Synthesis, Antiproliferative, and Antioxidant Evaluation of 2-Pentylquinazolin-4(3H)-one(thione) Derivatives with DFT Study

The current study was chiefly designed to examine the antiproliferative and antioxidant activities of some novel quinazolinone(thione) derivatives 6–14. The present work focused on two main points; firstly, comparing between quinazolinone and quinazolinthione derivatives. Whereas, antiproliferative (against two cell lines namely, HepG2 and MCF-7) and antioxidant (by two methods; ABTS and DPPH) activities of the investigated compounds, the best quinazolinthione derivatives were 6 and 14, which exhibited excellent potencies comparable to quinazolinone derivatives 5 and 9, respectively. Secondly, we compared the activity of four series of Schiff bases which included the quinazolinone moiety (11a–d). In addition, the antiproliferative and antioxidant activities of the compounds with various aryl aldehyde hydrazone derivatives (11a–d) analogs were studied. The compounds exhibited potency that increased with increasing electron donating group in p-position (OH > OMe > Cl) due to extended conjugated systems. Noteworthy, most of antiproliferative and antioxidant activities results for the tested compounds are consistent with the DFT calculations.


Introduction
Cancer is the second leading cause of death globally, and the contribution of cancer disease to the overall mortality rate is increasing. Economically, the total annual cost of cancer in 2010 was estimated at approximately US$ 1.16 trillion [1]. So that, more rational design, synthesis, and evaluation of new compounds as anticancer, with higher efficiency is considered as urgent mission in the medicinal chemistry field.
Afterwards, sulfuration of 2-pentylquinazolin-4(3H)-one 5 by utilizing of phosphorus pentasulfide in dry toluene afforded 2-pentylquinazoline-4(3H)-thione 6 (Scheme 2). The formation of compound 6 was unambiguously elaborated by the presence of intense band at 1236 cm −1 corresponding to υc=s and the absence of the stretching band of υc=o in the IR spectrum. On the other hand, the incorporation of β-d-glucose pentaacetate with quinazolinone derivative 5 at the nitrogen atom of the later awarded N-(β-d-glucopyranosyl-2,3,4,6-tetraacetate)-2-pentyl quinazolin-4(3H)-one 7 (Scheme 2), via attacking of the lone pair of nitrogen atom of quinazolinone derivative 5 at the anomeric carbon (C 1 ) of β-d-glucose pentaacetate, followed by ring opening and then ring closure with expulsion of acetate as a leaving group.
The chemical structure of compound 7 was explained by the IR spectrum, whereas it showed a band at 1746 cm −1 compatible with υ C=O of the acetate groups and lacked the absorption band for the NH group. Moreover, this structure was also interpreted by the 1 H-NMR spectrum which revealed seven signals at 5.92-3.51 ppm and four singlet signals at 1.97-1.91 ppm all of them corresponding to the protons of β-d-glucopyranosyl-2,3,4,6-tetraacetate moiety.
Curing of the benzoxazinone derivative 4 with ethanolamine under reflux for 3 h afforded 3-(2-hydroxyethyl)-2-pentylquinazolin-4(3H)-one 8 as the sole product. The IR spectrum of compound 8 showed a broad band at 3395 cm −1 corresponding to OH functionality. Furthermore, the 1 H-NMR spectrum appreciably emerged a triplet peak at 4.95 ppm exchangeable with D 2 O corresponding to OH proton, triplet, and quartet peaks at 4.11 and 3.65 ppm, respectively, compatible with ethyl protons of 2-hydroxyethyl moiety. As well, the 13 C-NMR spectrum exhibited two peaks at 58.8 and 46.1 ppm corresponding to the two carbons of 2-hydroxyethyl moiety.
3-Amino-2-pentylquinazolin-4(3H)-one 9 was commenced by refluxing of compound 4 with hydrazine monohydrate in absolute ethanol for 4 h (Scheme 2). The formation of compound 9 was confirmed by spectroscopic and elemental data. In particular, the 1 H-NMR spectrum of compound 9 manifested a singlet signal commutable in D 2 O at 5.70 ppm corresponding to NH 2 protons.
Molecules 2019, 24 3 Benzoxazinone derivative 4 was utilized in situ as a precursor to construct new quinazolinone derivatives. For instance, reaction of benzoxazinone derivative 4 with formamide afforded 2-pentylquinazolin-4(3H)-one 5 [41] (Scheme 2). The 1 H NMR spectrum of 5 exhibited a singlet peak at 12.13 ppm exchangeable with D2O corresponding to NH proton, two doublet and two triplet peaks in the aromatic region at 8.05-7.42 ppm corresponding to four aromatic protons, and four characteristic peaks upfield at 2.56-0.84 ppm for n-pentyl protons.
Afterwards, sulfuration of 2-pentylquinazolin-4(3H)-one 5 by utilizing of phosphorus pentasulfide in dry toluene afforded 2-pentylquinazoline-4(3H)-thione 6 (Scheme 2). The formation of compound 6 was unambiguously elaborated by the presence of intense band at 1236 cm −1 corresponding to υc=s and the absence of the stretching band of υc=o in the IR spectrum. On the other hand, the incorporation of β-D-glucose pentaacetate with quinazolinone derivative 5 at the nitrogen atom of the later awarded N-(β-D-glucopyranosyl-2,3,4,6-tetraacetate)-2-pentyl quinazolin-4(3H)-one 7 (Scheme 2), via attacking of the lone pair of nitrogen atom of quinazolinone derivative 5 at the anomeric carbon (C1) of β-D-glucose pentaacetate, followed by ring opening and then ring closure with expulsion of acetate as a leaving group.
The chemical structure of compound 7 was explained by the IR spectrum, whereas it showed a band at 1746 cm −1 compatible with υC=O of the acetate groups and lacked the absorption band for the NH group. Moreover, this structure was also interpreted by the 1 H-NMR spectrum which revealed seven signals at 5.92-3.51 ppm and four singlet signals at 1.97-1.91 ppm all of them corresponding to the protons of β-D-glucopyranosyl-2,3,4,6-tetraacetate moiety.
Curing of the benzoxazinone derivative 4 with ethanolamine under reflux for 3 h afforded 3-(2-hydroxyethyl)-2-pentylquinazolin-4(3H)-one 8 as the sole product. The IR spectrum of compound 8 showed a broad band at 3395 cm −1 corresponding to OH functionality. Furthermore, the 1 H-NMR spectrum appreciably emerged a triplet peak at 4.95 ppm exchangeable with D2O corresponding to OH proton, triplet, and quartet peaks at 4.11 and 3.65 ppm, respectively, compatible with ethyl protons of 2-hydroxyethyl moiety. As well, the 13 C-NMR spectrum exhibited two peaks at 58.8 and 46.1 ppm corresponding to the two carbons of 2-hydroxyethyl moiety.
3-Amino-2-pentylquinazolin-4(3H)-one 9 was commenced by refluxing of compound 4 with hydrazine monohydrate in absolute ethanol for 4 h (Scheme 2). The formation of compound 9 was confirmed by spectroscopic and elemental data. In particular, the 1 H-NMR spectrum of compound 9 manifested a singlet signal commutable in D2O at 5.70 ppm corresponding to NH2 protons. Scheme 2. Synthetic route to compounds 5-9.
Reaction of 3-amino-2-pentylquinazolin-4(3H)-one 9 with various aldehydes 10a-d gave Schiff bases 11a-d as the sole product in each case (Scheme 3). The 1 H-NMR spectra of compounds 11a-d Reaction of 3-amino-2-pentylquinazolin-4(3H)-one 9 with various aldehydes 10a-d gave Schiff bases 11a-d as the sole product in each case (Scheme 3). The 1 H-NMR spectra of compounds 11a-d exhibited the appearance of a singlet signal in the region between 8.81-8.69 ppm compatible with methine proton of N=CH group.
The thiazolidin-4-one moiety 12 was constructed by the reaction of Schiff base 11a with methyl thioglycolate in absolute ethanol including a small amount of piperidine as a catalyst for 3 h (Scheme 3). The prospective structure 12 is in keeping with its spectral and elemental analyses.
Additionally, the nucleophilicity of the amino group of compound 9 was also estimated by fusion of it with 4,5,6,7-tetrachloroisobenzofuran-1,3-dione in oil bath for an hour and that afforded phthalimido derivative 13 in an excellent yield (Scheme 3). The foreseeable structure of compound 13 was elucidated by their spectral data and elemental analysis. Obviously, its IR spectrum showed stretching absorption bands at 1788, and 1746 cm −1 corresponding to the carbonyl groups of phthalimido moiety and at 1707 cm −1 corresponding to carbonyl group of the quinazolinone moiety. The 1 H-NMR spectrum exhibited four peaks for four aromatic protons and another four peaks for n-pentyl protons. Furthermore, its 13 C-NMR spectrum emerged variant peaks, all of them fit with the proposed structure.
Eventually, the thione derivative 14 was obtained via sulfuration of compound 9 by utilizing phosphorus pentasulfide as the above pervious method (Scheme 3). The structure of 14 was unequivocally explained via the existence of a peak in the 13 C NMR spectrum at 182.1 ppm compatible with the carbon of the thione functional group.
Molecules 2019, 24 4 exhibited the appearance of a singlet signal in the region between 8.81-8.69 ppm compatible with methine proton of N=CH group. The thiazolidin-4-one moiety 12 was constructed by the reaction of Schiff base 11a with methyl thioglycolate in absolute ethanol including a small amount of piperidine as a catalyst for 3 h (Scheme 3). The prospective structure 12 is in keeping with its spectral and elemental analyses.
Additionally, the nucleophilicity of the amino group of compound 9 was also estimated by fusion of it with 4,5,6,7-tetrachloroisobenzofuran-1,3-dione in oil bath for an hour and that afforded phthalimido derivative 13 in an excellent yield (Scheme 3). The foreseeable structure of compound 13 was elucidated by their spectral data and elemental analysis. Obviously, its IR spectrum showed stretching absorption bands at 1788, and 1746 cm −1 corresponding to the carbonyl groups of phthalimido moiety and at 1707 cm −1 corresponding to carbonyl group of the quinazolinone moiety. The 1 H-NMR spectrum exhibited four peaks for four aromatic protons and another four peaks for n-pentyl protons. Furthermore, its 13 C-NMR spectrum emerged variant peaks, all of them fit with the proposed structure.
Eventually, the thione derivative 14 was obtained via sulfuration of compound 9 by utilizing phosphorus pentasulfide as the above pervious method (Scheme 3). The structure of 14 was unequivocally explained via the existence of a peak in the 13 C NMR spectrum at 182.1 ppm compatible with the carbon of the thione functional group.
The results listed in Table 1 and illustrated in Figure 3, demonstrate that compounds 6 and 11d have a very strong efficacy against HePG2 cell line with IC 50       Structure Activity Relationship (SAR) By comparing the antiproliferative efficacy of the thirteen synthesized compounds in this study to their chemical structures, it was concluded that the following structure activity relationship's (SAR's) is hypothesized: 1. Conversion of quinazolin-4(3H)-one derivative 5 to quinazolin-4(3H)-thione derivative 6 enhanced the antiproliferative activity against both cell lines from moderate activity to very strong activity.
4. Construction of the thiazolidinone ring in compound 12 decreased the antiproliferative activity comparable with the hydrazone derivative 11a, due to decreasing of the delocalization of n-π electrons after replacement of the C=N group (electron attracting group) by the thiazolidinone ring.

Antioxidant Activity Screening
One of the aims of this work is the screening of all synthesized compounds for antioxidant activity using two different methods, namely ABTS [2,2′-azino-bis(3-ethyl benzothiazoline-6-sulfonic acid)] and DPPH assays. DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical assay based on electron-transfer that produces a violet solution in ethanol. This free radical is stable at ambient temperature and reduced in the presence of an antioxidant molecule, leading to colorless ethanol solution. After investigation of these results as listed in Table 2 and Figure 6, it was realized that, compounds 6 and 11d have promising activity through using ABTS assay. Meanwhile, in the case of using DPPH assay, compounds 6, 11d and 14 have also very high activity. Ascorbic acid was used as a reference through the antioxidant activity screening.
The results depicted in Table 2 and Figure 6 demonstrated that, DPPH assay findings are very approximately related to those of ABTS assay with only one exception, compound 14 has an excellent antioxidant activity against DPPH (IC50 = 26.87 ± 0.23 μM) than that of the ABTS method (IC50 = 71.42 ± 0.52 μM). Noteworthy, all the screened compounds in the case of the DPPH method Structure Activity Relationship (SAR) By comparing the antiproliferative efficacy of the thirteen synthesized compounds in this study to their chemical structures, it was concluded that the following structure activity relationship's (SAR's) is hypothesized: 1. Conversion of quinazolin-4(3H)-one derivative 5 to quinazolin-4(3H)-thione derivative 6 enhanced the antiproliferative activity against both cell lines from moderate activity to very strong activity. 2.
4. Construction of the thiazolidinone ring in compound 12 decreased the antiproliferative activity comparable with the hydrazone derivative 11a, due to decreasing of the delocalization of n-π electrons after replacement of the C=N group (electron attracting group) by the thiazolidinone ring.

Antioxidant Activity Screening
One of the aims of this work is the screening of all synthesized compounds for antioxidant activity using two different methods, namely ABTS [2,2 -azino-bis(3-ethyl benzothiazoline-6-sulfonic acid)] and DPPH assays. DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical assay based on electron-transfer that produces a violet solution in ethanol. This free radical is stable at ambient temperature and reduced in the presence of an antioxidant molecule, leading to colorless ethanol solution. After investigation of these results as listed in Table 2 and Figure 6, it was realized that, compounds 6 and 11d have promising activity through using ABTS assay. Meanwhile, in the case of using DPPH assay, compounds 6, 11d and 14 have also very high activity. Ascorbic acid was used as a reference through the antioxidant activity screening.
The results depicted in Table 2 and Figure 6 demonstrated that, DPPH assay findings are very approximately related to those of ABTS assay with only one exception, compound 14 has an excellent antioxidant activity against DPPH (IC 50 = 26.87 ± 0.23 µM) than that of the ABTS method (IC 50  smaller than the corresponding ones of the same compounds in the case of the ABTS method, and it proposed that these compounds are more promising scavengers of the DPPH radical than those of the ABTS radical. By comparing the antioxidant efficacy of the thirteen synthesized compounds in this study to their chemical structures, it was concluded that the following structure antioxidant activity relationship's (SAR's) is hypothesized: 1. The presence of C=S enhanced antioxidant activity than the presence of C=O, as shown in compounds 6 and 14 comparable with compounds 5 and 9, respectively.
3. In compound 12, replacement of C=N group by the thiazolidinone ring decreased the antioxidant activity comparable with 11a, because of the lack of the conjugated system. Table 2. Antioxidant activities of all synthesized compounds by using 2,2 -azino-bis(3-ethyl benzothiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) methods. exhibited IC50 smaller than the corresponding ones of the same compounds in the case of the ABTS method, and it proposed that these compounds are more promising scavengers of the DPPH radical than those of the ABTS radical. By comparing the antioxidant efficacy of the thirteen synthesized compounds in this study to their chemical structures, it was concluded that the following structure antioxidant activity relationship's (SAR's) is hypothesized:

Compounds ABTS IC 50 (µM) DPPH IC 50 (µM)
1. The presence of C=S enhanced antioxidant activity than the presence of C=O, as shown in compounds 6 and 14 comparable with compounds 5 and 9, respectively.

Density Functional Theory
According to the frontier molecular orbital (FMO) theory, the highest occupied molecular orbital (HOMO) acts as an electron-donor and the lowest unoccupied molecular orbital (LUMO) acts as an electron-acceptor [45]. Meanwhile, both play remarkable roles in the electronic studies by using quantum chemical calculations and they are also of significant importance in modern biochemistry and molecular biology [46]. A molecule is considered as a softer and has an excellent chemical reactivity when it has a smaller energy gap. Meanwhile, a molecule is considered to have a higher chemical hardness and assumed to have good stability when it has a larger energy gap [47][48][49][50][51].
The quantum chemical calculations were implemented by the density functional theory (DFT) method by using the Gaussian(R) 09 program at the B3LYP level in conjunction with 6-31G(d,p) basis set and computed parameters are summarized in Table 3.
By computing and using the energy gap (∆E = E LUMO − E HOMO ) and dipole moment values beside another quantum chemical parameters such as ionization energy (I = −E HOMO ), electron affinity (A = −E LUMO ) [52], chemical hardness (η = (I − A)/2), chemical softness (S = 1/η) [53], and binding energy, we can rationally explicate the relation between the chemical structure and the antiproliferative activity (SAR's). Whereas, the energy gap of compound 6 (∆E = 3.98 eV) is smaller than that corresponding of compound 5 (∆E = 4.85 eV) and also compound 6 has a higher chemical softness value (S = 0.50 eV −1 ) than that corresponding of compound 5 (S = 0.41 eV −1 ). These results are matching with the results of the antiproliferative screening whereas, compound 6 has a higher potency comparable with compound 5 for both cell lines (HepG2 and MCF-7) as shown in Table 1 and Figure 3. Similarly, compound 14 has a smaller energy gap and a higher chemical softness than that corresponding to compound 9 as listed in Table 2. In addition, in vitro compound 14 showed a remarkable higher efficacy comparable with compound 9 as shown in Table 1 and Figure 3. Notably, the dipole moment values of compounds 6 (µ = 3.4641 D) and 14 (µ = 1.852 D) are higher than that of compounds 5 (µ = 3.2867 D) and 9 (µ = 1.764 D), respectively.
On the other hand, compounds 11a-d possess antiproliferative activity in the following order 11d > 11c > 11b > 11a, meanwhile, the energy gaps of these compounds increase in the following order 11a (∆E = 2.99 eV) < 11d (∆E = 3.05 eV) < 11c (∆E = 3.10 eV) < 11b (∆E = 3.13 eV). The lower of the antiproliferative activity of compound 11a may be explained by values of the dipole moment whereas; the dipole moment of compound 11a is smaller than that of compounds 11b-d as shown in Table 3. The distributions of the HOMO and LUMO orbitals of the selected compounds are computed at the same level of the DFT theory and are provided in Figures 7 and 8. The results manifested that possible reactive sites exist as shown below: 1. The HOMO of compounds 5 and 9 are nearly similar and the distribution of orbitals are mainly situated on the quinazolinone moiety, also, the LUMO of these compounds are situated on the same moiety.
2. The HOMO of compounds 6 and 14 are nearly similar and the distribution of orbitals are mainly situated on C=S, while, the LUMO of these compounds are mainly situated on the quinazolinthione moiety.
3. The HOMO of compounds 11a-d are nearly similar and the distribution of orbitals are mainly situated on the quniazolinone moiety, meanwhile, the LUMO of these compounds are mainly situated on the aryl aldehyde hydrazone system.
The distributions of the HOMO and LUMO orbitals of the selected compounds are computed at the same level of the DFT theory and are provided in Figures 7 and 8. The results manifested that possible reactive sites exist as shown below: 1. The HOMO of compounds 5 and 9 are nearly similar and the distribution of orbitals are mainly situated on the quinazolinone moiety, also, the LUMO of these compounds are situated on the same moiety.
2. The HOMO of compounds 6 and 14 are nearly similar and the distribution of orbitals are mainly situated on C=S, while, the LUMO of these compounds are mainly situated on the quinazolinthione moiety.
3. The HOMO of compounds 11a-d are nearly similar and the distribution of orbitals are mainly situated on the quniazolinone moiety, meanwhile, the LUMO of these compounds are mainly situated on the aryl aldehyde hydrazone system.

Chemistry
The melting point is uncorrected and was measured on a Stuart SMP 30 advanced digital electric melting point apparatus (Cole-Parmer, Staffordshire, UK). All reactions were monitored by TLC (Kieselgel 60 F254, Merck, Munchen, Germany) and spots were visualized using UV (254 nm), In the region (400−4000 cm −1 ), the IR spectrum was measured in the KBr phase by using the Nicolet iS10 FT-IR spectrometer (Shimadzu Corporation, Kyoto, Japan). The 1 H-NMR (at 400 MHz) and 13 C-NMR (at 100 MHz) spectra were performed at chemical warfare labs, Egypt, with a Varian Gemini Molecules 2019, 24 10 The distributions of the HOMO and LUMO orbitals of the selected compounds are computed at the same level of the DFT theory and are provided in Figures 7 and 8. The results manifested that possible reactive sites exist as shown below: 1. The HOMO of compounds 5 and 9 are nearly similar and the distribution of orbitals are mainly situated on the quinazolinone moiety, also, the LUMO of these compounds are situated on the same moiety.
2. The HOMO of compounds 6 and 14 are nearly similar and the distribution of orbitals are mainly situated on C=S, while, the LUMO of these compounds are mainly situated on the quinazolinthione moiety.
3. The HOMO of compounds 11a-d are nearly similar and the distribution of orbitals are mainly situated on the quniazolinone moiety, meanwhile, the LUMO of these compounds are mainly situated on the aryl aldehyde hydrazone system.

Chemistry
The melting point is uncorrected and was measured on a Stuart SMP 30 advanced digital electric melting point apparatus (Cole-Parmer, Staffordshire, UK). All reactions were monitored by TLC (Kieselgel 60 F254, Merck, Munchen, Germany) and spots were visualized using UV (254 nm), In the region (400−4000 cm −1 ), the IR spectrum was measured in the KBr phase by using the Nicolet iS10 FT-IR spectrometer (Shimadzu Corporation, Kyoto, Japan). The 1 H-NMR (at 400 MHz) and 13 C-NMR (at 100 MHz) spectra were performed at chemical warfare labs, Egypt, with a Varian Gemini

Chemistry
The melting point is uncorrected and was measured on a Stuart SMP 30 advanced digital electric melting point apparatus (Cole-Parmer, Staffordshire, UK). All reactions were monitored by TLC (Kieselgel 60 F 254 , Merck, Munchen, Germany) and spots were visualized using UV (254 nm), In the region (400−4000 cm −1 ), the IR spectrum was measured in the KBr phase by using the Nicolet iS10 FT-IR spectrometer (Shimadzu Corporation, Kyoto, Japan). The 1 H-NMR (at 400 MHz) and 13 C-NMR (at 100 MHz) spectra were performed at chemical warfare labs, Egypt, with a Varian Gemini spectrometer (Metrohim, California, United States) in DMSO-d 6 as a solvent by using tetramethylsilane (TMS) as a reference. Perkin-Elmer 2400 CHN elemental analyzer (Waltham, MA, USA) was used to record CHN elemental analysis at the Faculty of Science, Cairo University, Egypt. The mass spectrum was measured on Shimadzu GC-MS QP1000EX apparatus (Shimadzu Corporation, Kyoto, Japan) at the central analytical lab, Ain Shams University, Cairo, Egypt.

2-Hexanamidobenzoic Acid 3
Hexanoyl chloride 2 (1.39 mL, 0.01 mol) was added dropwise to anthranilic acid 1 (1.37 g, 0.01 mol) dissolved in dry pyridine (20 mL) at ambient temperature with stirring. The stirring was continued for an hour, and then the resulting emulsion was acidified with cold 10% HCl solution. The white solid which separated was collected by filtration and then recrystallized from benzene to give 3 [38]

2-Pentylquinazolin-4(3H)-one 5
A solution of benzoxazinone 4 (2.17 g, 0.01 mol) in formamide (15 mL) was refluxed for 7 h. After cooling, the reaction mixture was poured onto ice cold water, the obtained solid was filtered off, dried, and recrystallized from petroleum ether 60-80 • C to give 5 [41]  3.1.8. General Procedure for Synthesis of 11a-d A mixture of compound 9 (2.31 g, 0.01 mol) and the appropriate aldehydes 10a-d (0.01 mol) in absolute ethanol (30 mL) was refluxed for 4−6 h. The reaction mixture was evaporated under reduced pressure; the obtained residue was collected and recrystallized from the proper solvent to give the corresponding benzylidene derivatives 11a-d, respectively.

ABTS Method
By the bleaching of ABTS derived radical cations, the detections of antioxidant activities were estimated. The radical cation was prepared by the reaction of ABTS [2,2 -azino-bis(3-ethyl benzothiazoline-6-sulfonic acid)] (60 µL) with MnO 2 (3 mL, 25 mg/mL) in a phosphate buffer solution (10 µM, pH 7, 5 mL). The solution was shaken for 3 min, centrifuged, filtered, and recorded at λ max 734 nm the absorbance A (control) of the resulting ABTS radical solution (green-blue). Upon the addition of the tested sample solution (20 µl) with different concentrations of compounds such as 200, 100, 50, 25, and 12.5 µM in spectroscopic grade MeOH/buffer (1:1 v/v) to the ABTS solution, the absorbance A (test) was measured. The decreasing in the absorbance is expressed as % inhibition which was calculated according to the following equation [55]: where; the reference and standard antioxidant compound in this test is the ascorbic acid solution (20 µL, 2 mM) and the blank sample was performed by the solvent without ABTS.

DPPH Method
According to the methodology described by Brand-Williams et al. [56], the measurement of the DPPH radical scavenging activity was implemented. The samples with different concentrations of compounds such as 200, 100, 50, 25, and 12.5 µM were allowed to react with the stable DPPH radical in ethanol solution. Whereas, the reaction mixture consisted of sample (0.5 mL), absolute ethanol (3 mL), and DPPH radical solution (0.3 mL) 0.5 mM in ethanol. DPPH is reduced when it reacts with an antioxidant compound, which can donate hydrogen. The changes in color (from deep violet to light yellow) were recorded [absorbance (Abs)] at λ max 517 nm after 100 min of reaction using a UV-Vis spectrophotometer (Schimadzu Co., Tokyo, Japan). The blank solution was prepared by mixing ethanol (3.3 mL) and the sample (0.5 mL). Meanwhile, the mixture of ethanol (3.5 mL) and DPPH radical solution (0.3 mL) serve as a positive control.

Computational Procedures
All theoretical calculations and results of the studied compounds were implemented by utilizing Gaussian(R) 09 D.01 [58] (Semichem Inc., Shawnee Mission, KS, USA) by applying the DFT operation with the hybrid functional B3LYP level [59,60] in conjunction with the 6−31G(d,p) basis set. The visualization of these results was achieved using GaussView 6.0.16 software (Semichem Inc., Shawnee Mission, KS, USA).

Conclusions
In conclusion, this work focused on the study of the antiproliferative and antioxidant activities in vitro in addition to the theoretical calculation of the DFT theory of some novel quinazolinone(thione) derivatives 6-14. Two main points were the principal targets; firstly, by comparing the activities of quinazolinone and quinazolinthione derivatives. Secondly, comparing the activities of four series of Schiff bases, that have quinazolinone moiety. The results of this study imply that the quinazolinthione derivatives 6 and 14 have promising potent antiproliferative activity comparable with quinazolinone derivatives 5 and 9, respectively. According to the DFT study, compounds 6 and 14 have a smaller energy gap and a higher chemical softness than that of compounds 5 and 9, respectively. Additionally, screening of various aryl aldehyde hydrazone derivatives (11a-d) analogs exhibited that the potency increased with increasing the electron donating group in p-position due to increasing of the conjugated system, and that was supported by the DFT study.
On the other hand, compounds 6 and 11d showed promising antioxidant activity using ABTS assay. While in the DPPH assay, compounds 6, 11d, and 14 have showed potent activities comparable to the ascorbic acid which was used as a reference drug. Noteworthy, the results of both antiproliferative and antioxidant activities for each compound individually are nearly the same.

Conflicts of Interest:
The authors declare no conflict of interest.