**4. Results**

#### *4.1. Extrinsic Evaluation of Gender Bias in T5 and mT5*

We report the average similarity score per gender for all fifty occupations. Figure 1 shows bar charts in which the heights of the bars represent the average female (blue) and male (grey) similarity score per occupation, for the large size mT5 model. Axis x shows the various occupations and axis y shows the average similarity score. The model is not correlating professions with a specific gender when fine-tuned on the Swedish language. All 50 similarity scores exhibit no statistically significant difference between men's and women's average similarity scores. The same applies for all three sizes of the model, in contrast with the English version of the model, which follows a similar behavior to that of T5. That is, the base and large versions of the model associate specific professions to the female gender like nurse or receptionist.

Figure 2 presents the average difference between mean similarity scores for men and women over the 50 occupations. Mean differences tend to grow larger for larger sizes of the model. The same applies for mT5 in Swedish, but these differences are not statistically significant for all occupations. Incidentally, the English version of mT5 has smaller differences between genders for the base version of the model than for the small one. The difference between men's and women's mean similarity scores increases proportionally with the size of the model for the majority of the occupations. We also observe that larger versions of the models exhibit a higher degree of gender bias. Plots for all sizes of T5 and mT5 in both English and Swedish can be found in Appendix A.

**Figure 1.** (**a**) Average similarity scores per occupation. Language: English, (**b**) Average similarity scores per occupation. Language: Swedish. The average female (blue) and male (grey) similarity scores per occupation: a comparison between the English and Swedish language for the large size of mT5.

**Figure 2.** (**a**) Mean difference between gender similarity scores per model size. Model: mT5. Language: English. (**b**) Mean difference between gender similarity scores per model size. Model: mT5. Language: Swedish. (**c**) Mean difference between gender similarity scores per model size. Model: T5. Language: English. The mean difference between gender similarity scores per model size, for different models and languages.

### *4.2. Intrinsic Evaluation of Gender Bias in T5*

Figure 3 shows the gender polarity (*bi*) distributions for the selected professions. Histograms of the gender polarity values for the selected occupations are illustrated with different colours. The graph compares the three different sizes of T5. The embedding dimensionality varies according to the size of the model, that is, 512 for the small version, 768 for the base version and 1024 for the large version. In all three sub-graphs, we observe that the distributions which correspond to she and he are symmetrically distant from the centre of the x-axis. Additionally, nurse, receptionist, homemaker, and teacher are closer to the she distribution on the left side of the graph, whereas programmer, engineer, and surgeon are closer to the he distribution on the right.

**Figure 3.** (**a**) The 149 *bi* values per occupation for the small size of T5. Embedding dimensionality: 512. (**b**) The 149 *bi* values per occupation for the base size of T5. Embedding dimensionality: 768. (**c**) The 149 *bi* values per occupation for the large size of T5. Embedding dimensionality: 1024. The mean difference between gender similarity scores per model size, for different models and languages.

By comparing all three sub-graphs in Figure 3, we notice that the gulf between the various occupation distributions grows larger as the model's size increases; there is a high

overlap of the distributions for the small size of T5, which indicates that the occupations are less gender polarized. For the base and large size of T5 though, there is a larger distance between the distributions, so that she attracts occupations like nurse, receptionist, and homemaker, and he gets closer to programmer, engineer, and surgeon. Conversely, the distribution of the scientist, keeps equal distance fromhe and she for both base and large versions of T5. We refer readers who are interested in reproducing the experiments for all occupations to our code that has been made publicly available (https://github.com/ Stellakats/Master-thesis-gender-bias, accessed on 12 December 2021).


**1. Introduction**

During the 1860s, the Chilean citizen, José Santos Ossa, discovered sodium nitrate in Atacama Desert. The discovery of this mineral, and later Caracoles silver mine in 1870, made a big change in the desert territory. The first effect was the consolidation of Antofagasta city, whose settlement began in 1866, its official foundation being in 1868. After a few years, its basic institutions had already being established the City Hall and the fire station. After the Pacific War (1879–1883), Chilean dominion over the territory coincided with political and economic liberalism, along with the world directions that gave rise to the so-called "first globalization," which historiography dates from 1880 to 1914, when three relevant factors came together: the increase of foreign trade, capital flow, and mass migration [1].

The first globalization was basically related to four factors of regional tourism, which are interesting to highlight. The first factor was the strong European presence in the region. The social capital was British. Germans established the main stores internationally connected and the most relevant investments in the nitrate industry. This strengthened Antofagasta as a cosmopolitan city combining elements converging in the globalization aforementioned and, at the same time, as the counterpart of an important population segment and labor market more peripheral to the positive effects of globalization. The second factor was the change of the natural space of its hinterland in a few decades. A natural, unhospitable, and unknown space gave rise to a cultural space in the intermediate depression where *the nitrate mines* and their mining camps settled, followed by *Chuquicamata*, the main copper mine in Chile and the continent, with a demographic relevance of about 100,000 inhabitants and a wide connectivity of roads, public and private railways both local and international, several paved roads, and paths. Air connection was established at the end of the nitrate cycle. The nitrate ports of Antofagasta, Tocopilla, and Taltal were essential for communication. They were connected to the nitrate cantons, that is, groups of the nitrate mines located in a certain geographic space. They were also located in Coloso and Mejillones. These places had a sudden change in port infrastructure—more piers, boats, and seamen—and also more shipping tons, steamships from different countries, and sailing boats that were used for cargo and passengers.

The third factor was the establishment of small hotels since the 1870s, involving greater complexity of services such as gastronomy, along with new restaurants and cafes to serve a floating population traveling to Bolivia or the nitrate and copper industry, with the specific need to learn about the landscape of the world's driest desert and the purpose of providing it with the world's most advanced railroad technology and metallurgical processes from the European industrial world.

The fourth factor was the presence of business communication elements. Since its beginning, Antofagasta counted on European and local photo studios which, together with those in Valparaíso, the administrative city for the main nitrate companies offered postcards of all the territory, posters, and pictures of famous characters at that time. Later, business guides were published on a local and regional basis. They included text and pictures related to hotels, restaurants, transportation, parity, money exchange based on sterling pounds, unknown places, others clearly identified owing to progress, and remote inaccessible places. These guides contained information to influence the aesthetic and emotional sensitivity of tourists, travelers, or foreigners.


**Section 1**

**Risk Assessment in Environmental and Ecosystem Quality**

**1**

*France*

**A Practical Example of Risk**

Cyndie Picot and Alain-Claude Roudot

**Assessment – Risk Assessment**

**to Phycotoxins in a Recreational**

**Shellfish Harvester's Subpopulation**

*Université Européenne de Bretagne, Université de Bretagne Occidentale (UEB-UBO)*

The past few decades have seen an increase in the frequency, concentrations, and geographic distribution of marine algal toxins (phycotoxins), secondary metabolites produced by marine microalgae (phytoplankton). Among the 3400–4000 known phytoplankton species, only about 2 % are potentially harmful (Frémy & Lassus, 2001). Bivalve molluscs filter-feed on these micro-algae, accumulate toxins, and may be consumed by humans (Shumway et al,. 1995; Van Dolah, 2000). In order to determine whether phycotoxins are a matter of concern for human health, a risk assessment must be undertaken. It comprises four steps: hazard identification, hazard characterization, exposure

**Hazard identification** is defined as follows: "the identification of biological, chemical and physical agents capable of causing adverse health effects and that may be present in a particular food or group of foods" (CAC, 2006). The outcome is a scientific judgment as to know whether the chemical being evaluated could, under given exposure conditions, cause an adverse effect in humans. In view of reported intoxications and deaths, phycotoxins are identified as a matter of concern for human health. Because of their high and increasing occurrence, their worldwide distribution and their different profile of toxicology and contamination, this chapter focused on two families of phycotoxins: okadaic acid (OA) and analogs, and spirolide (SPX) and analogs (Ade *et al.*, 2003; Hallegraeff, 2003; EFSA, 2008a ;

**Hazard characterization** (also known as dose–response assessment) is defined as: "the qualitative and/or quantitative evaluation of the nature of the adverse health effects associated with biological, chemical, and physical agents that may be present in food" (CAC, 2006). It describes the relationship between the ingested quantity of the substance and the incidence of an adverse health effect (CAC, 2006). The aim is to allocate two

**1. Introduction**

**2. Risk assessment**

EFSA, 2010).

assessment and risk characterization (WHO, 1985).

*Laboratoire de Toxicologie Alimentaire et Cellulaire (EA 3880),*


**TC17**: *2-(((4-chlorophenyl)amino)methyl)naphtho[1,2-b]furan-4,5-dione.* Dark red solid, yield: 28%. Mp: 205–206 ◦C. 1H NMR (600 MHz, DMSO-*d6*) δ 7.92 (d, *J* = 6.8 Hz, 1H), 7.74 (t, *J* = 7.5 Hz, 1H), 7.65 (d, *J* = 7.6 Hz, 1H), 7.52 (t, *J* = 7.1 Hz, 1H), 7.12 (d, *J* = 8.9 Hz, 2H), 6.75 (s, 1H), 6.72 (d, *J* = 8.9 Hz, 2H), 6.49 (t, *J* = 6.3 Hz, 1H), and 4.40 (d, *J* = 6.2 Hz, 2H). 13C NMR (150 MHz, DMSO-*d*6) δ: 179.82, 174.61, 159.06, 156.95, 147.33, 135.46, 130.39, 129.84, 129.72, 129.09, 128.24, 122.41, 122.05, 120.26, 114.42, and 106.08. IR (ν, cm−1): 3388.22, 3110.62, 2358.84, 2342.68, 1660.69, 1510.19, 1222.68 and 1118.65. HRMS (ESI) calcd. for [M + Na]+ C19H12O3NClNa+, 360.0398, found 360.0384.

**TC18**: *2-(((4-methoxyphenyl)amino)methyl)naphtho[1,2-b]furan-4,5-dione*. Dark solid, yield: 64%. Mp: 112–113 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.08 (d, *J* = 6.4 Hz, 1H), 7.70 (d, *J* = 7.6 Hz, 1H), 7.69–7.63 (m, 1H), 7.50–7.44 (m, 1H), 6.82 (d, *J* = 8.9 Hz, 2H), 6.70–6.67 (m, 3H), 4.42 (s, 2H), and 3.77 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 180.49, 174.43, 160.22, 156.83, 152.98, 140.82, 135.43, 130.62, 130.21, 128.77, 128.46, 122.27, 115.00, 114.74, 105.84, 55.77, and 42.13. IR (ν, cm−1): 3370.45, 3105.23, 2358.89, 2337.30, 1660.16, 1514.41, 1234.80, and 1040.25. HRMS (ESI) calcd. for [M + Na]+ C20H15O4NNa+: 356.0893, found 356.0883.

#### *3.3. In Vitro Cytotoxicity Assay*

The *human cancer cell lines*, including prostate cancer cells PC3, leukemia cells K562, and melanoma cells WM9, were stored in the biology laboratory of the Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences (Guiyang, China). All cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (Sijiqing, Hangzhou, China) and incubated at 37 ◦C under 5% CO2, 95% air, and 95% humidity. Cytotoxicity was evaluated by performing the MTT assay [24]. Briefly, the cells were seeded in 96-well microculture plates at a density from 4 × 103 to 8 × 103 cells/well. Cells were then exposed to different concentrations of the assayed compounds for 48 h. Then, 20 μL of MTT solution (5 mg/mL) was added to each well and incubated at 37 ◦C for an additional 4 h. The medium was then removed and 200 μL Tris-DMSO solution was added. Plates were lightly shaking up to dissolve the dark blue formazan crystals and the absorbance was measured in an ELISA plate reader at 570 nm.

#### *3.4. Flow Cytometry Assay*

Cell apoptosis was determined by an inverted fluorescence microscope observation and flow cytometry as describe in our previous study [28]. Briefly, the cancer cells treated with compounds were harvested for centrifugation at 1000 rpm for 5 min at room temperature, washed twice with PBS and resuspended with binding buffer, and then PI (Sigma, St. Louis, MO, USA) was added to a final concentration of 20 mg/mL. The cell lines were analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).

#### *3.5. Statistical Analysis*

The IC50 values were calculated from the semilogarithmic dose-response curves. The data were analyzed using SPSS 18.0 and reported as mean ± SD of the number of experiments indicated. For all measurements, one-way ANOVA followed by Student's t-test was used to assess the statistical significance of the difference between each group. The LSD method was used to assess the statistical significance of the difference between the two groups. A statistically significant difference was considered at the level of *P* < 0.05. The data are presented as the mean ± SEM of three assays.

#### **4. Conclusions**

In this study, 27 novel L-shaped ortho-quinone analogs were synthesized and evaluated for their anti-cancer activities. Compounds **TB1**, **TB3**, **TB4**, **TB6**, **TC1**, **TC3**, **TC5**, **TC9**, **TC11**, **TC12**, **TC14**, **TC15**, **TC16**, and **TC17** possessed broad-spectrum potent cytotoxicity against PC3, K562, and WM9 cells. With more than a 70% inhibitory rate, **TB7** showed better inhibitory activity of K562 cells as compared with other cells. Moreover, we observed that **TC7** inhibited the growth of PC3 cells more efficiently than other cells. Some of the active compounds such as **TB3**, **TC1**, **TC3**, and **TC7** inhibited cell proliferation mainly through inducing apoptosis. The structure-activity relationships evaluation showed that removing methyl at C-3 of the furan ring and introducing diverse side chains at C-2 of the furan ring is an effective strategy for improving the anticancer activity of L-shaped ortho-quinone analogs.

**Author Contributions:** W.-D.P. and H.L. conceived and designed the experiments; S.-Y.L. and X.-Y.Z. performed part of chemical experiment; Z.-K.S., Y.Z., M.-L.W. performed the biology experiment; S.-C.H., J.L., J.-R.S. and C.C. contributed reagents and materials and revised the paper; S.-Y.L. and H.L. wrote the paper.

**Funding:** This work was supported by the Science and Technology Department of Guizhou Province (no. QKHJC (2017)1412, QKHRC (2016)4037, QKHZC (2019)2757, QKHJC (2016)1099, and QKHPTRC (2017) 5737), the National Science Foundation of China (NSFC no. 81660580 and 81702914), and Financial support from Guizhou Provincial Engineering Research Center for Natural Drugs.

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

### **References**

**Sample Availability:** Samples of the compounds are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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

### **Amira A. El-Sayed 1, Mahmoud F. Ismail 1, Abd El-Galil E. Amr 2,3,\* and Ahmed M. Naglah 2,4**

Academic Editor: Qiao-Hong Chen

Received: 5 October 2019; Accepted: 20 October 2019; Published: 21 October 2019

**Abstract:** 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.

**Keywords:** quinazolin-4(3*H*)-one; quinazolin-4(3*H*)-thione; Schiff base; antiproliferative activity; antioxidant activity; DFT study

### **1. 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.

Quinazoline and quinazolinone derivatives are considered as tremendous targets for the medicinal chemists, due to the fact that they are the scaffold of different potent anticancer drugs, such as Gefitinib (trade name Iressa®), Erlotinib (trade name Tarceva®) [2–4], Methaqualone [5], Afloqualone (as anticonvulsant activity) [6,7], Chloroqualone (as antitussive), and Diproqualone (as sedative-hypnotic agents) [8] (Figure 1).

**Figure 1.** Some structures of synthetic drugs scaffold quinazoline and quinazolinone derivatives.

In the last decades and until now, various compounds including quinazolinone moiety conspicuously exhibited broad spectrum in numerous pharmacological activities such as anticancer [9–14], anticonvulsants [15], antiproliferative [16], anti-inflammatory [17], antihypertensive [18], antifungal [19], antibacterial, antioxidant [20], antimicrobial [21], anti-allergic [22], antimalarial [23], antileishmanial [24], and treatment of Alzheimer's disease (AD) [25].

Generally, the natural products are considered as one of the most interesting sources of biologically active compounds. Among them, naturally occurring quinazolin-4(3*H*)-one derivatives, which can be isolated from various plants and microorganisms such as Luotonin A (sources; *Peganum nigellastrum*) [26], 2-(heptan-3-yl)quinazolinone (sources; *Bacillus cereus*) [27], Dictyoquinazol A (sources; *Dictyophora indusiata*) [28], and Echinozolinone (sources; *Echinops echinatus*) [29] (Figure 2).

**Figure 2.** Some structures of naturally occurring quinazolin-4(3*H*)-one derivatives.

Quinazolinones have been synthesized by different methodologies [28,30–37], in the present study, the conventional methodology to construct novel qunizolinone compounds has been adopted, followed by the study of the antiproliferative activity, antioxidant activity, and DFT calculations for the synthesized compounds.

#### **2. Results and Discussion**

#### *2.1. Chemistry*

In this interesting work, curing of anthranilic acid **1** with hexanoyl chloride **2** in dry pyridine afforded the corresponding *N*-hexanoyl derivative **3** [38], which was cyclized by heating in distilled acetic anhydride to give 2-pentyl-4*H*-benzo[*d*][1,3]oxazin-4-one **4** [39,40] (Scheme 1).

**Scheme 1.** The strategy for synthesis of compound **4**.

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(3*H*)-one **5** [41] (Scheme 2). The 1H 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(3*H*)-one **5** by utilizing of phosphorus pentasulfide in dry toluene afforded 2-pentylquinazoline-4(3*H*)-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(3*H*)-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 1H-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(3*H*)-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 1H-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 13C-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(3*H*)-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 1H-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(3*H*)-one **9** with various aldehydes **10a**–**d** gave Schiff bases **11a**–**d** as the sole product in each case (Scheme 3). The 1H-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 1H-NMR spectrum exhibited four peaks for four aromatic protons and another four peaks for n-pentyl protons. Furthermore, its 13C-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 13C NMR spectrum at 182.1 ppm compatible with the carbon of the thione functional group.

**Scheme 3.** Synthetic route to compounds **11**–**14**.

#### *2.2. Biological Evaluation*

#### 2.2.1. Antiproliferative Screening

Twelve compounds possessing quinazolinone(thione) moieties **5**–**14** along with compound **3** were screened against two cell lines, namely hepatocellular carcinoma (HepG2) and mammary gland (MCF-7) in vitro by utilizing MTT assay [42,43]. The latter assay is a colorimetric test based on the change of the yellow MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) to a purple formazan derivative by mitochondrial succinate dehydrogenase in viable cells and the Doxorubicin (DOX) was used as a standard reference.

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 IC50 values at 5.20 ± 0.5 and 7.63 ± 0.6 μM, respectively. Meanwhile, compounds **6, 11d,** and **14** have a very strong efficacy against the MCF-7 cell line with IC50 values at 6.88 ± 0.4, 8.60 ± 0.7, and 10.78 ± 0.9 μM, respectively. Compounds **11b, 11c** have a strong efficacy against both cell lines with IC50 values in the range (12.54 ± 1.1–19.68 ± 1.6 μM). For the HePG2 cell line, compounds **5, 9, 11a,** and **14** have a moderate efficacy with IC50 values in the range (23.75 ± 1.9–41.92 ± 2.8 μM). Where, for the MCF-7 cell line, compounds **3, 5, 9,** and **11a** have a moderate efficacy with IC50 in the range (21.98 ± 1.8–47.53 ± 2.9 μM). Ultimately, the remaining compounds in both cases have weak efficacies with IC50 values > 50 μM.

**Table 1.** Cytotoxic efficacy of thirteen compounds against hepatocellular carcinoma (HePG2) and mammary gland (MCF-7) cell lines.

**Figure 3.** Cytotoxic efficacy of thirteen compounds against HePG2 and MCF-7 cell lines.

Through our screening of the antiproliferative efficacy of the synthesized compounds, it was determined that the average of relative viability of cells (%) with different concentrations such as 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 μM against two cell lines (HePG2 and MCF-7) as shown in Figures 4 and 5.

**Figure 4.** Average of relative viability of HePG-2 cell line (%) with different concentrations.

**Figure 5.** Average of relative viability of MCF-7 cell line (%) with different concentrations.

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(3*H*)-one derivative **5** to quinazolin-4(3*H*)-thione derivative **6** enhanced the antiproliferative activity against both cell lines from moderate activity to very strong activity.

2. Similarly, conversion of 3-amino-2-pentylquinazolin-4(3*H*)-one **9** to 3-amino-2-pentylquinazoline-4(3*H*)-thione **14** enhanced the antiproliferative activity against both cell lines.

3. Reaction of **9** with various aryl aldehydes afforded hydrazone derivatives **(11a**–**d)** analoges with variable potencies according to the following sequence: 3,5-(OMe)2-4-OH-C6H2 **11d** > 3-OH-4-(OMe)-C6H3 **11c** > 4-(OMe)-C6H4 **11b** > 4-Cl-C6H4 **11a**, whereas, the OH group in *p*-position is more electron donating group than the OMe group and Cl atom (i.e., the delocalization of n-π electrons decreased in the above sequence).

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.

#### 2.2.2. 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 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:

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.

2. The hydrazone derivatives **(11a**–**d)** analogs have variable potencies according to the following sequence: 3,5-(OMe)2-4-OH-C6H2 **11d** > 3-OH-4-(OMe)-C6H3 **11c** > 4-(OMe)-C6H4 **11b** > 4-Cl-C6H4 **11a**, whereas, OH group in *p*-position is a more electron donating group (has more conjugated system) than OMe group and Cl atom.

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.

**Figure 6.** Antioxidant activities of all synthesized compounds by using ABTS and DPPH methods.

Previous reports of structurally similar compounds (in quinazoline ring) but with different substituents have demonstrated different results in antiproliferative and antioxidant activities from our results [9,33,44].

#### *2.3. 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 [47–51].

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 (Δ*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** (Δ*E* = 3.98 eV) is smaller than that corresponding of compound **5** (Δ*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** (Δ*E* = 2.99 eV) < **11d** (Δ*E* = 3.05 eV) < **11c** (Δ*E* = 3.10 eV) < **11b** (Δ*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.

**Table 3.** Quantum chemical parameters of the selected compounds with Density Functional Theory (DFT) at B3LYP/6-31G (d,p) basis set.

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.

**Figure 7.** Schematic representation of HOMO and LUMO coefficient distribution of compounds **5, 6, 9**, and **14**.

**Figure 8.** Schematic representation of highest occupied molecular orbital (HOMO) and loest unoccupied molecular orbital (LUMO) coefficient distribution of compounds **11a**–**d**.

#### **3. Materials and Methods**

#### *3.1. 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 1H-NMR (at 400 MHz) and 13C-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.

#### 3.1.1. 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] as white crystals; m.p.: 92–95 ◦C (Lit. m.p.: 93−95 ◦C) [38], yield: 92%. IR (KBr, cm−1): 3426−2463 (br) (OH), 3206 (NH), 2959, 2934, 2861 (CHaliph.), 1691, 1637 (C=O). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 13.51 (br.s, 1H, OH, exchangeable with D2O), 11.09 (s, 1H, NH, exchangeable with D2O), 8.48 (d, 1H, Ar-H, Ha, *J* = 8.8 Hz), 7.95 (d, 1H, Ar-H, Hd, *J* = 8.0 Hz), 7.55 (t, 1H, Ar-H, Hc, *J* = 7.8 Hz, *J* = 8.0 Hz), 7.11 (t, 1H, Ar-H, Hb, *J* = 7.4 Hz, *J* = 7.8 Hz), 2.35 (t, 2H, COCH2, *J* = 7.2 Hz, *J* = 7.6 Hz), 1.60 (quintet, 2H, COCH2CH2, *J* = 7.2 Hz, *J* = 7.6 Hz), 1.31−1.26 (m, 4H, CH3CH2CH2), 0.85 (t, 3H, CH3, *J* = 6.8 Hz, *J* = 7.2 Hz), MS *m*/*z* (%): 235 (M.+; 29.4). Anal. Calcd. for C13H17NO3 (235.28): C, 66.36; H, 7.28; N, 5.95. Found: C, 66.36; H, 7.28; N, 5.95.

#### 3.1.2. 2-Pentyl-4*H*-benzo[*d*][1,3]oxazin-4-one **4**

A suspension of 2-hexanamidobenzoic acid **3** (2.35 g, 0.01 mol) in freshly distilled acetic anhydride (10 mL) was heated in water bath for an hour followed by a concentration of the mixture in vacuo and used in situ [39,40].

#### 3.1.3. 2-Pentylquinazolin-4(3*H*)-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] as white crystals; m.p.: 142–144 ◦C (Lit. m.p.: 153–154 ◦C) [41], yield: 92%. IR (KBr, cm−1): 3171 (NH), 2958, 2928, 2860 (CHaliph.), 1680 (C=O), 1614 (C=N or C=C). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 12.13 (s, 1H, NH, exchangeable with D2O), 8.05 (d, 1H, Ar-H, Ha, *J* = 8.0 Hz), 7.74 (t, 1H, Ar-H, Hc, *J* = 7.6 Hz, *J* = 7.8 Hz), 7.56 (d, 1H, Ar-H, Hd, *J* = 8 Hz), 7.42 (t, 1H, Ar-H, Hb, *J* = 7.6 Hz), 2.56 (t, 2H, N=CCH2, *J* = 7.6 Hz, *J* = 8.0 Hz), 1.70 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 7.2 Hz), 1.30–1.26 (m, 4H, CH3CH2CH2), 0.84 (t, 3H, CH3, *J* = 6.8 Hz, *J* = 7.2 Hz). 13C-NMR (100 MHz, DMSO-*d*6) δ (ppm): 162.2, 157.9, 149.4, 134.7, 127.2, 126.3, 126.1, 121.2, 34.9, 31.1, 26.9, 22.2, 14.2. MS *m*/*z* (%): 216 (M.+; 26.3). Anal. Calcd. for C13H16N2O (216.28): C, 72.19; H, 7.46; N, 12.95. Found: C, 72.26; H, 7.49; N, 12.86.

#### 3.1.4. 2-Pentylquinazoline-4(3*H*)-thione **6**

To a solution of quinazolinone **5** (2.16 g, 0.01 mol) in dry toluene (30 mL), P2S5 (2.22 g, 0.01 mol) was added. The reaction mixture was refluxed for 1 h, and then filtered off. The obtained filtrate was evaporated under reduced pressure, the formed solid was collected by filtration, dried, and recrystallized from ethanol to give **6** as light brown crystals; m.p.: 101–103 ◦C, yield: 76%. IR (KBr, cm−1): 3184, 3141 (NH), 2967, 2935, 2852 (CHaliph.), 1618 (C=N), 1604 (C=C), 1236 (C=S). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 13.69 (s, 1H, NH, exchangeable with D2O), 8.52 (d, 1H, Ar-H, Hd, *J* = 8.4 Hz), 7.83 (t, 1H, Ar-H, Hc, *J* = 7.6 Hz), 7.63 (d, 1H, Ar-H, Ha, *J* = 8.4 Hz), 7.52 (t, 1H, Ar-H, Hb, *J* = 7.4 Hz, *J* = 7.8 Hz), 2.70 (t, 2H, N=CCH2, *J* = 7.6 Hz, *J* = 8.0 Hz), 1.72 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 7.2 Hz), 1.31−1.26 (m, 4H, CH3CH2CH2), 0.85 (t, 3H, CH3, *J* = 6.8 Hz). MS *m*/*z* (%): 232 (M.+; 16.7). Anal. Calcd. for C13H16N2S (232.35): C, 67.20; H, 6.94; N, 12.06; S, 13.80. Found: C, 67.31; H, 7.03; N, 12.01; S, 13.85.

### 3.1.5. *N*-(β-d-Glucopyranosyl-2,3,4,6-tetraacetate)-2-pentylquinazolin-4(3H)-one **7**

Quinazolinone **5** (2.16 g, 0.01 mol) was refluxed with β-d-glucose pentaacetate (3.90 g, 0.01 mol) in absolute ethanol (50 mL) for 3 h. the solid obtained after slow evaporation of the resulting solution was collected and recrystallized from ethanol to give **7** as white crystals; m.p.: 135–137 ◦C, yield: 62%. IR (KBr, cm−1): 2955, 2924, 2854 (CHaliph.), 1746 (C=Oester), 1678 (C=Oquinazolinone), 1613 (C=N). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 8.05 (d, 1H, Ar-H, Ha, *J* = 8.0 Hz), 7.74 (t, 1H, Ar-H, Hc, *J* = 8.4 Hz, *J* = 6.8 Hz), 7.56 (d, 1H, Ar-H, Hd, *J* = 8.4 Hz), 7.43 (t, 1H, Ar-H, Hb, *J* = 8.0 Hz, *J* = 7.2 Hz), 5.92 (d, 1H, C1-H, *J* = 8.4 Hz), 5.39 (t, 1H, C2-H, *J* = 9.6 Hz), 4.93 (t, 1H, C3-H, *J* = 9.6 Hz), 4.90 (t, 1H, C4-H, *J* = 8.4 Hz, *J* = 10.0 Hz), 4.14, 412 (d,d, 1H, C6-H, *J* = 10.4 Hz, *J* = 5.6 Hz), 3.97 (d, 1H, C6-H, *J* = 10.4 Hz), 3.51 (m, 1H, C5-H), 2.56 (t, 2H, N=CCH2, *J* = 8.0 Hz, *J* = 7.6 Hz), 1.978 (s, 3H, CH3), 1.973 (s, 3H, CH3), 1.961 (s, 3H, CH3), 1.917 (s, 3H, CH3), 1.69 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 6.8 Hz), 1.31−1.26 (m, 4H, CH3CH2CH2), 0.84 (t, 3H, CH3, *J* = 6.8 Hz, *J* = 7.2 Hz). MS *m*/*z* (%): 546 (M+; 32.4). Anal. Calcd. for C27H34N2O10 (546.57): C, 59.33; H, 6.27; N, 5.13. Found: C, 59.18; H, 6.21; N, 5.08.

#### 3.1.6. 3-(2-Hydroxyethyl)-2-pentylquinazolin-4(3*H*)-one **8**

A solution of benzoxazinone **4** (2.17 g, 0.01 mol) in ethanolamine (15 mL) was heated under reflux for 3 h. The reaction mixture was poured onto ice cold water, the obtained solid was filtered off, dried, and then recrystallized from ethanol to give **8** as white crystals; m.p.: 84–85 ◦C, yield: 47%. IR (KBr, cm−1): 3395 (OH), 2953, 2931, 2872 (CHaliph.), 1648 (C=O), 1611 (C=N). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 8.07 (d, 1H, Ar-H, Ha, *J* = 8.0 Hz), 7.75 (t, 1H, Ar-H, Hc, *J* = 7.8 Hz, *J* = 7.6 Hz), 7.57 (d, 1H, Ar-H, Hd, *J* = 8.0 Hz), 7.44 (t, 1H, Ar-H, Hb, *J* = 7.6 Hz, *J* = 7.2 Hz), 4.95 (t, 1H, OH, exchangeable with D2O, *J* = 5.6 Hz), 4.11 (t, 2H, CH2CH2OH, *J* = 5.6 Hz, *J* = 6.0 Hz), 3.65 (q, 2H, CH2OH, *J* = 6.0 Hz, *J* = 5.6 Hz), 2.93 (t, 2H, N=CCH2, *J* = 7.6 Hz, *J* = 8.0 Hz), 1.75 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 7.6 Hz), 1.40−1.32 (m, 4H, CH3CH2CH2), 0.88 (t, 3H, CH3, *J* = 7.2 Hz). 13C-NMR (100 MHz, DMSO-*d*6) δ (ppm): 161.7, 158.4, 147.4, 134.6, 127.1, 126.5, 126.4, 120.4, 58.8, 46.1, 34.6, 31.3, 26.3, 22.4, 14.3. MS *m*/*z* (%): 260 (M+; 41.2). Anal. Calcd. for C15H20N2O2 (260.34): C, 69.20; H, 7.74; N, 10.76. Found: C, 69.17; H, 7.68; N, 10.81.

#### 3.1.7. 3-Amino-2-pentylquinazolin-4(3*H*)-one **9**

A mixture of benzoxazinone **4** (2.17 g, 0.01 mol) and hydrazine hydrate (1.5 mL) in absolute ethanol (20 mL) was refluxed for 3 h. The mixture was poured onto ice cold water, the formed solid was filtered off, and recrystallized from ethanol to give **9** as buff crystals; m.p.: 58–60 ◦C, yield: 43%. IR (KBr, cm−1): 3306, 3263 (NH2), 2954, 2931, 2910, 2856 (CHaliph.), 1673 (C=O), 1630 (C=N). 1H NMR (400 MHz, DMSO-*d*6) δ (ppm): 8.08 (d, 1H, Ar-H, Ha, *J* = 7.8 Hz), 7.75 (t, 1H, Ar-H, Hc, *J* = 7.4 Hz, *J* = 7.8 Hz), 7.59 (d, 1H, Ar-H, Hd, *J* = 7.6 Hz), 7.45 (t, 1H, Ar-H, Hb, *J* = 7.2 Hz, *J* = 7.6 Hz), 5.70 (s, 2H, NH2, exchangeable with D2O), 2.90 (t, 2H, N=CCH2, *J* = 7.2 Hz, *J* = 8.0 Hz), 1.74 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 7.2 Hz), 1.37−1.30 (m, 4H, CH3CH2CH2), 0.87 (t, 3H, CH3, *J* = 6.4 Hz, *J* =7.2 Hz). 13C-NMR (400 MHz, DMSO-d6) δ (ppm): 160.9, 158.8, 147.0, 134.3, 127.2, 126.39, 126.31, 120.2, 34.0, 31.4, 26.0, 22.3, 14.3. MS *m*/*z* (%): 231 (M+; 41.1). Anal. Calcd. for C13H17N3O (231.30): C, 67.51; H, 7.41; N, 18.17. Found: C, 67.39; H, 7.34; N, 18.24.

#### 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.

#### 3-((4-Chlorobenzylidene)amino)-2-pentylquinazolin-4(3*H*)-one **11a**

Yellow crystals; m.p.: 176–178 ◦C (ethanol), yield: 72%. IR (KBr, cm−1): 2943, 2866 (CHaliph.), 1667 (C=O), 1624 (C=N). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 8.69 (s, 1H, N=CH), 7.95 (d, 1H, Ar-H, Ha, *J* = 7.8 Hz), 7.88 (d, 2H, Ar-H, HE + HF, *J* = 8.8 Hz), 7.66 (t, 1H, Ar-H, Hc, *J* = 8.4 Hz), 7.57 (d, 3H, Ar-H, Hd + HX + HZ), 7.46 (t, 1H, Ar-H, Hb, *J* = 8.4 Hz), 2.34 (t, 2H, N=CCH2, *J* = 7.6 Hz), 1.60 (quintet, 2H, N=CCH2CH2, *J* = 7.2 Hz), 1.31−1.26 (m, 4H, CH3CH2CH2), 0.85 (t, 3H, CH3, *J* = 6.8 Hz). MS *m*/*z* (%): 353 (M+; 4.0). Anal. Calcd. for C20H20ClN3O (353.85): C, 67.89; H, 5.70; Cl, 10.02; N, 11.88. Found: C, 67.78; H, 5.62; Cl, 9.89; N, 11.79.

#### 3-((4-Methoxybenzylidene)amino)-2-pentylquinazolin-4(3*H*)-one **11b**

White crystals; m.p.: 83–84 ◦C (petroleum ether 60–80 ◦C), yield: 64%. IR (KBr, cm−1): 2946, 2912, 2882, 2843 (CHaliph.), 1669 (C=O), 1606 (C=N or C=C). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 8.81 (s, 1H, N=CH), 8.12 (d, 1H, Ar-H, Ha, *J* = 7.8 Hz), 7.89 (d, 2H, Ar-H, HE + HF, *J* = 8.8 Hz), 7.80 (t, 1H, Ar-H, Hc, *J* = 8.2, Hz, *J* = 7.4 Hz), 7.65 (d, 1H, Ar-H, Hd, *J* = 8.0 Hz), 7.50 (t, 1H, Ar-H, Hb, *J* = 7.6, Hz, *J* = 7.4 Hz), 7.12 (d, 2H, Ar-H, HX + HZ, *J* = 8.8 Hz), 3.85 (s, 3H, OCH3), 2.80 (t, 2H, N=CCH2, *J* = 7.6 Hz, *J* = 8.0 Hz), 1.71 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 7.2 Hz), 1.33−1.26 (m, 4H, CH3CH2CH2), 0.81 (t, 3H, CH3, *J* = 7.2 Hz). MS *m*/*z* 7(%): 349 (M+; 11.). Anal. Calcd. for C21H23N3O2 (349.43): C, 72.18; H, 6.63; N, 12.03. Found: C, 72.29; H, 6.69; N, 11.88.

#### 3-((3-Hydroxy-4-methoxybenzylidene)amino)-2-pentylquinazolin-4(3*H*)-one **11c**

White crystals; m.p.: 150–152 ◦C (ethanol), yield: 57%. IR (KBr, cm−1): 3277 (OH), 2956, 2927, 2892, 2863, 2845 (CHaliph.), 1678 (C=O), 1603 (C=N or C=C). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 9.49 (s, 1H, OH, exchangeable with D2O), 8.70 (s, 1H, N=CH), 8.12 (d, 1H, Ar-H, Ha, *J* = 8.0 Hz), 7.79 (t, 1H, Ar-H, Hc, *J* = 8.0 Hz, *J* =8.4 Hz), 7.65 (d, 1H, Ar-H, Hd, *J* = 7.6 Hz), 7.49 (t, 1H, Ar-H, Hb, *J* = 8.0 Hz, *J* = 7.2 Hz), 7.42 (d, 1H, HF, *Jm* = 2 Hz), 7.30, 7.28 (d,d, 1H, Ar-H, HE, *Jo* = 8.4 Hz, *Jm* = 2 Hz), 7.07 (d, 1H, Ar-H, HX, *J* = 8.4 Hz), 3.85 (s, 3H, OCH3), 2.78 (t, 2H, N=CCH2, *J* = 7.6 Hz), 1.71 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 7.2 Hz), 1.31−1.28 (m, 4H, CH3CH2CH2), 0.81 (t, 3H, CH3, *J* = 6.8 Hz, *J* = 7.2 Hz). MS *m*/*z* (%): 365 (M+; 23.4). Anal. Calcd. for C21H23N3O3 (365.43): C, 69.02; H, 6.34; N, 11.50. Found: C, 68.88; H, 6.28; N, 11.62.

#### 3-((4-Hydroxy-3,5-dimethoxybenzylidene)amino)-2-pentylquinazolin-4(3*H*)-one **11d**

White crystals; m.p.: 148–150 ◦C (benzene), yield: 61%. IR (KBr, cm−1): 3408 (OH), 2952, 2911, 2844 (CHaliph.), 1668 (C=O), 1591 (C=N or C=C). 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.36 (br.s, 1H, OH, exchangeable with D2O), 8.72 (s, 1H, N=CH), 8.12 (d, 1H, Ar-H, Ha, *J* = 8.2 Hz), 7.79 (t, 1H, Ar-H, Hc, *J* = 8.0 Hz, *J* = 7.4 Hz), 7.65 (d, 1H, Ar-H, Hd, *J* = 7.6 Hz), 7.50 (t, 1H, Ar-H, Hb, *J* = 8.0 Hz, *J* = 7.2 Hz), 7.23 (s, 2H, HE + HF), 3.82 (s, 6H, 2OCH3), 2.81 (t, 2H, N=CCH2, *J* = 7.6 Hz, *J* = 8.0 Hz), 1.72 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 7.2 Hz), 1.36−1.26 (m, 4H, CH3CH2CH2), 0.81 (t, 3H, CH3, *J* = 6.8 Hz, *J* =7.2 Hz). 13C-NMR (100 MHz, DMSO-*d*6) δ (ppm): 169.8, 158.0, 156.5, 148.6 (2), 146.7, 140.8, 134.6, 127.4, 127.0, 126.7, 122.8, 121.3, 106.8 (2), 56.5 (2), 34.5, 31.3, 26.0, 22.2, 14.2. MS *m*/*z* (%): 395 (M+; 62.1), Anal. Calcd. for C22H25N3O4 (395.46): C, 66.82; H, 6.37; N, 10.63. Found: C, 66.95; H, 6.41; N, 10.58.

#### 3.1.9. 2-(4-Chlorophenyl)-3-(4-oxo-2-pentylquinazolin-3(4*H*)-yl)thiazolidin-4-one **12**

A mixture of compound **11a** (3.53 g, 0.01 mol) and methyl thioglycolate (0.89 mL, 0.01 mol) in absolute ethanol (30 mL) containing piperidine (0.5 mL) was refluxed for 3 h. The obtained solid after evaporation of the solvent was collected and recrystallized from petroleum ether 60–80 ◦C to give **12** as pale yellow crystals; m.p.: 78–80 ◦C, yield: 47%. IR (KBr, cm−1): 2951, 2925, 2868 (CHaliph.), 1736 (C=Othiazolidinone), 1671 (C=Oquinazolinone), 1608 (C=N or C=C). 1H-NMR (100 MHz, DMSO-*d*6) δ (ppm): 8.13 (d, 1H, Ar-H, Ha, *J* = 8.2 Hz), 7.96 (d, 2H, Ar-H, HE + HF, *J* = 8.0 Hz), 7.80 (t, 1H, Ar-H, Hc, *J* = 7.6 Hz), 7.65 (d, 1H, Ar-H, Hd, *J* = 8.0 Hz), 7.64 (d, 2H, Ar-H, HX + HZ, *J* = 8.4 Hz), 7.51 (t, 1H, Ar-H, Hb, *J* = 7.6 Hz, *J* = 7.8 Hz), 5.70 (s, 1H, SCH), 3.75, 3.67 (d,d, 2H, CH2(thiazolidinone), *J* = 23.6 Hz, *J* = 23.2 Hz), 2.81 (t, 2H, N=CCH2, *J* = 8.0 Hz, *J* = 7.6 Hz), 1.71 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 8.0 Hz), 1.35−1.17 (m, 4H, CH3CH2CH2), 0.80 (t, 3H, CH3, *J* = 7.2 Hz). MS *m*/*z* (%): 427 (M+; 11.8). Anal. Calcd. for C22H22ClN3O2S (427.95): C, 61.75; H, 5.18; Cl, 8.28; N, 9.82; S, 7.49. Found: C, 61.66; H, 5.12; Cl, 8.31; N, 9.75; S, 7.55.

#### 3.1.10. 4,5,6,7-Tetrachloro-2-(4-oxo-2-pentylquinazolin-3(4*H*)-yl)isoindoline-1,3-dione **13**

Compound **9** (2.31 g, 0.01 mol) was fused with 4,5,6,7-tetrachloroisobenzofuran-1,3-dione (2.85 g, 0.01 mol) in oil bath for an hour. The resulting solid was recrystallized from ethanol to give **13** as orange crystals; m.p.: 178–180 ◦C, yield: 86%. IR (KBr, cm−1): 2943, 2856 (CHaliph.), 1788, 1746 (C=Oimide), 1705 (C=Oquinazolinone), 1606 (C=N or C=C). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 8.08 (d, 1H, Ar-H, Ha, *J* = 8 Hz), 7.94 (t, 1H, Ar-H, Hc, *J* = 8.0 Hz, *J* = 7.2 Hz), 7.76 (d, 1H, Ar-H, Hd, *J* = 8.0 Hz), 7.60 (t, 1H, Ar-H, Hb, *J* = 7.6 Hz), 2.73 (t, 2H, N=CCH2, *J* = 6.8 Hz, *J* = 8.0 Hz), 1.70−1.60 (m, 2H, N=CCH2CH2), 1.29−1.27 (m, 4H, CH3CH2CH2), 0.81 (t, 3H, CH3, *J* = 6.8 Hz, *J* =7.2 Hz). 13C-NMR (100 MHz, DMSO-*d*6) δ (ppm): 161.5, 161.3, 157.9, 157.7, 147.4, 146.7, 140.0, 138.8, 136.6 (2), 128.2 (2), 128.0, 127.1, 126.8, 119.8, 32.6, 30.9, 26.0, 22.2, 14.2. MS *m*/*z* (%): 231 (M+; 41.1). Anal. Calcd. for C21H15Cl4N3O3 (499.17): C, 50.53; H, 3.03; Cl, 28.41; N, 8.42. Found: C, 50.61; H, 3.09; Cl, 28.37; N, 8.53.

#### 3.1.11. 3-Amino-2-pentylquinazoline-4(3*H*)-thione **14**

A mixture of compound **9** (2.31 g, 0.01 mol) and P2S5 (2.22 g, 0.01 mol) in dry toluene (15 mL) was heated under reflux for 4 h. The mixture was filtered off, the filtrate was evaporated under reduced pressure, the obtained solid was collected, dried, and recrystallized from ethanol to give **14** as yellow crystals; m.p.: 57–59 ◦C, yield: 53%. IR (KBr, cm−1): 3240, 3200 (NH2), 2925, 2855 (CHaliph.), 1591 (C=N or C=C), 1238 (C=S). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 8.51 (d, 1H, Ar-H, Ha, *J* = 8.2 Hz), 7.82 (t, 1H, Ar-H, Hc, *J* = 7.6 Hz, *J* = 7.8 Hz), 7.69 (d, 1H, Ar-H, Hd, *J* = 7.6 Hz), 7.57 (t, 1H, Ar-H, Hb, *J* = 7.6 Hz), 7.04 (s, 2H, NH2, exchangeable with D2O), 3.06 (t, 2H, N=CCH2, *J* = 7.2 Hz, *J* = 8.0 Hz), 1.81 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 7.2 Hz), 1.42−1.32 (m, 4H, CH3CH2CH2), 0.88 (t, 3H, CH3, *J* = 6.8 Hz, *J* =7.2 Hz). 13C NMR (100 MHz, DMSO-*d*6) δ (ppm): 182.1, 155.5, 142.0, 134.5, 130.6, 128.1 (2), 127.4, 34.4, 31.3, 25.5, 22.4, 14.3. MS *m*/*z* (%): 247 (M+; 74.3). Anal. Calcd. for C13H17N3S (247.36): C, 63.12; H, 6.93; N, 16.99; S, 12.96. Found: C, 63.19; H, 6.96; N, 17.11; S, 12.82.

#### *3.2. Cytotoxicity and Antiproliferative Evaluation*

#### MTT Assay

The implement of MTT methodology for the antiproliferative screening of quinazolinone derivatives **5**–**14** along with compound **3** against two cell lines, namely, hepatocellular carcinoma (HepG2) and mammary gland (MCF-7) were obtained from ATCC through the Holding company for biological products and vaccines (VACSERA), Cairo, Egypt. The reference anticancer drug used was Doxorubicin. The MTT assay was carried out at the pharmacology department, Faculty of pharmacy, Mansoura University, Egypt according to the reported literatures [42,43,54]. The cells were cultured in a RPMI-1640 medium with 10% fetal bovine serum, followed by the addition of antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin) at 37 ◦C in a 5% CO2 incubator. The cells were seeded in a 96-well plate at a density of (1.0 × 104 cells/well) at 37 ◦C for 48 h under 5% CO2. Treatment of cells with different concentrations of compounds such as 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 μM was carried out and placed in the incubator for 24 h. Then, 20 μL of MTT solution at 5 mg/mL was added and incubated for 4 h. DMSO (100 μL) was added into each well to dissolve the purple formazan formed. At 570 nm absorbance the colorimetric assay was measured and recorded by using a plate reader (BioTek EL ×800 Microplate Reader, BioTek Instruments, Inc, Winooski, VT, USA).

Calculation of the relative cell viability (%) = (A of treated samples /A of untreated sample) × 100.

#### *3.3. Antioxidant Assay*

#### 3.3.1. Antioxidant Activity Screening Assay

#### 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 MnO2 (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 λmax 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]:

$$^{0\text{\textquotedblleft}I\text{\textquotedblright}I\text{\textquotedblright}} = [A\_{\text{(control)}} - A\_{\text{(test)}} / A\_{\text{(control)}}] \times 100\tag{1}$$

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 λmax 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.

The scavenging activity percentage (*AA* %) was determined according to Mensor et al. [57]:

$$AA\ \%=100-\left[\left(Abs\_{\text{(sample)}}-Abs\_{\text{(blank)}}\right)A\text{bs}\_{\text{(control)}}\right]\times100\right] \tag{2}$$

#### *3.4. 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).

#### **4. 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.

**Author Contributions:** The listed authors contributed to this work as described in the following: A.A.E.-S., M.F.I., and A.E.-G.E.A. designed the research idea, implemented the synthesis and characterization of novel compounds, and contributed to the data interpretation. A.E.-G.E.A. and A.M.N. contributed to discuss the results, writing the original draft manuscript, and revisions. All authors read and approved the final manuscript.

**Funding:** The authors are grateful to the Deanship of Scientific Research, King Saud University for funding this work through research group project "RGP-172".

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

#### **References**

**Sample Availability:** Samples of the compounds are available from the authors.

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Acknowledgements

The author gratefully acknowledges the authorities of Royal Commission for Jubail and Yanbu and the authorities of Jubail Industrial College for the facilities offered to carry out the work. This work is part of the JRICH (Jubail Research and Innovation Cluster Hub) initiative of the Jubail Industrial College.

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