Hypoxia-Activated Prodrug Derivatives of Carbonic Anhydrase Inhibitors in Benzenesulfonamide Series: Synthesis and Biological Evaluation

Abstract

Hypoxia, a common feature of solid tumours' microenvironment, is associated with an aggressive phenotype and is known to cause resistance to anticancer chemo- and radiotherapies. Tumour-associated carbonic anhydrases isoform IX (hCA IX), which is upregulated under hypoxia in many malignancies participating to the microenvironment acidosis, represents a valuable target for drug strategy against advanced solid tumours. To overcome cancer cell resistance and improve the efficacy of therapeutics, the use of bio-reducible prodrugs also known as Hypoxia-activated prodrugs (HAPs), represents an interesting strategy to be applied to target hCA IX isozyme through the design of selective carbonic anhydrase IX inhibitors (CAIs). Here, we report the design, synthesis and biological evaluations including CA inhibition assays, toxicity assays on zebrafish and viability assays on human cell lines (HT29 and HCT116) of new HAP-CAIs, harboring different bio-reducible moieties in nitroaromatic series and a benzenesulfonamide warhead to target hCA IX. The CA inhibition assays of this compound series showed a slight selectivity against hCA IX versus the cytosolic off-target hCA II and hCA I isozymes. Toxicity and viability assays have highlighted that the compound bearing the 2-nitroimidazole moiety possesses the lowest toxicity (LC₅₀ of 1400 µM) and shows interesting results on viability assays.

Keywords: carbonic anhydrase; hypoxia-activated prodrug; hypoxic tumour; inhibitors; sulfonamides.

Scheme 1

Synthesis of hypoxia-activated prodrug carbonic anhydrase IX inhibitors (HAP-CAIs) 1b–4b and 2c–3c.

Figure 1

Relative cell viability (%) in (A) HT29 and (B) HCT116 cells exposed to increasing concentrations of the derivative 1b under normoxic (green) and anoxic (red) conditions. The data represent the average ± SEM of three independent biological repeats.

Figure 2

Clonogenic cell survival of (A) HT29 and (B) HCT116 cells during normoxia (white bars) and anoxia (black bars) when exposed to compound 1b. The data represent the average ± SEM of three independent biological repeats.

Figure 3

Lethal concentration 50 (LC₅₀) values of the prodrugs. The LC₅₀ doses of the compounds were calculated based on the 50% mortality of the developing larvae at the end of five days after the exposure of embryos to different concentrations of inhibitors. (A) The ranges of LC₅₀ values of compound 4b (<500 μM concentration). (B) The LC₅₀ value for compound 5b was below 1000 μM and (C) compound 1b showed an LC₅₀ value of about 1400 µM. The LC₅₀ doses were determined after three independent experiments with similar experimental conditions (N = 72 larvae).

Figure 4

Images of zebrafish larvae in the control and prodrug-treated groups. Representative images of 2–5 dpf zebrafish larvae exposed to different concentrations (80 μM–1000 μM) of 1b, 4b and 5b. The upper panel shows images for normal development of zebrafish larvae in the control group (not treated with inhibitors) and the 1% dimethyl sulfoxide (DMSO)-treated group. The lower panel shows the larvae treated with concentrations of the compounds at which they induced minimal or no phenotypic defects. Prodrug 4b showed (arrow) an absence of a swim bladder at a 250-μM concentration at 5 days. Prodrug 5b induced edema (arrow) as early as 2 days post exposure to the compound. Compound 1b showed an absence of a swim bladder (arrow) at a 1000-μM concentration at 5 days.

Figure 5

Effect of the prodrugs 4b, 5b, and 1b on the phenotypic parameters of zebrafish larvae. The plot graphs show the phenotypic abnormalities in the zebrafish larvae after 5 days of exposure to compounds. (A) Hatching, (B) edema, (C) swim bladder development, (D) yolk sac utilization and (E) body shape for each concentration (N = 72).