Introduction

In recent years, the exploration of novel synthetic compounds with antioxidant properties has become an active area of research. Free radicals and oxygen derivatives are constantly generated in vivo by specific metabolic processes [1]. These radicals can easily react with most biological molecules including proteins, lipids, lipoproteins, DNA and damage them. The damage of cells caused by free radicals might be responsible for the production of a wide range of human diseases such as arthritis, haemorrhagic shock, coronary artery diseases, cataract, cancer, AIDS as well as age-related degenerative brain diseases [2]. The production of these free radicals could be prevented by the addition of free radical scavengers like antioxidants. In this regard, there are some natural antioxidants such as beta-carotene, lycopene, vitamins A, C and E that are used to capture free radicals and neutralize them. Due to the expensive and lengthy isolation processes of the natural antioxidants from the various natural sources and their unprecise activity against many diseases, synthetic antioxidants play an important role in the treatment and prevention of major diseases that are associated with oxidative stress. Initially, various polyphenolic and thiol compounds were used as free radical scavengers [3, 4]. Recently, a variety of heterocyclic compounds such as quinazolinone, oxadiazole, thiadiazole, coumarin, pyrazole and pyrimidines also serve as antioxidants [5,6,7,8,9]. Thus, there is a constant need for identifying new and effective therapeutic agents.

Scheme 1
scheme 1

Synthesis of bis(2-phenyl-2,3-dihydroquinazolin-4(1H)-one) analogues

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2,3-Dihydroquinazolinone and its derivatives are a class of nitrogen containing heterocyclic compounds that exhibit a variety of pharmacological properties such as antimicrobial, antitumor, antibiotic, antipyretic, analgesic, antihypertonic, diuretic, antihistamine, antidepressant and vasodilating activities [10,11,12,13,14,15,16]. In addition to that, they comprise potent tubulin inhibitors with remarkable anti-proliferative activity against a variety of human cancer cells [17] and act as antimitotic agents [18]. Moreover, these 2,3-dihydroquinazolinone derivatives could be easily oxidized to produce quinazolin-4(3H)-one analogues [19] which are promising bioactive heterocyclic compounds [20,21,22] and are present in some natural products [23]. There are numerous reports available in the literature for the synthesis of mono 2,3-dihydroquinazolinone derivatives. Structurally complex quinazolinone-based natural product precursors have also been constructed indirectly via thioamide formation [24], oxidation of dehydroquinazolinone [25], aza-Wittig condensations [26] or from benzoxazinones [27]. However, only a few reports are available for the synthesis of bisquinazolinone [28,29,30,31,32] derivatives and, to the best of our knowledge, this is the first report for the biological and electrochemical properties of these compounds. Considering this limited background, we planned the synthesis of new 3,3\(^\prime \)-bis(dihydroquinazolin-4(1H)-one) and 2,2\(^\prime \)-bis(dihydroquinazolin-4(1H)-one) derivatives to investigate their DPPH radical scavenging and electrochemical redox properties.

Results and discussion

Chemistry

In continuation of our effort to develop novel heterocyclic compounds [33,34,35], herein we present a simple and efficient synthesis of novel bis(2-phenyl-2,3-dihydroquinazolin-4(1H)-one) derivatives from the one-pot five-component reaction of isatoic anhydride with aromatic aldehydes and p-phenylenediamine under refluxing conditions in glacial acetic acid (Scheme 1).

In order to optimize the reaction conditions, initially this cyclocondensation reaction was conducted in different solvents including ethanol, methanol, PEG-300, ethylene glycol, diethylene glycol, acetonitrile, DMF and dioxane in the presence of 30 mol% p-TSA as a catalyst (Table 1). When the reaction was refluxed in polar protic solvents such as ethanol and methanol, the expected compound 4c was not formed even after 10h (Table 1, Entry 1 and 2) and only imine 5 were observed. For the other solvents, moderate yields of compound 4c was observed (Table 1, Entry 3-5) except when using acetonitrile and 1,4-dioxane (Table 1, Entry 6 and 8). When glacial acetic acid was used, 4c was formed in excellent yields (Table 1, Entry 9). Furthermore, we have performed a control experiment by using glacial acetic acid as the reaction medium without the use of p-TSA as catalyst where 4c was also obtained in excellent yield (Table 1, Entry 10). This prompted us to use acetic acid as reaction medium for further reactions.

Table 1 Optimization of reaction conditions
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Table 2 Synthesis of 3,3\(^\prime \)-(1,4-phenylene)bis(2-phenyl-2,3-dihydroquinazolin-4(1H)-one) derivatives
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Scheme 2
scheme 2

Proposed mechanism for the formation of products 4(a–i)

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Table 3 Synthesis of 3,3\(^\prime \)-bisquinazolinone derivatives
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Scheme 3
scheme 3

Synthesis of 2,2\(^\prime \)-bisquinazolinone derivatives

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Table 4 Synthesis of 2,2\(^\prime \)-(1,4-phenylene)bis(3-phenyl-2,3-dihydroquinazolin-4(1H)-ones)
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Next, we explored the scope and generality of the reaction with respect to aromatic aldehydes 2(a–i) using the first reaction conditions (without p-TSA in glacial acetic acid). Aromatic aldehydes having both electron-donating and electron-withdrawing substituents are well tolerated, affording the corresponding compounds 4(a–i) in good to excellent yields (Table 2). From the Table 2, we observed that aldehydes having electron-withdrawing groups produced the corresponding products in higher yields than those aldehydes containing electron-donating groups. When an electron-withdrawing group was at the ortho-position of the aromatic aldehydes, longer reaction times were required to obtain the desired products; however, these were obtained in lower yields than those with groups present in either m- or p-positions. The structures of the products were confirmed by IR, \(^{1}\hbox {H}\) NMR, \(^{13}\hbox {C}\) NMR and LC-MS spectroscopic techniques.

The formation of compounds 4(a–i) can be explained by the proposed mechanism shown in Scheme 2. The carbonyl group of isatoic anhydride (1) could be protonated to give the intermediate (I), which might facilitate the nucleophilic attack of p-phenylenediamine (3) on the carbonyl group. Nucleophilic addition of p-phenylenediamine (3) to the intermediate (I) followed by decarboxylation produces 2-aminobenzamide (II). Condensation of II with protonated aldehyde (2) gives imine (III), which undergoes intramolecular cyclization to afford the final product 4(a–i).

In addition, we have also applied the same protocol for the synthesis of 3,3\(^\prime \)-bisquinazolinone derivatives (Table 3, Entries 1-4) using o-phenylenediamine, 1,5-diaminonaphthalene, 4,4’-diaminodiphenyl ether and 4,4’-diaminodiphenyl methane with isatoic anhydride and p-chlorobenzaldehyde and their results are shown in Table 3.

We have also tried to synthesize new 2,2\(^\prime \)-(1,4-phenylene)bis(3-phenyl-2,3-dihydroquinazolin-4(1H)-one) derivatives from the one-pot five-component reaction of isatoic anhydride and aromatic amine with terephthalaldehyde by refluxing the mixture in glacial acetic acid. It was observed that aromatic amines having electron-donating groups afforded products in higher yields faster than the electron-withdrawing counterparts (Scheme 3, Table 4).

Antioxidant study

We studied the antioxidant potential of the synthesized bis(2,3-dihydroquinazolin-4(1H)-one) compounds 4(a–i) and 8(a–k) by using the DPPH radical scavenging assay (RSA) [36, 37]. The radical scavenging activities of the test compounds were assessed by how the tested compounds are capable of stabilizing the DPPH radical, and the percentage of inhibition and 50% inhibitory concentration values are presented in Tables 5 and 6 and compared with those of the standards BHA and ascorbic acid. A stock solution of DPPH free radical in methanol (7.1 mg/300 mL) was prepared, and the absorbance was recorded at 517 nm. Then, a stock solution of the sample was prepared by dilution method to get solutions with 20, 40, 60, 80 100 \(\upmu \hbox {g}/\hbox {mL}\). The test solutions at different concentrations (1 mL) were mixed with the DPPH stock solution (2 mL) and were incubated in the dark for 30 min. After 30 min, the absorbance was measured at 517 nm. From these absorbance values, the percentage antioxidant activities were calculated according to the following equation,

$$\begin{aligned} \hbox {Radical scavenging activity}\,(\%)=[(\hbox {A}_\mathrm{c}-\hbox {A}_\mathrm{s})/\hbox {A}_\mathrm{c}] \times 100 \end{aligned}$$

where \(\hbox {A}_{\mathrm{c}}=\hbox {absorbance of DPPH }, \hbox {A}_{\mathrm{s}}=\hbox {absorbance of} \text { test sample}\).

Table 5 Antioxidant activities of synthesized compounds 4(a–i)
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The DPPH radical scavenging activity of the test compounds was found to be good to moderate when compared with the standards BHA and ascorbic acid. Initially, we chose compound 4a as our baseline for antioxidant activity and observed that the antioxidant activity remained within the range of 57–60% with increasing concentration. So, the antioxidant activities of the remaining compounds were evaluated at \(20\,\upmu \hbox {g}/\hbox {mL}\), and the results are presented in Table 5.

It was observed that all the compounds reduced the concentration of DPPH free radical and exhibited their antioxidant activities in the range of 57–71%. Compounds having electron-withdrawing groups showed 58–61% activities (Table 5, Entries 5, 6, 7 and 8), whereas compound possessing electron-donating groups showed a maximum (71%) antioxidant ability (Table 5, Entry 9). It is clear from Table 5 that compound 4i is the best antioxidant out of the analogues made.

The dihydroquinazolinone derivatives derived from terephthalaldehyde 10(a–k) also showed good antioxidant activities (Table 6). As evidenced from the data presented in Table 6, compound 10h was found to be the best antioxidant among the analogues studied (Table 6, Entry 8) which may be attributed to the –OH groups on the phenyl ring as they are known to scavenge DPPH.

Table 6 Antioxidant activities of synthesized compounds 10(a–k)
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Table 7 Cytotoxicity of compound 4f
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Fig. 1
figure 1

a Cytotoxicity on Vero cell lines, b cytotoxicity on MCF-7 cell lines

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Fig. 2
figure 2

Cyclic voltammogram of compound 4c

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Table 8 Formal redox potential of compounds 4c, 7(a–d), 10d and 11
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Scheme 4
scheme 4

Proposed electrochemical redox mechanism of compounds 4c, 7(a–d), 10d and11

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Anticancer activity

The in vitro anticancer activity of 4f was determined against both normal Vero cell line as well as human breast cancer cell line (MCF-7). The cell viability in the presence of test sample was measured by using the MTT assay and is presented in Table 7. The basic principle of this colorimetric assay is the reduction of water soluble yellow 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to the water insoluble purple-coloured formazan by mitochondrial succinate dehydrogenase. The resulting intracellular purple formazan could be solubilized and measured by spectrophotometric means [38]. The treatment of MCF-7 cells with 4f at different concentrations (0–200\(\,\upmu \hbox {M})\) for 24 h resulted in the decrease in number of MCF-7 cells corresponding to the concentration of the tested compound 4f (Fig. 1b). In contrast, cytotoxic effect of normal Vero cell was poorly affected by 4f (Fig. 1a). The cell viability of this compound was calculated from the percentage of viable 4f-treated cells and with untreated cells. Then we obtained the 50% of growth inhibition values \((\hbox {GI}_{50})\).

These results show that compound 4f possesses good growth inhibition towards human breast cancer cell (MCF-7). However, in the case of normal Vero cells, exposure to 4f at \(200\,\upmu \hbox {M}\) for 24 h only led to 17% of viable cells with maximum \(\hbox {GI}_{50}\) value \((84\,\upmu \hbox {M})\), which indicated that compound 4f is toxic only to cancer cells and not to normal cells. The \(\hbox {GI}_{50}\) value for compound 4f was found to be \(11.4\,\upmu \hbox {M}\), whereas in normal Vero cell it was \(84\,\upmu \hbox {M}\), indicating that compound 4f has anticancer activity.

Cyclic voltammetric studies

Finally, the synthesized bisquinazolinones 4c, 7(a–d) and 10d were screened for their redox behaviour using the cyclic voltammetric technique and their results are compared to 2,3-bis(4-chlorophenyl)-2,3-dihydroquinazolin-4(1H)-one 11. The representative cyclic voltammogram of compound 4c is shown in Fig. 2 and the major oxidation and reduction potential of the studied compounds are presented in Table 8.

It is clear from Fig. 2, the cyclic voltammogram of the studied compounds show three irreversible anodic signals and one cathodic signal during the reverse scan. In the cyclic voltammogram of compound 4c, the first anodic signal at −0.512 V may be due to the oxidation of secondary -NH group of quinazolinone ring. In addition, two more signals observed at more positive potentials (−0.063 V and +0.492 V) may be attributed to the oxidation of aromatic ring of quinazoline. In the reverse scan, we obtained a signal at −0.782 V that could be accounted for the reduction of the carbonyl group of the quinazolinone ring. Among the studied compounds, compound 7d was easily electrochemically oxidized (Table 8, Entry 5). However, all the compounds showed higher oxidation potential than that of monoquinazolinone 11 (Table 8, Entry 7). The relative ease of oxidation was expressed based on their first oxidation potential and the order is 7d > 4c > 10d > 7b > 7c > 7a > 11.

Based on these results, we propose a possible electrochemical mechanism as shown in Scheme 4.

Conclusions

In conclusion, bis(2,3-dihydroquinazolin-4(1H)-one derivatives have been successfully synthesized and characterized by IR, NMR and mass spectroscopic techniques. The antioxidant properties of all the synthesized compounds were evaluated using the DPPH radical scavenging method. In the series 4(a–i), compound 4i showed the highest \((\hbox {IC}_{50}=14\,\upmu \hbox {g}/\hbox {mL})\) and compound 4a showed the lowest \((\hbox {IC}_{50}=17.5\,\upmu \hbox {g}/\hbox {mL})\) radical scavenging activities, whereas in the series 10(a–k), compound 10h showed the highest \((\hbox {IC}_{50}=15.5\,\upmu \hbox {g}/\hbox {mL})\) and compound 10i showed the lowest \((\hbox {IC}_{50}=17.5\,\upmu \hbox {g}/\hbox {mL})\) radical scavenging activities. In addition to that, we have studied the anticancer activity of the synthesized compound 4f against human breast cancer cell line and the observed \(\hbox {GI}_{50}\) value was \(11.4\,\upmu \hbox {M}\).

Experimental

General remarks

All the reagents used for this study are commercially available and were freshly used after being purified by standard procedures. Reactions were monitored by TLC using silica gel-coated plates and chloroform/methanol (9:1) mixture as the mobile phase. Melting points are uncorrected and were measured using an electrothermal apparatus. The IR spectra (neat) were recorded on a Nicolet 6700 FT-IR spectrometer. NMR spectra were obtained on an FT-NMR Bruker Spectro Spin DRX-500 and 400 MHz instrument as DMSO solution, and the chemical shifts are expressed as \(\delta \) units with Me4Si as the internal standard. Multiplicities are abbreviated as follows: singlet (s), doublet (d), triplet (t), multiplet (m) and doublet of doublet (dd). The mass spectra were recorded on an LC-MS-Agilent 1100 series with MSD (Ion trap) using 0.1% aqueous TFA in acetonitrile system on C18-BDS column for 10 min duration.

General procedure for the synthesis of 3,3\(^\prime \)-bis(2-phenyl-2,3-dihydroquinazolin-4(1H)-one) derivatives 4(a–i)

A mixture of p-phenylenediamine (1 equiv), aromatic aldehydes (2 equiv) and isatoic anhydride (2 equiv) was refluxed in glacial acetic acid until the completion of the reaction as indicated by TLC. After completion, the reaction mixture was cooled to room temperature. Then, the solid was separated, washed with hot a methanol and chloroform mixture (1:1) and dried.

General procedure for the synthesis of 2,2\(^\prime \)-bis(3-phenyl-2,3-dihydroquinazolin-4(1H)-one) derivatives 10(a–k)

A mixture of terephthalaldehyde (1 equiv), aromatic amine (2 equiv) and isatoic anhydride (2 equiv) was refluxed in glacial acetic acid until the completion of the reaction as indicated by TLC. After completion, the reaction mixture was cooled to room temperature, the solid separated, washed with 1:1 mixture of hot methanol and chloroform and dried.

Cyclic voltammetric studies

Cyclic voltammetric experiments were carried out using a CHI 760C electrochemical workstation with a three-electrode system (platinum counter electrode, glassy carbon working electrode and Ag/AgCl reference electrode) at a scan rate of 50 \(\hbox {mVs}^{-1}\) in the presence of tetrabutylammonium perchlorate as a supporting electrolyte in DMSO.

Supplementary data

Spectral data, copies of \(^{1}\hbox {H}\), \(^{13}\hbox {C}\) NMR, mass spectra of novel compounds and cyclic voltammogram of tested compounds are given as a separate supplementary file.