6-Substituted amiloride derivatives as inhibitors of the urokinase-type plasminogen activator for use in metastatic disease

Abstract

The oral K+-sparing diuretic amiloride shows anti-cancer side-activities in multiple rodent models. These effects appear to arise, at least in part, through moderate inhibition of the urokinase-type plasminogen activator (uPA, K_i = 2.4 µM), a pro-metastatic trypsin-like serine protease that is upregulated in many aggressive solid malignancies. In applying the selective optimization of side-activity (SOSA) approach, a focused library of twenty two 6-substituted amiloride derivatives were prepared, with multiple examples displaying uPA inhibitory potencies in the nM range. X-ray co-crystal structures revealed that the potency increases relative to amiloride arise from increased occupancy of uPA's S1β subsite by the appended 6-substituents. Leading compounds were shown to have high selectivity over related trypsin-like serine proteases and no diuretic or anti-kaliuretic effects in rats. Compound 15 showed anti-metastatic effects in a xenografted mouse model of late-stage lung metastasis.

Keywords: Amiloride; Anti-metastatic; Cancer; Metastasis; Selective optimisation of side-activity; Trypsin-like serine protease; Urokinase plasminogen activator; uPA.

Fig. 1.

Chemical structures of amiloride 1 and 5-hexamethyleneamiloride (HMA).

Fig. 2.

X-ray co-crystal structures of A) 7 (PDB 6AG2; 1.8 Å), B) 8 (PDB 6AG3; 2.5 Å), C) 13 (PDB 6AG7; 1.9 Å) and D) 15 (PDB 6AG9; 1.6 Å) bound to the active site of uPA. A sulfate of crystallization is shown, where detected. X-ray statistics are provided in the Supplementary Material.

Fig. 3.

EnPlex assay showing comparative inhibition of 85 serine hydrolases by amiloride 1 and 6-substituted derivatives 7 and 15. Grey boxes indicate wells in which no fluorescence was detected. Wells where signal was detected at lower compound concentration without signal at higher concentrations are experimental artifacts. Black triangles indicate increasing compound concentrations in the range 5 nM-33 μM.

Fig. 4.

Inhibition of ENaCs by amiloride 1 and derivatives 7 and 15. All compounds were present at 10 μM. Data represent the mean ± SD (n = 4). Methods were as described in reference .

Fig. 5.

Effect of amiloride 1 and derivatives 7 and 15 on diuresis, naturesis and kaliuresis in Sprague Dawley rats. Rats were dosed with 25 mg/kg acetazolamide IV immediately prior to administration of 1.5 mg/kg of 1, 7 or 15 IP. Data represent the mean ± SEM (n = 8). **p = 0.001, ***p = 0.0005, ****p = 0.0001 relative to the acetazolamide control group (ACE). Methods were as described in reference .

Fig. 6.

Effects of 1 and 15 on experimental lung metastasis formed in mice following tail vein injection of HT-1080RedFluc cells. Compounds were administered at 7.5 mg/kg/day IP for 21 days. Lung metastases were quantified using an endpoint luciferase activity assay of whole lung homogenates. RLU = relative luminescence units. Data represent the mean ± SEM (vehicle and 1, n = 4; 15, n = 6), ns = not significant. Methods as described in reference .

Scheme 1.

Synthesis of 6-substituted amiloride derivatives. Reagents and conditions: (a) Ar-B(OH)₂, Pd(PPh₃)₄, K₂CO₃, Toluene/MeOH, reflux 0.5–48 h, (b) guanidine (2 M in MeOH), DMF, rt, 18 h or guanidine.HCl, Na_(s), ⁱPrOH, 1–4 h.