Anti-Cancer Activity of Phenyl and Pyrid-2-yl 1,3-Substituted Benzo[1,2,4]triazin-7-ones and Stable Free Radical Precursors

Cell viability studies for benzo[1,2,4]triazin-7-ones and 1,2,4-benzotriazinyl (Blatter-type) radical precursors are described with comparisons made with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO). All of the stable free radicals were several orders of magnitude less cytotoxic than the benzo[1,2,4]triazin-7-ones. The synthesis and evaluation of two new pyrid-2-yl benzo[1,2,4]triazin-7-ones are described, where altering the 1,3-substitution from phenyl to pyrid-2-yl increased cytotoxicity against most cancer cell lines, as indicated using National Cancer Institute (NCI) one-dose testing. COMPARE analysis of five-dose testing data from the NCI showed very strong correlations to the naturally occurring anti-cancer compound pleurotin. COMPARE is program, which analyzes similarities in cytotoxicity data of compounds, and enables quantitative expression as Pearson correlation coefficients. Compounds were also evaluated using the independent MTT assay, which was compared with SRB assay data generated at the NCI.

Incorporating pyridine is reported to significantly alter cytotoxicity against certain cancer cell lines, hence we made the choice of replacing phenyl in the parent structure 1 with the pyrid-2-yl substituent in 4a and 4b [6][7][8].

Development Therapeutic Program (DTP) National Cancer Institute (NCI) 60 Human Tumor Cell Line Screen and COMPARE Analysis
Radicals 3a and 3b were not selected by the NCI for cytotoxicity evaluation, although benzotriazinones 4a and 4b were used for one-dose (10 µM) screening against the DTP 60 cell line panel. Pyrid-2-yl-substituted compounds 4a and 4b exhibited similar variable cytotoxicity profiles, with overall cytotoxicity greater than the 1,3-diphenyl-substituted compound 1 [1] against most of the nine major histological tissue types at the NCI DTP ( Figure 2). There was particularly strong growth inhibition exhibited by 4a and 4b in certain cancer cell lines, NCI-H522 (non-small cell lung cancer), COLO 205 (colon cancer), HT29 (colon cancer), SW-620 (colon cancer), ACHN (renal cancer), TK-10 (renal cancer), T-47D (breast cancer), MDA-MB-468 (breast cancer), and MCF-7 (breast cancer). Pyridyl-substituted compounds 4a and 4b, however, showed little growth inhibition of the DU-145 (prostate cancer) cell line after one-dose testing in comparison to 1. However, IC50 values after NCI five-dose testing (see below) showed that 4b had comparable cytotoxicity to 1, with 4a being over Incorporating pyridine is reported to significantly alter cytotoxicity against certain cancer cell lines, hence we made the choice of replacing phenyl in the parent structure 1 with the pyrid-2-yl substituent in 4a and 4b [6][7][8].

Development Therapeutic Program (DTP) National Cancer Institute (NCI) 60 Human Tumor Cell Line Screen and COMPARE Analysis
Radicals 3a and 3b were not selected by the NCI for cytotoxicity evaluation, although benzotriazinones 4a and 4b were used for one-dose (10 µM) screening against the DTP 60 cell line panel. Pyrid-2-ylsubstituted compounds 4a and 4b exhibited similar variable cytotoxicity profiles, with overall cytotoxicity greater than the 1,3-diphenyl-substituted compound 1 [1] against most of the nine major histological tissue types at the NCI DTP ( Figure 2). There was particularly strong growth inhibition exhibited by 4a and 4b in certain cancer cell lines, NCI-H522 (non-small cell lung cancer), COLO 205 (colon cancer), HT29 (colon cancer), SW-620 (colon cancer), ACHN (renal cancer), TK-10 (renal cancer), T-47D (breast cancer), MDA-MB-468 (breast cancer), and MCF-7 (breast cancer). Pyridyl-substituted compounds 4a and 4b, however, showed little growth inhibition of the DU-145 (prostate cancer) cell line after one-dose testing in comparison to 1. However, IC 50 values after NCI five-dose testing (see below) showed that 4b had comparable cytotoxicity to 1, with 4a being over eight times less cytotoxic than 1 against the DU-145 cell line ( Table 1). The DU-145 and MCF-7 cell lines were available to us (see below), and the MTT assay was used to obtain independent IC 50 values.
lines were available to us (see below), and the MTT assay was used to obtain independent IC50 values.
The NCI selection of 4a and 4b for subsequent five-dose testing established key parameters used in the NCI COMPARE algorithm to determine closely matching cytotoxicity profiles [1,10] (complete one-and five-dose data for 4a and 4b can be found in the Supplementary Information accompanying this article). The COMPARE analysis facilitated comparisons of cytotoxicity with the NCI's vast database of over 250,000 synthetic compounds. The degree of similarity between two cytotoxicity profiles is described by the Pearson product-moment correlation coefficient (0 to ±1), with values above ±0.5 considered to be strong. On par with the Pearson correlation coefficient of 1 [1], very strong correlations to pleurotin of 0.84 and 0.73 were obtained for 4a and 4b, respectively (Table 2). Altering the 1,3-substitution from Ph to pyrid-2-yl in the benzo[1,2,4]triazin-7-one scaffold did not appear to alter the compound's mechanism of action, with cytotoxicity profiles closely matching that of the irreversible TrxR inhibitor pleurotin.   The NCI selection of 4a and 4b for subsequent five-dose testing established key parameters used in the NCI COMPARE algorithm to determine closely matching cytotoxicity profiles [1,10] (complete one-and five-dose data for 4a and 4b can be found in the Supplementary Information accompanying this article). The COMPARE analysis facilitated comparisons of cytotoxicity with the NCI's vast database of over 250,000 synthetic compounds. The degree of similarity between two cytotoxicity profiles is described by the Pearson product-moment correlation coefficient (0 to ±1), with values above ±0.5 considered to be strong. On par with the Pearson correlation coefficient of 1 [1], very strong correlations to pleurotin of 0.84 and 0.73 were obtained for 4a and 4b, respectively (Table 2). Altering the 1,3-substitution from Ph to pyrid-2-yl in the benzo [1,2,4]triazin-7-one scaffold did not appear to alter the compound's mechanism of action, with cytotoxicity profiles closely matching that of the irreversible TrxR inhibitor pleurotin.

Cytotoxicity against DU-145 and MCF-7 Cell Lines Using the MTT Assay
The DU-145 (prostate cancer) and MCF-7 (breast cancer) cell lines, available at the National University of Ireland Galway (NUI Galway), were used to independently determine IC 50 values. The cytotoxicity of benzotriazin-4-yl radicals 3a and 3b, TEMPO, and iminoquinones 4a and 4b was determined using the MTT assay. Radicals 3a and 3b exhibited very similar cytotoxicity profiles (see Supplementary Information), and were on average 13 and 105 times less cytotoxic towards the DU-145 and MCF-7 cell lines than oxidation products 4a and 4b (Table 3). Radicals 3a and 3b exhibited an approximate 10-fold greater cytotoxicity towards the prostate cancer compared to the breast cancer cell line. Benzotriazinones 4a and 4b gave similar submicromolar IC 50 values against both cell lines, which were similar in magnitude to that of the previously evaluated compound 1 using the MTT assay [1]. In comparison, TEMPO was found to be relatively non-toxic. This is perhaps not surprising given that high concentrations of TEMPO (of 2.5 mM for 24 h) were required in order to induce a 3.4-fold increase in both early apoptotic cells and late apoptotic/necrotic cells compared with untreated DU-145 cell controls [5]. Literature reports support the requirement for high concentrations (2.5-10 mM) of nitroxide radicals (such as TEMPO) to achieve a therapeutic dose against various breast and prostate cancer cell lines [4,5]. Nevertheless, all radicals were significantly more cytotoxic towards the DU-145 cell line than the MCF-7 cell line. Using the MTT assay (Table 3), 1, 4a, and 4b exhibited greater cytotoxicity towards DU-145 and MCF-7 cell lines than that shown by the NCI five-dose data (except for 1 against MCF-7 at the NCI Table 1). The discrepancies in IC 50 values were expected given the longer exposure time of cancer cells to the cytotoxic agent in the MTT assay (72 h versus 48 h at the NCI), as well as fundamental differences in the assays [11][12][13]. The SRB assay used by the NCI measures the amount of dye (sulforhodamine B) bound onto cellular protein, and is reported to be more sensitive than MTT [12,13]. In contrast, MTT relies on the ability of viable cells to reduce the tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) [11].