Discovery of a Slow Tight Binding LPA1 Antagonist (ONO-0300302) for the Treatment of Benign Prostatic Hyperplasia

Abstract

Scaffold hopping from the amide group of lead compound ONO-7300243 (1) to a secondary alcohol successfully gave a novel chemotype lysophosphatidic acid receptor 1 (LPA₁) antagonist 4. Wash-out experiments using rat isolated urethra showed that compound 4 possesses a tight binding feature to the LPA₁ receptor. Further modification of two phenyl groups of 1 to pyrrole and an indane moiety afforded an optimized compound ONO-0300302 (19). Despite its high i.v. clearance, 19 inhibited significantly an LPA-induced increase of intraurethral pressure (IUP) in rat (3 mg/kg, p.o.) and dog (1 mg/kg, p.o.) over 12 h. Binding experiments with [³H]-ONO-0300302 suggest that the observed long duration action is because of the slow tight binding character of 19.

Figure 1

Modification of lead compound 1. ^aIC₅₀ values were determined by nonlinear regression analysis of the dose–response curves (4 points) generated using GraphPad Prism ver. 5.04 with 95% confidence intervals in parentheses. The LPA₁ receptor stably expressed in CHO cells was used. ^brMS, rat microsomal stability; hMS, human microsomal stability; 0.5 mg/mL NADPH.

Figure 2

Wash-out experiments of compounds 1 and 4 using rat isolated urethras (y axis shows % inhibition of LPA induced rat isolated urethra contraction, means ± SD, n = 3). Ten micromolar LPA was added into a medium to measure the extent of a rat isolated urethra (100% control). After exchanging the medium, a test compound (1 μM) was added and incubated for 30 min. Ten micromolar LPA was added to record the extent of contraction (Compound). The urethra was washed out with media (no compound) three times every 10 min. Ten micromolar LPA was added to record the extent of contraction (Wash-out 1). The wash-out procedure was repeated (Wash-out 2). (A) Inhibition by compound 1 was reduced after the wash-out procedure. (B) Inhibition by compound 4 was unaffected after two wash-outs.

Figure 3

Docking results for compound 9 using the LPA₁ structure (PDB ID: 4z34). Hydrogen atoms are omitted for clarity.

Figure 4

In vivo efficacy and duration of action against LPA induced rat and dog IUP. (A) Compound or vehicle (20% STPG) was orally administered at 3 mg/kg in conscious rats. After the prescribed time (1, 3, 6, 9, 12, 18, and 24 h), LPA (300 μg/kg) was injected intravenously and the IUP measured in short-term anesthetized rats. (B) Compound was orally administered at 1 or 3 mg/kg in conscious dogs. LPA (300 μg/kg) was injected intravenously at each point (0.5, 1, 2, 4, 6, 9, 12, and 24 h), and the IUP was measured in conscious dogs. Each datum was shown as a % response, which is compared with the IUP of predosing (0 h) in each dog (%pre).

Figure 5

Binding experiments. Saturation binding for tritium labeled compounds to membranes of the LPA₁ receptor expressed in CHO cells. K_d values were determined by fitting curves with one-site binding (hyperbola) using GraphPad Prism ver. 5.04. (A) Specific binding curves for [³H]-compound 19 at different incubation times. (B) Specific binding curves for [³H]-compound 1 at different incubation times. (C) Effect of temperature. (D) Correlation between binding (K_i) and in vivo i.v. efficacy (ID₅₀). K_i values were measured at 37 °C for 2 h using 1 nM [³H]-ONO-0300302 and calculated with the Cheng–Prusoff equation (K_i = IC₅₀/(1 + [radioligand]/K_d)). ID₅₀ was estimated in the LPA induced rat IUP model via i.v. dosing (3 doses, n = 1).