Discovery and Optimization of 1-Phenoxy-2 ... - ACS Publications

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Discovery and Optimization of 1‑Phenoxy-2-aminoindanes as Potent, Selective, and Orally Bioavailable Inhibitors of the Na+/H+ Exchanger Type 3 (NHE3) Nils Rackelmann,* Hans Matter,* Heinrich Englert, Markus Follmann,† Thomas Maier, John Weston, Petra Arndt, Winfried Heyse, Katharina Mertsch, Klaus Wirth, and Laurent Bialy Sanofi-Aventis Deutschland GmbH, R&D, D-65926, Frankfurt am Main, Germany S Supporting Information *

ABSTRACT: The design, synthesis, and structure−activity relationship of 1-phenoxy-2-aminoindanes as inhibitors of the Na+/ H+ exchanger type 3 (NHE3) are described based on a hit from high-throughput screening (HTS). The chemical optimization resulted in the discovery of potent, selective, and orally bioavailable NHE3 inhibitors with 13d as best compound, showing high in vitro permeability and lacking CYP2D6 inhibition as main optimization parameters. Aligning 1-phenoxy-2-aminoindanes onto the X-ray structure of 13d then provided 3D-QSAR models for NHE3 inhibition capturing guidelines for optimization. These models showed good correlation coefficients and allowed for activity estimation. In silico ADMET models for Caco-2 permeability and CYP2D6 inhibition were also successfully applied for this series. Moreover, docking into the CYP2D6 X-ray structure provided a reliable alignment for 3D-QSAR models. Finally 13d, renamed as SAR197, was characterized in vitro and by in vivo pharmacokinetic (PK) and pharmacological studies to unveil its potential for reduction of obstructive sleep apneas.

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INTRODUCTION +

consequential and significant impact on intracellular pH values.6 Hypercapnia, a condition of abnormally elevated CO2 levels in the blood, and a lowered intracellular pH value are both known to increase the bioelectric activity of CO2/H+-sensitive neurons.7 Inhibition of NHE3 increases the activity of CO2/H+sensitive neurons upon intracellular acidification.8 Inhibition of NHE3 expressed in brain stem areas8b,c,9 is able to modulate the ventilation drive and therefore reduce central sleep apneas. In addition NHE3 inhibition leads to increased muscle tone of the upper airways. 10 Therefore, NHE3 inhibitors can potentially be used to treat muscle related respiratory impairments such as obstructive sleep apneas and snoring. These interesting properties prompted us to initiate a discovery program for novel inhibitors with a suitable profile for advanced pharmacological evaluations. The focus of our project was to identify orally bioavailable and potent small

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Sodium−hydrogen (Na /H ) exchanger type 3, also known as NHE3, sodium−hydrogen antiporter 3, or solute carrier family 9 member 3 (SLC9A3), is one of nine isoforms of the mammalian NHE gene family.1 These mammalian NHEs consist of isoforms that occur primarily in the plasma membrane and those that appear to primarily reside in intracellular organelles.2 The human NHE3 transporter is encoded by the SLC9A3 gene.3 NHE3 is primarily located in the nephron of the kidney, in the apical membrane of intestinal enterocytes, and in the brain stem cell area. A significant portion of proximal tubule sodium ion reabsorption is mediated by NHE3 and other membrane Na+/H+ exchangers.4,5 Immunochemical studies suggest that NHE3 is the principal exchanger isoform expressed on proximal tubular brush-border membranes, which might indicate a substantial role of this transporter for maintaining the sodium ion balance by sodium ion and fluid reabsorption in the proximal tubule with a © 2016 American Chemical Society

Received: April 22, 2016 Published: September 8, 2016 8812

DOI: 10.1021/acs.jmedchem.6b00624 J. Med. Chem. 2016, 59, 8812−8829

Journal of Medicinal Chemistry

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molecules without acylguanidine, thus complementing earlier NHE3 inhibitors like 1a (S3226).11,12 Guanidines and acylguanidines are important motifs of numerous active molecules. Depending on the protein environment, acylguanidines are less basic guanidine bioisosteres with potentially improved permeability and oral bioavailability. Despite lower basicity, they contain many polar atoms with high polar surface area (PSA), which still might cause lower permeability and therefore insufficient pharmacokinetic behavior in cases, where acylguanidines are not additionally decorated by lipophilic groups or cyclized. For interaction with NHE subtypes, unsubstituted N-acylguanidines are often required. In contrast, no general adverse effects are reported for this headgroup to our knowledge; several molecules were investigated in clinical studies. The goal of this project was to identify structurally unrelated compounds without polar acylguanidine to modulate molecular properties, which might also lead to different selectivity profiles among NHE isoforms. Figure 1 summarizes

Article

METHODS

Biological Assays. The primary assay used to determine NHE3 inhibition for both the high-throughput screening campaign and during lead optimization is described in detail in ref 10. Briefly, the recovery of the intracellular pH of LAP1 cells which stably express NHE3 after acidification is determined. The recovery occurs even under bicarbonate-free conditions with functional NHE3. To this end, the intracellular pH was determined using the pH-sensitive fluorescent dye BCECF (2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, employing the precursor BCECF-AM, from Molecular Probes, Eugene, OR, USA). The cells were initially incubated with BCECF-AM in NH4Cl buffer. The intracellular acidification was induced by washing the cells incubated with a NH4Cl-free buffer. After washing, 90 μL of the buffer was left on the cells. The pH recovery was initiated by ading 90 μL of Na+-containing buffer in a FLIPR instrument (fluorometric imaging plate reader, Molecular Devices, Sunnyvale, CA, USA). The BCECF fluorescence was determined with an excitation wavelength of 498 nm and an FLIPR emission filter (bandpass from 510 to 570 nm). Changes in fluorescence as a measure for pH recovery were recorded for 2 min. To evaluate the NHE3inhibition for each compound, the cells were initially investigated in buffers with which complete or no pH recovery took place. For complete recovery, the cells were incubated in Na+-containing buffer; for no recovery, the cells were incubated in Na+-free buffer. The recovery of the intracellular pH at each tested concentration of a substance was expressed as percent of the maximum recovery and used for IC50 determination. Each IC50 value was determined from 10 individual concentrations as duplicates. IC50 values for 9 more interesting, potent analogs out of 67 compounds described in this study were tested more than three times. For example, the best compound 13d was tested 10 times with a standard deviation of 0.011 μM for the IC50 value. For 8 compounds, three tests for IC50 values were performed, for 23 compounds two IC50 tests. For the remaining set of 27 compounds with lower activity mainly, only a single IC50 value was determined. The geometric mean was used for averaging. The standard deviation for IC50 determination ranges from 0.000 08 for very potent to 3.45 for very weak NHE3 inhibitors. There is a good correlation between the NHE3 inhibitory potency and standard deviation, as expected. For statistical model building, NHE3 inhibition (pIC50) is expressed as log(1/IC50). Permeability assays to assess membrane permeability potential were performed with Caco-2/TC7 cells.20 Conditions for apical compartment were the following: pH 6.5; 0.5% bovine serum albumin (BSA); compound concentration 20 μM; basolateral compartment pH 7.4; 5% BSA; 2 h incubation time. Efflux was determined with pH 7.4 in both compartments and 0.5% BSA with compound concentration of 2 μM. All analyses were performed with LC−MS/MS. Cytochrome P450 (CYP) inhibition assays21 were performed with pooled and phenotyped human hepatic microsomes (0.1 mg/mL for CYP3A4 and CYP2C9; 0.2 mg/mL for CYP2D6). As cofactors, 6 mM MgCl2, 0.5 mM EDTA, 1 mg/mL BSA, 1 mM NADPH were used; compound concentration tested was in the range of 0.001−200 μM (in duplicate). Results were expressed as % inhibition or IC50 values. As substrates, diclofenac was used for CYP2C9, dextromethorphan was used for CYP2D6, and both midazolam and testosterone

Figure 1. Known inhibitors for NHE3 and/or NHE1.

other chemical structures reported as NHE3 blockers like the aminobenzimidazole 1b (AVE0657)9d,13 and a series of tetrahydroisoquinolines, exemplified by 1c (THiQ).14,15 In addition to earlier acylguanidine-containing NHE3 inhibitors, blockers of NHE1 containing the same structural motif are described in addition, like 1d (HOE694) 12,16 and 1e (HOE642A/cariporide).17 Here we report the discovery, optimization, and structure− activity relationships (SARs) of a series of 1-phenoxy-2aminoindanes, which led to potent and selective NHE3 inhibitors. The manuscript is organized as follows. First we describe the optimization of a hit series from high-throughput screening (HTS) toward the identification of 13d as best compound. Next we describe 3D-QSAR (three-dimensional quantitative structure−activity relationship) models for NHE3 and CYP2D6 inhibition using comparative molecular field analysis (CoMFA)18 and comparative molecular similarity index analysis (CoMSIA).19 We then report studies to identify prerequisites for high in vitro Caco-2 cell permeability and low CYP2D6 inhibition by in silico ADMET (absorption, distribution, metabolism, excretion, toxicity) models. Then we summarize the in vitro characterization of 13d. Finally in vivo pharmacokinetic (PK) and pharmacological studies are discussed to highlight the potential of 13d for reduction of obstructive sleep apneas. 8813



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were employed for CYP3A4.22 In the CYP3A4 inhibition experiments, the control inhibitor ketoconazole was tested up to 0.25 μM and an IC50 value of 0.0285 μM was obtained (5 concentrations). Maximal concentration of 0.25 μM yielded 17% of remaining enzyme activity compared to control. In CYP2D6 experiments, the control inhibitor quinidine was tested up to 0.225 μM and resulted in an IC50 value of 0.0413 μM. The remaining activity of the enzyme was 21% at maximum 0.225 μM quinidine concentration compared to control. Both IC50 values are in agreement with literature. Lability assays were performed with pooled and phenotyped human liver microsomes taking samples at time 0 and time 20 min for LC−MS/MS analysis.21 Clearance in vitro was determined using primary human hepatocytes at 1.4 × 10−6 cells/mL.21 13d concentration varied from 0.5 μM to 5 μM. CYP contribution was determined by addition of either quinidine for CYP2D6 inhibition or ketoconazole for CYP3A4 inhibition to the assay plate. All bioanalytical analyses were performed by LC−MS/MS. Induction potential for CYP enzymes was tested with primary human hepatocytes and several batches of cryopreserved human hepatocytes. Incubation medium contained 0.1 μM dexamethasone; assays were performed on 48- or 96-well plates. 13d was tested in concentrations between 1 and 30 μM and compared to references β-naphtoflavone F (10 μM) and omeprazole (30 μM) for CYP1A1/2 and to rifampicin (10 μM) for CYP3A4 after 48 h of incubation. Analysis of CYP1A1/2, CYP1A2, and CYP3A4 was performed by RT-PCR.22a−c Pharmacokinetic parameters in pigs (male Duroc Landrace pigs) were determined after single intravenous and oral administrations of 13d. Dose, formulation, and route were used as follows: iv route with 1 mg/kg in 5% glucose/water in solution; po route with 3 mg/kg in agarose with sugar and crushed pellets as gelos formulation; sampling times of 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h intravenous route and 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h oral route. Analysis was performed with ESILC/MS−MS assay of 13d in plasma with a limit of quantitation (LOQ) of 5 ng/mL.23 All studies in animals were conducted in accordance with German laws for protection of animals. Furthermore, the investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of health.24 X-ray Structure Determination. The molecular and crystal structure of 13d was determined by single crystal Xray structure analysis. A single crystal (size: 0.50 × 0.05 × 0.02 mm3) obtained from dichloromethane/diethyl ether was sealed in a Lindemann glass capillary. X-ray diffraction data were collected on a Bruker/AXS three circle diffractometer, equipped with a SMART APEX area detector, a low temperature device, and a molybdenum-Kα rotating anode generator, operated at 50 kV/120 mA and adjusted to a fine focus of 0.5 × 5 mm2. Data frames were collected using the program SMART, version 5.628,25 applying ω-scans with step widths of 0.3° and an exposure time of 60 s. Data processing with the program SAINT + Release 6.4526 yielded 10 173 reflections (ϑmin = 1.98, ϑmax = 21.97; −18 < h < 20, −7< k < 7, −21< l < 22) of which 6343 reflections were unique (Rint = 0.0819, Rσ = 0.2271). Refinement of cell parameters was performed using 1101 reflections. The phase problem was solved with direct methods by the XS module of SHELXTL 6.14.27

The structure was refined by least-squares methods (minimization of (Fo2 − Fc2)2) using the XL module of SHELXTL 6.14 (Bruker AXS, 2000). The positions of all H atoms were calculated, except those bonded to the water molecules O1 and O4 to O7 which were experimentally determined from a difference Fourier synthesis map. Hydrogen atoms for oxygen atoms O2 and O3 could not be determined. Sgoodness of fit = 0.850, Rall data = 0.1912 (Robs data = 0.0607 for 2464 reflections with |Fobs| > 4σ, wR2all data = 0.1506, wR2obs data = 0.1161). The largest unassigned peaks in the difference map correspond to −0.248 versus +0.344 electrons per Å3. The average estimated standard deviation (esd) of a C−C bond is 0.014 Å, that of an O−C bond is 0.011 Å, and that of an N−C bond is 0.011 Å. The average esd of C−C−C bond angles is 1.1°, and that of C−C−C−C torsion angles is 1.4°. The Flack parameter has been determined to 0.0566 (esd of 0.1296), reference values are 0 (within 3 esd’s) for the correct and +1 for inverted absolute structure. Computational Procedures. The X-ray structure of 13d was used as template for aligning 1-phenoxy-2-aminoindanes. The ligand-based alignment was performed in a pairwise manner using tfit in QXP28 with a modified AMBER force field.29 The CYP2D6 X-ray structure 2f9q30 with a resolution of 3.0 Å from RCSB31 was used for docking. Molecules were minimized or automatically docked using QXP.28 Selection of poses was guided by consistent alignment of the indane. Selected side chains lining the substrate pocket were treated as flexible. The alignment for 27 molecules from Table 3 with CYP2D6 inhibition served as basis for 3D-QSAR. The program MOLCAD32 was used to visualize lipophilicity,33 hydrogen-bonding potential, and cavity depth on solvent accessible protein surfaces.34 Default settings were used for CoMFA and CoMSIA in Sybyl.35 For CoMFA steric and electrostatic energies are calculated at grid points with 2 Å spacing, a positively charged carbon atom and a distance-dependent dielectric constant with MMFF94 charges.36 The same alignments were used for CoMSIA steric, electrostatic, and hydrophobic similarity index fields19 using a probe with charge +1, a radius of +1, a hydrophobicity of +1, and 0.3 as attenuation factor α for Gaussian-type distance dependence. Cross-validated analyses37 were run using SAMPLS38 or two and five cross-validation groups with random selection of members. PLS (partial least-squares)39 analyses using two or five random cross-validation groups were averaged over 100 runs. For validation all affinities were randomized40 100 times, subjected to PLS, and the mean cross-validated r2 was calculated. 2D fingerprints were generated using UNITY41 for selection of training and test sets; their similarity is given as Tanimoto coefficient.42 In silico ADMET models were generated using data from multiple internal series.43 Model building for end points like human microsomal lability and PXR activation is described elsewhere.44 Pretreatment included removal of counterions and smaller fragments, neutralization plus canonization of structures. Canonical 3D geometries including hydrogen positions were generated using Corina.45 The 2D-QSAR models were based on MOE descriptors46 in combination with Cubist,47 which analyzes continuous variables by a regression tree. The principle was to construct a rule-based decision tree, where each rule was characterized by a multilinear regression (MLR) model describing the SAR in this node. A GA-based approach44 for variable selection maximized the sum of regression 8814



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Chemistry. To explore the SAR around the HTS hits 2a−c, a general, a robust, and enantioselective synthesis was established, as described below for representative members of this series. For example, the finally selected compound 13d (cf. Table 3) was synthesized in large scale starting from the propionic acid derivative 3a, which was converted in excellent yields into the corresponding indanone 4b following a intramolecular Friedel−Crafts acylation protocol, as outlined in Scheme 1. Alternatively, indanones 4 with other substitutions of the central core were commercially available or were synthesized as previous described.48 After reduction of the indanones 4 with NaBH4 and subsequent elimination of water in the presence of catalytic amounts of p-TsOH, the corresponding 1H-indenes 6 were isolated in good yields.49 The detailed synthesis of 5,7dichloro-1H-indene will serve as representative example for this synthesis sequence (cf. Experimental Section). By use of Jacobsen’s salen complex, indane oxides 7a−d were obtained in good yields with high enantioselectivity.50,51 The observed enantioselectivity was further increased by recrystallization of building blocks 7a−d from n-heptane. Racemic indane oxides 7g−f were obtained by direct oxidation of the indenes 6 with m-CPBA or using a stepwise protocol outlined in Scheme 2. Indane oxides 7 were regioselectively opened at position C1, with secondary amines in acetonitrile as solvent.52 By utilization of an rearrangement of the amino alcohol 9 in the presence of a phenol under Mitsunobu conditions, the trans configured 1phenoxy-2-aminoindanes 10−12 were obtained in good yields and excellent selectivity (Scheme 3).53 The syntheses of corresponding noncommercial phenols and secondary amines were performed as earlier described.10 In some cases, the final compounds 10−12 contained impurities after column chromatography (reagents from the Mitsunobu reaction; cf. Supporting Information). In those cases, the corresponding NHE3 inhibitors 10−12 were subjected to an additional preparative HPLC purification step. Starting from 11, NHE3 inhibitors containing an alcohol moiety 13 were isolated after using a standard TBAF deprotection protocol. Secondary amines 14 were obtained after treatment of Boc protected compounds 12 with HCl in dioxane. To further explore the SAR of secondary and tertiary amines, compounds 14 were reacted with alkylating reagents R4X in acetonitrile in the presence of potassium carbonate. SAR of 1-Phenoxy-2-aminoindanes. This section describes the optimization of the HTS hits to identify favorable substitutions at the indane scaffold. First, a pyrrolidine was introduced into the 2-amino position and combined with

coefficients for training and test sets. After validation, the best models were consensus models from five Cubist regression trees. The best model is selected from prediction and classification of internal and external test sets. Preliminary versions with smaller training sets were used and regularly updated during optimization. Therefore, the final correlation with the current model version herein included this series in training but serves to illustrate the workflow.

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RESULTS AND DISCUSSION Discovery of NHE3 Inhibitors. The 1-phenoxy-2-aminoindane series was identified following the analysis of a HTS campaign using the Sanofi screening library. The 1-phenoxy-2aminoindane hit series originally resulted from a parallel synthesis library targeted toward generic ion channel pharmacophore motifs. The series was identified by using a FLIPR assay. Three representative molecules 2a−c as hits from this campaign are shown in Table 1. These compounds exhibit NHE3 inhibitory activity ranging from 0.330 to 0.650 μM, respectively. Table 1. Representative NHE3 Inhibitors Identified from HTS

Hit structures 2a−c from the HTS were obtained as mixtures of diastereomers with defined trans-configuration at the indane C1 and C2 atoms. Further exploration then led to the observation that compounds with S,S-chirality at both result in more potent NHE3 inhibitors. Due to the presence of basic amino groups plus additional polar functionality, the in vitro membrane permeability, expressed as Caco-2 permeability (PTotal flux in 10−7 cm/s), was low, therefore suggesting a low in vivo intestinal absorption. Scheme 1. Synthesis of Indenesa

a

(a) (COCl)2, CH2Cl2; (b) AlCl3, CH2Cl2; (c) NaBH4, MeOH; (d) p-TsOH, toluene. bReference 48. 8815



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Scheme 2. Synthesis of Indane Oxidesa

a

(a) Jacobsen salen complex; (b) NBS, DMSO/H2O; (c) NaOH, THF; (d) m-CPBA, CH2Cl2.

Scheme 3. Synthesis of NHE3 Inhibitors 10−15a

a

(a) HNR2R3, CH3CN; (b) HOAr, DIAD, PPh3, THF; (c) TBAF, THF; (d) 4 M HCl, dioxane; (e) R4Br, K2CO3, CH3CN.

yl ring with a dimethylisoxazole ring with corresponding acceptor atom topology slightly lowers activity (cf. 10z, IC50 = 0.411 μM compared to 10m). This is partially attributed to unique electronic features of the 1,2,4-triazol-4-yl ring system. Next we varied halogen substituents on the indane. Introducing an indane-6-chloro substituent into 10v resulted in 10ab with an IC50 of 0.094. Interestingly CYP2D6 inhibition was reduced by this variation (IC50 = 2.500 μM) compared to 10v. Then we replaced the monobasic N2-pyrrolidine on the indane with dibasic moieties. Of particular interest is 10ac with the N-methyl-1,4-diazepane, which showed an IC50 of 0.093 μM. While this dibasic molecule also revealed a remarkable permeability in the Caco-2 assay, it is a strong CYP2D6 inhibitor (IC50 < 0.300 μM). Combining the N-methyl-1,4-diazepane with a 4-(3,5dimethyl-1,2,4-triazol-4-yl)phenoxy substituent then led us to identify the very potent compound 10ad (IC50 = 0.017 μM), again with a 4,6-dichloroindane scaffold. While this compound showed low CYP2D6 inhibition, it was found to be poorly permeable, suggesting that low permeability and lack of CYP2D6 inhibition are related and linked to physicochemical properties. Molecules with linear dibasic side chains also led to high NHE3 inhibition. One example was compound 10af with a Nmethyl-(N-2-dimethylaminoethyl)amino side chain (IC50 = 0.020 μM), again with CYP2D6 inhibition (IC50 < 0.300 μM). This led us to focus on less basic derivatives like 3fluoropyrrolidyl (e.g., 10ah, IC50 = 0.166 μM). It became clear that basicity, although favorable for NHE3, should be lowered for permeability and CYP2D6 inhibition.

phenoxy side chains at 1-position, as summarized in Table 2. With 3-acetylaminophenyl at O1, the derivative 10a with an IC50 of 0.438 μM for NHE3 was obtained. Replacing the 3acetylamino substituent by a 3-(1,3-dihydro-2-oxoimidazol-1yl) substituent further improves inhibition (10e, IC50 = 0.281 μM). Adding a 3-methyl group to the 3-(1,3-dihydro-2oxoimidazol-1-yl) substituent results in the potent compound 10l with an IC50 value of 0.197 μM. Testing selected compounds in the internal Caco-2 cell assay suggests reasonable permeability, as indicated by PTotal values of >20 × 10−7 cm/s as threshold. However, testing for cytochrome P450 (CYP) inhibition reveals that only the less potent 10e showed low CYP2D6 inhibition (IC50 = 3.80 μM), which subsequently was one key optimization parameter. No issues were observed for other cytochrome P450s tested. Introducing the 4-aminosulfonylphenyl-2,3-dichlorophenoxy side chain led to the potent compound 10b (IC50 = 0.123 μM), which still exhibited CYP2D6 inhibition (IC50 = 0.40 μM). Other substituents at the phenoxy group, heterobicyclic systems, or a second ring in 3- or 4-position of the phenoxy moiety did not improve NHE3 inhibition. Introducing a 3,5-dimethyl-4H-1,2,4-triazole ring into 4position of the phenoxy chain resulted in 10m with good NHE3 inhibition (IC50 = 0.345 μM). Introducing additional substituents to the phenoxy further improves NHE3 inhibition, e.g., 10v (IC50 = 0.078 μM). While it also showed favorable permeability, it inhibited CYP2D6 with an IC50 of 0.730 μM. Replacing the chlorine of 10v by trifluoromethyl lowered potency (10w, IC50 = 0.118 μM), as was also observed with two methyl substituents in positions 2 and 3 of the phenoxy side chain (10x, IC50 = 0.170 μM). Modulating the nature of both hydrogen bond acceptor atoms by replacing the 1,2,4-triazol-48816



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Table 2. NHE3 Inhibitors 10 from the Optimization Program

atom to the indane in 4-position resulted in improved NHE3 potency for 13d (IC50 = 0.034 μM) without significant CYP2D6 inhibition. On the other hand this increased lipophilicity of 13d with additional halogen decoration led to acceptable permeability (28 × 10−7 cm/s). On the basis of the entire in vitro profile, compound 13d was perceived as the best compound and was further investigated, as described below. This compound was also named SAR197. Replacing the 2-fluoro-4-(3,5-dimethyl-1,2,4-triazol-4-yl)phenoxy side chain from 13d by the 4-methylsulfonylphenoxy

We then pursued structure−activity studies with indanes carrying chlorine atoms in positions 4 and 6 together with 4methylsulfonylphenoxy or 4-(3,5-dimethyl-1,2,4-triazol-4-yl)phenoxy side chains. Those were combined with less basic amines (cf. Table 3), which led to the identification of 13a (IC50 = 0.090 μM). While only weak inhibition of CYP2D6 was observed (IC50 = 9.500 μM), this molecule still showed low permeability. Interestingly replacing the chlorine atom in 13a in the 4-(3,5-dimethyl-1,2,4-triazol-4-yl)phenoxy side chain by a smaller, lipophilic fluorine atom and adding another chlorine 8817



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Table 3. NHE3 Inhibitors 13−15 from the Optimization Program

of the 4-chloro substituent in 14j by a fluorine resulted in a slightly lower NHE3 inhibition (14n, IC50 = 0.168 μM). Replacing the other 6-chloro atom in 14j by a fluorine has the same detrimental effect on activity, as exemplified by 14o (IC50 = 0.123 μM). Adding a methyl group in 3-position to the piperazine in 14j led to the equipotent 14m (IC50 = 0.020 μM). Replacing piperazine by octahydropyrrolopyrrole did slightly affect NHE3 inhibition (14k, IC50 = 0.028 μM) but had a detrimental effect to permeability and CYP2D6 (IC50 = 0.900 μM). In contrast, replacing the piperazine by the spirocyclic diazaspirononane side chain slightly increases NHE3 inhibition (14l, IC50 = 0.011 μM). Some active molecules were obtained with 3-aminopiperidine side chains. Here the 3-aminopiperidine 14f (IC50 = 0.004 μM) was 5 times more active than the piperazine 14j. The activity of

side chain resulted in compound 13b with lower activity (IC50 = 0.588 μM), higher CYP2D6 inhibition of 2.400 μM, but higher permeability. Therefore, increased lipophilicity and less basic amino side chains produced interesting NHE3 inhibitors with 13d as best analogue. Further investigations were directed toward dibasic amines combined with lipophilic substitutions (cf. Table 3). As amino side chains, we have employed piperazine, 3-aminopiperidine, 1,4-diazepane, and bicyclic and spirocyclic systems (14a−s). The unsubstituted indane with piperazine and 4-methylsulfonylphenoxy side chains only led to moderate NHE3 inhibition (14a, IC50 = 6.680 μM). Adding a chlorine in 6-position increases activity (14b, IC50 = 0.540 μM), while two chlorines in 4- and 6-positions resulted in the potent 14j (IC50 = 0.022 μM). Although permeability was acceptable, it inhibited CYP2D6 with an IC50 value of 80% contribution) refer to regions, where steric bulk is favorable for affinity, while yellow contours (80% contribution) refer to regions where negatively charged substituents are unfavorable for affinity, and red contours (80% contribution) refer to regions where steric bulk is favorable, and yellow contours (80% contribution) refer to regions where negatively charged substituents are unfavorable, and red contours (80% contribution) refer to regions where hydrophilic substituents are favorable, and yellow contours (80% contribution). Those regions are located close to the 2,5-dimethyltriazolyl moiety, thus showing the influence of the polar nitrogen atoms in this ring. In addition, polarity is also favored around the hydroxy group of the pyrrolidine ring. The effect of favorable polarity appears to be correlated with electrostatic properties for these substituents and can be deduced by CoMSIA with its additional hydrophobic field. Additionally regions surrounding the aromatic triazole appear to be preferable for polar interactions. Preferred hydrophobic interactions are indicated by yellow contour regions (30% at a concentration of 10 μM. These targets included the GABA gated Cl-channel (55% inhibition at 10 μM, IC50 = 12 μM) and phosphodiesterase PDE4 (54% inhibition at 10 μM, IC50 = 6.6 μM), respectively. Moreover, 13d was profiled against a panel of 20 different ion channels and 30 different kinases. None of those channels and kinases were inhibited with relevant IC50 values of 50 μM for CYP2D6 and CYP3A4 and 0.9 μM for CYP2C9, low MBI potential, a very weak CYP gene induction potential, and no inhibitory potency toward P-gp, drug−drug interactions were considered to be unlikely at concentrations of the pharmacologically active dose (1 mg/kg in vivo in pigs).

CO of Leu213, is also favorable. This is also observed for positive charge close to the methyl of the 4-methylsulfonyl group, which is directed toward a more open region of this substrate binding site. Furthermore, red contour regions, indicating a preference for negative charge, surround the sulfone oxygen atoms. This potentially indicates favorable interactions of the −SO2Me group to the Ser217-OH, Arg221guanidine, and Phe483-NH. In this way, the interpretation of this 3D-QSAR model from a consistent alignment hypothesis provided reasonable explanations for SAR trends in this series and was useful to understand potential key ligand features associated with CYP2D6 inhibition. Physicochemical Profiling of 13d. For in vitro profiling of the best compound 13d, the crystalline mono-HCl salt was used, while the single X-ray crystal structure was obtained using the free base. Due to more favorable physicochemical properties, the monophosphate salt was later selected for further preclinical studies. Preliminary data on solid- and solution-state properties for the 13d monophosphate salt such as crystallinity, polymorphism, stability, and solubility in aqueous buffer suggested no obvious issues precluding further development. A log D7.4 value of 2.06 and a log P value of 2.05 were experimentally determined. Furthermore, pKa values of 2.9 and 5.9 were experimentally determined. From investigation of computed physicochemical descriptors, no violation of the ruleof-554 was found. In Vitro Profiling of 13d. 13d was experimentally tested against different NHE3 orthologues. IC 50 values were determined using the same FLIPR assay protocol as for the human orthologue. Experimental IC50 values of 0.0061 μM for cat NHE3, 0.0078 μM for pig NHE3, and 0.0102 μM for rat NHE3 were obtained. The identity of pairwise comparison of human, rat, cat, dog, pig, and rabbit NHE3 sequences is high and ranges from 87% to 98%. Selectivity toward human NHE1 and NHE2 subtypes is very high; a split of >5000 was observed after experimental testing. Experimental testing using the same FLIPR assay protocol results in an IC50 value of >100 μM for NHE1 (six replicates) and >100 μM for NHE2 (four replicates). Sequence identity between NHE3 and NHE1 or NHE2 is 44% and 49%, respectively. A factor of ∼9 in favor of NHE3 was observed, when testing selectivity against NHE5 with 59% sequence identity to NHE3. The IC50 value for NHE5 using the same assay from 11 replicates was determined as 0.291 μM. In addition, 13d was experimentally tested against a panel of 108 relevant, diverse targets. Only four targets out of this 8824



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Figure 9. Effect of 13d on pig upper airway collapsibility after intraduodenal administration of 1 and 3 mg/kg, respectively.

doses, while in a respective vehicle treated control group no effect on collapsibility was found (n = 3) (Figure 9). Concerning the onset of action, full inhibition of upper airway collapsibility was achieved 1 and 1.5 h after administration at 1 and 3 mg/kg, respectively. To evaluate whether NHE3 inhibition shows an unwanted effect on blood pressure or electrophysiology of the heart, 13d was subjected to a safety study in anesthetized mongrel dogs (intravenous, 10 mg/kg). 13d did not induce major hemodynamic or electrophysiological changes. Pharmacology and safety data confirm previous findings that NHE3 inhibition clearly has the potential for treating sleep apneas.

Furthermore, the compound was found to be negative in relevant safety assays, namely, AMES II (300 μg/mL), in vitro micronucleus test (MNT, 50 μg/mL), and FETAX assay (62.5 μg/mL). In Vivo Pharmacokinetics of 13d. After single oral administration of 10 mg/kg 13d (MC/Tween 80 formulation) to male Sprague Dawley rats, the pharmacokinetic parameters of 13d in plasma samples were determined. The maximum plasma concentration of 13d (0.57 μg/mL) was observed 1 h after drug administration. At 24 h after administration, the plasma concentration was below the lower limit of quantitation (LLOQ = 1 ng/mL). Its half-life was moderate (2.7 h) and the oral bioavailability was good (42%), as calculated from the area under the curve AUC(0−inf). By comparison of concentrations of 13d in plasma and different tissues, the order of exposure levels in terms of Cmax and AUC (area under the curve) was brain < plasma < heart < liver. The time profile of all investigated plasma and tissues was very similar. In a subsequent dose-escalation study, the increase in exposure for AUC and Cmax was overdose-proportional between low (10 mg/kg) and intermediate dose group (100 mg/kg) with a factor of 2.3. However, the increase of AUC exposure between the intermediate to the high dose group (300 mg/kg) was doseproportional (factor 1.1), while Cmax was saturated. The maximum mean plasma concentration of 13d in pigs (Duroc Landrace) after oral administration (3 mg/kg; agarose added with sugar and crushed pellets) was observed 0.5 h after administration and shows a high interindividual variability (Figure 8). In this experiment, the half-life was found to be very long (18 h, ranging from 8.1 to 33 h). Bioavailability calculated from the AUC(0−24) values was also extremely variable (ranging from 18% to 98% depending on the individual animal). Pharmacology of 13d. While previous studies8b,c,9c already indicated a potential for treating sleep apneas with NHE3 inhibitors, a more direct model for testing the potential of drug candidates for treating obstructive sleep apnea vs central apneas has been reported.24 In this model the effect of 13d was evaluated. Because of the impossibility for oral treatment in anesthetized pigs, the intraduodenal route was chosen. Upper airway collapses were induced by applying negative pressures of −50, −100, and −150 cmH2O to the upper airway of urethane−chloralose anesthetized pigs.24 Upper airway collapse tests in the pig were then performed before and at 0.5, 1, 2, 3, and 4 h after intraduodenal administration of the NHE3 inhibitor. 13d at 1 and 3 mg/kg (n = 3 in each group; bioavailability 61% for 3 mg/kg; bioavailability for 1 mg/kg was not determined) showed a full inhibition of upper airway collapsibility in every treated pig at all negative pressure levels that exceeded the duration of the experiment of 4 h at both

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CONCLUSIONS In this report, the identification of the potent NHE3 inhibitor 13d by optimization starting from 1-phenoxy-2-aminoindanes as HTS hits is described. During optimization, NHE3 inhibitory potency, in vitro permeability, and CYP2D6 inhibition were identified as key optimization parameters and then successfully addressed. The construction and interpretation of 3D-QSAR models for NHE3 and CYP2D6 plus global models for Caco-2 permeability and CYP2D6 inhibition were useful for property prediction and to identify potential chemical features linked to each biological property. The promising in vitro profile for 13d translated into favorable in vivo pharmacokinetics, which allowed pharmacological studies toward a treatment of sleep apneas to be carried out. Interestingly, 13d showed a full inhibition of upper airway collapsibility, which highlighted the potential of this molecule for further investigation and the value of targeting NHE3 as approach for treating sleep apneas.

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EXPERIMENTAL SECTION

Materials. Solvents and other reagents were used as received without further purification. Purification of Compounds. Normal phase column chromatography was carried out on Merck silica gel 60 (230−400 mesh). Reversed phase high pressure chromatography was conducted on an Agilent 1100 instrument using an Agilent Prep C18 column (10 μm, 30 mm × 250 mm). Varying ratios of acetonitrile and 0.1% trifluoroacetic acid in water were used as solvent systems. Thin-layer chromatography was carried out on TLC aluminum sheets with silica gel 60F254 from Merck. Analytic Methods. NMR spectra were recorded in CDCl3 or DMSO-d6 on either a Bruker ARX 500 or a Bruker DRX 400. Chemical shifts are reported as δ values from an internal tetramethylsilane standard. Purity and characterization of compounds were established by a combination of analytical UPLC−MS and NMR analytical techniques. All tested compounds were found to be >95% pure by analytical UPLC−MS analysis unless otherwise stated. UPLC−MS analyses were performed with a Waters ACQUITY UPLC system, column C18 1.7 μm, 2.1 mm × 50 mm; gradient (H2O + 0.05% FA)/(AcN + 0.035% FA) 95:5 (0 min) to 5:95 (2 min) to 8825



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5:95 (2.6 min) to 95:5 (2.7 min) to 95:5 (3 min), flow rate 0.9 mL/ min, temperature 55 °C; mass detector Waters SQD single quadrupole, ESI+ mode. Enantiomeric excess was determined by chiral analytical HPLC using a Waters 2695 HPLC system with a Waters 2996 PDA detector. Method a: column Chiralpak AS-H, 250 mm × 4.6 mm, isocratic eluent EtOH/MeOH (1:1), flow rate 0.5 mL/ min, temperature 30 °C. Method b: column Chiralpak AS-H, 250 mm × 4.6 mm, eluent EtOH, flow rate 0.5 mL/min, temperature 30 °C. Method c: columns 2 serial Chiralpak IA, 250 mm × 4.6 mm, isocratic eluent heptane/EtOH/MeOH (10:1:1) + 0.1%TFA, flow rate 1.25 mL/min, temperature 30 °C. General Procedures. General procedures for the synthesis of tested compounds 10 and 13−15 are given and illustrated by the representative synthesis of compound 13d. For the synthesis of all other compounds see Supporting Information. A: Indan-1-ones (4). Oxalyl chloride (3.4 equiv) was cautiously added to a solution of the propionic acid (1.0 equiv) in CH2Cl2 (1.4 mL/mmol of propionic acid) and DMF (0.01 mL/mmol of propionic acid) (intense gas formation). The resulting clear solution was stirred for a further 6 h, and then volatile components were removed under reduced pressure. The corresponding acid chloride was used in the next reaction without further purification. A solution of the acid chloride in CH2Cl2 (1.2 mL/mmol of propionic acid chloride) was added dropwise to a solution of AlCl3 (1.3 equiv) in CH2Cl2 (0.75 mL/mmol AlCl3) at 0 °C. After the addition was complete, the ice bath was removed and the reaction mixture heated under reflux for further 3 h. The mixture was poured into ice−water, and the aqueous layer was extracted with CH2Cl2. The combined organic layers were dried with Na2SO4 and filtered, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography. If the ring closure did not take place regioselectively, the regioisomers were separated by column chromatography. B: Indan-1-ols (5). NaBH4 (1 mmol/mmol of indanone) was cautiously added in portions to a solution of the indanone (1 equiv) in EtOH (4 mL/mmol of indanone) at 10 °C. After addition was complete, the solution was stirred at rt for 3−16 h and the reaction mixture was concentrated under reduced pressure. The suspension was poured on ice−water, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were dried with Na2SO4, filtered, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography. C: 1H-Indenes (6). A solution of the indanol (1 equiv) and ptoluenesulfonic acid monohydrate (0.1 equiv) in toluene (4 mL/mmol of indanol) was heated under reflux using a Dean−Stark water trap for 1−2 h. The solution was cooled to rt and washed with saturated aq NaHCO3 solution. The organic layer was dried with Na2SO4 and filtered, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography. D: (1aS,6aR)-6,6a-Dihydro-1aH-1-oxa-cyclopropa[a]indenes (7). 4-(3-Phenylpropyl)pyridine N-oxide (0.04 equiv) was added to a solution of the indene (1 equiv) in CH2Cl2 (1.2 mL/mmol of indene) and (S,S)-(+)-N,N′-bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexanediaminomanganese(III) chloride (0.01 equiv). The reaction solution was stirred for 10 min and cooled to −2 °C. Half-saturated aq K2CO3 solution (0.5 mL/mmol of indene) was added, and while stirring vigorously, aq NaOCl solution (1.25 mL/mmol of indene; 13% free chlorine) was slowly added dropwise. Immediately thereafter the pH was adjusted to pH 11−12 with 0.1 M phosphate buffer (pH 7.5). The two-layer system was stirred vigorously for additional 4 h, during which the temperature slowly rose to 5 °C. The layers were separated and the aq layer was extracted with CH2Cl2. The combined organic layers were washed with saturated aq Na2S2O3 solution and water, dried with Na2SO4 and filtered, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography. The obtained product was additionally recrystallized from heptane. E: Addition of Amines to Epoxides (7) To Yield Indan-2-ol (9). A solution of the epoxide 7 (1 equiv) and the appropriate secondary amine (1.05 equiv) in acetonitrile (1 mL/mmol of epoxide)

was heated at 80 °C for 1−6 h, monitoring by TLC. The solvent was removed under reduced pressure and the crude product was purified by column chromatography. F: Mitsunobu Reaction Products (11). A 1 M solution of DIAD (1.15 equiv) in THF was added dropwise to a solution/suspension of the amino alcohol (1 equiv), PPh3 (1.15 equiv), and the appropriate phenol (1.15 equiv) in THF (3 mL/mmol of amino alcohol). The solution was stirred at rt for 1−16 h, the reaction was monitored by TLC, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography. G: Deprotection of Silyl Ethers To Yield Final Compounds (13). To a solution of the silyl ether (1 equiv) in THF (4 mL/mmol silyl ether) at rt a 1 M solution of TBAF in THF (1.5 equiv) was added, and stirring at rt was continued for 4 h. The reaction mixture was diluted with water and ethyl acetate. The organic layer was washed with sat. aq NaHCO3 solution, dried with Na2SO4, and filtered. The solvent was removed under reduced pressure. The crude product was purified by column chromatography. 4,6-Dichloroindan-1-one (4b). Compound 4b was synthesized according to synthesis A. Yield 9.30 g, (100%). 1H NMR (DMSO-d6, 500 MHz) δ 7.96 (d, 1H, J = 1.9 Hz), 7.64 (d, 1H, J = 1.9 Hz), 3.04 (t, 2H, J = 6.0 Hz), 2.75 (t, 2H, J = 6.0 Hz). MS, m/z: 201 (M + H)+. 4,6-Dichloroindan-1-ol (5b). Compound 5b was synthesized according to synthesis B. Yield 1.90 g, (99%). 1H NMR (DMSO-d6, 500 MHz): δ 7.43 (s, 1H), 7.31 (s, 1H), 5.52 (d, 1H, J = 6.1 Hz), 5.09 (m, 1H), 2.91 (m, 1H), 2.70 (m, 1H), 2.39 (m, 1H), 1.81 (m, 1H). 5,7-Dichloro-1H-indene (6b). Compound 6b was synthesized according to synthesis C. Yield 1.47g, (87%). 1H NMR (DMSO-d6, 500 MHz): δ 7.52 (d, 1H, J = 1.8 Hz), 7.38 (d, 1H, J = 1.9 Hz), 6.95 (m, 1H), 6.80 (m, 1H), 3.47 (t, 2H, J = 1.9 Hz), 2.50 (m, 2H). MS, m/ z: 185 (M + H)+. (1aS,6aR)-3,5-Dichloro-6,6a-dihydro-1aH-1-oxacycloprop[a]indene (7c). Compound 7c was synthesized according to synthesis D. Yield 60.0 g, (57%), 98% ee after recrystallization. 1H NMR (DMSO-d6, 500 MHz): δ 7.66 (d, 1H, J = 1.8 Hz), 7.51 (d, 1H, J = 1.8 Hz), 4.45 (m, 1H), 4.23 (m, 1H), 3.09 (dd, 1H, J = 18.6, 1.0 Hz), 2.96 (dd, 1H, J = 18.6, 2.9 Hz), tR (method a): 8.48 (major), 8.90 min (minor). (1R,2R)-1-[(R)-3-(tert-Butyldiphenylsilanyloxy)pyrrolidin-1yl]-4,6-dichloroindan-2-ol (9k). Compound 9k was synthesized according to synthesis E. Yield 26.2 g, (97%). 1H NMR (DMSO-d6, 500 MHz): δ 7.60−7.56 (m, 4 H), 7.47−7.40 (m, 7H), 7.26 (s, 1H), 5.13 (d, 1H, J = 5.5 Hz), 4.39 (m, 1H), 4.29 (m, 1H), 3.93 (d, 1H, J = 2.4 Hz), 3.08 (dd, 1H, J = 16.6, 5.5 Hz), 2.73 (m, 1H), 2.63−2.58 (m, 3H), 2.54 (m, 1H), 1.89 (m, 1H), 1.67 (m, 1H), 0.99 (s, 9H). MS, m/ z: 527 (M + H)+. 4-(4-{(1S,2S)-2-[(R)-3-(tert-Butyldiphenylsilanyloxy)pyrrolidin-1-yl]-4,6-dichloroindan-1-yloxy}-3-fluorophenyl)3,5-dimethyl-4H-[1,2,4]triazole (11a). Compound 11a was synthesized according to synthesis F. Yield 2.50 g, (46%). 1H NMR (DMSO-d6, 400 MHz): δ 7.67 (t, 1H, J = 8.7 Hz), 7.63−7.53 (m, 6H), 7.46−7.34 (m, 6H), 7.28 (m, 1H), 7.20 (s, 1H), 5.96 (d, 1H, J = 5.6 Hz), 4.29 (m, 1H), 3.49 (m, 1H), 3.19 (dd, 1H, J = 16.6, 7.4 Hz), 2.84−2.76 (m, 2H), 2.68 (m, 2H), 2.58 (dd, 1H, J = 9.6, 4.3 Hz), 2.17 (s, 6H), 1.90 (m, 1H), 1.72 (m, 1H), 0.98 (s, 9H). MS, m/z: 716 (M + H)+. (R)-1-{(1S,2S)-4,6-Dichloro-1-[4-(3,5-dimethyl-1,2,4-triazol-4yl)-2-fluorophenoxy]indan-2-yl}pyrrolidin-3-ol (13d). Compound 13d was synthesized according to synthesis G. Yield 1.74 g (79%). 1H NMR (DMSO-d6, 500 MHz): δ 7.71 (t, 1H, J = 8.7 Hz), 7.61−7.57 (m, 2H), 7.33 (m, 1H), 7.23 (s, 1H), 5.98 (d, 1H, J = 5.6 Hz), 4.73 (d, 1H, J = 4.7 Hz), 4.18 (m, 1H), 3.49 (m, 1H), 3.19 (dd, 1H, J = 16.6, 7.4 Hz), 2.88−2.78 (m, 2H), 2.63−2.55 (m, 2H), 2.43 (dd, 1H, J = 9.6, 4.3 Hz), 2.18 (s, 6H), 1.95 (m, 1H), 1.56 (m, 1H), 0.98 (s, 9H). MS, m/z: 477 (M + H)+. 8826



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(6) Vallon, V.; Schwark, J.-R.; Richter, K.; Hropot, M. Role of Na+/ H+ exchanger NHE3 in nephron function: micropuncture studies with S3226, an inhibitor of NHE3. Am. J. Physiol.: Renal Physiol. 2000, 278, F375−F379. (7) Wiemann, M.; Schwark, J.-R.; Bonnet, U.; Jansen, H. W.; Grinstein, S.; Baker, R. E.; Lang, H.-J.; Wirth, K.; Bingmann, D. Selective inhibition of the Na+/H+ exchanger type 3 activates CO2/H +-sensitive medullary neurones. Pfluegers Arch. 1999, 438, 255−262. (8) (a) Wiemann, M.; Piechatzek, L.; Goepelt, K.; Kiwull-Schöne, H.; Kiwull, P.; Bingmann, D. The NHE3 inhibitor AVE1599 stimulates phrenic nerve activity in the rat. J. Physiol. Pharmacol. 2008, 59, 27−36. (b) Kiwull-Schöne, H.; Wiemann, M.; Frede, S.; Bingmann, D.; Wirth, K. J.; Heinelt, U.; Lang, H.-J; Kiwull, P. A novel inhibitor of the Na+/ H+ exchanger type 3 activates the central respiratory CO2 response and lowers the apneic threshold. Am. J. Respir. Crit. Care Med. 2001, 164, 1303−1311. (c) Kiwull-Schöne, H.; Kiwull, P.; Frede, S.; Wiemann, M. Role of brainstem sodium/proton exchanger 3 for breathing control during chronic acid-base imbalance. Am. J. Respir. Crit. Care Med. 2007, 176, 513−519. (9) (a) Kiwull-Schöne, H.; Teppema, L.; Wiemann, M.; Kalhoff, H.; Kiwull, P. Pharmacological impact on loop gain properties to prevent irregular breathing. J. Physiol. Pharmacol. 2008, 59, 37−45. (b) Wiemann, M.; Bingmann, D. Ventrolateral neurons of medullary organotypic cultures: Intracellular pH regulation and bioelectric activity. Respir. Physiol. 2001, 129, 57−70. (c) Abu-Shaweesh, J. M.; Dreshaj, I. A.; Martin, R. J.; Wirth, K. J.; Heinelt, U.; Haxhiu, M. A. Inhibition of Na+/H+ exchanger type 3 reduces duration of apnea induced by laryngeal stimulation in piglets. Pediatr. Res. 2002, 52, 459−464. (d) Wang, Q.; Zhou, R.; Zhang, C.; Dong, H.; Ma, J.; Wang, G. Inhibition of central Na+/H+ exchanger type 3 can alleviate sleep apnea in Sprague-Dawley rats. Chin. Med. J. (Beijing, China, Engl. Ed.) 2014, 127, 48−53. (e) Ribas-Salgueiro, J. L.; Matarredona, E. R.; Ribas, J.; Pasaro, R. Enhanced c-Fos expression in the rostral ventral respiratory complex and rostral parapyramidal region by inhibition of the Na+/H+ exchanger type 3. Auton. Neurosci. 2006, 126−127, 347− 354. (10) Rackelmann, N.; Bialy, L.; Englert, H.; Wirth, K.; Arndt, P.; Weston, J.; Heinelt, U.; Follmann, M. Preparation of substituted aminoindanes and their analogs as NHE3 inhibitors and use in the treatment of diseases. PCT Int. Appl. WO 2010025856, 2010. (11) Schwark, J. R.; Jansen, H. W.; Lang, H. J.; Krick, W.; Burckhardt, G.; Hropot, M. S3226, a novel inhibitor of Na+/H+ exchanger subtype 3 in various cell types. Pfluegers Arch. 1998, 436, 797−800. (12) Rajendran, V. M.; Nanda Kumar, N. S.; Tse, C. M.; Binder, H. J. Na-H Exchanger isoform-2 (NHE2) mediates butyrate-dependent Na+ absorption in dextran sulfate sodium (DSS) induced colitis. J. Biol. Chem. 2015, 290, 25487−25496. (13) Lang, H. J.; Heinelt, U.; Hofmeister, A.; Wirth, K.; Gekle, M.; Bleich, M. Preparation of N-thiophenyl-1H-benzimidazol-2-amines and related compounds as NHE-3 sodium-proton exchanger inhibitors. PCT Int. Appl. WO 2003101984, 2003. (14) Lang, H. J. Preparation of 4-phenyl-1,2,3,4-tetrahydroisoquinolines as NHE-3 and NHE-5 sodium-proton exchanger inhibitors. Ger. Offen. DE 102005044817, WO 2007033773, 2007. (15) Carreras, C.; Charmot, D.; Jacobs, J. W.; Labonte, E.; Lewis, J. G. Hydrogen ion-sodium-exchanging proteins (NHE3)-binding compounds as phosphate transport inhibitors and therapeutic uses thereof. PCT Int. Appl. WO 2014169094, 2014. (16) (a) Scholz, W.; Albus, U.; Lang, H. J.; Linz, W.; Martorana, P. A.; Englert, H. C.; Schoelkens, B. A. Hoe 694, a new sodium/hydrogen ion exchange inhibitor and its effects in cardiac ischemia. Br. J. Pharmacol. 1993, 109, 562−568. (b) Weichert, A.; Faber, S.; Jansen, H. W.; Scholz, W.; Lang, H.-J. Synthesis of the highly selective Na+/H+ exchange inhibitors cariporide mesilate and (3-methanesulfonyl-4piperidino-benzoyl) guanidine methanesulfonate. Arzneim. Forsch. 1997, 47, 1204−1207. (17) (a) Lang, H. J.; Weichert, A.; Kleemann, H. W.; Englert, H.; Scholz, W.; Albus, U. Preparation of benzoylguanidines as drugs, e.g., antiarrhythmic agents. Eur. Pat. Appl. EP 589336, 1994. (b) Scholz,

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00624. Details on crystal data from X-ray structure determination of 13d; experimental and predicted NHE3 inhibition; experimental and predicted ADME properties for selected compounds; NHE3 inhibition, permeability, CYP inhibition, lability, and induction of CYP enzyme assays; pharmacokinetic study protocols; materials, purification, analytical methods, and an experimental section for all synthesized compounds (PDF) Molecular formula strings and some data (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*N.R.: phone, +49 69 305 5277; e-mail, Nils.Rackelmann@ sanofi.com. *H.M.: phone, +49 69 305 84329; e-mail, Hans.Matter@sanofi. com. Present Address †

M.F.: Bayer Pharma AG, Aprather Weg 18A, D-42113 Wuppertal, Germany.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge discussions with R. Jäger and support with in silico ADMET modeling by C. Giegerich and K.F. Schmidt (all from Sanofi).

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ABBREVIATIONS USED DIAD, diisopropyl azodicarboxylate; esd, estimated standard deviation; FETAX, frog embryo teratogenesis assay: Xenopus; FLIPR, fluorometric imaging plate reader; GA, genetic algorithm; LLOQ, lower limit of quantification; LOQ, limit of quantification; MBI, mechanism based inhibition; MLR, multilinear regression; MMFF, Merck molecular force field; MNT, micronucleus test; MOE, molecular operating environment; NHE, sodium hydrogen exchanger; RCSB, research collaboratory for structural bioinformatics; RT-PCR, reverse transcription polymerase chain reaction; SD, standard deviation; std*coeff, standard deviation * coefficient

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REFERENCES

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