Synthesis and Evaluation of a Series of 2-Substituted-5-Thiopropylpiperazine (Piperidine)-1,3,4-Oxadiazoles Derivatives as Atypical Antipsychotics

Background It is important to develop novel antipsychotics that can effectively treat schizophrenia with minor side-effects. The aim of our work is to develop novel antipsychotics that act on dopamine D2 and D3, serotonin 5-HT1A and 5-HT2A receptors with low affinity for the serotonin 5-HT2C and H1 receptors, which can effectively cure positive symptoms, negative symptoms and cognitive impairment without the weight gain side-effect. Methodology/Principal Findings A series of 2-substituted-5-thiopropylpiperazine (piperidine) -1,3,4-oxadiazoles derivatives have been synthesized and the target compounds were evaluated for binding affinities to D2, 5-HT1A and 5-HT2A receptors. Preliminary results indicated that compounds 14, 16 and 22 exhibited high affinities to D2, 5-HT1A and 5-HT2A receptors among these compounds. Further binding tests showed that compound 22 had high affinity for D3 receptor, and low affinity for serotonin 5-HT2C and H1 receptors. In addition, compound 22 inhibited apomorphine-induced climbing behavior and MK-801-induced hyperactivity with no extrapyramidal symptoms liability in mice. Moreover, compound 22 exhibited acceptable pharmacokinetic properties. Conclusions/Significance Compound 22 showed an atypical antipsychotic activity without liability for extrapyramidal symptoms. We anticipate compound 22 to be useful for developing a novel class of drug for the treatment of schizophrenia.


Introduction
Schizophrenia is a serious mental disorder that significantly compromises the quality of life of those suffering from it. The early agents for the treatment of psychosis, the ''typical'' antipsychotics (haloperidol, Figure 1), were therapies for the positive symptoms of schizophrenia, but they failed to manage its negative symptoms and cognitive impairment [1]. Nevertheless, typical antipsychotics carry heavy side effects such as extrapyramidal symptoms (EPS) and hyperprolactinemia [2][3] [4].
A breakthrough in the pharmacotherapy of schizophrenia was achieved by the introduction of the ''atypical'' antipsychotics (e.g., clozapine, ziprasidone, risperidone, quetiapine and olanzapine) which combines a potent antagonism for serotonin 5-HT 2A with a dopamine D 2 receptors blockade [5]. A major advantage of atypical antipsychotics is their effectiveness in suppressing negative and cognitive symptoms [6] [7][8] [9] [10]. However, it has been proved that atypical antipsychotics cause numerous side effects, such as substantial weight gain and QT interval prolongation [11][12] [13]. Therefore, the discovery of novel antipsychotic agents that are effective and free of side effects with different chemical structures remains a challenging.
In the past decade, experimental evidence suggested that a complex binding profile is linked to the clinical efficacy of antipsychotic drugs. Indeed, the importance of designing multitarget G-protein-coupled receptors to deal with schizophrenia has been pointed out by many authors [14][15] [16]. The 5-HT 1A receptor plays crucial roles in regulating psychoemotional, cognitive and motor functions in the central nervous system [17] [18]. Many relevant preclinical data suggested that 5-HT 1A receptor activation may contribute to the improved activity of certain atypical antipsychotic drugs, such as treatment cognitive and negative symptoms, and decrease the development of EPS in schizophrenia [19]. Blockade of D 2 receptor was the key mechanism for controlling positive symptoms of schizophrenia [20]. The localization of D 3 receptor in the limbic regions of brain suggests that this receptor subtype may be a target for developing antipsychotics, and thus, some works suggested that D 3 antagonism may improve cognition [21] and reduce the risk of causing extrapyramidal side effect [22]. Compound S33138 (1) was shown to be a potent and selective dopamine D 3 receptor antagonist,which has been in Phase IIb clinical trials for schizophrenia [23]. Furthermore, two or more receptors may be involved in the weight gain associated with the treatment of schizophrenia via atypical antipsychotic drugs. Blockade of H 1 receptor by antipsychotics is more likely to be the primary cause of these adverse reactions [24] [25]. Although 5-HT 2C receptor blockade has been reported to counteract dopamine D 2 -mediated extrapyramidal side-effects (EPS) [26] and may also confer anxiolytic/antidepressant properties [27], 5-HT 2C receptor may be involved in the risk of obesity under chronic treatment [10][28] [29]. Thus,the aim of our work is to develop a novel antipsychotic that acts on dopamine D 2 and D 3 , serotonin 5-HT 1A and 5-HT 2A receptors with low affinity for the serotonin 5-HT 2C and H 1 receptors, so that it could effectively cure positive symptoms, negative symptoms and cognitive impairment without the weight gain side-effect.
In fact, some of the latest efforts in the development of novel antipsychotic drugs are aimed at obtaining compounds with binding affinities for a certain number of receptors [10] [30][31] [32]. To validate this multireceptor affinity profile approach to antipsychotics and to achieve an optimum interaction with dopamine and serotonin receptors, in this work, we report the synthesis and pharmacological evaluation of a new class of antipsychotic agents with a 1,3,4-oxadiazole system linked to the arylpiperazine (piperidine) group, which is one of the important kind of drugs for CNS-activity [33][34] [35]. This strategy led to the synthesis of compounds 7-26 ( Figure. 1) that allowed us to understand the SAR (structure-activity relationship) and to evaluate the pharmacological efficacy. The target compounds were subjected to preliminary pharmacological evaluation to determine their affinities for D 2 , D 3 , 5-HT 1A , 5-HT 2A , 5-HT 2C and H 1 receptors. Among the derivatives prepared, compound 22 exhibited high affinity to D 2 , D 3, 5-HT 1A and 5-HT 2A receptors, with low affinity for 5-HT 2C and H 1 receptors. In addition, Compound 22 inhibited apomorphine-induced climbing behavior and MK-801-induced hyperactivity without causing catalepsy in mice. In particular, compound 22 was more potent than clozapine.

Synthesis of Compounds 7-26
The general strategy for the synthesis of compounds 7-26 was summarized in Figure 2. Aromatic acids 2 were esterified with absolute ethanol using conc. sulfuric acid as catalyst and the resulting esters 3 were refluxed with hydrazine hydrate in ethanol to give aroyl hydrazines 4. The acid hydrazides were then subjected to cyclisation with carbon disulphide in the presence of potassium hydroxide in absolute alcohol to afford the corresponding 5-aryl-1,3,4-oxadiazol-2-thiones (5). Compounds 5 reacted with 1,3-dibromopropane, in acetone to give 6. Compounds 6 reacted with an arylpiperazine (piperidine) in acetonitrile, in the presence of K 2 CO 3 and a catalytic amount of potassium iodide, to give compounds 7-26 (Table 1) with good yields.
Furthermore, we investigated the effect of replacement of the Ar 1 phenyl ring with naphthalene and heterocyclic ( Table 2,    HT 1A , Ki = 141.6 nM; 5-HT 2A , Ki = 11.6 nM). Moreover, compound 22 displayed higher affinity to 5-HT 1A receptor than risperidone (Ki = 180 nM). However, the introduction of an aromatic heterocycle at Ar 1 (compounds 23-26) resulted in dramatic decrease of affinities for all the three receptors. These results pointed out the importance of the phenyl ring (Ar 1 ) for the affinities at the D 2 , 5-HT 1A and 5-HT 2A receptors.
In line with the multiple receptor-targeting approaches for the development of new antipsychotic agents, compounds 14, 16, and 22 were selected for further binding tests to D 3 , 5-HT 2C and H 1 receptors because they had high affinities for D 2 , 5-HT 1A and 5-H A receptors. Previously, the D 3 receptor was proposed for atypical antipsychotic drugs, and various pharmacological studies suggested that D 3 antagonism might improve cognitive symptoms [21] and reduce catalepsy [22]. Results showed that compounds 14, 16 and 22 displayed higher affinities to D 3 receptor than clozapine (Ki = 239.8 nM). In particular, compound 22 (Ki = 7.7 nM) displayed higher affinity than risperidone (Ki = 9.7 nM). Thus, these results suggested that compounds 14, 16 and 22 could reduce catalepsy in schizophrenia.
Treatment of schizophrenia with atypical antipsychotic drugs has been associated with weight gain. Two receptors, histamine H 1 and 5-HT 2C , have been suggested to be involved in this adverse event [24][25] [10][28] [29]. Several literatures have demonstrated that there is significant correlation between affinity for H 1 receptor and weight gain [24] [25]. As shown in Table 2, compound 22 had much lower affinity (Ki.10000 nM) for H 1 receptor than risperidone (Ki = 21.7 nM) and clozapine (Ki = 3.8 nM). More-over, compound 22 had lower affinity to the 5-HT 2C receptor (Ki.500 nM) in comparison to risperidone (Ki = 14.5 nM) and clozapine (Ki = 16.2 nM). These results suggested that compound 22 exhibited a low potential to elicit treatment-caused weight gain.

Acute Toxicity
The above results led to the conclusion that compound 22 exhibited high affinity for dopamine D 2 and D 3 , serotonin 5-HT 1A and 5-HT 2A receptors, with low affinity for the serotonin 5-HT 2C and H 1 receptors. We then assayed the acute toxicity of the new compound by determining their LD 50 value. Compound 22 showed good safety profiles even at the highest dose tested (LD 50 .2000 mg/kg).

In vivo Studies
An initial behavioral screening was performed on compound 22 based on their multiple receptors affinity profile. The atypical antipsychotics have been used for relieving positive symptoms at doses without EPS [10]. In this study, the side-effect liability was evaluated by the horizontal bar test, which is very sensitive for catalepsy induced by dopamine D 2 receptor blockade [10]. Antipsychotic potential of these compounds were assessed by apomorphine-induced climbing and dizocilpine (MK-801) induced hyperactivity. Apomorphine-induced climbing was potently reduced by D 2 receptor antagonists [36], while selective antagonism of the effect of the noncompetitive N-methyl-Daspartate (NMDA) antagonist MK-801 had been proposed as a Table 2. Binding affinities for D 2 , D 3 , 5-HT 1A , 5-HT 2A , 5-HT 2C and H 1 receptors of compounds 7-26 and reference antipsychotics. a

Compound
Binding data of compounds, Ki±SEM (nM) robust animal model for the negative and cognitive symptoms of schizophrenia [37]. The apomorphine-induced climbing model is based on the induction of a hyperdopaminergic state by apomorphine. This model has been classically linked to motor agitation and one of the schizophrenia positive symptoms [36]. In the apomorphineinduced climbing model, compound 22 produced the significant reversal of apomorphine-induced climbing, with ED 50 value of 3.68 mg/kg (Table 3). In comparison, risperidone, clozapine and haloperidol produced reversal of apomorphine-induced climbing with ED 50 values of 0.02, 7.99 and 0.09 mg/kg, respectively. These results suggested that compound 22 was slightly more potent at blocking the D 2 receptors in vivo than clozapine. This was also consistent with their estimated Ki values at the D 2 receptor.
The MK-801-induced hyperactivity model has been used to indirectly evaluate the ability of compounds to oppose cortical dopaminergic hypofunction induced by NMDA receptor blockade [37]. In this test, compound 22 significantly inhibited MK-801induced hyperactivity with ED 50 value of 3.58 mg/kg (Table 3). In comparison, risperidone, clozapine and haloperidol yielded ED 50 values of 0.01, 5.06 and 0.19 mg/kg, respectively. These results indicated that compound 22 was more potent than clozapine.
Catalepsy is often used as the method for predicting the incidence of extrapyramidal motor disorders. In this model (Table 3), it was clear that haloperidol had the highest propensity to induce catalepsy (ED 50 0.22 mg/kg), in agreement with the high capacity of this drug to block D 2 receptor [38]. In contrast, compound 22 exhibited a low potential to induce catalepsy with ED 50 value.300 mg/kg (Table 3), similar to those of risperidone and clozapine (ED 50 risperidone 0.3 mg/kg, clozapine 92.73 mg/ kg). Moreover, these results suggested that the therapeutic indices of compound 22 calculated between their efficacy (apomorphine or MK-801 models) and side effects (catalepsy) were in the range 81-83, while the therapeutic indices of risperidone and clozapine were roughly 11-30. Thus, in contrast to risperidone and clozapine, compound 22 had a high threshold for inducing catalepsy which might, by analogy, translate into lower clinical EPS liability.
Overall, compound 22 significantly inhibited apomorphineinduced climbing behavior and MK-801-induced hyperactivity without causing catalepsy. These results suggested a preferential ability of compound 22 to modulate mesolimbic instead of nigrostriatal dopaminergic neurotransmission, highlighting their atypicality and low propensity to induce unwanted extrapyramidal motor disturbances at therapeutically useful doses.

Pharmacokinetic Properties of Compound 22
Compound 22 was selected based on its in vitro profile for in vivo characterization. Table 4 highlights the pharmacokinetic parameters of compound 22 in the rat using both intravenous and oral administration. Intravenous administration of compound 22 to rats (5 mg/kg, n = 6) resulted in detectable plasma levels (half-life (t 1/2 ) = 9.3 h), and oral administration of compound 22 to rats (20 mg/kg, n = 6) resulted in a t 1/2 of 8.6 h. The area under the curve (AUC) value of compound 22 was 6239.0 ng6h/mL after intravenous administration versus 13602.7 ng6h/mL after oral administration. The C max value after oral dosing was 723.6 ng/ mL, and the T max value was 5.0 h. The bioavailability of compound 22 was 54.5%.
In summary, we described the synthesis and pharmacological evaluation of a series of 2-substituted-5-thiopropylpiperazine (piperidine)-1,3,4-oxadiazoles derivatives as potential multi-target antipsychotics. Among the derivatives synthesized, compound 22 showed high affinity for dopamine D 2 and D 3 , serotonin 5-HT 1A and 5-HT 2A receptors, with low affinity for the serotonin 5-HT 2C and H 1 receptors. In vivo animal models showed that compound 22 had high potential for treating symptoms of schizophrenia without causing catalepsy. Moreover, compound 22 exhibited acceptable pharmacokinetic properties.

Synthesis of Compounds 7-26
Melting points were determined in open capillary tubes and are uncorrected. 1 H NMR spectra were recorded at 400 MHz on a Varian Inova Unity 200 spectrometer in CDCl 3 solution. Chemical shifts were given in d values (ppm), using tetramethylsilane (TMS) as the internal standard; coupling constants (J) were given in Hz. Signal multiplicities were characterized as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad signal). Reagents were all of analytical grade or of chemical purity. Analytical TLC was performed on silica gel GF254. Column chromatographic purification was carried out using silica gel. General procedure for the synthesis of aroyl hydrazines 4 (a-h) [39]. A mixture of aromatic acids 2 (10 mmol), ethanol (20 mL) and a catalytic amount of conc. H 2 SO 4 were refluxed for 3 h. The reaction mixture was cooled and the formed solids were filtered to give ester 3, which was refluxed with 85% hydrazine hydrate (10 mL) in ethanol (20 mL) for 2 h. After completion of the reaction by TLC, the reaction mixture was cooled and the formed solids were filtered and washed with chilled ethanol (1 mL General procedure for the preparation of 5-aryl-1,3,4oxadiazol-2-thiones 5 (a-h) [44]. A mixture of 10 mmol of potassium hydroxide, 10 mmol of compounds 4 (a-h), and 15 mmol of carbon disulfide in 50 mL of absolute ethanol was refluxed for 8 h. After the solvent was evaporated in vacuum, the residue was dissolved in ice-cold water and acidified with dilute hydrochloric acid. The precipitate was filtered off, washed with water, and recrystallized from absolute ethanol to give compounds 5 (a-h). General procedure for the preparation of 5-aryl-2-((3bromopropyl)thio)-1,3,4-oxadiazole 6 (a-h). 1,3dibromopropane (3 mmol) was added to a solution of compounds 5 (a-h) (1 mmol) and potassium carbonate in acetone (50 mL), and the mixture was refluxed for 3 h. The progress of the reaction was monitored by TLC. After cooling to room temperature, the mixture was filtered, the solvent was evaporated and the residue was recrystallized from hexane/EtOH to yield compounds 6 (a-h). General procedure for the preparation of compounds 7-26. To a suspension of compounds 6 (0.32 mmol) and K 2 CO 3 (1.22 mmol) in acetonitrile (5.0 mL), arylpiperazine (piperidine) (0.32 mmol) and a catalytic amount of KI were added and the resulting mixture was refluxed for 12 h. After filtering, the resulting filtrate was evaporated to dryness under reduced pressure. The residue was suspended in water (10.0 ml) and extracted with dichloromethane (3625 mL). The combined organic layers were evaporated under reduced pressure, and the crude product was purified by means of chromatography (5% MeOH/CHCl 3 ) to yield compounds 7-26.

Ethics Statement
Chinese Kun Ming (KM) Mice (2062.0 g) and Sprague-Dawley (SD) rats (25065.0 g) were used as experimental animals in this study. Animals were housed under standardized conditions for light and temperature and received standard rat chow and tap water and libitum. Animals were randomly assigned to different experimental groups and each group was kept in a separate cage. All the research involving animals in this study follows the guidelines of the byelaw of experiments on animals, and has been approved by the Ethics and Experimental Animal Committee of Jiangsu Nhwa Pharmaceutical Co., Ltd.

In Vitro Binding Assays
General procedures. All the new compounds were dissolved in 5% DMSO. The following specific radioligands and tissue sources were used: (a) serotonin 5-HT 1A receptor, [ 3 H]8-OH-DPAT, rat brain cortex; (b) serotonin 5-HT 2A  Total binding was determined in the absence of no-specific binding and compounds. Specific binding was determined in the presence of compounds. Non-specific binding was determined as the difference between total and specific binding.
Blank experiments were carried out to determine the effect of 5% DMSO on the binding and no effects were observed. Compounds were tested at least three times over a 6 concentration range (10 25  5-HT 1A binding assay [49]. Rat cerebral cortex was homogenized in 20 volumes of ice-cold Tris-HCl buffer (50 mM, pH 7.7) using an ULTRA TURAX homogeniser, and was then centrifuged at 32000 g for 10 min. The resulting pellet was then resuspended in the same buffer, incubated for 10 min at 37 uC, and centrifuged at 32000 g for 10 min. The final pellet was resuspended in Tris-HCl buffer containing 10 mM Pargyline, 4 mM CaCl 2 and 0.1% ascorbic acid.
Total binding each assay tube was added 900 mL of the tissue suspension, 50 mL of 0. The tubes were incubated at 37uC for 30 min. The incubation was followed by a rapid vacuum filtration through Whatman GF/ B glass filters, and the filtrates were washed twice with 5mL cold buffer and transferred to scintillation vials. Scintillation fluid (3.0 mL) was added and the radioactivity bound was measured using a Beckman LS 6500 liquid scintillation counter.
5-HT 2A binding assay [49]. Rat cerebral cortex was homogenized in 20 volumes of ice-cold Tris-HCl buffer (50 mM, pH 7.7) using an ULTRA TURAX homogeniser, and centrifuged at 32000 g for 20 min. The resulting pellet was resuspended in the same quantity of the buffer centrifuged for 20 min. The final pellet was resuspended in 50 volumes of the Tris-HCl buffer.
Non-specific binding each assay tube was added 900 mL of the tissue suspension, 50 mL of 0.6 nM [ 3 H]ketanserin, 50 mL of 10 mM methisergide.
Specific binding each assay tube was added 900 mL of the tissue suspension, 50 mL of 0.6 nM [ 3 H]ketanserin, 150 mL of new compounds or reference drug.
The tubes were incubated at 37uC for 15 min. The incubation was followed by a rapid vacuum filtration through Whatman GF/ B glass filters, and the filtrates were washed twice with 5 mL cold buffer and transferred to scintillation vials. Scintillation fluid (3.0 mL) was added and the radioactivity bound was measured using a Beckman LS 6500 liquid scintillation counter.
5-HT 2C binding assay [49]. Rat cerebral cortex was homogenized in 20 volumes of ice-cold Tris-HCl buffer (50 mM, pH7.7) using ULTRA TURAX homogeniser, and centrifuged at 32000 g for 20 min. The resulting pellet was resuspended in the same quantity of the buffer centrifuged for 20 min. The final pellet was resuspended in 50 volumes of the Tris-HCl buffer.
Non-specific binding each assay tube was added 900 mL of the tissue suspension, 50 mL of 1 nM [ 3 H]mesulergine, 50 mL of 10 mM mianserin.
Specific binding each assay tube was added 900 mL of the tissue suspension, 50 mL of 1 nM [ 3 H]mesulergine, 50 mL of new compounds or reference drug.
The tubes were incubated at 37uC for 15 min. The incubation was followed by a rapid vacuum filtration through Whatman GF/ B glass filters, and the filtrates were washed twice with 5 mL cold buffer and transferred to scintillation vials. Scintillation fluid (3.0 mL) was added and the radioactivity bound was measured using a Beckman LS 6500 liquid scintillation counter. D 2 dopaminergic binding assay [49]. Rat striatum was homogenized in 20 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.7) using an ULTRA TURAX homogeniser, and centrifuged twice for 10 min at 48,000 g with resuspension of the pellet in fresh buffer. The final pellet was resuspended in 50 mM ice-cold Tris-HCl containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 0.1% ascorbic acid and 5 mM pargyline.
Specific binding each assay tube was added 900 mL of the tissue suspension, 50 mL of 0.5 nM [ 3 H]spiperone, 50 mL of new compounds or reference drug.
The tubes were incubated at 37uC for 15 min. The incubation was followed by a rapid vacuum filtration through Whatman GF/ B glass filters, and the filtrates were washed twice with 5 mL cold buffer and transferred to scintillation vials. Scintillation fluid (3.0 mL) was added and the radioactivity bound was measured using a Beckman LS 6500 liquid scintillation counter. D 3 Dopaminergic Binding Assay [37].
Rat olfactory tubercle was homogenized in 20 volumes of icecold 50 mM Hepes Na (pH 7.5) using an ULTRA TURAX homogeniser, and centrifuged twice for 10 min at 48,000 g with resuspension of the pellet in fresh buffer. The final pellet was resuspended in 50 mM Hepes Na, pH 7.5, containing 1 mM EDTA, 0.005% ascorbic acid, 0.1% albumin, 200 nM eliprodil.
Non-specific binding each assay tube was added 900 mL of membranes, 50 mL of [ 3 H]spiperone, 50 mL of 1 mM dopamine.
Specific binding each assay tube was added 900 mL of Membranes, 50 mL of [ 3 H]spiperone, 50 mL of new compounds or reference drug.
The tubes were incubated at 25uC for 60 min. The incubation was followed by a rapid vacuum filtration through Whatman GF/ B glass filters, and the filtrates were washed twice with 5 mL cold buffer and transferred to scintillation vials. Scintillation fluid (3.0 mL) was added and the radioactivity bound was measured using a Beckman LS 6500 liquid scintillation counter.
Histamine H 1 Binding Assay [50] Guinea pig cerebellum was homogenized in 20 volumes of icecold 50 mM phosphate buffer (pH 7.4) using an ULTRA TURAX homogeniser, and centrifuged twice for 10 min at 50,000 g with resuspension of the pellet in fresh buffer. The final pellet was resuspended in phosphate buffer.
Non-specific binding each assay tube was added 900 mL of membranes, 50 mL of [ 3 H]pyrilamine, 50 mL of 1 mM promethazine.
Specific binding each assay tube was added 900 mL of Membranes, 50 mL of [ 3 H]pyrilamine, 50 mL of new compounds or reference drug.
The tubes were incubated at 30uC for 60 min. The incubation was followed by a rapid vacuum filtration through Whatman GF/ B glass filters, and the filtrates were washed twice with 5 mL cold buffer and transferred to scintillation vials. Scintillation fluid (3.0 mL) was added and the radioactivity bound was measured using a Beckman LS 6500 liquid scintillation counter.
Acute toxicity study. Mice (5 mice in each group) were orally dosed with increasing doses of the compound 22 (200, 500, 1000, 1500 and 2000 mg/kg). The number of surviving animals was recorded after 24 h of drug administration, and the percent mortality in each group was calculated. The LD 50 value was calculated by using the program SPSS (Statistical Package for the Social Science).
Catalepsy test [38]. Mice (10 mice in each group) were orally dosed with vehicle or increasing doses of the haloperidol (0.18, 0.35, 0.75, 1.5 and 3.0 mg/kg), clozapine (25,50,100,150 and 200 mg/kg), risperidone (0.1, 0.6, 1.2, 2.5 and 5.0 mg/kg), compound 22 (50,150 and 300 mg/kg). Catalepsy was evaluated on a metal bar 0.6 cm in diameter positioned 4.5 cm above the tabletop. The test consisted in positioning the animal with its forepaws on the bar and recording how long it remained hanging onto the bar; the end-point was 60 s and an all-or-none criterion was used.
For routine compound 22 screening rats (n = 6/group) were dosed via the lateral tail vein at the indicated dose for intravenous administration (5 mg/kg, 100% saline) or via oral gavage (20 mg/ kg, suspension in 0.5% methylcellulose). At 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h and 24 h after administration, serial blood samples were collected from the lateral tail vein into heparinized collection tubes (approximately 0.25 mL). The plasma was separated by centrifugation, and the sample was prepared for analysis HPLC/MS by protein precipitation with acetonitrile. The plasma samples were analyzed for drug and internal standard via HPLC-MS/MS protocol.
Statistics. To estimate the potency of test and reference compounds, the ED 50 values and their 95% confidence limits were calculated by using the program SPSS (Statistical Package for the Social Science).