Design, Synthesis and Biological Investigation of Flavone Derivatives as Potential Multi-Receptor Atypical Antipsychotics

The design of a series of novel flavone derivatives was synthesized as potential broad-spectrum antipsychotics by using multi-receptor affinity strategy between dopamine receptors and serotonin receptors. Among them, 7-(4-(4-(6-fluorobenzo[d]isoxazol-3-yl) piperidin- 1-yl) butoxy)-2,2-dimethylchroman-4-one (6j) exhibited a promising preclinical profile. Compound 6j not only showed high affinity for dopamine D2, D3, and serotonin 5-HT1A, 5-HT2A receptors, but was also endowed with low to moderate activities on 5-HT2C, α1, and H1 receptors, indicating a low liability to induce side effects such as weight gain, orthostatic hypotension and QT prolongation. In vivo behavioral studies suggested that 6j has favorable effects in alleviating the schizophrenia-like symptoms without causing catalepsy. Taken together, compound 6j has the potential to be further developed as a novel atypical antipsychotic.


Animals
The experimental animal Chinese Kun Ming (KM) Mice (20 ± 2.0 g) and Sprague-Dawley (SD) rats (250 ± 5.0 g), and all the animals provided with standardized management. Animals were randomly selected and grouped; each group fed separate. All the animals used in the study obey the Ethical Guidelines for the Use of Animals in Research

General Procedures for the Binding Assays
All of the new compounds were dissolved in 50% (v/v) DMSO and the compound concentration was adjusted to 2 × 10 −3 M; dilution to the initial concentration of the new compound, 2 × 10 −4 M, contained 5% DMSO. Each specific binding (SB) was calculated as the total binding (TB) minus the nonspecific binding (NB) at a particular concentration of radioligand. Each percentage of inhibition (%) was calculated as follows: percentage of inhibition (%) = [(TB -CB)/(TB -NB)] × 100.
The rats were cut off the head in ice, take out the cerebral cortex and place in centrifuge tubes, and then homogenized in 20 volumes of ice-cold Trise HCl buffer (50 mM, pH 7.5) using an ULTRA TURAX homogenizer, then centrifuged at 48000 g for 10 min at 4 ℃, this procedure were repeated twice and the resulting precipitate was preserved and the supernatant discarded. The final precipitate was resuspended in 50 mM ice-cold Tris-HCl containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 0.1% ascorbic acid, and 5 mM pargyline. For total binding, to each assay tube was added 900 μL of the tissue suspension, 50 μL of 0.5 nM 3 H-spiperone (16.2 Ci/ mmol; PerkinElmer Life Sciences, Boston, MA, USA) and 50 μL Tris HCl buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 0.1% ascorbic acid, and 5 mM pargyline. For nonspecific binding, to each assay tube was added 900 μL of the tissue suspension, 50 μL of 3 H-spiperone, and 50 μL of 10 mM (t)-butaclamol. For compound binding, to each assay tube was added 900 μL of the tissue suspension, 50 μL of 3 H-spiperone, and 50 μL of new compounds or reference drug. The tubes were incubated at 37 °C for 30 min. The tubes were incubated at 37 ℃ for 30 min. the incubation filtered through Whatman GF/B glass filters, filtrates were washed twice with 5.0 mL cold buffer solution, then transferred to a 3.0 mL scintillation vials, 1.0 mL scintillation solution added and the mixture was detected by Beckman LS 6500 liquid scintillation counter.
The rats' heads were removed in ice, the rat olfactory system removed and placed in centrifuge tubes, and then homogenized in 20 volumes of ice-cold Tris HCl buffer (50 mM, pH 7.5) using an ULTRA TURAX homogenizer, followed by centrifugation at 48000 g for 10 min at 4 ℃, this procedure were repeated twice and the resulting precipitate was preserved and supernatant discarded. The final precipitate was resuspended in 50 mM Hepes Na, pH 7.5, containing 1 mM EDTA, 0.005% ascorbic acid, 0.1% albumin, and 200 nM eliprodil. For total binding, to each assay tube was added 900 μL of membranes, 50 μL of 0.6 nM 3 H-7-OH-DPAT (50 Ci/mmol; PerkinElmer Life Sciences), and 50 μL of 50 mM Hepes Na, pH 7.5, containing 1 mM EDTA, 0.005% ascorbic acid, 0.1% albumin, and 200 nM eliprodil. For nonspecific binding, to each assay tube was added 900 μL of membranes, 50 μL of 3 H-7-OH-DPAT (50 Ci/mmol; PerkinElmer Life Sciences), and 50 μL of 1 μM dopamine. For specific binding, to each assay tube was added 900 μL of membranes, 50 μL of 3 H-7-OH-DPAT (50 Ci/mmol; PerkinElmer Life Sciences), and 50 μL of new compounds or reference drug. The tubes were incubated at 25 °C for 60 min. The subsequent testing operation as described for the bending of dopamine D2 receptor. [1][2][3].

5-HT1A Receptor
The rats' heads were cut off in ice, the cerebral cortex removed and placed in centrifuge tubes, and then homogenized in 20 volumes of ice-cold Tris HCl buffer (50 mM, pH 7.5) using an ULTRA TURAX homogenizer, followed by centrifugation at 32000 g for 10 min at 4 ℃, the resulting precipitate was preserved and supernatant discarded. The precipitate was then resuspended in the Trise HCl buffer again, incubated for 10 min at 37 ℃, and centrifuged at 32000 g for 10 min. The precipitate was resuspended in Tris HCl buffer containing 10 mM Pargyline, 4 mM CaCl2 and 0.1% ascorbic acid. Total binding each assay tube was added 900 mL of the tissue suspension, 50 mL of 0.5 nM 3 H-8-OH-DPAT (187.4 Ci/mmol, Perkin Elmer Life Sciences), 50 mL Tris HCl buffer containing 10 mM pargyline, 4 mM CaCl2 and 0.1% ascorbic acid. Non-specific binding each assay tube was added 900 mL of the tissue suspension, 50 mL of 3 H-8-OH-DPAT, 50 mL of 10 mM serotonin. Specific binding each assay tube was added 900 mL of the tissue suspension, 50 mL of 3 H-8-OH-DPAT, 50 mL of new compounds or reference drug. The tubes were incubated at 37 ℃ for 30 min. The subsequent testing operation as described for the bending of dopamine D2 receptor. [1][2][3].

5-HT2A Receptor
The rats' heads were cut off in ice, the cerebral cortex removed and placed in centrifuge tubes, and then homogenized in 20 volumes of ice-cold Tris HCl buffer (50 mM, pH 7.5) using an ULTRA TURAX homogenizer, followed by centrifugation at 32000 g for 20 min at 4 ℃, the resulting precipitate was preserved and supernatant discarded. The precipitate was then resuspended in the Trise HCl buffer again, incubated for 10 min at 37 ℃, and centrifuged at 32000 g for 20 min. The final precipitate was resuspended in 50 volumes of the Tris HCl buffer. Total binding each assay tube was added 900 mL of the tissue suspension, 50 mL of 0.6 nM 3 H-ketanserin (60.0 Ci/mmol, Perkin Elmer Life Sciences), 50 mL Trise HCl buffer. Non-specific binding each assay tube was added 900 mL of the tissue suspension, 50 mL of 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 3 H-ketanserin, 150 mL of new compounds or reference drug. The tubes were incubated at 37 ℃ for 30 min. The subsequent testing operation as described for the bending of dopamine D2 receptor. [1][2][3].

5-HT2C Receptor
The rats' heads were cut off in ice, the cerebral cortex removed and placed in centrifuge tubes, and then homogenized in 20 volumes of ice-cold Tris HCl buffer (50 mM, pH 7.5) using an ULTRA TURAX homogenizer, followed by centrifugation at 32000 g for 20 min at 4 ℃, the resulting precipitate was preserved and supernatant discarded. The precipitate was then resuspended in 50 vol of Tris HCl buffer. For total binding, to each assay tube was added 900 μL of the tissue suspension, 50 μL of 3 H-mesulergine, 50 mL of spiperone, and 50 μL of Tris-HCl buffer. For nonspecific binding, to each assay tube was added 900 μL of the tissue suspension, 50 μL of 1 nM 3 H-mesulergine (85.4 Ci/mmol; PerkinElmer Life Sciences), 50 μL of spiperone, and 50 μL of 10 mM mianserin. For compound binding, to each assay tube was added 900 μL of the tissue suspension, 50 μL of 3 H-mesulergine, 50 μL of spiperone, and 50 μL of new compounds or reference drug. The tubes were incubated at 37 °C for 15 min. The subsequent testing operation as described for the bending of dopamine D2 receptor.
The rats' heads were cut off in ice, the cerebral cortex removed and placed in centrifuge tubes, and then homogenized in 20 volumes of ice-cold Tris HCl buffer (50 mM, pH 7.5) using an ULTRA TURAX homogenizer, followed by centrifugation at 44000 g for 20 min at 4 ℃, the resulting precipitate was preserved and supernatant discarded. The precipitate was then resuspended in 50 vol of Tris HCl buffer. For total binding, to each assay tube was added 900 μL of the tissue suspension, 50 μL of 1nM 3 H-prazosin (85.4 Ci/mmol; PerkinElmer Life Sciences), and 50 μL of Tris-HCl buffer. For nonspecific binding, to each assay tube was added 900 μL of the tissue suspension, 50 μL of 1 nM 3 H-prazosin, and 50 μL of 10 mM prazosin. For compound binding, to each assay tube was added 900 μL of the tissue suspension, 50 μL of 3 H-prazosin, and 50 μL of new compounds or reference drug. The tubes were incubated at 25 °C for 60 min. The subsequent testing operation as described for the bending of dopamine D2 receptor. [1][2][3].

H1 Receptor
The rats' heads were cut off in ice, the cerebral cortex removed and placed in centrifuge tubes, and then homogenized in 20 volumes of ice-cold Tris HCl buffer (50 mM, pH=7.5) using an ULTRA TURAX homogenizer, following centrifuged at 44000 g for 20 min at 4 ℃, this procedure were repeated twice and the resulting precipitate was preserved and supernatant discarded. The final precipitate was then resuspended in 50 vol of phosphate buffer. Total binding each assay tube was added 900 mL of membranes 50 mL of 1 nM 3 H-mepyramine (20.0 Ci/mmol; Perkin Elmer Life Sciences), 50 mL phosphate buffer. Non-specific binding each assay tube was added 900 mL of membranes, 50 mL of 3 H-mepyramine, 50 mL of 1 mM promethazine. Specific binding each assay tube was added 900 mL of membranes, 50 mL of 3 H-mepyramine, 50 mL of new compounds or reference drug. The tubes were incubated at 30 C for 60 min. The subsequent testing operation as described for the bending of dopamine D2 receptor.
For the agonist assay, 5 μL/well 5× working concentration of test compounds was added into cell plate using FLIPR and read with FLIPR (FLIPR Calcium 4, Molecular Devices) using the specified settings and saved data. The total assay volume was 25 μL including 20 μL/well dye loading buffer and 5 μL/well 5× working concentration of test compounds. For the antagonist assay, 6× working concentration of antagonist compound at 5 μL/well was added to cells and incubated plate at room temperature in the dark for 15 min, transferred to the assay plate to FLIPR, and 5 μL/well 6× working concentration of antagonist compound added was using FLIPR and read with FLIPR using the specified settings and saved data. The total assay volume was 30 μL including 20 μL/well dye loading buffer, 5 μL/well 6× work concentration of test compounds, and 5 μL/well 6× work concentration of agonist compound. Data analysis: FLIPR read the plate and got the maximal fluorescence signal data from the excitation light wavelength at 480 nm and emission light wavelength at 520 nm. All results for test compounds were test three times. According to the positive control (HPE) and negative control (ZPE) results, the effect (%) or inhibition (%) of reference and the test compounds was calculated by using Graph Pad Prism 5 to analyze the data and obtained the dose response curve and the value of EC50 and IC50. Effect (%) for agonist mode was calculated from the following equation: effect (%) =(value(raw data)-average(ZPE) )/ (value(HEP)-average(ZPE) ) ×100 The % effect was then plotted as a function of the log of the cumulative doses of compounds. Inhibition (%) for antagonist mode was calculated from the following equation: inhibition (%) = (average (HEP)-average(raw data) )/ (value(HEP)-average(ZPE) ) ×100 The % inhibition was then plotted as a function of the log of the cumulative doses of compounds.

hERG Affinity [2,3]
The ability to block hERG potassium channels was determined according to the related literatures our previous reported [1][2][3]. The test compound 6j was dissolved in 50% (w/v) DMSO and the initial concentration was 1 mM, and the test concentration for compound 6j was diluted to different drug concentrations (0.3, 1, 3, and 10 μM) by the bath solution. The holding membrane(HEK cells) potential was switched from −80 to +50 mV for 2 s, followed by return to −50 mV for 3 s (sampling rate of 4 kHz, low-pass filtered at 1 kHz) in intervals of 30 s. Tail currents were measured at −50 mV in control and in the presence of the drug at different concentrations determined empirically. All raw measurements were performed using Clamp fit (version 10.2), a part of pCLAMP software (version 10.1). The hERG inhibition experiments were tested for three independent experiments. Results were transferred to the program Statistical Package for the Social Sciences (SPSS) spreadsheets for further analysis.

Acute Toxicity
The mice were randomly divided into several groups(n=10), each group were treated with increasing doses of the compound 6j (250, 500, 1000, 1500, and 2000 mg/kg). The number of surviving animals was recorded until 24 h after giving 6j, and the relative mortality rates of each group was calculated. The LD50 values were calculated by using the Statistical Package for the Social Sciences (SPSS) program (SPSS Inc., Chicago, IL, USA). [1][2][3].

Catalepsy Test
The mice were randomly divided into several groups(n=10), and treated with increasing doses of the clozapine (1.0, 5.0, 15.0, 45.0 and 100.0 mg/kg), risperidone (0.1, 0.3, 1.0 and 3.0 mg/ kg), and compound 6j (1.0, 5.0, 15.0, 45.0 and 100.0 mg/kg), respectively. Catalepsy was evaluated on a metal bar 0.3 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. A mean immobility score of 30 s was used as the criterion for the presence of catalepsy.

1 H-NMR of Compound 6j
HR-MS of the compound 6j