Deep eutectic solvent for an expeditious sono-synthesis of novel series of bis-quinazolin-4-one derivatives as potential anti-cancer agents

Abstract

To produce a new category of anti-cancer compounds, a facile and environmentally sustainable method for preparing diversified bis-quinazolinones was demonstrated using recyclable deep eutectic solvent (DES) under ultrasonic irradiation. The reactions were performed smoothly with a wide scope of substrates affording the desired derivatives in good-to-excellent yields under an atom-economical pathway. Particularly, halogen substituents that are amenable for further synthetic elaborations are well tolerated. Furthermore, the ‘greenness’ of the protocol was assessed within the scope of several green metrics and found to display an excellent score in the specified parameters. Cytotoxic activity of all novel bis-quinazolinones was investigated utilizing two cancer cell lines: breast (MCF-7) and lung (A549) cell lines and their IC₅₀ values were determined. Most of the prepared derivatives displayed fascinating inhibitory activity with IC₅₀ values in a low micromolar range. Remarkably, the derivative 7e [3,3'-(sulfonylbis(4,1-phenylene))bis(2-methyl-6-nitroquinazolin-4(3H)-one)] showed superior potency against MCF-7 and A549 cancer cell lines, with IC₅₀ values of 1.26 µM and 2.75 µM, respectively. Moreover, this derivative was found to have low toxicity to the normal breast cell line (MCF-10A) and could serve as a promising lead candidate for further development.

1. Introduction

As an important category of nitrogen-containing heterocycles, quinazolines are prevalent in many naturally occurring alkaloids as well as marketed drugs [1–4]. Some quinazolines which are well known as drugs, such as mecloqualone (Casfen), mebroqualone and methaqualone (Quaalude) possessed anxiolytic, calmative and hypnotic properties and are used for treating insomnia [5,6]. Presently, the quinazoline moiety is recognized to have a broad scope of beneficial biological activities, for instance, anti-cancer, anti-inflammatory, anti-tumour, protein kinase inhibitor, anti-microbial cholinesterase inhibitor, antifolate, antiviral and are the essential moiety of HIV reverse transcriptase inhibitors [7–11]. Proportional with the importance of this heterocyclic motif, considerable synthetic protocols have been reported to develop access to quinazolines with diversified substituents [12–15]. Nevertheless, most of them are restricted with a multistep procedure and low atom-economy to prepare quinazoline compounds. Also, in several cases, the utilization of metal–catalyst may enhance the reaction efficiency; metal contaminations are still an issue, essentially when the outcomes are manufactured for the consumption of humans [16–19]. Latterly, many endeavours have been reported to demonstrate easy-to-handle, cost-effective and eco-friendly protocols for the preparation of quinazolinones [20–22]. Notwithstanding, to the best of our knowledge there have been restricted articles to date on the assembly of bis-quinazolinones [23,24]. Subsequently, the development of an effective and greener methodology for the construction of bis-quinazolinones is extremely in demand. Recently, more attempts have been made for designing eco-friendly solvents that could be reusable and/or smoothly biodegradable. For example, green solvents such as supercritical liquids [25], water [26], polyethylene glycol [27] and ionic liquids [28–30] have emerged to supersede numerous organic solvents. Nevertheless, in some cases, the utilization of these solvents is limited because of their poor stability and solubility of organic substances. Lately, deep eutectic solvents (DESs), the environmentally benign solvent systems, have attracted considerable attention [31,32]. DESs possess several noteworthy merits when compared with conventional solvents, comprising a broad range of liquid temperatures, renewability, water tolerance, low toxicity, biodegradability, low volatility and high solvation capacity for an extensive number of organic substances [33–35]. DESs are easily prepared from mixing an organic hydrogen bond donor (e.g. tartaric acid) and a hydrogen bond acceptor (e.g. choline chloride) [31,32]. The obtained mixture possesses high stability and remains in a high-entropy liquid state even at low temperatures; this may be attributed to the newly formed hydrogen-bond network between its components [31,32,36,37]. Numerous reports have demonstrated their utilization in several organic conversions [38–40]. Because of these special advantages, DESs opened novel perspectives to prepare new substances. The use of ultrasonic irradiation as the green energy source is of a significant utility in the field of pharmaceutical and green chemistry. The impacts of sonication on organic synthesis are imputed to cavitations, which generate extremely high local pressure and temperature inside the formed bubbles. When these bubbles collapse, a sufficient amount of energy was generated for performing the chemical reaction and allowing the process to occur easily with a high yield in a very short time [41,42]. In spite of that, to the best of our knowledge there have been no reports to date on the preparation of bis-quinazolinones using deep eutectic solvents under ultrasonic irradiation. In continuation to our ongoing efforts to develop greener synthetic pathways for organic conversions [43–46], herein I wish to report a DES-mediated protocol for greener and atom-economical preparation of novel series of bis-quinazolinones by using commercially available and inexpensive materials under appropriate catalyst-free and ultrasonic conditions.

2. Results and discussion

At the outset, the present study was commenced with optimization of the reaction conditions using 2-methyl-4H-benzo[d][1,3]oxazin-4-one (1a) and trans-cyclohexane-1,4-diamine (2a) as model substrates (table 1). The initial endeavour utilizing conventional refluxing conditions in CH₃CN and in the presence of K₂CO₃ as the basic catalyst [23,24] afforded the hitherto unreported 3,3′-((1R,4R)-cyclohexane-1,4-diyl)bis(2-methylquinazolin-4(3H)-one) (3a) but in a poor yield; instead, a considerable amount of starting materials remain unchanged (table 1, entry 1). Next, on performing the above reaction under ultrasonic irradiation (60 W) at 50°C for 20 min, furnished 40% of the product (table 1, entry 2). Anticipating further improvement in the yield, a series of base and acid catalysts, such as Cs₂CO₃, AcOH/NaOAc and p-toluenesulfonic acid (table 1, entries 3–5), were screened under ultrasonic irradiation (60 W); of these, p-TSA was found to be the most effective catalyst for this conversion (table 1, entry 5). Furthermore, a moderate yield was obtained under catalyst-free and ultrasound conditions, which suggest an apparent over-activity by the used catalyst (table 1, entry 6). Such an observation is also confirmed by literature studies, which proposed that ultrasonic irradiation may replace a catalyst under definite conditions [47,48]. In order to acquire more acceptable results, other reaction variables were assessed. First, the role of additional solvents was investigated and found that the yield of 3a was improved to be 70% when performing water as a solvent at 50°C (table 1, entry 7), and slightly increased to be 77% in a longer reaction time at 95°C (with table 1, entry 8). In addition, ethanol furnished a moderate yield at 50°C (table 1, entry 9), while other solvents such as i-PrOH and toluene were substantially less efficient and THF was ineffectual (table 1, entries 10–12). To evaluate the current protocol from the greener edge, deep eutectic solvents (DESs) were used as the reaction medium for this conversion. To acquire derivative 3a (table 1), we turned our venture by using certain types of DESs with the model substrates 1a (2.0 equiv.) and 2a (1.0 equiv). As displayed in table 1, only a small amount of the required product (3a) was obtained when the reaction proceeded in sucrose–choline chloride mixture (1 : 1) at its melting temperature (table 1, entry 13). Considering that the model reaction works well under acidic conditions, three acidic DESs were examined with a view to acquiring more satisfactory results. Interestingly, a considerable improvement was obtained when DESs such as lactic acid–choline chloride (1 : 2), oxalic acid–choline chloride (1 : 1) and l-(+)-tartaric acid–choline chloride (1 : 2) were used as the reaction solvent (table 1, entries 14–16). This screening disclosed that the utilization of l-(+)-tartaric acid–choline chloride (1 : 2) melt afforded the superior result (table 1, entry 16). In a quest to further improve the reaction yield, the model substrates were sonicated at two operating temperatures, i.e. 85 and 90°C and the required product (3a) was obtained in 89% and 99% yields, respectively (table 1, entries 16 and 17). Thus, the optimal temperature was established to be 90°C, which achieved the best yield of the desirable product. Decreasing the sonication time to 10 min diminished the isolated yield to 91% (table 1, entry 18).

Table 1.

Optimization of the reaction conditions for the preparation of compound 3a.

entry	catalyst	solvent	temp. (°C)/method	time (min)	yield (%)a
1	K2CO3	CH3CN	Refluxb	300	25
2	K2CO3	CH3CN	50/USc	20	40
3	Cs2CO3	CH3CN	50/US	20	43
4	NaOAc	AcOH	50/US	20	45
5	p-TSA	CH3CN	50/US	20	57
6	—	CH3CN	50/US	20	65
7	—	H2O	50/US	20	70
8	—	H2O	95/US	35	77
9	—	EtOH	50/US	20	68
10	—	i-PrOH	50/US	20	57
11	—	toluene	50/US	20	38
12	—	THF	50/US	20	15
13	—	sucrose–choline chloride (1 : 1)	80/US	20	17
14	—	lactic acid–choline chloride (1 : 2)	80/US	20	73
15	—	oxalic acid–choline chloride (1 : 1)	80/US	20	75
16	—	l-(+)-tartaric acid–choline chloride (1 : 2)	85/US	20	89
17	—	l-(+)-tartaric acid–choline chloride (1 : 2)	90/US	15	99
18	—	l-(+)-tartaric acid–choline chloride (1 : 2)	90/US	10	91

^aIsolated yield.

^bThe reaction was carried out under reflux conditions.

^cThe reactions were carried out under ultrasonic irradiation (US) conditions.

After optimization of the current protocol, the impact of several ultrasonic irradiation powers on the yield and rate of model reaction were also studied (table 2). The results display that the power of sonication has a considerable impact on the reaction system. Thus, the sonication of the model substrates in l-(+)-tartaric acid–choline chloride (1 : 2) at 30 W afforded the desired product (3a) in a moderate yield throughout 60 min (table 2, entry 1). While, both reaction rate and yield were improved by increasing the sonication power to 60 W (table 2, entries 2–4). Moreover, on sonication of model reactants at 70 W, neither the reaction rate nor the reaction yield was improved (table 2, entry 5). Noteworthy, on carrying out the model reaction under reflux conditions, the reaction effectiveness is obviously diminished (table 2, entry 6). The utilization of ultrasound technique reduces the reaction duration and enhances the reaction yield possibly through increasing the energy and collision speed of the reactants. Consequently, sonication (60 W) of 1a (2 mmol) and 2a (1 mmol) in l-(+)-tartaric acid–choline chloride (1 : 2) at 90°C for 15 min was established as the optimum conditions, affording the desired product (3a) in 99% yield (table 1, entry 17).

Table 2.

The impact of ultrasonic irradiation power on the synthesis of 3a.

entry	power (W)	time (min)	yield (%)a
1	30	60	76
2	40	50	80
3	50	30	82
4	60	15	99
5	70	15	99
6	silent (reflux)b	320	78

^aIsolated yield.

^bThe reaction was carried out under reflux conditions.

After achieving an optimized strategy for this new reaction, the substrates' versatility and scope with regard to diamines and benzoxazin-4-ones were then investigated for the assembly of various bis-quinazolinones (schemes 1 and 2). First, the electronic impacts of R groups on 2-methyl-4H-benzo[d][1,3]oxazin-4-ones (1a–e) were studied. Generally, the aryl motif linked to an electron donating substituent (e.g. –CH₃) afforded better yields than that linked to electron withdrawing substituents (e.g. –Cl, –F and –NO₂).

Scheme 1.

Reaction of derivatives 2a–c under the standard conditions.

Scheme 2.

Reaction of derivatives 2d and 2e under the standard conditions.

Furthermore, diverse diamines, comprising aromatic and aliphatic diamines, were well tolerated. Interestingly, all the reactions proceeded smoothly to provide the corresponding bis-quinazolinones depending on the reaction time. trans-Cyclohexane-1,4-diamine (2a) afforded the required products (3a–e) in short reaction time with high yields. Also, p-xylylenediamine (2b) and ethylene diamine (2c) could not reduce the reaction rate remarkably. Furthermore, p-phenylene diamine (2d) and dapsone (2e) produced the corresponding derivatives in moderate-to-high yields but in longer reaction time (scheme 2). Owing to the fact that heterocycles bearing halogens are significant building blocks in the assembly of plentiful pharmaceuticals and natural products [49,50], an assortment of halo-substituted bis-quinazolinones, e.g. chloro 3c (96%), 4c (95%), 5c (95%), 6c (95%), 7c (97%) and fluoro 3d (96%), 4d (92%), 5d (94%), 6d (97%), 7d (93%), were successfully prepared.

The steric impact of the utilized diamines had an obvious effect on the effectiveness of these reactions. For instance, when the present method was put in an application for o-phenylene diamine (2f), however, bis-quinazolinone derivative (9, 67%) was isolated along with the mono-quinazolinone derivative [23,24] (8, 33%). Whereas, by increasing the reaction time to 25 min and molar ratio of 1a to 2.5 mmol, the reaction yield was improved to 88% with the complete consumption of the mono-quinazolinone derivative 8 (monitored by TLC). The comparable result was acquired when m-phenylene diamine (2 g) was utilized as the substrate; the reaction afforded a complex mixture of compounds 10 [51] and 11 under optimum conditions. By increasing both time (30 min) and molar ratio of 1a (2.5 mmol), compound 10 disappeared and the reaction afforded derivative 11 in 92% yield (scheme 3). The structures of all the novel bis-quinazolinones were interpreted on the basis of spectral analyses (IR, NMR spectroscopy and mass spectrometry).

Scheme 3.

Reaction of derivatives 2f and 2g under the standard conditions.

In order to further display the practicality of the above-mentioned investigations, the current protocol was extended for large-scale preparation of bis-quinazolin-4(3H)-one (3a) (scheme 4). As outlined in scheme 4, the reaction of trans-cyclohexane-1,4-diamine (2a, 1.14 g, 10.0 mmol) with benzoxazine (1a, 3.22 g, 20.0 mmol), using l-(+)-tartaric acid–choline chloride (1 : 2) as the solvent, was scaled up. Under the optimal conditions, the required product 3a was obtained in 98% yield by crystallization from dioxane. Lastly, to assess the current protocol on the ‘greenness’ scale, green metrics [52,53] such as E-factor (EF), atom economy (AE), reaction mass efficiency (RME), process mass intensity (PMI), yield economy (YE) and carbon efficiency (CE) were studied. As summarized in scheme 4, the current protocol achieved a good combination of AE (91.74%), CE (100%), EF (0.02), RME (98.67%), PMI (1.11) and YE (6.53%), which makes it an ideal sustainable and green process. The results might be beneficial from environmental and industrial perspectives.

Scheme 4.

Calculated green metrics for the scaled-up preparation of 3,3'-((1R,4R)-cyclohexane-1,4-diyl)bis(2-methylquinazolin-4(3H)-one) 3a.

According to the aforementioned experiments and the reported literature [54], a proposed mechanism for the reaction between diamines (2a–g) and benzoxazines (1a–e) was established as presented in scheme 5. The first step could be the ring opening via nucleophilic attack of amine (NH₂) to carbonyl carbon under the presence of l-(+)-tartaric acid–choline chloride. Next, the formed intermediate underwent intramolecular nucleophilic attack of the nitrogen (–NH–) of the amide group to carbonyl carbon. Finally, the required product was formed through dehydration [54]. From the proposed mechanism, it is obviously concluded that, l-(+)-tartaric acid–choline chloride performs a dual function; as solvent and as catalyst.

Scheme 5.

A plausible mechanism for the preparation of bis-quinazolin-4-ones 3–11.

Recyclability of the solvent is a needful merit for industrial processes. The recyclability of l-(+)-tartaric acid–choline chloride in this investigation was proceeded in the model substrates; 2-methyl-4H-benzo[d][1,3]oxazin-4-one (1a) and trans-cyclohexane-1,4-diamine (2a) under optimal conditions (figure 1). After consumption of reactants, the reaction contents were cooled to ambient temperature and diluted with ethanol. The desired product was filtered and washed with ethanol. After removal of ethanol under vacuum, DES was recovered and re-used for the next cycles. The recyclability proceeded five times without considerably lowering the reaction efficiency (figure 1).

Figure 1.

Recyclability of DES for the synthesis of 3a.

3. Assessment of in vitro human cancer cell lines growth inhibition and the structure and activity relationships (SARs)

The unprecedented derivatives (3–7 and 11) were tested for their in vitro cytotoxicity against human MCF-7 (breast cancer), A549 (lung cancer) cancer cell lines and one type of normal cell line MCF10A (normal breast cell line) utilizing the standard MTT (3,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay according to Mosmann's methodology [55] (figure 2), whereas sorafenib was performed as a standard compound. Two concentrations (10 and 30 µM in triplicate) of the derivatives were utilized and after 48 h of drug treatment the results were analyzed. The obtained results were tabulated as mean ± s.e.m. With standard errors below 10%, the reproducibility between replicate wells is within the acceptable values. Concentrations less than 10 µM have negligible impacts on the cytotoxicity. The investigation of the SARs displayed that the substituent R groups in the quinazolinone moieties have a significant role in their biological properties. The results summed up in electronic supplementary material, S59, table S3 indicate that all of the tested derivatives have good-to-excellent cytotoxic activities, with the IC₅₀ values at the micromolar range (figure 2). Regarding the MCF-7 breast cancer cell line, compounds 7a–e elicited potent anti-breast cancer activity (IC₅₀ = 1.26–5.78 µM), compared with sorafenib (IC₅₀ = 4.03 µM). This reactivity could be related to the high potency of both dapsone and quinazolinone moieties, when compared with the other derivatives. Noteworthy, compounds 4a–e comprising p-xylylene moiety resulted in good-to-excellent activities with IC₅₀ of 2.75–6.73 µM. Bis-quinazolin-4-one derivatives 5a–e (IC₅₀ = 5.21–13.55 µM) were less active than the cyclohexane derivatives 3a–e (IC₅₀ = 4.55–12.70 µM). Whereas, 1,4-phenylene derivatives 6a–e (2.73–7.48 µM) were slightly more active than cyclohexane derivatives 3a–e. Similarly, derivative 11 possessed good activity with an IC₅₀ value of 5.42 µM. Moreover, substitution with electron withdrawing groups (Cl, F and NO₂) displayed good activity, while electron donating group (CH₃) reduced the activity. A comparison of the substituents on the quinazolinone ring indicated that nitro groups significantly enhanced the cytotoxic activity and determined the order of potency. For instance, derivative 7e with NO₂-substitution at the quinazolinone moiety was found to be the most potent in the series with IC₅₀ value of 1.26 µM against MCF-7, being approximately thrice as potent as sorafenib. Likewise, derivatives 7c and 7d possessed a more important activity than unsubstituted quinazolinone moiety (7a) and methyl substituted derivative (7b).

Figure 2.

In vitro studies of the synthesized compounds (3–7 and 11) against human cancer cell lines (MCF-7 and A549).

Concerning the A549 lung cancer cell line (electronic supplementary material, table S3), the alteration of substituents on 6-position of the quinazolinone ring could also influence the activities of these compounds. As summarized in figure 2, the potency of substituents was ordered as NO₂ > F > Cl > H > CH₃, indicating that those with the electron withdrawing group are more active than those with an electron donating substitute. Ten derivatives, 7a–e, 6c–e, 5e and 4e, possessed promising cytotoxic activities as indicated from their IC₅₀ values (2.78–5.77 µM) compared with sorafenib (IC₅₀ = 5.20 µM). Among them, derivative 7e, 3,3′-(sulfonylbis(4,1-phenylene))bis(2-methyl-6-nitroquinazolin-4(3H)-one), showed the best activity with IC₅₀ value of 2.75 µM against A549, being approximately twice as potent as sorafenib. The series of derivatives 6 and compound 11, with aryl motif, also afforded potent activities and displayed similar tendencies in the relationship between the structure and activity. Nevertheless, the series of compounds 3 and 5 afforded slightly weaker activities (figure 2). Figure 3 clarifies the structural activity relationship of the designed derivatives.

Figure 3.

Illustration of the structural activity relationship of the designed derivatives.

In order to decide whether the cytotoxic properties of the investigated derivatives were selective for cancerous cells in contrast to non-cancerous cells, the most active derivatives, viz. 4e, 6e, 7d and 7e, were subjected to in vitro assessment against the normal breast cell line (MCF-10A). The results of the non-malignant cell (MCF-10A) demonstrated that derivatives 7e (IC₅₀ = 7.17 ± 0.76 µM), 7d (IC₅₀ = 5.98 ± 1.86 µM), 6e (IC₅₀ = 5.65 ± 2.34 µM) and 4e (IC₅₀ = 5.43 ± 2.46 µM) exhibited promising results in comparison with sorafenib (IC₅₀ = 5.98 ± 0.49 µM) when tested under the same conditions. From the obtained results, we can conclude that these derivatives might be further used as promising anti-cancer agents.

In the present investigation, incorporation of two symmetrical pharmacophores into a single molecule resulted in designed multiple compounds possessing fascinating inhibition activities against clinically relevant objectives that are not obtainable by their monomeric analogues.

4. Conclusion

A straightforward, green and effective protocol for synthesizing new series of bis-quinazolin-4-ones from easily obtainable precursors under minutes of ultrasonic irradiation was demonstrated on a gram-scale. Noteworthy, the l-(+)-tartaric acid–choline chloride deep eutectic solvent displayed to be appropriate for these transformations as indicated by its recyclability, eco-friendly and inexpensive preparation, and simplicity of operation. Also, the sonochemical methodology performs well for both activating and deactivating starting substrates. The reaction yields are excellent and the protocol is devoid of column purification and any tedious work-up. An assortment of halo-substituted products was successfully synthesized via the current strategy that provides possibility for further cross-coupling reactions. As well, the protocol scored well in several green metrics and consequently may be of sustainable and practical benefit in the future. All the newly prepared derivatives possessed potential cytotoxic activities, with the strength of the impact depending on the substituent on the quinazolinone moiety. Among them, derivatives 7e, 3,3′-(sulfonylbis(4,1-phenylene))bis(2-methyl-6-nitroquinazolin-4(3H)-one), exhibited the most potent cytotoxic activities and possessed the ability to kill malignant cells more effectively than non-malignant cells.

5. Experiment

5.1. General information

¹H, ¹⁹F and ¹³C NMR spectra were recorded on Bruker Ultra Shield spectrometer at 400, 376 and 100 MHz, respectively. Mass spectra were determined on the GC-MS (QP/000 EX) Shimadzu spectrometer at an ionizing voltage of 70 eV. IR spectra were recorded on the Perkin-Elmer Spectrum One spectrometer using KBr pellets. Analytical thin layer chromatography (TLC) was employed on a silica gel plate (Merck^® 60F254). Sonication was performed in a SY5200DH-T ultrasound cleaner. Melting points were measured on Electrothermal IA9100 melting point apparatus (UK) using open capillary tubes and are uncorrected. All commercial reagents were used as received without any purification. Cytotoxic activities were performed by the Microanalysis Center, Faculty of Science, Cairo University. Derivatives 1a–e were synthesized by the sonication of anthranilic acid derivatives and acetic anhydride at 40°C for 10 min.

5.2. Synthesis of DESs

The DESs were synthesized by mixing choline chloride with several hydrogen bond donors such as sucrose, lactic acid, oxalic acid and tartaric acid, according to the molar ratio mentioned in table 1. The two components of each mixture were heated at 85°C under sonication until a clear liquid was obtained, which was directly used for bis-quinazolin-4(3H)-ones synthesis.

5.3. Typical procedure for the preparation of bis-quinazolin-4(3H)-ones 3–11

2-Methyl-4H-benzo[d][1,3]oxazin-4-ones (1a–e) (2 mmol), diamines (2a–g) (1 mmol) and l-(+)-tartaric acid–choline chloride (1 : 2) based DES (4 ml) were added to a 25 ml round bottom flask and the reaction mixture was sonicated (60 W) for 15 min (20–30 min in the case of using 2d–g) at 90°C. The progress of the reaction was monitored by TLC. After completion of the reaction, the mixture was cooled to room temperature and diluted with ethanol. The desired product was obtained by filtration and recrystallized from dioxane containing a few drops of DMF. The filtrate which mainly contained DES was dried under vacuum to get DES. DES so recovered was utilized for the next experiments. The recyclability study was performed five times and displayed no significant drop in the reaction yields.

5.3.1. 3,3′-((1R,4R)-Cyclohexane-1,4-diyl)bis(2-methylquinazolin-4(3H)-one) 3a

White crystals, yield 99%, m.p. 273–275°C; IR (KBr): ν/cm⁻¹ 1672 (C=O), 1620, 1595 (C=N, C=C); ¹H NMR (400 MHz, DMSO-d₆): δ 8.37–8.35 (d, J = 5.5 Hz, 2H, Ar-H), 7.87–7.85 (d, J = 5.6 Hz, 2H, Ar-H), 7.44–7.31 (m, 4H, Ar-H), 3.74–3.61 (m, 2H, Cyclohexane-H), 2.30 (s, 6H, CH₃), 1.65 (br, 4H, Cyclohexane-H), 1.44–1.31 (m, 4H, Cyclohexane-H); ¹³C NMR (100 MHz, DMSO-d₆): δ 170.1 (C=O), 147.6 (C=N), 146.0, 134.5, 128.1, 127.5, 127.1, 126.7 (Ar-C), 52.5 (Cyclohexane-C), 31.1 (Cyclohexane-C), 20.3 ppm (CH₃); MS (EI): m/z (%) 401 (M⁺+1, 4.6), 400 (M⁺, 100); Anal. Calcd for C₂₄H₂₄N₄O₂: C, 71.98; H, 6.04; N, 13.99%; Found: C, 71.93; H, 5.99; N, 13.90%.

5.3.2. 3,3′-((1R,4R)-Cyclohexane-1,4-diyl)bis(2,6-dimethylquinazolin-4(3H)-one) 3b

White crystals, yield 99%, m.p. 280–281°C; IR (KBr): ν /cm⁻¹ 1678 (C=O), 1613, 1590 (C=N, C=C); ¹H NMR (400 MHz, CDCl₃): δ 8.25 (s, 2H, Ar-H), 7.90–7.87 (d, J = 7.5 Hz, 2H, Ar-H), 7.59–7.55 (d, J = 7.8 Hz, 2H, Ar-H), 3.69 (br, 2H, Cyclohexane-H), 2.75 (s, 6H, CH₃), 2.30 (s, 6H, CH₃), 1.72 (br, 4H, Cyclohexane-H), 1.20 (m, 4H, Cyclohexane-H); ¹³C NMR (100 MHz, CDCl₃): δ 170.8 (C=O), 147.2 (C=N), 145.6, 138.7, 133.5, 128.0, 126.5, 122.1 (Ar-C), 52.4 (Cyclohexane-C), 31.0 (Cyclohexane-C), 20.8 ppm (CH₃), 20.2 ppm (CH₃); MS (EI): m/z (%) 429 (M⁺+1, 7.3), 428 (M⁺, 100); Anal. Calcd for C₂₆H₂₈N₄O₂: C, 72.87; H, 6.59; N, 13.07%; Found: C, 72.86; H, 6.62; N, 13.01%.

5.3.3. 3,3′-((1R,4R)-Cyclohexane-1,4-diyl)bis(6-chloro-2-methylquinazolin-4(3H)-one) 3c

White crystals, yield 96%, m.p. 311–313°C; IR (KBr): ν/cm⁻¹ 1670 (C=O), 1611, 1587 (C=N, C=C); ¹H NMR (400 MHz, CDCl₃): δ 8.11 (s, 2H, Ar-H), 7.92–7.90 (d, J = 7.7 Hz, 2H, Ar-H), 7.65–7.64 (d, J = 5.8 Hz, 2H, Ar-H), 3.29–3.27 (m, 2H, Cyclohexane-H), 2.31 (s, 6H, CH₃), 1.97–1.95 (m, 4H, Cyclohexane-H), 1.74–1.69 (m, 4H, Cyclohexane-H); ¹³C NMR (100 MHz, CDCl₃): δ 171.0 (C=O), 144.5 (C=N), 145.0, 133.8, 131.6, 128.3, 128.2, 122.6 (Ar-C), 51.7 (Cyclohexane-C), 31.3 (Cyclohexane-C), 20.4 ppm (CH₃); MS (EI): m/z (%) 472 (M⁺+4, 10.7), 470 (M⁺+2, 66.3), 468 (M⁺, 100); Anal. Calcd for C₂₄H₂₂Cl₂N₄O₂: C, 61.42; H, 4.72; N, 11.94%; Found: C, 61.39; H, 4.79; N, 11.88%.

5.3.4. 3,3'-((1R,4R)-Cyclohexane-1,4-diyl)bis(6-fluoro-2-methylquinazolin-4(3H)-one) 3d

White crystals, yield 96%, m.p. 262–264°C; IR (KBr): ν/cm⁻¹ 1682 (C=O), 1616, 1597 (C=N, C=C); ¹H NMR (400 MHz, CDCl₃): δ 8.12 (s, 2H, Ar-H), 7.81–7.80 (d, J = 5.5 Hz, 2H, Ar-H), 7.709–7.702 (m, 2H, Ar-H), 3.79 (br, 2H, Cyclohexane-H), 2.41 (s, 6H, CH₃), 1.71–1.64 (m, 4H, Cyclohexane-H), 1.33–1.26 (m, 4H, Cyclohexane-H); ¹³C NMR (100 MHz, CDCl₃): δ 171.1 (C=O), 144.5 (C=N), 159.8, 142.0, 123.8, 121.8, 118.8, 115.2 (Ar-C), 51.8 (Cyclohexane-C), 31.6 (Cyclohexane-C), 20.7 ppm (CH₃); ¹⁹F NMR (376 MHz, CDCl₃): δ -125.90; MS (EI): m/z (%) 437 (M⁺+1, 13.8), 436 (M⁺, 100); Anal. Calcd for C₂₄H₂₂F₂N₄O₂: C, 66.05; H, 5.08; N, 12.84%; Found: C, 66.11; H, 5.01; N, 12.80%.

5.3.5. 3,3′-((1R,4R)-Cyclohexane-1,4-diyl)bis(2-methyl-6-nitroquinazolin-4(3H)-one) 3e

Light yellow crystals, yield 96%, m.p. 301–304°C; IR (KBr): ν/cm⁻¹ 1675 (C=O), 1612, 1585 (C=N, C=C); ¹H NMR (400 MHz, DMSO-d₆): δ 8.80 (s, 2H, Ar-H), 8.13–8.11 (d, J = 7.5 Hz, 2H, Ar-H), 7.79–7.77 (d, J = 7.8 Hz, 2H, Ar-H), 3.71 (br, 2H, Cyclohexane-H), 2.63 (s, 6H, CH₃), 1.97–1.95 (m, 4H, Cyclohexane-H), 1.74–1.69 (m, 4H, Cyclohexane-H); ¹³C NMR (100 MHz, DMSO-d₆): δ 170.4 (C=O), 145.8 (C=N), 151.8, 143.3, 128.4, 124.8, 119.6, 117.4 (Ar-C), 52.3 (Cyclohexane-C), 31.9 (Cyclohexane-C), 20.4 ppm (CH₃); MS (EI): m/z (%) 491 (M⁺+1, 7.3), 490 (M⁺, 100); Anal. Calcd for C₂₄H₂₂N₆O₆: C, 58.77; H, 4.52; N, 17.13%; Found: C, 58.79; H, 4.48; N, 17.09%.

5.3.6. 3,3′-(1,4-Phenylenebis(methylene))bis(2-methylquinazolin-4(3H)-one) 4a

White crystals, yield 98%, m.p. 277–279°C; IR (KBr): ν/cm⁻¹ 1688 (C=O), 1616, 1605 (C=N, C=C); ¹H NMR (400 MHz, DMSO-d₆): δ 8.32–8.29 (d, J = 7.7 Hz, 2H, Ar-H), 7.91–7.90 (d, J = 5.6 Hz, 2H, Ar-H), 7.767–7.761 (m, 2H, Ar-H), 7.659–7.650 (d, J = 5.5 Hz, 2H, Ar-H), 7.30 (s, 4H, Ar-H), 4.78 (s, 4H, CH₂), 2.29 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 170.7 (C=O), 144.6 (C = N), 146.7, 134.8, 134.2, 128.2, 127.0, 126.9, 126.2, 122.5 (Ar-C), 62.5 (CH₂), 20.9 ppm (CH₃); MS (EI): m/z (%) 423 (M⁺+1, 8.9), 422 (M⁺, 100); Anal. Calcd for C₂₆H₂₂N₄O₂: C, 73.92; H, 5.25; N, 13.26%; Found: C, 73.89; H, 5.29; N, 13.21%.

5.3.7. 3,3′-(1,4-Phenylenebis(methylene))bis(2,6-dimethylquinazolin-4(3H)-one) 4b

White crystals, yield 99%, m.p. 285–287°C; IR (KBr): ν/cm⁻¹ 1682 (C=O), 1623, 1611 (C=N, C=C); ¹H NMR (400 MHz, DMSO-d₆): δ 8.39 (s, 2H, Ar-H), 7.98–7.96 (d, J = 7.7 Hz, 2H, Ar-H), 7.41–7.29 (m, 6H, Ar-H), 4.81 (s, 4H, CH₂), 2.67 (s, 6H, CH₃), 2.29 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 171.2 (C=O), 145.1 (C=N), 142.9, 136.7, 134.4, 132.6, 128.0, 127.5, 126.5, 121.2 (Ar-C), 62.7 (CH₂), 21.8 (CH₃), 20.6 ppm (CH₃); MS (EI): m/z (%) 451 (M⁺+1, 11.5), 450 (M⁺, 100); Anal. Calcd for C₂₈H₂₆N₄O₂: C, 74.65; H, 5.82; N, 12.44%; Found: C, 74.61; H, 5.87; N, 12.39%.

5.3.8. 3,3′-(1,4-Phenylenebis(methylene))bis(6-chloro-2-methylquinazolin-4(3H)-one) 4c

Light yellow crystals, yield 95%, m.p. 327–330°C; IR (KBr): ν/cm⁻¹ 1675 (C = O), 1615, 1602 (C = N, C = C); ¹H NMR (400 MHz, DMSO-d₆): δ 8.37 (s, 2H, Ar-H), 7.82–7.81 (d, J = 7.5 Hz, 2H, Ar-H), 7.29 (s, 4H, Ar-H), 7.13–7.06 (m, 2H, Ar-H), 4.81 (s, 4H, CH₂), 2.25 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 171.0 (C=O), 144.8 (C=N), 144.9, 133.6, 132.2, 128.2, 127.8, 127.4, 127.1, 121.2 (Ar-C), 62.8 (CH₂), 20.3 ppm (CH₃); MS (EI): m/z (%) 494 (M⁺+4, 11.1), 492 (M⁺+2, 65.3), 490 (M⁺, 100); Anal. Calcd for C₂₆H₂₀Cl₂N₄O₂: C, 63.55; H, 4.10; N, 11.40%; Found: C, 63.59; H, 4.05; N, 11.36%.

5.3.9. 3,3′-(1,4-Phenylenebis(methylene))bis(6-fluoro-2-methylquinazolin-4(3H)-one) 4d

White crystals, yield 92%, m.p. 217–219°C; IR (KBr): ν/cm⁻¹ 1680 (C = O), 1611, 1593 (C = N, C = C); ¹H NMR (400 MHz, DMSO-d₆): δ 8.01–8.00 (d, J = 5.6 Hz, 2H, Ar-H), 7.81–7.79 (m, 2H, Ar-H), 7.54–7.49 (m, 2H, Ar-H), 7.15 (s, 4H, Ar-H), 4.78 (s, 4H, CH₂), 2.29 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 171.0 (C = O), 145.4 (C = N), 160.8, 141.8, 134.6, 128.1, 127.9, 124.3, 120.2, 117.8 (Ar-C), 62.8 (CH₂), 20.3 ppm (CH₃); ¹⁹F NMR (376 MHz, DMSO-d₆): δ -125.91; MS (EI): m/z (%) 459 (M⁺+1, 11.8), 458 (M⁺, 100); Anal. Calcd for C₂₆H₂₀F₂N₄O₂: C, 68.11; H, 4.40; N, 12.22%; Found: C, 68.07; H, 4.48; N, 12.17%.

5.3.10. 3,3′-(1,4-Phenylenebis(methylene))bis(2-methyl-6-nitroquinazolin-4(3H)-one) 4e

Light yellow crystals, yield 93%, m.p. 350–353°C; IR (KBr): ν/cm⁻¹ 1675 (C = O), 1601, 1593 (C = N, C = C); ¹H NMR (400 MHz, DMSO-d₆): δ 8.69 (s, 2H, Ar-H), 8.36–8.35 (d, J = 5.6 Hz, 2H, Ar-H), 7.87–7.85 (d, J = 7.6 Hz, 2H, Ar-H), 7.29 (s, 4H, Ar-H), 4.78 (s, 4H, CH₂), 2.29 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 170.7 (C = O), 145.6 (C = N), 153.1, 142.8, 134.4, 128.4, 128.2, 123.1, 118.0, 117.4 (Ar-C), 62.5 (CH₂), 20.4 ppm (CH₃); MS (EI): m/z (%) 513 (M⁺+1, 2.6), 512 (M⁺, 100); Anal. Calcd for C₂₆H₂₀N₆O₆: C, 60.94; H, 3.93; N, 16.40%; Found: C, 61.00; H, 3.91; N, 16.38%.

5.3.11. 3,3′-(Ethane-1,2-diyl)bis(2-methylquinazolin-4(3H)-one) 5a

White crystals, yield 98%, m.p. 190–192°C; IR (KBr): ν/cm⁻¹ 1679 (C = O), 1612, 1590 (C = N, C = C); ¹H NMR (400 MHz, DMSO-d₆): δ 8.29–8.26 (d, J = 7.1 Hz, 2H, Ar-H), 7.78 (s, 2H, Ar-H), 7.59–7.48 (t, J = 7.4 Hz, 2H, Ar-H), 7.25–7.21 (d, J = 7.1 Hz, 2H, Ar-H), 3.78 (s, 4H, CH₂), 2.30 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 171.0 (C = O), 144.9 (C = N), 143.9, 132.7, 127.4, 126.4, 126.0, 121.3 (Ar-C), 42.5 (CH₂), 20.4 ppm (CH₃); MS (EI): m/z (%) 347 (M⁺+1, 1.8), 346 (M⁺, 100) [23,24].

5.3.12. 3,3′-(Ethane-1,2-diyl)bis(2,6-dimethylquinazolin-4(3H)-one) 5b

White crystals, yield 97%, m.p. 211–212°C; IR (KBr): ν/cm⁻¹ 1675 (C = O), 1608, 1590 (C = N, C = C); ¹H NMR (400 MHz, CDCl₃): δ 8.00 (s, 2H, Ar-H), 7.80–7.79 (d, J = 7.4 Hz, 2H, Ar-H), 7.50–7.49 (d, J = 7.4 Hz, 2H, Ar-H), 3.80 (s, 4H, CH₂), 2.80 (s, 6H, CH₃), 2.30 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ 171.2 (C = O), 144.6 (C = N), 143.3, 137.2, 134.6, 127.9, 126.2, 122.6 (Ar-C), 42.7 (CH₂), 21.9 (CH₃), 20.3 ppm (CH₃); MS (EI): m/z (%) 375 (M⁺+1, 3.2), 374 (M⁺, 100); Anal. Calcd for C₂₂H₂₂N₄O₂: C, 70.57; H, 5.92; N, 14.96%; Found: C, 70.60; H, 5.89; N, 14.91%.

5.3.13. 3,3′-(Ethane-1,2-diyl)bis(6-chloro-2-methylquinazolin-4(3H)-one) 5c

White crystals, yield 95%, m.p. 244–246°C; IR (KBr): ν/cm⁻¹ 1680 (C=O), 1618, 1603 (C=N, C=C); ¹H NMR (400 MHz, DMSO-d₆): δ 7.94 (s, 2H, Ar-H), 7.60–7.59 (d, J = 7.5 Hz, 2H, Ar-H), 7.20–7.19 (d, J = 7.4 Hz, 2H, Ar-H), 3.70 (s, 4H, CH₂), 2.29 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 170.5 (C=O), 144.9 (C=N), 133.0, 132.5, 127.6, 127.2, 122.0 (Ar-C), 42.9 (CH₂), 20.5 ppm (CH₃); MS (EI): m/z (%) 418 (M⁺+4, 11.2), 416 (M⁺+2, 64.6), 414 (M⁺, 100); Anal. Calcd for C₂₀H₁₆Cl₂N₄O₂: C, 57.85; H, 3.88; N, 13.49%; Found: C, 57.81; H, 3.91; N, 13.42%.

5.3.14. 3,3′-(Ethane-1,2-diyl)bis(6-fluoro-2-methylquinazolin-4(3H)-one) 5d

White crystals, yield 94%, m.p. 219–221°C; IR (KBr): ν/cm⁻¹ 1676 (C=O), 1609, 1601 (C=N, C=C); ¹H NMR (400 MHz, DMSO-d₆): δ 7.60–7.57 (d, J = 7.7 Hz, 2H, Ar-H), 7.53–7.51 (d, J = 7.6 Hz, 2H, Ar-H), 7.39–7.35 (d, J = 7.7 Hz, 2H, Ar-H), 3.73 (s, 4H, CH₂), 2.34 ppm (s, 6H, CH₃); ¹⁹F NMR (376 MHz, DMSO-d₆): -125.96; ¹³C NMR (100 MHz, DMSO-d₆): δ 170.8 (C=O), 145.5 (C=N), 160.8, 143.1, 124.5, 122.1, 121.1, 113.9 (Ar-C), 42.8 (CH₂), 20.5 ppm (CH₃); MS (EI): m/z (%) 383 (M⁺+1, 4.9), 382 (M⁺, 100); Anal. Calcd for C₂₀H₁₆F₂N₄O₂: C, 62.82; H, 4.22; N, 14.65%; Found: C, 62.87; H, 4.18; N, 14.60%.

5.3.15. 3,3′-(Ethane-1,2-diyl)bis(2-methyl-6-nitroquinazolin-4(3H)-one) 5e

Yellow crystals, yield 94%, m.p. 278–279°C; IR (KBr): ν/cm⁻¹ 1683 (C=O), 1613, 1603 (C=N, C=C); ¹H NMR (400 MHz, DMSO-d₆): δ 8.61 (s, 2H, Ar-H), 8.41–8.40 (d, J = 7.7 Hz, 2H, Ar-H), 7.809–7.802 (d, J = 7.8 Hz, 2H, Ar-H), 3.60 (s, 4H, CH₂), 2.29 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 171.3 (C=O), 144.8 (C=N), 152.6, 143.0, 128.3, 125.3, 120.0, 115.9 (Ar-C), 42.8 (CH₂), 20.5 ppm (CH₃); MS (EI): m/z (%) 437 (M⁺+1, 6.2), 436 (M⁺, 100); Anal. Calcd for C₂₀H₁₆N₆O₆: C, 55.05; H, 3.70; N, 19.26%; Found: C, 54.98; H, 3.74; N, 19.22%.

5.3.16. 3,3′-(1,4-Phenylene)bis(2-methylquinazolin-4(3H)-one) 6a

Yellow crystals, yield 98%, m.p. 178–180°C; IR (KBr): ν/cm⁻¹ 1687 (C=O), 1604, 1598 (C=N, C=C); ¹H NMR (400 MHz, DMSO-d₆): δ 8.30–8.28 (d, J = 7.5 Hz, 2H, Ar-H), 8.09–8.01 (m, 2H, Ar-H), 7.78–7.74 (d, J = 7.6 Hz, 2H, Ar-H), 7.61–7.54 (m, 2H, Ar-H), 7.28 (s, 4H, Ar-H), 2.29 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 170.1 (C=O), 145.2 (C=N), 136.8, 134.3, 130.8, 129.2, 127.3, 126.9, 122.3, 117.2 (Ar-C), 21.2 ppm (CH₃); MS (EI): m/z (%) 395 (M⁺+1, 11.2), 394 (M⁺, 100) [23,24].

5.3.17. 3,3′-(1,4-Phenylene)bis(2,6-dimethylquinazolin-4(3H)-one) 6b

Yellow crystals, yield 97%, m.p. 263–265°C; IR (KBr): ν/cm⁻¹ 1676 (C=O), 1610, 1591 (C=N, C=C); ¹H NMR (400 MHz, DMSO-d₆): δ 7.99 (s, 2H, Ar-H), 7.80–7.79 (d, J = 7.6 Hz, 2H, Ar-H), 7.42–7.39 (m, 2H, Ar-H), 7.23 (s, 4H, Ar-H), 2.63 (s, 6H, CH₃), 2.26 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 170.9 (C=O), 144.5 (C=N), 143.1, 138.3, 133.9, 133.0, 128.1, 126.3, 121.9 (Ar-C), 23.6 (CH₃), 21.3 ppm (CH₃); MS (EI): m/z (%) 423 (M⁺+1, 6.2), 422 (M⁺, 100); Anal. Calcd for C₂₆H₂₂N₄O₂: C, 73.92; H, 5.25; N, 13.26%; Found: C, 73.96; H, 5.22; N, 13.21%.

5.3.18. 3,3′-(1,4-Phenylene)bis(6-chloro-2-methylquinazolin-4(3H)-one) 6c

Yellow crystals, yield 95%, m.p. 287–290°C; IR (KBr): ν/cm⁻¹ 1660 (C = O), 1615, 1597 (C=N, C=C); ¹H NMR (400 MHz, DMSO-d₆): δ 8.09 (s, 2H, Ar-H), 7.55–7.51 (d, J = 7.6 Hz, 2H, Ar-H), 7.46 (s, 4H, Ar-H), 7.12–7.10 (m, 2H, Ar-H), 2.30 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 170.0 (C=O), 144.7 (C=N), 144.5, 134.2, 133.7, 127.7, 127.2, 122.9, 121.7 (Ar-C), 21.5 ppm (CH₃); MS (EI): m/z (%) 464 (M⁺+2, 62.8), 462 (M⁺, 100); Anal. Calcd for C₂₄H₁₆Cl₂N₄O₂: C, 62.22; H, 3.48; N, 12.09%; Found: C, 62.18; H, 3.51; N, 12.03%.

5.3.19. 3,3′-(1,4-Phenylene)bis(6-fluoro-2-methylquinazolin-4(3H)-one) 6d

Yellow crystals, yield 97%, m.p. 269–272°C; IR (KBr): ν/cm⁻¹ 1674 (C=O), 1622, 1599 (C=N, C=C); ¹H NMR (400 MHz, DMSO-d₆): δ 7.98–7.96 (d, J = 7.7 Hz, 2H, Ar-H), 7.66–7.61 (m, 2H, Ar-H), 7.32 (s, 4H, Ar-H), 7.22–7.19 (m, 2H, Ar-H), 2.20 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 170.2 (C=O), 144.0 (C = N), 162.2, 143.3, 134.0, 125.7, 122.7, 121.2, 120.9, 112.9 (Ar-C), 21.8 ppm (CH₃); MS (EI): m/z (%) 431 (M⁺+1, 6.4), 430 (M⁺, 100); Anal. Calcd for C₂₄H₁₆F₂N₄O₂: C, 66.97; H, 3.75; N, 13.02%; Found: C, 66.95; H, 3.78; N, 12.98%.

5.3.20. 3,3′-(1,4-Phenylene)bis(2-methyl-6-nitroquinazolin-4(3H)-one) 6e

Yellow crystals, yield 93%, m.p. 307–310°C; IR (KBr): ν/cm⁻¹ 1683 (C=O), 1614, 1593 (C=N, C=C); ¹H NMR (400 MHz, DMSO-d₆): δ 8.68 (s, 2H, Ar-H), 8.22–8.20 (d, J = 5.5 Hz, 2H, Ar-H), 7.81–7.80 (d, J = 5.5 Hz, 2H, Ar-H), 7.33 (s, 4H, Ar-H), 2.26 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 170.9 (C=O), 144.2 (C = N), 152.2, 144.7, 134.6, 128.5, 124.2, 121.8, 121.5, 119.5 (Ar-C), 21.6 ppm (CH₃); MS (EI): m/z (%) 485 (M⁺+1, 3.1), 484 (M⁺, 100); Anal. Calcd for C₂₄H₁₆N₆O₆: C, 59.51; H, 3.33; N, 17.35%; Found: C, 59.48; H, 3.39; N, 17.31%.

5.3.21. 3,3′-(Sulfonylbis(4,1-phenylene))bis(2-methylquinazolin-4(3H)-one) 7a

Yellow crystals, yield 98%, m.p. 294–297°C; IR (KBr): ν/cm⁻¹ 1689 (C=O), 1615, 1597 (C=N, C=C), 1321 (S=O); ¹H NMR (400 MHz, DMSO-d₆): δ 8.10–8.07 (m, 4H, Ar-H), 7.88–7.83 (m, 4H, Ar-H), 7.58–7.54 (m, 4H, Ar-H), 7.44–7.40 (m, 4H, Ar-H), 2.22 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 170.2 (C=O), 144.8 (C=N), 145.7, 144.6, 138.5, 134.0, 128.5, 128.1, 126.1, 123.5, 121.3 (Ar-C), 21.9 ppm (CH₃); MS (EI): m/z (%) 535 (M⁺+1, 1.3), 534 (M⁺, 100); Anal. Calcd for C₃₀H₂₂N₄O₄S: C, 67.40; H, 4.15; N, 10.48%; Found: C, 67.44; H, 4.12; N, 10.43%.

5.3.22. 3,3′-(Sulfonylbis(4,1-phenylene))bis(2,6-dimethylquinazolin-4(3H)-one) 7b

Light yellow crystals, yield 98%, m.p. 310–313°C; IR (KBr): ν/cm⁻¹ 1680 (C=O), 1622, 1611 (C=N, C=C), 1308 (S=O); ¹H NMR (400 MHz, DMSO-d₆): δ 7.92–7.90 (m, 2H, Ar-H), 7.72 (s, 2H, Ar-H), 7.60–7.52 (m, 2H, Ar-H), 7.42–7.39 (d, J = 7.5 Hz, 4H, Ar-H), 7.306–7.301 (m, 4H, Ar-H), 2.40 (s, 6H, CH₃), 2.21 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 170.5 (C=O), 144.2 (C=N), 144.0, 143.7, 137.5, 133.7, 128.2, 128.0, 127.8, 126.2, 123.1, 121.8 (Ar-C), 23.3 (CH₃), 21.6 ppm (CH₃); MS (EI): m/z (%) 563 (M⁺+1, 1.9), 562 (M⁺, 100); Anal. Calcd for C₃₂H₂₆N₄O₄S: C, 68.31; H, 4.66; N, 9.96%; Found: C, 68.27; H, 4.69; N, 9.91%.

5.3.23. 3,3′-(Sulfonylbis(4,1-phenylene))bis(6-chloro-2-methylquinazolin-4(3H)-one) 7c

Yellow crystals, yield 97%, m.p. 332–335°C; IR (KBr): ν/cm⁻¹ 1689 (C=O), 1629, 1607 (C=N, C=C), 1327 (S=O); ¹H NMR (400 MHz, DMSO-d₆): δ 8.36 (s, 2H, Ar-H), 7.92–7.90 (m, 2H, Ar-H), 7.71–7.62 (m, 2H, Ar-H), 7.47–7.41 (d, J = 7.8 Hz, 4H, Ar-H), 7.39–7.32 (m, 4H, Ar-H), 2.29 (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 171.1 (C=O), 144.0 (C=N), 144.2, 143.9, 138.1, 133.9, 132.8, 128.5, 127.8, 122.8, 121.6 (Ar-C), 21.9 ppm (CH₃); MS (EI): m/z (%) 604 (M⁺+2, 62.9), 602 (M⁺, 100); Anal. Calcd for C₃₀H₂₀Cl₂N₄O₄S: C, 59.71; H, 3.34; N, 9.28%; Found: C, 59.74; H, 3.32; N, 9.25%.

5.3.24. 3,3′-(Sulfonylbis(4,1-phenylene))bis(6-fluoro-2-methylquinazolin-4(3H)-one) 7d

Yellow crystals, yield 93%, m.p. 301–303°C; IR (KBr): ν/cm⁻¹ 1692 (C=O), 1621, 1602 (C=N, C=C), 1312 (S=O); ¹H NMR (400 MHz, DMSO-d₆): δ 7.82–7.80 (m, 2H, Ar-H), 7.69–7.59 (m, 2H, Ar-H), 7.36–7.30 (m, 4H, Ar-H), 7.16–7.15 (m, 2H, Ar-H), 7.14–7.08 (m, 4H, Ar-H), 2.25 (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 171.1 (C=O), 144.0 (C=N), 161.9, 143.6, 142.7, 138.0, 128.9, 125.6, 122.7, 120.8, 112.9 (Ar-C), 21.8 ppm (CH₃); MS (EI): m/z (%) 571 (M⁺+1, 2.5), 570 (M⁺, 100); Anal. Calcd for C₃₀H₂₀F₂N₄O₄S: C, 63.15; H, 3.53; N, 9.82%; Found: C, 63.11; H, 3.57; N, 9.78%.

5.3.25. 3,3′-(Sulfonylbis(4,1-phenylene))bis(2-methyl-6-nitroquinazolin-4(3H)-one) 7e

Yellow crystals, yield 95%, m.p. 316–319°C; IR (KBr): ν/cm⁻¹ 1688 (C=O), 1601, 1589 (C=N, C=C), 1318 (S=O); ¹H NMR (400 MHz, DMSO-d₆): δ 8.66 (s, 2H, Ar-H), 8.31–8.29 (m, 2H, Ar-H), 8.00–7.98 (m, 4H, Ar-H), 7.66–7.65 (d, J = 5.5 Hz, 4H, Ar-H), 7.45–7.40 (m, 2H, Ar-H), 2.21 (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 171.1 (C=O), 144.2 (C=N), 152.9, 143.2, 137.3, 128.6, 125.0, 122.4, 122.2, 120.6, 118.5 (Ar-C), 21.8 ppm (CH₃); MS (EI): m/z (%) 625 (M⁺+1, 0.9), 624 (M⁺, 100); Anal. Calcd for C₃₀H₂₀N₆O₈S: C, 57.69; H, 3.23; N, 13.46%; Found: C, 57.72; H, 3.20; N, 13.41%.

5.3.26. 3-(2-Aminophenyl)-2-methylquinazolin-4(3H)-one 8

White crystals, yield 33%, m.p. 109–111°C; IR (KBr): ν/cm⁻¹ 3324–2984 (NH₂), 1694 (C=O), 1611, 1594 (C=N, C=C); ¹H NMR (400 MHz, DMSO-d₆): δ 8.208–8.205 (d, J = 5.7 Hz, 1H, Ar-H), 7.93–7.91 (d, J = 7.8 Hz, 1H, Ar-H), 7.73–7.70 (d, J = 5.7 Hz, 1H, Ar-H), 7.66–7.63 (m, 2H, Ar-H), 7.48–7.44 (m, 2H, Ar-H), 7.28–7.24 (m, 1H, Ar-H), 6.50 (br, 2H, NH₂), 2.29 ppm (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 171.1 (C=O), 154.3 (C=N), 141.7, 137.8, 135.0, 130.9, 130.1, 128.9, 127.7, 127.4, 126.7, 121.9, 117.4, 115.0 (Ar-C), 21.0 ppm (CH₃); MS (EI): m/z (%) 252 (M⁺+1, 100), 251 (M⁺, 2.6) [23,24].

5.3.27. 3,3′-(1,2-Phenylene)bis(2-methylquinazolin-4(3H)-one) 9

Yellow crystals, yield 88%, m.p. 187–190°C; IR (KBr): ν/cm⁻¹ 1685 (C=O), 1621, 1588 (C=N, C=C); ¹H NMR (400 MHz, DMSO-d₆): δ 8.11–8.08 (d, J = 5.7 Hz, 2H, Ar-H), 7.90–7.88 (d, J = 7.7 Hz, 2H, Ar-H), 7.72–7.68 (m, 4H, Ar-H), 7.33–7.30 (d, J = 9.8 Hz, 4H, Ar-H), 2.24 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 171.7 (C=O), 148.9 (C=N), 142.7, 133.9, 133.7, 127.0, 126.9, 126.6, 123.4, 121.2, 115.7 (Ar-C), 24.1 ppm CH₃); MS (EI): m/z (%) 395 (M⁺+1, 7.9), 394 (M⁺, 100) [23,24].

5.3.28. 3-(3-Aminophenyl)-2-methylquinazolin-4(3H)-one 10

White crystals, yield 33%, m.p. 121–123°C; IR (KBr): ν/cm⁻¹ 3365–2990 (NH₂), 1687 (C=O), 1609, 1587 (C=N, C=C); ¹H NMR (400 MHz, DMSO-d₆): δ 8.18–8.15 (d, J = 7.5 Hz, 1H, Ar-H), 7.86–7.81 (t, J = 7.8 Hz, 1H, Ar-H), 7.76–7.70 (m, 2H, Ar-H), 7.36–7.31 (m, 1H, Ar-H), 6.98–6.94 (m, 2H, Ar-H), 6.78–6.74 (m, 1H, Ar-H), 2.26 ppm (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 170.4 (C=O), 154.0 (C=N), 145.7, 144.8, 133.2, 129.9, 128.0, 127.5, 126.6, 122.4, 120.0, 118.9, 115.4, 111.6 (Ar-C), 21.4 ppm (CH₃); MS (EI): m/z (%) 252 (M⁺+1, 17.0), 251 (M⁺, 100) [51].

5.3.29. 3,3′-(1,3-Phenylene)bis(2-methylquinazolin-4(3H)-one) 11

Light yellow crystals, yield 92%, m.p. 236–238°C; IR (KBr): ν/cm⁻¹ 1679 (C=O), 1616, 1607 (C=N, C=C); ¹H NMR (400 MHz, DMSO-d₆): δ 7.92–7.90 (m, 2H, Ar-H), 7.72 (s, 1H, Ar-H), 7.34–7.28 (m, 4H, Ar-H), 7.10–7.08 (d, J = 7.5 Hz, 2H, Ar-H), 7.03–6.99 (m, 3H, Ar-H), 2.30 ppm (s, 6H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 171.2 (C=O), 154.3 (C=N), 144.6, 134.4, 133.4, 129.6, 127.8, 127.4, 126.1, 122.4, 121.7, 114.1 (Ar-C), 21.7 ppm (CH₃); MS (EI): m/z (%) 395 (M⁺+1, 22.5), 394 (M⁺, 100); Anal. Calcd for C₂₄H₁₈N₄O₂: C, 73.08; H, 4.60; N, 14.20%; Found: C, 73.11; H, 4.58; N, 14.19%.

5.4. In vitro anti-cancer screening

The cytotoxic activities of all newly prepared derivatives were assessed in monolayer cultures by utilizing MTT assay. Two human cancer cell lines (MCF-7 and A549) and one normal cell line (MCF-10A), were maintained in the minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2 ml glutamine and 100 units ml⁻¹ penicillin in a CO₂ incubator in a humidified atmosphere of 5% CO₂ and 95% air. The tested derivatives were prepared prior to the experiment by dissolving in 0.1% DMSO and diluted with medium. The cells were then exposed to two concentrations of drugs (10 and 30 µM) in the volume of 100 µM/well. Cells in the control wells received the same volume of medium comprising 0.1% DMSO. After 24 h, the medium was removed and cell cultures were incubated with 100 ml MTT reagent for 5 h at 37°C. The known number of cells was incubated in a 5% CO₂ incubator at 37°C in the presence of different concentrations of test compounds. After 48 h of drug incubation, the MTT solution was added in each well and the absorbance was measured by using a microplate reader at 490 nm. The experiment was performed in triplicate. Cell survival was calculated as the percentage of MTT inhibition as % growth inhibition = 100 − (mean OD of individual test group/mean OD of each control group) × 100. The IC₅₀ values of the synthesized compounds for two cell lines are summarized in electronic supplementary material, S59, table S3).

Data accessibility

The datasets supporting this article have been uploaded as part of the electronic supplementary material.

Competing interests

I declare I have no competing interests.

Funding

This study was financially supported by the Jouf University, project no. 634/39.