicating bacterial growth inhibition, as described earlier [36]. All the experiments were performed in eight replicates represented by one column in the 96-well plates.

3.2.3. Determination of Minimum Bactericidal Concentration (MBC)

The MBC, which represents the lowest concentration that completely eliminates the bacteria, was determined by sub-culturing 20 µL of the suspension from MIC wells onto sterile tryptic soya agar plates [38]. The MBC was determined by transferring 20 microliters of suspension from each well of overnight incubated MIC plates and inoculated on sterile tryptic soya agar in fresh plates with continuous shaking with sterilized glass beads (0.4 mm) and incubated at 37 °C for 24 h. The MBC-causing bactericidal effect was identified on the basis of colony absence on the agar plates [39].

3.3. In Silico Studies

The molecular docking studies were performed for a set of quinazolin-2,4-diones and a standard drug and reported antibacterial drug against the targets dihydrofolate reductase (PDB ID: 2W9S) and eukaryotic initiation factor 2 α (eIF2α) (PDB ID: 1Q46) utilizing the PyRx-virtual screening tool [33]. The crystal structures of the target enzymes were obtained from the RCSB Protein Data Bank web server. Subsequently, the target files were optimized by removing the ligands and water molecules. Their energies were minimized using CHARMm Force Field [40] in Discovery Studio 3.5 Visualizer. In addition, the prepared molecules, methotrexate, and the reported antibacterial drug were sketched in cdx format (2D structures) using ChemDraw Ultra 8.0 and then were converted to sdf files (3D structures) by using Open Babel GUI 2.4.1 tool [41]. The energy of the synthesized molecules was minimized in the PyRx tool with default parameters (UFF force field) [42] and then docked flexibly to the targets. The visualizations of docking results were performed using Discovery Studio 3.5. Finally, the ADMET properties of the best-docked molecules and standard drugs were investigated using the AdmetSAR and SwissADME web servers.

4. Conclusions

A series of new quinazolin-2,4-dione derivatives were prepared with from good to excellent yields. Chemical structures and purity were proven from spectral data and elemental analysis. Antibacterial efficacies for new derivatives were assessed in vitro against two Gram-positive bacterial strains (S. aureus and S. haemolyticus). The molecules 2b and 2c exhibited good antibacterial activity, which may be due to the presence of quinazolin-2,4-dione, –CH₂CN, amide, and/or thia/tri/tetr-azocine moieties, respectively.

Additionally, computer-aided drug design (CADD) was carried out to screen the new quinazolin-2,4-dione derivatives, standard drug, and reported antibacterial drug against the target enzymes to establish the mechanism by which the molecules inhibit the growth of S. aureus and S. haemolyticus. It is noteworthy that the data obtained from the in silico docking study were in excellent agreement with the in vitro antibacterial results.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules29235529/s1. Figure S1: IR spectrum of compound 1; Figure S2: ¹H-NMR spectrum of compound 1; Figure S3: ¹³C-NMR spectrum of compound 1; Figure S4: Mass spectrum of compound 1; Figure S5: IR spectrum of compound 2a; Figure S6: ¹H-NMR spectrum of compound 2a; Figure S7: ¹³C-NMR spectrum of compound 2a; Figure S8: Mass spectrum of compound 2a; Figure S9: IR spectrum of compound 2b; Figure S10: ¹H-NMR spectrum of compound 2b; Figure S11: ¹³C-NMR spectrum of compound 2b; Figure S12: Mass spectrum of compound 2b; Figure S13: IR spectrum of compound 2c; Figure S14: ¹H-NMR spectrum of compound 2c; Figure S15: ¹³C-NMR spectrum of compound 2c; Figure S16: Mass spectrum of compound 2c; Figure S17: IR spectrum of compound 3a; Figure S18: ¹H-NMR spectrum of compound 3a; Figure S19: ¹³C-NMR spectrum of compound 3a; Figure S20: Mass spectrum of compound 3a; Figure S21: IR spectrum of compound 3b; Figure S22: ¹H-NMR spectrum of compound 3b; Figure S23: ¹³C-NMR spectrum of compound 3b; Figure S24: Mass spectrum of compound 3b; Figure S25: IR spectrum of compound 3c; Figure S26: ¹H-NMR spectrum of compound 3c; Figure S27: ¹³C-NMR spectrum of compound 3c; Figure S28: Mass spectrum of compound 3c; Figure S29: IR spectrum of compound 3d; Figure S30: ¹H-NMR spectrum of compound 3d; Figure S31: ¹³C-NMR spectrum of compound 3d; Figure S32: Mass spectrum of compound 3d; Figure S33: IR spectrum of compound 4a; Figure S34: ¹H-NMR spectrum of compound 4a; Figure S35: ¹³C-NMR spectrum of compound 4a; Figure S36: Mass spectrum of compound 4a; Figure S37: IR spectrum of compound 4b; Figure S38: ¹H-NMR spectrum of compound 4b; Figure S39: ¹³C-NMR spectrum of compound 4b; Figure S40: Mass spectrum of compound 4b; Figure S41: IR spectrum of compound 4c; Figure S42: ¹H-NMR spectrum of compound 4c; Figure S43: Mass spectrum of compound 4c; Figure S44: IR spectrum of compound 4d; Figure S45: ¹H-NMR spectrum of compound 4d; Figure S46: Mass spectrum of compound 4d; Figure S47: 3D interactions of the other docked compounds against 2W9S; Figure S48: 3D interactions of the other docked compounds against 1Q46.

Author Contributions

Methodology, M.E.-N., A.O.A.I., A.S.A. and I.A.S.; Formal analysis, A.H.A., M.E.-N. and I.A.S.; Writing—original draft, A.H.A., A.O.A.I., A.S.A., A.M.M. and A.K.; Writing—review & editing, A.H.A.; Supervision, A.H.A., A.M.M. and A.K. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in the Supplementary Information File.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Funding Statement

This research received no external funding.