Studies on 1,4-Quinone Derivatives Exhibiting Anti-Leukemic Activity along with Anti-Colorectal and Anti-Breast Cancer Effects

Colorectal cancer (CRC), breast cancer, and chronic myeloid leukemia (CML) are life-threatening malignancies worldwide. Although potent therapeutic and screening strategies have been developed so far, these cancer types are still major public health problems. Therefore, the exploration of more potent and selective new agents is urgently required for the treatment of these cancers. Quinones represent one of the most important structures in anticancer drug discovery. We have previously identified a series of quinone-based compounds (ABQ-1-17) as anti-CML agents. In the current work, ABQ-3 was taken to the National Cancer Institute (NCI) for screening to determine its in vitro antiproliferative effects against a large panel of human tumor cell lines at five doses. ABQ-3 revealed significant growth inhibition against HCT-116 CRC and MCF-7 breast cancer cells with 2.00 µM and 2.35 µM GI50 values, respectively. The MTT test also showed that ABQ-3 possessed anticancer effects towards HCT-116 and MCF-7 cells with IC50 values of 5.22 ± 2.41 μM and 7.46 ± 2.76 μM, respectively. Further experiments indicated that ABQ-3 induced apoptosis in both cell lines, and molecular docking studies explicitly suggested that ABQ-3 exhibited DNA binding in a similar fashion to previously reported compounds. Based on in silico pharmacokinetic prediction, ABQ-3 might display drug-like features enabling this compound to become a lead molecule for future studies.


Introduction
Colorectal cancer (CRC) is a very malignant and prevalent tumor worldwide. Age, local inflammatory changes, genetic components, and a substantial number of environmental and lifestyle factors account for important risks of CRC development. The great majority of patients with CRC are generally diagnosed at late stages with metastases requiring the administration of radiation therapy and chemotherapy as the leading therapeutic strategies for controlling the disease. Although these therapeutic options have evolved to targeted therapy and immunotherapy, the prognosis of CRC has never reached to the desired rate beyond a delay of disease progression so far. The complexity of the underlying mechanisms of the disease and the development of resistance are the major challenges for the treatment failure [1][2][3][4][5][6][7].  In order to enhance biological activity, we additionally studied the lead molecules to determine further activity against HCT-116 CRC and MCF7 breast cancer cells. Moreover, the most effective anticancer compound was subjected to further mechanistic experiments, such as the in vitro determination of levels of apoptosis in both cell lines and the in silico analysis of the DNA binding mode. Several pharmacokinetic determinants of this compound were also anticipated in silico.

In Vitro Anticancer Screening
In our previous study, PQ analogs ( Figure 3) were documented to show anticancer activities against leukemia cell lines [23]. This was followed by other reports claiming the anticancer potential of PQ analogs against breast and colon cancers as evidenced by their cytotoxic activities [22,39,40]. In the current work, the NCI initially screened three PQ analogs (ABQ-3, NCI: D-827196/1, ABQ-11, NCI: D-827197/1, and ABQ-12, NCI: D-827198/1) towards 60 cancer cell lines [41] at a single dose concentration (10 µM) on different cancer types, namely CRC, breast cancer, leukemia, melanoma, central nervous system (CNS), non-small cell lung cancer (NSCLC), ovarian, and renal and prostate cancer cell lines [42,43]. After this evaluation, ABQ-3 (2-chloro-3-((4-methoxyphenyl)amino)-5,6dimethyl-1,4-benzoquinone) was appointed as a lead PQ analog because of its significant selective anticancer potential compared to ABQ-11 and ABQ-12 for five-dose in vitro anticancer activity assessment in the range of 0.01-100 µM. In the current work, GI 50 (growth inhibitory activity), TGI (cytostatic activity), and LC 50 (cytotoxic activity)) [44] were used to evaluate the biological potential of the selected PQ analog ABQ-3. The GI 50 is an indicative concentration with a 50% growth inhibition, whereas TGI refers to the total growth inhibitory activity, and LC 50 is an indicative concen-tration in which 50% of cancer cells died. These parameters were calculated for each cell line from the log concentration versus % growth inhibition curves on 60 human cancer cell lines to create dose-response curves [43,45].
The selected PQ analog ABQ-3 showed high anticancer effects against all leukemia cell lines with GI 50 values of around 2.50 µM. ABQ-3 revealed sensitivity towards all leukemia cell lines in concordance with our previous encouraging results that the IC 50 values of ABQ-3 against K562, Jurkat, and MT-2 cells were found as 0.82 ± 0.07 µM, 1.51 ± 0.29 µM and 5.41 ± 0.95 µM, respectively. ABQ-3 also exerted low cytotoxicity against the healthy cell line (Table 1).   Table 2). This analog displayed excellent cytotoxicity towards HL-60, K-562, RPMI-8226, and SR cells with TGI ranging from 5.54 to 9.59 µM. In addition, LC 50

MTT Assay on HCT-116 and MCF-7 Cells
ABQ-3 displayed significant sensitivity towards HCT-116 and MCF-7 cell lines based on NCI GI 50 , TGI, and LC 50 parameters. In addition, CRC and breast cancer are important research platforms that we have been working on for a long time. Therefore, we further examined the anticancer effects of ABQ-3 on these two cell lines via an MTT (3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) assay at five dose concentrations (1, 3, 10, 30, and 100 µM) in comparison with cisplatin. Cisplatin was selected as a control because cisplatin has been used for the treatment of various cancers such as CRC and breast cancer [46][47][48][49][50].
According to the results, ABQ-3 inhibited the cell viability of HCT-116 and MCF-7 with notable IC 50    Since ABQ-3 displayed notable anticancer effects on CRC and breast cancer cells, we further searched for its apoptotic effects in HCT-116 and MCF-7 cells using the annexin V/ethidium homodimer III staining assay, which was detected by a fluorescence microscope representing apoptosis, necrosis, or late apoptosis/necrosis with green, yellow and red staining, respectively ( Figure 6A). ABQ-3 was found to possess similar apoptotic behavior in HCT-116 cells (61.80%) with cisplatin (62.30%). In addition, ABQ-3 showed 22.70% late apoptotic/necrotic and 15.50% necrotic activity in HCT-116 cells when compared with cisplatin (20.20% and 17.50%, respectively) ( Figure 6B). The difference in apoptosis enhancement between ABQ-3 and cisplatin treatment in HCT-116 cells was found to not be significant; conversely, it was found to be significant in MCF-7 cells, as shown in Figure 6C. MCF-7 cells underwent apoptosis with a higher percentage compared to HCT-116 cells (69.70%) after ABQ-3 exposure. ABQ-3 led to 16.67% late apoptosis/necrosis and 13.60% necrosis in MCF-7 cells ( Figure 6B).

The Conjecture of Pharmacokinetic Determinants
The pharmacokinetic features of ABQ-3 were estimated by projecting compounds on the QikProp algorithm [57]. Moreover, the inhibitory effects of ABQ-3 on cytochrome P450 (CYP) enzymes, the bioavailability, passive gastrointestinal absorption, and brain penetration of ABQ-3 were in silico anticipated with the help of the SwissADME web service [58,59]. ABQ-3 served a drug-like character that crucial pharmacokinetic determinants, including the octanol/water partition coefficient (QPlogPo/w), aqueous solubility (QPlogS), human serum albumin binding (QPlogKhsa), and brain/blood partition coefficient (QPlogBB) were coherent within specified ranges (Table 5). ABQ-3 exhibited an excellent % of absorption (100%) and followed Lipinski's rule of five and Jorgensen's rule of three without any violation. The demonstration of physicochemical determinants of ABQ-3 based on the Swis-sADME tool (Figure 9) indicated the components of the pink area, including saturation, size, polarity, solubility, lipophilicity, and flexibility abbreviated as INSATU, SIZE, POLAR, INSOLU, LIPO, and FLEX, respectively. The red hexagonal line must be entirely in this area for the optimal pharmacokinetic profile. This line of ABQ-3 was involved in this pink area just out of INSATU with a little range. This compound was able to inhibit CYP1A2, CYP2C19, CYP2C9, and CYP3A4 but matched no CYP2D6 inhibition. The BOILED-egg model ( Figure 10) refers to the potential of a compound for passive gastrointestinal absorption and BBB permeation. ABQ-3 was observed in the egg yolk (yellow region), implying its high penetration through the BBB. ABQ-3 was also detected with a red dot, explaining that it was not a substrate for P-glycoprotein [59][60][61].

Discussion
There are many gaps to fill in the treatment of CRC and breast cancer, albeit to the advancements in the screening programs and in the therapeutic options related to prognostic biomarkers. The discovery of new potential anticancer agents to be effective in both cancers may hold the key to the enhancement of treatment responses for patients with CRC and breast cancer. Several lines of evidence have documented that quinone-based compounds, in particular PQ analogs, stand out as promising candidates for anticancer drug discovery [62,63].
Our research group also demonstrated that PQ analogs were endowed with anti-CRC and/or anti-breast cancer properties. Compound 1 (Figure 11) [22] and compound 2 ( Figure 11) [39] showed anti-breast cancer effects against the MCF-7 cell line with IC 50 values of 6.58 µM and 6.53 ± 0.71 µM, respectively, whereas compound 3 ( Figure 11) [40] revealed anti-CRC effects towards HCT-116 cells with an IC 50 value of 4.97 ± 1.93 µM. In the recent study, ABQ-3 was selected regarding the protocol of NCI for the evaluation of its antiproliferative effects against a broad range of cancer cell types, including HCT-116 and MCF-7 cells, at five doses. ABQ-3 exerted notable anticancer effects on HCT-116 and MCF-7 cells with significant GI 50 , TGI, and LC 50 values. This finding points out that p-methoxy substitution on the anilino ring contributed to the anticancer effects of ABQ-3. When compared with our aforementioned studies [22,39,40], ABQ-3 revealed a similar anticancer potential against both cell lines.
Abnormalities in apoptosis can also deteriorate the pathogenesis of CRC and breast cancer and decrease the treatment success causing resistance to current therapy options [64][65][66][67][68][69][70][71]. ABQ-3 induced apoptosis in both cell lines compared to cisplatin. Comparing our previous results, it was also concluded that ABQ-3 displayed a similar apoptotic pattern with compound 3 (Figure 10) in HCT-116 cells [40].
Our research group previously reported the DNA binding potential of PQ analogs [40,[51][52][53] in the minor groove of DNA (PDB IDs: 2GWA [54] and 2GB9 [55]). ABQ-3 presented high affinity with a significant docking score value with important hydrogen bonding through quinone moiety. The 4-methoxy moiety made no contribution to the docking interactions of ABQ-3.
The determination of the theoretical prediction of the physicochemical properties of a drug candidate has enormously affected successful drug discovery. Several absorptions, distribution, metabolism, and excretion (ADME) parameters of ABQ-3 were estimated. Based on these parameters, ABQ-3 was determined to show significant lipophilicity and water solubility, indicating its ability to penetrate the cell membranes and distribute properly in aqueous compartments, respectively. ABQ-3 could possess an appropriate distribution volume and half-life at an acceptable dose and dose frequency related to its QPlogKhsa value. In particular, the penetration of a drug molecule from the blood into the brain is essential for the treatment of brain metastases of other cancer types. Both QikProp and SwissADME results showed that ABQ-3 could cross this barrier easily. ABQ-3 served a dug-likeness property, albeit to be out of the limit with a little rate for saturation in the bioavailability chart of SwissADME. ABQ-3 was not a substrate of P-glycoprotein: a membrane protein that causes less drug concentration in the cell and triggers the development of resistance. ABQ-3 matched the inhibition of CYP enzymes apart from CYP2D6, indicating that drug-drug interactions could emerge with molecules that undergo metabolism with these enzymes [72][73][74].

Chemistry
The synthetic experiments of ABQ-3, ABQ-11, and ABQ-12 were carried out previously. Their structures were characterized by spectral analysis previously [23].

NCI Single Dose Screening
The PQs were investigated for their growth inhibitory activity by the NCI (Bethesda, MD, USA) protocol at a 10 µM concentration in DMSO for 60 cancer cell lines. Compounds were added to the microtiter culture plates and incubated for 48 h at 37 • C. Sulforhodamine B (SRB) was applied for end-point detection. The percentage of the growth of the exposed cells was measured in comparison to the nonexposed control cells, and the findings of each tested compound were calculated [43,44,74].

NCI Five-Dose Anticancer Screening
Serial 5 × 10-fold dilution was carried out from an initial DMSO stock solution before incubation at each individual concentration. The Developmental Therapeutics Program (DTP)-NCI screened the most effective compound (ABQ-3) for a higher testing level to determine GI 50 , TGI, and LC 50 for each cell line after generating a dose-response curve, including concentrations of 0.01, 0.1, 1, 10, and 100 µM for ABQ-3 [44,45,75] [40]. The stock solution of ABQ-3 was prepared in DMSO (Wako Pure Chemical Industries, Osaka, Japan) and cisplatin in DMF (Wako Pure Chemical Industries, Osaka, Japan) at concentrations in the range of 0.1-10 mM and further was diluted with fresh culture medium. The concentration of DMSO and DMF in the final culture medium was 1% without affecting the cell viability [23,40].
MTT (Dojindo Molecular Technologies, Kumamoto, Japan) assay was used as previously explained [23,40] for the investigation of the effects of ABQ-3 and cisplatin on cell viability. HCT-116 and MCF-7 cell lines were subjected to ABQ-3 and cisplatin at 1, 3, 10, 30, and 100 µM concentrations at 37 • C for 48 h before being stained with an MTT solution and further incubated for 4 h. After the removal of supernatants, 100 µL DMSO and DMF (for cisplatin) were added to each well. For the analysis of the absorbance of the solution, a plate reader Infinite M1000 (Tecan, Mannedorf, Switzerland) was used. All experiments were applied with three repeats, and IC 50 values were identified as the drug concentrations, which decreased the absorbance to 50% of the control values.

Apoptosis Detection
HCT-116 and MCF-7 cells were incubated with ABQ-3 and cisplatin at an IC 50 concentration for 12 h before the cell death detection kit (PromoKine, Heidelberg, Germany) was carried out with some modifications to the manufacturer's protocol [40]. Both cell lines were subjected to a binding buffer and staining solution and were then analyzed by fluorescence microscope Biorevo Fluorescence BZ-9000 (Keyence, Osaka, Japan) [76].

Statistical Data
All findings were exhibited as means ± SD. Data were screened using a one-way analysis of variance, and diversity was accepted 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.

Absorption, Distribution, Metabolism, and Excretion (ADME) Estimation
The QikProp [57] and SwissADME web tool [58] were used for the determination of several important pharmacokinetic parameters of ABQ-3.

Conclusions
The quinone-based compound ABQ-3 was selected for NCI-60 during in vitro screening at five doses towards a huge panel of cancer cells, including HCT-116 and MCF-7 cell lines. ABQ-3 displayed 2.00 µM GI 50 , 4.37 µM TGI, and 9.55 µM LC 50 values against HCT-116 cells, whereas these values were detected as 2.35 µM, 6.05 µM and 30.60 µM, respectively, against MCF-7 cells, demonstrating the growth inhibitory, cytostatic, and cytotoxic effects of ABQ-3 on these cell lines. Based on MTT screening, this compound also showed significant cytotoxicity against HCT-116 and MCF-7 cells. Furthermore, the cell death assay demonstrated that ABQ-3 enhanced apoptotic activity in HCT-116 and MCF-7 cell lines when compared with cisplatin. Molecular docking studies suggested the strong DNA binding of ABQ-3. In silico ADME prediction indicated that ABQ-3 afforded positive drug-likeness values, thus, making this titled compound a potentially good and orally active anticancer agent for future studies. Funding: This work was financially supported by the Scientific Research Projects Coordination Unit of Istanbul University. This publication has been produced benefiting from TUBITAK 2236 CoCirculation2, grant number 121C063. However, the entire responsibility of the publication belongs to the authors. The financial support received from TUBITAK does not mean that the content of the publication is approved in a scientific sense by TUBITAK.
Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.