From orexin receptor agonist YNT-185 to novel antagonists with drug-like properties for the treatment of insomnia
Abstract
YNT-185 is the first identified small molecule functioning as an orexin 2 receptor (OX2R) agonist with potential applications in narcolepsy treatment. It served as a structural scaffold for the development of a small set of seven compounds predicted to have affinity for OX2R. The design of these novel molecules primarily focused on enhancing the physicochemical properties of YNT-185 while incorporating in silico studies to optimize binding interactions within the OX2R active site. Seven potential OX2R binders were synthesized and evaluated in vitro for central nervous system (CNS) availability, cytotoxicity, and receptor modulation characteristics. Among them, compound 15 exhibited the strongest modulatory effect on OX2R but, in contrast to YNT-185, demonstrated an inverse mode of action, functioning as an antagonist. Further in vivo testing of compound 15 revealed its ability to cross the blood-brain barrier (BBB) and reach the brain, albeit with a short half-life.
Introduction
Orexin A and orexin B are neuropeptides distributed throughout the central and peripheral nervous systems, playing key roles in food intake stimulation, sleep-wake cycle regulation, glucose metabolism, reward-seeking behavior, drug addiction, neuroendocrine functions, and energy homeostasis. They exert their effects via G-protein coupled receptors, specifically orexin receptor type 1 (OX1R) and type 2 (OX2R). These receptors share a moderate degree of amino acid similarity (64%) but have distinct distributions in the central nervous system, with OX1Rs prevalent in the locus coeruleus and OX2Rs predominantly expressed in the tuberomammillary nucleus. Research has linked deficiencies in the orexin system to narcolepsy. Because the orexin/OXRs system promotes wakefulness, antagonists that block OXRs may be used to treat insomnia by facilitating sleep and suppressing wakefulness. Recent findings indicate that both OX1R and OX2R signaling influence sleep-stage transitions, and dual antagonists could provide a more comprehensive treatment approach for insomnia.
Insomnia is one of the most prevalent sleep disorders, characterized by difficulty initiating and maintaining sleep. It can lead to cognitive impairment, physical deterioration, emotional disturbances, and conditions such as depression and anxiety. While insomnia directly affects approximately 6% of the population, its symptoms are common in nearly one-third of the global adult population. Pharmacological treatments for insomnia include benzodiazepines, hypnotic agents like zolpidem and zaleplon, antihistamines, antidepressants, antipsychotics, anticonvulsants, and melatonin receptor agonists, though their long-term efficacy remains uncertain. In 2014, the US Food and Drug Administration approved suvorexant, the first small-molecule drug designed as a dual orexin receptor antagonist (DORA) that non-selectively targets OX1R and OX2R.
Building on the success of suvorexant and knowledge gained from YNT-185, the first non-peptide selective OX2R agonist, seven novel potential OX2R modulators were designed and synthesized. A small virtual database of over 160 compounds was established using knowledge-based and structure-based drug design approaches, incorporating essential structural features necessary for OX2R activation based on YNT-185. These features included a sulfonamide moiety between two aromatic cores, a basic appendage, and a central 1,3-disubstituted aromatic ring—all crucial for receptor activation. Before synthesis, molecular modeling studies were conducted to evaluate the potential binding of these compounds within the OX2R active site, with a focus on interactions involving the sulfonamide group near key aromatic residues Thr111, Tyr354, and His350. Docking studies confirmed that these interactions play a critical role in OX2R activation, and all newly designed compounds met these structural requirements.
Additionally, improvements were made to address the physicochemical limitations of YNT-185—such as inappropriate logP, molecular weight, and solubility—that may have restricted its central nervous system availability and in vivo efficacy. A multi-parameter optimization score was calculated to predict the central activity of these new compounds, alongside solubility predictions. To ensure feasible synthesis, commercially available starting materials were prioritized. Collectively, the optimization of physicochemical properties and receptor-binding patterns led to the generation of seven novel and promising OX2R modulators. This study provides an evaluation of their in vitro activity on OX2R, cytotoxicity profile, blood-brain barrier permeability prediction using parallel artificial membrane permeation assay (PAMPA), and in vivo pharmacokinetic profile for the most promising compound.
Results and discussion
In silico studies
To predict whether the proposed ligands with CNS drug-like properties can bind to OX2R, molecular docking studies were conducted using the crystal structure of human OX2R bound to suvorexant. The putative binding orientation of compound 15 within the receptor was examined. The methyl ester group of 15 is positioned near the carbonyl moiety of Cys210, forming double hydrogen bonds at distances of 3.0 and 3.3 Å within the transmembrane helix 3 region of OX2R. Additionally, Cys210 appears to participate in π-sulfur interactions with the N-methylpyrrole moiety. The central phenyl ring engages in multiple hydrophobic interactions, including cation-π interactions with His350, van der Waals forces with Gln134, and alkyl-π interactions with Val353. The (piperidin-4-yl)methanol tail contributes a classical hydrogen bond with Thr231 at a distance of 2.1 Å.
An essential observation from in silico studies is that the sulfonamide moiety of 15 forms hydrogen bonds with the hydroxyl groups of Tyr354 and Thr111, as well as the ketone oxygen of Gln134, at distances of 2.5 Å and 3.4 Å, respectively. This suggests that the binding orientation of 15 may facilitate receptor activation in a manner similar to YNT-185. The overall topology of 15 closely resembles that of YNT-185 rather than suvorexant. While suvorexant adopts a π-stacked horseshoe-like conformation, the binding pose of 15 aligns more with the structural properties of YNT-185 within OX2R. A detailed comparison of suvorexant’s binding mode reveals that its horseshoe conformation is stabilized by the presence of a boat-shaped diazepane ring. The suvorexant binding pattern primarily consists of van der Waals interactions with OX2R and a direct hydrogen bond with Asn324—an interaction absent in the 15-OX2R complex. Additionally, the crystal structure of suvorexant in OX2R indicates a water-mediated bridge to His350, whereas compound 15 engages His350 via a direct hydrogen-bond interaction. Notably, Cys210 is critically involved in anchoring the 15-OX2R complex, a feature not observed in suvorexant’s binding mode. These findings were further investigated through in vitro experiments.
Chemistry
The reaction of sulfonyl chlorides (1, 2, or 3) with amines (4, 5, 6, 7, or 10) in the presence of trimethylamine (TEA) as a base in acetonitrile (CH3CN) yielded sulfonamides 11–17. The synthesized compounds were subsequently converted into hydrochlorides with yields ranging from 73% to 98% over two steps.
Derivative 17 was obtained through a two-step process. Initially, amide 10 was synthesized from m-phenylenediamine (8) and 4-(2-oxopyrrolidin-1-yl)butanoic acid (9) using N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC·HCl) and 1-hydroxybenzotriazole hydrate (HOBt) as coupling agents. In the final step, amide 10 was reacted with sulfonyl chloride 1, leading to the formation of sulfonamide 17.
For comparison, the OX2R agonist YNT-185 was synthesized as a reference compound following previously published methods, achieving an overall yield of 8%. All final compounds were thoroughly characterized using 1H NMR, 13C NMR, HRMS, and LC-MS analysis, confirming a purity level exceeding 95%.
Ligands effect on the OX2R
The discovery of orexin receptor (OXR) modulators and their potential applications in treating narcolepsy and insomnia have marked significant progress in sleep research. Both OX1R and OX2R interact with orexin A and orexin B, but they exhibit different binding affinities. While orexin A binds non-selectively to both receptor subtypes, orexin B displays 10- to 100-fold higher potency for OX2R compared to OX1R. Activation of OXRs leads to multiple calcium signaling mechanisms across native and recombinant cell types.
In this study, a Chinese hamster ovary (CHO-K1) cell line stably expressing OX2R was used to evaluate receptor activation by measuring calcium release from intracellular storage and influx. The fluorescence probe Fluo-3 was utilized to detect changes in calcium levels. The results from the Fluo-3 assay indicated that the tested compounds influenced OX2R activity upon orexin A stimulation. As a reference compound, the non-peptide OX2R agonist YNT-185—previously reported for its ability to enhance wakefulness in wild-type mice and suppress cataplexy-like episodes in orexin-deficient models—was included in the analysis.
Contrary to YNT-185 (tested at 5–100 µM) and the reference peptide agonist orexin A (0.1 and 0.5 µM), none of the tested compounds (11–17) displayed substantial agonistic effects, even at 100 µM. Consequently, their potential antagonistic activity was examined. Calcium release assays demonstrated that compounds 11–17 significantly and dose-dependently reduced the orexin A-evoked response (0.2 µM). Among them, compounds 12 and 15 exhibited the strongest antagonistic effects, completely blocking OX2R at 1 µM. Suvorexant (0.5 µM), a well-established nonselective OXR antagonist, was used as a positive control. The concentration of suvorexant was selected to exceed its reported IC50 value (IC50 = 0.055 µM), aligning with previously reported findings (IC50 = 0.071 µM).
Based on the obtained data, all tested compounds except compound 17 effectively inhibited orexin A-induced responses at the evaluated concentrations. There is compelling evidence that OX2R antagonism is beneficial for sleep induction. Among existing sleep-promoting molecules that target OX2R, EMPA, TCS-OX2-29, and JNJ-10397049 have demonstrated greater efficacy in reducing wakefulness compared to OX1R antagonists. EMPA, a selective OX2R antagonist, has been shown to increase cumulative non-rapid eye movement (nREM) sleep following intraperitoneal administration. Similarly, JNJ-10397049, administered intraperitoneally, resulted in a significant increase in total sleep time, influencing both REM and nREM sleep. Moreover, intracerebroventricular infusion of TCS-OX2-29 in rats led to an observed increase in total sleep duration.
These findings suggest that the newly synthesized compounds could be valuable for insomnia treatment rather than narcolepsy, contributing to further advancements in sleep disorder therapies.
In vitro BBB permeation and CHO-K1 cytotoxicity
One of the key requirements for novel OX2R modulators is their ability to cross the blood-brain barrier (BBB). To assess this property, compounds 11–17 were evaluated using the parallel artificial membrane permeability assay (PAMPA), a method that simulates transcellular absorption processes in vivo. The results indicated that all tested compounds, except for 15, demonstrated a high likelihood of CNS permeation. Compound 15 showed uncertain BBB permeability; however, this result does not necessarily preclude its potential for further in vivo testing, as the PAMPA assay has certain limitations. These include differences between human phospholipid bilayers and the lipid porcine model used in this study, exclusion of compounds that are actively transported by influx mechanisms or removed via efflux transporters such as glycoprotein P, and the inability to account for paracellular transport, which primarily applies to small hydrophobic molecules.
Additionally, the toxicity profile of the tested ligands was examined using an in vitro MTT colorimetric assay to evaluate their effects on cell viability. Cytotoxicity measurements were conducted after 24 hours of incubation, and the IC50 values were recorded. Among the compounds, 14 exhibited the lowest cytotoxicity. However, all tested compounds demonstrated relatively low cytotoxicity overall, with IC50 values in the range of hundreds of micromoles.
Experimental section
General chemistry methods
All chemical reagents and solvents were obtained from Sigma-Aldrich and used in the highest available purity without further purification. Reaction monitoring was conducted using thin-layer chromatography (TLC) on silica gel plates, with spot visualization under ultraviolet light at 254 nm. Crude products were purified using silica gel column chromatography.
Nuclear magnetic resonance (NMR) spectra were recorded in deuterated chloroform (CDCl3), deuterated acetone ((CD3)2CO), or deuterated methanol (CD3OD) using a Varian S500 spectrometer. Chemical shifts (δ) were expressed in parts per million (ppm), and spin multiplicities were classified as singlet (s), broad singlet (bs), doublet (d), doublet of doublets (dd), doublet of doublet of doublets (ddd), triplet (t), triplet of doublets (td), triplet of triplets (tt), quartet (q), or multiplet (m). Coupling constants (J) were reported in Hertz (Hz).
High-performance liquid chromatography (HPLC) combined with mass spectrometry (MS) was employed to determine high-resolution mass spectra (HRMS) and assess purity. All final compounds exhibited a purity exceeding 95% based on HPLC-UV analysis at 254 nm.
In silico studies
Docking experiments were carried out following the previously described protocol [22,45]. For more details, readers are kindly referred to Supplementary Material.
Cytotoxicity evaluation
The cytotoxic effect of the studied compounds was assessed using standard MTT (3-[4–dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay on the CHO-K1 cell line [32]. For more details, readers are kindly referred to Supplementary Material.
PAMPA assay
The parallel artificial membrane permeability assay (PAMPA) was carried out as described previously.[29] For more details, readers are kindly referred to Supplementary Material.
In vitro evaluation of OX2R activity
Determination of antagonist properties of novel OX2R was in accordance with previously described method [22]. A more detailed description of OX2R agonist assay can be found in Supplementary Material.
Pharmacokinetic study of 15 in rats
For the pharmacokinetic study, 30 male albino Wistar rats (weighing 230 ± 20 g) were obtained from Velaz, Czech Republic. They were housed in an air-conditioned environment with a light cycle from 7:00 a.m. to 7:00 p.m. and provided unrestricted access to standard food and tap water. All procedures involving animal handling were conducted under the supervision of the Ethics Committee of the Faculty of Military Health Sciences, Czech Republic.
Following intraperitoneal (i.p.) administration of compound 15 (75 mg/kg—50% MTD; 0.1 mL/100 g body weight in saline), blood samples were collected under deep terminal anesthesia via cardiac puncture into heparinized tubes at time points of 0, 5, 10, 20, 30, 40, 60, 90, 120, and 240 minutes (n = 3). The collected samples were immediately centrifuged at 3000g for 10 minutes at 10°C to obtain plasma. Subsequently, the animals were perfused transcardially with 0.9% NaCl saline solution for 5 minutes at a rate of 10 mL/min. After perfusion, the brain was carefully extracted, and both plasma and brain samples were stored at −80°C until LC-MS analysis.
Brains were weighed, and phosphate-buffered saline (PBS) was added at a volume four times the brain weight. The homogenization process was carried out using a T-25 Ultra Turrax disperser, followed by transfer into microcentrifuge tubes and sonication with a UP 50H needle homogenizer. The homogenized samples were stored at −80°C before extraction.
A total of 190 µL of brain homogenate or plasma was mixed with 10 µL of an internal standard (IS; donepezil in methanol) at a final concentration of 2 µM. The mixture was vortexed briefly, and 1000 µL of acetone was added. The samples were then shaken for 10 minutes at 1200 RPM and centrifuged at 4000 RPM for 5 minutes. An 800 µL aliquot of the supernatant was transferred to a microtube and evaporated to dryness in a CentriVap concentrator. Before analysis, samples were reconstituted in 100 µL of 50% (v/v) acetonitrile.
Calibration samples were prepared by adding 10 µL of a methanol solution containing compound 15 (final concentration range: 0.1–50 µM for plasma and 0.5–5000 nM for brain homogenate) and 10 µL of the internal standard (final concentration: 2 µM) to 180 µL of blank plasma or brain homogenate. These samples were processed using the same extraction protocol. The final samples were analyzed via LC-MS.
Liquid chromatography-mass spectrometry
LC-MS instrumentation
The study utilized a Dionex Ultimate 3000 UHPLC system, equipped with an RS LPG quaternary pump, RS column compartment, RS autosampler, and diode array detector, all controlled by Chromeleon software. Mass spectrometric detection was carried out using a Q Exactive Plus Orbitrap mass spectrometer, managed through Thermo Xcalibur software. The electrospray ionization source operated in positive mode, with settings including a spray voltage of 3.5 kV, capillary temperature of 220°C, sheath gas at 55 arbitrary units, auxiliary gas at 15 arbitrary units, spare gas at 3 arbitrary units, probe heater temperature at 220°C, maximum spray current of 100 µA, and an S-lens RF level of 50.
HRMS and purity determination
High-resolution mass spectrometry (HRMS) and sample purity assessments were conducted using high-performance liquid chromatography (HPLC) with UV and mass spectrometry (MS) gradient methods. The study employed a Waters Atlantis dC18 column (2.1 × 100 mm, 3 μm). Mobile phase A consisted of ultrapure ASTM I water (resistance 18.2 MΩ.cm at 25°C) prepared using a Barnstead Smart2Pure 3 UV/UF apparatus, with 0.1% (v/v) formic acid. Mobile phase B was acetonitrile containing 0.1% (v/v) formic acid. The flow rate was maintained at 0.4 mL/min.
The method initiated with an isocratic flow at 5% B for 1 minute, followed by a gradient increase to 100% B over 3 minutes, which remained constant for 1 minute before reverting to 5% B and equilibrating for 2.5 minutes. Samples were dissolved in methanol at a concentration of 1 mg/mL, with a sample injection volume of 1 µL. Purity was determined via UV spectra at a wavelength of 254 nm. HRMS measurements were performed in total ion current spectra using a mass spectrometer in positive mode.
Pharmacokinetic study sample analysis
Data were acquired using a reverse-phase gradient elution method with a Kinetex C18 polar column (3 × 150 mm, 2.6 µm, Phenomenex, Japan). Mobile phase A consisted of 0.1% (v/v) formic acid in ultrapure water, while mobile phase B contained 0.1% (v/v) formic acid in acetonitrile. The flow rate was maintained at 0.4 mL/min.
The gradient method commenced with an isocratic flow of 5% B for 1 minute, followed by a gradual increase to 100% B over 3 minutes. This concentration remained constant for 1 minute before returning to 5% B, followed by an equilibration period of 5 minutes. The column temperature was maintained at 27°C, with an injection volume of 1 µL.
Samples were analyzed using a mass spectrometer under parallel reaction monitoring (PRM) conditions. The calibration curve for plasma included seven points ranging from 0.5 to 50 µM, while the brain homogenate calibration curve contained nine points spanning 0.5 to 5000 nM, both demonstrating linearity across the measured ranges TAK-861.
Statistical analysis
Calculation was performed using GraphPad Prism 6.05 (GraphPad Software, San Diego, USA) and Microsoft Excel 2010 (Microsoft Corporation, Redmond, USA) with PKsolver extension [33].