Identification, Structure-Activity Relationship, and Biological Characterization of 2,3,4,5-Tetrahydro-1 H-pyrido[4,3- b]indoles as a Novel Class of CFTR Potentiators

Abstract

Cystic fibrosis (CF) is a life-threatening autosomal recessive disease, caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR modulators have been reported to address the basic defects associated with CF-causing mutations, partially restoring the CFTR function in terms of protein processing and/or channel gating. Small-molecule compounds, called potentiators, are known to ameliorate the gating defect. In this study, we describe the identification of the 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole core as a novel chemotype of potentiators. In-depth structure-activity relationship studies led to the discovery of enantiomerically pure 39 endowed with a good efficacy in rescuing the gating defect of F508del- and G551D-CFTR and a promising in vitro druglike profile. The in vivo characterization of γ-carboline 39 showed considerable exposure levels and good oral bioavailability, with detectable distribution to the lungs after oral administration to rats. Overall, these findings may represent an encouraging starting point to further expand this chemical class, adding a new chemotype to the existing classes of CFTR potentiators.

Figure 1

Chemical structure of known CFTR potentiators.

Figure 2

Discovery of mutant CFTR potentiators by HTS. (A) Summary of results obtained by screening the entire chemical library on FRT cells expressing F508del-CFTR and HS-YFP, followed by 24 h of incubation at 32 °C to rescue the trafficking defect. The graphs report the normalized HS-YFP quenching rate (QR), reflecting CFTR-dependent iodide influx, for cells treated with forskolin (10 μM) plus test compounds (5 μM; gray dots), VX-770 (1 μM; red dots), or vehicle [dimethyl sulfoxide (DMSO), black dots]. (B) Ordered distribution of the QR scores measured, for each test compound, in the screening and displayed in (A).

Figure 3

Identification of novel potentiators of mutant CFTR. Dose–response relationships for selected compounds on (A) F508del-CFTR FRT cells rescued at 32 °C for 24 h and (B) on G551D-CFTR FRT cells, compared to VX-770 (1 μM for F508del and 5 μM for G551D). (C) Structures of potentiators Hit-7 (1), Hit-8 (2), and Hit-9 (3), selected for further SAR evolution. The data are expressed as mean ± standard deviation (SD) (n = 9; from three independent experiments, each one having three biological replicates). Statistical significance was tested by parametric analysis of variance (ANOVA), followed by the Dunnett multiple comparisons test (all groups against the control group). Symbols indicate statistical significance vs control (DMSO-treated): ***p < 0.001, **p < 0.01, and *p < 0.05.

Figure 4

CFTR activation by tetrahydro-γ-carbolines on bronchial epithelial cells. (A) Dose–response relationships for selected compounds, compared to VX-770 (1 μM), on F508del-CFTR CFBE41o-cells rescued at 32 °C for 24 h. The data are expressed as mean ± SD (n = 9; from three independent experiments, each one having three biological replicates). Statistical significance was tested by parametric ANOVA, followed by the Dunnett multiple comparisons test (all groups against the control group). Symbols indicate statistical significance vs control (DMSO-treated): ***p < 0.001 and **p < 0.01. (B) Representative traces show the response of CFTR to stimulation with the indicated concentrations of CPT-cAMP and compound 3. The currents stimulated by compound 3 and CPT-cAMP were blocked by the selective CFTR inhibitor-172. Each experimental condition was tested in three independent experiments, each one performed with three biological replicates.

Figure 5

Sites of chemical modifications on the structure of 3 to explore the SAR of the tetrahydro-γ-carboline class.

Figure 6

Activity of selected tetrahydro-γ-carboline potentiators (3, 20, 25, 30, and 39) and, for comparison, VX-770 on (A) F508del-CFTR FRT and (B) on G551D-CFTR FRT cells. The data are expressed as mean ± SD (n = 9; from three independent experiments, each one having three biological replicates). Statistical significance was tested by parametric ANOVA, followed by the Dunnett multiple comparisons test (all groups against the control group). Symbols indicate statistical significance vs control (DMSO-treated): ***p < 0.001, **p < 0.01, and *p < 0.05.

Figure 7

Potentiator 39 does not influence mutant F508del rescue by correctors VX-809 and ARN23765. The graphs report the (A) values of normalized QR measured in the YFP-based functional assay on CFBE41o-expressing F508del-CFTR treated for 24 h with VX-809 (1 μM) or ARN23765 (10 nM) in the absence or presence of compound 39 (5 μM) and (B) equivalent short-circuit current (calculated from TEER/PD measurements) in F508del/F508del bronchial epithelial cells treated for 24 h with VX-809 (1 μM) or ARN23765 (10 nM) in the absence or presence of compound 39 (0.5 μM). The data are expressed as mean ± SD (n = 9; from three independent experiments, each one having three biological replicates). Statistical significance was tested by parametric ANOVA, followed by the Tukey test (for multiple comparisons). Symbols indicate statistical significance: ***p < 0.001, n.s. (not significant) indicates p > 0.05.

Figure 8

Efficacy of potentiator 39 on primary bronchial epithelial cells from non-CF individuals. (A) Representative traces of short-circuit current measurements showing the response of CFTR to stimulation with the indicated concentrations of CPT-cAMP in the absence or presence of potentiators 39 or VX-770 (1 μM for both compounds). (B) Bar graph showing the currents stimulated by submaximal concentration of CPT-cAMP measured in experiments performed as in (A). The data are expressed as mean ± SD (n = 9; from three independent experiments, each one having three biological replicates). Statistical significance was tested by parametric ANOVA, followed by the Dunnett multiple comparisons test (all groups against the control group). Symbols indicate statistical significance: **p < 0.01 and *p < 0.05.

Scheme 1. Synthesis of Tetrahydro-γ-carboline Amide Analogues 1–11 and 13–31

Reagents and reaction conditions: (i) HCl (36%), 0 °C, then NaNO₂, H₂O, SnCl₂ in HCl (6 M), 0 °C to r.t., 24 h; (ii) protocol A: tert-butyl 4-oxopiperidine-1-carboxylate, HCl (36%), EtOH, 80 °C, and 16 h; protocol B: tert-butyl 4-oxopiperidine-1-carboxylate, EtOH, r.t., then 2,4,6-trichloro-1,3,5-triazine, 90 °C, 8 h; protocol C: tert-butyl 4-oxopiperidine-1-carboxylate, EtOH, 30 min, r.t., then removal of the solvent and addition of BF₃·Et₂O, AcOH, 90 °C, 16 h; (iii) protocol A: carboxylic acid, HATU, DIPEA, DMF, 0 °C, 10–30 min, then 45a,h,j,p–r r.t., 16 h, 22–95%; protocol B: carboxylic acid, 45a–h,k-m,o–s, Et₃N, EDC·HCl, CH₂Cl₂, r.t., 16 h, 6–77%.

Scheme 2. Synthesis of N⁵-Methyl-tetrahydro-γ-carboline Analogue 12

Reagents and reaction conditions: (i) (BOC)₂O, DIPEA, CH₂Cl₂, 0 °C to r.t., 1 h; (ii) NaH (60% dispersion in mineral oil), CH₃I, DMF, 0 °C to r.t., 16 h; (iii) HCl (4.0 M) in dioxane, DCM, r.t., and 20 h; and (iv) 5-trifluoromethyl-1H-pyrazole-3-carboxylic acid, HATU, DIPEA, DMF, 10 min, then 48, r.t., 16 h, and 24%.

Scheme 3. Synthesis of 6-Fluoro-9-methyl-Substituted γ-Carbolines 32–37 and 39–42 with Modified Tetrahydro-pyridine Ring

Reagents and reaction conditions: (i) Protocol A: tert-butyl 2-methyl-4-oxo-piperidine-1-carboxylate [rac-49, (R)-50, and (S)-51] or tert-butyl 2,2-dimethyl-4-oxo-piperidine-1-carboxylate (56), toluene, 50 °C, then TsOH, 120 °C, and 24 h; protocol B: tert-butyl 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (58), HCl (36%), EtOH, 80 °C, and 16 h; (ii) 5-methyl-1H-pyrazole-3-carboxylic acid, HATU, DIPEA, DMF, 0 °C, 10–30 min, then 52–55, 57, 59, r.t., 16 h, and 5–47%; (iii) semipreparative chiral separation: ChiralPak AD, n-heptane–EtOH (75:25) [for 36, 37, 41, and 42: absolute configuration not determined and arbitrary drawn].

Scheme 4. Synthesis of 7-Fluoro-10-methyl-2,4,5,6-tetrahydro-1H-azepino[4,5-b]indol-3-yl Amide 38

Reagents and reaction conditions: (i) tert-butyl 4-oxo-azepane-1-carboxylate (60), HCl (36%), EtOH, 80 °C, and 16 h; (ii) 5-methyl-1H-pyrazole-3-carboxylic acid, HATU, DIPEA, dry DMF, 0 °C to r.t., 16 h, and 7%.