1-BENZYLSPIRO[PIPERIDINE-4,1'-PYRIDO[3,4-b]indole] 'co-potentiators' for minimal function CFTR mutants

Abstract

We previously identified a spiro [piperidine-4,1-pyrido [3,4-b]indole] class of co-potentiators that function in synergy with existing CFTR potentiators such as VX-770 or GLGP1837 to restore channel activity of a defined subset of minimal function cystic fibrosis transmembrane conductance regulator (CFTR) mutants. Here, structure-activity studies were conducted to improve their potency over the previously identified compound, 20 (originally termed CP-A01). Targeted synthesis of 37 spiro [piperidine-4,1-pyrido [3,4-b]indoles] was generally accomplished using versatile two or three step reaction protocols with each step having high efficiency. Structure-activity relationship studies established that analog 2i, with 6'-methoxyindole and 2,4,5-trifluorobenzyl substituents, had the greatest potency for activation of N1303K-CFTR, with EC₅₀ ∼600 nM representing an ∼17-fold improvement over the original compound identified in a small molecule screen.

Keywords: CFTR; Cystic fibrosis; Modulator; N1303K-CFTR; Potentiator; c.3700A>G.

Figure 1.

Structural variants 1–6 of the spiro[piperidine-4,1’-pyrido[3,4-b]indole] scaffold.

Figure 2.

Optimized geometries of the lowest energy conformers of 2a, 2c, 2e and 4a–4c.Distances (Å) are measured from the centroid of Ring i, to those of Ring ii and Ring iii. The lowest energy conformers for 4a–c preferred a close from conformation, where one face of Ring i is blocked from potential interactions with the protein interior.

Figure 3.

Superposition of optimized energetically relevant conformers for 2a, 2c, 2e, 4a-4c.

Figure 4.

Structures of 6a and 6b. Based on quantum calculations at SMD(chloroform)-B3LYP-D3(BJ)/6–31+G(d,p). 6b is 2.33 kcal/mol more thermodynamically favorable than 6a.

Figure 5.

Lowest energy conformers optimized at SMD(chloroform)6–31+G(d,p) with D3(BJ) for 6a and 6b respectively (top); the distance between centroids of Rings i and ii and Rings i and iii were measured. Superimposed images of energetically relevant conformers for 6a and 6b (bottom); 6a has more open form conformers in equilibrium.

Figure 6.

Structural determinants of spiro[piperidine-4,1’-pyrido[3,4-b]indole] scaffold. See text for explanation.

Figure 7.

Short-circuit current measurement of mutant CFTR activation by spiro[piperidine-4,1-pyrido[3,4-b]indoles]. A. (left) Short-circuit current data in FRT cells expressing N1303K-CFTR in response to 20 μM forskolin (fsk), 5 μM VX-770, indicated concentration of 2i and 2e, and 10 μM CFTR_inh-172. (right) Summary of concentration-dependence data (n=3, mean ± S.E.M.). B. Measurements done as in A, but with 20 μM GLPG1837 instead of VX-770. C. Short-circuit current in FRT cells expressing I1234del-CFTR in response to 20 μM forskolin, 5 μM VX-770, indicated concentration of 2i, and 10 μM CFTR_inh-172. D. Short-circuit current in FRT cells expressing R347P-CFTR in response to 20 μM forskolin, 5 μM VX-770, 20 μM 2i and 10 μM CFTR_inh-172. In all studies, cells were corrected with 3 μM VX-661 for 18–24 hours prior to measurement. All traces are representative of three replicates.

Figure 8.

Activity of 2i in human airway epithelial cell cultures. A. Short-circuit current in gene-edited 16HBE14o- cells expressing N1303K-CFTR. B. Short-circuit current data in gene-edited 16HBE14o- cells expressing I1234del-CFTR. C. Summary of changes in short-circuit current (ΔIsc; mean ± S.E.M., n = 3, *P < 0.05). Concentrations: 20 μM forskolin, 5 μM VX-770, 10 μM 2i, and 10 μM CFTR_inh-172. 16HBE14o- cell models expressing I1234del-CFTR were corrected with 18 μM VX-661 and 3 μM VX-445 for 18–24 hours prior to measurement.

Scheme #1.

Synthetic route to spiro[piperidine-4,1’-pyrido[3,4-b]indole] analogs 1–6.

Scheme #2.

Synthesis of constrained analog 6.