Discovery of NVP-LDE225, a Potent and Selective Smoothened Antagonist

Abstract

The blockade of aberrant hedgehog (Hh) signaling has shown promise for therapeutic intervention in cancer. A cell-based phenotypic high-throughput screen was performed, and the lead structure (1) was identified as an inhibitor of the Hh pathway via antagonism of the Smoothened receptor (Smo). Structure-activity relationship studies led to the discovery of a potent and specific Smoothened antagonist N-(6-((2S,6R)-2,6-dimethylmorpholino)pyridin-3-yl)-2-methyl-4'-(trifluoromethoxy)biphenyl-3-carboxamide (5m, NVP-LDE225), which is currently in clinical development.

Keywords: Hedgehog signaling pathway; Smoothened; medulloblastoma.

Figure 1

Structures of cyclopamine, GDC-0449, and IPI-926.

Figure 2

Structure of 1, a screening hit.

Scheme 1. General Synthetic Scheme

Reagents and conditions: (a) Anilines or aminopyridines, HATU, Et₃N, DMF. (b) Boronic acids, cat. Pd(PPh₃)₄, Na₂CO₃, DME−H₂O.

Figure 3

Antitumor activity upon treatment with 5m diphosphate salt or vehicle in a Ptch^+/−p53^−/− medulloblastoma subcutaneous allograft model in nude mice. Treatment started on day 8 postimplantation (5 million cells/animal). Compound 5m was administered po at 5 (filled triangle), 10 (open triangle), and 20 (filled diamond) mg/kg/day qd for 13 days total. All doses are expressed as free base equivalents. Vehicle control (open square) of 5m: 0.5% methylcellulose and 0.5% Tween 80 in water. All treatment groups consisted of eight animals. The vehicle group was taken down 7−9 days after treatment due to excessive tumor size (greater than 10% of mouse body weight). The body weight change observed was <±5% for all treated groups.

Figure 4

Gli1 mRNA inhibition (open circle), tumor PK (filled squares), and plasma PK (filled triangles) in Ptch^+/−p53^−/− medulloblastoma model after treatment with 5m. Plasma and tumors were harvested at 4, 8, 16, and 24 h after a single oral dose of 5m at 5, 10, or 20 mg/kg. Gli1 mRNA levels were analyzed by real-time PCR and normalized to β-actin expression. Data shown are percent inhibition relative to vehicle-treated control tumors. Compound 5m concentrations were determined in plasma and tumor by LC-MS/MS. N = 3 per time point.

Figure 5

Antitumor activity in an orthotopic Ptch^+/−p53^−/− medulloblastoma allograft model in nude mice upon treatment with 5m diphosphate salt dosed at 40 mg/kg/day po bid or vehicle at equal dose volume. Athymic nude mice were implanted with 100000 tumor cells 17 days before the start of dosing. Daily treatment (vehicle or 5m) was initiated on day 0. Eight animals were enrolled into each group. MRI scans were performed at baseline and day 4 postinitiation of treatment. The bar graph represents the group mean ± SEM, with crosses indicating data from individual animals. At each time point, a representative 3D rendering and paired MRI are shown of an animal representative of the group mean. To enable longitudinal comparisons, the selected MRI is from the same animal over time point.