Charles S. Matthews

Synthesis and biological properties of benzothiazole, benzoxazole, and chromen-4-one analogues of the potent antitumor agent 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (PMX 610, NSC 721648).

New fluorinated 2-aryl-benzothiazoles, -benzoxazoles, and -chromen-4-ones have been synthesized and their activity against MCF-7 and MDA 468 breast cancer cell lines compared with the potent antitumor benzothiazole 5. Analogues such as 9a, b and 12a, d yielded submicromolar GI50 values in both cell lines; however, none of the new compounds approached 5 in terms of antitumor potency. For 5, binding to the aryl hydrocarbon receptor appeared to be necessary but not sufficient for growth inhibition.

Elucidation of thioredoxin as a molecular target for antitumor quinols

Heteroaromatic quinols 4-(benzothiazol-2-yl)-4-hydroxycyclohexa-2,5-dienone (1) and 4-(1-benzenesulfonyl-1H-indol-2-yl)-4-hydroxycyclohexa-2,5-dienone (2) exhibit potent and selective antitumor activity against colon, renal, and breast carcinoma cell lines in vitro (GI50 < 500 nmol/L). In vivo growth inhibition of renal, colon, and breast xenografts has been observed. Profound G2-M cell cycle block accompanied down-regulation of cdk1 gene transcription was corroborated by decreased CDK1 protein expression following treatment of HCT 116 cells with growth inhibitory concentrations of 1 or 2. The chemical structure of the quinol pharmacophore 4-(hydroxycyclohexa-2,5-dienone) suggested that these novel agents would readily react with nucleophiles in a double Michael (beta-carbon) addition. Indeed, COMPARE analysis within the National Cancer Institute database revealed a number of chemically related quinone derivatives that could potentially react with sulfur nucleophiles in a similar manner and suggested that thioredoxin/thioredoxin reductase signal transduction could be a putative target. Molecular modeling predicted covalent irreversible binding between quinol analogues and cysteine residues 32 and 35 of thioredoxin, thereby inhibiting enzyme activity. Binding has been confirmed, via mass spectrometry, between reduced human thioredoxin and 1. Microarray analyses of untreated HCT 116 cells and those exposed to either 1 (1 micromol/L) or 2 (500 nmol/L and 1 micromol/L) determined that of > or =10,000 cancer-related genes, expression of thioredoxin reductase was up-regulated >3-fold. Furthermore, quinols 1 and 2 inhibited insulin reduction, catalyzed by thioredoxin/thioredoxin reductase signaling in a dose-dependent manner (IC50 < 6 micromol/L). Results are consistent with a mechanism of action of novel antitumor quinols involving inhibition of the small redox protein thioredoxin.

Mechanisms of acquired resistance to 2-(4-aminophenyl)benzothiazole (CJM 126, NSC 34445)

2-(4-aminophenyl)benzothiazole (CJM 126) elicits potent growth inhibition in human-derived breast carcinoma cell lines, including oestrogen receptor-positive (ER+) MCF-7wt cells. Analogues substituted in the 3′ position with I (DF 129), CH3 (DF 203), or CI (DF 229) possess an extended profile of antitumour activity with remarkable selective activity in cell lines derived from solid tumours associated with poor prognosis, e.g. breast, ovarian, renal and colon. Growth inhibition occurs via unknown, possibly novel mechanism(s) of action. Two cell lines have been derived from sensitive MCF-7wt breast cancer cells (IC50 value < 0.001 μM) following long-term exposure to 10 nM or 10 μM CJM 126, MCF-710 nM 126 and MCF-710 μM 126 respectively, which demonstrate acquired resistance to this agent (IC50 > 30 μM) and cross-resistance to DF 129, DF 203 and DF 229. Sensitivity to tamoxifen, benzo[a]pyrene (BP), mitomyin C, doxorubicin and actinomycin D is retained. Resistance may, in part, be conferred by the constitutively increased expression of bcl-2 and p53 proteins detected in MCF-710 nM 126 and MCF-710 μM 126 lysates. Significantly decreased depletion of CJM 126 (30 μM) from nutrient medium of MCF-710 nM 126 cells was observed with predominantly cytoplasmic drug localization and negligible DNA strand breaks. N-acetyl transferase (NAT)1 and NAT2 proteins were expressed by all three MCF-7 sub-lines, but significantly higher expression of NAT2 was accompanied by enhanced acetylation efficacy in MCF-710 nM 126 cells. In contrast, CJM 126 (30 μM) was rapidly depleted from nutrient medium of MCF-710 μM 126 culture and accessed nuclei of these cells exerting damage to DNA. The major biotransformation product of CJM 126 in MCF-710 μM 126 cells was 2-(4-aminophenyl)-6-hydroxybenzothiazole (6-OH 126). This metabolite possessed no antitumour activity. Accordingly, in this sub-line, low constitutive expression and activity of cytochrome P450 (CYP) 1A1 was detected.

Synthesis and antitumour evaluation of novel 2-phenylbenzimidazoles

A new series of fluorinated and non-fluorinated 2-phenylbenzimidazoles bearing oxygenated substituents on the phenyl ring has been synthesized. Synthesis of the new series was based on our previous discovery of 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (PMX 610) as a potent and selective antitumour agent in vitro (sub-nanomolar GI50 in sensitive human cancer cell lines), but with poor aqueous solubility and lack of a definitive cellular target limiting further development. In this study we test the hypothesis that 2-phenylbenzimidazoles with similar substitution patterns to PMX 610 would retain potent antitumour activity but with potentially superior pharmaceutical properties. In general the new compounds were less active than the former benzothiazole series in vitro when tested against the breast cancer cell lines MCF-7 and MDA 468; however the two most active compounds in the present series (3j and 3k) exhibit low micromolar GI50 values in both cell lines and provide the opportunity for further chemical derivatization with a view to target identification.

Synthesis of antitumour (1H-1,2,3-triazol-4-yl)-4-hydroxycyclohexa-2,5-dien-1-ones by copper-catalysed Huisgen cycloadditions

4-Ethynyl-4-hydroxycyclohexa-2,5-dien-1-one 5 undergoes cycloaddition reactions with a range of substituted azides in the presence of copper salts to form 1,4-disubstituted triazoles 8-11 bearing the 4-hydroxycyclohexa-2,5-dien-1-one (quinol) pharmacophore; one example of an isomeric 1,5-disubstituted triazole 12 was formed from 5 and benzyl azide in the presence of a ruthenium catalyst. Compounds were screened for growth-inhibitory activity against five cancer cell lines of colon, breast and lung origin, but were overall less potent than the benzothiazolyl- and indolyl-substituted quinols 2 and 3.

N3-Substituted Temozolomide Analogs Overcome Methylguanine-DNA Methyltransferase and Mismatch Repair Precipitating Apoptotic and Autophagic Cancer Cell Death

Glioblastoma multiforme (GBM) treatment includes temozolomide (TMZ) chemotherapy. O6-Methylguanine lesions are repaired by methylguanine-DNA methyltransferase (MGMT). Response to TMZ requires low MGMT and functional mismatch repair (MMR); resistance, conferred by MGMT or MMR deficiency, represents a barrier to successful treatment. TMZ analogs were synthesized, substituting N3-methyl with propargyl (1) or sulfoxide (2). MTT assays were conducted in SNB19 and U373 isogenic glioma cell lines (V = vector control; M = MGMT-transfected). TMZ potency was reduced >5-fold in SNB19M and U373M cells; in contrast, MGMT-expressing cells were equisensitive as vector controls to analogs 1 and 2. GI50 values <50 VM of analogs 1 or 2 were detected in V cells possessing acquired TMZ resistance: SNB19VR (hMSH6 loss) and U373VR (MGMT upregulation). Analogs 1 and 2 inhibited MMR-deficient colorectal carcinoma cell growth (irrespective of p53); G2/M cell cycle arrest preceded apoptosis. GH2AX foci inferred the generation of DNA double-strand breaks by analogs 1 and 2. Acridine orange-stained vesicles, intracellular punctate GFP-LC3 protein and double-membraned autophagosomes indicate that TMZ, 1 and 2 induce autophagy in apoptotis-resistant GBM cells. Analogs 1 and 2 elicit in vitro antitumor activity irrespective of MGMT, MMR and p53. Such imidazotetrazines may treat MGMT+ GBM and possess broader spectrum activity causing apoptosis and autophagy in malignancies which evade apoptosis. i 2014 S. Karger AG, Basel

Antitumor imidazo[5,1-d]-1,2,3,5-tetrazines: compounds modified at the 3-position overcome resistance in human glioblastoma cell lines

Synthetic routes to 3-substituted imidazo[5,1-d]-1,2,3,5-tetrazines structurally related to temozolomide were explored. Interaction of 4-diazoimidazole-5-carboxamide with an isocyanate afforded high product yields when the isocyanate was available in acceptable purity. Alternatively, alkylation of the nor-temozolomide anion afforded high yields of new imidazotetrazines. Several compounds, evaluated against a panel containing matched MGMT± glioma cell lines, showed equal inhibitory activity irrespective of MGMT status; the N3-propargyl-imidazotetrazine (10m) was prioritised as an alternative to temozolomide able to bypass drug-resistance mechanisms. In Taq polymerase assays 10m, like temozolomide and its ring-opened counterpart MTIC, alkylated DNA at clusters of three and five guanine residues; covalent modification of N-7 sites of guanine were detected in piperidine cleavage assays. Compound 10m did not cross-link DNA but induced double-strand breaks evidenced by γ-H2AX detection. Propargyl-substituted imidazotriazene (13g), showed comparable activity to 10m indicating that ring-opening of the bicyclic nucleus of novel imidazotetrazine is probably required for activity.

Abstract B164: Role of endoplasmic reticulum stress in mechanism of action of antitumor quinols

The novel quinol PMX 290 is an experimental anti‐tumor agent with potent activity against colon, renal and breast tumor cell lines in vitro (GI50 Treatment of HCT116 colon or TK‐10 renal carcinoma cells with PMX 290 leads to the induction of a distinct vacuolar phenotype 4 – 6h after addition of the compound. These perinuclear vacuoles were identified as swollen endoplasmic reticulum (ER) as their membranes stain positively with a fluorescent ER dye. Furthermore when a fluorescent analogue of PMX 290 is added to cells it co‐localized with the ER dye. Microarray data from HCT116 cells treated with PMX 290 demonstrated the induction of a panel of mRNAs for genes involved in ER stress. These included GADD34, TRIB3, DNAJB9, BiP and CHOP as well as several genes upregulated by ATF4. Uncharacteristically for a quinol there was no increase in thioredoxin reductase mRNA levels although other markers of oxidative stress were upregulated (e.g. heme oxygenase 1). The gene array results suggest that the PERK‐eIF2alpha ER stress pathway is activated by PMX 290. Upon activation by ER stress PERK phosphorylates eIF2alpha which switches off general translation and allows the expression of the transcription factor ATF4. This switch to reduced protein synthesis and expression of ATF4 transcribed chaperone proteins lowers the load on ER and increases protein folding efficiency thus alleviating ER stress. A transient increase in eIF2aplha phosphorylation and a concurrent increase in BiP levels were detected byWestern blot in response to PMX 290 in HCT116 cells. In conclusion, ER stress appears to play an important role in the anti‐tumor activity of PMX 290 and represents a novel mechanism of action for this class of compound. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B164.