Nanomolar-Potency Aminophenyl-1,3,5-triazine Activators of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Chloride Channel for Prosecretory Therapy of Dry Eye Diseases

Abstract

Dry eye disorders are a significant health problem for which limited therapeutic options are available. CFTR is a major prosecretory chloride channel at the ocular surface. We previously identified, by high-throughput screening, aminophenyl-1,3,5-triazine CFTR_act-K089 (1) that activated CFTR with EC₅₀ ≈ 250 nM, which when delivered topically increased tear fluid secretion in mice and showed efficacy in an experimental dry eye model. Here, functional analysis of aminophenyl-1,3,5-triazine analogs elucidated structure-activity relationships for CFTR activation and identified substantially more potent analogs than 1. The most potent compound, 12, fully activated CFTR chloride conductance with EC₅₀ ≈ 30 nM, without causing cAMP or calcium elevation. 12 was rapidly metabolized by hepatic microsomes, which supports its topical use. Single topical administration of 25 pmol of 12 increased tear volume in wild-type mice with sustained action for 8 h and was without effect in CFTR-deficient mice. Topically delivered 12 may be efficacious in human dry eye diseases.

Figure 1

Structure–activity analysis of aminophenyl-1,3,5-triazine CFTR activators. (A) Chemical structure of 1. (B) Preliminary SAR analysis based on commercial analogs (see Supporting Information Table 1 for data on all commercial analogs).

Figure 2

Short-circuit current measurement of CFTR activation by 6k and 12. (A) Measurements done in FRT cells expressing human wild-type CFTR showing responses to indicated concentrations of forskolin, 6k, or 12 and 10 μM CFTR inhibitor CFTR_inh-172. (B) Concentration-dependent activation of CFTR (mean ± SEM, n = 3).

Figure 3

Characterization of 6k and 12. (A) Cellular cAMP in FRT cells following incubation for 10 min with 10 μM 6k or 12, without or with 90 nM forskolin (fsk), as well as forskolin alone (90 nM and 20 μM) and forskolin (20 μM) + IBMX (100 μM) (mean ± SEM, n = 4). (B) Cytoplasmic calcium measured by Fluo-4 fluorescence. FRT cells were pretreated for 5 min with 10 μM 6k or 12 (or control), with 100 μM ATP added as a calcium agonist as indicated. (C) TMEM16A activity measured in FRT cells expressing YFP showing no inhibition (left, iodide + ATP addition) or activation (right, iodide addition) by 10 μM 6k or 12. (D) CaCC activity measured in HT-29 cells expressing YFP showing no activation (iodide addition) or inhibition (iodide + ATP addition) by 10 μM 6k or 12. (E) Short circuit current in primary cultures of human bronchial epithelial cells in response to agonists and inhibitors that target key ion transport processes: 20 μM amiloride (ami); 20 μM forskolin (fsk); 10 μM CFTR_inh-172; 100 μM ATP. Experiments were done following a 10 min preincubation with 10 μM 6k or 12. (F) CFTR activation by indicated concentration of 12 without forskolin (top) and after 100 nM forskolin (bottom) addition. Studies in parts B–F are representative of two to four separate sets of experiments.

Figure 4

Compound pharmacology. (A) Cytotoxicity was measured by Alamar Blue assay in FRT cells incubated for 8 h with 10 μM 6k or 12, with 33% DMSO as positive control (mean ± SEM, n = 8). (B) In vitro metabolic stability. Compounds at 5 μM were incubated for indicated times with 1 mg/mL hepatic microsomes in the presence of NADPH and parent compound assayed by LC/MS (mean ± SEM, n = 3). LC/MS profile of 12 is shown on the right with elution time on the x-axis for incubation times of 0, 15, and 60 min.

Figure 5

Tear fluid volume in mice following ocular delivery of 1 or 12. (A) Tear volume was measured just before and at the indicated times after single ocular delivery of vehicle, 1 (250 pmol), or 12 (250 pmol) in a 2.5 μL volume. (B) Study as in part A but in CF mice lacking functional CFTR. (C) Single dose study as in part A with different amounts of 12. Data are reported as the mean ± SEM, 5 mice, 10 eyes per condition, (*) p < 0.05, (**) p < 0.01 compared to vehicle control.

Chart 1

Structures of N,N-Diethyl-N′-aryl-1,3,5-triazine Diamine Analogs and Their EC₅₀ (μM)

Chart 2

Substitution with Bicyclic Anilines and Their EC₅₀ (μM)

Chart 3

N-Methyl-N′-phenyl-1,3,5-triazine Diamine Analogs and Their EC₅₀ (μM)

Scheme 1. Synthesis of N,N-Diethyl-1,3,5-triazine Amine Analogs^a

^aReagents and conditions: (a) (CF₃)₂CHOH, K₂CO₃, THF, −80 °C, 62% for 2; (b) CHF₂CF₂CH₂OH, K₂CO₃, THF, −80 °C, 68% for 3; (c) Et₂NH, DIPEA, THF, 0 °C, 55%; (d) DIPEA, THF, reflux, 25–81%.

Scheme 2. Synthesis of N-Methyl-N′-phenyl-1,3,5-triazine Diamine Analogs^a

^aReagents and conditions: (a) DIPEA, THF, 0 °C, 75%; (b) (CF₃)₂CHOH, K₂CO₃, THF, 0 °C, 43% for 9; (c) CHF₂CF₂CH₂OH, K₂CO₃, THF, 0 °C, 66% for 10; (d) DIPEA, THF, reflux, 74% for 12; 68% for 11.