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. 2023 Nov 25;8(48):45606–45615. doi: 10.1021/acsomega.3c05839

Synthesis and Evaluation of Ivacaftor Derivatives with Reduced Lipophilicity

Melissa Iazzi †, Phillip Junor †, Jitesh Doshi ‡, Saujanya Acharya †, Roxana Sühring †, Russell D Viirre †,*, Gagan D Gupta †,*
PMCID: PMC10702196  PMID: 38075767

Abstract

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Mutations in the unique ATP-binding cassette anion channel, the cystic fibrosis conductance regulator (CFTR), lead to the inherited fatal disease known as cystic fibrosis (CF). Ivacaftor enhances channel gating of CFTR by stabilizing its open state and has been approved as monotherapy for CF patients with CFTR gating mutations (e.g., G551D) and as part of combination therapy with lumacaftor for CFTR folding mutations (e.g., ΔF508). However, in the latter context, ivacaftor may destabilize folding-rescued ΔF508-CFTR and membrane-associated proteins and attenuate lumacaftor pharmacotherapy. Here, we tested the hypothesis that the high lipophilicity of ivacaftor may contribute to this effect. We describe the synthesis of three glutamic acid ivacaftor derivatives with reduced lipophilicity that bear different charges at neutral pH (compounds 2, 3, 4). In a cellular ion flux assay, all three restored G551D-CFTR channel activity at comparable or better levels than ivacaftor. Furthermore, unlike ivacaftor, compound 3 did not attenuate levels of folding-rescued ΔF508 at the cell surface. Molecular modeling predicts that the increased polarity of compound 3 allows engagement with polar amino acids present in the binding pocket with hydrogen bonding and ionic interactions, which are collectively higher in strength as compared to hydrophobic interactions that stabilize ivacaftor. Overall, the data suggests that reduced lipophilicity may improve the efficacy of this class of CFTR potentiators when used for folding-rescued ΔF508-CFTR.

1. Introduction

Mutations in the unique ATP-binding cassette anion channel, cystic fibrosis transmembrane conductance regulator (CFTR), lead to the inherited fatal disease known as cystic fibrosis (CF).1−4 CF is commonly associated with mucus obstruction of the airways, persistent bacterial infections, and gradual loss of lung function.5,6 The major cause of morbidity and mortality in people with CF is the lack of functional CFTR at the apical membrane of epithelial cells.7 CFTR is composed of five domains: two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. The discovered small-molecule CFTR modulators include correctors (i.e., lumacaftor or VX-809; tezacaftor or VX-661; elexacaftor or VX-445) to increase cell surface abundance of CFTR, and potentiators (i.e., ivacaftor or VX-770, 1) to increase the ion flux of mutant CFTR.8−13 Ivacaftor (1) was discovered by screening compounds in cell models that increased the conductance of G551D-CFTR. 1 binds CFTR at the protein–lipid interface within TMD2, docking into a cleft formed by TM helices 4, 5, and 8.14 The binding site coincides with a hinge region in TM8, a structural feature of CFTR not found in other ABC transporters.15 The extracellular segment of TM8 rotates around this hinge upon ATP binding, and the conformational stability requirement of this event may explain the drug’s efficacy.151 enhances both ATP-dependent and ATP-independent channel gating of CFTR, possibly by stabilizing the TMDs in the pore-open configuration.15−17

Graphical Representation of Compounds 1–41.

1 was the first US Food and Drug Administration-approved medicine to treat patients with CF and is also approved for use in combination with available correctors (ORKAMBI – lumacaftor + ivacaftor; and TRIKAFTA – tezacaftor + elexacaftor + ivacaftor).9,11,18,191 is known to exhibit low solubility in water (<50 ng/mL) with a relatively high logP of 5.6, and is normally taken with a high-fat meal to improve absorption.12 A common finding for oral drugs in the early stage of development is that high lipophilicity (log P > 5) results in a rapid metabolic turnover, low solubility, and poor absorption. Also, there is an increased likelihood of in vitro receptor promiscuity and in vivo toxicity. On the contrary, if lipophilicity is too low, a drug can display poor pharmacokinetic properties.20 Lipinski’s rule of 5 states that a compound for oral administration should have a log P ≤ 5.21

The introduction of 1 has undoubtedly advanced healthcare for patients with CF. However, a contributing factor for marginal rescue levels of potentiation of mutants in cell models, and the variable response patients experience in the clinical setting, could be the destabilizing effects that 1 exerts on corrected CFTR.11,22−24

Previous work studied the effect of the lipophilicity of derivatives of 1 on the membrane stability of CFTR.11 It was found that the lipophilicity decreases through the removal of one or both t-butyl groups from the phenyl ring resulting in improved protein stability, while the addition of alkyl, aryl, and other lipophilicity-increasing substituents at the quinolinone 6-position was deleterious to stability. These authors further found that these modifications did not abrogate the ability to potentiate CFTR channel activity. It was proposed that there is a correlation between the lipophilicity of derivatives of 1 and their destabilizing effect on corrected CFTR protein.11

In order to test this hypothesis, in this study, we have prepared new derivatives of ivacaftor (2–4), lacking the hydrophobic t-butyl groups on the phenyl ring but also incorporating hydrophilicity-enhancing ionic groups at the quinolinone 6-position, and tested their ability to potentiate CFTR channel function, as well as their effect on the protein’s membrane levels.

2. Results

2.1. Ivacaftor Derivative Synthesis

Given the relative ease of preparing a quinolinone derivative with an iodine substituent at the 6-position (see below), we envisioned the incorporation of hydrophilicity via Sonogashira coupling with a glutamic acid bearing a propargylamide side chain. Simple manipulations of the amino and carboxyl groups would then allow for rapid access to derivatives that would be cationic, zwitterionic, or anionic at a neutral pH.

The syntheses of the derivatives used for this study are described in Scheme 1. Commercial 4-iodoaniline was reacted with diethyl ethoxymethylenemalonate via Michael addition/elimination to give an excellent yield of the enamine product 5, which was then cyclized in a Gould–Jacobs reaction to give quinolinone ester 6. After saponification, acid 7 was coupled with tert-butyldimethylsilyl-protected 3-hydroxyaniline to afford amide 8, mediated by the peptide coupling reagent HBTU (O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate).

Scheme 1. Synthesis of Polar Ivacaftor Derivatives.

Scheme 1

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Reagents and conditions: (a) C2H5OCHC(COOC2H5)2, EtOH, reflux, 4 h, 96%; (b) diphenyl ether, reflux 45 min, 86%; (c) (i) NaOH, H2O, reflux, 4 h, then (ii) H2SO4(aq), 97%; (d) (i) 3-aminophenol, TBDMSCl, NEt3, DMAP rt, 24 h, quant, then (ii) 7, HBTU, DIPEA, DMF, rt, 16 h, 40%; (e) SOCl2, MeOH, 0 °C → rt, 40 min, quant.; (f) Boc2O, NaHCO3, dioxane/H2O (2:1), 0 °C → rt, 4 h, 78%; (g) propargylamine, HBTU, DIPEA, THF, rt, 6 h, 79%; (h) 8, Pd(PPh3)2Cl2, CuI, DIPEA, THF, 55 °C, 16 h, 66%; (i) TFA/CH2Cl2 (1:1), 40 °C, 3 h, 65%; (j) (i) 5% NaOH/H2O/THF, rt, 2 h, then (ii) TFA/CH2Cl2 rt, 1 h, 94%; (k) (i) TFA/CH2Cl2 (1:1), rt, 3 h, quant., then, (ii) Ac2O, NaHCO3(aq), rt, 1 h, 83%; (l) 8, Pd(PPh3)2Cl2, CuI, DIPEA, THF, 55 °C, 3 h, 70%; (m) 5% NaOH(aq), THF/H2O, rt, 2 h, 34%.

Using slightly modified Fischer esterification conditions, in which anhydrous HCl is generated in situ by the addition of thionyl chloride to methanol resulted in the selective monoesterification of the side chain carboxyl group of commercial l-glutamic acid.25 Under these conditions, at low temperatures, presumably the protonation of the amino group electrostatically disfavors the further protonation of the nearby α-carboxyl group needed to initiate the acid-catalyzed esterification to proceed, thereby favoring reaction at the more remote δ-carboxyl group. The ester intermediate was then N-Boc-protected, and HBTU coupled with propargylamine at the α-carboxyl group to give the fully protected glutamic acid propargylamide 9. Sonogashira coupling of 8 and 9 proceeded smoothly to afford fully protected compound 10. Treatment of 10 with trifluoroacetic acid (TFA) simultaneously removed the tert-butyldimethylsilyl (TBDMS) and Boc protecting groups to yield the target compound 2, bearing an ester and free amino group. Saponification of 10 followed by TFA treatment yielded the target free amino acid, compound 3. Attempts to acetylate either 2 or 3, toward 4, led to complex mixtures. To circumvent this, 9 was subjected to BOC deprotection, acetylation, and Sonogashira reaction with 8. Application of saponification conditions to product 11 simultaneously hydrolyzed the ester and TBDMS groups to afford target compound 4, bearing a free carboxylic acid and N-acetamide.

2.2. Measuring Lipophilicity

The differences between the new compounds 2–4 and the parent compound were intentionally chosen to decrease the lipophilicity of the compound relative to 1. Intuitively, the removal of two hydrophobic t-butyl groups and the addition of ionizable amino, carboxyl (or both) groups achieve this goal, but we sought to quantify the magnitude of the effect of the changes. The octanol–water partition coefficients of 1–4 were determined experimentally by a reversed-phase HPLC method (OECD Test Guideline No. 11726). In brief, a calibration graph was generated using the retention times of reference substances with known partition coefficients, and then, the retention times of the test compounds were interpolated onto this graph (which is approximately linear when the coefficients are expressed as a logarithm) (Figure S1). Table 1 gives the measured logP values of the compounds, as well as the computed (C log P) values generated using the consensus method in MarvinSketch software version 20.7. By either measure, all three of the new compounds were found to be considerably less lipophilic than 1, having values 3.8–4.5 logarithmic units lower.

Table 1. Measured and Computed Octanol-Water Partition Coefficients.

sample log P C log P
1 5.6 5.76
2 1.8 1.20
3 1.1 1.30
4 1.7 1.52
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2.3. Ivacaftor Derivatives Retain Potentiator Ability

Potentiator activity was measured using a fluorescent imaging plate reader (FLIPR) assay, which has been validated for measuring channel activity of CFTR upon drug exposure in vitro.27,28 In this assay, the biological response of CFTR to an agonist as reflected through membrane depolarization is measured using a fluorescent membrane potential dye. WT or G551D-CFTR expression in engineered human embryonic kidney (HEK293) cell lines29 was induced with tetracycline for 24 h at 37 °C. The choice of HEK293 cells was dictated by past studies indicating that CFTR expression, function, and localization were not compromised in these cells.30 Cells were preloaded with blue membrane potential dye and then treated with 1–4 (1 μM) and 10 μM forskolin (FSK). Dye fluorescence was monitored for several minutes, followed by the addition of a specific CFTR inhibitor, CFTRinh-172, to identify the CFTR-specific response.27,37 As demonstrated in Figure 1a, stimulation of WT-CFTR (blue trace) cells with the FSK agonist led to membrane depolarization, which resulted in increased fluorescence, and the addition of CFTRinh-172 rapidly abolished this increase. As expected, an increase in fluorescence was noted only in HEK cells expressing WT-CFTR but not in those expressing G551D-CFTR (gray trace). Mild hyperpolarization was noted here possibly due to nonspecific activation of other ion channels or receptors by the agonist. Notably, the addition of 1 (black trace) to G551D-CFTR cells resulted in an ∼51% increase in agonist-induced fluorescence above the baseline fluorescence (Figure 1b). 2 (orange trace) and 4 (red trace) achieved comparable peak rescue to 1 (1 vs 2, p = 0.88; 1 vs 4p = 0.35 by Dunnett’t test) but exhibited a shorter duration of stimulation. 3 (green trace) achieved better peak rescue than 1 (1 vs 3, p = 0.02 by Dunnett’s test) and exhibited the highest average maximum peak (167%) upon FSK stimulation relative to the other tested compounds (1 (black trace): 151%; 2 (orange trace): 155%; 4 (red trace): 144%) (Figure 1b).

Figure 1.

Figure 1

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Glutamic acid derivatives potentiation on G551D-CFTR. (a) FLIPR channel function studies of WT-CFTR (blue trace) and G551D-CFTR (gray trace) stimulated with 10 μM cAMP agonist, forskolin (FSK), and then inhibited with 10 μM CFTRinh-172. Averaged traces (n = 3) of response of G551D-CFTR to chronic treatment of 1 μM 1 (black trace), and glutamic acid derivatives of 1:2 (orange trace), 3 (green trace), and 4 (red trace). (b) Quantification of the maximum peak of FSK stimulation in the FLIPR assay relative to the baseline shows that acute treatment with 1 μM 1–4 shows significantly potentiated CFTR chloride flux compared to 10 μM FSK alone (n = 3 for all conditions). Results are presented as mean ± SD and analyzed by one-way ANOVA and Dunnett’s post hoc test (**P < 0.02; ***P < 0.0002; ****P < 0.0001 compared with 10 μM FSK).

2.4. Effect of Ivacaftor Derivatives on Surface Levels of CFTR

To test the destabilizing activity of 1–4, we examined surface levels of corrected-ΔF508-CFTR after prolonged exposure to suprapharmacological concentrations of all compounds.11 CFBE cells expressing a reporter construct (Flag-ΔF508-CFTR-mCherry31) that generates both an internal marker (mCherry) and a surface exposed epitope (FLAG) on CFTR were treated with lumacaftor (3 μM) in addition to 10 μM 1 (black) or derivatives 2 (orange), 3 (green), or 4 (red) for 24 h, at folding permissive temperature (27 °C). Cells were subsequently imaged at high resolution, and cellular surface and internal CFTR label were quantified using image analysis. In this assay, lumacaftor addition alone at the permissive temperature enhanced the steady-state levels of folding-rescued ΔF508-CFTR at the cell surface, after normalizing to expression levels of the construct31 (Figure S6). The resultant CFTR surface:total ratio was denoted as the surface rescue efficiency of the drug treatment. We observed a significant decrease in the rescue efficiency of Flag-ΔF508-CFTR after prolonged exposure to 1 when normalized to lumacaftor alone (mean %rescue = 83% vs 100%; p = 0.0001). By contrast, there was no significant difference in rescue efficiency of ΔF508-CFTR with the inclusion of 2–4 when compared with lumacaftor alone (mean %rescue = 96%, 98 and 105%, respectively; p = n.s.) (Figure 2). When compared against each other, the response of compound 3 with lumacaftor was statistically different from that of 1 with lumacaftor (mean %rescue = 83% vs 105%; p = 0.0002).

Figure 2.

Figure 2

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Levels of surface-localized lumacaftor (VX-809) rescued ΔF508-CFTR when combined with 1–4 treatment. Quantification of surface:total fluorescence ratios for cellular ΔF508-CFTR upon treatment with 10 μM compounds 1–4 in combination with 3 μM lumacaftor. Steady-state surface CFTR levels were normalized to the control lumacaftor-only condition and represented as % rescue. Data is represented as mean ± SD between at least two experiments of n > 400 cells each, analyzed by Dunnett’s post hoc test (***P < 0.0001; ns = not significant; compared with VX-809).

2.5. Molecular Docking of Ivacaftor Derivatives

Using the known structure of CFTR-bound 1,15 we used molecular docking followed by energy minimization of the protein–ligand complex to evaluate binding of 2–4 to CFTR. Complexes were prioritized by their energetic complementarity and ability to make favorable polar interactions. Scoring of minimum-energy poses indicated that 3 had the best scoring pose with binding affinity measured using the Vina approach32 of −8.433 kcal/mol, which was better than the parent compound (1, −7.5 kcal/mol) (Table 2). This was further supported by the improved Vinardo scoring function,33,34 which held true even after local minimization of the top poses.

Table 2. Molecular Docking Scores of Compounds 1–4 Using Vinaa.

compound Vina score Vinardo optimized median score of top 9 poses
1 –7.5 –6.462 –6.801
2 –7.969 –6.406 –6.81
3 –8.433 –6.539 –6.958
4 –7.904 –6.285 –7.118
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a

Scores are given in kcal/mol, with more negative values indicating stronger binding affinity.

Docked complexes of ligands and CFTR were further examined to explore the chemical nature of these interactions (Figure 3). As has been previously reported,15 binding of 1 to CFTR (Figure 3a) is largely supported by hydrophobic interactions between the di-t-butylphenyl ring and a pocket created by Phe305 and Phe931, with one hydrogen bond between the amide oxygen atom of 1 and the N-H of Phe931, and an aromatic π–π interaction between the quinolinone ring and the side chain phenyl ring of Phe312. The binding of 2 is enhanced primarily by an ionic interaction between its cationic protonated amino group and the side chain carboxylate of Glu837 (Figure 3b).

Figure 3.

Figure 3

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Two-dimensional interaction map of docked complexes. (a) The published cryo-EM structure of 1 complexed with CFTR;15 (b) 2-CFTR; (c) 3-CFTR; (d) 4-CFTR. The three-dimensional models are appended as PDB Molecular Structure files in the Supporting Information.

Likewise, anionic derivative 4 experiences a beneficial ionic interaction between its carboxylate and the guanidine side chain of Arg933 (Figure 3d). The strongest binding, observed for the zwitterionic 3, arises from the combination of both of the ionic interactions described above for 2 and 4 (Figure 3c).15

3. Discussion and Conclusions

The cryo-electron microscopy (EM)-derived structure of 1 bound to CFTR indicates that about half of 1 is buried against CFTR, while the remaining is exposed to the hydrophobic region of the membrane.15 The destabilizing effect of 1 has been linked to its lipophilic property.11 From the results of that study, it could be inferred that the two t-butyl groups on the anilide in 1 are not strictly necessary for potentiation activity, so we proposed derivatives omitting these in order to decrease lipophilicity. Furthermore, it was shown that substituents at the quinolinone 6-position were also tolerated. We thus sought to prepare considerably less lipophilic derivatives by incorporating (through a side chain propargylamide) glutamic acid at the 6-position, which could easily be manipulated to reveal a polar amino group in 2, or carboxyl group in 4, or both as in 3.

We observed retained potentiator capabilities of all three derivatives when tested on G551D-CFTR expressing cells, although 2 and 4 exhibited a shorter duration of stimulation of channel activity compared to that of 1 and 3. The reason for this difference is unclear and may relate to differences in binding dynamics during the forskolin stimulatory period. Compound 3 experienced the highest peak activity of all of the tested compounds, including 1. For this reason, we considered 3 as an excellent candidate for further characterization.

Chronic exposure to 1 on lumacaftor-rescued ΔF508-CFTR protein inhibits the positive stabilizing effects of lumacaftor.11,23,35 Even at high concentrations of the much less lipophilic 3, we did not observe attenuation of (lumacaftor and low temperature rescued) ΔF508-CFTR protein surface levels. Lumacaftor binds to a deep hydrophobic pocket spanning TM helices 1, 2, 3, and 6 on TMD1.14,15,36,37 This is distant to the binding site of 1, which is on TMD2.36,37 While TMDs are initially separate, they undergo a conformational transition to come into close contact following R domain phosphorylation and NBD dimerization.15,17,38 NBD dimerization is allosterically linked to channel opening, operating through a long-range structural connection that spans TM8 and TM9, as well as the potentiator binding site of 1.17,38 It is possible then that the destabilizing effect of 1 on ΔF508-CFTR may relate to a deleterious event post-NBD dimerization when the TMDs are closer in proximity. While we cannot rule out factors extrinsic to the CFTR protein, our data predict that the glutamic derivatives of 1 preserve the allosteric link between the NBD dimer and channel pore opening. At higher concentrations (>1 μM), 1 is also known to have an off-target destabilizing effect on other membrane-localized proteins, including members of the SLC family proteins.11 The concentration of 1 in the tissues of CF-treated patients is uncertain,39 but 1 has been shown to accumulate in the lung and tracheal epithelial tissues.11 Notably, concentrations of >18 μM have been reported in primary cultures of CF bronchial epithelial cells treated with ivacaftor for 14 days followed by a 14-day wash period.40 When bound to CFTR, the hydrophobic end of 1 that is lipid-exposed may interfere with cholesterol-organized microdomain rafts, and/or with lipid–CFTR interactions directly.41 For the glutamic derivatives, our modeling predicts that several of these lipid-facing interactions of 1 are replaced by charged or hydrogen bonds oriented toward CFTR instead. Our results may be explained by these derivatives exhibiting lower lipid environment engagement and disruption around CFTR. Further work to test this hypothesis could help understand and improve the efficacy of potentiators for CF patients.

4. Experimental Section

4.1. Synthesis

4.1.1. General Considerations

All chemicals were obtained from commercial sources and used without further purification. Thin-layer chromatography (TLC) was performed on silica gel with an aluminum-backed plate. Column chromatography was carried out on silica gel 40–63 μm, eluting with the indicated solvent system. 1H and 13C spectroscopy was performed on a Bruker Avance instrument at 400 MHz (1H) and 100.6 MHz (13C) in deuterated solvents. Chemical shifts are recorded in parts per million (δ) in reference to the residual solvent peaks (CHCl3 7.26 ppm; DMSO-d5 2.50 ppm; MeOD-d3 3.31 ppm). Standard abbreviations indicating multiplicity are used as follows: s = singlet, d = doublet, t = triplet, and br = broad.

4.1.2. Diethyl 2-(((4-Iodophenyl)amino)methylene)malonate (5)

To a stirred solution of 4-iodoaniline (8.5000 g, 38.8 mmol) in EtOH (25 mL) was added diethyl ethoxymethylenemalonate (8 mL, 38.8 mmol). The reaction was heated to reflux for 4 h and then cooled to rt. The precipitate formed was filtered, washed with cold EtOH, and dried under vacuum. Yield (14.500 g, 96%); Rf = 0.22 (10% EtOAc/heptane)

1H NMR (400 MHz, CDCl3) δ 10.98 (d, J = 13.4, 1H), 8.46 (d, J = 13.5, 1H), 7.66 (d, J = 8.6, 2H), 6.90 (d, J = 8.6, 2H), 4.30 (q, J = 7.1 Hz, 2H), 4.24 (q, J = 7.1 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H) and 1.32 (t, J = 7.1 Hz, 3H) ppm. 13C NMR (100.6 MHz, CDCl3) δ: 168.97, 165.55, 151.21, 139.05, 138.74, 94.39, 87.96, 60.56, 60.26, 14.42, 14.28 ppm. NMR data matches the literature.42

4.1.3. Ethyl 6-Iodo-4-oxo-1,4-dihydroquinoline-3-carboxylate (6)

A round-bottom flask equipped with a magnetic stir bar was charged with diphenyl ether (21.3000 g, 125.1 mmol) and 5 (4.4143 g, 11.3 mmol). The reaction was heated to reflux between 240 and 260 °C for 45 min. The precipitate formed after cooling to room temperature was filtered and washed with heptane to afford the respective compound. Yield (3.3326, 86%).

1H NMR (400 MHz, DSMO-d6) δ 12.40 (s, 1H), 8.57 (s, 1H), 8.42 (d, J = 2 Hz, 1H), 7.99 (dd, J = 8.6, 2 Hz, 1H), 7.44 (d, J = 8.7 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H) ppm. NMR data corresponds with the literature.42 Due to the low solubility, no 13C NMR was obtained.

4.1.4. 6-Iodo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (7)

To a stirring suspension of 6 (1.8133 g, 5.3 mmol) in water (30 mL) was added NaOH (0.6341, 15.9 mmol), and the reaction mixture was heated to reflux for 4 h. The solution was then cooled to room temperature and acidified with 2 M H2SO4 (pH ∼3). The precipitate formed filtered and washed with a small portion of cold water, and the product dried under vacuum. Yield (1.6085 g, 97%).

1H NMR (400 MHz, DSMO-d6) δ 15.04 (brs, 1H), 13.49 (s, 1H), 8.93 (s, 1H), 8.56 (d, J = 1.9 Hz, 1H), 8.17 (dd, J = 8.7 Hz, 1.9 Hz, 1H) and 7.62 (d, J = 8.7 Hz, 1H) ppm. 13C NMR (100.6 MHz, DSMO-d6) δ: 177.45, 166.45, 146.10, 142.45, 139.20, 133.82, 126.55, 122.28, 108.57, 92.09 ppm. NMR data is in accordance with the literature.42

4.1.5. 3-((tert-Butyldimethylsilyl)oxy)aniline

To a solution of 3-aminophenol (2.5401 g, 23.28 mmol) in DCM (50 mL) were added TEA (10 mL, 69.83 mmol), DMAP (0.2844 g, 2.33 mmol), and TBDMSCl (10.5251 g, 69.83 mmol). The reaction mixture was stirred at RT for 24 h and then quenched with water (70 mL). The organic layer was separated and washed with an additional 70 mL of water, dried over MgSO4, filtered, and concentrated. Column chromatography (EtOAc/heptane 2:8, v/v) was used to purify the crude to obtain a yellow oil in quantitative yield (5.1899 g, 99%).

1H NMR (400 MHz, CDCl3) δ 7.01 (t, J = 8.0 Hz, 1H), 6.32–6.26 (m, 2H), 6.21–6.20 (m, 1H), 0.99 (s, 9H) and 0.21 (s, 6H) ppm. 13C NMR (100.6 MHz, CDCl3) δ: 156.64, 147.62, 129.92, 110.51, 108.56, 107.19, 25.73, 18.21, −4.36 ppm.

4.1.6. N-(3-((tert-Butyldimethylsilyl)oxy)phenyl)-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxamide (8)

To a stirred solution of 7 (1.2970 g, 4.1 mmol) in DMF (8 mL) and DIPEA (2 mL, 11.5 mmol) was added HBTU (2.3419 g, 6.2 mmol). The reaction mixture was stirred for 10 min, and then 3-((tert-butyldimethylsilyl)oxy)aniline (0.9645 g, 4.3 mmol) was added. The reaction was stirred at room temperature for 16 h (based on TLC), then diluted with 60 mL of DCM, and washed 40 mL of H2O (×2). The organic layer was dried over MgSO4 filtered, and concentrated. The crude was purified by flash column chromatography. Yield (0.8659 g, 40%); Rf = 0.54 (60% EtOAc/heptane).

1H NMR (400 MHz, DSMO-d6) δ 13.05 (brs, 1H), 12.29 (s, 1H), 8.91 (s, 1H), 8.60 (s, 1H), 8.08 (d, J = 8.1 Hz, 1H), 7.57–7.54 (m, 2H), 7.22 (t, J = 8.0 Hz, 1H), 7.10 (d, J = 8.1 Hz, 1H), 6.58 (d, J = 7.7 Hz, 1H), 0.97 (s, 9H) and 0.22 (s, 6H) ppm. 13C NMR (100.6, DSMO-d6) δ 175.40, 162.99, 156.02, 145.08, 141.51, 140.28, 138.87, 134.41, 130.31, 128.05, 121.95, 115.41, 113.11, 111.69, 111.54, 90.87, 26.05, 18.43, and −4.03 ppm.

4.1.7. Glutamic Acid, Side Chain Methyl Ester, Hydrochloride Salt

Thionyl chloride (4 mL, 54.8 mmol) was slowly added to MeOH (50 mL) at −10 °C followed by L-glutamic acid (5.9720 g, 40.6 mmol). The reaction was warmed to rt and stirred for 40 min, and then about half the volume of MeOH was removed by reduced pressure. Cold diethyl ether (50 mL) was added to the remaining volume, and the resultant precipitate was filtered and dried under vacuum to obtain the title compound as a white solid. Yield (6.5431 g, 99%). 1H NMR (400 MHz, DMSO-d6) δ: 8.62 (s, 3H), 3.89 (brs, 1H), 3.59 (s, 3H), 2.63–2.44 (m, 2H), 2.10–2.04 (m, 2H) ppm. 13C NMR (100.6 MHz, DMSO-d6) δ: 172.66, 170.86, 51.97, 51.58, 29.57, 25.58 ppm. NMR data is consistent with the literature.43

4.1.8. N-Boc Glutamic Acid, Side Chain Methyl Ester

To a solution of the methyl ester hydrochloride salt obtained previously (5.0712 g, 31.5 mmol) in water (25 mL) and dioxane (50 mL) at 0 °C were added di-tert-butyl dicarbonate (6.7439 g, 30.9 mmol) and NaHCO3 (5.8128 g, 69.2 mmol). The reaction was stirred at rt for 4 h before the dioxane was removed under reduced pressure. The aqueous layer was washed with Et2O (×2), and the organic layer was discarded. The aqueous phase was acidified with 2 M HCl to pH ∼4 and extracted ethyl acetate (×3). The combined organic layers were dried over MgSO4, and the solvent was removed in vacuo to afford the Boc-protected methyl ester as oil, which solidifies on standing. Yield (6.2497 g, 78%).

1H NMR (400 MHz, CDCl3) δ 5.31 (d, J = 7.4 Hz, 1H), 4.32 (d, J = 4.5 Hz, 1H), 3.65 (s, 3H), 2.48–2.37 (m, 2H), 2.22–2.18 (m, 1H), 2.00–1.95 (m, 1H), 1.41 (s, 9H) ppm. 13C NMR (100.6 MHz, CDCl3) δ: 175.85, 173.57, 155.70, 80.32, 52.76, 51.84, 30.12, 28.24, 27.49 ppm. NMR data is consistent with the literature.44,45

4.1.9. Methyl 4-((tert-Butoxycarbonyl)amino)-5-oxo-5-(prop-2-yn-1-yl-amino)pentanoate (9)

A 150 mL RBF equipped with a magnetic stir bar was charged THF (45 mL) and DIPEA (4.5 mL, 25.8 mmol), then the Boc-protected methyl ester (6.1551 g, 23.6 mmol) and HBTU (9.8949 g, 26.1 mmol) were, respectively added, and the mixture was stirred for 10 min at rt, then propargylamine (2 mL, 26 mmol) was added. The reaction mixture was stirred for 6 h at rt and then diluted with 70 mL of DCM. The organic layer was washed twice with 0.1 M HCl and then sat. NaHCO3 (×2) and H2O. The organic layer was dried over MgSO4, filtered, and the solvent was removed under reduced pressure. Flash column chromatography (6:4, EtOAc/heptane) was used to purify the crude product to obtain the title compound. Yield (5.5442 g, 79%); Rf = 0.55 (EtOAc/heptane (6:4), KMnO4 stain).

1H NMR (400 MHz, CDCl3) δ 7.18 (s, 1H), 5.55 (d, J = 4.4 Hz, 1H), 4.19 (s, 1H), 3.98–3.96 (m, 2H), 3.62 (s, 3H), 2.45–2.34 (m, 2H), 2.18 (t, J = 2.5 Hz, 1H), 2.12–2.04 (m, 1H), 1.94–1.85 (m, 1H), 1.38 (s, 9H) ppm. 13C NMR (100.6 MHz, CDCl3) δ: 173.68, 171.59, 155.81, 80.10, 71.55, 53.41, 51.79, 30.13, 29.05, 28.28, 27.91 ppm.

4.1.10. Methyl 4-((tert-Butoxycarbonyl)amino)-5-((3-(3-((3-((tert-butyldimethylsilyl)oxy)phenyl)carbamoyl)-4-oxo-1,4-dihydroquinolin-6-yl)prop-2-yn-1-yl)amino)-5-oxopentanoate (10)

A 50 mL round-bottom flask equipped with a magnetic stir bar was charged with 8 (0.7502 g, 1.4 mmol) and 9 (0.4712 g, 1.6 mmol). THF (5 mL) and DIPEA (0.3 mL, 1.7 mmol) were added, respectively, and the reaction mixture was sparged for 10 min with N2 before Pd(PPh3)2Cl2 (61.9 mg, 0.09 mmol) and CuI (27 mg, 0.14 mmol) were added sequentially. After stirring at 55 °C for 16 h, the solvent was removed and the crude was purified by column chromatography (80% EtOAc/heptane). Yield (0.6537 g, 66%); Rf = 0.43.

1H NMR (400 MHz, MeOD-d4) δ 8.79 (s, 1H), 8.35 (s, 1H), 7.72 (d, J = 8.6 Hz, 1H), 7.56 (d, J = 8.6 Hz, 1H), 7.43 (s, 1H), 7.23–7.15 (m, 2H), 6.61 (d, J = 7.8 Hz, 1H), 4.25 (d, J = 6.6 Hz, 2H), 3.67 (s, 3H), 2.45 (t, J = 7.32 Hz, 2H), 2.12–2.09 (m, 1H), 1.97–1.89 (m, 1H), 1.45 (s, 9H), 1.02 (s, 9H), 0.24 (s, 6H) ppm. 13C NMR (100.6 MeOD-d4) δ: 176.48, 173.44, 163.44, 156.10, 143.93, 139.35, 138.68, 135.45, 129.30, 128.81, 125.99, 120.11, 118.89, 115.51, 113.10, 111.98, 111.37, 50.83, 37.48, 29.72, 27.30, 24.79, 17.69, −5.66 ppm.

4.1.11. Methyl 4-Amino-5-((3-(3-((3-hydroxyphenyl)carbamoyl)-4-oxo-1,4-dihydroquinolin-6-yl)prop-2-yn-1-yl)amino)-5-oxopentanoate (2)

A mixture of CH2Cl2/TFA (10 mL, 1:1 v/v) was added to 10 (0.2751 g, 0.4 mmol), and the mixture was heated to 40 °C for 3 h. The solvent was removed by reduced pressure, then the residue was triturated in water for about 30 min. The precipitate was filtered and washed with ice-cold water to afford the title compound. Yield (0.1247 g, 65%).

1H NMR (400 MHz, MeOD-d4) δ 8.80 (s, 1H), 8.32 (s, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.57 (d, J = 8.6 Hz, 1H), 7.30 (s, 1H), 7.16 (t, J = 8.0 Hz, 1H), 7.06 (9d, J = 7.8 Hz, 1H), 6.82 (dd, J = 8.0 Hz, 1.8 Hz, 1H), 4.31 (s, 2H), 3.97 (t, J = 6.4 Hz, 1H), 3.70 (s, 3H), 2.54–2.43 (m, 2H), 2.16–1.91 (m, 2H) ppm. 13C NMR (100.6 MHz, MeOD-d4) δ: 176.44, 172.69, 168.06, 163.34, 157.70, 144.01, 139.21, 138.76, 135.30, 129.36, 128.80, 125.94, 119.78, 119.01, 111.41, 111.28, 110.95, 107.23, 85.54, 81.39, 52.41, 52.30, 29.11, 28.53, 26.32, 26.18 ppm. HRMS (ESI) m/z calculated for C25H25N4O6+ (M + H)+: 477.1769; found: 477.1768.

4.1.12. 4-Amino-5-((3-(3-((3-hydroxyphenyl)carbamoyl)-4-oxo-1,4-dihydroquinolin-6-yl)prop-2-yn-1-yl)amino)-5-oxopentanoic Acid (3)

To a stirred solution of 10 (0.2678 g, 0.4 mmol) in THF (20 mL) was added 5% NaOH in H2O (8 mL). The reaction was stirred for 2 h at rt and then acidified using 1 M HCl to pH ∼ 3–4. The acidic layer was extracted with 25 mL of EtOAc (×2). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product (0.1914 g) was solubilized in DCM (10 mL), and then 3 mL of TFA was added and stirred for 1 h at rt. The solvent was removed in vacuo to obtain 3. Yield (0.1746 g, 94% over 2 steps).

1H NMR (MeOD-d4 some signals split due to rotamerism) δ 8.83 (s, 1H), 8.39 (t, J = 2.2 Hz, 1H), 7.91(s, 1H), 7.77–7.75 (m, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.32–7.31 (m, 1H), 7.16 (t, J = 8.0 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.58 (dd, J = 7.3 Hz, 1.6 Hz, 1H), 4.35 (s, ∼0.5 × 2H), 4.28 (s, ∼0.5 × 2H), 4.08 (t, J = 6.4 Hz, ∼0.5 × 1H), 3.98 (t, J = 6.4 Hz, ∼0.5 × 1H), 2.58–2.50 (m, 2H), 2.26–2.14 (m, 2H) ppm. 13C NMR (100.6 MHz, MeOD-d4 some signals split due to rotamerism): δ 176.40, 174.18, 172.55, 168.17, 163.32, 157.68, 143.97, 139.21, 138.71, 138.63, 135.30, 132.41, 132.38, 131.71, 131.62, 129.35, 128.76, 125.88, 119.99, 119.76, 118.98, 111.28, 110.93, 107.23, 86.05, 85.53, 81.40, 81.01, 78.07, 52.42, 30.90, 29.12, 28.99, 28.72, 26.31, 25.71 ppm.

4.1.13. Methyl 4-Acetamido-5-oxo-5-(prop-2-yn-1-yl-amino)pentanoate

To a round-bottom flask equipped with a magnetic stir bar was added 9 (1.0978 g, 3.8 mmol). A mixture of DCM/TFA (10 mL; 1:1) was added to the flask, and the reaction was stirred at rt for 3 h; then, the solvent was removed under reduced pressure. The crude product was added to a saturated solution of NaHCO3 (2.1708 g, 25.8 mmol) in 3 mL of water. At 0 °C, Ac2O (1 mL, 10.6 mmol) was added dropwise to the stirring mixture. After stirring for 1 h at rt, the solvent was evaporated, and the crude was purified by column chromatography (10% MeOH/DCM) to obtain a liquid that solidifies on standing. Yield (0.7608 g, 83%); Rf = 0.46 (10% MeOH/DCM, KMnO4 stain).

1H NMR (400 MHz, CDCl3) δ 7.44–7.41 (m, 1H), 7.13 (d, J = 7.9 Hz, 1H), 4.62–4.56 (m, 1H), 4.02–4.01 (m, 2H), 3.67 (s, 3H), 2.52–2.36 (m, 2H), 2.22 (t, J = 2.4 Hz, 1H), 2.16–2.08 (m, 2H), 2.03 (s, 3H) ppm. 13C NMR (100.6 MHz, CDCl3) δ: 173.79, 171.30, 78.92, 71.74, 52.28, 51.95, 30.14, 29.24, 27.75, 22.88 ppm.

4.1.14. Methyl 4-Acetamido-5-((3-(3-((3-((tert-butyldimethylsilyl)oxy)phenyl)carbamoyl)-4-oxo-1,4-dihydroquinolin-6-yl)prop-2-yn-1-yl)amino)-5-oxopentanoate (11)

Compound 8 (0.2496 g, 0.5 mmol) and methyl 4-acetamido-5-oxo-5-(prop-2-yn-1-yl-amino)pentanoate (0.1562 g, 0.7 mmol) were solubilized in THF (5 mL) and DIPEA (0.3 mL, 1.7 mmol) and then sparged with N2 for 10 min. Pd(PPh3)2Cl2 (44.5 mg, 0.06 mmol) and CuI (17.5 mg, 0.09 mmol) were added sequentially. The flask was sealed under N2, and the reaction mixture was heated to 55 °C for 3 h. The volatiles were removed in vacuo, and the crude was absorbed onto silica and purified by column chromatography (10% MeOH/DCM). Yield (0.2215 g, 70%).

1H NMR (400 MHz, MeOD-d4) δ 8.71 (s, 1H), 8.21 (d, J = 1.6 Hz, 1H), 7.61–7.58 (m,1H), 7.46 (d, J = 8.6 Hz, 1H), 7.40 (t, J = 2.0 Hz, 1H), 7.20–7.11 (m, 2H), 6.60–6.57 (m, 1H), 4.44 −4.41 (m, 1H), 4.22 (s, 2H), 3.66 (s, 3H), 2.16–2.13 (m, 2H), 2.02 (s, 3H), 2.00–1.93 (m, 2H), 1.01 (s, 9H), 0.23 (s, 6H) ppm. 13C NMR (100.6 MHz, MeOD-d4) δ: 176.30, 173.41, 172.17. 172.09, 163.36, 156.06, 143.83, 139.36, 138.53, 135.31, 131.72, 131.62, 129.29, 128.71, 125.82, 119.91, 118.82, 115.46, 113.10, 111.93, 111.30, 86.13, 81.01, 52.59, 50.87, 29.70, 28.98, 26.95, 21.16, 17.70, −5.61 ppm.

4.1.15. 4-Acetamido-5-((3-((3-hydroxyphenyl)carbamoyl)-4-oxo-1,4-dihydroqinolin-6-yl)prop-2-yn-1-yl)amino-5-oxopentanoic acid (4)

To a stirred solution of 11 (0.2898 g, 0.5 mmol) in THF (20 mL) was added 5% NaOH in H2O (8 mL). The reaction was stirred at rt for 2 h and then acidified to pH ∼ 3–4 using 1 M HCl. The aqueous layer was extracted with 25 mL of EtOAc (×2), and then the combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. Yield (0.0869 g, 34%).

1H NMR (400 MHz, MeOD-d4) δ 8.79 (s, 1H), 8.35–8.34 (m, 1H), 7.75–7.71 (m, 1H), 7.58–7.55 (m, 1H), 7.30 (t, J = 2.0 Hz, 1H), 7.16 (t, J = 8.0 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.57 (dd, J = 8.0 Hz, 1.6 Hz, 1), 2.44–2.36 (m, 2H), 4.44–4.40 (m, 1H), 4.25–4.23 (m, 2H), 2.26–2.23 (m, 2H), 2.01 (s, 3H) ppm. 13C NMR (100.6 MHz, MeOD-d4) δ: 176.41, 173.50, 173.10, 172.09, 163.34, 157.68, 143.86, 139.23, 138.61, 135.42, 129.34, 128.70, 125.88, 120.09, 118.90, 111.27, 110.92, 107.20, 86.23, 80.93, 51.84, 31.69, 29.48, 28.92, 27.12, 21.03 ppm. HRMS (ESI) m/z calculated for C26H25N4O7+ (M + H)+: 505.1718; found: 505.1711.

4.2. Biological Evaluation Experiments

4.2.1. Cell Culture and Reagents

Cells were maintained in modified Eagle’s medium (DMEM) supplemented with 10% FBS, 100 g/mL penicillin/streptomycin at 5% CO2 at 37 °C. Human Embryonic Kidney (HEK) 293 T-REx cells were stably transfected with tetracycline-inducible pcDNA5 FRT/TO BirA-R118G-FLAG (BirA*FLAG) expression vectors, expressing either wild-type (WT) or G551D-CFTR. Doxycycline inducible CFBE mCherry-Flag-ΔF508-CFTR cells31 obtained from Dr. Maragarida Amaral (Lisbon, Portugal) were maintained in MEM supplemented with 10% FBS, 100 g/mL penicillin/streptomycin at 5% CO2 at 37 °C. CFBEs are a CF human bronchial epithelial cell line (CFBE41o-), derived from a CF patient homozygous for the ΔF508-CFTR mutation, and immortalized with the origin-of-replication defective SV40 plasmid.46,47

4.2.2. FLIPR Membrane Potential Assay

WT or G551D-CFTR expressing HEK293 T-REx cells29 were seeded in 96-well plates (Costar, Corning) and induced with tetracycline for 24 h at 37 °C with 5% CO2. The cells were then loaded with blue FLIPR membrane potential dye27 dissolved in chloride-free buffer (136 mM sodium gluconate, 3 mM potassium gluconate, 10 mM glucose, 20 mM HEPES, pH 7.35, 300 mOsm, at a concentration of 0.5 mg/mL) for 30 min at 37 °C. CFTR function was determined using a BioTek Synergy HTX Multi-Mode Reader at 37 °C. After establishing a baseline fluorescence read (excitation 530nm/emission 560 nm) for 3 min, CFTR was stimulated by Forskolin (FSK) (10 μM, MedChemExpress, Princeton, NJ), 10 μM FSK + 1 μM 1, or 10 μM FSK + 1 derivatives for 8 min. Stock solutions of 1-4 were prepared in DMSO and used at final concentrations of 0.1% DMSO, which were verified to have no effect on membrane potential activity. CFTR-mediated depolarization of the plasma membrane was detected as an increase in fluorescence. CFTR activity was then inhibited with the CFTR inhibitor CFTRinh-172 (10 μM, MedChemExpress, Princeton, NJ). The changes in fluorescence to CFTR agonists were normalized relative to the baseline fluorescence (ΔF/F0).48 Curves were normalized to the average of the baseline fluorescence reads, and the maximal points of stimulation were plotted with GraphPad Prism 7 (San Diego, CA).

4.2.3. Membrane Potential Analysis

Results were plotted and analyzed on GraphPad Prism 7. Results were presented as ± SD with each replicated representing one biological replicate from an individual experiment. For all statistical analyses, one-way ANOVA and Dunnett’s post hoc tests were conducted on GraphPad Prism 7 for multiple comparisons of conditions to the control. The p values were automatically adjusted by GraphPad Prism 7 for the multiple comparisons, and p < 0.05 was considered significantly different from the control.

4.2.4. Surface Expression Assay and Image Analysis

CFBE mCherry-Flag-ΔF508-CFTR cells29 were incubated for 24 h at 27 °C with 3 μM lumacaftor and 10 μM 1–4. Extracellular FLAG-epitopes on rescued mCherry-Flag-ΔF508-CFTR were immunolabeled in nonpermeabilized cells. Immunolabeling, imaging, and analysis were carried out using the protocol established previously.29 Briefly, after culture medium removal, the cells were washed once in ice-cold PBS and incubated for 45 min on ice with anti-FLAGm2 antibody. Then, cells were washed 3 times with ice-cold PBS, incubated for 10 min with 4% PFA on ice, and transferred to room temperature for the remaining staining procedure. The cells were washed with PBS and incubated for 30 min with Alexa Fluor 488 donkey anti-mouse before mounting onto glass slides. Fluorescence images were acquired on an automated DeltaVision Microscope with a 60× 1.4 NA objective and 2 × 2 binning (GE Healthcare). For every well, 25 fields of Z-stacks encompassing 8 μm were deconvolved, projected, and exported as 16-bit TIFF images prior to analysis. On average, each field sampled 25–40 cells. Using the MATLAB image analysis toolbox, we estimated dark noise and background using demarcated regions in several images from each data set. The background was calculated using the most populated pixel bin from histograms of these regions (for each channel) and subtracted from the corresponding channels. For every field, each channel was thresholded using a stringent cutoff (7× and 20× over background for the “surface” and “total” channels, respectively) to select only pixels corresponding to cellular contents, and all other pixel values were discarded from subsequent calculations. The mean thresholded pixel intensity for each channel was then calculated. The ratio of surface/total was calculated by dividing the two background subtracted and thresholded “surface” and “total” channels for every field, thereby generating 25 ratios for each set. Outlier fields with out-of-focus images or sparsely populated fields were discarded. All imaging experiments were performed three times and displayed similar trends. MATLAB scripts are available upon request.

4.3. Molecular Docking of Ivacaftor Derivatives with CFTR

The 3-dimensional atomistic structure of ivacaftor-bound CFTR was obtained from Protein Data Bank (PDB) [PDB ID: 6O2P].49 The structure chosen for the molecular docking was in the ATP-bound state with the E1371Q mutation retained,15 as the mutant structure is the target of ivacaftor. Also, the mutation site and ATP binding site were situated away from the grid map of protein atoms used to calculate interactions for the docking studies. The structure was prepared using UCSF Chimera to be ready for molecular docking.50 Hydrogens were added to the structure at physiological pH and partial atomic charges were assigned using the Gasteiger method in AutoDock tools. The binding site was identified using the PDB records and interacting residues of ivacaftor. Coordinates for 1–4 were prepared using the Datawarrior tool and were further optimized with the MMFF94S+ force field using a random, low-energy bias algorithm to obtain stable, minimum-energy conformers for each ligand. To perform molecular docking, a grid box of volume 22 Å was used with the spacing of 0.375 Å between each grid point and it was centered around the center of the ivacaftor binding site. Ligands were docked into the protein structure using AutoDock Vina v1.2.3, which uses a Monte Carlo algorithm to sample the conformers and scores each pose using the Vina empirical scoring function.32,51 Top poses were also optimized and rescored using the Vinardo scoring function within the AutoDock Vina software. Molecular interactions between the CFTR protein and ligands were evaluated using the Protein Plus molecular modeling server (https://proteins.plus).51,52

Acknowledgments

The authors acknowledge the gift of cell lines and reagents from Profs. Garry Cutting, Margarida Amaral, and Christine Bear. They also thank Eric Fries for assistance with HPLC-MS data.

Glossary

Abbreviations

CFTR

cystic fibrosis transmembrane conductance regulator

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c05839.

  • HPLC-MS and log P determination; representative data for surface level quantitation; 1H and 13C NMR spectra; and HMRS (PDF)

  • 3D structure of 2 docked on CFTR (PDB)

  • 3D structure of 3 docked on CFTR (PDB)

  • 3D structure of 4 docked on CFTR (PDB)

Author Contributions

§ M.I. and P.J. contributed equally. P.J. synthesized the glutamic acid ivacaftor derivatives and wrote the chemistry evaluation, analysis, and methods portion of this manuscript. M.I. coordinated and performed all biology evaluation and analysis and wrote the biology evaluation, analysis, and methods portion. J.D. and S.A. performed the molecular docking and modeling. R.S. performed the HPLC experiments to calculate c log P. G.D.G. and R.D.V. wrote the manuscript with contributions from all authors. The project was supervised by G.D.G. and R.D.V. All authors have read and agreed to the published version of the manuscript.

G.D.G. is funded by a Cystic Fibrosis Canada ECI Grant 1008549.

The authors declare no competing financial interest.

Supplementary Material

ao3c05839_si_001.pdf (4.3MB, pdf)
ao3c05839_si_002.pdb (1.5MB, pdb)
ao3c05839_si_003.pdb (1.5MB, pdb)
ao3c05839_si_004.pdb (1.5MB, pdb)

References

  1. De Boeck K. Cystic fibrosis in the year 2020: A disease with a new face. Acta Paediatr. 2020, 109 (5), 893–899. 10.1111/apa.15155. [DOI] [PubMed] [Google Scholar]
  2. De Boeck K.; Amaral M. D. Progress in therapies for cystic fibrosis. Lancet Respir. Med. 2016, 4 (8), 662–674. 10.1016/S2213-2600(16)00023-0. [DOI] [PubMed] [Google Scholar]
  3. Cutting G. R. Treating Specific Variants Causing Cystic Fibrosis. JAMA 2017, 318 (21), 2130. 10.1001/jama.2017.16823. [DOI] [PubMed] [Google Scholar]
  4. Veit G.; Avramescu R. G.; Chiang A. N.; Houck S. A.; Cai Z.; Peters K. W.; Hong J. S.; Pollard H. B.; Guggino W. B.; Balch W. E.; Skach W. R.; Cutting G. R.; Frizzell R. A.; Sheppard D. N.; Cyr D. M.; Sorscher E. J.; Brodsky J. L.; Lukacs G. L. From CFTR biology toward combinatorial pharmacotherapy: expanded classification of cystic fibrosis mutations. Mol. Biol. Cell 2016, 27 (3), 424–433. 10.1091/mbc.e14-04-0935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Laselva O.; Qureshi Z.; Zeng Z.-W.; Petrotchenko E. V.; Ramjeesingh M.; Hamilton C. M.; Huan L.-J.; Borchers C. H.; Pomès R.; Young R.; Bear C. E. Identification of binding sites for ivacaftor on the cystic fibrosis transmembrane conductance regulator. iScience 2021, 24 (6), 102542 10.1016/j.isci.2021.102542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Graeber S. Y.; Mall M. A. The future of cystic fibrosis treatment: from disease mechanisms to novel therapeutic approaches. Lancet 2023, 402, 1185–1198. 10.1016/S0140-6736(23)01608-2. [DOI] [PubMed] [Google Scholar]
  7. Lopes-Pacheco M. CFTR Modulators: Shedding Light on Precision Medicine for Cystic Fibrosis. Front. Pharmacol. 2016, 7, 275 10.3389/fphar.2016.00275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Van Goor F.; Hadida S.; Grootenhuis P. D. J.; Burton B.; Stack J. H.; Straley K. S.; Decker C. J.; Miller M.; McCartney J.; Olson E. R.; Wine J. J.; Frizzell R. A.; Ashlock M.; Negulescu P. A. Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (46), 18843–18848. 10.1073/pnas.1105787108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bear C. E. A Therapy for Most with Cystic Fibrosis. Cell 2020, 180 (2), 211. 10.1016/j.cell.2019.12.032. [DOI] [PubMed] [Google Scholar]
  10. Davis P. B. Cystic Fibrosis Since 1938. Am. J. Respir. Crit. Care Med. 2006, 173 (5), 475–482. 10.1164/rccm.200505-840OE. [DOI] [PubMed] [Google Scholar]
  11. Chin S.; Hung M.; Won A.; Wu Y.-S.; Ahmadi S.; Yang D.; Elmallah S.; Toutah K.; Hamilton C. M.; Young R. N.; Viirre R. D.; Yip C. M.; Bear C. E. Lipophilicity of the Cystic Fibrosis Drug, Ivacaftor (VX-770), and Its Destabilizing Effect on the Major CF-causing Mutation: F508del. Mol. Pharmacol. 2018, 94 (2), 917–925. 10.1124/mol.118.112177. [DOI] [PubMed] [Google Scholar]
  12. Fohner A. E.; McDonagh E. M.; Clancy J. P.; Whirl Carrillo M.; Altman R. B.; Klein T. E. PharmGKB summary: ivacaftor pathway, pharmacokinetics/pharmacodynamics. Pharmacogenet. Genomics 2017, 27 (1), 39–42. 10.1097/FPC.0000000000000246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Van Goor F.; Hadida S.; Grootenhuis P. D.; Burton B.; Cao D.; Neuberger T.; Turnbull A.; Singh A.; Joubran J.; Hazlewood A.; Zhou J.; McCartney J.; Arumugam V.; Decker C.; Yang J.; Young C.; Olson E. R.; Wine J. J.; Frizzell R. A.; Ashlock M.; Negulescu P. Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (44), 18825–18830. 10.1073/pnas.0904709106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Yeh H. I.; Qiu L.; Sohma Y.; Conrath K.; Zou X.; Hwang T. C. Identifying the molecular target sites for CFTR potentiators GLPG1837 and VX-770. J. Gen. Physiol. 2019, 151 (7), 912–928. 10.1085/jgp.201912360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Liu F.; Zhang Z.; Levit A.; Levring J.; Touhara K. K.; Shoichet B. K.; Chen J. Structural identification of a hotspot on CFTR for potentiation. Science 2019, 364 (6446), 1184–1188. 10.1126/science.aaw7611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hadida S.; Van Goor F.; Zhou J.; Arumugam V.; McCartney J.; Hazlewood A.; Decker C.; Negulescu P.; Grootenhuis P. D. J. Discovery of N -(2,4-Di- tert -butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (VX-770, Ivacaftor), a Potent and Orally Bioavailable CFTR Potentiator. J. Med. Chem. 2014, 57 (23), 9776–9795. 10.1021/jm5012808. [DOI] [PubMed] [Google Scholar]
  17. Levring J.; Terry D. S.; Kilic Z.; Fitzgerald G.; Blanchard S. C.; Chen J. CFTR function, pathology and pharmacology at single-molecule resolution. Nature 2023, 616 (7957), 606–614. 10.1038/s41586-023-05854-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Middleton P. G.; Mall M. A.; Drevinek P.; Lands L. C.; McKone E. F.; Polineni D.; Ramsey B. W.; Taylor-Cousar J. L.; Tullis E.; Vermeulen F.; Marigowda G.; McKee C. M.; Moskowitz S. M.; Nair N.; Savage J.; Simard C.; Tian S.; Waltz D.; Xuan F.; Rowe S. M.; Jain R. Elexacaftor-Tezacaftor-Ivacaftor for Cystic Fibrosis with a Single Phe508del Allele. N. Engl. J. Med. 2019, 381 (19), 1809–1819. 10.1056/NEJMoa1908639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wainwright C. E.; Elborn J. S.; Ramsey B. W.; Marigowda G.; Huang X.; Cipolli M.; Colombo C.; Davies J. C.; De Boeck K.; Flume P. A.; Konstan M. W.; McColley S. A.; McCoy K.; McKone E. F.; Munck A.; Ratjen F.; Rowe S. M.; Waltz D.; Boyle M. P. Lumacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR. N. Engl. J. Med. 2015, 373 (3), 220–231. 10.1056/NEJMoa1409547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Arnott J. A.; Planey S. L. The influence of lipophilicity in drug discovery and design. Expert Opin. Drug Discovery 2012, 7 (10), 863–875. 10.1517/17460441.2012.714363. [DOI] [PubMed] [Google Scholar]
  21. Chagas C. M.; Moss S.; Alisaraie L. Drug metabolites and their effects on the development of adverse reactions: Revisiting Lipinski’s Rule of Five. Int. J. Pharm. 2018, 549 (1–2), 133–149. 10.1016/j.ijpharm.2018.07.046. [DOI] [PubMed] [Google Scholar]
  22. Boyle M. P.; Bell S. C.; Konstan M. W.; McColley S. A.; Rowe S. M.; Rietschel E.; Huang X.; Waltz D.; Patel N. R.; Rodman D. A CFTR corrector (lumacaftor) and a CFTR potentiator (ivacaftor) for treatment of patients with cystic fibrosis who have a phe508del CFTR mutation: a phase 2 randomised controlled trial. Lancet Respir. Med. 2014, 2 (7), 527–538. 10.1016/S2213-2600(14)70132-8. [DOI] [PubMed] [Google Scholar]
  23. Cholon D. M.; Quinney N. L.; Fulcher M. L.; Esther C. R.; Das J.; Dokholyan N. V.; Randell S. H.; Boucher R. C.; Gentzsch M. Potentiator ivacaftor abrogates pharmacological correction of ΔF508 CFTR in cystic fibrosis. Sci. Transl. Med. 2014, 6 (246), 246ra96 10.1126/scitranslmed.3008680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Matthes E.; Goepp J.; Carlile G. W.; Luo Y.; Dejgaard K.; Billet A.; Robert R.; Thomas D. Y.; Hanrahan J. W. Low free drug concentration prevents inhibition of F508del CFTR functional expression by the potentiator VX-770 (ivacaftor): Interaction between CF drugs. Br. J. Pharmacol. 2016, 173 (3), 459–470. 10.1111/bph.13365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hjelmgaard T.; Tanner D. Copper(I) mediated cross-coupling of amino acid derived organozinc reagents with acid chlorides. Org. Biomol. Chem. 2006, 4 (9), 1796–1805. 10.1039/b601996j. [DOI] [PubMed] [Google Scholar]
  26. OECD . Test No. 117: Partition Coefficient (n-Octanol/Water): HPLC Method, 2022.
  27. Ahmadi S.; Bozoky Z.; Di Paola M.; Xia S.; Li C.; Wong A. P.; Wellhauser L.; Molinski S. V.; Ip W.; Ouyang H.; Avolio J.; Forman-Kay J. D.; Ratjen F.; Hirota J. A.; Rommens J.; Rossant J.; Gonska T.; Moraes T. J.; Bear C. E. Phenotypic profiling of CFTR modulators in patient-derived respiratory epithelia. npj Genomic Med. 2017, 2, 12 10.1038/s41525-017-0015-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Maitra R.; Sivashanmugam P.; Warner K. A rapid membrane potential assay to monitor CFTR function and inhibition. J. Biomol. Screen. 2013, 18 (9), 1132–1137. 10.1177/1087057113488420. [DOI] [PubMed] [Google Scholar]
  29. Iazzi M.; Astori A.; St-Germain J.; Raught B.; Gupta G. D. Proximity Profiling of the CFTR Interaction Landscape in Response to Orkambi. Int. J. Mol. Sci. 2022, 23 (5), 2442. 10.3390/ijms23052442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Domingue J. C.; Ao M.; Sarathy J.; George A.; Alrefai W. A.; Nelson D. J.; Rao M. C. HEK-293 cells expressing the cystic fibrosis transmembrane conductance regulator (CFTR): a model for studying regulation of Cl- transport. Physiol. Rep. 2014, 2 (9), e12158 10.14814/phy2.12158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Botelho H. M.; Uliyakina I.; Awatade N. T.; Proenca M. C.; Tischer C.; Sirianant L.; Kunzelmann K.; Pepperkok R.; Amaral M. D. Protein traffic disorders: an effective high-throughput fluorescence microscopy pipeline for drug discovery. Sci. Rep. 2015, 5, 9038 10.1038/srep09038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Trott O.; Olson A. J. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010, 31 (2), 455–461. 10.1002/jcc.21334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Quiroga R.; Villarreal M. A. Vinardo: A Scoring Function Based on Autodock Vina Improves Scoring, Docking, and Virtual Screening. PLoS One 2016, 11 (5), e0155183 10.1371/journal.pone.0155183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Meli R.; Morris G. M.; Biggin P. C. Scoring Functions for Protein-Ligand Binding Affinity Prediction using Structure-Based Deep Learning: A Review. Front. Bioinform. 2022, 2, 885983 10.3389/fbinf.2022.885983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Veit G.; Avramescu R. G.; Perdomo D.; Phuan P. W.; Bagdany M.; Apaja P. M.; Borot F.; Szollosi D.; Wu Y. S.; Finkbeiner W. E.; Hegedus T.; Verkman A. S.; Lukacs G. L. Some gating potentiators, including VX-770, diminish DeltaF508-CFTR functional expression. Sci. Transl. Med. 2014, 6 (246), 246ra97 10.1126/scitranslmed.3008889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Fiedorczuk K.; Chen J. Molecular structures reveal synergistic rescue of Delta508 CFTR by Trikafta modulators. Science 2022, 378 (6617), 284–290. 10.1126/science.ade2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Fiedorczuk K.; Chen J. Mechanism of CFTR correction by type I folding correctors. Cell 2022, 185 (1), 158–168. 10.1016/j.cell.2021.12.009. [DOI] [PubMed] [Google Scholar]
  38. Vergani P.; Lockless S. W.; Nairn A. C.; Gadsby D. C. CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains. Nature 2005, 433 (7028), 876–880. 10.1038/nature03313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Trittler R.; Hug M. PKP-017 Monitoring of ivacaftor serum levels. Eur. J. Hosp. Pharm. 2014, 21 (Suppl 1), A143–A144. 10.1136/ejhpharm-2013-000436.352. [DOI] [Google Scholar]
  40. Guhr Lee T. N.; Cholon D. M.; Quinney N. L.; Gentzsch M.; Esther C. R. Accumulation and persistence of ivacaftor in airway epithelia with prolonged treatment. J. Cystic Fibrosis 2020, 19 (5), 746–751. 10.1016/j.jcf.2020.04.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Cottrill K. A.; Farinha C. M.; McCarty N. A. The bidirectional relationship between CFTR and lipids. Commun. Biol. 2020, 3 (1), 179 10.1038/s42003-020-0909-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Chen Y. L.; Tang J.; Kesler M. J.; Sham Y. Y.; Vince R.; Geraghty R. J.; Wang Z. The design, synthesis and biological evaluations of C-6 or C-7 substituted 2-hydroxyisoquinoline-1,3-diones as inhibitors of hepatitis C virus. Bioorg. Med. Chem. 2012, 20 (1), 467–479. 10.1016/j.bmc.2011.10.058. [DOI] [PubMed] [Google Scholar]
  43. Buckingham F.; Kirjavainen A. K.; Forsback S.; Krzyczmonik A.; Keller T.; Newington I. M.; Glaser M.; Luthra S. K.; Solin O.; Gouverneur V. Organomediated Enantioselective (18)F Fluorination for PET Applications. Angew. Chem., Int. Ed. 2015, 54 (45), 13366–13369. 10.1002/anie.201506035. [DOI] [PubMed] [Google Scholar]
  44. More S. S.; Vince R. Inhibition of glyoxalase I: the first low-nanomolar tight-binding inhibitors. J. Med. Chem. 2009, 52 (15), 4650–4656. 10.1021/jm900382u. [DOI] [PubMed] [Google Scholar]
  45. Yang T.; Liu Z.; Li X. D. Developing diazirine-based chemical probes to identify histone modification ’readers’ and ’erasers’. Chem. Sci. 2015, 6 (2), 1011–1017. 10.1039/C4SC02328E. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Illek B.; Maurisse R.; Wahler L.; Kunzelmann K.; Fischer H.; Gruenert D. C. Cl transport in complemented CF bronchial epithelial cells correlates with CFTR mRNA expression levels. Cell. Physiol. Biochem. 2008, 22 (1–4), 57–68. 10.1159/000149783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Bruscia E.; Sangiuolo F.; Sinibaldi P.; Goncz K. K.; Novelli G.; Gruenert D. C. Isolation of CF cell lines corrected at DeltaF508-CFTR locus by SFHR-mediated targeting. Gene Ther. 2002, 9 (11), 683–685. 10.1038/sj.gt.3301741. [DOI] [PubMed] [Google Scholar]
  48. Laselva O.; Ardelean M. C.; Bear C. E. Phenotyping Rare CFTR Mutations Reveal Functional Expression Defects Restored by TRIKAFTA(TM).. J. Pers. Med. 2021, 11 (4), 301 10.3390/jpm11040301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Berman H. M. The Protein Data Bank. Nucleic Acids Res. 2000, 28 (1), 235–242. 10.1093/nar/28.1.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Pettersen E. F.; Goddard T. D.; Huang C. C.; Couch G. S.; Greenblatt D. M.; Meng E. C.; Ferrin T. E. UCSF Chimera?A visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25 (13), 1605–1612. 10.1002/jcc.20084. [DOI] [PubMed] [Google Scholar]
  51. Trott O.; Olson A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010, 31 (2), 455–461. 10.1002/jcc.21334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Schöning-Stierand K.; Diedrich K.; Ehrt C.; Flachsenberg F.; Graef J.; Sieg J.; Penner P.; Poppinga M.; Ungethüm A.; Rarey M. Proteins Plus: a comprehensive collection of web-based molecular modeling tools. Nucleic Acids Res. 2022, 50 (W1), W611–W615. 10.1093/nar/gkac305. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao3c05839_si_001.pdf (4.3MB, pdf)
ao3c05839_si_002.pdb (1.5MB, pdb)
ao3c05839_si_003.pdb (1.5MB, pdb)
ao3c05839_si_004.pdb (1.5MB, pdb)

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