Discovery of GLPG2451, a Novel Once Daily Potentiator for the Treatment of Cystic Fibrosis

Steven E. Van der Plas,* Hans Kelgtermans, Oscar Mammoliti, Christel Menet, Giovanni Tricarico,

Abstract

Mutations in CFTR affect various tissues including the lung, pancreas, intestine, reproductive tract, and sweat ducts, making cystic fibrosis a systemic disease. Nevertheless, the most severe effects come from the reduced chloride secretion in the airways. Subsequent lung surface dehydration results in increased thickness of mucus in the lungs, resulting in impaired airway mucociliary clearance, which in turns leads to airway obstruction, chronic bacterial infection, and excessive ion channel. There are currently four approved CFTR modulators available for CF patients, namely the potentiator Ivacaftor (Kalydeco) or VX-770, the correctors Lumacaftor or VX-809, Tezacaftor or VX-661, and Elexacaftor or VX-445.8 For patients homozygous for the F508del CFTR mutation, combination therapies exist that combine one or two corrector molecules with the potentiator Ivacaftor. Patients with gating mutations such as G551D already have sufficient CFTR on the inflammation. Eventually, lung damage leads to a progressive decline in pulmonary function.3

Introduction

More than 2000 mutations of the CFTR gene are known.4 These defects have been grouped in 6 classes based on their defective mechanisms.5 The most common mutation, F508del, belongs to the class II trafficking defects, where folding of CFTR is impaired, resulting in a reduced amount of ion cell membrane and can therefore be treated with Ivacaftor as a mono therapy.9
Recently, we described the discovery of a novel potentiator 2 (GLPG1837).10 Although chemically distinct to Ivacaftor, it was shown that both Ivacaftor and 2 compete for the same binding site.11 Both of these compounds require twice daily dosing strategies to give sufficient exposure of compound in plasma for the full effect of the compounds to be seen in patients. Evaluation of 2 in CF patients with at least one G551D mutation showed that 2 had a significant effect on clinical parameters such as sweat chloride level and most importantly improving the predicted forced expiratory volume (FEV1).12 In this paper, the discovery and optimization of a novel potentiator molecule with pharamacokinetics allowing a once daily dosing regime is described.

■ RESULTS AND DISCUSSION

To measure compound dependent restoration of chloride transport, a CFTR dependent yellow fluorescent protein (YFP) halide potentiator assay (YHA) was developed. A bronchial epithelial cell line derived from a CF patient (CFBE41o-cells) was used to overexpress both F508del CFTR and yellow fluorescent protein.13 To increase the amounts of F508del CFTR at the membrane, cells were incubated at 27 °C, which partially rescues the protein. In other forms of the assay, activity on different class III and IV mutants (eg G551D) was measured in HEK293 cells overexpressing the respective mutant CFTR and YFP. In these assays, CFTR activity is proportional to the quenching of the YFP related fluorescence, and the percentage activation is a measure for the extent or efficacy of channel opening.
The search for novel potentiators started by performing a screen on 589 commercial compounds, selected based on similarity around existing potentiators (Figure 1). From this screen, the most active hits were found around compound 3, a potentiator described by Verkmann et al.14 As already described, optimization of the cyclohexyl ring lead to the discovery of the tetramethyl-THP scaffold from which the clinical candidate 2 was identified. Additionally, it was found that the cyclohexyl ring could also be replaced by a sulfonamide group (Figure 2). Initial exploration of this new series lead to the discovery of 4, which had an EC50̈ of 17 nM on the F508del mutant.
Interestingly, as was the case for 2, compound 4 was also able to potentiate the G551D CFTR channel to a higher extent than 1; the efficacy of 4 in the YHA assay overexpressing G551D CFTR, was determined to be 115% compared to 53% for 1. These data were confirmed in an Ussing chamber experiment using primary human bronchial epithelium cells (HBE) from patients carrying the G551D/F508del mutation (Figure 3).15 Compound 4 was able to induce an increase in current almost twice as high as compound 1. This experiment confirmed that higher efficacies in the YHA could lead to increased response in patient derived cells.
With 4 identified as a lead, a drug discovery optimization campaign was initiated. The clearance of the compounds was optimized using the rat microsomal assay, as rat was the first preclinical species considered for toxicological evaluation. Compound 4 had a high turnover in rat microsomes, however. Replacing the benzylic sulfonamide by a secondary piperidine group (5, Table 1) resulted in a lower clearance. Further improvement in clearance was made by replacing the sulfonamide by a sulfone (6). As typically increasing lipophilicity leads to increased clearance, the results obtained with 6 suggest that the sulfone linker is intrinsically more stable.16
As shown in Table 1, compounds bearing a sulfone or sulfonamide in combination with the pyrazolo amide suffered from low passive permeability and high efflux ratio (ER). Although less potent than 6, compound 7 demonstrated that a saturated substituent could be tolerated (Table 2). The loss in potency was attributed to the lack of an H bond donor and acceptor on the amide. Therefore, several alcohol-bearing amides were explored to address this, resulting in an improvement in potency. Overall, the best combination of potency and clearance were obtained with the substituted glycolic acid derivatives 8 and 9 (Table 2). We anticipated that the alcohol could serve as a potential site for glucoronidation; however, testing of the compound in rat hepatocytes showed no significant difference in clearance to that seen in microsomes.
Interestingly, the passive permeability and especially the efflux ratio for these two compounds differed. This could not be attributed to differences in the physicochemical properties of the molecules. Conformational modeling of the two compounds in the gas phase (better representing the inner part of a phospholipid bilayer) was performed to explain the difference (Supporting Information). This approach suggested that for compound 9 the alcohol could form a hydrogen bond to the carbonyl, effectively shielding the polar centers.18 This internal hydrogen bond was not observed with 8, possibly due to the highly constrained nature of the cyclopropyl ring. Testing in rat pharmacokinetics confirmed the more interesting profile of 9, showing it to have a 10-fold higher exposure than 8.
Having optimized the amide, further fine-tuning of the sulfone group was performed. Whereas the activity on the F508del mutant was used as the main driver for SAR for progression of the series overall, further differentiation between most advanced compounds was done based on the activity on the G551D CFTR. It was found that potency improvement in the latter assay could be obtained by varying the substitution on the phenyl ring of the sulfone. Replacing the fluorine in 9 with a trifluoromethoxy group gave compound 10 which showed a twofold improvement in potency on G511D CFTR. Although this change increased the lipophilicity, 10 still showed low clearance rates in rat and human hepatocytes (Table 3), and evaluation in rat pharmacokinetics showed resulted in a very attractive profile. The total plasma clearance was 0.123 L/h/kg or less than 3% of the liver blood flow in rat, and when correcting for plasma protein binding, the resulting unbound clearance was determined to be 10 L/h/kg. Together with the volume of distribution of 2.7 L/kg, this resulted in an IV half-life of greater than 15 h. When dosed orally at 5 mg/kg, the bioavailability was determined to be 46% and obtained exposure was more than double that for compound 9 at the same dose. In addition to improving the potency on G551D CFTR, the replacement of the fluorine with a trifluoromethoxy group had resulted in a PK profile suited for once daily dosing. Because of the overall interesting profile, compound 10 was characterized further to assess the potential as a preclinical candidate. However, multiple analogues in the series, including 10, were found to be positive in an in vitro micronucleus assay (MNT). Confronted with this setback, it was decided to not initiate in vivo derisking experiments but instead explore new chemical matter devoid of any genotoxic liabilities.
Reflecting on the SAR developed during the project, it was clear that significant knowledge was generated that could be used in any follow up project. During the exploration of the SO2 linked thiophene series more than 450 analogues had been made. Both the left-hand side (SO2 linker) and right-hand side (amide) had been extensively explored. As such, the groups providing good potency were well understood and these points could be fixed. It was therefore decided to start a scaffold hopping exercise to find an alternative series with designs based on these SAR insights (Figure 4). In addition to the SO2 linker and amide groups defined above, we established that the upper carboxamide could be replaced with a group containing at least one hydrogen bond donor. Modeling had shown that due to the extensive intramolecular contacts, the central part could considered to be essentially flat. New scaffolds were then designed that either altered presentation of these features by locking conformations covalently or replaced the thiophene ring with different aromatic structures. In all cases, the sulfone group was used as a fishing lure as this group was considered as a key driver for activity. Synthetically, the sulfone appeared straightforward to install on different scaffolds. Additionally, the interaction of the alcohol was retained via a hydrogen bond donor in that region of the molecule.
A number of different ideas were evaluated, leading to the synthesis of seven different new scaffolds. From this exercise, the most promising result was obtained by replacing the acylated 2-aminothiophene-3-carboxamide with a 3-aminopicolinic amide scaffold. Compound 11 was the first analogue, and it showed a potency of 33 nM on F508del CFTR, together withlow clearance in the rat microsomal assay. Compound 11 can be considered as a matched molecular pair (MMP) of 10, showing that the extensive intramolecular interactions of the acylated 2-aminothiophene-3-carboxamide scaffold could be replaced by the H bonding network present in the 3-amino picolinic amide derivatives, retaining the planarity of the central part of the molecules (Figure 5).
More importantly, the scaffold change resulted in the absence of any genotoxic signal as 11 was found to be negative in both the MNT and AMES in vitro tests. Encouraged by these data, the series was expanded to fully explore its potential.
Several β-amino-alcohols were incorporated on the righthand side (Table 4). In general, these compounds were found to combine a good potency with low clearance in the rat microsomal assay. It seemed that the initial dimethyl substitution of 11 was the least potent seeing that 12, 13 and the cyclopropyl analogue 14 had improved potency on F508del CFTR. When replacing the trifluoromethoxy group on the arylsulfone with a fluorine (15), the lipophilicity was reduced with a subsequent improvement in clearance.
Alkylation of the amide (16) resulted in loss of activity as was expected based on the pharmacophore model. This was further confirmed by 17, but unexpectedly, the azetidine derivative 18 was very potent with an IC50 of 1.1 nM on F508del CFTR. It can be hypothesized that the small and constrained azetidine still allows the molecule to obtain the required flat conformation.
Another interesting result was obtained with compound 19, which has the trifluoromethoxy group in the ortho position of the arylsulfone. This isomer of 12 showed subnanomolar potency, although the intrinsic clearance was higher.
A similar trend was observed upon replacing the aromatic sulfones with saturated sulfonamides, as in the 2-aminothiophene-3-carboxamide series. However, although the desired level of potency could be obtained, the sulfonamides suffered uniformly from a higher clearance (Supporting Information). For example, when comparing 12 with 20 (Table 4), a significant reduction in lipophilicity was obtained; however, the in intrinsic clearance was higher. It was therefore decided to stop exploration of this subseries.
Based on the interesting combination of potency and low clearance, compound 12 was selected for evaluation in rat PK (Table 5). A half-life of >50 h was obtained, which can be linked to the total low clearance and the Vss of 6.0 L/kg. When taking into account the plasma protein binding, the unbound plasma clearance was determined to be 5.2 L/h/kg. This showed that the compound is intrinsically stable, and moreover, the clearance can be well-predicted based on the in vitro data. By the same method, compounds 13 and 15 were shown to have a similar plasma clearance in rat when compared to 12. However, the lower Vss for 13 and 15 reduced the half-lives after IV dosing, which were now 8.9 and >15 h, respectively. Additionally, IV rat PK confirmed the higher clearances for 19 and 20. Whereas the half-life of the latter was suboptimal, compound 19 still had a half-life of 6.7 h. In combination with its subnanomolar potency, this compound had a highly attractive profile.
During the course of the project, multiple compounds beyond those detailed in Table 5 were profiled in rat PK. We were satisfied to note that the scaffold hop from the carboxamide-thiophene series into the 3-amino picolinic amides resulted in a lower unbound plasma clearance (Figure 6 A) for the series as a whole. Importantly, the rat in vivo unbound plasma clearance for the amino picolinic amides was predicted rather well using the in vitro intrinsic clearance in microsomes (Figure 6 B), and the intrinsic human clearance in microsomes followed the same trend as the rat (Figure 6 C).
Targeting the largest patient population in CF (F508del), requires that a potentiator molecule is combined with one or more corrector molecules. For successful combination therapy, it is important to consider potential drug−drug interactions (DDI). Potential interactions with the CYP P450 enzymes are generally considered as being the most relevant interactions,19 therefore the compounds were profiled for CYP inhibition and induction. CYP inhibition was not a general issue for the series. On the other hand, it was noticed that in both the thiophenecarboxamides as the 3-amino picolinic amide subseries, some compounds with CYP induction were present, as determined by the PXR reporter assay.20
As seen in Table 6, the dimethylalcohol side chain bearing compound 11 had moderate PXR activation; however, removing a methyl group to give compound 13 resulted in low PXR activation of only 12% compared to the positive control. Interestingly, compound 12, which contains the trifluoromethyl side chain also had a low PXR activation. However, this compound also suffered from a low kinetic solubility (<19 μM at pH 7.4), rendering the data obtained more challenging to interpret. It was noted that compound 19, which shows a higher kinetic solubility, also showed a significant increase in the PXR activation.
Based on the low CYP induction potential for 13 and its overall interesting profile in terms of potency and rat PK, the compound was characterized further to understand its full potential. The potency was confirmed in a trans-epithelial clamp circuit (TECC)21 assay using HBE cells from patients carrying the F508del mutation. In this assay, an EC50 of 18 nM was obtained.
The compound was found to have thermodynamic aqueous solubility of 83 μM in the Fassif medium and showed good permeability in the MDCK assay. Low intrinsic clearance was obtained in the dog and monkey microsomal assay. The compound did not show any significant CYP inhibition. Confirming the trend observed with previous compounds from the same series, 13 was also negative in both the AMES and micronucleus assay, and no hERG inhibition was measured.
In PK testing (Tables 7 and 8), 13 was found to show slow clearance in dog and monkey, leading to half-lives of 15 or 16 h in the species, respectively. Moreover, bioavailability after oral dose of 5 mg/kg was >90% in both species.
Based on the overall profile, 13 was selected for preclinical safety evaluation and nominated as GLPG2451. After successful completion of this process, it was progressed into a phase I study to evaluate its PK and tolerability in humans. The administered doses ranged from 5 to 80 mg. 13 was generally well tolerated at all doses, and the as-predicted from the in vitro results the pharmacokinetic profile was doseproportional and allows for once-daily treatment. Next, the efficacy of 13 in combination with novel correctors GLPG222222 and GLPG273723 was evaluated in patients being homozygous for the F508del mutation in the FALCON trial (NCT03540524), and the results of this study will be discussed in future papers.

CHEMICAL SYNTHESIS OF COMPOUNDS 10 AND 13

Scheme 1 displays the synthetic route to obtained compound 10. The commercially available 2-aminothiophene-3-carboxamide 21 was first acylated with acetoxyisobutyryl chloride to give 22. Next, a bromine was installed via electrophilic aromatic substitution using NBS. Subsequently, the thioether IV parameters were determined using a 1 mg/kg dose. F% was determined after a PO dose of 1 mg/kg. For dog PK, six animals were used. For monkey PK, three animals were used. 24 was formed using a Pd catalyzed transformation. Oxidation of the thioether bond to a sulfone was performed with hydrogen peroxide, yielding 25. Finally, the alcohol was deprotected in basic conditions to give compound 10.
Scheme 2 outlines the research synthesis of compound 13. The synthesis was performed in 3 steps. To make the thioether bond, 3-amino-5-bromo-pyridine-2-carboxylic acid 26 was reacted with 4-trifluoromethoxy-benzenethiol in the presence of DBU to give 27. Next, oxidation of the thioether to the corresponding sulfone was achieved by adding H2O2 to a mixture of 28 in TFA. Finally, the amide bond with (2S)-1aminopropan-2-ol was formed by using HATU as a coupling agent in the presence of Et3N in NMP.

■ CONCLUSION

A novel potentiator 13 was discovered by replacing the thiophene amide scaffold into a picolinic amide derived series. This scaffold hop was needed to remove the in vitro genotoxic liabilities observed with some of the thiophene derivatives. Although the thiophene amide series already had overall an attractive PK profile, the scaffold hop resulted in a lower clearance and longer half-life. Final optimization was needed to minimize the CYP induction potential as the developed potentiator would be used in combination with other CFTR modulators to enhance the efficacy. Although being derived from the same starting point as 2, compound 13 is chemically distinct and, in addition, has the potential of once daily dosing compared to twice a day regimen for 2 and 1.

■ EXPERIMENTAL SECTION

Yellow Fluorescent Protein Halide Assays (YHA). CFBe41o cells were cultured in Eagle’s minimal essential medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamine, and 500 μg/mL hygromycin B. The cells were grown on culture flasks coated with 0.01% bovine serum albumin (BSA) (Sigma), 30 μg/mL Purecol (Nutacon) and 0.001% human fibronectin (Sigma). CFBe41o cells were transduced with adenoviruses containing F508del CFTR and YFP (H148Q/I152L/F47L). HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) supplemented with 10% FBS and 1% penicillin/streptomycin. HEK293 cells were transfected with plasmids containing G551D, G178R, S549N, R117H, CFTR, and YFP (H148Q/I152L/F47L).
Directly after transfection, the HEK293 cells were seeded in black 96well plates coated with poly-D-lysine at a density of 70 000 cells per well. The next day, cells were incubated for 24 h at 27 °C (CFBE41o) or 37 °C (HEK293). Then, cells were treated for 10 min with 10 μM forskolin and the desired concentration of potentiator at room temperature. The YFP fluorescence was recorded during 7 s (CFBE41o) or 2 min (HEK293), starting immediately before addition of NaI buffer (CFBE41o: 137 mM NaI, 2.7 mM KI, 1.7 mM K H2PO4, 10.1 mM Na2HPO4, 5 mM D-glucose, HEK923: (375 mM NaI, 7.5 mM KI, 1.76 mM KH2PO4, 10.1 mM Na2HPO4, 13.75 mM glucose) to the wells using a fluorescence reader. The capacity of potentiators to increase CFTR channel function was expressed as 1(fluorescence after NaI addition (F)/fluorescence before NaI addition (F0)).
Ussing Chamber Experiments. Ussing chamber experiments were performed using chambers developed by physiological instruments. During the recording, the epithelial cells were kept at 37 °C and bathed on the basolateral side in 4 mL of a NaCl−Ringer solution (120 mM NaCl, 25 mM NaHCO3, 1.2 mM CaCl2, 1.2 mM MgCl2, 0.8 mM KH2PO4, 0.8 mM K2HPO4, 5 mM glucose, pH 7.4) and 4 mL of a low chloride solution on the apical side (120 mM Naglutamate, 25 mM NaHCO3, 1.2 mM CaCl2, 1.2 mM MgCl2, 0.8 mM KH2PO4, 0.8 mM K2HPO4, 5 mM glucose, pH 7.4). Apical amiloride was used to inhibit the endogenous ENaC currents (100 μM) while forskolin (10 μM) was applied on both the apical and basolateral sides to stimulate CFTR. Potentiator compounds were tested after apical and basolateral application. All triggers and compounds used during the experiment were first dissolved in DMSO to a 1000× concentrated solution just prior to treatment. Measurements of the potentiator response were done during a 20 min time frame with recordings every second. The short circuit current (Isc) was measured and the increase in Ieq (ΔIeq, the difference in current before and after potentiator treatment) was used as a measure for the increased CFTR activity. CFTRInh-172 was used at 10 μM to assess the specificity of the response.
Chemistry. All reagents were of commercial grade and were used as received without further purification, unless otherwise stated. Commercially available anhydrous solvents were used for reactions conducted under inert atmosphere. Reagent grade solvents were used in all other cases unless otherwise specified. Column chromatography was performed on silica gel 60 (35−70 μm). Thin layer chromatography was carried out using precoated silica gel F-254 plates (thickness 0.25 mm). 1H NMR spectra were recorded on a Bruker DPX 400 NMR spectrometer (400 MHz) or a Bruker Advance 300 NMR spectrometer (300 MHz). Chemical shifts (δ) for 1H NMR spectra are reported in parts per million (ppm) with the appropriate residual solvent peak, i.e. CHCl3 (δ 7.27), as internal reference. Multiplicities are given as singlet (s), doublet (d), triplet (t), quartet (q), quintuplet (quin), multiplet (m), and broad (br). Microwave heating was performed with a Biotage Initiator.
Ultraviolet and electrospray MS spectra were obtained on a Waters Acquity H-Class UPLC which was coupled to a Waters Mass detector QDA or ZQ2000 and an Acquity PDA Detector (210−400 nm). All final compounds reported were analyzed using one of these analytical methods and were at least 95% pure. Columns used: Waters Acquity UPLC BEH C18 1.7 μm, 2.1 mm ID × 50 mm L or Waters Acquity UPLC CSH C18 2.1 × 50 mm 1.7 μm. The methods are using MeCN/H2O gradients (H2O contains either 0.1% TFA or 15 mM NH3).
Racemic mixtures were separated on an Agilent HP1100 system with UV detection. Column used: Chiralpak 1A (10 × 250 mm, 5 μm). Solvents used: iPrOH and tBME. Alternatively, separation was done using a SFC2 system. Column used: Lux Cellulose-4. Solvents used: CO2 and MeOH. Enantiomeric purity was determined on an Agilent HP1100 system with UV detection. Column used: Chiralpak IA (4.6 × 250 mm, 5 μm). Solvents used: iPrOH and tBME.
1-((3-Carbamoylthiophen-2-yl)amino)-2-methyl-1-oxopropan2-yl acetate (22). 2-Aminothiophene-3-carboxamide (3.9 g, 27.4 mmol) was taken up in DCM (45 mL); pyridine (3.77 mL, 45.8 mmol) was added, and the mixture was cooled to 0 °C. Next, a solution of 2-acetoxyisobutyryl chloride (5 g, 30.5 mmol) in DCM (25 mL) was added dropwise. After complete addition, the resulting mixture was stirred at room temperature. After 2 h, the reaction was quenched with water, and the organic layer was separated. After drying the organic layer with Na2SO4, it was concentrated, affording a white solid that was further triturated with water. Freeze-drying of the resulting white solid yielded (23) (6.89 g, 93% yield). LC−MS: m/z 254.0 [M − NH3]+.
1-((5-Bromo-3-carbamoylthiophen-2-yl)amino)-2-methyl-1-oxopropan-2-yl acetate (23). Compound 23 (4.05 g, 15.0 mol) was taken up in acetic acid (60 mL) and stirred at room temperature. Next, NBS (2.8 g, 15.75 mmol) was added portionwise. After 1 h, the suspension was diluted with water and subsequently extracted with EtOAc. The combined organic layers were washed with saturated Na2CO3, dried over Na2SO4, and concentrated to afford 23 (5.12 g, 98% yield) that was used as such in the next step. LC−MS: m/z 349− 351 [M + H]+.
1-((3-Carbamoyl-5-((4-(trifluoromethoxy)phenyl)thio)thiophen2-yl)amino)-2-methyl-1-oxopropan-2-yl acetate (24). Compound 23 (250 mg, 0.72 mmol) was taken up in dioxane (3 mL) in a microwave vial. 4-trifluoromethoxy-benzenethiol (181 mg, 0.93 mmol), Pd(OAc)2 (8.1 mg, 0.036 mmol), DiPPF (9.7 mg, 0.043 mmol), and NaOtBu (83 mg, 0.86 mmol) were added. The reaction mixture was flushed with nitrogen and the vial was closed and heated at 100 °C. After 16 h, the reaction mixture was filtered through a silica plug that was rinsed with EtOAc. The obtained filtrate was reduced, affording 24 that was used as such in the next step. 1H NMR (400 MHz, DMSO-d6) δ 12.86 (s, 1H), 7.98 (s, 1H), 7.75 (s, 1H), 7.62 (s, 1H), 2.09 (s, 3H), 1.59 (s, 6H). LC-MS: m/z= 463 [M + H].
1-((3-Carbamoyl-5-((4-(trifluoromethoxy)phenyl)sulfonyl)thiophen-2-yl)amino)-2-methyl-1-oxopropan-2-yl acetate (25). Compound 24 (330 mg, 0.72 mmol) was taken up in acetic acid (10 mL). H2O2 (0.25 mL, 2.88 mmol) was then added, and the reaction was stirred at 60 °C. After 3.5 h, the mixture was diluted with water and subsequently extracted with EtOAc. The combined organic layers were washed with Na2CO3, dried over MgSO4, and concentrated to afford 25 as a crude solid (360 mg) that was used as such in the next step. LC-MS: m/z = 495 [M + H]. 2-(2-Hydroxy-2-methylpropanamido)-5-((4-(trifluoromethoxy)phenyl)sulfonyl)thiophene-3-carboxamide (10). Compound 25
(360 mg, 0.72 mmol) was mixed with K2CO3 (200 mg, 1.44 mmol) in MeOH (10 mL). The resulting mixture was stirred at 60 °C. After 15 h, the reaction was diluted with water and extracted with EtOAc. The combined organic layers were dried over MgSO4 and concentrated to give a crude that was purified by preparative HPLC, yielding compound 10 (22 mg, 6.7% yield). 1H NMR (DMSO-d6): 12.96 (s, 1H), 8.36 (s, 1H), 8.31 (br. s, 1H), 8.06 (d, 2H), 7.74 (br. s, 1H), 7.64 (d, 2H), 6.15 (s, 1H), 1.34 (s, 6H). LC-MS: m/z = 453 [M + H]+.
3-Amino-5-(4-trifluoromethoxy-phenylsulfanyl)-pyridine-2-carboxylic acid (27). A solution of 3-amino-5-bromo-pyridine-2carboxylic acid (3.26 g, 15 mmol), 4-trifluoromethoxy-benzenethiol (3.5 g, 18 mmol), and DBU (2.22 mL, 15 mmol) was prepared in DMA (15 mL). This mixture was heated at 140 °C for 45 min in the MW. Next, the mixture was diluted with a mixture of 1% AcOH in water. A suspension was obtained that was subsequently filtered to give a precipitate. This precipitate was washed with a 1% AcOH/ water mixture followed by washing with petroleum ether. After drying in a vacuum oven, 27 (2.75 g, 70% yield) was obtained. LC-MS: m/z = 331 [M + H]+.
3-Amino-5-(4-trifluoromethoxy-benzenesulfonyl)-pyridine-2carboxylic acid (28). Compound 27 (2.75 g, 10.5 mmol) was dissolved in TFA (20 mL), and the resulting mixture was cooled at 0 °C with an ice bath. Next, H2O2 (3.7 mL, 42 mmol) was added, and the mixture was stirred at 0 °C until the reaction was finished. For the work up, the mixture was diluted with a mixture of 1% AcOH in water. A suspension was obtained that was subsequently filtered to give a precipitate. This precipitate was washed with 1% AcOH/water mixture followed by washing with petroleum ether. After drying in a vacuum oven, 28 was obtained (2.76 g, 90% yield). LC-MS: m/z = 363 [M + H]+.
3-Amino-N-[(2S)-2-hydroxypropyl]-5-[4-(trifluoromethoxy)phenyl]sulfonyl-pyridine-2-carboxamide (13). Compound 28 (50 mg, 0.14 mmol) was taken up in N,N-dimethylacetamide (552 μL). (2S)-1-Aminopropan-2-ol (10 mg, 0.14 mmol), HATU (5 mg, 0.14 mmol), and triethylamine (39 μL, 0.28 mmol) were subsequently added. The reaction mixture was stirred at room temperature for 15 h. The reaction mixture was diluted with water, and the resulting mixture was extracted with EtOAc. The combined organic layers were dried over MgSO4 and concentrated to give an oil. After purification by preparative HPLC, compound 13 was obtained (15 mg, 26%). 1H NMR (DMSO-d6): δ 8.39 (t, 1H), 8.24 (d, 1H), 8.13 (d, 2H), 7.74 (d, 1H), 7.65 (d, 2H), 7.27 (br.s, 2H), 3.75 (m, 1H), 3.13 (m, 1H), 1.03 (d, 3H). LC-MS: m/z = 420 [M + H]+.

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(21) The TECC instrument is developed and sold by EP Design (Bertem, Belgium) and measures the potential difference (PD) and transmembrane resistance (Rt) over epithelial cells, and these values are used to calculate the apparent short circuit current (Ieq) using Ohms law. The Ieq can be interpreted as the charge flow per time when the tissue is short circuited, i.e. the transepithelial voltage Vt is clamped to 0 mV and changes when ion channels in the apical or basolateral membrane of epithelial cells are opened or closed.
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