In Vitro Cytotoxicity Evaluation of Plastoquinone Analogues against Colorectal and Breast Cancers along with In Silico Insights

Colorectal cancer (CRC) and breast cancer are leading causes of death globally, due to significant challenges in detection and management. The late-stage diagnosis and treatment failures require the discovery of potential anticancer agents to achieve a satisfactory therapeutic effect. We have previously reported a series of plastoquinone analogues to understand their cytotoxic profile. Among these derivatives, three of them (AQ-11, AQ-12, and AQ-15) were selected by the National Cancer Institute (NCI) to evaluate their in vitro antiproliferative activity against a panel of 60 human tumor cell lines. AQ-12 exhibited significant antiproliferative activity against HCT-116 CRC and MCF-7 breast cancer cells at a single dose and further five doses. MTT assay was also performed for AQ-12 at different concentrations against these two cells, implying that AQ-12 exerted notable cytotoxicity toward HCT-116 (IC50 = 5.11 ± 2.14 μM) and MCF-7 (IC50 = 6.06 ± 3.09 μM) cells in comparison with cisplatin (IC50 = 23.68 ± 6.81 μM and 19.67 ± 5.94 μM, respectively). This compound also augmented apoptosis in HCT-116 (62.30%) and MCF-7 (64.60%) cells comparable to cisplatin (67.30% and 78.80%, respectively). Molecular docking studies showed that AQ-12 bound to DNA, forming hydrogen bonding through the quinone scaffold. In silico pharmacokinetic determinants indicated that AQ-12 demonstrated drug-likeness with a remarkable pharmacokinetic profile for future mechanistic anti-CRC and anti-breast cancer activity studies.


Introduction
Colorectal cancer (CRC), the third most common cancer type and the fourth leading cause of cancer-related death, comprises nearly 10% of all annually diagnosed tumors across the world [1][2][3][4]. Male gender, old age, dietary habits, and environmental factors affect the pathogenesis of CRC as well as the genetic background [5]. In spite of advancements in CRC screening and treatment options such as surgery, radiotherapy, local ablative therapy, chemotherapy, targeted therapy, and immunotherapy, most of cases in particular diagnosed at an advanced stage with metastases, result in subsequent cancer-related deaths. The size, stage, and metastasis of tumor whether the therapy will be curative or palliative. Among CRC treatment options, chemotherapy incorporates single-agent therapy (primarily fluoropyrimidine, oxaliplatin, irinotecan, and capecitabine) or combined regimens of these agents. Targeted therapy is another approach, which has been reported to extend survival of patients with CRC. However, the resistance and toxicity problems restrict the success of the therapy. Therefore, there is an urgency to find more effective and safer drugs [2,6].
The history of breast cancer was shown to be a risk factor for CRC in several studies, implying low or high relative risks. These risks were considered due to the common etiologic factors associated with the development of both cancers and administration of antihormone drugs in breast cancer treatment, which alter sex hormone levels and contribute to the development of CRC [7][8][9][10][11]. New efficacious therapeutic options have been also developed for the battle with breast cancer related to its clinical stage, histopathologic properties, and biomarker profiling. These options include traditional, personalized, neoadjuvant, and targeted therapies. The treatment still remains limited mainly in the breast cancer metastasis owing to heterogeneity of the disease, acquired and primary resistance, and toxicity problems during the treatment. New agents should be also developed to overcome or prevent these problems in breast cancer treatment [12][13][14].
Much effort to design and discover efficient and safe drug candidates led to identifying several hit compounds and analogues of natural products. In silico analyses were exploited to improve molecules with greater potential efficacy to cope with the adverse toxicological outcomes by emphasizing physicochemical parameters [15]. In addition, the study of the structure-activity relationship (SAR) has provided valuable information on the design of safe drug candidates with continuity about how structural changes can improve potency and bioavailability [16].
1,4-Quinones have been explored as attractive anticancer hit molecules to their multitargeted mode of actions [17][18][19][20][21][22]. In this field, a small library of amino-quinones based on bioactive natural products (fifty Plastoquinone (PQ) [23][24][25] and thirty-four LY83583 analogues [26,27]) that specifically target leukemia cancer cell lines, in the period 2017-2021, was generated for the purpose of discovering the SAR of various substituents in aminoquinones for their further mechanistic anticancer potential. Knowing that the PQ analogues are active toward some cancer cell lines, and considering our previous findings that demonstrated greater activity by the introduction of a chlorine atom in the quinone moiety, we also designed and evaluated the effect on inhibitory activity against some cancer cell lines caused by replacing the chlorine with a bromine atom in PQ analogues [28]. Regarding all PQ analogues, including halogenated (brominated and chlorinated) and non-halogenated analogues, a major breakthrough was the discovery of AQ-11, AQ-12, and AQ-15 [25], as illustrated in Figure 1. These analogues showed consistent growth-inhibitory activities with low IC 50 value against K562 human chronic myelogenous leukemia (CML) cell line and low toxicity toward human peripheral blood mononuclear cells (PBMCs) (healthy) ( Table 1) [25].   Considering the encouraging results of the target PQ analogues, further studies were assessed aiming at the identification of new analogues for their antiproliferative activity against HCT-116 human CRC and MCF-7 human breast cancer cell lines. In addition, apoptosis-inducing activity in both cell lines, DNA binding characteristics, and a number of pharmacokinetic descriptors of the most effective anticancer analogue were examined.

Anticancer Activity Assessment
2.1.1. In Vitro Screening of Tumor Cell Growth Inhibition at One Dose Continuing our efforts on anticancer drug discovery, most effective PQ analogues from our previous study [25] were submitted to the National Cancer Institute (NCI) of Bethesda within the Developmental Therapeutics Program (DTP) for their in vitro anticancer activity with the protocol of the Drug Evaluation Branch, NCI. A single dose (10 μM) of all tested PQ analogues was used in the panel of 60 human cancer cell lines, including nine tumor subpanels, namely: leukemia, lung, CRC, central nervous system (CNS), melanoma, ovarian, renal, prostate, and breast cancer cell lines [29][30][31]. The in vitro growth inhibition and lethality were ascertained as percentages as follows: growth inhibition (G%) (values between 0 and 100) and lethality (values less than 0). Herein, three PQ analogues (AQ-11, NCI: D-827199/1; AQ-12, NCI: D-827200/1; and AQ-15, NCI: D- Considering the encouraging results of the target PQ analogues, further studies were assessed aiming at the identification of new analogues for their antiproliferative activity against HCT-116 human CRC and MCF-7 human breast cancer cell lines. In addition, apoptosis-inducing activity in both cell lines, DNA binding characteristics, and a number of pharmacokinetic descriptors of the most effective anticancer analogue were examined.

Results
2.1. Anticancer Activity Assessment 2.1.1. In Vitro Screening of Tumor Cell Growth Inhibition at One Dose Continuing our efforts on anticancer drug discovery, most effective PQ analogues from our previous study [25] were submitted to the National Cancer Institute (NCI) of Bethesda within the Developmental Therapeutics Program (DTP) for their in vitro anticancer activity with the protocol of the Drug Evaluation Branch, NCI. A single dose (10 µM) of all tested PQ analogues was used in the panel of 60 human cancer cell lines, including nine tumor subpanels, namely: leukemia, lung, CRC, central nervous system (CNS), melanoma, ovarian, renal, prostate, and breast cancer cell lines [29][30][31]. The in vitro growth inhibition and lethality were ascertained as percentages as follows: growth inhibition (G%) (values between 0 and 100) and lethality (values less than 0). Herein, three PQ analogues (AQ-11, NCI: D-827199/1; AQ-12, NCI: D-827200/1; and AQ-15, NCI: D-827201/1) were selected by the NCI for in vitro disease-oriented human-cell-screening panel assay.
The results of each tested PQ analogue were reported in terms of percent growth inhibition (GI% = 100 − G%) and lethality [32] (Table 2) and were also depicted as bars in the single-dose mean graphs (Supplementary Materials, Figures S1-S3). Overall, consistent with the previous data, PQ analogues showed the most notable anticancer activity against leukemia cancer cell lines. AQ-11 and AQ-15 were found ineffective against the other cancer cell lines except for significant anticancer effects of AQ-11 on MDA-MB-231 breast cancer cell line with 84.65% inhibition percent. On the other hand, NCI-60 data suggested that AQ-12 revealed prominent anticancer activity toward the subpanel cell line of CRC (HCT-116 cells, 66.14% inhibition; SW-620 cells, 82.93% inhibition) and breast cancer (MCF-7 cells, 64.64% inhibition; MDA-MB-231 cells, 81.04% inhibition). Additionally, this analogue also showed promising anticancer effects against NCI-H522 lung cancer cells. From the single-dose assay data from the NCI screen, AQ-12 was selected as a lead PQ analogue because of its pronounced anticancer selectivity compared with other PQ analogues. AQ-12 exhibited the threshold inhibition criterion in the single-dose screening and was qualified for the evaluation in the full-panel five-dose in vitro anticancer screening at 10-fold dilutions in the range 0.01-100 µM. Three response parameters (50% cell growth inhibition (GI 50 ) (growth inhibitory activity), total cell growth inhibition (TGI) (cytostatic activity), and 50% cell death (LC 50 ) (cytotoxic activity)) [33] were used to establish biological potential of the tested AQ-12. The GI 50 is an indicative concentration at 50% growth inhibitory activity, whereas TGI reflects total growth inhibition, and LC 50 is an indicative concentration at which 50% of cancer cells are killed. In this assay, three parameters were calculated for each cell line from log concentration versus percent growth inhibition curves on nine panels of human cancer cell lines to generate dose response curves [31,34]. GI 50 is the concentration of the test drug where 100 × (T − T0)/(C − T0) = 50. Herein, T explains the optical density of the test well after a 48 h period of treatment with the test drug; T0 explains the optical density at time zero; ultimately, C is the control (nondrug) optical density. The "50" is called the GI50PRCNT, a T/C-like parameter that can have values from +100 to −100. The TGI is the concentration of test drug where 100 × (T − T0)/(C − T0) = 0. LC 50 is the concentration of the drug where 100 × (T − T0)/T0 = −50 [31].
The GI 50 , TGI, and LC 50 (in µM) values against subpanel cell lines are illustrated in Table 3, indicating that AQ-12 displays high anticancer activity against all leukemia cell lines with GI 50 values ranging from 1.32 to 2.59 µM. This compound also demonstrated superior cytotoxic activity against HL-60(TB) and RPMI-8226 cell lines with TGI values 6.54 and 7.32 µM, respectively. On the other hand, LC 50 values were more than 100 µM against the entire panel of cancer cell lines. Moderate cytotoxicity was recorded against non-small cell lung cancer cells, except for EKVX cell line with a GI 50 Figure 2 and Table 3, which include nine tumor subpanels, namely: leukemia, melanoma, CRC, lung, CNS, ovarian, renal, prostate, and breast cancer cell lines.

Cell Viability Assay on CRC and Breast Cancer Cells
AQ-12 was found to possess a superior sensitivity profile toward H MCF-7 breast cancer cell lines with a higher growth inhibitory percent c 11 and AQ-15. AQ-12 also displayed sensitivity toward MDA-MB-468 H522 lung cancer cell lines. However, AQ-11 also presented significant g effects on MDA-MB-468 breast cancer cells, restricting the selectivity of A cell line, and GI50, TGI, and LC50 parameters were found very high agains cancer cells. Satisfactory results obtained from both single and five screening toward HCT-116 CRC and MCF-7 breast cancer cells encoura investigate the anticancer effects of AQ-12 against these two cell lines dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) assay at five do (1, 3, 10, 30, and 100 μM) in comparison with cisplatin, the reference age Cisplatin, a metallic coordination compound leading to DNA dam quently apoptosis induction in cancer cells, is one of the most important c agents, which has been approved for the treatment of different fatal can ing CRC and breast cancer [35][36][37][38][39][40][41].
Results indicated that AQ-12 showed cytotoxic effects on HCT-116 with IC50 values of 5.11 ± 2.14 μM and 6.06 ± 3.09 μM when compared w = 23.68 ± 6.81 μM for HCT-116 cells and 19.67 ± 5.94 μM for MCF-7 ce decline in percentage of viable cells was detected between 3 and 10 μM a sure while a similar decline was observed between 10 and 30 μM after mentation ( Figure 3, Table 4).  Cisplatin, a metallic coordination compound leading to DNA damage and subsequently apoptosis induction in cancer cells, is one of the most important chemotherapeutic agents, which has been approved for the treatment of different fatal cancer types, including CRC and breast cancer [35][36][37][38][39][40][41].

Cell Death Investigation
Based on significant anticancer activity results of AQ-12 on HCT-116 and MCF-7 cells, we also further investigated potential effects of this compound on apoptosis in both cell lines using the annexin V/ethidium homodimer III staining procedure, which was observed by fluorescence microscopy, indicating apoptosis, necrosis or late apoptosis, and necrosis with green, yellow, and red staining, respectively ( Figure 4A). AQ-12 induced apoptotic behavior of HCT-116 cells (62.30%) in a similar manner with cisplatin (67.30%). This compound exhibited 21.30% late apoptotic/necrotic and 16.40% necrotic effects in HCT-116 cells compared to cisplatin (12.30% and 20.40%, respectively) ( Figure 4B). The difference of apoptosis induction between AQ-12 and cisplatin treatment in HCT-116 cells was found not significant, contrary to that of MCF-7 cells, which was found significant ( Figure 4C

Cell Death Investigation
Based on significant anticancer activity results of AQ-12 on HCT-116 and MCF-7 cells, we also further investigated potential effects of this compound on apoptosis in both cell lines using the annexin V/ethidium homodimer III staining procedure, which was observed by fluorescence microscopy, indicating apoptosis, necrosis or late apoptosis, and necrosis with green, yellow, and red staining, respectively ( Figure 4A). AQ-12 induced apoptotic behavior of HCT-116 cells (62.30%) in a similar manner with cisplatin (67.30%). This compound exhibited 21.30% late apoptotic/necrotic and 16.40% necrotic effects in HCT-116 cells compared to cisplatin (12.30% and 20.40%, respectively) ( Figure 4B). The difference of apoptosis induction between AQ-12 and cisplatin treatment in HCT-116 cells was found not significant, contrary to that of MCF-7 cells, which was found significant ( Figure 4C   The percentage of apoptosis, late apoptosis/necrosis, and necrosis (green, yellow, and red, respectively) cells (C) was determined by analyzing 100 randomly selected stained cells in each experiment (ns: not statistically significant). Data from three independent experiments were expressed as means ± standard deviation and p values were determined using Student's test.

Molecular Docking
In our previous studies, we manifested that PQ analogues were able to bind DNA significantly [26,27,42]. In this study, the DNA binding effects of AQ-11, AQ-12, and AQ-15 were also searched with molecular docking studies in the minor groove of the double helix of DNA (PDB ID: 2GWA) [43] via Maestro software [44]. Results corresponded to previous DNA cleavage outcomes, implying that AQ-15 showed the most promising DNA binding potential through a key π-π interaction between DG-4 with its 4-methyl substituent. However, AQ-11 and AQ-12 displayed less binding capacity compared to AQ-15, forming hydrogen bonding with DT-5 and DG-4, respectively ( Figure 5A,B). The docking scores were determined as −4.641 kcal/mol, −5.087 kcal/mol, and −5.097 kcal/mol for AQ-11, AQ-12, and AQ-15, indicating higher binding capacity of AQ-15 compared to AQ-11 and AQ-12. The percentage of apoptosis, late apoptosis/necrosis, and necrosis (green, yellow, and red, respectively) cells (C) was determined by analyzing 100 randomly selected stained cells in each experiment (ns: not statistically significant). Data from three independent experiments were expressed as means ± standard deviation and p values were determined using Student's test.

Molecular Docking
In our previous studies, we manifested that PQ analogues were able to bind DNA significantly [26,27,42]. In this study, the DNA binding effects of AQ-11, AQ-12, and AQ-15 were also searched with molecular docking studies in the minor groove of the double helix of DNA (PDB ID: 2GWA) [43] via Maestro software [44]. Results corresponded to previous DNA cleavage outcomes, implying that AQ-15 showed the most promising DNA binding potential through a key π-π interaction between DG-4 with its 4-methyl substituent. However, AQ-11 and AQ-12 displayed less binding capacity compared to AQ-15, forming hydrogen bonding with DT-5 and DG-4, respectively ( Figure 5A,B). The docking scores were determined as −4.641 kcal/mol, −5.087 kcal/mol, and −5.097 kcal/mol for AQ-11, AQ-12, and AQ-15, indicating higher binding capacity of AQ-15 compared to AQ-11 and AQ-12.

Estimation of Pharmacokinetic Parameters
AQ-12 was profiled in silico for various pharmacokinetic properties of interest such as octanol/water partition coefficient (QPlogPo/w), aqueous solubility (QPlogS), human serum albumin binding (QPlogKhsa), brain/blood partition coefficient (QPlogBB), and compliance to Lipinski's rule of five and Jorgensen's rule of three using the QikProp algorithm [45]. We also checked the in silico inhibitory potential of AQ-12 on several cytochrome P450 (CYP) enzymes such as CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4, along with the evaluation of bioavailability and passive gastrointestinal absorption and brain penetration using the SwissADME web service [46,47].
AQ-12 represented a remarkable pharmacokinetic profile in which all the descriptors were found in appropriate ranges: QPlogPo/w, QPlogS, QPlogKhsa, and QPlogBB were computed with the values of 3.513, −5.035, 0.197, and 0.140, respectively, within the limits (−2 to 6.5, −6.5 to 0.5, −1.5 to 1.5, and −3 to 1.2, respectively). Additionally, AQ-12 revealed robust human oral absorption (100%) and was found to possess of all the conditions of drug-likeness characters without any violation of Lipinski's rule of five and Jorgensen's rule of three.
The pink region of bioavailability radar ( Figure 6) identifies the values of saturation (INSATU), size (SIZE), polarity (POLAR), solubility (INSOLU), lipophilicity (LIPO), and flexibility (FLEX) for oral bioavailability. AQ-12 was found only beyond the saturation value for other values it was participating in, as shown in the pink area. AQ-12 matched with CYP1A2, CYP2C19, CYP2C9, and CYP3A4 inhibition, apart from CYP2D6 inhibition, indicating that AQ-12 could cause possible drug-drug or drug-food interactions. The boiled-egg model (Figure 7) explains whether a molecule has properties for the passive gastrointestinal absorption and blood-brain barrier (BBB) permeation. According to the results, AQ-12 was predicted as brain-penetrant (in the yellow area) and not a substrate for P-glycoprotein (red dot), which decreased the possibility of its resistance by tumor cell lines through efflux [47][48][49][50].

Discussion
Worldwide, CRC and breast cancer are prevalent and deadly cancers. The complete cure for both cancers is still far from success, albeit to increase overall survival rate obtained with new therapeutic options. Therefore, the discovery of new and better thera-

Discussion
Worldwide, CRC and breast cancer are prevalent and deadly cancers. The complete cure for both cancers is still far from success, albeit to increase overall survival rate obtained with new therapeutic options. Therefore, the discovery of new and better therapeutics is still needed [51,52]. In spite of numerous efforts in the search for more effective

Discussion
Worldwide, CRC and breast cancer are prevalent and deadly cancers. The complete cure for both cancers is still far from success, albeit to increase overall survival rate obtained with new therapeutic options. Therefore, the discovery of new and better therapeutics is still needed [51,52]. In spite of numerous efforts in the search for more effective anticancer agents, quinone moiety still remains one of the most versatile members against cancer cell lines in drug discovery [53,54].
In our previous studies, we also reported the significant outcomes of quinone derivatives against CRC or breast cancer cell lines. We showed that compound PQ11, PQ analogue with N-phenylpiperazine (Figure 8), exhibited the most potent anticancer activity against MCF-7, MDA-MB-231, and UACC-2087 cell lines, with the IC 50 values of 6.58, 16.66, and 38.52 µM [28]. In our recently published studies, we also confirmed anti-CRC and anti-breast cancer effects of PQ analogues. In the first study [42], the most significant cytotoxic effects were observed with PQ2, amino-1,4-benzoquinone (Figure 8), against HCT-116 CRC cells with an IC 50 value of 4.97 ± 1.93 µM. In the latter one [55], compound ClPQ1, quinone-benzocaine hybrid molecule, (Figure 8) was found as the most effective anti-breast cancer agent against T47D and MCF-7 breast cancer cells, with IC 50 values of 2.35 ± 0.30 and 6.53 ± 0.71 µM, respectively. In the current work, PQ analogues (AQ-11, AQ-12, and AQ-15) were selected by the NCI in vitro disease-oriented antitumor screening to be evaluated for their anticancer effects. Testing of the PQ analogues against the NCI-60 cell line panel revealed valuable information on their inhibitory activity across a broad variety of human cancer cell lines. In particular, AQ-12 displayed potential growth inhibitory activity against HCT-116 and MCF-7 cell lines at a single dose and a super-sensitivity profile with low micromolar GI50, TGI, and LC50 values against both cell lines at five doses. These findings indicated that meta trifluoromethyl substitution of AQ-12 played an important role in its significant anti-CRC and anti-breast cancer activity when compared with the para methyl substitution of AQ-15 and non-substitution of AQ-11. Moreover, AQ-12 exerted similar cytotoxic effects against both cell lines in comparison with our aforementioned studies [28,42,55]. Current results once more confirmed that the presence of PQ moiety played an important role in anti-CRC and anti-breast cancer activity.
Genetically encoded programmed cell death (apoptosis) leads to elimination of cancer cells, and DNA degradation is one of the crucial indicators of apoptosis. Aberrant apoptotic activity can increase not only the pathogenesis of CRC and breast cancer, but also their resistance to current therapy options [56][57][58][59]. Regarding the anticancer efficacy of AQ-12 in CRC and breast cancer cells, it was ascertained that AQ-12 led to apoptosis in both cells with similar apoptotic pattern with PQ2 ( Figure 8) [42].
Molecular docking studies were carried out for AQ-12 in order to discover its binding efficacy in the minor groove of the double helix of DNA (PDB ID: 2GWA). We previously showed that PQ analogues occupied this region with key interactions [26,27,42]. The 3,5dimethyl phenyl [26] and benzodioxole [27] moieties were determined to be crucial in binding with DNA, forming π-π stacking interactions with DA-17 and DG-16, and DA-5 and DG-4, respectively. In other our previous study [27], the methoxy substitution was also found to be important for high interaction between PQ2 and DT-5 in the minor groove of DNA. In the current study, AQ-12 was found less capable of binding DNA compared to AQ-15, albeit to hydrogen bonding with DG-4 through quinone moiety. The trifluoromethyl substitution of AQ-12 played no significant role in binding with DNA. AQ-15 In the current work, PQ analogues (AQ-11, AQ-12, and AQ-15) were selected by the NCI in vitro disease-oriented antitumor screening to be evaluated for their anticancer effects. Testing of the PQ analogues against the NCI-60 cell line panel revealed valuable information on their inhibitory activity across a broad variety of human cancer cell lines. In particular, AQ-12 displayed potential growth inhibitory activity against HCT-116 and MCF-7 cell lines at a single dose and a super-sensitivity profile with low micromolar GI 50 , TGI, and LC 50 values against both cell lines at five doses. These findings indicated that meta trifluoromethyl substitution of AQ-12 played an important role in its significant anti-CRC and anti-breast cancer activity when compared with the para methyl substitution of AQ-15 and non-substitution of AQ-11. Moreover, AQ-12 exerted similar cytotoxic effects against both cell lines in comparison with our aforementioned studies [28,42,55]. Current results once more confirmed that the presence of PQ moiety played an important role in anti-CRC and anti-breast cancer activity.
Genetically encoded programmed cell death (apoptosis) leads to elimination of cancer cells, and DNA degradation is one of the crucial indicators of apoptosis. Aberrant apoptotic activity can increase not only the pathogenesis of CRC and breast cancer, but also their resistance to current therapy options [56][57][58][59]. Regarding the anticancer efficacy of AQ-12 in CRC and breast cancer cells, it was ascertained that AQ-12 led to apoptosis in both cells with similar apoptotic pattern with PQ2 ( Figure 8) [42].
Molecular docking studies were carried out for AQ-12 in order to discover its binding efficacy in the minor groove of the double helix of DNA (PDB ID: 2GWA). We previously showed that PQ analogues occupied this region with key interactions [26,27,42]. The 3,5dimethyl phenyl [26] and benzodioxole [27] moieties were determined to be crucial in binding with DNA, forming π-π stacking interactions with DA-17 and DG-16, and DA-5 and DG-4, respectively. In other our previous study [27], the methoxy substitution was also found to be important for high interaction between PQ2 and DT-5 in the minor groove of DNA. In the current study, AQ-12 was found less capable of binding DNA compared to AQ-15, albeit to hydrogen bonding with DG-4 through quinone moiety. The trifluoromethyl substitution of AQ-12 played no significant role in binding with DNA. AQ-15 bound to DG-4 through its p-methyl moiety, forming π-π stacking interactions. The docking score with the lowest energy (high negative scores) was found to pertain to AQ-15, followed by AQ-12 and AQ-11, indicating their binding affinities. Compare to our previous studies, it can be concluded that CH 3 substitution (−σ effect), OCH 3 substitution (−σ effect), and (-CH 2 -O-CH 2 -) (−σ effect) [26,27,42] were found to increase the binding capacity of the tested compounds, whereas CF 3 substitution (+σ effect) was not detected to contribute to binding capacity of AQ-12. The higher docking score and the binding capacity of p-methylsubstituted AQ-15 also complied with the previous data. This finding also suggested that the high apoptotic effect of AQ-12 might be independent from DNA cleavage-associated cell death.
Absorption, distribution, metabolism, and excretion (ADME) parameters of a drug molecule have an enormous impact for successful drug discovery. Some of these essential parameters were predicted in silico for AQ-12. Lipophilicity is crucial for absorption, which is the process of movement of a drug into the systemic circulation crossing the lipid bilayers of cell membranes. On the other hand, optimum water solubility is also necessary since the active ingredient must be dissolved in aqueous compartments to some extent before drug absorption. The human serum albumin binding is directly associated with the volume of distribution and half-life of drugs. The transition of drugs from blood into brain is particularly important for brain metastases of other cancer types. According to the results of the QikProp module, AQ-12 was endowed with drug-like properties. The outcomes of SwissADME web server signified that AQ-12 was predicted not orally bioavailable. This was due to the out-of-limits for saturation, as shown in the bioavailability chart, in which a molecule must be entirely included in the pink area. AQ-12 exerted inhibition against all tested CYP enzymes, except for CYP2D6, which had a higher risk for drug-drug interactions [60][61][62][63].

In Vitro Single-Dose Anticancer Screening by NCI
The PQ analogues were submitted to NCI, Bethesda, USA, and screened based on the procedures of NCI; all compounds were investigated for their cancer cell growth inhibitory activity at 10 µM concentration against a wide range of cancer cell lines stemming from leukemia, melanoma, CRC, non-small cell lung, CNS, ovarian, renal, prostate, and breast cancers. Tested compounds were added to the microtiter culture plates followed by incubation for 48 h at 37 • C. SRB was used for end point detection. The percent of growth of the treated cells was observed compared to the untreated control cells. Data from one-dose experiments corresponded to the percentage growth at 10 µM [29][30][31]34,64].

In Vitro Five-Dose Anticancer Screening by NCI
Initial DMSO stock solution was carried out for serial 5 × 10-fold dilution before incubation at each individual concentration. AQ-12 was selected for a higher testing level by DTP-NCI to identify GI 50 , TGI, and LC 50 for each cell line after generating a dose response curve from 5 different concentrations (0.01, 0.1, 1, 10, and 100 µM). The definite protocol for the latter assay was explained in detail previously. The cells were assayed by using the SRB method. The optical densities were measured by a plate reader and a microcomputer processed the optical densities into the special concentration parameters, as defined above [29,34,64,65].

Cell Culture, Drug Treatment, and MTT Assay
The HCT-116 cell line (provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT/AMED, Japan (RCB2979)) and MCF-7 cell line (Precision Bioservices, Frederick, MD, USA) were incubated in Dulbecco's modified Eagle's medium (DMEM) (Wako Pure Chemical Industries, Osaka, Japan) and RPMI 1640 (Wako Pure Chemical Industries, Osaka, Japan), respectively. Ten percent fetal bovine serum (FBS) (Sigma Aldrich, St. Louis, MO, USA) and 89 µg/mL streptomycin (Meiji Seika Pharma, Tokyo, Japan) were added to total media (Wako Pure Chemical Industries) at 37 • C and 5% CO 2 atmosphere. HCT-116 and MCF-7 cells were cultured for 48 h in a 24-well plate (Iwaki brand Asahi Glass Co., Chiba, Japan) at 4 × 10 4 cells/mL concentration [42]. The stock solution of AQ-12 and cisplatin in concentrations were prepared in DMSO (Wako Pure Chemical Industries, Osaka, Japan) (0.1 to 10 mM), and fresh culture medium was used for further dilution. The final DMSO concentration was set at 1% to prevent any effect of it on cell viability. MTT (Dojindo Molecular Technologies, Kumamoto, Japan) was used to examine the cytotoxic effects of AQ-12 and cisplatin, as previously indicated [66]. HCT-116 and MCF-7 cells were treated with AQ-12 and cisplatin at five dose concentrations (1, 3, 10, 30, and 100 µM) at 37 • C for 48 h, and then treated with MTT solution and incubated for 4 h. Eventually, 100 µL DMSO was added to each well following removal of supernatants. Infinite M1000 (Tecan, Mannedorf, Switzerland) was used for the analysis of the absorbance of the solution. All experiments were repeated three times, and IC 50 values were calculated as the drug concentrations that diminished absorbance to 50% of control values.

Cell Death Analysis
The HCT-116 and MCF-7 cell lines were incubated with AQ-12 and cisplatin at IC 50 concentration for 12 h before the apoptotic/necrotic/detection kit (PromoKine, Heidelberg, Germany) was applied, with some alterations to the manufacturer's guidance [42]. HCT-116 and MCF-7 cells, treated with appropriate content including binding buffer and staining solution, were analyzed by an all-in-one fluorescence microscope, Biorevo Fluorescence BZ-9000 (Keyence, Osaka, Japan). Numbers of apoptotic, late apoptotic/necrotic, and necrotic cells were determined based on the staining with annexin V and ethidium homodimer III, as previously explained [66].

Statistical Analyses
All results were reported as means ± SD. One-way analysis of variance was used for the analysis of data. Differences were defined as significant at * p < 0.05, ** p < 0.01, and *** p < 0.001. GraphPad Prism7 (GraphPad Software, San Diego, CA, USA) was used for the determination of the IC 50 values.

Molecular Docking
AQ-11, AQ-12, and AQ-15 were prepared with energy minimization by applying the OPLS_2005 force field at physiological pH using the LigPrep module. The crystallographic structure of DNA was downloaded from the PDB server (PDB ID: 2GWA) [43,44] and prepared for the docking assessment by the PrepWizard module of Maestro. Then, the determined grid by Grid Generation was used for molecular docking with Glide/XP docking procedures [26,27,42].