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

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


■ INTRODUCTION
Cystic fibrosis (CF) is the most frequent life-threatening autosomal recessive disease in Caucasians, caused by loss-offunction mutations in the CF transmembrane conductance regulator (cftr) gene, encoding for CFTR protein. 1,2 CFTR is a cAMP-regulated chloride channel expressed at the apical membrane of epithelial cells, where it provides a route for electrogenic anion flux, thus regulating the composition and volume of epithelial secretions. 3,4 CF is a multiorgan disease, affecting the lungs, pancreas, liver, and other organs. 2 More than 2000 mutations have been described in the cftr gene; however, the pathogenicity has been demonstrated only for approximately 300 mutations. 5 CF mutations cause the loss of function of CFTR protein by affecting its synthesis, trafficking, or its function as an anion channel. 6 According to the mechanism causing CFTR dysfunction, CF mutations have been grouped into seven different classes: mutations introducing a premature stop codon (class I), mutations causing protein misfolding (class II), mutations causing defective channel gating (class III), mutations causing defective channel conductance (class IV), mutations leading to aberrant mRNA splicing (class V), mutations causing reduced stability at the plasma membrane (class VI), and mutations resulting in no mRNA expression (class VII). 7 Despite this clear classification, the majority of CF mutations cause CFTR dysfunction by multiple mechanisms, 8 as in the case of the deletion of phenylalanine 508 (F508del), the most frequent mutation among CF patients. 9 Indeed, F508del causes a folding defect, leading to premature protein degradation. 10,11 In addition, when F508del-CFTR is forced to traffic to the plasma membrane, for example, by rescue maneuvers, the mutant protein shows reduced stability because of the peripheral protein quality control mechanisms 12 and defective channel gating. 13,14 Other mutations, however, are associated with a single mechanism of CFTR dysfunction, as in the case of the class III mutation G551D, which causes a severe CFTR channel gating defect. 15 Druglike small molecules, known as "CFTR modulators", can target these specific defects caused by CFTR mutations restoring, at least partially, the CFTR function. 16 The maturation defect can be rescued by small molecules called correctors, such as VX-809 17 or VX-661, 18 while the gating defect can be corrected by small molecules called potentiators, such as VX-770 ( Figure 1). 19 Together with VX-770, other small-molecule potentiators have been reported ameliorating gating defects of mutant CFTR. 20−23 However, only a few have progressed to the stage of evaluation in clinical trials. Among them, the corresponding nona-deuterated (VX-561) 24 and bis-(trimethylsilyl) (PTI-808) 25 analogues of VX-770 or AbbVie-Galapagos GLPG-1837 26,27 and GLPG-2451 27 compounds have successfully entered clinical trials in CF patients with different gating mutations ( Figure 1).
Presently, VX-770 is the only potentiator drug that has been approved for monotherapy of different mutants displaying a gating defect. 28 Aiming to add new small-molecule compounds to the existing classes of CFTR potentiators, expanding the portfolio of modulators available to CF patients, we embarked on a drug discovery effort to the identification of novel chemotypes endowed with a promising pharmacological profile. After a highthroughput screening (HTS) campaign, few structurally diverse small-molecule hits were identified; among them, the most promising ones shared the 2,3,4,5-tetrahydro-1H-pyrido [4,3b]indole (or 1,2,3,4-tetrahydro-γ-carboline) core structure.
In this work, we disclose the identification and an extended structure−activity relationship (SAR) study of tetrahydro-γcarboline derivatives, which led to the discovery of novel CFTR potentiators, characterized by a nanomolar activity.

■ RESULTS AND DISCUSSION
A screening collection of 11,334 maximally diverse smallmolecule compounds was assembled starting from a set of ca. 300,000 commercially available molecules belonging to the diversity subsets of major vendors. A series of more and more stringent filters were applied in order to discard compounds with suboptimal druglike properties, and those containing chemically reactive moieties, unstable and known cytotoxic groups, and frequent hitters (e.g., PAINS). 29−32 A subsequent stepwise clustering protocol based on an unweighted pair group method with arithmetic mean (UPGMA) hierarchical agglomerative algorithm 33 allowed for the selection of the final set of molecules suited for HTS.
The chemical library was screened in duplicate on a Fischer rat thyroid (FRT) cell line, stably coexpressing F508del-CFTR and the halide-sensitive yellow fluorescent protein, HS-YFP. 23,34,35 To overcome the F508del trafficking defect, cells were initially incubated for 24 h at 32°C and then acutely treated (15−30 min) with a single compound (5 μM) in combination with forskolin (10 μM). After stimulation, the F508del-CFTR activity in the plasma membrane was calculated by measuring the rate of fluorescence quenching arising from iodide influx. 34 This activity was compared to that of wells containing forskolin alone (negative control) or forskolin plus VX-770 (1 μM, as a positive control) (Figure 2A). Analysis of screening performance with the Z′ method gave a score of 0.6, which can be considered optimal for this type of assay. The activity scores for each compound were then plotted as an ordered distribution ( Figure 2B). All compounds whose average activity calculated from the two rounds of screening was greater than 185% with respect to the negative control (i.e., forskolin alone) were considered as primary hits. The screening detected 104 putative potentiators, with some compounds showing a promising initial activity comparable to VX-770 ( Figure 2B).
The primary hits were confirmed by testing them at different concentrations, in order to extrapolate the dose−response relationship. First, we focused on the F508del mutant. The compounds were tested in the low micromolar range, in the presence of forskolin (10 μM), on F508del-CFTR FRT cells following rescue of the trafficking defect by low-temperature incubation, as done for the primary screening. The data for selected test compounds are shown in Figure 3A. The most interesting hits were subsequently tested for their ability to overcome the more severe gating defect of pure class III mutant using FRT cells stably expressing G551D-CFTR ( Figure 3B).  Interestingly, some compounds were effective on both types of mutants with a strong increase in CFTR activity that in some cases approached the effect of VX-770 ( Figure 3A,B).
This finding, besides being a promising preliminary data in this screening campaign, confirmed that this specific chemotype  (2), and Hit-9 (3), selected for further SAR evolution. The data are expressed as mean ± standard deviation (SD) (n = 9; from three independent experiments, each one having three biological replicates). Statistical significance was tested by parametric analysis of variance (ANOVA), followed by the Dunnett multiple comparisons test (all groups against the control group). Symbols indicate statistical significance vs control (DMSO-treated): ***p < 0.001, **p < 0.01, and *p < 0.05. , on F508del-CFTR CFBE41o-cells rescued at 32°C for 24 h. The data are expressed as mean ± SD (n = 9; from three independent experiments, each one having three biological replicates). Statistical significance was tested by parametric ANOVA, followed by the Dunnett multiple comparisons test (all groups against the control group). Symbols indicate statistical significance vs control (DMSO-treated): ***p < 0.001 and **p < 0.01. (B) Representative traces show the response of CFTR to stimulation with the indicated concentrations of CPT-cAMP and compound 3. The currents stimulated by compound 3 and CPT-cAMP were blocked by the selective CFTR inhibitor-172. Each experimental condition was tested in three independent experiments, each one performed with three biological replicates. could be considered a reliable starting point to further evolve a newly identified chemical class. In particular, derivatives 1 and 3 showed a good efficacy and an interesting sub-micromolar potency ( Figure 3).
The preliminary activity of these three hits was further evaluated in secondary screening assays. Although featuring the same common scaffold, in order to possibly discriminate the most interesting hit for further structural investigations, the compounds belonging to the family of tetrahydro-γ-carbolines were tested on more relevant cell models. First, the hits were tested on an immortalized bronchial epithelial cell line (CFBE41o-) stably expressing F508del-CFTR and HS-YFP. Acute stimulation with the compounds, in the presence of forskolin to increase the intracellular cAMP content, resulted in a dose-dependent activation of mutant CFTR ( Figure 4A), with compounds 1 and 3 being the most effective.
Subsequently, the ability of compounds to elicit CFTRmediated chloride secretion was verified on primary human bronchial epithelial (HBE) cells, from non-CF individuals, in short-circuit current experiments. In this respect, it should be noticed that potentiators are also able to stimulate wild-type CFTR, provided that a submaximal cAMP-dependent stimulation is applied. 20,22 Accordingly, cells were stimulated with a submaximal concentration of the cAMP analogue, CPT-cAMP, followed by increasing concentrations of the potentiator hit. 20,22 In such experimental conditions, compound 3 was able to stimulate CFTR-mediated chloride current of similar amplitude as that elicited by maximal cAMP stimulation, as confirmed by using the specific CFTR inhibitor-172 ( Figure 4B).
Based on these initial, promising findings achieved in HBE experiments, compound 3 was selected as the reference molecule to explore the SAR around this chemotype, aiming to improve potency and efficacy of this novel class of CFTR potentiators. The SAR study focused on the investigation of the role of the heteroaromatic carboxylic acid (A) acylating the position N 2 of the tetrahydro-γ-carboline, the substitution pattern of the phenyl ring (B), and the modification of the tetrahydropyridine portion (C) ( Figure 5).
The role of the heteroaryl group (A) acylating the position 2 of the tetrahydro-γ-carboline was initially explored by modifying the substituent on the pyrazolyl residue and replacing the pyrazole with other rings, while keeping the 8-methoxy carboline moiety unmodified. The activity of each compound is described in terms of normalized maximal efficacy (E max ), the maximum fold increase in the rescue of F508del-CFTR activity with respect to hit 3, and potency (EC 50 ), the concentration producing half-maximal efficacy. The chemical structures and the data of the first set of compounds are reported in Table 1.
Having demonstrated the importance of the 5′-trifluoromethyl-pyrazol-3′-yl residue as an ideal acylating group of the 8methoxy-tetrahydro-1H-pyrido [4,3-b]indole derivative in targeting the gating defects caused by CF mutations, the importance of the substitution pattern on the phenyl ring (B, Figure 5) was investigated by the synthesis of a number of phenyl-substituted analogues (Table 2).
Having demonstrated that position 8 of the γ-carboline moiety could affect positively both efficacy and potency, a number of new analogues of compounds 3 (8-OMe) and 14 (8-Me) were synthesized in order to gain additional information on the SAR of this class. The investigation of the importance of the substituent at position 8 was broadened by preparing derivatives where the methoxy residue was replaced by an isopropyl (18), a fluoro (19), a trifluoromethyl (21), a trifluoromethoxy (22), a cyano (23), and a methylsulfonyl (24) moiety. While compounds 19 and 21 showed potency and efficacy comparable to 3, a relevant drop in the overall activity was observed with derivatives featuring a sterically demanding isopropyl (18) and methylsulfonyl (24) groups or the polar linear cyano residue (23). On the contrary, the 8-trifluoromethoxy (22) derivative showed improved potency (EC 50 : 0.16 μM) and a comparable normalized efficacy (E max = 0.89) with respect to 3. Interestingly, compound 20, bearing a fluorine atom at position 6, turned out to be the most potent analogue within this small set of monosubstituted γ-tetrahydro-carbolines, displaying a double-digit nanomolar potency (EC 50 = 0.096 μM), while retaining a similar efficacy (E max = 0.96) to analogue 3.
As a further step in the exploration of SAR within this chemotype, disubstituted phenyl derivatives were also explored. Based on the promising effect shown by 20 in dose−response data in F508del-CFTR FRT cells, a small set of disubstituted compounds bearing a fluorine atom at position 6 was prepared and tested. The introduction of a second substituent on the phenyl ring proved in general to be beneficial, leading to some compounds with efficacy comparable or superior to 3 and potency in the double-digit nanomolar range. Trying to possibly gain an additive effect by introducing previously identified substituents, the influence on the activity of 6-fluoro (as in 20) was combined with a 8-methoxy, a 8-methyl, and a 8-fluoro group, as in disubstituted analogues 25, 26, and 27 (Table 2). Unfortunately, these modifications turned out to be not so beneficial in terms of overall activity, showing in all cases a significant drop in potency when compared to monosubstituted analogue 20. A similar effect was displayed by the insertion of a trifluoromethyl group at position 8, as in 28, which caused a marked 6-fold drop in potency (EC 50 : 0.57 μM) with respect to 20 (Table 2).
While retaining a fluorine atom at position 6, as in 20, the SAR study around this scaffold was further expanded by modification of the substitution pattern on the phenyl ring of the tetrahydroγ-carboline core. Accordingly, whereas the insertion of another fluorine in position 9 (29) negatively influenced the potency (EC 50 = 0.15 μM), the replacement of a hydrogen with a methyl group in the same position resulted in a double-digit nanomolar active disubstituted derivative 30 (EC 50 = 0.06 μM), with more than 6-fold increase in potency and comparable efficacy to hit 3.
To firmly prove that the 6-fluoro-9-methyl disubstitution pattern, as in 30, was convenient to maintain the activity, we swapped the position of fluorine and methyl group, leading to compound 31. This modification resulted to be detrimental for efficacy and led to more than 15-fold decrease in potency (EC 50 = 0.94 μM) ( Table 2).
As the next step in the exploration of SAR of the class, modifications in the tetrahydropyridine ring (C, Figure 5) were investigated, retaining the optimal 6-fluoro-9-methyl substitution on the phenyl ring. Remarkably, the introduction of a methyl group at position 1 or 3 of the tetrahydropyridine ring, as in racemic 32 and 33, resulted in a considerable boost in activity, as shown by the good efficacy and low double-digit nanomolar potency (EC 50 = 0.03 and 0.022 μM, respectively) ( Table 3). On the contrary, the insertion of a gem-dimethyl moiety at 3position of the tetrahydropyridine ring, as for compound 34, negatively affected both potency (EC 50 = 1.91 μM) and efficacy (E max = 0.79) with respect to derivative 30.
A more sterically demanding modification of the γ-carboline nucleus was explored by incorporating an ethylene bridge in the tetrahydropyridine ring, as for racemic compound 35. This modification leads to a more rigid skeleton, possibly resulting in increased affinity at the target binding site. 36,37 Unfortunately, this structural change was detrimental for both efficacy and potency with respect to 30. The separation of the racemate into the two pure enantiomers did not result in any improvement because only one of them (37) retained some activity, whereas the opposite isomer 36 was completely inactive (Table 3).
Trying to evaluate the effect on the activity of the size of the heterocyclic ring, the corresponding tetrahydro-azepino-indole derivative 38 was synthesized and tested; the compound showed a marked 38-fold drop in potency with respect to the tetrahydropyridine analogue 30.
Based on the promising data in improving the gating of mutant F508del-CFTR in FRT cells shown by racemic tetrahydro-γ-carbolines 32 and 33, the corresponding pure enantiomers were synthesized and tested. Notably, a strong difference (>100-fold) in the biological activity was displayed by these chiral analogues. While (S)-enantiomer 40 (distomer) resulted in a marked loss in potency but similar efficacy (E max : 1.31, EC 50 : 1.1 μM) to racemic 33, the corresponding (R)enantiomer 39 (eutomer) showed a comparable activity with respect to racemate, retaining a low double-digit nanomolar potency (EC 50 : 0.017 μM) ( Table 3).  Figure 3A. Data are expressed as mean ± SD (n = 3−6). c Racemic compound. d Absolute configuration not determined and arbitrary drawn. e Absolute configuration known. f n.a.: not active (up to 20 μM).
A slightly different pattern of activity was observed with the 1methyl-substituted enantiomers 41 and 42, which showed no major differences in terms of efficacy and potency, also when compared to racemic 32.
Within this small set of alkyl-substituted analogues, the results observed with compounds 41 and 42, along with the likelihood of 1-alkyl substituted γ-carboline analogues possibly undergoing epimerization in acidic aqueous media, 38 convinced us to convey our attention primarily on compound 39.
A small set of selected potentiators with good efficacy and potency on F508del-CFTR FRT cells ( Figure 6A) was also tested at three concentrations on G551D-CFTR FRT cells Figure 6. Activity of selected tetrahydro-γ-carboline potentiators (3, 20, 25, 30, and 39) and, for comparison, VX-770 on (A) F508del-CFTR FRT and (B) on G551D-CFTR FRT cells. The data are expressed as mean ± SD (n = 9; from three independent experiments, each one having three biological replicates). Statistical significance was tested by parametric ANOVA, followed by the Dunnett multiple comparisons test (all groups against the control group). Symbols indicate statistical significance vs control (DMSO-treated): ***p < 0.001, **p < 0.01, and *p < 0.05. . The data are expressed as mean ± SD (n = 9; from three independent experiments, each one having three biological replicates). Statistical significance was tested by parametric ANOVA, followed by the Tukey test (for multiple comparisons). Symbols indicate statistical significance: ***p < 0.001, n.s. (not significant) indicates p > 0.05.
( Figure 6B). With the only exception of compound 3, the potentiators displayed efficacy comparable to VX-770 at the highest concentration (20 μM), with analogue 39 being the most potent one, although the affinity was lower than that of VX-770.
In F508del-CFTR FRT cells, the selected potentiators displayed efficacy comparable to VX-770. Interestingly, enantiomer 39 resulted to be the most potent analogue within this set of novel tetrahydro-γ-carbolines, although showing a slightly lower activity than VX-770 when tested at 10 and 1 nM ( Figure 6A). For these selected compounds, a very similar trend was also observed in G551D-CFTR FRT cells, where 39 showed a comparable pattern of activity at the highest concentrations (20−3.3 μM) and a decrease in activity at 0.56 μM when compared to VX-770 ( Figure 6B).
Recent studies have shown that most potentiators have an undesired activity on F508del-CFTR protein processing/ trafficking. 39,40 According to these studies, chronic incubation with potentiators results in decreased activity of VX-809 as a corrector. To test the effect of our potentiators, we incubated F508del-CFTR CFBE41o-cells for 24 h with tetrahydro-γcarboline 39 (5 μM) together with VX-809 (1 μM) and ARN23765 (10 nM), the picomolar affinity corrector recently reported by our research team. 35 Interestingly, cotreatment with potentiator 39 did not affect the rescue efficacy of VX-809 or ARN23765 ( Figure 7A).
A similar combination study was conducted on primary HBE cells from an F508del/F508del CF patient. The effect of the compound was assessed with the transepithelial electrical resistance and potential difference (TEER/PD) technique. 41 Epithelia were treated for 24 h with a vehicle, a corrector ARN23765 alone (10 nM), or ARN23765 (10 nM) plus the potentiator 39 (0.5 μM). TEER and PD values were taken at resting, after the addition of apical amiloride, after maximal stimulation of F508del-CFTR activity (with forskolin plus genistein), and after blocking with PPQ-102. For each epithelium, we measured the difference in the short-circuit current, I eq (calculated from the TEER and PD values), before and after blocking with PPQ-102 (ΔI eq ). The results obtained on primary epithelia confirmed that the rescue of CFTR activity by the corrector ARN23765 was not affected by coincubation with the novel potentiator 39 ( Figure 7B).
Finally, potentiator 39 and VX-770 were further evaluated in primary HBE cells from non-CF individuals, using short-circuit current measurements, to assess the ability of compounds to maximally activate CFTR function in the presence of a . The data are expressed as mean ± SD (n = 9; from three independent experiments, each one having three biological replicates). Statistical significance was tested by parametric ANOVA, followed by the Dunnett multiple comparisons test (all groups against the control group). Symbols indicate statistical significance: **p < 0.01 and *p < 0.05.  submaximal cAMP stimulation. After the addition of amiloride to inhibit sodium absorption through the ENaC channel, the potentiators were added at the maximal effective concentration (1 μM) after which epithelia were stimulated with a submaximal concentration of CPT-cAMP (5 μM), followed by a maximal concentration of the cAMP analogue (100 μM) ( Figure 8). We then measured the extent of CFTR-mediated chloride current elicited by submaximal cAMP stimulation in the presence of a potentiator and compared it to total CFTR-mediated chloride current, determined as the current inhibited by CFTR inhibitor-172. In parallel, other epithelia were stimulated only with the two concentrations of the cAMP analogue, in the absence of a potentiator. Interestingly, the fraction of CFTR-mediated chloride current activated by submaximal cAMP stimulation was significantly increased by more than 2-fold when epithelia were prestimulated with a potentiator (Figure 8). In this respect, VX-770 and compound 39 were similarly effective ( Figure 8B). The most interesting potentiators (3,20,25,29,30, and 39), selected on the basis of their efficacy and potency in the HS-YFP assay on F508del-CFTR FRT cells, were profiled in vitro for their druglike properties. Kinetic solubility and metabolic stability in rat, dog, and human liver microsomes, in the presence of NADPH and UDPGA (only for human) as cofactors, were assessed along with an indication of potential for hepatotoxicity in HepG2 cells (Table 4).
In general, the selected compounds showed a low kinetic solubility (<40 μM), with the exception of the hit 3, which exhibited high solubility (237 μM). The metabolic stability (phase I metabolism) in the presence of liver microsomes was generally quite good in both dog (t 1/2 > 60 min) and human (t 1/2 > 55 min), with the exception of compounds 3 and 25, featuring both a methoxy group on the phenyl ring. Indeed, although quite promising in terms of overall activity (Table 2), derivative 25, along with hit 3, turned out to be the least stable compounds among the selected novel potentiators. However, their metabolic stability to oxidative metabolism was generally higher in the presence of human liver microsomes compared to rat (Table 4). Tetrahydro-γ-carbolines 30 and 39 turned out to be quite stable to phase II conjugation reactions, showing half-life values (t 1/2 > 60 min) in human liver microsomes, with UDPGA as a cofactor. In addition, a considerable amount (>75%) of parent compound remained at the last time point (Table 4).
In order to assess a possible liver toxicity liability, the selected analogues were also tested in HepG2 cells at two concentrations (2.0 and 20 μM) for 24 h, along with a reference compound (rotenone). A reduction of cell viability to less than 80% was set as the threshold for estimation of cytotoxicity. 42 None of the compounds induced a decrease in cell viability at the highest dose tested (20 μM), resulting in a cell survival >80% (Table 4).
Based on both its biological profile, showing a good efficacy in primary and secondary assays, and preliminary in vitro ADME properties, potentiator 39 was further evaluated in in vivo studies. The compound was dosed in Sprague−Dawley rats by intravenous (i.v.) administration, at a dose of 3 mg/kg, and by oral gavage (p.o.), at a dose of 10 mg/kg, to determine its pharmacokinetic profile (Table 5).
After i.v. administration, the maximal plasma concentration was ca. 7.3 μM, with a volume of distribution (V d ) of 2.39 L/kg, indicating an overall good distribution to the tissues. The low clearance (19 mL/min/kg) was in accordance with the good stability of 39 as shown in the rat liver microsomal stability assays. After oral administration, the compound reached the maximal plasma concentration at 2 h, and considerable levels of compound (>450 ng/mL, corresponding to a ca. 1.2 μM) were still present at 6 h postadministration, showing a relatively slow elimination phase (see Figure S1, Supporting Information). The exposure (area under the curve, AUC) over the time interval 0− 4 h was 6.1 μM·h, resulting in a ca. 30% oral bioavailability, calculated over the same time interval.
Taking into account both the encouraging results in rescuing the gating defect in mutant CFTR in both primary and secondary biological assays, and the promising in vivo PK profile, we quantified the amount of compound 39 in rat lung tissue, the main target organ for CF treatment. The lung tissue distribution of potentiator 39 was investigated following administration of a single oral dose of 10 mg/kg to Sprague−Dawley rats. Supported by the data obtained in the pharmacokinetic study after oral administration, two time points (2 and 4 h) were selected for collecting plasma and lung tissue samples. The mean concentration vs. time profiles of compound 39 in plasma and lung tissue are reported in Figure S2 (see the Supporting Information). In this study, while the plasma levels at 2 and 4 h (1026 and 675 ng/mL, respectively) turned out to be in accordance with those observed in the PK experiment, the concentration of 39 in the lungs was quantified to be 4.5 and 2.2 ng/mg tissue at the two selected time points. Overall, this study demonstrated that tetrahydro-γ-carboline 39 distributed to the lung following oral administration, although with a low concentration.
Finally, based on both its biological and pharmacological profile, potentiator 39 could be fairly considered as a lead compound and a valuable starting point for further optimization of 1,2,3,4-tetrahydro-γ-carbolines as novel F508del-CFTR potentiators.
Enantiomerically pure analogues 36 and 37 were obtained by semipreparative chiral separation starting from racemic 35. The reaction with (R)-and (S)-2-methyl-piperidin-4-ones 50 and 51 afforded the corresponding methyl-substituted tetrahydropyridoindoles, as mixtures of 1-methyl and 3-methyl regioisomers. Chromatographic purification allowed to obtain pure regioisomers, which upon amide coupling with pyrazolyl carboxylic acid led to the desired substituted compounds. Whereas 3-methyl analogues (R)-39 and (S)-40 were afforded  enantiomerically pure from the corresponding chiral intermediates 54 and 55, a careful investigation revealed for 1-methyl substituted γ-carbolines a complete racemization of the stereogenic center, probably because of the relatively acidic character of allylic proton in C1-position, allowing a enamine− imine equilibrium during Fischer indole synthesis. 38 Therefore, the corresponding 1-methyl-substituted compounds (S)-41 and (R)-42 were isolated as enantioenriched compounds after semipreparative chiral separation starting from racemic 32 (Scheme 3).
Compound 38, featuring a substituted tetrahydroazepinoindole scaffold, was synthesized starting from Boc-protected azepan-4-one 60 with an analogous approach, 48 as seen for the previously described tetrahydropyridine-indole analogues, though slightly forcing the reaction conditions (Scheme 4). Probably because of conformational constraints, Fischer indole synthesis of hydrazine 44a with ketone 60 led only to hexahydroazepino-indole 61 with complete regioselectivity. The intermediate 61 underwent classical amide coupling with 5-trifluoromethyl-1H-pyrazole-3-carboxylic acid (C) to obtain final compound 38.

■ CONCLUSIONS
CF, the most frequent autosomal recessive disease, is a multiorgan disease, primarily affecting the lungs. CF mutations cause CFTR protein dysfunction by multiple mechanisms, affecting its expression, stability, or its function as an anion channel. CFTR modulators have been reported to address the basic defects caused by CF mutations restoring, at least partially, the CFTR function. In particular, small-molecule compounds, called potentiators, are known to ameliorate the gating defect.
In the present work, we describe the identification of a novel chemotype of CFTR potentiators. The screening of a library of compounds provided a few hits featuring a common 2,3,4,5tetrahydro-1H-pyrido[4,3-b]indole core, which were able to rescue the activity of F508del-and G551D-CFTR in an effective manner.
1,2,3,4-Tetrahydro-γ-carbolines represent a chemical class of well-studied heterocycles, extensively characterized for their chemical and biological properties; therefore, as reported by Ivashchenko,49 such tricyclic compounds could be regarded as typical "privileged structures". 50 The sustained interest in this scaffold is due to the fact that tetrahydro-γ-carbolines and their derivatives have shown a broad spectrum of biological activities, 49,51−53 primarily for the treatment of central nervous system diseases. 36,54−56 Although in the last few years substituted derivatives of tetrahydro-γ-carbolines have been reported to be active toward different biological targets, 52 to the best of our knowledge, this type of heterocyclic small molecules has never been described as pharmacologically active compounds for the treatment of CF or related conditions. The initial hits were validated and further explored in SAR studies, leading to the discovery of novel potentiators active in the mid-to-low nanomolar range. Among them, the enantiomerically pure compound 39 turned out to be quite promising being able to rescue the gating defect of both F508del-CFTR (in FRT and CFBE41o-cells) and G551D-CFTR (in FRT cells) with good potency and efficacy, similarly to VX-770. Notably, the tetrahydro-γ-carboline 39 did not affect the rescue efficacy of correctors VX-809 or ARN23765 in immortalized bronchial CFBE41o-cells and in primary HBE cells from an F508del/ F508del CF patient and increased by more than 2-fold the fraction of CFTR-mediated chloride current activated by submaximal cAMP stimulation in HBE cells from non-CF individuals.
Furthermore, potentiator 39 showed good in vitro druglike properties and was therefore evaluated in vivo for its pharmacokinetic profile in rats. Following oral administration, significant exposure levels were obtained, leading to good oral bioavailability. A subsequent study showed that potentiator 39 distributed to the lung after oral administration to rats, with compound levels also detectable at 4 h postdosing.
To conclude, this study allowed the identification of N 2 -acylsubstituted 2,3,4,5-tetrahydro-1H-pyrido [4,3-b]indoles as novel CFTR potentiators endowed with a good efficacy in rescuing the gating defect of F508del-and G551D-CFTR and a preliminary promising druglike profile. These findings represent a promising starting point to further improve and develop this chemical class, adding a new chemotype to the existing classes of CFTR potentiators, possibly expanding the current portfolio of therapeutic solutions for the treatment of CF.
■ EXPERIMENTAL SECTION Chemistry. Synthetic Materials and Methods. Solvents and reagents were obtained from commercial suppliers and were used without further purification. Automated column chromatography purifications were performed on a Teledyne ISCO apparatus (CombiFlash Rf) with prepacked silica gel columns of different sizes (RediSep). NMR experiments were run at 300 K on a Bruker AVANCE III 400 system (400.13 MHz for 1 H and 100.62 MHz for 13 C), equipped with a BBI probe and Z-gradients, and Bruker FT NMR AVANCE III 600 MHz spectrometer equipped with a 5 mm CryoProbe QCI 1 H/ 19 F− 13 C/ 15 N−D quadruple resonance, a shielded Z-gradient coil and the automatic sample changer SampleJet NMR system (600 MHz for 1 H, 151 MHz for 13 C, and 565 MHz for 19 F). Chemical shifts for 1 H and 13 C spectra were reported in parts per million (ppm), calibrating the residual nondeuterated solvent peak for 1 H and 13 C, respectively, to 7.26 and 77.16 ppm for CDCl 3 and 2.50 and 39.52 ppm for DMSO-d 6 , whereas spectra in D 2 O were referred to trimethylsilylpropanoic acid peak set at 0.00 ppm. Ultra performance liquid chromatography−mass spectrometry (UPLC/MS) analyses were performed on a Waters ACQUITY UPLC/MS system consisting of a single quadrupole detector (SQD) mass spectrometer equipped with an electrospray ionization interface and a photodiode array detector. Electrospray ionization in positive and negative mode was applied in the mass scan range 100−500 Da. The PDA range was 210−400 nm. The mobile phase was 10 mM NH 4 OAc in H 2 O at pH 5 adjusted with AcOH (A) and 10 mM NH 4 OAc in CH 3 CN−H 2 O (95:5) at pH 5 (B) with 0.5 mL/min as the flow rate. For intermediates, the analyses were run on an ACQUITY UPLC BEH C 18 column (100 × 2.1 mm ID, particle size 1.7 μm) with a VanGuard BEH C 18 precolumn (5 × 2.1 mm ID, particle size 1.7 μm). A linear gradient was applied: 0−0. with a XBridge Prep C 18 (10 × 19 mm ID, particle size 5 μm) Guard cartridge with a flow rate of 20 mL/min. The analytical chiral separations were performed on a Waters Alliance HPLC instrument consisting of an e2695 Separation Module and a 2998 Photodiode Array Detector. The PDA range was 210−400 nm. The analyses were run in isocratic mode on Daicel ChiralPak AD column (250 × 4.6 mm ID, particle size 10 μm) with a flow rate of 1.0 mL/min. The semipreparative chiral separations were performed on a Waters Alliance HPLC instrument consisting of a 1525 Binary HPLC Pump, a Waters Fraction Collector III, and a 2998 Photodiode Array Detector. The separations were run in the isocratic mode on a Daicel ChiralPak AD column (250 × 10 mm ID, particle size 10 μm) with a ChiralPak AD Semi-Prep. Guard precolumn (50 × 10 mm ID, particle size 10 μm) at room temperature (r.t.), with a flow rate of 5.0 mL/min. Highresolution mass spectrometry (HRMS) measurements were performed on a Waters SYNAPT G2 Q-ToF mass spectrometer equipped with an electrospray ionization interface and coupled to a Waters ACQUITY UPLC. Leucine enkephalin (2 ng/mL) was used as the lock mass reference compound for spectral recalibration. The analyses were run on an ACQUITY UPLC BEH C 18 column (100 × 2.1 mm ID, particle size 1.7 μm) with a VanGuard BEH C 18 precolumn (5 × 2.1 mm ID, particle size 1.7 μm). The mobile phase was H 2 O + 0.1% HCOOH (A) and CH 3  Purity of the initial hits (Hit 1−Hit 6) and final compounds was determined by UPLC/MS and quantitative 1 H NMR (qNMR, see the Supporting Information) and was equal to or greater than 95% for all of the compounds, except for Hit-6 (80% purity) and analogue 15 (93% purity).
Synthesis of Phenylhydrazine Hydrochlorides 44a,b. General Procedure 1b (Gp1b). A solution of hydrazine of type I (1.0 equiv) and ketone of type II (1.0 equiv) in EtOH (0.5 M) was stirred at r.t., until the formation of hydrazone intermediate. 2,4,6-Trichloro-1,3,5-triazine (0.4 equiv) was added and the reaction mixture was heated to 90°C for 8 h. The reaction mixture was cooled to r.t. and the obtained precipitate was filtered and washed with cold EtOH. The crude product of type III was used as such in the next step without further purification.
General Procedure 1c (Gp1c). Hydrazine of type I (1.0 equiv) and ketone of type II (1.0 equiv) were dissolved in EtOH (0.2 M) and the reaction mixture was stirred at r.t. for 30 min, until the complete formation of hydrazone. The solvent was removed under reduced pressure and the crude mixture was dissolved in AcOH (0.1 M), followed by addition of trifluoroborate diethyletherate (2.0 equiv). The reaction was stirred at 90°C for 16 h. The solvent was removed and the crude mixture was poured into an aq 2.0 M NaOH solution and extracted with dichloromethane (DCM), dried over Na 2 SO 4 , filtered, and concentrated in vacuo to afford the crude product of type III, which was used in the next step without any further purification.
General Procedure 1d (Gp1d). A suspension of hydrazine of type I (1.0 equiv) and ketone of type II (1.0 equiv) in toluene (0.2 M) was stirred at 50°C until the formation of hydrazone intermediate. p-Toluenesulfonic acid (1.5 equiv) was added and the reaction mixture was heated to 120°C for 24 h. The reaction mixture was cooled to r.t. and sat. aq Na 2 CO 3 solution was added until pH 8. The crude was extracted with DCM, dried over Na 2 SO 4 , filtered, and concentrated under vacuo. The compound of type III was isolated by chromatography on alumina using DCM/MeOH·NH 3 (1.0 N) as an eluent.
Biology. Cell Models and Cell Culture procedures. FRT cells stably expressing mutant F508del-CFTR or G551D-CFTR and the halidesensitive yellow fluorescent protein (HS-YFP) YFP-H148Q/I152L and CFBE41o-cells stably expressing F508del-CFTR and HS-YFP were generated as previously described. 21,57 FRT cells were cultured using the Coon's modification of Ham's F12 medium, while CFBE41o-cells were cultured in the modified Eagle's medium. Media were supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ mL of penicillin, and 100 mg/mL of streptomycin. For functional assays of CFTR activity based on the HS-YFP assays, CFBE41o-or FRT cells were plated (50,000 cells/well) on clear-bottom 96-well black microplates (Corning Life Sciences, Acton, MA). The following day, cells were assayed. In the case of FRT cells expressing F508del-CFTR, plates were kept at 32°C (or treated with correctors at 37°C where indicated) for an additional 24 h to rescue the mutant trafficking defect, before assays.
Primary bronchial epithelial cells were cultured as previously described. 58 In brief, epithelial cells were cultured in a serum-free medium (LHC9 mixed with RPMI 1640, 1:1) supplemented with hormones and supplements to support cell number amplification. Then, the cells were seeded at high density on porous membranes (500,000 cells for 1 cm 2 Snapwell inserts, for Ussing chamber studies; 200,000 cells for 0.33 cm 2 Mini-Transwell inserts, for TEER/PD measurements). After 24 h, the serum-free medium was replaced with Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 containing 2% fetal bovine serum (FBS) plus hormones and supplements. Differentiation of cells to form a tight epithelium was monitored by measuring transepithelial electrical resistance and potential difference with an epithelial voltohmmeter (EVOM1, World Precision Instruments). After 8−10 days, the apical medium was removed, and the cells received nutrients only from the basolateral side (air−liquid interface, ALI) to promote further differentiation of the epithelium. Cells were maintained under ALI for 2−3 weeks before experiments.
HS-YFP-Based Assay for CFTR Activity. Prior assay, cells were washed with phosphate-buffered saline (PBS) containing (in mM) 137 NaCl, 2.7 KCl, 8.1 Na 2 HPO 4 , 1.5 KH 2 PO 4 , 1 CaCl 2 , and 0.5 MgCl 2 . Cells were then incubated for 25 min with 60 μL of PBS plus forskolin (20 μM) and test compounds (at the desired concentration) to stimulate mutant CFTR. Cells were then transferred to microplate readers (FluoStar Optima; BMG Labtech, Offenburg, Germany) for CFTR activity determination. The plate readers were equipped with high-quality excitation (HQ500/20X: 500 ± 10 nm) and emission (HQ535/30M: 535 ± 15 nm) filters for YFP (Chroma Technology). The assay consisted of a continuous 14 s fluorescence reading, 2 s before and 12 s after injection of 165 μL of an iodide-containing solution (PBS with Cl − replaced by I − ; final I − concentration 100 mM). Data were normalized to the initial background-subtracted fluorescence. To determine the I − influx rate, the final 10 s of the data for each well was fitted with a linear function to extrapolate the initial slope (dF/dt).
Each experimental condition was tested in three independent experiments, each one performed with three biological replicates (n = 9).
TEER/PD Measurements. Differentiated bronchial epithelia were treated with compounds included in the appropriate culture medium at the indicated concentrations for 24 h at 37°C and 5% CO 2 , before measuring the TEER and/or PD by means of an epithelial voltohmmeter (EVOM1, World Precision Instruments).
The electrical measurements were done in Coon's modified Ham's F-12 medium, where NaHCO 3 was replaced with 20 mM Na−HEPES (pH 7.3). TEER and PD were measured in each well under basal conditions, after ENaC inhibition with apical amiloride (10 μM), after CFTR stimulation with forskolin (10 μM) plus test compounds (at the desired concentration) on both sides, and after CFTR inhibition with apical PPQ102 (30 μM). After each treatment, we waited 10 min before recording the electrical parameters. The TEER and PD values for each well were converted into short-circuit current equivalent by Ohm's law.
Each experimental condition was tested in three independent experiments, each one performed with three biological replicates (n = 9).
Short-Circuit Current Recordings. Differentiated bronchial epithelia on Snapwell inserts were mounted in a Ussing chamber with internal fluid circulation. Apical and basolateral hemichambers were filled with 5 mL of a solution containing (in mM) 126 NaCl, 0.38 KH 2 PO 4 , 2.13 K 2 HPO 4 , 1 MgSO 4 , 1 CaCl 2 , 24 NaHCO 3 , and 10 glucose, and both sides were continuously bubbled with a 5% CO 2 −95% air mixture, with the temperature of the solution maintained at 37°C. The transepithelial voltage was short-circuited with a voltage clamp (DVC-1000, World Precision Instruments) connected to the apical and basolateral chambers via Ag/AgCl electrodes and agar bridges (1 M KCl in 1% agar). The offset between voltage electrodes and the fluid electrical resistance were set to zero before each set of experiments. The shortcircuit current was recorded with a PowerLab 4/25 (ADInstruments) analog-to-digital converter connected to a personal computer.
Each experimental condition was tested in three independent experiments, each one performed with three biological replicates (n = 9).
Statistical Analysis. Each experimental condition was tested in three independent experiments, each one performed with three biological replicates (n = 9). The Kolmogorov−Smirnov test was used to evaluate the assumption of normality. The statistical significance of the effect of single treatments on CFTR activity or expression was tested by parametric one-way ANOVA, followed by the Dunnett multiple comparisons test (all groups against the control group) as a post-hoc test. In the case of a combination of treatments, statistical significance was verified by ANOVA, followed by the Tukey test (for multiple comparisons) as the post-hoc test. Normally distributed data are expressed as mean ± SD, and significances are two-sided. Differences were considered statistically significant when P was less than 0.05.
In Vitro ADMET. Aqueous Kinetic Solubility Assay. The aqueous kinetic solubility was determined from a 10 mM DMSO stock solution of test compound in PBS at pH 7.4. The study was performed by incubation of an aliquot of 10 mM DMSO stock solution in PBS (pH 7.4) at a target concentration of 250 μM (2.5% DMSO). The incubation was carried out under shaking at 25°C for 24 h, followed by centrifugation at 21,100g for 30 min. The supernatant was further diluted (4:1) with CH 3 CN and the dissolved test compound was quantified by UV at 215 nm on a Waters ACQUITY UPLC/MS system consisting of an SQD mass spectrometer equipped with an electrospray ionization interface and a photodiode array detector. Electrospray ionization in the positive mode was used in the mass scan range of 100− 500 Da. The PDA range was 210−400 nm. The analyses were run on an ACQUITY UPLC BEH C 18 column (50 × 2.1 mm ID, particle size 1.7 μm) with a VanGuard BEH C 18 precolumn (5 × 2.1 mm ID, particle size 1.7 μm) using 10 mM NH 4 OAc in H 2 O at pH 5 adjusted with AcOH (A) and 10 mM NH 4 OAc in CH 3 CN−H 2 O (95:5) at pH 5 (B) as the mobile phase. The aqueous kinetic solubility (in μM) was calculated by dividing the peak areas of the dissolved test compound and the test compound in the reference (250 μM of test compound in CH 3 CN) and multiply by the target concentration and dilution factor.
Liver Microsomal Stability Assay. Phase I: 10 mM DMSO stock solution of the test compound was preincubated at 37°C for 15 min with rat, dog, or human liver microsomes in 0.1 M Tris−HCl buffer (pH 7.5) with 10% DMSO. The final concentration was 4.6 μM. After preincubation, the cofactors (NADPH, G6P, G6PDH, and MgCl 2 predissolved in 0.1 M Tris−HCl) were added to the incubation mixture and the incubation was continued at 37°C for 1 h.
Phase II: 10 mM DMSO stock solution of the test compound was preincubated at 37°C for 15 min with human liver microsomes added alamethicin in 0.1 M Tris−HCl buffer (pH 7.5) with 10% DMSO. The final concentration was 4.6 μM. After preincubation, the cofactors (UDPGA, D-saccharic acid lactone, and MgCl 2 predissolved in 0.1 M Tris−HCl) were added to the incubation mixture and the incubation was continued at 37°C for 1 h.
For both phase I and II studies: At each time point (0, 5, 15, 30, and 60 min), 30 μL of the incubation mixture was diluted with 200 μL of cold CH 3 CN spiked with 200 nM of an appropriate internal standard, followed by centrifugation at 3270g for 15 min. The supernatant was further diluted with H 2 O (1:1) for analysis. A reference incubation mixture (microsomes without cofactors) was prepared for each test compound and analyzed at t = 0 and 60 min in order to verify the compound's stability in the matrix. The two time points were diluted as for the time points of the incubation mixture above. The supernatants were analyzed by LC/MS−MS on a Waters ACQUITY UPLC/MS TQD system consisting of a TQD (triple quadrupole detector) mass spectrometer equipped with an electrospray ionization interface and a photodiode array eλ detector. Electrospray ionization was applied in positive mode. Compound-dependent parameters as MRM transitions and collision energy were developed for each compound. The analyses were run on an ACQUITY UPLC BEH C 18 (50 × 2.1 mm ID, particle size 1.7 μm) with a VanGuard BEH C 18 precolumn (5 × 2.1 mm ID, particle size 1.7 μm) at 40°C, using H 2 O + 0.1% HCOOH (A) and CH 3 CN + 0.1% HCOOH (B) as the mobile phase. The percentage of test compound remaining at each time point relative to t = 0 was calculated by the response factor on the basis of the internal standard peak area. The percentage of test compound versus time was plotted and fitted by GraphPad Prism (GraphPad Software, Version 5 for Windows, CA, USA, www.graphpad.com) to estimate the compound's half-life (t 1/2 ), which was reported as mean value along with the standard deviation (n = 3).
HepG2 Cell toxicity Assay. Cell Culture Conditions. To increase the detection of drug-induced mitochondrial effects in a preclinical cellbased assay, HepG2 hepatocellular carcinoma cells (ATCC HB-8065) were forced to rely on mitochondrial oxidative phosphorylation rather than glycolysis by substituting galactose (10 mM) for glucose (25 mM) in the growth media (DMEM, Life Technologies).
Media Composition. High-glucose media: high-glucose DMEM (Invitrogen 11995-065) containing 25 mM glucose, 1.0 mM sodium pyruvate, supplemented with 5 mM N-(2-hydroxyethyl)piperazine-N′- Cell Viability Assessment. For the cytotoxicity assay, cells were plated at 20,000 cells/well in 100 μL of cell culture media in 96-well plates and allowed to grow overnight. The cells were treated for 24 h with 2.0 or 20 μM of each compound. All compounds were dissolved in DMSO with a stock concentration of 4 mM. The first dilution step of compounds was prepared in DMSO (200× stock solutions), while the second dilution step was carried out in a complete cell culture medium (5% DMSO). Of this dilution, 10 μL were added to the wells of the 96well plate, with a final DMSO concentration of 0.5%. Rotenone, a wellknown mitochondrial inhibitor, was used as a reference compound. After treatment, cellular viability was assessed by using two different assays, run on independent plates: the CellTiter-Glo (CTG) Luminescent Cell Viability Assay (Promega), which determines the number of viable cells based on the quantitation of the ATP present, and the thiazolyl blue tetrazolium blue (MTT) dye (Aldrich), which is converted to water-insoluble MTT formazan crystals by mitochondrial dehydrogenases of living cells. Each experimental condition (i.e., control, reference, and compounds' doses) has been tested in three technical replicates.
In Vivo Pharmacology. Animals. Male Sprague−Dawley rats, 2 month old and weighing 175−200 g (Charles River, Calco, Italy), were used. Animals were group-housed in ventilated cages and had free access to food and water. They were maintained under a 12 h light/dark cycle (lights on at 8:00 am) at a controlled temperature (21 ± 1°C) and relative humidity (55 ± 10%). All experiments were carried out in accordance with the guidelines established by the European Communities Council Directive (Directive 2010/63/EU of September 22, 2010) and approved by the National Council on Animal Care of the Italian Ministry of Health. All efforts were made to minimize animal suffering and to use the minimal number of animals required to produce reliable results.
Pharmacokinetic Methods. Compound 39 was administered intravenously (i.v.) and orally (p.o.) to cannulated Sprague−Dawley rats at doses of 3 and 10 mg/kg, respectively. PEG400/Tween 80/saline solution was used as a vehicle at 10/10/80% in volume, respectively. Three animals per dose were treated. Blood samples at 0, 15, 30, 60, 90, 120, 240, and 360 min after administration were collected for p.o. arm. Blood samples at 0, 5, 15, 30, 60, 90, 120, and 240 min after administration were collected for i.v. arm. Plasma was separated from blood by centrifugation for 15 min at 3500 rpm at 4°C, collected in an Eppendorf tube, and frozen (−80°C). Control animals treated with vehicle only were also included in the experimental protocol.
Sample Preparation for Lung Exposure Analysis. Three animals per dose and timing were treated. Compound 39 was dissolved in PEG400/Tween80/saline solution at 10/10/80% in volume and administered orally at a dose of 10 mg/kg. After 120 and 240 min, rats were sacrificed and lungs were immediately dissected, frozen on dry ice, and stored at −80°C until analysis. Lung samples were homogenized in RIPA buffer (150 mM NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, and pH 8.0) and then split into two aliquots kept at −80°C until analysis. An aliquot was used for compound lung-level evaluations. The second aliquot was kept for protein content evaluation using the bicinchoninic acid assay (Thermo Scientific, Rockford, IL, USA).
Bioanalytical Analyses. Plasma samples were centrifuged at 21,100g for 15 min at 4°C, while homogenized lung samples were vigorously whirled. An aliquot of each sample was extracted (1:3) with cold CH 3 CN containing 200 nM of an appropriate internal standard being a close analogue of the parent compound. A calibration curve was prepared in both blank mouse plasma and naive lung homogenate over a 1 nM to 10 μM range. Three quality controls were prepared by spiking the parent compound in both blank mouse plasma and naive lung homogenate to 20, 200, and 2000 nM as final concentrations. The calibrators and quality controls were extracted (1:3) with the same extraction solution as the plasma and lung samples. The plasma and lung samples, the calibrators, and quality controls were centrifuged at 3270g for 15 min at 4°C. The supernatants were further diluted (1:1) with H 2 O and analyzed by LC/MS−MS on a Waters ACQUITY UPLC/MS TQD system consisting of a TQD mass spectrometer equipped with an electrospray ionization interface and a photodiode array eλ detector. Electrospray ionization was applied in the positive mode. Compound-dependent parameters such as MRM transitions and collision energy were developed for the parent compound and the internal standard. The mobile phase was H 2 O + 0.1% HCOOH (A) and CH 3 CN + 0.1% HCOOH (B) at a flow rate of 0.5 mL/min. For plasma samples, the analyses were run on an ACQUITY UPLC BEH C 18 (50 × 2.1 mm ID, particle size 1.7 μm) with a VanGuard BEH C 18 precolumn (5 × 2.1 mm ID, particle size 1.7 μm) at 40°C. A linear gradient was applied starting at 30% B with an initial hold for 0.2 min and then 30− 100% B in 2 min. For lung samples, the analyses were run on an ACQUITY UPLC BEH C 18 (100 × 2.1 mm ID, particle size 1.7 μm) with a VanGuard BEH C 18 precolumn (5 × 2.1 mm ID, particle size 1.7 μm) at 40°C. A linear gradient was applied starting at 30% B with an initial hold for 0.2 min and then 30−100% B in 6 min. All samples (plasma and lung samples, calibrators, and quality controls) were quantified by MRM peak area response factor in order to determine the levels of the parent compound in plasma and lung. The concentrations versus time data were plotted and the profiles were fitted using PK Solutions Excel Application (Summit Research Service, USA) in order to determine the pharmacokinetic parameters. Pure regioisomer 52 was obtained as a racemate starting from enantiopure (S)-51 because of the epimerization process during Fischer indole synthesis.