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. 2022 Feb 1;5(2):80–88. doi: 10.1021/acsptsci.1c00238

Development of Inhaled GABAA Receptor Modulators to Improve Airway Function in Bronchoconstrictive Disorders

Nicolas M Zahn †, M S Rashid Roni †, Gene T Yocum ‡, Michelle J Meyer †, Daniel A Webb †, Md Yeunus Mian †, James M Cook †, Douglas C Stafford §, Charles W Emala ‡, Leggy A Arnold †,§,*
PMCID: PMC8844962  PMID: 35187417

Abstract

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We report the modification of MIDD0301, an imidazodiazepine GABAA receptor (GABAAR) ligand, using two alkyl substituents. We developed PI310 with a 6-(4-phenylbutoxy)hexyl chain as used in the long-acting β2-agonist salmeterol and PI320 with a poly(ethylene glycol) chain as used to improve the brain:plasma ratio of naloxegol, a naloxone analogue. Both imidazodiazepines showed affinity toward the GABAAR binding site of clonazepam, with IC50 values of 576 and 242 nM, respectively. Molecular docking analysis, using the available α1β3γ2 GABAAR structural data, suggests binding of the diazepine core between the α1+/γ2– interface, whereas alkyl substituents are located outside the binding site and thus interact with the protein surface and solvent molecules. The physicochemical properties of these compounds are very different. The solubility of PI310 is low in water. PEGylation of PI320 significantly improves aqueous solubility and cell permeability. Neither compound is toxic in HEK293 cells following exposure at >300 μM for 18 h. Ex vivo studies using guinea pig tracheal rings showed that PI310 was unable to relax the constricted airway smooth muscle. In contrast, PI320 induced muscle relaxation at organ bath concentrations as low as 5 μM, with rapid onset (15 min) at 25 μM. PI320 also reduced airway hyper-responsiveness in vivo in a mouse model of steroid-resistant lung inflammation induced by intratracheal challenge with INFγ and lipopolysaccharide (LPS). At nebulized doses of 7.2 mg/kg, PI320 and albuterol were equally effective in reducing airway hyper-responsiveness. Ten minutes after nebulization, the lung concentration of PI320 was 50-fold that of PI310, indicating superior availability of PI320 when nebulized as an aqueous solution. Overall, PI320 is a promising inhaled drug candidate to quickly relax airway smooth muscle in bronchoconstrictive disorders, such as asthma. Future studies will evaluate the pharmacokinetic/pharmacodynamic properties of PI320 when administered orally.

Keywords: airway function, asthma, GABA, bronchoconstrictive disorders

Asthma is a common chronic respiratory condition that affects about 7.8% of the US population.1 Wheezing, chest tightness, and coughing are caused by reversible airway constriction, excess of mucus, and airway inflammation.2 Currently, approved medications are based on β2-adrenoreceptor agonists, corticosteroids, leukotriene receptor antagonists, muscarinic receptor antagonists, and biologic therapies.3 A long-term, affordable oral medication to control asthma symptoms with negligible adverse effects is still unavailable, prompting further research to validate new therapeutic modalities. Among those, we have investigated imidazodiazepines, which relax constricted human airway smooth muscle and reduce airway hyper-responsiveness (AHR) in several murine asthma models.4−9 The compounds have a high affinity for GABAA receptors (GABAAR), which are expressed on airway smooth muscle10,11 and inflammatory cells.9 To avoid central nervous system (CNS) adverse effects, we engineered pharmacokinetic properties of these compounds to prevent blood–brain barrier transit. We determined compound efficacy when nebulized and showed a reduction of AHR at doses equivalent or lower than albuterol.12

The addition of a hydrophobic group to a catecholamine scaffold resulted in the generation of long-acting β2-agonists (LABA), such as formoterol and salmeterol.13,14 Other LABAs, such as vilanterol, have been approved in combination with inhaled corticosteroids such as fluticasone.15 The long-acting properties of salbutamol have been attributed to microkinetics related to lipophilicity and exosite binding.16 Procopiou et al. proposed exosite binding for vilanterol, as well (Figure 1).17

Figure 1.

Figure 1

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Compound structures of conjugated drugs.

We describe the synthesis and characterization of new imidazodiazepines that have long alkyl chain substituents like salmeterol and vilanterol for the alleviation of AHR. The first strategy included the incorporation of the hydrophobic group found in salbutamol. The second strategy includes the addition of a poly(ethylene glycol) chain. PEGylation has been used extensively for improving the stability and half-life of biological molecules and has been applied recently for small-molecule drugs.18 Naloxegol, for example, is naloxone with a heptaethylene glycol substitution, which has been approved for opioid-induced constipation.19 The CNS effects of naloxegol are minimal with a low brain:plasma ratio. PEGylation also increases the water solubility of hydrophobic small molecules, which still retain oral availability dependent on the number of ethylene glycol units. Distribution and clearance of PEGylated small molecules in the lung have not been described, which prompted investigations of MIDD0301 analogues using constricted tracheal rings ex vivo and murine in vivo asthma models.

Results and Discussion

PI310 and PI320 were synthesized using parent compound MIDD0301 and corresponding amines, as illustrated in Scheme 1.

Scheme 1. Synthesis of PI310 and PI320.

Scheme 1

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MIDD0301 was coupled with 2,5,8,11,14-pentaoxahexadecan-16-amine in the presence of N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) and N-methylmorpholine resulting in PI320 at 62% yield as a viscous oil. For the synthesis of PI310, 6-(4-phenylbutoxy)hexan-1-amine was used, which has been described as an unstable amine.20 Therefore, 6-(4-phenylbutoxy)hexan-1-amine was generated from tert-butyl (6-(4-phenylbutoxy) hexyl)carbamate with trifluoroacetic acid and used as crude product for the next reaction after neutralization and extraction. The coupling was achieved with the acid chloride of MIDD0301 in the presence of N-methylmorpholine, resulting in PI310 at a 32% overall yield. Like all chiral imidazodiazepines in this class, PI310 and PI320 have two stable configurations that can be distinguished by nuclear magnetic resonance.21

Next, we investigated the affinity of these compounds at the GABAAR. Here, rat brain homogenate was used in combination with 3H-flunitrazepam. GABAAR subtypes that bind flunitrazepam consist of α1–3,5,6 β1–3 γ1–3/δ GABAARs.22−25 Relative expression of GABAARs in the brain consist of 43% α1β2γ2, 15% α2β3γ2 plus 8% α2βγ1, 10% α3β3γ2, 6% α4βγ/δ, 4% α5β3γ2, and 4% α6β2γ2/δ.26 Thus, compounds such as clonazepam with high activity in this assay bind predominately α1–3β2–3γ1–2 GABAARs (Figure 2).

Figure 2.

Figure 2

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Competition assay of GABAAR ligand 3H-flunitrazepam with PI310 and PI320 using rat brain extract.

An IC50 of 576 nM was observed for PI310, which is significantly higher than the reported IC50 of 72 nM for MIDD0301.27 Thus, the conjugation to 6-(4-phenylbutoxy)hexan-1-amine reduced binding 8-fold. PI320 shows better binding at GABAARs with an IC50 of 242 nM. Overall, these data confirm that even large substituents are tolerated at the carboxyl position for imidazobenzodiazepines; however, the hydrophobic nature of the substituents has an influence on binding affinity. To investigate possible binding modes of these ligands, we used the reported crystal structure of α1β3γ2 GABAAR to dock both ligands (Figure 3).

Figure 3.

Figure 3

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Docking study with PI310 and PI320. Overlay of docked conformations of PI310 (magenta) and PI320 (green) in the complex with the α1β3γ2L GABAA receptor using the structure 6HUO.28 The α1+/γ2– interface is indicated as α1 (red) and γ2 (cyan). Hydrogen and halogen bonds are indicated as dashed lines.

The ligands were docked at the alprazolam binding site, which is located at the α1+/γ2– interface. The bromine atom is near Asn60 and His102. Experiments with isocyanate-substituted diazepam have resulted in alkylation of these residues.29 Halogen bonding with Phe100 located on α1 subunit is possible. Hydrogen bond interactions have been reported for other imidazodiazepines such as Hz166 with Ser205.30 Amides PI310 and PI320 can additionally interact with Glu189 located on γ2 subunit. The alkyl chain of both compounds is located on the periphery of the protein complex and is mostly exposed to water and hydrophilic amino acid residues. In contrast to the 6-(4-phenylbutoxy)hexyl side chain, the 2,5,8,11,14-pentaoxahexadecanyl side chain has multiple hydrogen-accepting groups that can interact via hydrogen bond interactions with charged residues on the protein surface, resulting in a higher affinity for GABAARs.

Next, we investigated the lung smooth muscle-relaxing effects of PI310 and PI320. Here, guinea pig tracheal rings in an ex vivo organ bath were contracted with substance P or acetylcholine and compounds applied at indicated concentrations. The resulting effects of compounds on the contractile force are depicted in Figure 4 as % peak contraction versus time. Representative tracings of contractile force versus time can be found in the Supporting Information.

Figure 4.

Figure 4

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Relaxation of tracheal rings constricted ex vivo. (A,B) Guinea pig trachea rings were contracted with 1 μM substance P and indicated compounds and concentrations. The vehicle contains 0.1% DMSO; (C) guinea pig trachea rings were treated with vehicle or gabazine (200 μM), then contracted with an EC50 concentration of acetylcholine, and subsequently relaxed with PI320. The percentage of muscle force with respect to the initial muscle contraction is shown as determined at various time points. A two-way analysis of variance (ANOVA) repeated measure analysis was applied to determine significance with *, **, ***, and **** equals p < 0.05, 0.01, 0.001, or 0.0001, respectively.

PI310 was unable to relax airway smooth muscle at 100 μM during a 1 h exposure (Figure 4A). In contrast, 100 μM PI320 induced strong relaxation similar to 10 μM formoterol. Both compounds showed quick onset of action in reducing the contractile force by almost 70% within the first 15 min. The parent compound, MIDD0301, exhibited a slower onset of action. The contractile force was reduced by 30% after 15 min at 100 μM. No statistical differences in contractile force reductions were observed for all three compounds after 30 min. The application of different concentrations of PI320 showed that 5 μM was sufficient to significantly reduce the contractile force after 30 min (Figure 4B). Twenty-five micromolar PI320 reduced the contractile force significantly at 15 min. In comparison, MIDD0301 reduced the contractile force at 25 μM but not at 10 μM.7 Additionally, we used acetylcholine as a preconstrictor because its induced contraction is more sustained than substance P (Figure 4C). As expected, a more stable plateau in muscle force was observed for a period of 60 min. One hundred micromolar of PI320 induced ASM relaxation already at 15 min but did not fully relax acetylcholine-constricted trachea rings. Importantly, pretreatment with GABAAR antagonist gabazine partially reversed the effect of PI320. Thus, the pharmacological effect of PI320 is at least in part mediated by the GABAAR. For MIDD0301, we demonstrated previously that tissue penetration is limited when using ex vivo tissue.7 Only 10% of the organ bath concentration was measured in the organ tissue itself; thus, diffusion is limited under those conditions. We anticipate that for PI310 and PI320 the tissue concentration is also lower than the organ bath concentration. To further characterize the physicochemical properties of PI320 and PI310, solubility, permeability, and toxicity assays were conducted (Table 1).

Table 1. Physicochemical Data for MIDD0301, PI310, and PI320.

compound solubility (mg/L)a log Pe (cm/s)b log Dc viability HEK293 LD50 (μM)d
PI310 3.9 ± 0.3 insoluble 5.95 ± 0.08 >300
PI320 1542 ± 55 –5.90 ± 0.01 5.12 ± 0.02 >300
MIDD0301 3220 ± 102 (pH = 6.75)7 –7.12 ± 0.13 (pH = 7.2) 0.33 ± 0.05 (pH = 7)7 >40027
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a

Shake flask assay.

b

PAMPA assay (Pion).

c

Octanol:water coefficient.

d

Cell-based viability assay (18 h).

Due to the limited aqueous solubility of PI310 of 3.9 mg/L, we were not able to achieve a sufficient concentration to carry out the corresponding permeability assay. In contrast, the aqueous solubility of PI320 was excellent at 1542 mg/L. Importantly, the permeability of PI320 (−5.90) was similar to control compound verapamil (−5.76). Verapamil is considered to be a very permeable drug. The parent compound MIDD0301 is negatively charged under neutral pH, which results in not only high solubility (3.2 g/L) but also lower permeability (−7.1 cm/s). The partition coefficient between water and n-octanol is very high for PI310, which might negatively influence passive diffusion. PI320 has a lower log D than PI310 and is more water solubility. MIDD0301 has a low log D at neutral pH, which increased to 2.0 at pH 4.21 Finally, all test compounds have very low toxicity, with LD50 values in excess of 300 μM using embryonic kidney cell line HEK293.

Next, we quantified the time-dependent lung concentration of PI310 and PI320 after single nebulized doses (Figure 5).

Figure 5.

Figure 5

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Pharmacokinetics of nebulized PI310 and PI320 in Swiss Webster mice. Groups of four mice were dosed with 7.2 mg/kg of nebulized compound (20 μL). Lungs were harvested at indicated time points, and PI310 and PI320 were quantified by liquid chromatography–mass spectrometry (LCMS)/MS. Data are depicted as means ± standard deviation (SD). Data were analyzed using nonlinear regression (one-phase decay).

The lung concentrations of PI310 and PI320 were very different after 10 min, showing 42 and 2160 nmol/kg, respectively. Both compounds were soluble in water at 3.2 mg/mL with the addition of 0.17% Tween-20. It can be hypothesized that the nebulizing PI310 solution was not homogeneous and that nonspecific binding to the nebulizer itself could have reduced the administered dose. Another possibility for different lung concentrations could involve binding to the tissue of the nasal cavity and trachea. Therefore, compounds like PI310 with long alkyl chain substituents, such as salmeterol, were administered by a dry powder inhaler, overcoming their challenging physicochemical properties. However, once administered, both compounds showed good half-life of 11.8 and 19.4 min, respectively. This is an improvement compared to the parent compound MIDD0301, which exhibited a half-life of 5.2 min.12 PI320 also has a greater AUC, which was almost 10 times as high as MIDD0301.

Next, we determined the in vivo activity of PI320 in relaxing airway smooth muscle. Here, mouse inflammatory AHR was induced with INFγ and bacterial lipopolysaccharide (LPS) via intratracheal installation (Figure 6). Elevated levels of IFNγ in the lung were observed in neutrophilic asthma patients who responded poorly to steroid treatment.31 One contributing factor was the impaired nuclear translocation of the liganded glucocorticoid receptor in pulmonary macrophages.32 LPS models a microbial infection leading to a macrophage-mediated inflammatory response.33

Figure 6.

Figure 6

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Pharmacological effect of PI320 in a steroid-resistant asthma model. Female Swiss Webster mice received an intratracheal solution of LPS and INFγ 1 day before the measurement. Specific airway resistance (sRaw) was measured repeatedly after nebulization of vehicle, followed by drug in a vehicle and nebulized methacholine using a NAM instrument. Data are depicted as means ± standard error of the mean (SEM) of n = 10. * and *** indicate p < 0.05 and p < 0.001 significance between vehicle and drug-treated groups determined by two-way ANOVA with Bonferroni correlation.

The intratracheal installation of INFγ/LPS markedly increased the sRaw value, a measurement of AHR that is based on phase delay between the nasal and thoracic air flows (Figure 6, black bar). After the first nebulized methacholine challenge (3 mg/mL, 20 μL), the sRaw increased from 3.7 to 4.8 cmH2O*s. Further methacholine challenges increased the sRaw to 8.0 cmH2O*s. Animals that received nebulized albuterol (7.2 mg/kg) prior to the methacholine challenge showed reductions of AHR for the second and third methacholine administrations. Similar results were observed for PI320 at the same dose. PI320 also reduced AHR for the third methacholine challenge at 3 mg/kg and therefore performed better than MIDD0301, which showed no efficacy in this model at 3 mg/kg.12 The precise measurement of lung function parameters such as airway resistance and elastance alternatively can be conducted using an invasive forced oscillation technique.34 Accordingly, using a flexiVent apparatus, we found that 1.5 mg/kg of MIDD0301 was sufficient to reduce airway resistance.12 Future studies with the flexiVent apparatus will be performed to quantify the dose-dependent changes of airway resistance for PI320.

Further investigations with PI320 were conducted with A/J mice that display severe AHR to methacholine without a pre-existing allergic state due to their unique genetic profile.35

The repeated nebulized administration of methacholine induced severe AHR resulting in sRaw values of more than 7.0 cmH2O*s after five applications (Figure 7A). The nebulized administration of albuterol (7.2 mg/kg) prior to the methacholine challenge reduced AHR for the second and subsequent methacholine applications. Similar effects were observed for nebulized 7.2 mg/kg PI320 for the third and subsequent methacholine applications. The reduction of AHR by PI320 was not statically different from albuterol and could be reversed by GABAA receptor antagonist gabazine (Figure 7B). The nebulized application of gabazine (15 mg/kg) increased the sRaw from 7.0 to 9.1 cmH2O*s for the last methacholine application, which is consistent with the previously reported increase of contractile force of acetylcholine-contracted guinea pig tracheal rings.36 Other studies with gabazine included the reversal of PI320 analogue SH-053–2′F-R-CH3 induced negative membrane potential and bradykinin-induced [Ca2+]i of ASM.11

Figure 7.

Figure 7

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Airway smooth muscle relaxation in female A/J mice. (A) sRaw was measured after the nebulized vehicle or PI320 treatment (7.2 mg/kg) followed by repeated treatment with nebulized methacholine. (B) sRaw was measured after nebulized GABAA receptor antagonist gabazine (15 mg/kg) or gabazine followed by PI320 (7.2 mg/kg) and repeated nebulized methacholine applications. Data are depicted as means ± SEM of n = 10. * and *** indicate p < 0.05 and p < 0.001 significance between vehicle and drug-treated animals using a two-way ANOVA with Bonferroni correlation.

We can conclude that the conjugation of MIDD0301 at the carboxyl group can generate amides that retain GABAAR affinity. Other examples of MIDD0301-derived amides and esters have been reported.37−102 Furthermore, compounds like PI320 have excellent physicochemical properties, quick onset of smooth muscle relaxation, and potently alleviate AHR. PI320 can be nebulized as an aqueous solution achieving high lung concentrations and a good half-life. Several hypotheses have been reported to explain how positive allosteric GABAAR modulators affect constricted airways.8 For instance, in the contracted state, the flux of chloride via the GABAAR may reverse, resulting in a relative hyperpolarization that inhibits calcium influx via voltage-gated channels.36 Alternatively, GABAARs are permeant to bicarbonate, which changes the intercellular pH upon activation.11 In turn, this can reduce calcium entry via the STIM-Orai1 complex.39 Further mechanistic studies have to be conducted to confirm such hypotheses. To our knowledge, PI320 is the first example of a PEGylated benzodiazepine, with significant affinity to the GABAARs expressed in the lungs. The development of a new asthma medication targeting receptors other than the β2-adrenoreceptor is especially important for asthma patients with an Arg/Arg-16 genotype, which increases the risk of having an exacerbation in patients taking ICS in conjunction with salmeterol.40 Furthermore, full agonists such as formoterol are known to downregulate the β2-adrenoreceptor in inhaled corticosteroid-treated patients with asthma.41 We are currently investigating if compounds like PI320 are available orally and if brain exposure is limited due to the poly(ethylene glycol) chain.

Methods

Chemistry

Chemicals and solvents were purchased from commercial vendors and used as received. Reaction progress was analyzed by thin-layer chromatography (Polygram Sil UV254, Macherey-Nagel, Bethlehem, PA). 1H, 13C, and 19F NMR spectra were recorded on a Bruker 500 MHz instrument with cryoprobe. The chemical shifts in δ (ppm) are reported with reference to solvent signals for DMSO-d6 (δ = 2.50 ppm for 1H NMR and δ = 39.52 ppm for 13C NMR) and for CDCl3 (δ = 7.20 ppm for 1H NMR and δ = 77.00 ppm for 13C NMR). High-resolution mass spectrometry was performed with a Q-TOF spectrometer (Shimadzu, Kyoto, Japan). High-performance liquid chromatography (Nexara, Shimadzu, Kyoto) coupled with a photodiode array detector (SPD-M30A, Shimadzu Kyoto, Japan) and a single quadrupole mass analyzer (2020, Shimadzu, Kyoto, Japan) was used for purity analysis (absolute area %). Optical purity was determined by chiral chromatography (1100 high-performance liquid chromatography (HPLC) system with a DAD detector, Agilent, Santa Clara, CA) using a Chiralpak IB-N3 column (4.6 mm × 15 cm, 3 μm).

(R)-8-Bromo-6-(2-fluorophenyl)-4-methyl-N-(6-(4-phenylbutoxy)hexyl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxamide (PI310)

MIDD030142 (498 mg, 1.2 mmol) was dissolved in dichloromethane (10 mL) and thionyl chloride (1 mL) and refluxed for 8 h. The solvent was evaporated, and the residue was stripped consecutive times with dichloromethane (3 × 5 mL), resulting in a yellow solid. In a separate flask, tert-butyl (6-(4-phenylbutoxy) hexyl)carbamate (0.32 g, 0.91 mmol) was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (1.70 mL, 10 mmol) added dropwise. After stirring at room temperature for 90 min, the solution was cooled to 0 °C and treated with concentrated aqueous ammonia (until pH = 10–11). The mixture was extracted with dichloromethane (2 × 15 mL), dried over MgSO4, and filtered. The solution was concentrated to 8 mL and cooled to 0 °C. To this solution, N-methylmorpholine (0.219 mL, 2.0 mmol) was added dropwise, followed by the addition of the prepared acid chloride in dichloromethane (5 mL). Stirring was continued for 18 h at room temperature, followed by quenching with a saturated solution of ammonium chloride (10 mL). The organic layer was separated, and the aqueous phase was extracted with dichloromethane (2 × 15 mL). The combined organic layers were washed with brine and dried over MgSO4. Column chromatography (0–100% ethyl acetate in hexanes) resulted in PI310 as a white solid at 32% yield (189 mg). TLC: 1:1 ethyl acetate/hexanes, RF = 0.52. HPLC purity was 99.1%, and optical purity was 98.5% ee. HRMS C35H38N4O2FBr [M + H]+ predicted 647.2221 found 647.2237.

1H NMR (500 MHz, CDCl3) δ 7.72 (s, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.56 (t, J = 7.5, 1H), 7.39–7.32 (m, 3H) 7.22–7.15 (m, 2H), 7.13–7.08 (m, 4H), 6.95 (t, J = 7.5 Hz, 1H), 6.83 (q, J = 7.0 Hz, 0.73H), 4.24 (m, 0.27H), 3.34 (t, J = 6.5 Hz, 2H), 3.31 (t, J = 6.5 Hz, 2H), 2.56 (t, J = 7.5 Hz), 2.16 (m, 0.38H), 1.64–1.58 (m, 2H), 1.57–1.47 (m, 6H), 1.35–1.28 (m, 4H), 1.20 (d, J = 7.0 Hz, 2.62H); 13C NMR (126 MHz, CDCl3) δ 162.51, 162.18, 161.07 (d, 1JCF = 250.8 Hz), 142.49, 138.68, 134.55, 133.81, 133.22, 132.92, 131.91(d, 3JCF = 8.2 Hz), 131.83, 131.33, 131.28, 128.56 (d, 2JCF = 12.6 Hz), 128.38, 128.20, 125.59, 124.48 (d, 4JCF = 3.8 Hz), 123.46, 120.65, 116.06 (d, 2JCF = 21.4 Hz), 70.80, 70.64, 53.40, 49.89, 38.78, 35.70, 29.74, 29.62, 29.37, 28.03, 26.83, 25.91, 15.00; 19F NMR (471 MHz, CDCl3) δ −112.0 (minor rotamer), −112.26 (major rotamer).

(R)-8-Bromo-6-(2-fluorophenyl)-N-(2,5,8,11,14-pentaoxahexadecan-16-yl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxamide (PI320)

MIDD030142 (207 mg, 0.5 mmol), HBTU (280 mg, 0.52 mmol), and N-methylmorpholine (0.202 mL, 2.0 mmol) were stirred in dry acetonitrile (5 mL) under nitrogen at room temperature for 10 min, resulting in an almost clear solution. 2,5,8,11,14-Pentaoxahexadecan-16-amine (125 mg, 0.5 mmol) was added, and stirring was continued for 18 h. The solvent was removed under reduced pressure and the residue was dissolved in chloroform (15 mL) and treated with a saturated solution of ammonium chloride (10 mL, pH 7.0). After separation, the aqueous layer was again extracted with chloroform (15 mL). The combined organic layers were washed with brine and dried over MgSO4. The residue was purified by column chromatography with a gradient of 0–10% methanol in chloroform over 30 column volumes. The product was obtained as a viscous oil at a 62% yield (201 mg). TLC: 8% methanol in chloroform, RF = 0.52. HPLC purity was 99.3%, and optical purity was 98.1% ee. HRMS C30H36N4O6FBr [M + H]+ predicted 649.1860 found 649.1877.

1H NMR (500 MHz, DMSO-d6) δ 8.42 (s, 1H), 8.06 (t, J = 7.5 Hz, 1H), 7.93 (d, J = 7.5, 1H), 7.88 (d, J = 7.5, 1H), 7.58 (t, J = 7.5, 1H), 7.54 (q, J = 7.5, 1H), 7.32 (t, J = 7.5, 1H), 7.21 (t, J = 7.5, 1H), 6.66 (q, J = 7.5 Hz, 0.69H), 4.29 (m, 0.31), 3.53–3.46 (m, 16H), 3.41–3.38 (m, 4H), 3.32 (NH, 1H), 3.21 (s, 3H), 1.99 (m, 0.31), 1.16 (d, J = 7.0 Hz, 2.45H); 13C NMR (126 MHz, d6 DMSO) δ 162.28, 161.75, 159.34 (d, 1JCF = 248.2 Hz), 137.56, 135.34, 135.0 (d, 3JCF = 8.2 Hz), 133.60, 132.18, 131.93, 131.43, 130.63, 130.41, 128.37 (d, 2JCF = 12.6 Hz), 125.08 (d, 4JCF = 3.8 Hz), 124.69, 119.66, 115.89 (d, 2JCF = 21.2 Hz), 71.25, 69.79, 69.76, 69.75, 69.74, 69.73, 69.73, 69.72, 69.54, 68.96, 58.01, 49.01, 14.72; 19F NMR (471 MHz, DMSO-d6) δ −109.0 (minor rotamer), −109.48 (major rotamer).

GABAA Receptor Binding

Rat brain membranes were prepared from frozen tissue that was thawed on ice and homogenized on ice in 10 volumes of cold lysis buffer (50 mM Tris HCl, pH 7.4, containing protease inhibitor cocktail; Roche) using a Polytron homogenizer (6 pulses and 10 s per pulse). The homogenate was centrifuged at 1000g for 10 min at 4 °C to obtain the supernatant. The supernatant was then centrifuged at 40 000g for 20 min, and the resulting supernatant was decanted and replaced with the same ice-cold lysis buffer. Two or three additional rounds of homogenization-centrifugation were performed to ensure thorough homogenization and wash out endogenous ligands. The final pellet was resuspended in the same buffer and homogenized one last time. The rat brain suspension was diluted in buffer (50 mM Tris HCl, 2.5 mM CaCl2, pH 7.4), followed by the addition of [3H]-flunitrazepam (0.6–4.0 nM in DMSO) and PI320 or PI310 in DMSO at different concentrations to reach a final volume of 125 μL per well. Total binding and nonspecific binding were determined with reference compound clonazepam. In brief, plates are usually incubated at room temperature and in the dark for 90 min. Reactions are stopped by vacuum filtration onto 0.3% polyethyleneimine (PEI) soaked 96-well filter mats using a 96-well Filtermate harvester, followed by three washes with cold phosphate-buffered saline (PBS) buffer. The scintillation cocktail was then melted onto microwave-dried filters on a hot plate, and radioactivity was counted in a Microbeta counter. The data (n = 6) were analyzed by nonlinear regression.

Molecular Docking

Molecular docking analysis was performed with MOE 2015.1001 (Molecular Operating Environment, Montreal, Canada). The 6HUO(28) protein database file was downloaded via https://www.ncbi.nlm.nih.gov and prepared for docking studies using the available MOE feature. The pharmacophore query editor was used to determine location and annotations for alprazolam bound to the α1β3γ2L GABAAR. Docking was performed with both thermodynamic rotamers21 of PI310 and PI320 using the pharmacophore of alprazolam for placement and affinity dG for scoring. Refinement was determined with a rigid receptor structure.

Experimental Animals

Female A/J mice were purchased from Jackson Laboratory (Bar Harbor, ME) and female Swiss Webster mice from Charles River Laboratory (Wilmington, MA). For ex vivo muscle relaxation studies, adult male Hartley guinea pigs were purchased from Charles River Laboratories (Wilmington, MA). Animals were housed in a pathogen-free and 12 h light and dark cycle environment. Animals had ad libitum access to food and water. UW-Milwaukee and Columbia University confirmed that all animal experiments were performed in compliance with their Institutional Animal Care and Use Committees.

Ex Vivo Airway Smooth Muscle Relaxation Assay

Relaxation of constricted guinea pig trachea rings in the presence of PI320 was determined as reported.7 Briefly, epithelium-denuded trachea rings were attached to a force transducer and suspended in an organ bath with buffer consisting of 118 mM NaCl, 5.6 mM KCl, 0.5 mM CaCl2, 0.2 mM MgSO4, 25 mM NaHCO3, 1.3 mM NaH2PO4, 5.6 mM, and 10 μM indomethacin with the addition of 5% carbon dioxide and 95% oxygen. N-vanillylnonanamide (10 μM) was added to the bath (to deplete nonadrenergic, noncholinergic nerves) followed by two cycles of increasing concentrations of acetylcholine (0.1–100 μM). One micromolar tetrodotoxin and 10 μM pyrilamine were added to reduce the confounding effects of airway nerves and histamine. Substance P (1 μM) or an EC50 concentration of acetylcholine was then added, followed by experimental compounds or vehicles once peak contraction was reached. In some experiments, tracheal rings were pretreated with 200 μM gabazine before contraction with acetylcholine. Contraction force (presented as a percentage of peak contraction force) was measured for 1 h after compound addition.

Drug Formulation

For nebulization, a 3.0 mg/mL solution of albuterol was prepared in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.2). PI320 was prepared as a 3.2 mg/mL solution in water with 0.17% Tween-20.

PAMPA, Solubility, log P, Cytotoxicity, and Pharmacokinetic (PK)

Detailed protocols are given in the Supporting Information.

Airway Hyper-Responsiveness Measurements

Measurement of sRaw was performed using a double-chamber phethysmographic noninvasive airway mechanics instrument (DSI, St. Paul, MN) as reported.12 Briefly, animals were trained for 15 min on each of 5 days to remain calm in the measuring chambers. Calibration of the instrument was carried out before data collection. Specific airway resistance (sRaw) was computed with FinePoint software using ventilation parameters recorded during the experiment from a nasal chamber and a thoracic chamber. Formulated compounds were aerosolized for 1 min followed data recording and a 1 min pause. This iterative protocol was completed as indicated for each study.

Acknowledgments

The authors thank Dr. Adrienne Allen and Jennifer L. Nemke (Animal Resource Center at UWM) for their guidance and support. This work was supported by the National Institutes of Health R41HL147658 (L.A.A.), R01HL118561 (J.M.C., L.A.A., D.C.S.), R35GM140880 (C.W.E., J.M.C., G.T.Y.), K08HL140102 and Louis V. Gerstner, Jr., Scholar Award (G.T.Y.), as well as the University of Wisconsin-Milwaukee, University of Wisconsin-Milwaukee Research Foundation (Catalyst grant), the Lynde and Harry Bradley Foundation, and the Richard and Ethel Herzfeld Foundation. In addition, this work was supported by grant CHE-1625735 from the National Science Foundation, Division of Chemistry.

Glossary

Abbreviations

AHR

airway hyper-responsiveness

GABAAR

gamma amino butyric acid type A receptor

LABA

long-acting β2-agonists

NAM

noninvasive airway mechanics instrument

SEM

standard error of the mean

sRaw

specific airway resistance

IFNγ

interferon gamma

LPS

bacterial lipopolysaccharide

PBS

phosphate-buffered saline

NMR

nuclear magnetic resonance

HPLC

high-performance liquid chromatography

HRMS

high-resolution mass spectrometry

TOF

time of flight

TLC

thin-layer chromatography

HBTU

N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.1c00238.

  • NMR and purity analysis of PI310 and PI320; representative tracings of guinea pig tracheal ring contractile force versus time; and protocols for analysis of pharmacokinetics, compound purity, cell viability, aqueous solubility, log P, and PAMA (PDF)

Author Contributions

Conceptualization, D.C.S. and L.A.A.; methodology, N.M.Z., M.S.R.R., G.T.Y., and L.A.A.; synthesis, L.A.A. and M.Y.M.; design of compound, L.A.A.; writing—original draft preparation, N.M.Z. and L.A.A.; writing—review and editing, N.M.Z., M.S.R.R., G.T.Y., M.Y.M., J.M.C., C.W.E., D.C.S., and L.A.A.; project administration, D.C.S. and L.A.A.; and funding acquisition, J.M.C., D.C.S., C.W.E., and L.A.A. All authors have read and agreed to the published version of the manuscript.

The authors declare the following competing financial interest(s): L.A.A. and D.C.S. are employees of Pantherics Incorporated. L.A.A. and D.C.S. have an ownership interest in Pantherics, which has acquired rights to the technology reported in this publication. Some of the research was funded by R41HL147658, which was awarded to Pantherics. Pantherics did not finance this research directly. The funders indicated in the acknowledgment section they had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Supplementary Material

pt1c00238_si_001.pdf (1.5MB, pdf)

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Supplementary Materials

pt1c00238_si_001.pdf (1.5MB, pdf)

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