N-Phenethyl Substitution in 14-Methoxy-N-methylmorphinan-6-ones Turns Selective µ Opioid Receptor Ligands into Dual µ/δ Opioid Receptor Agonists

Morphine and structurally-derived compounds are µ opioid receptor (µOR) agonists, and the most effective analgesic drugs. However, their usefulness is limited by serious side effects, including dependence and abuse potential. The N-substituent in morphinans plays an important role in opioid activities in vitro and in vivo. This study presents the synthesis and pharmacological evaluation of new N-phenethyl substituted 14-O-methylmorphinan-6-ones. Whereas substitution of the N-methyl substituent in morphine (1) and oxymorphone (2) by an N-phenethyl group enhances binding affinity, selectivity and agonist potency at the µOR of 1a and 2a, the N-phenethyl substitution in 14-methoxy-N-methylmorphinan-6-ones (3 and 4) converts selective µOR ligands into dual µ/δOR agonists (3a and 4a). Contrary to N-methylmorphinans 1–4, the N-phenethyl substituted morphinans 1a–4a produce effective and potent antinociception without motor impairment in mice. Using docking and molecular dynamics simulations with the µOR, we establish that N-methylmorphinans 1–4 and their N-phenethyl counterparts 1a–4a share several essential receptor-ligand interactions, but also interaction pattern differences related to specific structural features, thus providing a structural basis for their pharmacological profiles. The emerged structure-activity relationships in this class of morphinans provide important information for tuning in vitro and in vivo opioid activities towards discovery of effective and safer analgesics.

morphine (1) and oxymorphone 2 13 . Herein, we have evaluated their binding profile at the human µOR expressed in CHO cells, and made similar observations with 1a and 2a displaying ca. 13-and 12-fold increased affinity at the human µOR than 1 and 2, respectively (Table 1). In this study, comparison of the in vitro binding at the µOR of 14-methoxy-N-methylmorphinan-6-ones to their N-phenethyl analogues revealed that introduction of a phenethyl group at position 17 does not influence affinity at the µOR when relating 14-OMO (3) vs. 3a, and 14-MM (4) vs. 4a (P > 0.05, t-test). Furthermore, all N-phenethyl derivatives 1a-4a have higher affinities (5-to 13-fold) than their N-methyl counterparts 1-4 at the human δOR (P < 0.05, t-test). Particularly, 3a and 4a showed very low nanomolar affinities at the δOR (K i = 1.81 nM and 1.45 nM, respectively). While affinities of 1a, 2a and 3a at the human κOR were in the range of their parent molecules 1-3, a ca. 3-fold decrease in the κOR affinity was noted for 4a vs. 4 (Table 1). We have also observed that replacement of the N-methyl group in 1 and 2 with an N-phenethyl group enhanced not only µOR affinity but also µOR selectivity vs. δOR and vs. κOR of 1a and 2a. In the case of 14-methoxy-N-phenethylmorphinan-6-ones 3a and 4a, a reduction in µOR vs. δOR selectivity was noticed, while selectivity for µOR vs. κOR was higher than that of 14-OMO (3) and 14-MM (4), respectively (Table 1).
In vitro opioid activities of targeted compounds at the human µOR and δOR were determined in the guanosine-5′-O-(3-[ 35 S]thio)-triphosphate ([ 35 S]GTPγS) binding (Table 2) and forskolin-induced cAMP accumulation assays (Table 3), performed as described 13,21 . The κOR-mediated G protein activation was assessed using [ 35 S]GTPγS binding assays with CHO cell membranes expressing the human κOR (Table 2). Previous work from our laboratory on the introduction of a phenethyl group at the nitrogen in morphine (1) and oxymorphone (2) showed an increase in agonist potency by 2-to 3-fold and full efficacy for 1a and 2a in inducing µOR-mediated G protein signaling as assessed by [ 35 S]GTPγS binding and calcium mobilization assays 13 . Similarly, enhanced µOR agonist potencies by 4-to 5-fold were measured for N-phenethyl analogues 1a and 2a as compared to morphine and oxymorphone, respectively, in the cAMP accumulation assay, while the δOR agonism remained unchanged (Table 3). In the series of 14-O-methylmorphinan-6-ones, exchanging the N-methyl by an N-phenethyl substituent did not largely influence agonist potency and full efficacy at the µOR of 14-OMO (3) vs. 3a, and 14-MM (4) vs. 4a (P > 0.05, t-test). These findings reveal that the N-phenethyl substitution in 14-methoxy-N-methylmorphinan-6-ones does not cause any change in binding affinity nor in vitro agonism at the µOR. All compounds displayed full efficacies at the δOR with different levels of potencies, while at the κOR   13 . *P < 0.05, **P < 0.01 and ***P < 0.001 for N-methylmorphinans vs. respective N-phenethyl analogues (unpaired t-test).
a partial agonist profile with very low potencies was noted (Tables 2 and 3). In the [ 35 S]GTPγS binding assay, the 14-methoxy-N-phenethylmorphinan-6-ones 3a and 4a displayed the highest agonist potencies at the δOR (EC 50 = 9.34 nM and 9.54 nM, respectively), which were higher (4-fold) than potencies of their N-methyl counterparts 14-OMO (3) and 14-MM (4). The same observation was made when comparing agonist activity of 3 vs. 3a and 4 vs. 4a at the δOR in the cAMP accumulation assay, with a significant increase in potency (P < 0.05, t-test) for the N-phenethylmorphinan-6-ones 3a and 4a (Table 3). Thus, the outcomes from the [ 35 S]GTPγS functional and cAMP accumulation assays are in agreement with the results on increased binding affinity at the δOR of 3a and 4a compared to 3 and 4, respectively. Additionally, 3a and 4a have a functional profile in vitro as dual µ/δOR full agonists, a class of ligands nowadays targeted as new analgesics with reduced unwanted side effects. Numerous pharmacological and biochemical reports and studies with opioid receptor knockout mice have provided evidence on the modulatory interactions between the µOR and δOR systems [22][23][24][25][26] . While the mechanisms are still unknown, several studies have established that the therapeutic profile of opioids could be improved by simultaneous modulation of the µOR and δOR, with compounds designed to target both receptors based on peptidic structures, non-peptidic structures or utilize the morphinan scaffold [25][26][27][28][29][30][31][32] . We 13 and others 11,12 have reported that the N-phenethyl substituted morphinans 1a and 2a exhibit increased antinociceptive potencies than their respective N-methyl analogues morphine (1) and oxymorphone (2) in mouse models of acute thermal nociception after subcutaneous (s.c.) administration, which is in line with findings from binding and functional in vitro assays. We have shown that 1a was 22-and 28-fold more effective than morphine (1) in the hot-plate and tail-flick tests, respectively 13 . Further, the N-phenethyl analogue of oxymorphone (2a) was found to be highly active with about 2-fold higher potency than oxymorphone (1) ( Table 4) 13 . In this study, we have also evaluated if targeted structural changes in 14-OMO (3) and 14-MM (4) also affects antinociceptive activities. Antinociceptive effects of N-phenethyl substituted 3a and 4a were assessed in the tail-flick assay in mice after s.c. administration as described 13 . Antinociceptive potencies (ED 50 values) were determined at the peak of action and compared to those of N-methyl counterparts 3 and 4 ( Table 4). All compounds increased tail-withdrawal latencies to thermal stimulation in a time-and dose-dependent manner with a peak effect generally occurring at 30 min (Fig. 2). As shown in Table 4 and Fig. 3, 3a and 4a display similar antinociceptive activities to their analogues 14-OMO (3) and 14-MM (4), respectively (P > 0.05, two-way ANOVA), indicating that exchanging the N-methyl by an N-phenethyl group in 14-O-methylmorphinan-6-ones does not affect the in vivo agonism.  Table 3. Agonist potencies and efficacies at the human µOR and δOR of N-methylmorphinans 1-4 and their respective N-phenethy analogues 1a-4a in the cAMP accumulation assay. a Determined in the forskolin-induced cAMP accumulation assay using CHO cells co-expressing the human opioid receptors and the cAMP biosensor GloSensor-22F (CHO-hµOR-p22F or CHO-hδOR-p22F cells). Percentage stimulation (% stim.) relative to the agonist DAMGO (µOR) or DPDPE (δOR). Values represent the mean ± SEM (n = 3-4). *P < 0.05 and **P < 0.01 for N-methylmorphinans vs. respective N-phenethyl analogues (unpaired t-test).  www.nature.com/scientificreports www.nature.com/scientificreports/ Clinically used opioid analgesics, such as morphine, oxycodone or fentanyl, are known to produce sedation and motor dysfunction, side effects that limits their clinical usefulness [33][34][35] . With literature evidence that mixed µOR/δOR agonists are efficacious analgesics with reduced side effects 25-32 , we have evaluated the effect of 3a and 4a as mixed µ/δOR agonists, and behavioral consequences of the replacement of the N-methyl group in N-methylmorphinans 1-4 by an N-phenethyl group in 1a-4a on motor coordination in mice using the rotarod assay, a well-established model for evaluating loss of coordinated locomotion 36 . The first behavioral data on motor function following systemic s.c. administration of N-phenethyl substituted derivatives of morphine and oxymorphone, 1a and 2a, respectively are presented. Mice were s.c. treated with the respective compound at doses equivalent to a 3-to 4-fold the antinociceptive ED 50 dose. Rotarod experiments demonstrate the lack of the mixed µ/ δOR agonists 3a and 4a to induce motor dysfunction, having an improved profile than their parent µOR selective agonists 14-OMO (3) and 14-MM (4), respectively (Fig. 4). Whereas morphine (1) and oxymorphone (2) caused a significant deficit in rotarod performance, their N-phenethyl substituted 1a and 2a did not affect the evoked locomotor activity of mice (Fig. 4). In this study, we show that N-phenethyl substituted morphinans 1a-4a elicit effective and potent antinociception without motor impairment in mice.  www.nature.com/scientificreports www.nature.com/scientificreports/ Molecular modeling. The µOR was the first opioid receptor type resolved in an inactive (PDB ID: 4DKL) 37 and an active conformation (PDB ID: 5C1M) 38 . The access to crystal structures of the µOR provides essential knowledge on key aspects of the µOR pharmacology and its function [37][38][39] . All investigated morphinans (Fig. 1) bind and are agonists at the µOR. The observed similarities or differences in their in vitro and in vivo activity profiles incited exploration of their binding modes at the µOR. Molecular docking investigations were performed with N-methylmorphinans 1-4 and their N-phenethyl counterparts 1a-4a, where a 3D-pharmacophore approach based on the LigandScout program 29 was applied to analyze shared and distinct receptor-ligand interactions. Docking studies using the active conformation of the µOR (PDB ID: 5C1M) 38 revealed comparable binding orientations for all targeted morphinans, which are in accordance with BU72 27 in its co-crystallized conformation. An overview of detected interactions is presented in Figs. 5 and S1.
Although all investigated compounds show a comparable binding mode to the µOR in which the morphinan moiety was found to adopt a similar orientation and to share several essential receptor-ligand interactions, we have also observed interaction pattern differences related to specific structural features (Fig. 5). The tertiary amine forms an essential charge interaction with D147 and a π-cation interaction with Y148 residue. The crucial role of D147 for the binding to the µOR of morphinan ligands, as well as other chemotypes (i.e. mitragynine pseudoindoxyl) and peptides (i.e. DAMGO) has been described 37,38,[41][42][43][44] . The interaction with Y148 is also recognized as an important requirement for ligands (small molecules and peptides) to bind to the µOR 37,38,[41][42][43][44] . In this study, the oxygen of the partially saturated furan ring (E-ring) of the morphinan system serves as a hydrogen bond acceptor for Y148 in both series of N-methyl (1)(2)(3)(4) and N-phenethyl substituted morphinans (1a-4a). The 14-O-methylmorphinan-6-ones 3, 4, 3a and 4a show a lipophilic contact of the 14-methoxy group with I322 (Fig. 5C,D). The phenolic substructure lies opposite to M151, V236 and V300 residues. The 14-hydroxyl group of oxymorphone (2) and its N-phenethyl analogue 2a forms hydrogen bonds to both D147 and Y326 residues (Fig. 5B). Compounds 1 and 1a exhibited the same interaction pattern with the only difference in the additional lipophilic contacts of the N-phenethyl moiety of 1a with a lipophilic subpocket (Fig. 5A). For all ligands with an N-phenethyl group, this moiety is embedded in a lipophilic pocket formed by A117, W293 and Y326 residues.
The recent crystal structure of the δOR (PDB ID: 6PT3) 45 with the co-crystallized agonist DPI-287 supports our proposed binding mode of 4a at the δOR, since DPI-287 also has a phenyl ring which is filling the beforementioned lipophilic subpocket (Fig. 6). The active κOR structure (PDB ID: 6B73) was also determined in complex with the epoxymorphinan agonist MP1104 46 . Compared to the µOR, the size and shape of this subpocket was found to be highly similar for the δOR (Figs. 6A and S2), but different for the κOR (Figs. 6 and S2). Whereas µOR and δOR have an alanine residue in position 2.53 (according to Ballesteros-Weinstein nomenclature), a valine  N-methylmorphinans 1-4 and their respective N-phenethyl analogues 1a-4a in the mouse rotarod assay. Mice were tested 30 min after s.c. administration of control (saline) or test compounds. Data depicts latencies to fall from the rotarod as the mean percent changes from baseline performance ± SEM (n = 6 mice per group). *P < 0.05, **P < 0.01 and ***P < 0.001 vs. saline group; # P < 0.05 and ### P < 0.001 vs. Nmethylmorphinan treated group; one-way ANOVA followed by Tukey's post hoc test.
Several studies have evidenced MD simulations as an effective approach to examine binding modes between opioid receptors and their ligands 39,[41][42][43][44] . In order to further validate the binding modes of morphinans 1-4 and 1a-4a depicted using molecular docking, we performed all-atoms MD simulations of the µOR as described 47 .  All investigated ligands are full agonists at the µOR, in accordance with the observation that all structures provide a full constriction of the orthosteric binding site as a key feature for receptor activation. The additional lipophilic contacts of the N-phenethyl substituent deep in the core region of the receptor are supposed to enhance ligand binding. This effect is less prominent for morphinans in which the parent compound already has a binding affinity in the subnanomolar range to the µOR (Table 1). This may be explained by additional interactions with the receptor, such as additional hydrogen bonds for the 14-hydroxymorphinans 2 and 2a, or an additional lipophilic contact with I322 residue for the 14-O-methylmorphinans, 14-OMO (3), 14-MM (4), 3a and 4a. The latter interaction is of particular importance for orientation of the ligand in the binding site, and is visualized in Fig. 7. Interestingly, N-methyl substituted 1 and 2 show a slightly different orientation compared with their related N-phenethyl analogues (Fig. S4). This effect is not observable for 3, 3a, 4 and 4a, which suggests that an increased µOR affinity of morphinan analogs can be achieved by either a methoxy substitution at position 14 or by an N-phenethyl group. Since the combination of the two strategies does not show additive effects, we assume that the optimal orientation can be sufficiently triggered by only one of the two substitutions. Furthermore, a direct comparison of the active δOR 45 and the µOR 38 crystal structures unveils the high similarity of their binding pockets. While all residues forming key interactions are identical, a major difference was observed at the beginning of helix seven at position 7.35 (Fig. S5). The tryptophan residue in µOR was found to be optimal in forming lipophilic contacts with the morphinan moiety, whereas this receptor-ligand contact is missing for the δOR, due to a leucine at this position. This might explain why all studied compounds are have higher affinity for the µOR compared to the δOR.

Conclusions
The results of the present study provide SAR evidence on the consequences of an N-methyl substitution in morphinan opioids 1-4 by an N-phenethyl in 1a-4a on in vitro and in vivo activities, with molecular docking and MD simulations studies offering a structural basis for the observed pharmacological profiles at the opioid receptors. Pharmacological findings are supported by docking and MD simulations analysis with N-methyl substitute morphine (1) and oxymorphone (2) showing a slightly different orientation in the binding pocket of the µOR compared to their related N-phenethyl analogues, 1a and 2a, respectively. This was not noticed for 14-OMO (3) vs. 3a, and 14-MM (4) vs. 4a, indicating that an increased µOR affinity can be achieved by either a 14-methoxy or by an N-phenethyl substitution, as key sites to be targeted in modulating the binding affinity and efficacy of morphinans to the µOR. Whereas replacement of the N-methyl substituent in morphine (1) and oxymorphone (2) by an N-phenethyl group enhanced binding affinity, selectivity and agonist potency at the µOR of 1a and 2a, the N-phenethyl substitution in 14-methoxy-N-methylmorphinan-6-ones (3 and 4) turned selective µOR ligands into dual µ/δOR agonists (3a and 4a), a profile that currently emerges as a promising approach to opioid analgesic drug discovery [26][27][28][29][30][31][32] . Furthermore, we have demonstrated that the N-phenethyl substituted morphinans 1a-4a are effective and potent antinociception agents without causing unwanted motor impairment in mice after s.c. administration. Altogether, these data offer important insights on the SARs in the morphinan class of opioid ligands, by increasing the current understanding of the impact of different substituents at the nitrogen and position 14 on ligand-µOR binding, receptor activation and the link between antinociception and side effects (i.e. motor function).

Materials and Methods
Chemistry. General chemical and analytical methods were performed according to protocols as described previously 20 . All chemicals were of reagent grade and obtained from standard commercial sources. Melting points were determined on a Kofler melting point microscope and are uncorrected. 1 H and 13 C NMR spectra were recorded on a Bruker Avance II + spectrometer operating at 600 MHz and equipped with a Prodigy TCI probe. IR spectra were taken on a Bruker Alpha FT-IR spectrometer (for detection, an ATR sensor was used). Mass spectra were recorded on a Varian MAT 44 S apparatus. Elemental analyses were performed at the Microanalytic Laboratory of the University of Vienna, Austria. For column chromatography (MPLC), silica gel 60 (0.040-0.063 mm, Fluka, Switzerland) was used. Compounds 3a and 4a were used as bases for testing. The combustion analysis values were found to be within ± 0.4% of the calculated values, confirming a purity of the tested compounds of >95%. -3-hydroxy-14-methoxy-N-phenethylmorphinan-6-one (3a). A mixture 4,5α-epoxy-3-hydroxy-14-methoxymorphinan-6-one hydrochloride (5 • HCl) (100 mg, 0.296 mmol), prepared according to the described procedure 19 , phenethyl bromide (76.7 mg, 0.40 mmol), and NaHCO 3 (67.3 mg, 0.8 mmol) in 3 mL DMF was stirred at 80 °C for 48 h. The cooled mixture was filtered, the filtrate evaporated to dryness and purified by column chromatography (CH 2 Cl 2 /MeOH/NH 4
Competition binding assays. In vitro binding assays were conducted on human opioid receptors stably transfected into CHO cells according to the published procedures 20 . Briefly, CHO-hµOR, CHO-hδOR and CHO-hκOR cells grown at confluence were removed from the culture plates by scraping, homogenized in 50 mM Tris-HCl Scientific RepoRtS | (2020) 10:5653 | https://doi.org/10.1038/s41598-020-62530-w www.nature.com/scientificreports www.nature.com/scientificreports/ buffer (pH 7.4), using a Polytron homogenizer, then centrifuged once and washed by an additional centrifugation at 27,000 × g for 15 min, at 4 °C. The final pellet was resuspended in 50 mM Tris-HCl buffer (pH 7.4), and cell membranes (15-20 µg) were incubated with various concentrations of test compound and the appropriate radioligand [ 3 H]DAMGO or [ 3 H]Diprenorphine for 60 min at 25 °C, or [ 3 H]HS665 for 30 min at 0 °C. Non-specific binding was determined using 1-10 µM of the unlabeled counterpart of each radioligand. Reactions were terminated by rapid filtration through Whatman glass GF/C fiber filters. Filters were washed three times with 5 mL of ice-cold 50 mM Tris-HCl buffer (pH 7.4) using a Brandel M24R cell harvester (Gaithersburg, MD). Radioactivity retained on the filters was counted by liquid scintillation counting using a Beckman Coulter LS6500 (Beckman Coulter Inc., Fullerton, CA). All experiments were performed in duplicate, and repeated at least three times with independently prepared samples.
[ 35 13,20 . Cell membranes were prepared in Buffer A (20 mM HEPES, 10 mM MgCl 2 and 100 mM NaCl, pH 7.4) as described for competitive radioligand binding assays. Cell membranes (5-10 µg) in Buffer A were incubated with 0.05 nM [ 35 S]GTPγS, 10 µM GDP and various concentrations of test peptides in a final volume of 1 mL, for 60 min at 25 °C. Non-specific binding was determined using 10 µM GTPγS, and the basal binding was determined in the absence of test ligand. Samples were filtered over Whatman glass GF/B fiber filters and counted as described for competitive binding assays. All experiments were performed in duplicate, and repeated at least three times with independently prepared samples.
cAMP accumulation assay. Inhibition of the forskolin-stimulated intracellular cAMP accumulation in CHO cells co-expressing the hµOR and the cAMP biosensor GloSensor-22F (CHO-hµOR-p22F cells) and CHO cells co-expressing the hδOR and the cAMP biosensor GloSensor-22F (CHO-hδOR-p22F cells) was performed using the Glo-Sensor cAMP assay (Promega) according to the published procedure 21 . Cells were seeded in growth medium into 384-well plates at a density of 5,000 cells in 30 μL per well and incubated overnight. On the day of assay, culture media was removed, and cells were pre-equilibrated for 90 min with 4% v/v of the GloSensor cAMP reagent in reaction medium (20 mM HEPES, 1 x HBSS, pH 7.4) at 37 °C and 5% CO 2 . Cells were then treated with various concentrations of test compounds for 15 min at room temperature. Forskolin (10 μM) was added to each well, and luminescence was measured after 20 min using PerkinElmer Wallac Victor 1420 Mulitlable Counter. All experiments were performed in triplicate, and repeated at least three times with independently prepared samples.
In Vivo assays. Animals and Drug Administration. In vivo studies were performed as described previously 20 . Male CD1 mice (30-35 g, 7-8 weeks old) were obtained from the Center of Biomodels and Experimental Medicine (CBEM) (Innsbruck, Austria). Mice were group-housed in a temperature controlled room with a 12 h light/dark cycle and with free access to food and water. All animal studies were conducted in accordance with ethical guidelines and animal welfare standards according to Austrian regulations for animal research, and were approved by the Committee of Animal Care of the Austrian Federal Ministry of Science and Research. Test compounds or vehicle (saline) were administered by s.c. route in a volume of 10 µL/1 g of body weight. Each experimental group included five to six animals. Separate groups of mice received the respective dose of compound, and individual mice were only used once for behavioral testing.
Tail-flick assay. The radiant heat tail-flick test was used to assess antinociceptive effects of test compounds after s.c. administration in mice, according to the original procedure of D' Amour and Smith 51 . The tail-flick test was performed using an UB 37360 Ugo Basile analgesiometer (Ugo Basile s.r.l., Varese, Italy. The reaction time required by the mouse to remove its tail after application of the radiant heat was measured and defined as the tail-flick latency (in seconds). Tail-flick latencies were measured before (basal latency, BL) and after drug or saline (control) s.c. administration (i.e. 30, 60 and 120 min) and (test latency, TL). A cut-off time of 10 s was used in order to minimize tissue damage.
Rotarod assay. Possible motor dysfunction or sedative effects of test compounds were assessed in mice using the rotarod test, as earlier described 52,53 . The accelerating rotarod treadmill (Acceler Rota-Rod 7650, Ugo Basile s.r.l., Varese, Italy) for mice (diameter 3.5 cm) was used. Animals were habituated to the equipment in two training sessions (30 min apart) one day before testing. On the experimental day, mice were placed on the rotarod, and treadmill was accelerated from 4 to 40 rpm over a period of 5 min. The time spent on the drum was recorded for each mouse before (baseline) and at 30 min after s.c. administration of saline (control) or test compound. Decreased latencies to fall in the rotarod test indicate impaired motor performance. A 300 s cut-off time was used. Data analysis. Data were analysed and graphically processed using GraphPad Prism 5.0. software (GraphPad Prism Software Inc., San Diego, CA, USA) and are presented as mean ± SEM. The K i (nM), potency EC 50 (nM), and efficacy E max (%) values were determined from concentration-response curves by nonlinear regression analysis. The K i values were determined by the method of Cheng and Prusoff 54 . In the [ 35 S]GTPγS binding assays, efficacy was determined relative to the reference full opioid agonists, DAMGO (µOR), DPDPE (δOR), and U69,593 (κOR). In the cAMP accumulation assay, efficacy was determined relative to the reference µOR agonist DAMGO. The antinociceptive effect (as percentage of Maximum Possible Effect, %MPE) was calculated according to the formula = [(TL -BL)/(cut-off time -BL)] × 100, and the dose necessary to produce a 50% MPE (ED 50 ) and 95% confidence limits (95% CL) were determined using the method of Litchfield and Wilcoxon 55 . In the rotarod test, percentage (%) changes from the rotarod latencies obtained before (baseline, B) and after drug administration (test, T) were calculated as: 100 × (T/B). Data were statistically evaluated using unpaired t-test, one-way ANOVA with Tukey's multiple comparison post hoc test, or two-way ANOVA with significance set at P < 0.05.