Antioxidant and Cytotoxic Activity of New Polyphenolic Derivatives of Quinazolin-4(3H)-one: Synthesis and In Vitro Activities Evaluation

The development of hybrid molecules with significant human therapeutic properties is one of the main approaches of pharmaceutical research. One of the most important pharmacophores is the quinazolin-4(3H)-one heterocycle moiety, due to its wide range of biological activities. By its derivatization with polyphenolic compounds, in our previous research, it proved to possess a good antiradical activity of ortho-diphenolic derivatives of quinazolin-4(3H)-one. In this study, we developed two new series of compounds, with an additional phenolic group or with a methyl group on the thioacetohydrazone fragment. The methods used to evaluate the activity of the compounds were radical scavenging, reduction of oxidizing reagents and transition metals’ ions chelation assays. Quantum descriptors were also calculated in order to evaluate the influence of substituents and their position on the activity of the compounds. The cytotoxic activity was evaluated using normal human foreskin fibroblast cells (BJ) and two cancerous cell lines, lung adenocarcinoma cells (A549) and prostate carcinoma cells (LNCaP). The results obtained for the pyrogallol derivatives showed a high antioxidant activity compared to ascorbic acid and Trolox. All the synthesized compounds displayed a higher cytotoxicity against the cancerous cell types and a high cytocompatibility with the normal cells. The antioxidant activity was deeply influenced by the addition of the third phenolic group in the synthesized molecules.

Today, the medical and pharmaceutical fields are subject to huge progress, with the help of increasingly advanced technology. An interesting observation emphasizes oxidative stress and its effects on the human body. Continuous oxidative stress can be harmful to human cells and can ultimately lead to chronic inflammation. By activating transcription In the in vitro evaluation of the antioxidant capacity, several antiradical, electron transfer, ferrous and cupric ions chelation assays were used. Cytotoxicity was evaluated using normal human foreskin fibroblasts (BJ) in order to establish the safety of the compounds and two cancerous cell types, namely A549 (lung adenocarcinoma) and LNCaP (prostate carcinoma), to establish a possible anticancerous activity. The theoretical quantum and thermodynamical calculations were also studied for these newly synthesized compounds.
Therefore, we present the chemical synthesis, design, quantum studies and in vitro antioxidant and cell toxicity activities of the new quinazolin-4-one polyphenolic derivatives.

Chemistry
For all synthesis, purification, structural analysis, and in vitro biological activity evaluation, the reagents were purchased from local suppliers and used in accordance with the instructions.
Melting points were measured using a melting point device MPM-H1 (Schorpp Gerätetechnik, Überlingen, Germany), based on the glass capillary method.
The IR spectra were recorded in KBr pellets, under vacuum, with a FT/IR 6100 spectrometer (Jasco, Cremella, Italy).
An Agilent Ion Trap SL mass spectrometer (70 eV) instrument (Agilent Technologies, Santa Clara, CA, USA) was used in negative ionization mode, for the final compounds 5ad and 6a-d. 1 H-NMR and 13 C-NMR spectra were recorded using an Avance NMR spectrometer (Bruker, Karlsruhe, Germany), in dimethylsulfoxide-d6 (DMSO-d6). The calibration of the spectrometer was made using tetramethylsilane. The following abbreviations for peak patterns were used to identify the multiplicity of the signals in the 1 H-NMR spectra: brbroad, s-singlet, d-doublet, dd-double doublet, t-triplet, td-triplet of doublets, q-quartet, Scheme 1. Development of newly polyphenolic derivatives of quinazolin-4(3H)-one.

Chemistry
For all synthesis, purification, structural analysis, and in vitro biological activity evaluation, the reagents were purchased from local suppliers and used in accordance with the instructions.
Melting points were measured using a melting point device MPM-H1 (Schorpp Gerätetechnik, Überlingen, Germany), based on the glass capillary method.
The IR spectra were recorded in KBr pellets, under vacuum, with a FT/IR 6100 spectrometer (Jasco, Cremella, Italy).
An Agilent Ion Trap SL mass spectrometer (70 eV) instrument (Agilent Technologies, Santa Clara, CA, USA) was used in negative ionization mode, for the final compounds 5a-d and 6a-d. 1 H-NMR and 13 C-NMR spectra were recorded using an Avance NMR spectrometer (Bruker, Karlsruhe, Germany), in dimethylsulfoxide-d 6 (DMSO-d 6 ). The calibration of the spectrometer was made using tetramethylsilane. The following abbreviations for peak patterns were used to identify the multiplicity of the signals in the 1 H-NMR spectra: brbroad, s-singlet, d-doublet, dd-double doublet, t-triplet, td-triplet of doublets, q-quartet, quint-quintet, sext-sextet and m-multiplet, respectively. For the signals given by the hydrogen or carbon atoms, to describe the location of the atom in a specific region of the molecule, some abbreviations were used: Q-quinazolin-4(3H)-one, Bz-benzyl and Ar-phenolic benzene ring.

Synthesis of Intermediate Compounds 1a-d, 2a-d, 3a-d
The protocol followed for the synthesis of the intermediate compounds 1a-d, 2a-d, 3ad was previously reported by our group. Characterization of the intermediate compounds synthesized in order to obtain the final compounds 5a-d and 6a-d that were previously reported in the literature was consistent with the original papers [21][22][23][24]. The intermediate hydrazides 3a-d used in the present research were obtained during our previous research and were used in the current study without being resynthesized [16].

Synthesis of Compounds 5a-d
Two mmol of the appropriate quinazoline-4-one acetohydrazide 3a-d were suspended in 8 mL of ethanol 96%, in a glass flask. Two mmol of 2,3,4-trihydroxybenzaldehyde and a drop of glacial acetic acid were added. The mixture was refluxed gently, for 3 h, under a condenser. The resulting precipitate was filtered under a vacuum, dried and recrystallized from dioxane.

In Vitro Antioxidant, Antiradical and Chelation Assays
The stock solutions (1 mg/mL) of the tested compounds and the controls were obtained, by dissolving the substances in dimethyl sulfoxide (DMSO). Using Jasco V-530, an UV-VIS spectrophotometer (Jasco International Co., Ltd., Tokyo, Japan), we performed the colorimetric determinations in low-volume single-use 10 mm width cuvettes. Solutions of the test compounds did not have absorption maxima near the wavelengths at which these assays were performed.
The assays were performed in triplicate and the results are presented as averages for each assay.

Antiradical Assays
By monitoring the absorbance of reagent in the presence of various amounts of evaluated compounds and controls at λ = 734 nm, a green ABTS˙+ (2,2 -azinobis-(3ethylbenzothiazoline-6-sulfonic acid) discoloration assay was performed. The previous protocols reported by our group are based on the initial report of Re et al. [16,25,26]. Using Equation (1), we calculated the ABTS˙+ scavenging activity of the compounds 5a-d and 6a-d: The DPPH˙radical scavenging assay is based on the transfer of one hydrogen from the analyzed compound to the violet stable free radical DPPH˙(2,2-diphenyl-1-picrylhydrazyl), as we convert it into a yellow compound. The absorbance of the reagent mixed with samples presented inversely proportional values to the amount of DPPH˙neutralized. The absorbance of the reagent was measured at λ = 517 nm, according to our previous reports [16,26]. The assay was made in the presence of various concentrations of samples. The used protocol is based on the report of Brand-Williams et al. [27]. Using Equation (2), we calculated the DPPH˙radical scavenging activity of the compounds 5a-d and 6a-d: The scavenging of the NO˙radical was performed by applying previously reported protocols [16,26]. The in vitro protocol describes the decomposition of sodium nitroprusside in phosphate buffered saline (PBS) at pH = 7.4. The NO˙radicals would be involved in a Griess reaction if they were not captured by the tested compounds. This reaction results from the formation of an azo dye, with maximum absorption at 546 nm. This azo dye is formed from sulfanilic acid and naphthyl ethylenediamine dichloride [28][29][30]. Using Equation (3), we calculated the NO˙radical scavenging activity of the compounds 5a-d and 6a-d: NO. scavenging (%) = control absorbance − sample absorbance control absorbance × 100

Electron Transfer Assays
In the ferric reducing antioxidant potential (FRAP) assay, in comparison with a blank sample prepared from DMSO mixed with FRAP reagent, the absorbance of the solutions was measured at λ = 593 nm. The compounds were tested according to a modified method proposed initially by Benzie and Strain [31]. Using Equation (4), we calculated the reducing antioxidant potential of 5a-d and 6a-d as a percentage of the activity of the reference compounds: % of control activity = sample absorbance reference absorbance × 100 The reduction capacity of the ferric ion displayed by the tested compounds was determined with the use of a supplementary method. Herein, the iron (III) is not free in the solution as it was in the FRAP assay and is complexed by cyanide ions. The evaluation of the reducing power (RP) was performed at a pH closer to neutral (pH = 6.6); this is different from the FRAP test, which is performed in an acidic environment. Against a blank sample, the absorbances were measured at λ = 700 nm. The followed protocol was based on the adaptation of previously reported protocols [30] and was previously reported by  [16,32,33]. Using Equation (4), we calculated the reducing power of the 5a-d and 6a-d compounds as a percentage of the reference compounds activity.
Using the phosphomolybdate assay, we determined the total antioxidant capacity (TAC) of the tested compounds. We measured the solutions' absorbances against a blank sample at λ = 695 nm, using a previously reported procedure by our group [16,32,33], based on initial literature reports [30,34]. Using Equation (4), we calculated the TAC of 5a-d and 6a-d compounds.
The ability to donate electrons was evaluated for the compounds 5a-d and 6a-d, by the CUPRAC (CUPric reducing antioxidant capacity) method. The absorbance was measured against a blank sample, at λ = 450 nm. The assay was performed based on our group's previous report [16] by the adaptation of the initial report of Alam et al., Özyürek et al. and Apak et al. [30,35,36]. With the use of Equation (4), the cupric ion reduction by compounds 5a-d and 6a-d was expressed as a percentage of the reference compound activity.

Transition Metals' Ions Chelation Assays
In the protocol used for evaluating the Fe 2+ chelating ability of the compounds, the absorbances of the solutions were determined at λ = 562 nm, against a blank sample. The method was adapted from the initial report of Benzie and Strain et al. [31], which was previously reported by our group [16,37]. Using Equation (5), we calculated the iron chelation capacities of the 5a-d and 6a-d compounds: iron chelation (%) = control absorbance − sample absorbance control absorbance × 100 With murexide as a chromophoric chelator, we performed the protocol followed for the evaluation of Cu 2+ chelating activities of the compounds using an adaptation of the method presented by Cesari et al. and Wu et al. [38,39]. Using Equation (6), we calculated the copper chelation capacities of the 5a-d and 6a-d compounds:

Theoretical Quantum and Thermodynamical Calculations
The importance of in silico studies has increased over the past few years in the medicinal chemistry research field. By using modern technology, with the help of computers, we can calculate some molecular descriptors, to obtain information of particular significance regarding the explanation or prediction of some experimental results. Through this method, the characteristics of the synthesized compounds (5a-d and 6a-d) can be determined.
The main molecular descriptors used in the literature are the energy levels of frontier orbitals such as HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital). They are joined by some thermodynamic calculations, which evaluate the ease of releasing hydrogen atoms from a molecule that acts as an antioxidant compound. When a compound releases hydrogen, the resulting radical must stabilize by internal conjugation, expressed by a lower energy state. This can be calculated as the BDE (bond dissociation enthalpy) of the hydrogen-releasing groups.
The antioxidant activity can be manifested in the case of these new chemically synthesized compounds through the polyphenolic structure, because they present a multitude of phenolic groups.
All theoretical in silico assays reported in this paper were performed following a previously reported protocol [33,40,41]. Using Chimera 1.10.2 (University of California, San Francisco, CA, USA), we generated the depictions of the lowest energy conformation of the compounds [42]. Normal human foreskin (BJ) fibroblasts and two types of cancer cells, namely A549 (lung adenocarcinoma) and LNCaP (prostate carcinoma) purchased from American Type Culture Collection (Manassas, VA, USA), were used in the experiments. Each cell line was maintained in a different culture medium, depending on the metabolic necessities of the cells. BJ cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with low glucose. A549 cells were maintained in DMEM with high glucose (1 g/L), while LNCaP cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium with high glucose (5 g/L). All media used were supplemented with 10% fetal bovine serum (FBS) and were refreshed every two days. Cells were used in experiments or subcultured when they reached 70-80% confluence.

Experimental
In 100 µL in 96-well plates, a number of 30,000 LNCaP cells, 15,000 A549 cells, and 7500 normal (BJ) cells were seeded. Because the cells have different sizes and volumes, the purpose of the different seedings was to achieve a confluency of 70-80% after 24 h. All types of cells were further exposed to the synthesized compounds (5a-d and 6a-d) for 24 h, at a concentration ranging from 12.5 to 100 µg/mL. Post-exposure, the media were removed, and the cells were washed with phosphate buffer saline (PBS). Their viability was evaluated using Alamar blue (AB) assay as previously described [43]. Briefly, the cells were incubated with a 200 µM resazurin solution for 4 h. Using a Synergy 2 multi-mode microplate reader, we measured the fluorescence. The measurement was performed at λ excitation = 530/25 nm; λ emission = 590/35 nm. We conducted the experiments using three biological replicates, each one including 6 technical replicates. As negative controls (NC), cells exposed to culture medium containing 0.2% DMSO were used. Compared to the NC (100%), the results were expressed as relative values. We used IC 50 values for the potency evaluation, and calculated them for each cell line, using the dose-effect curves obtained by fitting the experimental data with a 4-parameter logistic curve.
Mean values ± standard deviation (SD) values of at least three biological replicates were used to calculate experimental data. One-way analysis of variance (ANOVA) with a post hoc Holm-Sidak test was used to statistically examine the data. Using SigmaPlot 11.0 software (Systat, Software Inc., Chicago, IL, USA), we performed the graphical display and data analysis. From a statistical point of view, the results were considered different, at values of p < 0.05.

Molecular Properties with Influence on the Pharmacokinetics of Compounds
In order to obtain preliminary information regarding molecular properties which could influence the pharmacokinetics of the compounds, we obtained them using Swis-sADME [44]. The evaluated molecular properties were the topological polar surface area (TPSA) [45], octanol-water partition coefficient expressed as Moriguchi's LogP [46], water solubility [47] and violations of the Lipinski's rule of five [46].
The spectral data resulting from the analysis were consistent with the proposed structures. In the IR spectra for all the synthesized compounds 5a-d and 6a-d, the desired signals were revealed.
The spectral data resulting from the analysis were consistent with the proposed structures. In the IR spectra for all the synthesized compounds 5a-d and 6a-d, the desired signals were revealed.
For the compounds 5a-d, two strong νC=O stretching signals were found between 1637.27-1696.57 cm −1 : one from the quinazolin-4(3H)-one heterocycle and another one from the thioacetohydrazone linker. The νC=N stretching, from the quinazolin-4(3H)-one heterocycle, had a specific signal between 1550.97-1558.68 cm −1 . The νN-H stretching bands, from the hydrazone derivate, were between 3414.83-3508.85 cm −1 . The νO-H stretching, from the phenolic groups, were as wide bands between 2918.25-3445.69 cm −1 . All the identified signals proved that the condensation took place successfully.
For the compounds 6a-d, between 1548.56-1697.53 cm −1 , three strong νC=O stretching signals were found: one from the quinazolin-4(3H)-one heterocycle, one from the thioacetohydrazone linker and another from the 2,4-dihydroxy-acetophenone. Between 1550.49-1570.46 cm −1 , the νC=N stretching had a specific signal from the quinazolin-4(3H)one heterocycle. Between 3469.79-3481.36 cm −1 , the νN-H stretching bands had a signal from the hydrazone derivate. Between 3238.38-3420.14 cm −1 , the νO-H stretching bands were wide due to the presence of phenolic groups. All the signals were in accordance with the proposed structures.
In the MS spectra of the synthesized compounds 5a-d and 6a-d, the corresponding molecular mass peaks were identified.
In the 1 H-NMR spectra of the 5a-d and 6a-d compounds, all the expected signals of all protons were identified, with the corresponding multiplicity. In the 13 C-NMR spectra of compounds, the expected signals corresponding to the carbon atoms were identified. All the signals were in the expected region of each spectrum.
For compounds 5a-d and 6a-d, the graphic depictions of the recorded spectra are provided in the Supplementary Materials ( Figures S1-S32).

In Vitro Antioxidant, Antiradical and Chelation Assays
The antioxidant potentials of compounds 5a-d and 6a-d were evaluated based on their direct antiradical activity and reduction of oxidized reagents and, complementarily on their transition metals' chelation activity. The in vitro protocols applied for the current evaluations were performed at a semi-microscale level, according to our group's previous reports and using reference compounds [32,33]. All determinations were performed in triplicate and results are presented as averages.

Antiradical Assays
The antiradical potential of compounds 5a-d and 6a-d was evaluated spectrophotometrically as a capacity to scavenge the ABTS˙+, DPPH˙and NO˙radicals.

ABTS˙+ Radical Scavenging Assay
Compounds 5a-d and 6a-d were screened for their ABTS˙+ radical scavenging capacity and the results obtained are presented in Table 1. Trolox and ascorbic acid were used as positive controls. The most active compounds were 5a, 5c and 5d, having lower IC 50 values than those of the antioxidant reference drugs.

DPPH˙Radical Scavenging Assay
The DPPH˙scavenging activity of the compounds 5a-d and 6a-d was evaluated spectrophotometrically and the obtained results are presented in Table 2. The reference antioxidants were ascorbic acid and Trolox. Compounds 5a, 5c and 5d had the best activity. -represents less than 5%; + represents more than 95%.

NO˙Radical Scavenging Assay
The NO˙antiradical potential of compounds 5a-d and 6a-d and gentisic acid was evaluated spectrophotometrically, based on the Griess reaction; the obtained results are presented in Table 3. Compounds 5a and 5c had the best ability to scavenge this radical.

Electron Transfer Assays
Using the FRAP, TAC, RP and CUPRAC spectrophotometric assays, we determined the antioxidant capacity of compounds 5a-d and 6a-d as the capacity of donating electrons.

Ferric Reducing Antioxidant Power (FRAP)
Ferrous ions resulting from the reduction of ferric ions in the presence of an antioxidant compound form a blue-colored complex (Fe 2+ -TPTZ) at pH = 3.6 with tripyridyltriazine (2,4,6-tris(2-pyridyl)-s-triazine). The intensity of the blue complex formed is proportional to the number of ferrous ions. The results obtained for the FRAP assay are presented in Table 4. Compounds 5a, 5c and 5d expressed a good electron donation capacity.

Phosphomolybdate Assay for Total Antioxidant Capacity (TAC)
At acidic pH, the tested compounds transfer one electron to Mo 6+ to reduce it to Mo 5+ . The higher amount of Mo 5+ green phosphate complex that results, the more active the tested compound is. The results of the TAC assay are presented in Table 4. The obtained results of the TAC assay proved the excellent electron-donating capacity of compounds 5a, 5b and 5c. Reducing Power (RP) Assay The evaluated compounds can reduce ferricyanide to ferrocyanide, in the presence of ferric ions, resulting in the Perl's Prussian which gives blue. The higher the percentage of reducing power, the higher was the absorbance that was measured due to a higher amount of colored complex resulting from the reaction. The results are presented in Table 4. Compounds 5a, 5b and 5c displayed a good activity.

Cupric Reducing Antioxidant Capacity (CUPRAC) Assay
The Cu 2+ ions are reduced to Cu + ions by the electron-donating compounds. The Cu + ions are chelated by neocuproine, giving a colored complex with absorbance proportional to the quantity of Cu + ions that resulted. The results of the CUPRAC assay are presented in Table 4. Compounds 5a, 5c and 5d displayed excellent activity.

Transition Metals' Ions Chelation Assays
Fe 2+ Chelation Assay The chelating capacity of the tested compounds of Fe 2+ was evaluated based on their competition for Fe 2+ with ferrozine. A decrease in the resulting absorbance indicated that the ferrous ions were sequestered by the evaluated compounds. The result of the Fe 2+ chelation assay is presented in Table 5. The most active compounds were 5a, 5c and 5d.  2 1.32 20.59 42.89 95.10 + + + + + -represents less than 5%; + represents more than 95%.

Cu 2+ Chelation Assay
The evaluation of the Cu 2+ chelation activity of compounds was made based on the competition for Cu 2+ with murexide. The results are presented in Table 6. The chelating activity of compounds for the cupric ions found in the current assay is significant for the pyrogallol derivatives, being quite close to EDTA-Na 2 .

Theoretical Quantum and Thermodynamical Calculations
The highest occupied molecular orbital (HOMO) is the parameter that indicates a good capacity for electron donation of the molecule which is due to the susceptibility of a molecule to be attacked by electrophilic species. The energy of the lowest unoccupied molecular orbital (LUMO) is the parameter that is related to its susceptibility to be attacked by nucleophilic species and to its electron affinity. The general structure representing the possible sites of 5a-d and 6a-d molecules to release hydrogen atoms is shown in Figure 1.

Cu 2+ Chelation Assay
The evaluation of the Cu 2+ chelation activity of compounds was made based on the competition for Cu 2+ with murexide. The results are presented in Table 6. The chelating activity of compounds for the cupric ions found in the current assay is significant for the pyrogallol derivatives, being quite close to EDTA-Na2.

Theoretical Quantum and Thermodynamical Calculations
The highest occupied molecular orbital (HOMO) is the parameter that indicates a good capacity for electron donation of the molecule which is due to the susceptibility of a molecule to be attacked by electrophilic species. The energy of the lowest unoccupied molecular orbital (LUMO) is the parameter that is related to its susceptibility to be attacked by nucleophilic species and to its electron affinity. The general structure representing the possible sites of 5a-d and 6a-d molecules to release hydrogen atoms is shown in Figure 1. The energy levels of HOMO and LUMO are presented in Table 7, together with the bond dissociation energies (BDEs). The BDEs of the sites in the molecules can yield hydrogen atoms (numbered H1-H5) and are derived from the in-silico calculations. The depiction of HOMO and LUMO as well as the depiction of the spin density maps of the phenol group radicalization are presented in Supplementary Materials (Tables S1 and S2). The energy levels of HOMO and LUMO are presented in Table 7, together with the bond dissociation energies (BDEs). The BDEs of the sites in the molecules can yield hydrogen atoms (numbered H 1 -H 5 ) and are derived from the in-silico calculations. The depiction of HOMO and LUMO as well as the depiction of the spin density maps of the phenol group radicalization are presented in Supplementary Materials (Tables S1 and S2).
Regarding HOMO energy levels, the identified values were lower for the pyrogallolderived molecules, compounds 5a, 5b, 5c and 5d, (between −5.53 eV and −5.51 eV) and slightly higher for compounds 6a, 6b, 6c and 6d (between −5.42 eV and −5.39 eV). In the case of the 5b and 5c molecules, the lowest HOMO orbital was identified, with an energy level of −5.53 eV. The highest HOMO orbital was identified in the case of the 6b and 6d molecules at −5.39 eV.
The lowest values for the LUMO energy levels were identified in 5b and 6b (−1.64 eV). Both compounds were substituted at the N 3 atom from the quinazolin-4(3H)-one ring with a benzyl moiety. The LUMO energy level in the 5b and 6b compounds was significantly influenced by this substituent, because in all the other compounds the values were quite similar (between −1.60 eV and −1.55 eV), energies being a little smaller in the compounds 5a, 5b, 5c and 5d compared to the compounds 6a, 6b, 6c and 6d. Table 7. The energy of the frontier orbitals and the BDE for the H 1 -H 5 sites in 5a-d and 6a-d compounds, according to the numeration presented in Figure 1. The HOMO-LUMO gap was found to be higher in the case of the pyrogallol-derived compounds 5a, 5b, 5c and 5d (between 3.89 eV and 3.93 eV), while in the case of the 2,4-dihydroxy-acetophenone derivatives 6a, 6b, 6c and 6d, the energy difference between the two frontier orbitals was found to be between 3.75 eV and 3.84 eV.

Compound Frontier Orbitals (eV) X-H BDE (kcal/mol
Overall, the most susceptible group to release hydrogen atoms is the meta-hydroxy (site H 3 ) found in compounds 5a, 5b, 5c and 5d (pyrogallol-derived compounds), with a BDE ranging from 71.98 kcal/mol to 72.00 kcal/mol. The next type of groups in ascending order of BDE that could yield hydrogen atoms are ortho-hydroxy (site H 1 ) in the 5a, 5b, 5c and 5d compounds, with a BDE ranging from 77.95 kcal/mol to 78.08 kcal/mol. The next type of group that releases hydrogen atoms is the para-hydroxy (site H 3 ), with similar values in both series of compounds, ranging between 78.26 kcal/mol and 79.49 kcal/mol.
Interestingly, the ortho-OH group (site H 1 ) in the case of 6a, 6b, 6c and 6d (dihydroxyacetophenone derivatives) had a significantly higher BDE compared to those in pyrogallolderived compounds 5a, 5b, 5c and 5d, relative to the same site H 1 , values ranging from 84.44 kcal/mol to 84.64 kcal/mol. Most likely, in the case of the dihydroxy-acetophenone derivatives compounds, compared to the pyrogallol-derived compounds, this is a consequence of the insertion of the methyl group on the azomethine carbon atom and the disappearance of the phenolic group from the meta position.
On the next level, in terms of ease of release of hydrogen atoms, is the N-H group (site 4). Hydrogen atoms could be more easily released from this site by the compounds from the dihydroxy-acetophenone derivatives compounds, having a BDE ranging from 83.12 kcal/mol to 83.39 kcal/mol, lower than those from the pyrogallol-derived compounds, which had a significantly higher BDE for the N-H bond, ranging from 86.01 kcal/mol to 89.91 kcal/mol. Abstraction of hydrogen atoms from the azomethine group (site H 5 ) would be difficult, because the BDE of the C-H bond was much higher, with values ranging from 100.98 kcal/mol to 106.64 kcal/mol. The BDE computed for the C-H bonds from the allyl group of compounds 5a and 6a was found to be more than 105 kcal/mol, which makes the allyl group inert in terms of hydrogen atom abstraction for neutralization of an external radical.
In all compounds, the ortho-hydroxy phenol made a hydrogen bond as a donor to the imine nitrogen atom from the hydrazone as an acceptor. The hydrazide−hydrazone group had a negative effect on the hydrogen release from the ortho-OH groups, due to the intramolecular hydrogen bonding, the BDE of OH from H 1 site being higher than ones from H 2 or H 3 . On the other hand, compounds from the pyrogallol-derived series were strongly favorized by the internal hydrogen bonding. The pseudo-polycyclic rings that appear due to the internal hydrogen bonding favored the stabilization by the extended internal conjugation of the resulting radicals after hydrogen atom release.
The conformation with the lowest energy of the 5a-d and 6a-d compounds with the intramolecular hydrogen bonds is presented in Table 8. Table 8. The conformation with the lowest energy of the 5a-d and 6a-d compounds with the depiction of the resulting hydrogen bonds.

Compound
Conformation Compound Conformation 5a the intramolecular hydrogen bonding, the BDE of OH from H1 site being higher than ones from H2 or H3. On the other hand, compounds from the pyrogallol-derived series were strongly favorized by the internal hydrogen bonding. The pseudo-polycyclic rings that appear due to the internal hydrogen bonding favored the stabilization by the extended internal conjugation of the resulting radicals after hydrogen atom release. The conformation with the lowest energy of the 5a-d and 6a-d compounds with the intramolecular hydrogen bonds is presented in Table 8. the intramolecular hydrogen bonding, the BDE of OH from H1 site being higher than ones from H2 or H3. On the other hand, compounds from the pyrogallol-derived series were strongly favorized by the internal hydrogen bonding. The pseudo-polycyclic rings that appear due to the internal hydrogen bonding favored the stabilization by the extended internal conjugation of the resulting radicals after hydrogen atom release. The conformation with the lowest energy of the 5a-d and 6a-d compounds with the intramolecular hydrogen bonds is presented in Table 8. from H2 or H3. On the other hand, compounds from the pyrogallol-derived series were strongly favorized by the internal hydrogen bonding. The pseudo-polycyclic rings that appear due to the internal hydrogen bonding favored the stabilization by the extended internal conjugation of the resulting radicals after hydrogen atom release. The conformation with the lowest energy of the 5a-d and 6a-d compounds with the intramolecular hydrogen bonds is presented in Table 8. from H2 or H3. On the other hand, compounds from the pyrogallol-derived series were strongly favorized by the internal hydrogen bonding. The pseudo-polycyclic rings that appear due to the internal hydrogen bonding favored the stabilization by the extended internal conjugation of the resulting radicals after hydrogen atom release. The conformation with the lowest energy of the 5a-d and 6a-d compounds with the intramolecular hydrogen bonds is presented in Table 8.

In Vitro Cytotoxicity Activity
The cytotoxic effects of the synthesized compounds 5a-d and 6a-d were evaluated on two cancerous cell lines (A549, LNCaP), in parallel with normal fibroblast cells (BJ). Independently of the concentration tested, all the compounds presented higher cytotoxicity towards the cancerous cell phenotypes (Figure 2). The citotoxiciy of doxorubicin, used as a standard agent, is presented in Figure 3.
The IC50 values were calculated for all the compounds towards doxorubicin ( Table  9). The results indicated that at all concentrations tested, the type of cells and the compounds tested had a statistically significant contribution to the recorded viability.

In Vitro Cytotoxicity Activity
The cytotoxic effects of the synthesized compounds 5a-d and 6a-d were evaluated on two cancerous cell lines (A549, LNCaP), in parallel with normal fibroblast cells (BJ). Independently of the concentration tested, all the compounds presented higher cytotoxicity towards the cancerous cell phenotypes (Figure 2). The citotoxiciy of doxorubicin, used as a standard agent, is presented in Figure 3.
The IC50 values were calculated for all the compounds towards doxorubicin ( Table  9). The results indicated that at all concentrations tested, the type of cells and the compounds tested had a statistically significant contribution to the recorded viability.

In Vitro Cytotoxicity Activity
The cytotoxic effects of the synthesized compounds 5a-d and 6a-d were evaluated on two cancerous cell lines (A549, LNCaP), in parallel with normal fibroblast cells (BJ). Independently of the concentration tested, all the compounds presented higher cytotoxicity towards the cancerous cell phenotypes (Figure 2). The citotoxiciy of doxorubicin, used as a standard agent, is presented in Figure 3.
The IC50 values were calculated for all the compounds towards doxorubicin ( Table  9). The results indicated that at all concentrations tested, the type of cells and the compounds tested had a statistically significant contribution to the recorded viability.

In Vitro Cytotoxicity Activity
The cytotoxic effects of the synthesized compounds 5a-d and 6a-d were evaluated on two cancerous cell lines (A549, LNCaP), in parallel with normal fibroblast cells (BJ). Independently of the concentration tested, all the compounds presented higher cytotoxicity towards the cancerous cell phenotypes (Figure 2). The citotoxiciy of doxorubicin, used as a standard agent, is presented in Figure 3.
The IC50 values were calculated for all the compounds towards doxorubicin ( Table  9). The results indicated that at all concentrations tested, the type of cells and the compounds tested had a statistically significant contribution to the recorded viability.

In Vitro Cytotoxicity Activity
The cytotoxic effects of the synthesized compounds 5a-d and 6a-d were evaluated on two cancerous cell lines (A549, LNCaP), in parallel with normal fibroblast cells (BJ). Independently of the concentration tested, all the compounds presented higher cytotoxicity towards the cancerous cell phenotypes (Figure 2). The citotoxiciy of doxorubicin, used as a standard agent, is presented in Figure 3.

Molecular Properties with Influence on the Pharmacokinetics of Compounds
For the compounds 5a-d and 6a-d, molecular properties were computed in silico and are presented in Table 10. The molecular weight of compounds is found between 412.46 and 476.50, all of them being under 500, respecting the rule proposed by Lipinski [46]. Compounds 5b and 6b possessing a bulky benzylidene ring (R1) have the highest molecular weight in the present series. Compounds from the a series that have an extra phenol group have a molecular weight two units higher than those from the b series, which have an extra methyl group. The number of rotatable bonds in all compounds are under 10, respecting the rule proposed by Lipinski. No differences between the compounds from the two series are identified by the proposed structural modifications.
The number of hydrogen bond acceptors (HBA) and hydrogen bond donors (HBD) is in direct relationship with the structure of the compounds in the two series. Insertion of The IC 50 values were calculated for all the compounds towards doxorubicin ( Table 9). The results indicated that at all concentrations tested, the type of cells and the compounds tested had a statistically significant contribution to the recorded viability. In case of A549, the potency of the compound varied in the order 5a > 6d > 6a > 5d > 6b > 5c > 5b > 6c, while in the case of LNCaP the order was 6d > 6b > 5c > 6a > 5a > 5d > 5b > 6c. A three-way ANOVA with the cell type (A549, LNCaP, and BJ), exposure dose (12.5, 25, 50, and 100 µg/mL), and type of compounds (5a-d and 6a-d) as variables and the measured viability as a response was performed to evaluate if there are any statistical differences between the conditions. Except for compound 6c which presented the lowest toxicity on all three types of cell lines, all compounds induced statistically significant cytotoxicity starting from the lowest dose of 12.5 µg/mL on all three cellular lines.
Interestingly, different anticancerous potencies were observed for the synthesized compounds between the two cell lines employed. For the A549 cells, compounds 5a, 5d, 6a and 6d which shared the same substituents (R 1 ) (-CH 2 -CH=CH 2 and -CH 2 -CH 2 -CH 2 -CH 3 ) were more cytotoxic, while in the case oF LNCaP cells no such observations could be made because the four most cytotoxic compounds had different substituents. Compounds 5b and 6c had a similar toxicity pattern on both cancerous cell types.
All compounds presented higher cytotoxicity on the LNCaP cells than on the A549 cells at an exposure dose of 12.5 µg/mL. This effect could be related to the weak attachment of the LNCaP cells to the plastic substrate, the presence of the substances favoring the detachment of the cells and thus inducing an apparent decrease in the recorded viabilities.

Molecular Properties with Influence on the Pharmacokinetics of Compounds
For the compounds 5a-d and 6a-d, molecular properties were computed in silico and are presented in Table 10. The molecular weight of compounds is found between 412.46 and 476.50, all of them being under 500, respecting the rule proposed by Lipinski [46]. Compounds 5b and 6b possessing a bulky benzylidene ring (R 1 ) have the highest molecular weight in the present series. Compounds from the a series that have an extra phenol group have a molecular weight two units higher than those from the b series, which have an extra methyl group. The number of rotatable bonds in all compounds are under 10, respecting the rule proposed by Lipinski. No differences between the compounds from the two series are identified by the proposed structural modifications.
The number of hydrogen bond acceptors (HBA) and hydrogen bond donors (HBD) is in direct relationship with the structure of the compounds in the two series. Insertion of the supplementary phenol group in the a series of compounds compared with the b series of compounds leads to their increase by one unit. Regarding the HBA and HBD parameters, all compounds pass the rule proposed by Lipinski.
Insertion of the supplementary phenol group led to the increase in the polar surface in the case of compounds from series a (162.34 Å 2 ), compared to those from series b (142.11 Å 2 ). The R 1 substituent has no influence on the polar surface of the compound, because of its lipophilic properties, without any polar groups. It influences the lipophilicity of the compounds, in the order ethyl < butyl < allyl < benzyl. When we corroborate all the molecular properties of compounds 5a-d and 6a-d, it can be concluded that none of them violates any of Lipinski's rules.
Solubility in water is strongly influenced by the chemical structure of the compounds in the present study. The supplementary phenol group from the series a of compounds increases the solubility of the compounds in water, compared to the b series of compounds. For the compounds that possess the same R 1 , the solubility in water of the compounds from series a is more than double that of those from series b. R 1 strongly influences the solubility of compounds in water. The most soluble are those in which R 1 is small (ethyl), and with its increase, the solubility of the compounds decreases, the most insoluble being those where R 1 is benzyl.

Chemical Synthesis
The synthesis and characterization of the intermediate compounds 1a-d, 2a-d and 3a-d were previously reported by our group [16].
In the final step of the chemical synthesis, compounds 5a-d and 6a-d were synthesized by refluxing in ethanol the previously obtained 4-quinazolinon-2-thioacetohydrazides 3a-d with 2,3,4-trihydroxybenzaldehyde or 2,4-dihydroxy-acetophenone. The spectral analysis performed indicated successful obtention of the desired compounds.

In Vitro Antioxidant, Antiradical and Chelation Assay
The results of the in vitro evaluations performed indicated that the 5a-d series of compounds (pyrogallol derivatives) exhibit a higher antioxidant activity than the 6a-d series of compounds (resorcinol derivatives). It can be concluded that the insertion of the third phenol group (in the 5a-d series of compounds) was much more effective than the insertion of the supplementary methyl group on the azomethine carbon (in the 6a-d series of compounds).

Antiradical Assays
In the antiradical assays (ABTS˙+, DPPH˙and NO˙), the obtained results displayed compounds 5a, 5c and 5d as the most active of the two synthesized series of compounds. In comparison with the radical scavenging activity of the reference antioxidants used, the activity of 5a, 5c and 5d was higher.

Electron Transfer Assays
The pyrogallol derivatives 5a, 5b, 5c and 5d were the most active compounds. The results of this assay revealed a better antioxidant potential for these derivatives than that of ascorbic acid and Trolox, used as reference antioxidants.

Transition Metal Ions Chelation Assays
The chelating activity of compounds for the ferrous ions was higher for the 5a, 5c and 5d compounds. The Cu 2+ chelation activity was significant for the 5a, 5b and 5d compounds, being quite close to that of EDTA-Na 2 .
According to the structure-activity relationship, the pyrogallol derivatives had better chelating properties.

Theoretical Quantum and Thermodynamical Energy Calculations
The BDE (bond dissociation enthalpy) of the hydrogen-releasing groups to neutralize external radicals was the second theoretical important descriptor analyzed in this research, because the energy of the frontier orbitals failed to make a correlation with the antioxidant activity of the compounds. The evaluation of the BDE indicated that the most susceptible compounds to release a hydrogen atom are those from the 5a-d series (pyrogallol-derived compounds) from the newly introduced phenol group from meta. This supplementary group influences the antioxidant activity of the compounds not just per se, but it reduces the ease of release of hydrogen atoms from the phenol groups from ortho and para, from the 5a-d compounds, compared to the 6a-d compounds, that lack the supplementary OH group in meta, but possess a supplementary methyl group on the azomethine. It can be concluded that in terms of antioxidant activity expressed as hydrogen atoms release, the beneficial effect of the insertion of the third phenol group is much higher than the insertion of the supplementary methyl group.

In Vitro Cytotoxicity Activity
As a next step in the biological activity evaluation of the synthesized compounds, their possible antitumoral activity towards two different cancerous cell lines in parallel with normal cells was determined. All compounds (5a-d and 6a-d) were evaluted in the concentration range of 12.5-100 µg/mL after a 24 h exposure, by the Alamar blue assay as previously described [43]. The normal cell line was included to verify if the synthesized compounds display a selective toxicity towards the cancerous phenotype. The cytotoxic effects of the compounds were compared with those of a reference anticancerous substance, namely doxorubicin.
The compounds exhibited lower cytotoxicity on the normal fibroblast cell line and higher cytotoxicity on the cancerous cell lines. With the exception of compound 5c that decreased the cellular viability in BJ cells by approximately 40% at the highest concentration tested, all the other compounds displayed high cytocompatibility on normal cells. At the highest concentration tested, the recorded viabilities for BJ ranged from 50 to 100%, while in the case of A549 and LNCaP the viabilities ranged between 0 and 60% and 5 and 60%, respectively.
Different cytotoxicity patterns were observed for the two series of compounds (5ad and 6a-d), with compounds 6a-d inducing more pronounced toxicity than the other compounds at lower concentrations. Compounds 5a-d induced higher cytotoxicity at intermediary and high doses.
As the IC 50 values of doxorubicin are much lower than the IC 50 values of the 5a-d and 6a-d compounds (approximately 100 times), a potential anticancerous effect of the tested compounds is not promising.

Molecular Properties with Influence on the Pharmacokinetics of Compounds
The increase in the size of the lipophilic substituent at the level of the nitrogen atom of quinazolone (R 1 ) leads to an increase in the molecular weight, an increase in lipophilicity and a decrease in the water solubility of the compounds. On the contrary, the smaller the R 1 lipophilic substituent, the lower the molecular mass, the lower the lipophilicity and the higher the solubility in water. It can be seen that the insertion of the third phenolic OH group in compounds 5a-d leads to an increase in the polarity of the compounds, a decrease in lipophilicity and an increase in their solubility in water, compared to the series of compounds 6a-d.
The way in which the substitution influences the properties influencing the pharmacokinetics of the compounds in the present study follows a normal trend for medicinal chemistry and the design of new bioactive compounds. The variation in the molecules of R 1 and arylidene can bring a significant variation in the properties of compounds. For example, the compound 5c is 24 times more soluble in water than 6b. On the other hand, compound 6b being more lipophylic and less polar than compound 5c can have a better penetration through cell membranes.

Conclusions
Through this study of some new polyphenolic derivatives of quinazolin-4(3H)-one, the synthesis, characterization and in-vitro antioxidant and cytotoxic activity evaluation are presented.
Compounds 5a, 5b, 5c, and 5d were the most active, following the evaluation of the antioxidant activity. Their antiradical potential was better than that of ascorbic acid and Trolox, used as reference antioxidants, for these pyrogallol derivatives.
The HOMO-LUMO gap was higher in the case of the pyrogallol-derived compounds 5a, 5b, 5c and 5d, while in the 2,4-dihydroxy-acetophenone derivatives 6a, 6b, 6c and 6d, the energy difference between the two frontier orbitals was lower. The most susceptible group to release hydrogen atoms was meta-hydroxy (site H 3 ), which is found in the 5a, 5b, 5c and 5d compounds (pyrogallol-derived compounds).
The cytotoxic activity was evaluated on three different cell lines (BJ, A549 and LNCaP). The compounds 6a-d induced a higher toxicity than the other compounds on A549 cells and LNCaP cells at lower concentrations, while the compounds 5a-d induced higher cytotoxicity at intermediary and high doses. In comparison with cancerous cells, higher viabilities were observed on the normal cells after exposure to the synthesized compounds. Thus, in a therapeutic hypothesis, the new synthesized compounds are cytocompatible towards normal cells and healthy tissue.
The results of the in-vitro studies performed on these compounds confirm that the antioxidant activity is strongly influenced by the presence of the third phenolic-OH group in the molecular structure. The trihydroxilated hybrid derivatives exhibited lower cytotoxicity on the normal fibroblast cell line and activity on the cancerous cells lines depending of the concentrations of the compounds. The methyl group inserted in the arylidene carbon did not increase the antioxidant potential of the compounds, but influenced the cytotoxicity activity on cell lines.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics15010136/s1. Figures S1-S8: The IR spectrums for the compounds 5a-d and 6a-d; Figures S9-S16: The MS spectrums for the compound 5a-d and 6a-d; Figures S17-S24: The 1 H-NMR spectrum for the compound 5a-d and 6a-d; Figures S25-S32: The 13 C-NMR spectrum for the compound 5a-d and 6a-d; Table S1: The depiction of HOMO and LUMO for the compounds 5a-d and 6a-d; Table S2: The depiction of the spin density maps of the phenol group radicalization for the compounds 5a-d and 6a-d.