Clifford W Fong Eigenenergy
email: cwfong@internode.on.net

Role of stable free radicals in conjugated antioxidant and cytotoxicity treatment of triple negative breast cancer

Keywords: Triple negative breast cancer, oxidative stress, BRCA1 mutations, stable free radicals, antioxidants, Tempol conjugates, cytotoxicity, mitochondrial dysfunction, quantum mechanics, HOMO-LUMO gap

TNBC triple negative breast cancer, BRCA1 human tumour suppressor gene and protein, MnSOD manganese superoxide dismutase, ΔGdesolv,CDS free energy of water desolvation, ΔGlipo,CDS lipophilicity free energy, CDS cavity dispersion solvent structure of the first solvation shell, HOMO highest occupied molecular orbital, LUMO lowest unoccupied molecular orbital , R 2 multiple correlation coefficient, the F test of significance, SEE standards errors for the estimate, standard errors of the variables SE(ΔGdesolCDS), SE(ΔGlipoCDS), SE(Dipole Moment), SE (Molecular Volume, ROS reactive oxygen species

Introduction

Oxidative stress is well known to be involved in many breast cancers, including triple negative breast cancer (TNBC). TNBC are breast cancers that do not have receptors for estrogen (ER), progesterone (PR), human epidermal growth factor receptor 2 (HER2) commonly found in most breast cancers. [START_REF] Vera-Ramirez | Biological bases to develop oxidative-based therapies[END_REF][START_REF] S Ríos-Arrabal | Involvement of free radicals in breast cancer[END_REF][START_REF] Bl Gilmore | Molecular Analysis of BRCA1 in Human Breast Cancer Cells Under Oxidative Stress[END_REF][START_REF] Valko | Free radicals, metals and antioxidants in oxidative stress-induced cancer[END_REF][START_REF] Sinha | Synergistic enhancement of topotecan-induced cell death by ascorbic acid in human breast MCF-7 tumour cells[END_REF][START_REF] Panis | stress and hematological profiles of advanced breast cancer patients subjected to paclitaxel or doxorubicin chemotherapy[END_REF][START_REF] Ue Martinez-Outschoorn | BRCA1 mutations drive oxidative stress and glycolysis in the tumor microenvironment. Implications for breast cancer prevention with antioxidant therapies[END_REF][START_REF] Yi | BRCA1 and Oxidative Stress[END_REF][START_REF] Pourahmad | Role of Oxygen Free Radicals in Cancer Development and Treatment[END_REF][START_REF] Chen | Reactive Oxygen Species and Drug Resistance in Cancer Chemotherapy[END_REF][START_REF] Kovacic | Unifying mechanism for anticancer agents involving electron transfer and oxidative stress: clinical implications[END_REF][START_REF] Sainz | Radical Decisions in Cancer: Redox Control of Cell Growth and Death[END_REF][START_REF] Li | Chemopreventive Effects of ROS Targeting in a Murine Model of BRCA1-Deficient Breast Cancer[END_REF] There are also well known relationships between TNBC and BRCA mutations with patients having BRCA mutations having a higher risk of TNBC. There are also known linkages between TNBC and ovarian cancer. [START_REF] Celik | Relationship of Breast Cancer with Ovarian Cancer, A Concise Review of Molecular Pathology of Breast Cancer[END_REF][START_REF] Belwalkar | Oxidative stress and chemotherapy-induced ovarian ablation in premeno-pausal women with breast cancer[END_REF] The breast cancer susceptibility protein, BRCA1, functions as a tumor suppressor, but mutations in BRCA1 decrease in its ability to repair DNA and cells are more likely to become cancerous. In human cancer cells it is known that mutated BRCA1 proteins are destroyed under stressful, oxidative cellular conditions by ROS. [START_REF] Bl Gilmore | Molecular Analysis of BRCA1 in Human Breast Cancer Cells Under Oxidative Stress[END_REF] It has also been found that limiting the production of reactive oxygen species (ROS) reduces DNA lesions and delay tumorigenesis in a murine model of BRCA1-deficient breast cancer. [START_REF] Li | Chemopreventive Effects of ROS Targeting in a Murine Model of BRCA1-Deficient Breast Cancer[END_REF] TNBC are considered aggressive cancers and prognosis is generally poor. Treatment is hampered by the heterogeneity of TNBC and the failure to identify biomarkers which can be monitored to gauge the efficacy of chemotherapy. Some biomarkers which have been assessed include TP53, BRCA1/2, EGFR, PIK3CA and PTEN, but results have been ambiguous. Growth factor receptors such as EGFR, VEGFR and FGFR have also been targeted. [START_REF] Ha Wahba | Current approaches in treatment of triple-negative breast cancer[END_REF][START_REF] Gautam | Identification of selective cytotoxic and synthetic lethal drug responses in triple negative breast cancer cells[END_REF][START_REF] Pommier | DNA Topoisomerase I Inhibitors: Chemistry, Biology and Interfacial Inhibition[END_REF] Common chemotherapy treatments are also known to induce oxidative stress in breast tumours, including TNBC [START_REF] Ka Conklin | Chemotherapy-Associated Oxidative Stress: Impact on Chemotherapeutic Effectiveness[END_REF][START_REF] Chen | Clair, Collateral damage in cancer chemotherapy. Oxidative stress in non-targeted tissue[END_REF][START_REF] Al Gomes | Serum Oxidative Stress Markers and Genotoxic Profile Induced by Chemotherapy in Patients with Breast Cancer: A Pilot Study, Oxidative Medicine and Cellular Longevity[END_REF][START_REF] Timur | The effect of Topotecan on oxidative stress in MCF-7 human breast cancer cell line[END_REF][START_REF] Amoury | Photoimmunotheranostic agents for triple-negative breast cancer diagnosis and therapy that can be activated on demand[END_REF][START_REF] Ck Anders | Poly(ADP-Ribose) Polymerase Inhibition: "Targeted" Therapy for Triple-Negative Breast Cancer[END_REF][START_REF] Lopez | Mitochondrial Fuel Metabolic Differences in Triple Negative Breast Cancer[END_REF] The Topoisomerase I inhibitors (topotecan and camptothecin) and II inhibitors have shown promise against TNBC. [START_REF] Gautam | Identification of selective cytotoxic and synthetic lethal drug responses in triple negative breast cancer cells[END_REF][START_REF] Pommier | DNA Topoisomerase I Inhibitors: Chemistry, Biology and Interfacial Inhibition[END_REF] Oxidative stress involving reactive oxygen species (ROS) are known to be involved in anti-cancer treatment of breast cancers. [START_REF] Timur | The effect of Topotecan on oxidative stress in MCF-7 human breast cancer cell line[END_REF][START_REF] Amoury | Photoimmunotheranostic agents for triple-negative breast cancer diagnosis and therapy that can be activated on demand[END_REF][START_REF] Ck Anders | Poly(ADP-Ribose) Polymerase Inhibition: "Targeted" Therapy for Triple-Negative Breast Cancer[END_REF][START_REF] Lopez | Mitochondrial Fuel Metabolic Differences in Triple Negative Breast Cancer[END_REF] Topotecan, like camptothecin, has also been shown to cause oxidative stress in MCF-7 human breast cancer cells thus inducing cytotoxicity. [START_REF] Timur | The effect of Topotecan on oxidative stress in MCF-7 human breast cancer cell line[END_REF] A novel photoimmunotheranostic technique in which antibodies deliver nontoxic photosensitizers that emit light and produce cytotoxic ROS can induce apoptosis and/or necrosis in cancer cells. Photosensitized antibodies against epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM) have been shown to be effective against TNBC. [START_REF] Amoury | Photoimmunotheranostic agents for triple-negative breast cancer diagnosis and therapy that can be activated on demand[END_REF] Manganese superoxide dismutase (MnSOD) is a major antioxidant protein that serves to maintain the tight balance between intracellular O 2 • -and H 2 O 2 . MnSOD has been shown to regulate the switch between epithelial-like and mesenchymal-like phenotypes in breast carcinoma, by influencing the cellular redox environment via its effect on the intracellular ratio between O 2 • -and H 2 O 2 . Higher MnSOD levels were shown in the highly invasive MDA-MB-231 cell lines (TNBC) compared with the noninvasive (MCF-7 and T47D) or non-tumorigenic cell lines. Patients with TNBC also had higher MnSOD levels. [START_REF] Ck Anders | Poly(ADP-Ribose) Polymerase Inhibition: "Targeted" Therapy for Triple-Negative Breast Cancer[END_REF] TNBC is characterized by mitochondrial dysfunction, elevated glycolysis, and increased generation of reactive oxygen species (ROS) as compared with receptor positive breast cancer cells. MDA-MB-231 has a higher dependency for glycolysis and fatty acid oxidation for fueling mitochondria than non TNBC cells. Target inhibition of mitochondrial oxidative damage by conjugating antioxidants such as tempol or related nitroxides coupled with the cationic lipophilic triphenylphosphonium moiety has been shown to be an effective therapeutic process for diseases driven by ROS. These conjugates are transported across most cell membranes, including the blood-brain barrier and muscle cells, and can therefore accumulate in tissues most affected by mitochondrial oxidative damage. [START_REF] Lopez | Mitochondrial Fuel Metabolic Differences in Triple Negative Breast Cancer[END_REF][START_REF] Sanabria-Barrera | Effects Beyond Mitochondria in Triple Negative Breast Cancer of Mitochondria-targeted SG1 Nitroxide[END_REF][START_REF] Jr Jhan | Triple-negative breast cancer and the potential for targeted therapy[END_REF] The effect of the triphenylphosphonium positive charge on the membrane potential allows high accumulation of these drugs within the mitochondria due to the large negative membrane potential (150-180 mV) across the inner membrane (cancerous cells also have a higher membrane potential than normal cells). [START_REF] Cheng | Mitochondria-targeted nitroxides exacerbate fluvastatin-mediated cytostatic and cytotoxic effects in breast cancer cells[END_REF][START_REF] Cheng | Antiproliferative effects of mitochondria-targeted cationic antioxidants and analogs: Role of mitochondrial bioenergetics and energy-sensing mechanism[END_REF] Molecular targets for TNBC include BRCA1/2, PARP, cyclin-dependent kinases, CHK1 and WEE1, various growth factors (EGFR, HER2, VEGF, VEGFR2, VEGFR/FGFR) and angiogenesis, androgen receptor, PI3K/AKT/mTOR pathway, and Src and WNT signalling. Many anticancer treatments are currently undergoing clinical trials. [START_REF] Murphy | RAJ Smith Targeting Antioxidants to Mitochondria by Conjugation to Lipophilic Cations[END_REF] A systematic metaanalysis of current TNBC treatments found that bevacizumab, carboplatin, and paclitaxel treatment was the most likely to improve the overall response rate in TNBC patients and in advanced metastatic TNBC patients. Bevacizumab binds VEGF, whereas carboplatin and paclitaxel are known to include free radical and other cytotoxic mechanisms. [START_REF] Tian | Effect of antitumor treatments on triplenegative breast cancer patients A PRISMA-compliant network meta-analysis of randomized controlled trials[END_REF] Tempol is a stable free radical and a powerful biological antioxidant, with 'SOD mimetic' properties, and has strong destructive effects on oxidative stress and ROS reactions. Tempol functions as an antioxidant which can undergo facile one electron redox cycles. Nitroxides such as tempol can also have proxidant properties in cells similar to other antioxidants such as flavanoids and vitamins and are mainly reduced by ascorbic acid and thiols. Nitroxide free radical reduction is faster under anerobic conditions, which are prevalent in many tumours. [START_REF] Beigrezaei | Tempol as an antioxidant; an updated review on current knowledge[END_REF][START_REF] Lewandowski | Nitroxides as Antioxidants and Anticancer Drugs[END_REF] Tempol increases the apoptotic rates of MDA-MB-231 cells (which have a wild-type BRCA1), but only during co-culture with fibroblasts. BRCA1, the human tumour suppressor gene and its protein, normally functions as an endogenous antioxidant, so helping prevents the onset of hereditary breast cancers. The loss of BRCA1 functionality (common in sporadic breast cancers) increases hydrogen peroxide generation in both epithelial breast cancer cells and neighbouring stromal fibroblasts, and promotes the onset of a reactive glycolytic stroma, with increased monocarboxylate transporter 4 and decreased caveolin-1expression. These glycolytic cancerassociated fibroblasts in turn provide mitochondrial fuels (such as L-lactate) to epithelial cancer cells, to burn via oxidative mitochondrial metabolism. [START_REF] Ue Martinez-Outschoorn | BRCA1 mutations drive oxidative stress and glycolysis in the tumor microenvironment. Implications for breast cancer prevention with antioxidant therapies[END_REF] Cancer cells produce high levels of ROS, which promotes cancer mutagenesis. By acting on cells producing ROS in the local microenvironment, tempol antioxidant therapy appears to be synthetically lethal with a BRCA1deficiency in breast cancer cells. [7,7(a)] There is much reviewed evidence that BRCA1 regulates oxidative stress. [START_REF] Yi | BRCA1 and Oxidative Stress[END_REF] Tempol and doxorubicin have been shown to be synergistically cytotoxic to breast cancer cells (MCF-7 wildtype and their MDR variant MCF-7 Adr(R) cells) when exposure to tempol preceded or was simultaneous with doxorubicin treatment in MCF-7 Adr(R) cells. The effect of tempol was thought to be due to its ability to increase peroxide levels and to deplete cellular glutathione pools. Doxorubicin is known to involve free radicals in its cytotoxic effect. [START_REF] Lewandowski | Nitroxides as Antioxidants and Anticancer Drugs[END_REF] Mito-CP 11 a tempol analogue, has been shown to be effective in breast cancer cells MCF-7 but had no impact on normal MCF-10A epithelial cells. Similar effects were observed for MDA-MB-231 breast cancer cells. Mito-CP 11 accumulates in the mitochondria where ROS are present. When co-administered with fluvastatin, Mito-CP 11 was 80% effective against MCF-7 cells compared with only 25% with Mito-CP 11 alone. The close analogue of Mito-CP 11 , Mito-CP 11 -Ac which has a non-radical -N-C(=O)-Me group instead of a free radical -N-O• group also showed potent anti-tumour cell efficacy by also altering mitochondrial and glycolytic function, depleting intracellular ATP and causing apoptosis. [START_REF] Cheng | Mitochondria-targeted nitroxides exacerbate fluvastatin-mediated cytostatic and cytotoxic effects in breast cancer cells[END_REF][START_REF] Cheng | Antiproliferative effects of mitochondria-targeted cationic antioxidants and analogs: Role of mitochondrial bioenergetics and energy-sensing mechanism[END_REF] Objective: 1. To examine the effect of drugs containing the antioxidant moiety tempol (and closely related compounds) conjugated with other anti-cancer compounds to see if there are coupled synergistic interactions against TNBC cell lines 2. To determine whether the energy gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) can predict the TNBC cytotoxicity of conjugated drugs.

Results and Discussion

(a) {Camptothecin-Tempol} conjugates

Zhao et al [START_REF] Zhao | Design and synthesis of novel spin-labeled camptothecin derivatives as potent cytotoxic agents[END_REF] have studied the cytotoxicity of a series of cancer cell lines using a closely related series of drugs containing the anti-cancer camptothecin moiety conjugated with the R 3 substituent using the antioxidant tempol (or closely related compounds), and where R 1 , R 2 and R 3 are other substituents as given in Table 1. The cell lines studied include the human tumour cell lines MDA-MB-231 (TNBC) which is the focus of this study, plus A549 (lung carcinoma), KB (epidermoid carcinoma of the nasopharynx), and KBvin (vincristine-resistant KB subline). The IC 50 values (μM) are shown in Table 3 (with the relevant calculated molecular parameters) for the {camptothecin-tempol} analogues plus the IC 50 value for topotecan which was used as a reference anti-cancer drug. Topotecan is similar in structure and anti-cancer efficacy to camptothecin, and both are topoisomerase I inhibitors, but also cause apoptotic cytotoxicity by an oxidative stress mechanism in breast cancer cells. [START_REF] Gautam | Identification of selective cytotoxic and synthetic lethal drug responses in triple negative breast cancer cells[END_REF][START_REF] Pommier | DNA Topoisomerase I Inhibitors: Chemistry, Biology and Interfacial Inhibition[END_REF][START_REF] Timur | The effect of Topotecan on oxidative stress in MCF-7 human breast cancer cell line[END_REF] However, it is not known what is the molecular target(s) that are operating in the {Camptothecin-Tempol} TNBC cytotoxicity studies. However, there is a possibility that the camptothecin moiety (which is similar to topotecan) of these {Camptothecin-Tempol} conjugates might involve a cytotoxic oxidative stress mechanism, [START_REF] Pommier | DNA Topoisomerase I Inhibitors: Chemistry, Biology and Interfacial Inhibition[END_REF][START_REF] Timur | The effect of Topotecan on oxidative stress in MCF-7 human breast cancer cell line[END_REF] while the tempol moiety might target BRCA1, since BRCA1 is involved in increased cytotoxicity in cells with mutated BRCA1 involving increased oxidative stress and ROS production. [START_REF] S Ríos-Arrabal | Involvement of free radicals in breast cancer[END_REF][START_REF] Bl Gilmore | Molecular Analysis of BRCA1 in Human Breast Cancer Cells Under Oxidative Stress[END_REF][START_REF] Ue Martinez-Outschoorn | BRCA1 mutations drive oxidative stress and glycolysis in the tumor microenvironment. Implications for breast cancer prevention with antioxidant therapies[END_REF][START_REF] Yi | BRCA1 and Oxidative Stress[END_REF][START_REF] Li | Chemopreventive Effects of ROS Targeting in a Murine Model of BRCA1-Deficient Breast Cancer[END_REF] We have previously shown that drug transport and binding to molecular targets can be analysed by equation 1 or variations. [START_REF] Fong | Drug discovery model using molecular orbital computations: tyrosine kinase inhibitors[END_REF][START_REF] Fong | Permeability of the Blood-Brain Barrier: Molecular Mechanism of Transport of Drugs and Physiologically Important Compounds[END_REF][START_REF] Fong | The effect of desolvation on the binding of inhibitors to HIV-1 protease and cyclin-dependent kinases: Causes of resistance[END_REF][START_REF] Fong | Predicting PARP inhibitory activity -A novel quantum mechanical based model[END_REF][START_REF] Fong | A novel predictive model for the anti-bacterial, anti-malarial and hERG cardiac QT prolongation properties of fluoroquinolones[END_REF][START_REF] Fong | Statins in therapy: Cellular transport, side effects, drug-drug interactions and cytotoxicity -the unrecognized role of lactones[END_REF][START_REF] Fong | Physiology of ionophore transport of potassium and sodium ions across cell membranes: Valinomycin and 18-Crown-6 Ether[END_REF][START_REF] Fong | Statins in therapy: Understanding their hydrophilicity, lipophilicity, binding to 3-hydroxy-3-methylglutaryl-CoA reductase, ability to cross the blood brain barrier and metabolic stability based on electrostatic molecular orbital studies[END_REF] The free energy of water desolvation (ΔG desolv,CDS ) and the lipophilicity free energy (ΔG lipo,CDS ) where CDS represents the non-electrostatic first solvation shell solvent properties, may be a better approximation of the cybotactic environment around the drug approaching or within the protein receptor pocket, or the cell membrane surface or the surface of a drug transporter, than the bulk water environment outside the receptor pocket or cell membrane surface. The CDS includes dispersion, cavitation, and covalent components of hydrogen bonding, hydrophobic effects. Desolvation of water from the drug (ΔG desolv,CDS ) before binding in the receptor pocket is required, and hydrophobic interactions between the drug and protein (ΔG lipo,CDS ) is a positive contribution to binding. ΔG lipo,CDS is calculated from the solvation energy in n-octane. The molecular volumes of the various drugs is an important factor in transport and binding processes, particularly where steric factors may be involved as they are in this study of the conjugated analogues. The HOMO-LUMO gap has been chosen to focus on the relationship between the conjugated camptothecin and tempol moieties as they interact during binding with the (unknown) molecular target. The HOMO-LUMO gap has been used to predict drug-protein target interaction. [START_REF] Hn Banavath | Identification of novel tyrosine kinase inhibitors for drug resistant T315I mutant BCR-ABL: a virtual screening and molecular dynamics simulations study[END_REF][START_REF] Qian | Molecular Modeling Studies of 11_-Hydroxysteroid Dehydrogenase Type 1 Inhibitors through Receptor-Based 3D-QSAR and Molecular Dynamics Simulations[END_REF][START_REF] Balajee | In Silico Screening, Alanine Mutation, and DFT Approaches for Identification of NS2B/NS3 Protease Inhibitors[END_REF] The cytotoxic effect of drugs on cancer (and non-cancer) cells depends on two dominant factors, the transport of the drug into the cell and the interaction of the drug with its molecular target in the cellular environment. In this study, the molecular target of the {camptothecin-tempol} conjugates in the MDA-MB-231 TNBC cells is unknown, but the tempol is known to target the BRCA1 gene or protein. [START_REF] Ue Martinez-Outschoorn | BRCA1 mutations drive oxidative stress and glycolysis in the tumor microenvironment. Implications for breast cancer prevention with antioxidant therapies[END_REF] It is likely then that the tempol moiety of the {camptothecin-tempol} conjugates may be the dominant factor inducing cytotoxicity, if it can be shown that the camptothecin moiety is not necessary to induce cell death in all the studied conjugates by identifying the molecular properties of the conjugates that are effective indicators of cytotoxicity. It is clear from eqs 2 and 3 that for the TNBC cells and lung carcinoma cells that the three equally dominant factors influencing cytotoxicity are the ΔG desolv,CDS and ΔG lipo,CDS and HOMO-LUMO energy gap of the conjugates. Molecular volumes are insignificant factors. We have previously showed that drug cellular transport processes are often dominated by ΔG desolv,CDS and ΔG lipo,CDS , but drug-target cytotoxicity interactions (independent of transport processes) are often highly dependent on the hydrophobic interaction (ΔG lipo,CDS ) and polar-electronic interactions. [START_REF] Fong | Drug discovery model using molecular orbital computations: tyrosine kinase inhibitors[END_REF][START_REF] Fong | Permeability of the Blood-Brain Barrier: Molecular Mechanism of Transport of Drugs and Physiologically Important Compounds[END_REF][START_REF] Fong | The effect of desolvation on the binding of inhibitors to HIV-1 protease and cyclin-dependent kinases: Causes of resistance[END_REF][START_REF] Fong | Predicting PARP inhibitory activity -A novel quantum mechanical based model[END_REF][START_REF] Fong | A novel predictive model for the anti-bacterial, anti-malarial and hERG cardiac QT prolongation properties of fluoroquinolones[END_REF][START_REF] Fong | Statins in therapy: Cellular transport, side effects, drug-drug interactions and cytotoxicity -the unrecognized role of lactones[END_REF][START_REF] Fong | Physiology of ionophore transport of potassium and sodium ions across cell membranes: Valinomycin and 18-Crown-6 Ether[END_REF][START_REF] Fong | Statins in therapy: Understanding their hydrophilicity, lipophilicity, binding to 3-hydroxy-3-methylglutaryl-CoA reductase, ability to cross the blood brain barrier and metabolic stability based on electrostatic molecular orbital studies[END_REF] Thus eq 2 and 3 can be examined further by seeking correlations between IC 50 and the ΔG lipo,CDS and HOMO-LUMO energy gap as shown in eq 4 and 5.

Equation 4

Cytotoxicity values for {Camptothecin-Tempol} conjugates with MDA-MB-231 TNBC cells (18 conjugates, excluding 9K outlier, Table 2) Eqs 4 and 5 show strong correlations between IC50 and ΔG lipo,CDS and the HOMO-LUMO energy gap for both the MDA-MB-231 TNBC cells and the A549 lung carcinoma cells. These equations are consistent with the conjugate drugs interacting with their molecular targets via a hydrophobic electron transfer mechanism, which may occur after desolvation of the drugs occur in the binding pocket of the target.

IC
In all cases except one (9J) the HUMO is located on the tempol moiety, and the LUMO is located largely on the A,B,C and D rings of the camptothecin group of the {Camptothecin-Tempol} conjugates in water. The HOMO-LUMO gap is largely invariant to the conformations as shown in Figures 5 and6 where the rotation of the tempol and camptothecin groups away from each other has only a very small effect on the HOMO-LUMO gap: for 9A and 9F a difference of 0.002a.u. and 0.0009a.u. The large R 2 = -(CH 2 ) 3 -CH 3 group of 9F results in preferred conformations where the tempol and camptothecin groups are far apart to allow the R 2 group to pass between them. Figure 7 shows compound 9C which has R 1 = i-Pr which shows the greatest steric interaction, where the bulky i-Pr forces the tempol and camptothecin moieties apart to allow dynamic rotation of the i-Pr group between the "unhindered" and "hindered" conformations. It is noteworthy that 9C has a high IC 50 > 1, indicating no detectable interaction between the 9C conjugate and the molecular target in the TNBC cells.

The relative orientation of HOMO and LUMO orbitals to each other provides an explanation of how the IC 50 values vary for the various conjugates. An examination of the various conformations of the {Camptothecin-Tempol} conjugates shows that large steric factors influence how close the HOMO orbitals can dynamically orient to the LUMO orbitals. The molecular target in the TNBC cells can interact with the HOMO of the tempol moiety, as well as accept electrons from the target to the camptothecin moiety. Clearly, the distance between the two conjugated moieties will be crucial to drug-receptor binding via the coupled HOMO and LUMO interaction.

The exception to the binding of the {Camptothecin-Tempol} conjugates to the molecular target receptor in the TNBC cells is compound 9J which has R 2 = 4-NO 2 -C 6 H 4 -moiety, whereas the directly comparable drugs 9G, 9H, 9I have R 2 = 4-MeO-C 6 H 4 -, 4-Cl-C 6 H 4 -and 4-F-C 6 H 4 -groups respectively. However the IC 50 values are 9J = 0.1, 9G = 0.27, 9H = 0.19, 9I = 0.27, showing that the 4-NO 2 substituent is 2-3 times as cytotoxic as the other 4-substituents, which on the surface seems inexplicable. However, the explanation is clear when it is shown that the LUMO resides on the 4-NO 2 -C 6 H 4 -moiety, not the camptothecin moiety as in all the other conjugates (see Figure 8).The HOMO-LUMO gap for 9J is 0.0651a.u. compared to 0.1295a.u. for drugs 9G, 9H, 9I which is in agreement with the enhanced cytotoxicity of 9J over 9G, 9H, 9I. This exception clearly demonstrates that it is the antioxidant properties of the tempol moiety of the {Camptothecin-Tempol} conjugates that is the determining factor in the cytotoxicity against the TNBC cells.

To further explore ramifications of the LUMO residing on the 4-NO Topotecan has been used as a reference drug for the {Camptothecin-Tempol} conjugates, and has a HOMO-LUMO gap of 0.1358 a.u. which is very similar to most of the {Camptothecin-Tempol} conjugates with values about 0.13 a.u. The HOMO and LUMO of topotecan both reside on the A,B,C and D rings. This observation suggests that topotecan is effective against TNBC cells via a free radical oxidative stress mechanism, [START_REF] Timur | The effect of Topotecan on oxidative stress in MCF-7 human breast cancer cell line[END_REF] similar to tempol and the {Camptothecin-Tempol} conjugates.

Examination of the IC 50 values for the A549, KB and KBvin indicates that the steric effect exerted by the R 1 and R 2 groups is greater in the MDA-MB-231 (TNBC) cells. The molecular volumes shown in Table 2 show the relative effect of changes to R 1 and R 2 groups when solvated in water. The isotropic polarizabilities are also shown in Table 2 as they are a measure of the ability to induce a dipole in a drug-receptor binding process.

(b) Mito-CP 11 and Mito-CP 11 -Ac

The cytotoxicity of Mito-CP 11 and its non-radical analogue Mito-CP 11 -Ac (see Figure 3) have also been examined to shed light on the possible target and mechanism of the {camptothecin-tempol} series. Mito-CP 11 contains the nitroxide CP moiety conjugated to the (C 6 H 5 ) [34] It is known that it is the (C 6 H 5 ) 3 P + group that allows high accumulation of these conjugates in the mitochondria, so their anti-cancer ability are expected to differ from the {camptothecin-tempol} conjugates discussed above.

Figure 9 shows the HOMO and LUMO on Mito-CP

(c) {5-Fluorouracil-Tempol} conjugates

The {5-Fluorouracil-Tempol} conjugates have been cursorily examined to compare with the {Camptothecin-Tempol} conjugates and Mito-CP 11 conjugates as they are structurally very similar. Substitution of the R group (see Figure 4) has revealed a steric effect (larger R groups generally increase IC 50 values) which can affect the distance between the HOMO (located on the tempol moiety) and the LUMO (located on the 5FU moiety). The two exceptions are where R = benzyl and proline where the IC 50 values are ca 5 times lower than the other conjugates. [START_REF] Yang | Synthesis of novel spinlabeled derivatives of 5-FU as potential antineoplastic agents[END_REF] In Figure 10 it can be seen that the proline group constrains the dynamic flexibility of the tempol group relative to the 5FU group, which may explain the lower IC 50 value. The lower IC 50 for the {5FU-Tempol-Benzyl} conjugate appears anomalous, since its gap energy is 0.1671a.u. compared to the proline analogue value of 0.1439a.u., and rotation of the benzyl group closer to the tempol moiety does not significantly change the HOMO-LUMO gap nor location of the orbitals.

The {5-Fluorouracil-Tempol} conjugates display similar behaviour to those shown by the {Camptothecin-Tempol} and Mito-CP 11 conjugates. Though not tested against TNBC cells, the {5FU-Tempol} conjugates showed comparable activity against A-549, KB and KBvin as did the {Camptothecin-Tempol} conjugates.

[50]

Conclusions

It has been shown that the {Camptothecin-Tempol} conjugates, the Mito-CP 11 and Mito-CP 11 -Ac conjugates and the {5-Fluorouracil-Tempol} conjugates are driven by the antioxidant properties of the tempol moieties in inducing cytotoxicity in TNBC cells, and as such are consistent with oxidative stress mechanisms. Martinez-Outschoorn et al [START_REF] Ue Martinez-Outschoorn | BRCA1 mutations drive oxidative stress and glycolysis in the tumor microenvironment. Implications for breast cancer prevention with antioxidant therapies[END_REF] have demonstrated that BRCA1 mutations in breast cancer drive oxidative stress and glycolytic transformation of the tumour stroma expression. Antioxidant drugs such as Tempol can reverse stromal transformation and induce epithelial cancer cell apoptosis. Tempol treatment increases the apoptotic rates of MDA-MB-231 cells, which have wild-type BRCA1. BRCA1 is a plausible molecular target of the {Camptothecin-Tempol} conjugates since these conjugates act dominantly as tempol equivalents in TNBC cells.

The HOMO-LUMO energy gaps, water desolvation and lipophilicity are of equal importance in their correlation with IC 50 values, and can be used to predict the binding of these conjugates to the molecular target in TNBC cells. The HOMO in these conjugates always resides on the tempol moiety with the LUMO residing on other moieties (Camptothecin, Triphenylphosphonium, or 5-Fluorouracil). Steric factors influence the distance between the tempol and camptothecin moieties in these conjugates and thereby affect the cytotoxicity in TNBC cells. It has been shown that the cytotoxicity of the {Camptothecin-Tempol} conjugates can be tailored by identifying appropriate substituent groups that can accommodate the LUMO or vary the LUMO energy. It is possible to design such conjugates by use of the HOMO-LUMO gaps and their effect on cytotoxicity without explicit knowledge of the actual molecular targets.

The HOMO-LUMO energy gaps may also be a good predictor of synergistic adjunct co-therapy, as illustrated with Mito-CP 11 co-administered with fluvastatin.

Experimental

All calculations were carried out using the Gaussian 09 package. Energy optimisations were at the DFT/B3LYP/6-31G(d) (6d, 7f) level of theory for all atoms. Selected optimisations at the DFT/B3LYP/6-31 + G(d,p) (6d, 7f) level of theory gave very similar results to those at the lower level. Optimized structures were checked to ensure energy minima were located, with no negative frequencies. Energy calculations were conducted at the DFT/B3LYP/6-31G(d,p) (6d, 7f) level of theory with optimised geometries in water, using the IEFPCM/SMD solvent model. Energy calculations were also conducted at the DFT/B3LYP/6-311G(d,p) (6d, 7f) and DFT/B3LYP/6-31+G(d,p) (6d, 7f) levels of theory to verify the delocalized HOMO and LUMO locations and energy levels were not significantly different from those conducted at the DFT/B3LYP/6-31G(d,p) (6d, 7f) level. HOMO and LUMO energies are in a.u. (1 a.u. = 27.212 eV).

With the 6-31G* basis set, the SMD model achieves mean unsigned errors of 0.6 -1.0 kcal/mol in the solvation free energies of tested neutrals and mean unsigned errors of 4 kcal/mol on average for ions. [START_REF] Av Marenich | Universal Solvation Model Based on Solute Electron Density and on a Continuum Model of the Solvent Defined by the Bulk Dielectric Constant and Atomic Surface Tensions[END_REF] The 6-31G** basis set has been used to calculate absolute free energies of solvation and compare these data with experimental results for more than 500 neutral and charged compounds. The calculated values were in good agreement with experimental results across a wide range of compounds. [START_REF] Rayne | Accuracy of computational solvation free energies for neutral and ionic compounds: Dependence on level of theory and solvent model[END_REF][START_REF] Rizzo | Estimation of Absolute Free Energies of Hydration Using Continuum Methods: Accuracy of Partial Charge Models and Optimization of Nonpolar Contributions[END_REF] Adding diffuse functions to the 6-31G* basis set (ie 6-31 + G**) had no significant effect on the solvation energies with a difference of less than 1% observed in solvents, which is within the literature error range for the IEFPCM/SMD solvent model.

It is noted that high computational accuracy for each species in different environments is not the focus of this study, but comparative differences between various species is the aim of the study. The literature values for IC 50 used to develop the multiple regression LFER equations have much higher experimental uncertainties than the calculated molecular properties. The statistical analyses include the multiple correlation coefficient R (i) 9C Hind (i) = stericly hindered conformation of 9C, (ii) 9F Rotated (ii) = conformation with camptothecin moiety rotated away from the tempol moiety (a) for compound 9J the R 2 group has a 2,3,5,6-tetrafluoro-4-nitrophenyl instead of the 4-nitrophenyl group at R 2 , (b) for compound 9J the R group has a 2,3,4,5,6-pentafluorophenyl instead of the 4-nitrophenyl group at R 2 , (c) for compound 9J the R 2 group has a 2,4,6-trinitrophenyl instead of the 4-nitrophenyl group at R 2 ; the 2,4 nitro groups are tilted at 52 degrees from planar due to steric hindrance at the ortho positions, the 4-nitro group is coplanar with the R 2 phenyl ring (d) R=proline in Figure 4, (e) R=benzyl in Figure 4, (f) R=iso-propyl in Figure 4.

Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .

 23456 Figure 2. Structures of Camptothecin (left) with ring nomenclature, Topotecan (centre) and Tempol (left)

Figure 7 .

 7 Figure 7. Compound 9C (left) (HOMO-LUMO gap 0.1295a.u.) showing the "hindered" conformation and 9C "unhindered" (right) (HOMO-LUMO gap 0.1268a.u.) showing HOMO on the tempol moiety and LUMO on the camptothecin moiety with the bulky R 1 = i-Pr group.

Figure 8 .

 8 Figure 8. Compound 9J (left) (HOMO-LUMO gap 0.0651a.u.) showing the HOMO on the tempol moiety and the LUMO on the R 2 = 4-NO 2 -C 6 H 4 -moiety.

Figure 9 .

 9 Figure 9. Compound Mito-CP 11 (left) (HOMO-LUMO gap 0.148a.u.) showing the HOMO on the tempol moiety and the LUMO on the (C 6 H 5 ) 3 P + moiety and Mito-CP 11 -AC (right) (HOMO-LUMO gap 0.1543a.u.) showing the HOMO on the CP-Ac moiety and the LUMO on the (C 6 H 5 ) 3 P + moiety.

Figure 10 .

 10 Figure 10. Compound {5FU-Tempol-Benzyl} (left) (HOMO-LUMO gap 0.1671a.u.) showing the HOMO on the tempol moiety and the LUMO on the 5FU moiety and {5FU-Tempol-Proline} (right) (HOMO-LUMO gap 0.1439a.u.) showing the HOMO on the tempol moiety and the LUMO on the 5FU moiety.

Table 2

 2

shows the IC 50 values (μM) for MDA-MB-231, A549 (lung carcinoma), KB (epidermoid carcinoma of the nasopharynx), KBvin (vincristine-resistant KB subline) and the ΔG desolv,CDS , ΔG lipo,CDS, molecular volume, (HOMO-LUMO) gap, isotropic polarizability of the various {Camptothecin-Tempol} conjugates shown in Table

1

and Figures

1-4

. The A549 and KB IC 50 data are shown as comparative examples for the TNBC data since these cell lines are also known to involve oxidative stress in cytotoxicity.

[START_REF] Wang | Anticancer effect of salidroside on A549 lung cancer cells through inhibition of oxidative stress and phospho-p38 expression[END_REF][START_REF] Sn Tan | Potential role of oxidative stress-induced apoptosis in mediating chromosomal rearrangements in nasopharyngeal carcinoma[END_REF][START_REF] Lu Ling | Role of reactive oxygen species in the synergistic cytotoxicity of safingol-based combination regimens with conventional chemotherapeutics[END_REF]

Equation 1 has been used to evaluate the transport of the {Camptothecin-Tempol} conjugates into the TNBC cells using the ΔG desolv,CDS , ΔG lipo,CDS , molecular volume properties of the conjugates, coupled with the HOMO-LUMO energy gap as the molecular indicator which describes the potential binding interaction of the conjugates with the molecular target in the cell. The raw values for molecular volume (cm 3 /mol) have been normalized by dividing by 25 and the HOMO-LUMO energy gaps (a.u.) multiplied by 100 to allow direct quantitative comparison with the ΔG desolv,CDS and ΔG lipo,CDS coefficients in the equations derived from eq 1.

Equation 1 Transport or Binding IC 50 = ΔG desolv,CDS + ΔG lipo,CDS + Molecular Volume + (HOMO- LUMO) Equation 2

  Cytotoxicity values for {Camptothecin-Tempol} conjugates with MDA-MB-231 TNBC cells (18 conjugates, excluding 9K as an outlier,

Table 2 ) IC 50 = 0.066ΔG desolv,CDS -0.068ΔG lipo,CDS + 0.015Molec Vol + 0.009(HOMO-LUMO) -0.442

 2

	Where R 2 = 0.577, SEE = 0.128, SE(ΔG desolvCDS ) = 0.036, SE(ΔG lipoCDS ) = 0.033, SE(Molec Vol) = 0.001, SE(HOMO-LUMO) =
	2.218, F=4.42, Significance=0.018
	Equation 3 Cytotoxicity values for {Camptothecin-Tempol} conjugates with A549 lung
	carcinoma cells (16 conjugates, excluding 9N,9B,9K as outliers, Table 2)
	IC 50 = -0.002ΔG desolv,CDS -0.005ΔG lipo,CDS + 0.001Molec Vol + 0.004(HOMO-LUMO) -0.109
	Where R 2 = 0.489, SEE = 0.023, SE(ΔG desolvCDS ) = 0.007, SE(ΔG lipoCDS ) = 0.006, SE(Molec Vol) = 0.000, SE(HOMO-LUMO) =
	0.410, F=2.63, Significance=0.092

  2 -C 6 H 4 -moiety in 9J, the following drugs were examined where R 2 = 4-NO 2 -C 6 F 4 , 2,4,6-(NO 2 ) 3 -C 6 H 2 and -C 6 F 5. It was found that the 2,4,6-(NO 2 ) 3 -C 6 H 2 gave the best result, with a HOMO-LUMO gap of 0.0449 a.u. compared with 0.0651 a.u. for 4-NO 2 -C 6 F 4 and is thus predicted to have a lower IC 50 value against the MDA-MB-231 cells.

  3 P + group separated by 11 methylene groups: neither the CP or (C 6 H 5 ) 3 P + groups separately show any activity, and the Mito-CP 4 analogue (where the CP and (C 6 H 5 ) 3 P + groups are separated by 4 methylene groups) is also inactive against TNBC cells. In addition, Mito-CP 11 -Ac is similar in activity against TNBC cells as Mito-CP 11 but Mito-CP 11 -Ac lacks the ability to dismutate mitochondrial superoxide O 2 • -, suggesting that Mito-CP 11 and Mito-CP 11 -Ac induce apoptosis by disrupting mitochondrial energetic pathways, not by ROS scavenging or superoxide dismutation.

  [START_REF] Kovacic | Unifying mechanism for anticancer agents involving electron transfer and oxidative stress: clinical implications[END_REF] and Mito-CP 11 -Ac. The HOMO is located on the tempol or CP moiety, with the LUMO on the (C 6 H 5 ) 3 P + group, with HOMO-LUMO gaps of Mito-CP 11 and Mito-CP 11 -Ac are 0.1480 and 0.1543a.u., indicating similar binding capacities, despite Mito-CP 11 being a stable free radical and Mito-CP 11 -Ac being a neutral species. It is noted that HOMO (and LUMO) are strongly delocalized over much of the tempol (and CP) moieties, indicating that Mito-CP 11 is not a traditional strongly localized free radical. It is also noteworthy that Mito-CP 4 which has only 4 methylene spacer groups has a gap of 0.1447a.u. and has the same locations for the HOMO and LUMO is inactive against TNBC cells whereas the Mito-CP 11 is active. This observation supports the conclusion reached above for the {Camptothecin-Tempol} conjugates where the distance between the HOMO and LUMO determines the binding of the drugs to the molecular target as well as the HOMO-LUMO gap itself. It is also noted that Mito-CP 11 and Mito-CP 11 -Ac have very similar ΔG desolv,CDS, ΔG lipo,CDS , molecular volumes and polarizabilities indicating that their drug cellular transport properties are very similar.

When co-administered with fluvastatin, Mito-CP 11 was 80% effective against MCF-7 cells compared with only 25% with Mito-CP 11 alone. Fluvastatin (anionic at physiological pH) has a HOMO-LUMO gap of 0.1592a.u. compared to Mito-CP 11 0.1480a.u. This similarity in reactivity and the closeness of the Mito-CP 11 and fluvastatin HOMOs (-0.1971 and -0.2012a.u.) and LUMOs (-0.0491 and -0.042a.u.) can explain why a synergistic effect may occur.

Figure 1 {Camptothecin -Tempol} conjugate analogues where R 1 , R 2 and R 3 are given in Table 1

  2 , the F test of significance, standards errors for the estimates (SEE) and each of the variables SE(ΔG desolCDS ), SE(ΔG lipoCDS ), SE(Dipole Moment), SE (Molecular Volume), SE (AEA) as calculated from "t" distribution statistics. Residual analysis was used to identify outliers.The literature values for IC 50 (μM) for the {Camptothecin-Tempol} conjugates, Mito-CP 11 and Mito-CP 11 -Ac and 5-Fluorouracil-Tempol} conjugates were taken from references35,33,34, and 50.

(taken from ref 35).

Table 1 .

 1 Substituents for Figure1(taken from ref[START_REF] Zhao | Design and synthesis of novel spin-labeled camptothecin derivatives as potent cytotoxic agents[END_REF]

Table 2 .

 2 Drug structures are shown inTable1, Figures 1-4 ΔG W = ΔG desolv,CDS , ΔG O = ΔG lipo,CDS kcal/mol, Molecular Volumes water cm 3 /mol, Isotropic Polarizabilities for W= 0.000000 Bohr 3 HOMO= Highest occupied molecular orbital, LUMO = Lowest unoccupied molecular orbital, a.u. (1 a.u. = 27.212 eV)

			MDA-
			MB-					Isotropic
		A549	231	KB	KBvin		Molec	Polar-	HOMO-
	Drug	IC 50	IC 50	IC 50	IC 50	ΔG W ΔG O	Volume	izability HOMO LUMO	LUMO
	9A Alt	0.076	0.45 0.21	0.11 13.69 -17.65	554	773 -0.2041 -0.0766 0.1275
	9A Alt1	0.076	0.45 0.21	0.11 13.5 -17.91	529	770 -0.2065 -0.0773 0.1293
	9A	0.076	0.45 0.21	0.11 13.6 -18.05	592	770 -0.2066 -0.0771 0.1295
	9B	0.45	0.53 0.56	0.25 13.96 -18.18	581	786 -0.2066 0.0771 0.1295
	9C	>1	>1	>1	>1	14.66 -18.59	556	816 -0.2066 -0.0771 0.1295
	9C Hind (i) >1	>1	>1	>1	14.04 -17.44	612	823 -0.2045 -0.0778 0.1268
	9D	0.059	0.11 0.071	0.09 11.73 -13.24	424	681 -0.2004 -0.0787 0.1216
	9E	0.055	0.08 0.059 0.057 11.75 -14.07	490	697 -0.2003 -0.0787 0.1216
	9F	0.062	0.23 0.075 0.095 12.22 -15.25	534	727 -0.2003 -0.0787 0.1216

Footnotes:


Clifford W Fong
email: cwfong@internode.on.net

Free radical anticancer drugs and oxidative stress: ORAC and CellROX-colorectal cancer cells by quantum chemical determinations

Keywords: colorectal cancer, oxidative stress, free radicals, CellROX, ORAC, antineoplastic drugs, quantum mechanics

Free radical anticancer drugs and oxidative stress: ORAC and CellROX-colorectal cancer cells by quantum chemical determinations

Introduction

There is extensive evidence supporting involvement of electron transfer (ET), reactive oxygen species (ROS) and oxidative stress (OS) in the mechanism of many anticancer drugs. These free radical ET agents function catalytically in redox cycling with formation of ROS from oxygen. [START_REF] Kovacic | Unifying mechanism for anticancer agents involving electron transfer and oxidative stress: clinical implications[END_REF][START_REF] Sainz | Radical Decisions in Cancer: Redox Control of Cell Growth and Death[END_REF][START_REF] Wardman | Electron transfer and oxidative stress as key factors in the design of drugs selectively active in hypoxia[END_REF] The metabolism of a drug may generate a reactive intermediate that can reduce molecular oxygen directly to generate ROS. [START_REF] Dg Deavall | Drug-Induced Oxidative Stress and Toxicity[END_REF] OS is known to be involved both tumour development and responses to anticancer therapies. The metabolism of reactive oxygen species is linked to tumorigenesis through signalling, and it is known that high ROS levels are generally detrimental to cells, and the redox properties of cancer cells differs from that of normal cells. The cellular mitochondrial electron transport system is a critical source of ROS and OS. Cancer cells show elevated ROS levels which are counter balanced by an increased antioxidant capacity. While high ROS levels may constitute a barrier to tumorigenesis, conversely ROS can also promote tumour formation by inducing DNA mutations and pro-oncogenic signalling pathways. At low to moderate levels, ROS may contribute to tumour formation either by acting as signalling molecules or by promoting the mutation of genomic DNA. Cancer cells characteristically have a high antioxidant capacity that regulates ROS to levels that are compatible with cellular biological functions but still higher than in normal cells. Targeting these high antioxidant defence mechanisms is a strategy to kill cancer cells but not normal cells. [START_REF] Gorrini | Modulation of oxidative stress as an anticancer strategy[END_REF][START_REF] Liu | Increased Oxidative Stress as a Selective Anticancer Therapy, Oxidative Medicine and Cellular Longevity[END_REF][START_REF] Sitthimonchai | Mechanism-based in vitro screening of potential cancer chemopreventive agents[END_REF][START_REF] De | Oxidative Therapy Against Cancer, Oxidative Stress and Diseases[END_REF] Many anticancer drugs are known to produce unwanted OS in patients during chemotherapy. However, all antineoplastic agents generate some ROS as they induce apoptosis in cancer cells, because one of the pathways of drug-induced apoptosis involves the release of cytochrome C from mitochondria. When this occurs, electrons are diverted from the electron transfer system to oxygen by NADH dehydrogenase and reduced coenzyme Q10, resulting in the formation of superoxide radicals. [START_REF] Gorrini | Modulation of oxidative stress as an anticancer strategy[END_REF] Clearly the chemotherapeutic efficacy of antineoplastic drugs is a tradeoff of tumour cytotoxicity and unwanted side effects elsewhere in the body. High levels of ROS can inhibit apoptosis at a caspase level and change the antineoplastic therapy from apoptosis to necrosis. The rupture of necrotic cells in solid tumours release enzymes that degrade nearby tissues, whereas apoptotic cells do not release their contents and are phagocytosed by macrophages. [START_REF] De | Oxidative Therapy Against Cancer, Oxidative Stress and Diseases[END_REF] Many currently prescribed antineoplastic drugs induce high levels of free radicals and OS, with patients showing ROS-induced lipid peroxidation in their plasma, have reduced blood levels of vitamin E, vitamin C and β-carotene, and decreased tissue glutathione levels. Amongst the common antineoplastic drugs, the severity of OS varies from: (a) very high, anthracyclines: such as doxorubicin, epirubicin [START_REF] Davies | Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracycline radical formation by NADH dehydrogenase[END_REF][START_REF] Jh Doroshow | Redox cycling of anthracyclines by cardiac mitochondria. II. Formation of superoxide anion, hydrogen peroxide, and hydroxyl radical[END_REF] (b) moderately high, Pt-complexes: such as cisplatin, carboplatin, oxaliplatin, alkylating agents: such as cyclophosphamide, dicarbazine, temozolomide, cisplatins, nimustine, epipodophyllotoxins: such as etoposide and teniposide, camptothecins: such as irinotecan, topotecan, and (c) low, purines/pyrimidines: such as 5fluorouracil, gemcitabine, carmofur, antimetabolites: such as 2-methoxyestradiol, cytarabine, mercaptopurine, taxanes: such as paclitaxel, vinca alkaloids: such as vinblastin, vincristine, vinorelbine. [START_REF] Ka Conklin | Chemotherapy-Associated Oxidative Stress: Impact on Chemotherapeutic Effectiveness[END_REF][START_REF] Gerhäuser | Mechanism-based in vitro screening of potential cancer chemopreventive agents[END_REF][START_REF] Jomova | Advances in metal-induced oxidative stress and human disease[END_REF][14][15][START_REF] Sinha | Free radicals in anticancer drug pharmacology[END_REF][START_REF] Fong | Platinum based radiochemotherapies: Free radical mechanisms and radiotherapy sensitizers[END_REF][START_REF] Fong | Platinum anti-cancer drugs: Free radical mechanism of Pt-DNA adduct formation and anti-neoplastic effect[END_REF] Other antineoplastic drugs or therapies [START_REF] Gorrini | Modulation of oxidative stress as an anticancer strategy[END_REF][START_REF] Liu | Increased Oxidative Stress as a Selective Anticancer Therapy, Oxidative Medicine and Cellular Longevity[END_REF][START_REF] Sitthimonchai | Mechanism-based in vitro screening of potential cancer chemopreventive agents[END_REF][START_REF] De | Oxidative Therapy Against Cancer, Oxidative Stress and Diseases[END_REF] which involve free radicals and OS in their therapies include: (i) Cytotoxic antibiotics: such as mitomycin C, mitoxantrone, and actinomycin. Mitomycin C has been shown to induce apoptosis through DNA strand breakage and ROS induction and has been used in treating a wide variety of cancers including ovarian cancer. [START_REF] Fang | Tumor-targeted induction of oxystress for cancer therapy[END_REF][START_REF] Sinha | Free radicals and anticancer drug resistance: oxygen free radicals in the mechanisms of drug cytotoxicity and resistance by certain tumors[END_REF] Actinomycin D causes breaks in DNA, and actinomycin treatment sensitizes subsequent treatment (eg TRAIL,TNF-alpha) to apoptosis by elevating reactive oxygen species concentration. [START_REF] Brodská | Generation of Reactive Oxygen Species during Apoptosis Induced by DNA-Damaging Agents and/or Histone Deacetylase Inhibitors[END_REF] (ii) PARP inhibitors: such as olaparib, rucaparib, veliparib, niraparib inhibit the action of the enzyme PARP, and reduce the capacity to repair ROS-induced DNA damage. [START_REF] Hou | Increased oxidative stress mediates the antitumor effect of PARP inhibition in ovarian cancer[END_REF] (iii) Nitroxides: such as Tempol and its analogues, camptothecins, which are stable free radicals, can act as antioxidants and anticancer drugs. The antioxidant nature is associated with their redox cycles, and the ability to engage in single electron oxidation reduction reactions. As anticancer drugs they are only toxic to tumour cells, not normal cells. Studies have confirmed their efficacy against breast, liver, lung, thyroid, ovarian and lymphatic cancers. [START_REF] Lewandowski | Nitroxides as Antioxidants and Anticancer Drugs[END_REF] (iv) Immunotherapy: Adoptive T cell immunotherapy can change the metabolism of tumour cells, and tumour necrosis factor alpha can act directly on tumour cells and induce ROS inside them. Higher ROS levels correlated with high tumour cell death. It was found that tumor necrosis factor alpha synergizes with chemotherapy to increase OS and cancer cell death. Pro-oxidants drugs known to raise ROS levels can replicate the tumour-killing benefit of adoptive T cell therapy. [START_REF] Habtetsion | Alteration of Tumor Metabolism by CD4 T Cells Leads to TNF-α-Dependent Intensification of Oxidative Stress and Tumor Cell Death[END_REF] (v) Radiotherapy also is another exogenous source of free radicals and OS.

There are few systematic studies of known antineoplastic drugs and their ability to induce OS in cells. Yokoyama [START_REF] Yokoyama | Induction of oxidative stress by anticancer drugs in the presence and absence of cells[END_REF] has examined a wide range of currently prescribed anti-cancer drugs for induced OS in DLD-1 human colorectal cancer cells using CellROX, as well as using oxygen radical absorbance capacity assays (ORAC and ORAC/Cu) to estimate the oxidative activities of the anticancer drugs in the absence of cells. It was found that vinorelbine, vinblastin, camptothecin, paclitaxel, doxorubicin, actinomycin D, mitomycin C, mercaptopruine and carmofur induced OS in the colorectal cancer cells as judged by CellROX. However the no consistent conclusion could be reached when comparing the CellROX results with the ORAC and ORAC/Cu assays, though some drugs showed consistent oxidative properties in both the CellROX and ORAC or ORAC/Cu assays.

Results

The CellROX data for DLD-1 human colorectal cancer cells and the in vitro ORAC or ORAC/Cu assays for a series of antineoplastic drugs taken from Yokoyama [START_REF] Yokoyama | Induction of oxidative stress by anticancer drugs in the presence and absence of cells[END_REF] has been analysed using a previously described model that has been shown to apply to a wide range of drug transport, binding, metabolic and cytotoxicity properties of cells and tumours (Equation 1).

The model is based on establishing linear free energy relationships between the four drug properties and various biological processes. Equation 1 has been previously applied to passive and facilitated diffusion of a wide range of drugs crossing the blood brain barrier, the active competitive transport of tyrosine kinase inhibitors by the hOCT3, OATP1A2 and OCT1 transporters, and cyclin-dependent kinase inhibitors and HIV-1 protease inhibitors. The model also applies to PARP inhibitors, the anti-bacterial and anti-malarial properties of fluoroquinolones, and active organic anion transporter drug membrane transport, and some competitive statin-CYP enzyme binding processes. There is strong independent evidence from the literature that ΔG desolvation , ΔG lipophilicity , the dipole moment and molecular volume are good inherent indicators of the transport or binding ability of drugs. [START_REF] Fong | The extravascular penetration of tirapazamine into tumours: a predictive model of the transport and efficacy of hypoxia specific cytotoxic analogues and the potential use of cucurbiturils to facilitate delivery[END_REF][START_REF] Fong | Permeability of the Blood-Brain Barrier: Molecular Mechanism of Transport of Drugs and Physiologically Important Compounds[END_REF][START_REF] Fong | The effect of desolvation on the binding of inhibitors to HIV-1 protease and cyclin-dependent kinases: Causes of resistance[END_REF][START_REF] Fong | Physiology of ionophore transport of potassium and sodium ions across cell membranes: Valinomycin and 18-Crown-6 Ether[END_REF][START_REF] Fong | Statins in therapy: Understanding their hydrophilicity, lipophilicity, binding to 3-hydroxy-3-methylglutaryl-CoA reductase, ability to cross the blood brain barrier and metabolic stability based on electrostatic molecular orbital studies[END_REF][START_REF] Fong | Predicting PARP inhibitory activity -A novel quantum mechanical based model[END_REF][START_REF] Fong | A novel predictive model for the anti-bacterial, anti-malarial and hERG cardiac QT prolongation properties of fluoroquinolones[END_REF][START_REF] Fong | Statins in therapy: Cellular transport, side effects, drug-drug interactions and cytotoxicity -the unrecognized role of lactones[END_REF][START_REF] Fong | Drug discovery model using molecular orbital computations: tyrosine kinase inhibitors[END_REF] Equation 1:

Transport or Binding or Cytotoxicity = ΔG desolv,CDS + ΔG lipo,CDS + Dipole Moment + Molecular Volume or Electron Affinity Or

Transport or Binding or Cytotoxicity = ΔG desolv,CDS + ΔG lipo,CDS + Dipole Moment + Electron Affinity

Or Equation 2: used in this study

Oxidative Stress or Oxidative Properties = ΔG desolv,CDS + ΔG lipo,CDS + Dipole Moment + Electron Affinity

Equation 1 uses the free energy of water desolvation (ΔG desolv,CDS ) and the lipophilicity free energy (ΔG lipo,CDS ) where CDS represents the non-electrostatic first solvation shell solvent properties. CDS may be a better approximation of the cybotactic environment around the drug approaching or within the protein receptor pocket, or the cell membrane surface or the surface of a drug transporter, than the bulk water environment outside the receptor pocket or cell membrane surface. The CDS includes dispersion, cavitation, and covalent components of hydrogen bonding, hydrophobic effects. Desolvation of water from the drug (ΔG desolv,CDS ) before binding in the receptor pocket is required, and hydrophobic interactions between the drug and protein (ΔG lipo,CDS ) is a positive contribution to binding. ΔG lipo,CDS is calculated from the solvation energy in n-octane. In some biological processes, where biological reduction may be occurring, and the influence of molecular volume is small, the reduction potential (electron affinity) has been included in place of the molecular volume. In other processes, the influence of some of the independent variables is small and can be eliminated to focus on the major determinants of biological activity.

We have recently used this model to develop a predictive model of the transport and efficacy of hypoxia specific cytotoxic analogues of tirapazmine and the effect on the extravascular penetration of tirapazamine into tumours. [START_REF] Fong | The extravascular penetration of tirapazamine into tumours: a predictive model of the transport and efficacy of hypoxia specific cytotoxic analogues and the potential use of cucurbiturils to facilitate delivery[END_REF] It was found that the multiparameter model of the diffusion, antiproliferative assays IC 50 and aerobic and hypoxic clonogenic assays for a wide range of neutral and radical anion forms of tirapazamine (TPZ) analogues showed: (a) extravascular diffusion is governed by the desolvation, lipophilicity, dipole moment and molecular volume, similar to passive and facilitated permeation through the blood brain barrier and other cellular membranes, (b) hypoxic assay properties of the TPZ analogues showed dependencies on the electron affinity, as well as lipophilicity and dipole moment and desolvation, similar to other biological processes involving permeation of cellular membranes, including nuclear membranes, (c) aerobic assay properties were dependent on the almost exclusively on the electron affinity, consistent with electron transfer involving free radicals being the dominant species.

The model has been recently applied to triple negative breast and ovarian cancers where transient and stable free radicals are involved in the cytotoxic oxidative stress processes. The electron affinity of the various drugs, along with the water desolvation, lipophilicity and dipole moment, has been shown to be an important predictor of cytotoxic efficacy. [START_REF] Fong | Free radicals in chemotherapy induced cytotoxicity and oxidative stress in triple negative breast and ovarian cancers under hypoxic and normoxic conditions[END_REF][START_REF] Fong | The role of free radicals in the effectiveness of anti-cancer chemotherapy in hypoxic ovarian cells and tumours[END_REF][START_REF] Fong | Role of stable free radicals in conjugated antioxidant and cytotoxicity treatment of triple negative breast cancer[END_REF] CellROX Oxidative Stress Reagents are proprietary fluorogenic probes designed to reliably measure ROS in live cells. The cell-permeable reagents are non-fluorescent or very weakly fluorescent while in a reduced state and upon oxidation exhibit strong fluorogenic signal. CellROX Green Reagent is a DNA dye, and upon oxidation, it binds to DNA; thus, its signal is localized primarily in the nucleus and mitochondria.

The ORAC assay involves free radical damage to a fluorescent probe, (usually fluorescein), caused by an oxidizing reagent, resulting in a time dependent decrease of fluorescent intensity, which can be correlated with the amount of oxidant present. Conversely, inhibition of oxidative damage to the fluorescent probe can be correlated with the antioxidant capacity of a compound acting as a free radical scavenger. Free radical sources usually involve AAPH which produce ROO• or RO• radicals.

Reactions containing antioxidants and blanks are typically run in parallel with a known equivalent of a ROS generator and fluorescent probe. Reactions are typically run to completion allowing the determination of the area under the resultant kinetic curve (AUC). Antioxidant protection can then be quantified by subtraction of AUC of the blank reaction from those reactions containing antioxidant. The resultant difference is considered to be the antioxidant protection conferred by the sample compound.

The antioxidant properties of Trolox remain a popular standard against which the antioxidant capacity of a range of substances can be related. Thus, ORAC results are commonly referred to as Trolox equivalents (TE) as calculated from comparison to a Trolox calibration curve. The AUC calculation combines both the inhibition time as well as inhibition percentage of free radical damage by the antioxidant into a single value.

The Table shows the molecular properties of 20 commonly used anticancer drugs calculated by quantum mechanical methods and their CellROX, ORAC and ORAC/Cu experimental values. The drugs are calculated as free radicals which assumes that single electron transfer has occurred under the CellROX and ORAC or ORAC/Cu experimental conditions. The pKa of some drugs indicates that some degree of protonation or diprotonation can exist at neutral pH levels for certain drugs under the experimental conditions. [START_REF] Yokoyama | Induction of oxidative stress by anticancer drugs in the presence and absence of cells[END_REF] The calculated molecular properties of Trolox are also given as a reference point. The oxidant properties of other pure anticancer drugs in the ORAC assay have been previously examined. [START_REF] Gerhäuser | Mechanism-based in vitro screening of potential cancer chemopreventive agents[END_REF] General equation 2 has been used to analyse the CellROX oxidative stress data in the Table :   Eq 3(a) Oxidative stress (OS) for 25 anion radical drugs = .

desol,CDS -. lipo,CDS + .

-. + .

Where R 2 = 0.658, SEE = 0.715, SE(ΔG desol,CDS ) = 0.05, SE(ΔG lipo,CDS ) = 0.05, SE(DM) = 0.01, SE(EA) = 0.10, F=11.12, Significance F = 0.0000 Eq 3(a) shows no significant dependence on ΔG desolv,CDS and DM as the magnitude of the coefficients are smaller than the SE. It is also noted that the robustness of eq 3(a) and 3(b) is only moderate as the OS values as measured by CellROX have only unitary precision values.

Eq 3(b) Oxidative stress (OS) for 25 anion radical drugs = -. lipo,CDS -. + .

Where R 2 = 0.651, SEE = 0.722, SE(ΔG lipo,CDS ) = 0.03, SE(EA) = 0.10, F=20.53, Significance F = 0.0000

Analyses of the ORAC data in the

Discussion

The The CellROX results must differ from the in vitro ORAC results since cellular membrane transport processes are involved in permeating the cell membrane and then the nucleus membrane before mitochondrial redox reactions can lead to formation of radical drug species.

Redox imbalance between oxidants such as ROS and antioxidants such as anticancer drugs is known to be important in many cancers, particularly colorectal cancer. The regulation of ROS levels by anticancer drugs may involve mitochondrial or endoplasmic reticulum processes. For example cisplatin is thought to induce a mitochondrial dependent ROS generation, but carboplatin is thought to induce ROS in the endoplasmic reticulum. Mitochondrial electron transfer to the drug is a complex process, and appears to involve some lipophilic drug-enzyme interaction. This may explain why the dependence of lipophilicity is about the same as the dependence on EA in eq 3(b). [START_REF] Liu | Redox Imbalance in the Development of Colorectal Cancer[END_REF][START_REF] Aa Farooqi | Anticancer drugs for the modulation of endoplasmic reticulum stress and oxidative stress[END_REF][START_REF] Mandic | Cisplatin Induces Endoplasmic Reticulum Stress and Nucleus-independent Apoptotic Signaling[END_REF] The mechanism by which some anticancer drugs (such as taxane, vinxa alkaloids or antimetabolites) can regulate oxidative stress is through the promotion of cytochrome C from the mitochondria, while other drugs such as cisplatin, carboplatin and anthracyclines (such as doxorubicin) generate very high ROS levels. Doxorubicin interferes with coenzyme Q10 in the mitochondria to induce ROS production to exert its anticancer effect. [START_REF] Gorrini | Modulation of oxidative stress as an anticancer strategy[END_REF] The bioreduction of anticancer drugs in the nucleus or endoplasmic reticulum is also known to occur via NADPH:cytochrome P450 reductase and cytochrome b 5 etc. [1-5, 20, 43] It is known that there is a strong relationship between OS and ROS and colorectal cancer (CRC). Chronic OS in patients is a known risk factor for CRC and that OS may begin in the polyp stage of CRC. Modulation of OS is an accepted anticancer strategy in CRC. 5-Fluorouracil is commonly used in the clinic for stage II and III CRC, and in addition to damaging DNA, also elevates ROS, thereby positively regulating p53 proteins and inducing cancer cell apoptosis.

There is evidence that antioxidants such as vitamin D and E, selenium and β-carotene decrease the risk and mortality of CRC. [START_REF] Liu | Redox Imbalance in the Development of Colorectal Cancer[END_REF] The much smaller dependence on drug lipophilicity compared to EA in eq 4(b) and 5(b) in the ORAC assays possibly reflects some radical-drug interaction during the dominant electron transfer reaction that results in the formation of the Drug• species.

Eqs 3(b), 4(b) and (5b) are entirely consistent with the involvement of the radical form of these antineoplastic drugs in oxidative stress and redox processes, and are consistent with the known literature on oxidative stress for these drugs.

Eqs 4(b) and 5(b) also validate that radicals are involved in the in vitro ORAC reactions. The presence of radicals in ORAC assays has been shown by the ORAC/EPR method. [START_REF] Kohri | An oxygen radical absorbance capacity-like assay that directly quantifies the antioxidant's scavenging capacity against AAPHderived free radicals[END_REF][START_REF] Kohri | Reactive oxygen radical-scavenging activity assay by photolysis of azoradical initiator without exposing samples to ultraviolet light: A preliminary feasibility study of a modified assay[END_REF]

Conclusions

It has been shown that the radical form of a wide range of current antineoplastic drugs is involved in oxidative stress processes in human DLD-1 human colorectal cancer cells as determined by the CellROX assay. The oxidative stress capability of these drugs is dependent on the lipophilicity and electron affinity of the drugs in water. All of the drugs examined are known to be involved in oxidative processes to some degree according to literature sources.

The antioxidant properties of the radical drug species in the ORAC or ORAC/Cu assays are also dependent on the lipophilicity and electron affinity properties of the drugs.

Linear equations have been derived which can predict the oxidative stress in colorectal cancer cells and the redox behavior in the ORAC assays.

Materials and Methods

All calculations were carried out using the Gaussian 09 package. Energy optimizations were at the DFT/B3LYP/6-31G(d,p) (6d, 7f) or DFT/B3LYP/3-21G (for larger molecules) level of theory for all atoms. Selected optimizations at the DFT/B3LYP/6-311 + G(d,p) (6d, 7f) level of theory gave very similar results to those at the lower level. Optimized structures were checked to ensure energy minima were located, with no negative frequencies. Energy calculations were conducted at the DFT/B3LYP/6-31+G(d,p) (6d, 7f) level of theory with optimized geometries in water, using the IEFPCM/SMD solvent model. With the 6-31G* basis set, the SMD model achieves mean unsigned errors of 0.6 -1.0 kcal/mol in the solvation free energies of tested neutrals and mean unsigned errors of 4 kcal/mol on average for ions. [START_REF] Av Marenich | Universal Solvation Model Based on Solute Electron Density and on a Continuum Model of the Solvent Defined by the Bulk Dielectric Constant and Atomic Surface Tensions[END_REF] The 6-31G** basis set has been used to calculate absolute free energies of solvation and compare these data with experimental results for more than 500 neutral and charged compounds. The calculated values were in good agreement with experimental results across a wide range of compounds. [START_REF] Rayne | Accuracy of computational solvation free energies for neutral and ionic compounds: Dependence on level of theory and solvent model[END_REF][START_REF] Rizzo | Estimation of Absolute Free Energies of Hydration Using Continuum Methods: Accuracy of Partial Charge Models and Optimization of Nonpolar Contributions[END_REF] Adding diffuse functions to the 6-31G* basis set (ie 6-31 + G**) had no significant effect on the solvation energies with a difference of less than 1% observed in solvents, which is within the literature error range for the IEFPCM/SMD solvent model. HOMO and LUMO calculations included both delocalized and localized orbitals (NBO).

Electron affinities (EA) in eV in water were calculated by the SCF difference between the optimised/relaxed neutral and optimised radical species method as previously described. [START_REF] Fong | The extravascular penetration of tirapazamine into tumours: a predictive model of the transport and efficacy of hypoxia specific cytotoxic analogues and the potential use of cucurbiturils to facilitate delivery[END_REF] It has been shown that the B3LYP functional gives accurate electron affinities when tested against a large range of molecules, atoms, ions and radicals with an absolute maximum error of 0.2 eV. [START_REF] Peverati | The quest for a universal density functional: the accuracy of density functionals across a broad spectrum of databases in chemistry and physics[END_REF][START_REF] Jc Rienstra-Kiracofe | Atomic and molecular electron affinities: Photoelectron experiments and theoretical computations[END_REF][START_REF] Ke Riley | Critical Assessment of the Performance of Density Functional Methods for Several Atomic and Molecular Properties[END_REF][START_REF] Cg Zhan | Ionization Potential, Electron Affinity, Electronegativity, Hardness, and Electron Excitation Energy: Molecular Properties from Density Functional Theory Orbital Energies[END_REF] It is noted that high computational accuracy for each species in different environments is not the focus of this study, but comparative differences between various species is the aim of the study. The literature values for CellROX and ORAC used to develop the multiple regression LFER equations have much higher experimental uncertainties than the calculated molecular properties. The statistical analyses include the multiple correlation coefficient R 2 , the F test of significance, standards errors for the estimates (SEE) and each of the variables SE(ΔG desolCDS ), SE(ΔG lipoCDS ), SE(Dipole Moment), SE (EA) as calculated from "t" distribution statistics. Residual analysis was used to identify outliers.

  results from the CellROX in vivo determinations in eq 3(b) are very similar to those shown for the in vitro ORAC in eq 4(b) and ORAC/Cu in eq 5(b). Eqs 4(b) and 5(b) are very similar, indicating that the addition of Cu 2+ to the ORAC assay has little or no effect. Both eq 4(b) and 5(b) show that the EA is the dominant dependent over the much smaller lipophilicity effect. Eq 3(b) for the colorectal cancer cells shows about an equal effect for the EA and lipophilicity, but the lipophilicity has an opposite effect compared to the in vitro ORAC assays. The reactions processes for the DLD-1 cellular CellROX processes and ORAC can be portrayed by the following: CellROX assay ORAC Assay: AAPH is the free radical (peroxy• or alkoxy•) generator (i) Drug + {AAPH: peroxy• or alkoxy• radicals}  Drug• (electron transfer) (ii) Fluoroscein + {AAPH: peroxy• or alkoxy• radicals}  Degraded Fluoroscence (iii) Calculated difference between (ii) with (i) and without (i) = measure of antioxidant capacity of Drug•

Table gives

 gives Where R 2 = 0.150, SEE = 22.63, SE(ΔG desol,CDS ) = 1.42, SE(ΔG lipo,CDS ) = 1.83, SE(DM) = 0.33, SE(EA) = 3.42, F=0.87, Significance F = 0.50 Eq 4(a) is a very poor equation, with several large outliers as shown by residual analysis, and no significant dependence on ΔG desolv,CDS and DM.

	Eq 4(b) ORAC for 22 anion radical drugs, excluding paclitaxel, vinorelbine, and vinorelbineH + H +
		!"# = .	lipo,CDS -. &	+	.
	Where R 2 = 0.346, SEE = 13.65, SE(ΔG lipo,CDS ) = 0.63, SE(EA) = 2.22, F=5.03, Significance F = 0.017
	Analyses of the ORAC/Cu data in the Table gives:
	Eq 5(a) ORAC/Cu for 25 anion radical drugs
	!"#/#( = -.	desol,CDS + . % lipo,CDS -.		-. %	+ % . &&
	Where R 2 = 0.080, SEE = 31.99, SE(ΔG desol,CDS ) = 2.00, SE(ΔG lipo,CDS ) = 2.50, SE(DM) = 0.47, SE(EA) = 4.83,
	F=0.43, Significance F = 0.78
	Eq 5(a) is a very poor equation, with several large outliers as shown by residual analysis, and no
	significant dependence on ΔG desolv,CDS and DM.
	Eq 5(b) ORAC for 19 anion radical drugs, excluding gemcitabine, gemcitabineH + , nimustine,
	nimustineH + , vinorelbine, and vinorelbineH + H +
	!"#/#( = .	lipo,CDS -&.	+	. &
			:
	Eq 4(a) ORAC for 25 anion radical drugs
	!"# = .	desol,CDS + .	lipo,CDS -.	-. $% +	. $

Where R

2

= 0.294, SEE = 19.65, SE(ΔG lipo,CDS ) = 0.88, SE(EA) = 3.26, F=3.35, Significance F = 0.061

  H + and H + H + refer the protonated and diprotonated species of the drug; MercaptopurineSH refers to the thiol tautomer of Mercaptopurine; CellROX, ORAC and ORAC/Cu data from reference 25

	Table
		CellROX ORAC	ORAC/Cu	ΔG desolv,CDS	ΔG lipo,CDS	Electron	Dipole
		%		%	kcal/mol	kcal/mol	Affinity	Moment
							eV	D
	Nimustine	1	58.2	42.7	-3.66	-5.29	3.42	3.25
	NimustineH +	1	58.2	42.7	-5.4	-5.94	2.23	17.62
	Dacarbazine	1	98.9	99.8	-0.9	-3.38	3.18	16.32
	Cyclophosphamide	1	99.5	103	-4.28	-4.92	0.48	7.45
	Temozolomide	1	102.4	106	-4.37	-5.16	2.96	5.03
	MitoxantroneH + H +	1	51.8	32.2	-12.54	-10.87	7.26	16.23
	Doxorubicin	3	83.5	89.2	-10.96	-10.05	3.46	22.8
	Mitomycin C	2	102.6	98	-4.54	-3.64	3.61	9.9
	Mercaptopurine	2	99.4	78.4	0.27	-7.14	1.89	9.69
	MercaptopurineSH	2	99.4	78.4	1.39	-6.19	1.79	4.77
	Carmofur	2	85.8	90.5	-8.49	-7.52	2.13	5.38
	Fluorouracil	1	104.3	98.9	-5.74	-2.87	1.91	6.72
	Cytarbine	1	106.1	110.4	-4.46	-5.6	1.4	5.52
	Carboplatin	1	104.2	94.2	-8.64	-1.54	1.305	18.66
	Oxaliplatin	1	103.3	101.6	-6.88	-3.2	2.72	24.67
	Vinblastin	4	79.3	49.1	-16.82	-13.68	1.43	4.35
	VinblastinH + H +	4	79.3	49.1	-17	-13.59	2.08	75.74
	Camptothecin	3	98.9	99.5	-6.24	-9.35	2.59	10.49
	Etoposide	1	96.9	109.8	-13.48	-8.48	1.25	27.2
	Actinomycin	3	88.2	92.3	-27.15	-22.5	3.38	6.66
	Paclitaxel	3	110.3	101.1	-18.43	-16.17	1.93	16.49
	Vinorelbine	4	33	19.9	-11.99	-13.09	0.92	7.31
	VinorelbineH + H +	4	33	19.9	-17.97	-13.31	0.97	19.56
	Gemcitabine	1	93.2	44.5	-5.34	-3.47	1.49	5.78
	GemcitabineH +	1	93.2	44.5	-4.46	-3.65	3.05	22.39
	Trolox				-6.56	-5.93	1.07	7.95

Footnotes: