New Quinoxaline-Based Derivatives as PARP-1 Inhibitors: Design, Synthesis, Antiproliferative, and Computational Studies

Herein, 2,3-dioxo-1,2,3,4-tetrahydroquinoxaline was used as a bio-isosteric scaffold to the phthalazinone motif of the standard drug Olaparib to design and synthesize new derivatives of potential PARP-1 inhibitory activity using the 6-sulfonohydrazide analog 3 as the key intermediate. Although the new compounds represented the PARP-1 suppression impact of IC50 values in the nanomolar range, compounds 8a, 5 were the most promising suppressors, producing IC50 values of 2.31 and 3.05 nM compared to Olaparib with IC50 of 4.40 nM. Compounds 4, 10b, and 11b showed a mild decrease in the potency of the IC50 range of 6.35–8.73 nM. Furthermore, compounds 4, 5, 8a, 10b, and 11b were evaluated as in vitro antiproliferative agents against the mutant BRCA1 (MDA-MB-436, breast cancer) compared to Olaparib as a positive control. Compound 5 exhibited the most significant potency of IC50; 2.57 µM, whereas the IC50 value of Olaparib was 8.90 µM. In addition, the examined derivatives displayed a promising safety profile against the normal WI-38 cell line. Cell cycle, apoptosis, and autophagy analyses were carried out in the MDA-MB-436 cell line for compound 5, which exhibited cell growth arrest at the G2/M phase, in addition to induction of programmed apoptosis and an increase in the autophagic process. Molecular docking of the compounds 4, 5, 8a, 10b, and 11b into the active site of PARP-1 was carried out to determine their modes of interaction. In addition, an in silico ADMET study was performed. The results evidenced that compound 5 could serve as a new framework for discovering new potent anticancer agents targeting the PARP-1 enzyme.


Introduction
Poly (ADP-ribose) polymerases (PARPs) constitute a group of at least 17 enzymes that are correlated to the DNA damage repair process. PARP-1is the most abundant member of this group and has emerged as one of the most auspicious molecular targets for cancer management in the past decade [1- 4]. PARP-1 acts as a "molecular nick sensor" to DNA single-strand (ssDNA) breaks and catalyzes the transference of ADP-ribose units (utilizing nicotinamide adenine dinucleotide (NAD + ) as a substrate) to acceptor proteins, facilitating the recruitment of the damaged DNA and promoting cell survival. It is an important stage in the base excision repair (BER) of single-strand DNA breaks [4], which is linked to the resistance that typically develops following traditional cancer treatments [5][6][7]. PARP-1 suppression enhances the damage of injured DNA resulting in synthetic lethality in DNA-repairing-deficient cancer cells, such as BRCA1/2-deficient cells. Thus, PARP-1 suppression synergizes the impact of various antiproliferative drugs such as topoisomerase-I inhibitors and DNA alkylating drugs in addition to ionizing radiation. Moreover, It has been reported that the catalytic pocket of PARP1 is divided into three subpockets that are occupied by the substrate NAD + . The first sub-pocket is the nicotinamideribose binding site (NI site), the second is the phosphate-binding site (PH site), and the third is the adenine-ribose binding site (AD site) [16] (Figure 2). It has been reported that most of the PARP-1 suppressors bind with the NI site via H-binding and π-π stacking interactions, and some of them produce further interactions in the adenine-ribose binding (AD) site, which is large enough to fit a variety of molecules, It has been reported that the catalytic pocket of PARP1 is divided into three sub-pockets that are occupied by the substrate NAD + . The first sub-pocket is the nicotinamide-ribose binding site (NI site), the second is the phosphate-binding site (PH site), and the third is the adenine-ribose binding site (AD site) [16] (Figure 2). lethality in DNA-repairing-deficient cancer cells, such as BRCA1/2-deficient cells. Thus, PARP-1 suppression synergizes the impact of various antiproliferative drugs such as topoisomerase-I inhibitors and DNA alkylating drugs in addition to ionizing radiation. Moreover, some PARP suppressors are effective as single agents against cancers bearing BRCA1-or BRCA2-mutations [8][9][10][11].
The US FDA recently approved four PARP suppressors, Olaparib, Rucaparib, Niraparib, and Talazoparib, for curing BRCA-mutated, HER2-negative advanced, metastatic ovarian, or breast cancer. In addition, there are a number of PARP suppressors that are under study in various clinical phases such as Veliparib, Pamiparib, Simmiparib, and Fluzoparib [12,13] (Figure 1). Furthermore, recent studies investigated the therapeutic potential of various PARP-1 suppressors for other refractory diseases such as Alzheimer's disease (AD) [14,15]. Accordingly, the development of effective PARP-1 inhibitors plays an important role in medicinal chemistry communities. It has been reported that the catalytic pocket of PARP1 is divided into three subpockets that are occupied by the substrate NAD + . The first sub-pocket is the nicotinamideribose binding site (NI site), the second is the phosphate-binding site (PH site), and the third is the adenine-ribose binding site (AD site) [16] (Figure 2). It has been reported that most of the PARP-1 suppressors bind with the NI site via H-binding and π-π stacking interactions, and some of them produce further interactions in the adenine-ribose binding (AD) site, which is large enough to fit a variety of molecules, It has been reported that most of the PARP-1 suppressors bind with the NI site via H-binding and π-π stacking interactions, and some of them produce further interactions in the adenine-ribose binding (AD) site, which is large enough to fit a variety of molecules, leading to enhancing their effectiveness and pharmacokinetic characteristics [17,18]. Many studies have determined that the design of PARP-1 inhibitors is based on the nicotinamide section of NAD + to imitate the ligand-protein binding of NAD + with PARP-1 [19]. Accordingly, PARP-1 suppressors shar common pharmacophoric features, which are an aromatic ring and a carboxamide core. The critical bindings between them are the H-bonding networks initiated between the carboxamide moiety and Gly863 (NH to Gly C=O and C=O to Gly NH) and Ser904 (C=O to Ser OH). Additionally, the phenyl ring of Tyr907 induces the π-π stacking interaction with the aryl ring. Additionally, an auxiliary appendage with a linking side chain is commonly conjugated with the polycyclic core as a solvent accessory region in the AD site [20][21][22][23] (Figure 3). leading to enhancing their effectiveness and pharmacokinetic characteristics [17,18].
Many studies have determined that the design of PARP-1 inhibitors is based on the nicotinamide section of NAD + to imitate the ligand-protein binding of NAD + with PARP-1 [19]. Accordingly, PARP-1 suppressors shar common pharmacophoric features, which are an aromatic ring and a carboxamide core. The critical bindings between them are the Hbonding networks initiated between the carboxamide moiety and Gly863 (NH to Gly C=O and C=O to Gly NH) and Ser904 (C=O to Ser OH). Additionally, the phenyl ring of Tyr907 induces the π-π stacking interaction with the aryl ring. Additionally, an auxiliary appendage with a linking side chain is commonly conjugated with the polycyclic core as a solvent accessory region in the AD site [20][21][22][23] (Figure 3). Plenty of research has shown that the improvement of PARP inhibitors' binding affinity by restriction of the carboxamide's free rotation greatly enhances the PARP1 inhibitory activity. The carboxamide moiety can be locked into the required confirmation by either inserting the aromatic ring heteroatoms or functionalities that can form an intramolecular hydrogen bond with the amide NH or conjugating the amide group in a bicyclic system [4,6,24].
Quinoxaline is a privileged scaffold and one of the main blocks of different anticancer agents as it has been proven to be selective adenosine triphosphate (ATP) competitive as well as a bioisostere to benzimidazole, quinazolinones, isoquinolinones, phenanthridone, or phthalazinones, which are the basic scaffolds of the plurality of PARP-1 inhibitors [25][26][27]. In addition, sulfonyl and sulfonamide moieties conjugated to different heterocyclic ring systems have been reported as one of the most privileged scaffolds to inhibit the Plenty of research has shown that the improvement of PARP inhibitors' binding affinity by restriction of the carboxamide's free rotation greatly enhances the PARP1 inhibitory activity. The carboxamide moiety can be locked into the required confirmation by either inserting the aromatic ring heteroatoms or functionalities that can form an intramolecular hydrogen bond with the amide NH or conjugating the amide group in a bicyclic system [4,6,24].
Quinoxaline is a privileged scaffold and one of the main blocks of different anticancer agents as it has been proven to be selective adenosine triphosphate (ATP) competitive as well as a bioisostere to benzimidazole, quinazolinones, isoquinolinones, phenanthridone, or phthalazinones, which are the basic scaffolds of the plurality of PARP-1 inhibitors [25][26][27]. In addition, sulfonyl and sulfonamide moieties conjugated to different heterocyclic ring systems have been reported as one of the most privileged scaffolds to inhibit the growth of various human cancer cell lines via different modes of action [28,29]. Cancer treatment is still a challenge due to the development of cancer cell resistance, toxicity, and the lack of selectivity of most commercialized anticancer medications. As a result, and in view of the continuation of our efforts in discovering new heterocyclic compounds of potential anticancer activity targeting the PARP-1 enzyme [30][31][32], the strategy of this study was focused on the design and synthesis of the new compounds based on the quinoxaline core to occupy the NI site of PARP-1 hybridized at its position-6 with different heterocycles, such as pyrrole, pyrazole, thiazole, imidazolidinone, and pyrimidine via sulfonyl, sulfonamide, and sulfonohydrazide linkers aiming to engage with the PARP-1 enzyme through different binding modes of action. The quinoxaline nucleus bears two carboxamide moieties that engage with the enzyme through additional hydrogen bonding ( Figure 3). All new compounds were examined as PARP-1 inhibitors. Since PARP-1 inhibitors result in synthetic lethal effects, specifically in BRCA-mutated cells, MDA-MB-436 (BRCA-1-mutated breast cancer cell line) was selected to conduct a cell proliferation assay for the analogs that exhibited the most active inhibition effect against the PARP-1 enzyme. Thereafter, the safety margin of the most potent members was evaluated against WISH normal cells. A molecular docking study was also employed for the promising PARP-1 inhibiting candidates to rationalize and emphasize their mechanisms of binding with the active pocket of the target enzyme. Furthermore, in silico ADMET prediction was performed for the new compounds to explore their drug-likeness characteristics.

Chemistry
This study was directed toward the design and construction of new quinoxaline compounds using various synthetic pathways illustrated in Schemes 1 and 2. The synthesis was initiated by reacting the starting material o-phenylenediamine with oxalic acid in the presence of HCl to provide quinoxaline-2,3(1H,4H)-dione (1), which was treated with chlorosulfonic acid to provide the corresponding key intermediate 6-sulfonyl chloride derivative 2 according to the reported methodology [33,34]. The latter derivative served as a facile intermediate for the nucleophilic substitution reaction with hydrazine hydrate to afford the 6-sulfonohydrazide derivative 3, which was utilized as a precursor for the ring closure reaction with different active methylene reagents, namely, ethyl-acetoacetate, acetylacetone, and diethyl malonate, to accomplish the corresponding pyrazole derivatives 4-6, respectively. Furthermore, the reaction of 6-sulfonohydrazide derivative 3 with 4-methoxybenzene isothiocyanate and/or benzoyl isothiocyanate in refluxing DMF in the presence of a few drops of triethylamine resulted in the formation of the thiosemicarbazide derivatives 8a,b, respectively. 1 H NMR spectra of the latter derivatives 8a,b represented singlet signals at δ 10.71-11.97 ppm exchangeable with D2O affordable to NH groups, multiplet sig-Scheme 1. Synthesis of different new quinoxaline-2,3-dione -based derivatives. All the new target quinoxaline compounds 3-12a,b were evaluated as PARP-1 inhibitors to gain clear insight into the structure-activity relationship using the colorimetric 96well PARP-1 assay kit [6,35]. The IC50 values of all the tested compounds against PARP-1 were expressed in nM concentrations utilizing Olaparib as a reference drug and are summarized in Table 1. Despite all the obtained IC50 values being in the nanomolar range, they showed a wide variation in PARP-1 inhibitory activity (IC50 range; 2.31-57.35 nM). Therefore, it can be supposed that the terminal position of the molecule can tolerate a wide variety of substituents and this point can be explained if these side chains are approaching the solvent surface and do not bind significantly with the enzyme. The key starting intermediate 6-sulfonohydrazide derivative 3 displayed 3-fold less inhibitory activity against PARP-1 than that of the reference drug Olaparib of IC50s; 12.86, 4.40 nM, respectively. We detected the direct conjugation of the parent 6-sulfonoquinoxaline core with a 3,5-dimethylpyrazole ring as compound 5 exhibited PARP-1 inhibitory activity higher than that of the control drug by 1.5 folds (IC50s = 3.05 nM). Conversely, the replacement of either one or both methyl groups of the pyrazole ring of compound 5 with OH or 2C=O groups as compounds 4 and 6, respectively, decreased the suppression impact by nearly 2-and 3fold of IC50s = 8.73, 13.27 nM, respectively. This result indicated that the hydrophobic residues are favorable for PARP-1 inhibition activity. IR spectra of compounds 3-6 demonstrated stretching bands at approximately 3441-3315 cm −1 corresponding to the NH 2 and NH groups, other bands ranging from 1750 to 1678 cm −1 due to C=O groups, and at 1392-1138 corresponding to SO 2 groups. 1 H NMR spectra of compounds 3-6 represented D 2 O exchangeable signals of NH 2 and NH functionalities in the range of δ 11.78-11.83 ppm, alongside multiplet signals at the region of δ 7.01-8.03 ppm related to the aromatic protons. The sulfonohydrazide compound 3 showed an additional D 2 O exchangeable singlet at δ 7.85 ppm assignable to the NH 2 group, while the target pyrazole derivatives 4, 5, and 6 represented new singlets at δ 2.12-2.74 ppm related to CH 3 , 2CH 3 , and CH 2 functionalities, respectively, at δ 6.78-6.31 ppm due to the pyrazole-H 4 of compounds 4 and 5, and at 12.35 ppm exchangeable with D 2 O related to the OH group of compound 4. Furthermore, 13 C NMR spectra of compounds 4, 5, and 6 exhibited singlet signals at δ 12.53, 11.21, and 55.92 ppm assignable to CH 3 , 2CH 3 , and CH 2 groups, respectively, at δ 102.07-155.73 related to the aromatic carbons, and at δ 154.82-167.89 ppm due to C=O groups.
The further condensation reaction of 6-sulfonohydrazide derivative 3 with different acid anhydrides, namely succinic, maleic, and/or phthalic anhydride in glacial acetic acid, resulted in the achievement of the corresponding analogs 7a-c, respectively. 1 H NMR spectra of the latter derivatives 7a-c revealed multiplet signals in the range of δ 7.32-7.93 ppm, contributing to the aromatic protons, as well as three D 2 O exchangeable signals at the region δ 9.61-12.01 ppm due to 3NH groups. Compound 7a represented a singlet signal at δ 2.73 ppm corresponding to the dioxopyrrolidine-2CH 2 function, 7b exhibited a doublet signal at δ 7.20 ppm attributed to its vinylic protons, while 7c revealed an increase in the integration values in the aromatic region due to the phthalic protons. Moreover, 13 C NMR spectra of compounds 7a-c exhibited singlet signals in the range of δ 115.13-138.42 ppm assigned to the aromatic carbons and in the range of δ 154.34-170.81 ppm representing C=O groups. A singlet signal appeared at δ 30.07 ppm due to the two methylene carbons of the pyrrolidine-2CH 2 of compound 7a (Scheme 1).
Furthermore, the reaction of 6-sulfonohydrazide derivative 3 with 4-methoxybenzene isothiocyanate and/or benzoyl isothiocyanate in refluxing DMF in the presence of a few drops of triethylamine resulted in the formation of the thiosemicarbazide derivatives 8a,b, respectively. 1 H NMR spectra of the latter derivatives 8a,b represented singlet signals at δ 10.71-11.97 ppm exchangeable with D 2 O affordable to NH groups, multiplet signals at δ 6.90-8.14 ppm related to the aromatic protons, and a singlet signal at δ 3.81 ppm assignable to the methoxy protons (-OCH 3 ) in the case of compound 8a. Furthermore, their 13 C NMR spectra revealed the parent carbons of both derivatives in companion with a singlet signal at δ 55.44 ppm related to OCH 3 of 8a and at δ 163 ppm due to ph-C=O of the benzoyl compound 8b.
Thiosemicarbazide congeners are reported to be valuable intermediates in organic chemistry since they act as building blocks for the preparation of various heterocyclic compounds possessing biological importance [35,36]. Accordingly, the treatment of compounds 8a,b with diethyl malonic acid in refluxing ethanol furnished the corresponding 2-thioxo-3,4-dihydropyrimidine derivatives 9a,b, respectively. IR spectra of compounds 9a,b displayed different absorption bands at 3441-3160, 1710-1645, 1415, 1338, and 1196 cm −1 related to 3NH, 3C=O, C=S, and SO 2 groups. 1 H NMR spectra of compounds 9a,b represented an additional signal at δ 10.51 ppm exchangeable with D 2 O referring to the OH group, while the pyrimidine-H 5 appeared as a singlet signal in the aromatic region at δ 7.12-7.35 ppm alongside the parent protons, which appeared in their expected regions. Furthermore, 13 C NMR spectra of compounds 9a,b represented singlet signals at the region of δ 82.50-155.47 ppm related to the aromatic carbons, at the region of δ 155.40-155.50 due to C=O groups, and at δ 170.1, 189.68 ppm due to C=S groups. The methoxy carbon of compound 9a appeared as a singlet signal at δ 55.80 ppm.
Moreover, the nucleophilic reaction of various αhalo ketones, namely chloroacetone and 3-chloroacetylacetone with compounds 8a,b, was carried out in the presence of sodium acetate to give the corresponding thiazolines, 10a,b, and 11a,b, respectively. 1 H NMR spectra of compounds 10a,b exhibited a singlet signal in the region of δ 1.85-2.31 ppm contributing to the thiazoline-CH 3 protons, while the thiazoline-H 5 appeared as a singlet signal at δ 5.45 ppm, in addition to the precursor protons, which was presented in the correct regions. Furthermore, 1 H NMR spectra of 11a,b exhibited two singlet signals at δ 2.33 and 2.51 ppm assigned to thiazoline-CH 3 and COCH 3 , respectively, alongside the signals of the parent protons. Similarly, 13 C NMR spectra of 10a,b, and 11a,b represented CH 3 carbons as singlet signals in the region of δ 14.56-19.77 ppm, in addition to the parent carbons, which were presented in their correct regions. The acetyl carbon of compounds 11a,b was represented as a singlet signal at δ 25.13 and 25.62 ppm, respectively.
Moreover, the treatment of 8a,b with ethyl bromoacetate in absolute ethanol containing a catalytic amount of sodium acetate accomplished the corresponding thiazolidine derivatives 12a,b, respectively. 1 H NMR spectra of the target derivatives 12a,b showed an up-field signal in the region of δ 4.67 ppm corresponding to the thiazolidine-CH 2 methylene protons alongside the parent protons, which appeared at their expected regions. 13 C NMR of the latter derivatives showed a singlet signal at δ 25.13 assignable to the thiazolidine-CH 2 , and singlet signals in the regions of δ 114.66-155.68 ppm and 163.27-176.33 due to the aromatic and C=O carbons, respectively. The methoxy carbon of 12a appeared as a singlet signal at δ 63.09 ppm (Scheme 2). Mass spectra of the newly prepared compounds exhibited correct molecular ion peaks, which were in accordance with their molecular formulae. 96-well PARP-1 assay kit [6,35]. The IC 50 values of all the tested compounds against PARP-1 were expressed in nM concentrations utilizing Olaparib as a reference drug and are summarized in Table 1. Despite all the obtained IC 50 values being in the nanomolar range, they showed a wide variation in PARP-1 inhibitory activity (IC 50 range; 2.31-57.35 nM). Therefore, it can be supposed that the terminal position of the molecule can tolerate a wide variety of substituents and this point can be explained if these side chains are approaching the solvent surface and do not bind significantly with the enzyme. The key starting intermediate 6-sulfonohydrazide derivative 3 displayed 3-fold less inhibitory activity against PARP-1 than that of the reference drug Olaparib of IC 50 s; 12.86, 4.40 nM, respectively. We detected the direct conjugation of the parent 6-sulfonoquinoxaline core with a 3,5-dimethylpyrazole ring as compound 5 exhibited PARP-1 inhibitory activity higher than that of the control drug by 1.5 folds (IC 50s = 3.05 nM). Conversely, the replacement of either one or both methyl groups of the pyrazole ring of compound 5 with OH or 2C=O groups as compounds 4 and 6, respectively, decreased the suppression impact by nearly 2-and 3-fold of IC 50s = 8.73, 13.27 nM, respectively. This result indicated that the hydrophobic residues are favorable for PARP-1 inhibition activity. Moreover, hybridization of the 6-sulfonoquinoxaline scaffold with the p-methoxyphenyl ring via the thiosemicarbazide linker as compound 8a represented a promising impact on the PARP-1 suppression effect, nearly 2-fold higher than that of Olaparib of IC 50 = 2.31 nM. On the other hand, the inhibitory activity was 2.5 times less than the reference drug upon conjugation of the thiosemicarbazide side chain with the CO-unsubstituted phenyl ring as compound 8b of IC 50 = 11.06 nM. The p-substitution of the phenyl ring with the electron-donating group OCH 3 signified the inhibitory potency as depicted by compound 8a.
On the other hand, the hybridization of the parent 6-sulfonoquinoxaline with 3-benzoyl-4-methylthiazoline and 5-acetyl-3-benzoyl-4-methylthiazoline moieties via the hydrazide linker as congeners 10b, 11b produced a slight reduction in the inhibitory activity compared to the reference drug of IC 50s = 6.35, 8.25 nM, respectively. A detectable decrease in the activity was further observed by the 4-methoxyphenyl analogs 10a and 11a of IC 50s = 21.63 and 19.45, respectively. It could be noted that the decrease in the thiosemicarbazide length is not favorable for the potency of the desired activity. With respect to the series of pyrrole and isoindoline derivatives 7a-c, the 2-thioxopyrimidine derivatives 9a,b and the 4-oxothiazolidine derivatives 12a,b exhibited the lowest activity of IC 50 values ranging from 35.82-57.14 nM. The SAR study of the most potent active congeners is depicted in Figure 4.

Antiproliferative Activity
In order to find out the relationship between the anticancer potency and the PARP-1 suppression effect, the most effective compounds as PARP-1 inhibitors (4, 5, 8a, 10b, 11b) were further evaluated for their in vitro cytotoxicity against the mutant BRCA1 (MDA-MB-436, breast cancer) using an MTT assay [37]. Olaparib was used as a positive control.
The IC50 values of all examined compounds are tabulated in Table 1. The resultant data exhibited that the dimethyl pyrazole compound 5 exhibited the best antiproliferative activity against the examined cancer cell line, being approximately 4 times more potent than the reference drug with IC50 values of 2.57, 8.90 µM, respectively. Furthermore, the tested compounds 8a, 10b, and 11b exhibited an approximately equal activity to that of Olaparib with IC50 values of 10.70, 9.62, and 11.50 µM, respectively. On the contrary, compound 4 displayed the weakest antitumor activity with an IC50 value of 30.30µM.
This result represented an outstanding correlation between PARP-1 suppression activity and the anticancer activity of the tested compounds.
It has been reported that the frequency and severity of the side effects on normal healthy cells at therapeutic levels are deemed to be critical factors that distinguish different anticancer drugs from each other. Accordingly, the cytotoxic activity of the potent members 5, 8a, 10b, and 11b was evaluated against the normal WI-38 cell line via an MTT assay to determine their safety profiles. It is worth mentioning that the IC50 values of all the representative compounds against the normal cells range from 70.46-81.67 µM, which are 7-8-fold higher than their IC50s values against the cancer cell line, confirming their promising safety profile (Table 1).

Cell Cycle Analysis in MDA-MB-436
Based on its well-balanced biological activity, i.e., promising PARP-1 inhibition and high antiproliferative activity, compound 5 was chosen as a representative example for further examining cellular mechanisms with respect to its impact on cell cycle progression and induction of apoptosis in MDA-MB-436 cells by using the flow cytometric technique

Antiproliferative Activity
In order to find out the relationship between the anticancer potency and the PARP-1 suppression effect, the most effective compounds as PARP-1 inhibitors (4, 5, 8a, 10b, 11b) were further evaluated for their in vitro cytotoxicity against the mutant BRCA1 (MDA-MB-436, breast cancer) using an MTT assay [37]. Olaparib was used as a positive control. The IC 50 values of all examined compounds are tabulated in Table 1. The resultant data exhibited that the dimethyl pyrazole compound 5 exhibited the best antiproliferative activity against the examined cancer cell line, being approximately 4 times more potent than the reference drug with IC 50 values of 2.57, 8.90 µM, respectively. Furthermore, the tested compounds 8a, 10b, and 11b exhibited an approximately equal activity to that of Olaparib with IC 50 values of 10.70, 9.62, and 11.50 µM, respectively. On the contrary, compound 4 displayed the weakest antitumor activity with an IC 50 value of 30.30µM.
This result represented an outstanding correlation between PARP-1 suppression activity and the anticancer activity of the tested compounds.
It has been reported that the frequency and severity of the side effects on normal healthy cells at therapeutic levels are deemed to be critical factors that distinguish different anticancer drugs from each other. Accordingly, the cytotoxic activity of the potent members 5, 8a, 10b, and 11b was evaluated against the normal WI-38 cell line via an MTT assay to determine their safety profiles. It is worth mentioning that the IC 50 values of all the representative compounds against the normal cells range from 70.46-81.67 µM, which are 7-8-fold higher than their IC 50s values against the cancer cell line, confirming their promising safety profile (Table 1).

Cell Cycle Analysis in MDA-MB-436
Based on its well-balanced biological activity, i.e., promising PARP-1 inhibition and high antiproliferative activity, compound 5 was chosen as a representative example for further examining cellular mechanisms with respect to its impact on cell cycle progression and induction of apoptosis in MDA-MB-436 cells by using the flow cytometric technique [38,39]. In the present work, MDA-MB-436 cells were treated with compound 5 at its IC 50

Apoptosis Assay in MDA-MB-436 Cells
Annexin V-FITC and PI staining coupled with flow cytometry was utilized for further investigation of the apoptotic impact of compound 5 on MDA-MB-436. Treatment of MDA-MB-436 cells with the IC50 concentration of 5 induced both apoptosis and necrotic effects (Figures 7 and 8 ). The early apoptotic cell population increased from 0.14% (control) to 0.27% (5-treated cells), the percentage of the late apoptotic cell population increased from 0.98% (control) to 1.02% (5-treated cells), and the necrotic cell population increased from 1.11% (control) to 1.72% (5-treated cells).    (Figures 7 and 8 ). The early apoptotic cell population increased from 0.14% (con trol) to 0.27% (5-treated cells), the percentage of the late apoptotic cell population in creased from 0.98% (control) to 1.02% (5-treated cells), and the necrotic cell population increased from 1.11% (control) to 1.72% (5-treated cells).   (Figures 7 and 8). The early apoptotic cell population increased from 0.14% (control) to 0.27% (5-treated cells), the percentage of the late apoptotic cell population increased from 0.98% (control) to 1.02% (5-treated cells), and the necrotic cell population increased from 1.11% (control) to 1.72% (5-treated cells).

Autophagy Assay
It has been reported that autophagy-induced programmed cell death is a hot topic in the scientific community. It was of interest to study the effect of compound 5 on the autophagy process within MDA-MB-436 cells utilizing Cyto-ID autophagy detection dye coupled with flow cytometry [40,41]. Treatment of the latter cells with 5 increased autophagic cell death by 68.65% (Figure 9).

Autophagy Assay
It has been reported that autophagy-induced programmed cell death is a hot topic in the scientific community. It was of interest to study the effect of compound 5 on the au tophagy process within MDA-MB-436 cells utilizing Cyto-ID autophagy detection dy coupled with flow cytometry [40,41]. Treatment of the latter cells with 5 increased au tophagic cell death by 68.65% (Figure 9).

Autophagy Assay
It has been reported that autophagy-induced programmed cell death is a hot topic in the scientific community. It was of interest to study the effect of compound 5 on the autophagy process within MDA-MB-436 cells utilizing Cyto-ID autophagy detection dye coupled with flow cytometry [40,41]. Treatment of the latter cells with 5 increased autophagic cell death by 68.65% (Figure 9).

Autophagy Assay
It has been reported that autophagy-induced programmed cell death is a hot topic in the scientific community. It was of interest to study the effect of compound 5 on the autophagy process within MDA-MB-436 cells utilizing Cyto-ID autophagy detection dye coupled with flow cytometry [40,41]. Treatment of the latter cells with 5 increased autophagic cell death by 68.65% (Figure 9).

Molecular Docking
To find out the interaction modes of the most promising quinoxaline congeners 4, 5, 8a, 10b, and 11b, a standard docking protocol was used where Olaparib was utilized as the reference frame of the docking grid. The crustal structure of Olaparib in complex with the catalytic domain of PARP1 was downloaded from the protein databank (www.rcsb.org accessed 15 April 2021). The complex structure was processed with the Protein Preparation Wizard in Maestro to add missing atoms, sidechains, and residues, complete loops, add hydrogen atoms, and adjust bond orders for amino acids and ligands [42][43][44][45][46].
The five compounds were docked with high affinity, and the prime MM-GBSA free energy of binding was computed as −93 kcal/mol for Olaparib, −79.3 kcal/mol for compound 8a, −63.9 for compound 10b, −60.9 kcal/mol for compound 5, −54.4 kcal/mol for compound 11b, and −54.3 kcal/mol for compound 4. The compounds fit well in the binding pocket and demonstrated several favorable interactions with the surrounding amino acids. Olaparib is complex with PARP1 in the crystal structure, and it showed the following interactions: Hydrogen bonds with Ser904, Gly863, Ser864, and Tyr896, waterbridged hydrogen bonds with Arg878, Ile879, and π-π contacts with Tyr896 and Tyr907. The compounds showed the following interactions: Compound 8a interacts with hydrogen bonds with Ser904, His862, Asp766, Ser864, π-π contacts with Tyr907 and His862, and cation-π contacts with Arg848; compound 5 showed hydrogen bonds with Gly863 and Ser904, and π-π contacts with Tyr907; compound 10b interacts through hydrogen bonds with Tyr896 and Ser894, a water-bridged hydrogen bond with Arg873, and π-π contacts with Tyr907 and Tyr889; compound 11b showed hydrogen bonds with Ser904, Asn906, and Lys903, water-bridged hydrogen bonds with Met890 and Glu988, π-π contacts with Tyr907, and cation-π contacts with Lys903; and compound 4 interacted through hydrogen bonds with Ser904, Gly863, and Asp766, and π-π contacts with Tyr907 and Tyr889 (Figures 10 and 11 and Table 2).

Prediction ADME parameters
The SwissADME tool was used to calculate the physicochemical properties of the tested compounds 4, 5, 8a, 10b, and 11b ( Figure 12) [47][48][49][50][51][52][53]. The compounds showed low to moderate water solubility. Compound 4 showed the highest predicted solubility. Only compound 11b violated Lipinski's role of five having more than 10 NH and OH groups. All the compounds were computed to have low GIT absorption with the exception of compound 5, which has a promising property. All the compounds are not expected to cross the BBB.

Chemistry
The instruments used for measuring the melting points, spectral data (IR, Mass, 1 H NMR, and 13 C NMR), and elemental analyses are provided in detail in Supplementary Material.

PARP-1 Inhibition Assay
PARP-1 enzyme inhibition activity was evaluated using a colorimetric 96-well PARP-1 assay kit (catalog no. 80580) (BPS Bioscience), according to the manufacturer's protocol. More details are provided in Supplementary Material.

In Vitro Anticancer Screening
The in vitro cytotoxicity potency was screened against the MDA-MB-436 cancer cell line by MTT assay. The cytotoxicity was estimated as IC50 in µM for the tested compounds and the reference drug Olaparib. More details are provided in Supplementary Material.

Cell Cycle Analysis
The pre-calculated IC50 of compound 5 was applied to MDA-MB-436 breast cancer cells for 48h. The cells were treated with trypsin, rinsed two times in PBS, fixed in ice-cold 60% ethanol at 40 °C, and washed again in PBS. More details are provided in Supplementary Material.

Chemistry
The instruments used for measuring the melting points, spectral data (IR, Mass, 1 H NMR, and 13 C NMR), and elemental analyses are provided in detail in Supplementary Material.

PARP-1 Inhibition Assay
PARP-1 enzyme inhibition activity was evaluated using a colorimetric 96-well PARP-1 assay kit (catalog no. 80580) (BPS Bioscience), according to the manufacturer's protocol. More details are provided in Supplementary Material.

In Vitro Anticancer Screening
The in vitro cytotoxicity potency was screened against the MDA-MB-436 cancer cell line by MTT assay. The cytotoxicity was estimated as IC 50 in µM for the tested compounds and the reference drug Olaparib. More details are provided in Supplementary Material.

Cell Cycle Analysis
The pre-calculated IC 50

Apoptosis Analysis
MDA-MB-436 cells were treated with compound 5 for 48 h, then treated with trypsin and rinsed twice in PBS. Apoptosis assessment was performed via the "Annexin V-FITC/PI Apoptosis Detection Kit", "BD Biosciences, San Diego, CA, USA", as stated by the manufacturer. More details are provided in Supplementary Material.

Autophagy Analysis
To further confirm the cell death mechanism induced by the drugs, autophagic cell death was quantitatively analyzed using a Cyto-ID Autophagy Detection Kit (Abcam Inc., Cambridge Science Park, Cambridge, UK). More details are provided in Supplementary Material.

Docking Methodology
The crustal structure of Olaparib in complex with the catalytic domain of PARP1 [42] was downloaded from the protein databank (www.rcsb.org accessed on 15 April 2021). The complex structure was processed with the Protein Preparation Wizard [43,44] in Maestro [45] to add missing atoms, sidechains, and residues, complete loops, add hydrogen atoms, and adjust bond orders for amino acids and ligands. More details are provided in Supplementary Material. The starting compound 1,4-quinoxaline-2,3-dione (1) (1.90 g, 10 mmol) was added portion-wise to chlorosulfonic acid (2 mL, 3 mmol) at 65-90 • C over 3 h. The reaction mixture was cooled to room temperature and poured slowly onto the ice/water mixture. The formed precipitate was collected by filtration and washed with water and dried. The obtained product was crystallized from benzene/petroleum ether  to give the desired 6-sulfonyl chloride product as a yellowish-white solid according to the reported method [34]. Yield 75%, m.p. 280 (decomposed).

Conclusions
The current study deals with the design and synthesis of a novel set of derivatives 3-12a,b bearing the quinoxaline scaffold that is hybridized with various heterocyclic ring systems via a sulfonamide linkage. The new compounds were assessed for their suppression impact against the PARP-1 enzyme using Olaparib as a positive reference drug. Among the examined compounds, 4, 5, 8a, 10b, and 11b displayed the highest PARP-1 inhibitory suppression effect with IC 50 values ranging from 2.31 to 8.25 nM, compared to IC 50Olaparib of 4.40 nM. The latter compounds were further examined as antiproliferative agents in MDA-MB-436 in comparison with Olaparib as a reference drug. The compounds 5, 8a, 10b, and 11b exhibited promising inhibitory activity with IC 50 values ranging from 2.57 to 11.50 µM, compared to IC 50Olaparib of 4.40 µM, and confirmed a safety profile against the normal cells' WI-38 cell lines. Due to the well-balanced activity of compound 5 as a promising PARP-1 inhibitor, as well as the antiproliferative agent, it was chosen as a representative example for further cellular mechanistic investigation regarding its impact on the cell cycle progression and induction of apoptosis in the MDA-MB-436 cell line. Treatment of the latter cells with compound 5 led to cell cycle arrest at the G2/M phase and demonstrated apoptotic and necrotic effects in comparison to the untreated control cells and increased the autophagic cell death (68.65%).
Molecular docking of the newly synthesized hybrids 4, 5, 8a, 10b, and 11b in the PARP-1 active sites involved their good accommodation interacting with the various amino acid residues through hydrogen bonding and π-π contacts. The SwissADME tool represented the good GIT absorption of compound 5 and the inability of all the compounds to cross the BBB.