Alicyclic[c] benzopyrone derivatives and uses thereof

7-{3-[4-(2,3-dimethylphenyl)piperazin-1-yl]-propoxy}-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

1) 7.3 g of adipic acid (50 mmol) and 40 ml of anhydrous methanol were added to a flask, and 2 ml of concentrated sulfuric acid was added slowly and dropwise under stirring with heating, and the reaction was carried out under reflux for 5 hours. The mixture was cooled to room temperature and the solvent was distilled off under reduced pressure. The mixture was then diluted with 30 ml of dichloromethane and 25 ml of water. The aqueous layer was extracted with dichloromethane (30 ml×2). The organic layers were combined, and washed to neutral with saturated sodium bicarbonate solution and then with saturated NaCl, dried with anhydrous magnesium sulfate, and filtrated. The solvent was distilled off under reduced pressure to give 8 g of dimethyl adipate. The yield was 91.9%.

2) 8.7 g (50 mmol) of dimethyl adipate, anhydrous aluminum trichloride (20 g) and 100 ml of dry dichloromethane were cooled to 0° C. under an ice bath. At 0° C., triethylamine (dried with sodium hydroxide, 45 ml) was added dropwise and slowly. After addition, the ice bath was removed and the reaction was carried out at room temperature for 4 hours, being monitored with TLC. After the reaction was completed, the reaction liquid was poured into an ice-water mixture slowly, which was stirred for 30 min. The organic layer was separated and the aqueous layer was extracted with dichloromethane (50 ml×2). The organic layers were combined and washed with water and then with saturated NaCl, dried with anhydrous magnesium sulfate, and filtrated. The solvent was distilled off under reduced pressure to give 5.1 g of 2-oxocyclopentanecarboxylate methyl ester. The yield was 71.8%.

3) 7.1 g (50 mmol) of 2-oxocyclopentanecarboxylate methyl ester, 5.5 g (50 mmol) of resorcinol and bismuth nitrate pentahydrate were heated to 80° C. and the reaction was carried out for 4 hours. After the reaction was completed, the reaction mixture was cooled to room temperature and the reaction liquid was poured into water and yellowish solid was precipitated. Stirring was performed for 30 min and filtration lead to solid. Recrystallization with 95% ethanol gave 5.8 g of white solid. The yield was 57.4%.

4) 5 g of the product from step 3), 6 g of anhydrous potassium carbonate, 50 ml of acetone and 8 g of 1,3-dibromopropane were heated under reflux for 6 hours and then cooled to room temperature and filtrated. The solvent was distilled off to give yellowish oil, which was pass through a column to give 5 g of white solid.

5) To 0.51 g of the product from step 4) was added 0.61 g of 2,3-dimethylphenylpiperazine hydrochloride, 2 g of anhydrous potassium carbonate, 0.2 g of potassium iodide and 25 ml of acetonitrile and the mixture was heated under reflux for 12 hours and then cooled to room temperature. The solvent was distilled off. Appropriate amount of dichloromethane was added to the reaction mixture, which was washed with water. The aqueous layer was separated and the organic layer was dried with anhydrous magnesium sulfate. The solvent was distilled off to give yellowish oil, which was pass through a chromatograph column to give 0.48 g of white solid.

1 H NMR (CDCl 3 ) δ 2.03-2.26 (m, 10H), 2.59-2.66 (m, 6H), 2.89-3.04 (m, 8H), 4.11 (t, 2H, J=12.8 Hz), 6.84-6.94 (m, 4H), 7.06-7.08 (m, 1H), 7.33 (d, 1H, J=8.4 Hz)

MS (ESI) m/z 433.3 ([M+H] + ).

7-{3-[4-(2,3-dichlorophenyl)piperazin-1-yl]-propoxy}-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

The target compound was prepared according to the procedures of Example 1, using 2,3-dichlorophenylpiperazine hydrochloride instead of 2,3-dimethylphenylpiperazine hydrochloride.

1 H NMR (CDCl 3 ) δ 2.03-2.23 (m, 4H), 2.60-2.68 (m, 6H), 2.89 (t, 2H, J=15.2 Hz), 3.02-3.08 (m, 6H), 4.11 (t, 2H, J=12.4 Hz), 6.84-6.98 (m, 3H), 7.14-7.16 (m, 2H), 7.33 (d, 1H, J=8.4 Hz)

MS (ESI) m/z 473.2 ([M+H] + ).

7-{3-[4-(3-trifluoromethylphenyl)piperazin-1-yl]-propoxy}-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

The target compound was prepared according to the procedures of Example 1, using 3-trifluoromethylphenylpiperazine hydrochloride instead of 2,3-dimethylphenylpiperazine hydrochloride.

1 H NMR (CDCl 3 ) δ 2.03-2.23 (m, 4H), 2.59-2.66 (m, 6H), 2.89 (t, 2H, J=14.8 Hz), 3.04 (t, 2H, J=15.8 Hz), 3.26 (t, 4H, J=10 Hz), 4.11 (t, 2H, J=12.4 Hz), 6.84-6.87 (m, 2H), 7.06-7.11 (m, 3H), 7.32-7.34 (m, 2H)

MS (ESI) m/z 473.3 ([M+H] + ).

7-{3-[4-(2-methoxy)piperazin-1-yl]-propoxy}-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

The target compound was prepared according to the procedures of Example 1, using 2-methoxyphenylpiperazine hydrochloride instead of 2,3-dimethylphenyl piperazine hydrochloride.

1 H NMR (CDCl 3 ) δ 2.05-2.21 (m, 4H), 2.62 (t, 2H, J=14.4 Hz), 2.70 (s, br, 4H), 2.87-2.89 (m, 2H), 3.02-3.12 (m, 6H), 3.87 (s, 3H), 4.10 (t, 2H, J=12.4 Hz), 6.84-7.00 (m, 6H), 7.32-7.34 (m, 1H)

MS (ESI) m/z 435.3 ([M+H] + ).

7-{3-[4-(3-(6-fluoro-benzisoxazole)-1-piperidyl)-n-propoxy]}-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

The target compound was prepared according to the procedures of Example 1, using 6-fluoro-3-(4-piperidyl)-1,2-benzisoxazole hydrochloride instead of 2,3-dimethylphenyl piperazine hydrochloride.

1 H NMR (CDCl 3 ) δ 1.90-2.20 (m, 10H), 2.61 (t, 2H, J=14.8 Hz), 2.88-2.91 (m, 2H), 3.03-3.11 (m, 5H), 4.11 (t, 2H, J=12.4 Hz), 6.85-6.87 (m, 2H), 7.06-7.07 (m, 1H), 7.23-7.35 (m, 2H), 7.70-7.73 (m, 1H)

MS (ESI) m/z 463.3 ([M+H] + ).

7-{3-[4-(2-methoxyphenyl)piperazin-1-yl]-propoxy}-6-methyl-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

The target compound was prepared according to the procedures of Example 4, using 2-methylresorcinol as starting material.

1 H NMR (CDCl 3 ) δ 2.06-2.21 (m, 4H), 2.33 (s, 3H), 2.65-3.12 (m, 14H), 3.87 (s, 3H), 4.13 (t, 2H, J=12.8 Hz), 6.83-7.01 (m, 5H), 7.22-7.27 (m, 1H)

MS (ESI) m/z 449.3 ([M+H] + )

7-{4-[4-(2-methoxyphenyl)piperazin-1-yl]-butoxy}-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

The target compound was prepared according to the procedures of Example 4, using 1,4-dibromobutane instead of 1,3-dibromopropane.

1 H NMR (CDCl 3 ) δ 1.74-1.89 (m, 4H), 2.19-2.21 (m, 3H), 2.52 (t, 2H, J=14.4 Hz), 2.71 (s, br, 4H), 2.87-2.90 (m, 2H), 3.02-3.13 (m, 5H), 3.86 (s, 3H), 4.05 (t, 2H, J=12.4 Hz), 6.83-7.00 (m, 6H), 7.32-7.34 (m, 4H)

MS (ESI) m/z 449.3 ([M+H] + )

7-{4-[4-(4-methoxyphenyl)piperazin-1-yl]-butoxy}-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

The target compound was prepared according to the procedures of Example 7, using 4-methoxyphenylpiperazine hydrochloride instead of 2-methoxyphenylpiperazine hydrochloride.

1 H NMR (CDCl 3 ) δ 1.71-1.87 (m, 4H), 2.19 (t, 2H, J=14.8 Hz), 2.47 (t, 2H, J=14.8 Hz), 2.62-2.64 (m, 4H), (t, 4H, J=10 Hz), 2.89 (t, 2H J=15.2 Hz), 3.04 (t, 2H, J=15.2 Hz), 3.09-3.11 (m, 4H), 3.77 (s, 3H), 4.05 (t, 2H, J=12.4 Hz), 6.82-6.92 (m, 6H), 7.32 (d, 1H, J=8.4 Hz)

MS (ESI) m/z 449.3 ([M+H] + )

7-{4-[4-(4-fluorophenyl)piperazin-1-yl]-butoxy}-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

The target compound was prepared according to the procedures of Example 7, using 4-fluorophenylpiperazine hydrochloride instead of 2-methoxyphenylpiperazine hydrochloride.

1 H NMR (CDCl 3 ) δ 1.71-1.89 (m, 4H), 2.19 (t, 2H, J=15.2 Hz), 2.47 (t, 2H, J=14.8 Hz), 2.62 (t, 4H, J=10 Hz), 2.88 (t, 2H, J=14.8 Hz), 3.03 (t, 2H, J=15.2 Hz), 3.12 (t, 2H, J=9.6 Hz), 4.05 (t, 2H, J=12.4 Hz), 6.82-6.97 (m, 6H), 7.32 (d, 1H, J=9.2 Hz)

MS (ESI) m/z 437.2 ([M+H] + )

7-{4-[4-(3-(6-fluoro-benzisoxazole)-1-piperidyl)-butoxy]}-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

The target compound was prepared according to the procedures of Example 7, using 6-fluoro-3-(4-piperidyl)-1,2-benzisoxazole hydrochloride instead of 2-methoxyphenylpiperazine hydrochloride.

1 H NMR (CDCl 3 ) δ 1.74-1.88 (m, 4H), 2.06-2.21 (m, 8H), 2.48 (t, 2H, J=14.8 Hz), 2.87-2.89 (m, 2H), 3.02-3.10 (m, 5H), 4.06 (t, 2H, J=12.4 Hz), 6.83-6.86 (m, 2H), 7.04-7.05 (m, 1H), 7.22-7.27 (m, 1H), 7.33 (d, 1H, J=9.2 Hz), 7.68-7.71 (m, 1H)

MS (ESI) m/z 477.3 ([M+H] + )

7-{4-[4-(2-methoxyphenyl)piperazin-1-yl]-butoxy}-6-methyl-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

The target compound was prepared according to the procedures of Example 6, using 1,4-dibromobutane instead of 1,3-dibromopropane.

1 H NMR (CDCl 3 ) δ 1.76-1.90 (m, 4H), 2.16-2.20 (m, 2H), 2.33 (s, 3H), 2.51 (t, 2H, J=14.8 Hz), 2.67 (s, br, 4H), 2.87-2.90 (m, 2H), 3.00-3.10 (m, 5H), 3.86 (s, 3H), 4.08 (t, 2H, J=12 Hz), 6.80-6.99 (m, 5H), 7.21-7.23 (m, 1H)

MS (ESI) m/z 463.3 ([M+H] + )

7-{4-[4-(3-(1,2-benzisothiazole)-1-piperazinyl)-butoxy]}-6-methyl-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

The target compound was prepared according to the procedures of Example 11, using 3-(1-piperazinyl)-1,2-benzisothiazole hydrochloride instead of 2-methoxyphenylpiperazine hydrochloride.

1 H NMR (CDCl 3 ) δ 1.78-1.92 (m, 4H), 2.17-2.21 (m, 2H), 2.34 (s, 3H), 2.54 (t, 2H, J=14.4 Hz), 2.69-2.72 (m, 4H), 2.89-3.03 (m, 4H), 3.57-3.59 (m, 4H), 4.10 (t, 2H, J=12.4 Hz), 6.81-6.83 (m, 1H), 7.22-7.27 (m, 1H), 7.34-7.37 (m, 1H), 7.45-7.49 (m, 1H), 7.80-7.82 (m, 1H), 7.90-7.92 (m, 1H)

MS (ESI) m/z 490.3 ([M+H] + )

7-{4-[4-(2-methoxyphenyl)piperazin-1-yl]-butoxy}-6-chloro-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

The target compound was prepared according to the procedures of Example 11, using 2-chlororesorcinol instead of 2-methylresorcinol.

1 H NMR (CDCl 3 ) δ 1.78-1.96 (m, 4H), 2.18-2.22 (m, 2H), 2.51 (t, 2H, J=14.8 Hz), 2.67 (s, br, 4H), 2.87-2.90 (m, 2H), 3.01-3.10 (m, 5H), 3.86 (s, 3H), 4.16 (t, 2H, J=12.4 Hz), 6.87-6.93 (m, 5H), 7.26-7.28 (m, 1H)

MS (ESI) m/z 483.3 ([M+H] + )

7-{4-[4-(3-(1,2-benzisothiazole)-1-piperazinyl)-n-butoxy]}-6-chloro-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

The target compound was prepared according to the procedures of Example 13, using 3-(1-piperazinyl)-1,2-benzisothiazole hydrochloride instead of 2-methoxyphenylpiperazine hydrochloride.

1 H NMR (CDCl 3 ) δ 1.76-2.20 (m, 6H), 2.54 (t, 2H, J=14.4 Hz), 2.68-2.70 (m, 4H), 2.85-2.99 (m, 2H), 3.01-3.03 (m, 2H), 3.55 (s, br, 4H), 4.17 (t, 2H, J=12 Hz), 6.85-6.88 (m, 1H), 7.24-7.36 (m, 2H), 7.43-7.47 (m, 1H), 7.77-7.80 (m, 1H), 7.88-7.91 (m, 1H)

MS (ESI) m/z 510.2 ([M+H] + )

7-{4-[4-(3-(6-fluoro-benzisoxazole)-1-piperidyl)-butoxy]}-6-chloro-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

The target compound was prepared according to the procedures of Example 13, using 6-fluoro-3-(4-piperidyl)-1,2-benzisoxazole hydrochloride instead of 2-methoxyphenylpiperazine hydrochloride.

1 H NMR (CDCl 1 ) δ 1.76-2.23 (m, 12H), 2.51 (t, 2H, J=14.8 Hz), 2.87-2.90 (m, 2H), 3.02-3.11 (m, 5H), 4.17 (t, 2H, J=12.4 Hz), 6.88-6.91 (m, 1H), 7.04-7.05 (m, 1H), 7.22-7.30 (m, 2H), 7.68-7.72 (m, 1H)

MS (ESI) m/z 511.3 ([M+H] + )

7-{4-[4-(3-(6-fluoro-benzisoxazole)-1-piperidyl)-butoxy]}-8-chloro-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

The target compound was prepared according to the procedures of Example 15, using 4-chlororesorcinol instead of 2-chlororesorcinol as starting material.

1 H NMR (CDCl 3 ) δ 1.79-2.23 (m, 12H), 2.51 (t, 2H, J=14.4 Hz), 2.88-2.92 (m, 2H), 3.00-3.09 (m, 5H), 4.12 (t, 2H, J=12.4 Hz), 6.88-6.89 (m, 1H), 7.03-7.05 (m, 1H), 7.23-7.27 (m, 1H), 7.42 (s, 1H), 7.68-7.72 (m, 1H)

MS (ESI) m/z 511.3 ([M+H] + )

7-{4-[4-(2-methoxyphenyl)piperazin-1-yl-butoxy]}-8-chloro-2,3-dihydro-1H-cyclopenteno[c]benzopyran-4-one

The target compound was prepared according to the procedures of Example 16, using 2-methoxyphenylpiperazine hydrochloride instead of 6-fluoro-3-(4-piperidyl)-1,2-benzisoxazole hydrochloride.

1 H NMR (CDCl 3 ) δ 1.69-1.95 (m, 6H), 2.19-2.23 (m, 2H) 2.51 (t, 2H, J=14.8 Hz), 2.68 (s, br, 3H), 2.88-2.91 (m, 2H), 3.00-3.10 (m, 5H), 3.86 (s, 3H) 4.12 (t, 2H, J=12.4 Hz), 6.85-7.00 (m, 5H), 7.41 (s, 1H)

MS (ESI) m/z 483.3 ([M+H] + )

3-{4-[4-(3-(6-fluoro-benzisoxazole)-1-piperidyl)-butoxy]}-7,8,9,10-tetrahydrobenzo[c]benzopyran-6-one

The target compound was prepared according to the procedures of Example 10, using pimelic acid instead of adipic acid.

1 H NMR (CDCl 1 ) δ 1.67-1.87 (m, 10H), 2.06-2.13 (m, 4H), 2.47 (t, 2H, J=14.8 Hz), 2.56 (t, 2H, J=8.8 Hz), 2.75 (t, 2H, J=8.8 Hz), 3.07-3.10 (m, 3H), 4.05 (t, 2H, J=12.8 Hz), 6.80-6.85 (m, 2H), 7.04-7.05 (m, 1H), 7.22-7.25 (m, 1H), 7.45 (d, 1H, J=8.4 Hz),

7.68-7.71 (m, 1H)

MS (ESI) m/z 491.3 ([M+H] + )

3-{4-[4-(2-methoxyphenyl)piperazin-1-yl]-butoxy}-7,8,9,10-tetrahydrobenzo[c]benzopyran-6-one

The target compound was prepared according to the procedures of Example 18, using 2-methoxyphenyl piperazine hydrochloride instead of 6-fluoro-3-(4-piperidyl)-1,2-benzisoxazole hydrochloride.

1 H NMR (CDCl 3 ) δ 1.73-1.87 (m, 8H), 2.47-2.74 (m, 10H), 3.11 (s, br, 4H), 3.86 (s, 3H) 4.04 (t, 2H, J=12.4 Hz), 6.78-6.99 (m, 6H), 7.43-7.15 (m, 1H)

MS (ESI) m/z 463.3 ([M+H] + )

3-{4-[4-(2-methoxyphenyl)piperazin-1-yl]-butoxy}-4-methyl-7,8,9,10-tetrahydrobenzo[c]benzopyran-6-one

The target compound was prepared according to the procedures of Example 19, using 2-methylresorcinol instead of resorcinol.

1 H NMR (CDCl 3 ) δ 1.76-1.88 (m, 8H), 2.32 (s, 3H), 2.48-2.75 (m, 10H), 3.11 (s, br, 4H), 3.87 (s, 3H), 4.08 (t, 2H, J=12.4 Hz), 6.80-7.00 (m, 5H), 7.35 (d, 1H, J=8.8 Hz)

MS (ESI) m/z 477.3 ([M+H] + )

3-{4-[4-(3-(6-fluoro-benzisoxazole)-1-piperidyl)-butoxy]}-4-methyl-7,8,9,10-tetrahydrobenzo[c]benzopyran-6-one

The target compound was prepared according to the procedures of Example 18, using 2-methylresorcinol instead of resorcinol as starting material.

1 H NMR (CDCl 3 ) δ 1.78-1.88 (m, 8H), 2.07-2.08 (m, 6H), 2.32 (s, 3H), 2.48 (t, 2H, J=7.6 Hz), 2.57-2.75 (m, 4H), 3.08-3.10 (m, 3H), 4.08 (t, 2H, J=12 Hz), 6.81-6.83 (m, 1H), 7.05-7.06 (m, 1H), 7.23-7.26 (m, 1H), 7.35-7.37 (m, 1H), 7.68-7.71 (m, 1H)

MS (ESI) m/z 505.3 ([M+H] + )

5HT 1A Receptor Assay

Preparation of 5HT 1A Membrane

Rats were sacrificed by cervical dislocation on ice. Cerebral cortex was rapidly taken, and 2 cerebral cortexes were combined into a centrifuge tube, to which 3 ml of buffer (0.05 M Tris-HCl buffer, containing 0.1% ascorbic acid, 10 μm pargyline and 4 mM CaCl 2 ) was added. Homogenization was conducted for 3-4 s at level 4 for four times, and then 5 ml of buffer was added (0.05 M Tris-HCl buffer, containing 0.1% ascorbic acid, 10 μm pargyline and 4 mM CaCl 2 ). Incubation at 37° C. was conducted for 10 min, the weight of the tubes were adjusted using a balance after incubation. Centrifugation was conducted at 12000 r, 4° C. for 20 min, the supernatant was discarded, and 3 ml of buffer was added. Vortex mixer was used for blending, and then 5 ml of buffer was added. Centrifugation was conducted and repeated 3 times. After the centrifugations, the supernatant was discarded, and the pellets were stored at −80° C. for future use.

Materials for the Receptor Binding Assay

Isotope ligand 3 H-8-OH-DPAT (67.0 Ci/mmol) was purchased from PerkinElmer Company; 5-HT was purchased from RBI Company; GF/C glass fiber filter paper was purchased from Whatman Company; Tris was imported and divided into aliquots; PPO, POPOP were purchased from Shanghai No. 1 Reagent Factory; lipid-soluble scintillation solution. Beckman LS-6500 Multi-function Liquid Scintillation Counter was used.

Procedures

(1) The prepared membrane was first applied with appropriate amount of buffer, and homogenizer was used for evenly dispersing. 15 tubes were mixed into a 100 ml container, and appropriate amount of buffer was added to give 50 ml of membrane suspension, which was reserved for future use.

(2) 100 μL of membrane preparation and 100 μL of homogenized liquid were added into each reaction tube.

(3) 100 μL of homogenized liquid was added into the total binding tube (TB), 100 μL of 5-HT (final concentration 10 −5 M) was added into the non-specific binding tube (NB), and 100 μL of the test compound (final concentration 10 −5 M) was added into the specific binding tube (SB) for each compound.

(4) 10 μL of radioactive ligand 3 H-8-OH-DPAT was respectively added into each reaction tube (2 parallel tubes were used for each reaction tube, and each of them was placed on ice when adding sample).

(5) Each reaction tube was incubated at 37° C. for 10 min; after the reaction was completed, the bound ligands were rapidly filtered under reduced pressure, and the ice-chilled assay buffer was used for adequate washing. The filter was taken out and put into a 3 ml scintillation vial, and 2 ml of toluene scintillation solution was added and blended.

(6) The scintillation vials were put into Liquid Scintillation Counter for counting.

Inhibition rate ( I %)=(Total binding tube cpm−compound cpm)/(Total binding tube cpm−non-specific binding tube cpm)×100%

Each assay for the compounds was conducted in duplicate.

The results are listed in Table 2.

5HT 2A Receptor Assay

Preparation of 5HT 4 Membrane

Rats were sacrificed by cervical dislocation on ice. Cerebral cortex was rapidly taken, and 2 cerebral cortexes were combined into a centrifuge tube, to which 3 ml of buffer (0.05 M Tris-HCl buffer: 6.05 g of Tris was dissolved in 1000 ml of double-distilled water, and concentrated HCl was used to adjust to pH 7.5) was added, homogenization was conducted for 3-4 s at level 4 for four times, and then 5 ml of buffer was added. Incubation at 37° C. was conducted for 10 min, the weight of the tubes were adjusted using a balance after incubation. Centrifugation was conducted at 12000 r, 4° C. for 20 min, the supernatant was discarded, and 3 ml of A solution was added. Vortex mixer was used for blending, and then 5 ml of buffer was added. Centrifugation was conducted (repeated 3 times). After the centrifugations, the supernatant was discarded, and the pellets were stored at −80° C. for future use.

Materials for the Receptor Binding Assay

Isotope ligand [ 3 H]-Ketanserin (67.0 Ci/mmol) was purchased from PerkinElmer Company; Methysergide was purchased from RBI Company; GF/C glass fiber filter paper was purchased from Whatman Company; Tris was imported and divided into aliquots; PPO, POPOP were purchased from Shanghai No. 1 Reagent Factory; lipid-soluble scintillation solution. Beckman LS-6500 Multi-function Liquid Scintillation Counter was used.

Procedures

(1) The prepared membrane was first applied with appropriate amount of buffer, and homogenizer was used for evenly dispersing. 15 tubes were mixed into a 100 ml container, and appropriate amount of buffer was added to give 50 ml of membrane suspension, which was reserved for future use.

(2) 100 μL of membrane preparation and 100 μL of homogenized liquid were added into each reaction tube.

(3) 100 μL of homogenized liquid was added into the total binding tube (TB), 100 μL of Methysergide (final concentration 10 −5 M) was added into the non-specific binding tube (NB), and 100 μL of the test compound (final concentration 10 −5 M) was added into the specific binding tube (SB) for each compound.

(4) 10 μL of radioactive ligand 3 H-Ketanserin was respectively added into each reaction tube (2 parallel tubes were used for each reaction tube, and each of them was placed on ice when adding sample).

(5) Each of the reaction tubes was incubated at 37° C. for 15 min. After the reaction was completed, the bound ligands were rapidly filtered under reduced pressure, and the ice-chilled assay buffer was used for adequate washing. The filter was taken out and put into a 3 ml scintillation vial, and 2 ml of toluene scintillation solution was added and blended.

(6) The scintillation vials were put into Liquid Scintillation Counter for counting.

Inhibition rate ( I %)=(Total binding tube cpm−compound cpm)/(Total binding tube cpm−non-specific binding tube cpm)×100%

Each assay for the compounds was conducted in duplicate.

The results are listed in Table 2.

D 2 Receptor Inhibition

Rats were sacrificed by cervical dislocation on ice. Cerebral cortex was rapidly taken, and 2 cerebral cortexes were combined into a centrifuge tube, to which 3 ml of buffer (0.05 M Tris-HCl buffer, containing NaCl 120 mM, KCl 5 mM, MgCl 2 1 mM, CaCl 2 1 mM) was added, homogenization was conducted for 3-4 s at level 4 for four times, and then 5 ml of buffer was then added. The weight of the homogenized tubes were adjusted using a balance, and centrifugation was conducted at 12000 r, 4° C. for 20 min. The supernatant was discarded, and 3 ml of B solution was added. Vortex mixer was used for blending, and then 5 ml of B solution was added. Centrifugation was conducted and repeated 3 times. After the centrifugations, the supernatant was discarded, and the pellets were stored at −80° C. for future use.

Materials for the Receptor Binding Assay

Isotope ligand 3 H-Spiperone (67.0 Ci/mmol) was purchased from PerkinElmer Company; Butaclamol was purchased from RBI Company; GF/C glass fiber filter paper was purchased from Whatman Company; Tris was imported and divided into aliquots; PPO, POPOP were purchased from Shanghai No. 1 Reagent Factory; lipid-soluble scintillation solution. Beckman LS-6500 Multi-function Liquid Scintillation Counter was used.

Procedures

(1) The prepared membrane was first applied with appropriate amount of homogenized liquid, and homogenizer was used for evenly dispersing. 15 tubes were mixed into a 100 ml container, and appropriate amount of homogenized liquid was added to give 50 ml of membrane suspension, which was reserved for future use.

(2) 100 μL of membrane preparation and 100 μL of buffer were added into each reaction tube.

(3) 100 μL of homogenized liquid was added into the total binding tube (TB), 100 μL of Butaclamol (final concentration 10 −5 M) was added into the non-specific binding tube (NB), and 100 μL of the test compound (final concentration 10 −5 M) was added into the specific binding tube (SB) for each compound.

(4) 10 μL of radioactive ligand 3 H-Spiperone was respectively added into each reaction tube (2 parallel tubes were used for each reaction tube, and each of them was placed on ice when adding sample).

(5) Each of the reaction tubes was incubated at 37° C. for 20 min. After the reaction was completed, the bound ligands were rapidly filtered under reduced pressure, and the ice-chilled assay buffer was used for adequate washing. The filter was taken out and put into a 3 ml scintillation vial, and 2 ml of toluene scintillation solution was added and blended.

(6) The scintillation vials were put into Liquid Scintillation Counter for counting.

Inhibitory rate ( I %)=(Total binding tube cpm−compound cpm)/(Total binding tube cpm−non-specific binding tube cpm)×100%

Each assay for the compounds was conducted in duplicate.

The results are listed in Table 2.

D 3 Receptor Assay

Cell

In HEK-293 cells, after 48-72 hours, receptor proteins were expressed on membrane in large amount. After the cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell pellet was collected and stored in a −20° C. fridge for reservation. It was re-suspended with Tris-Cl (pH 7.4) in the assay.

Materials for the Assay

D 3 receptor isotope ligand [3H]-Spiperone was purchased from Amersham Company; (+) Butaclamol was purchased from RBI Company; GF/C glass fiber filter paper was purchased from Whatman Company; lipid-soluble scintillation solution. Tris was divided into aliquots by Genetimes Technology Inc.

Procedures

Competitive binding test for receptors: 20 μl of each of the test compounds and 20 μl of the radioactive ligand together with 160 μl of the receptor proteins were added into the reaction tubes, and the final concentrations of the test compound and the positive drug were all 10 μmol/L. After 50 min of incubation in 30° C. water bath, the tubes were immediately moved to ice bath to terminate the reactions. GF/C glass fiber filter papers were used for rapid sucking filtration on a Millipore cell sample collector, elution buffer (50 mM Tris-HCl, PH 7.4) was applied for 3 ml×3 times, and microwave was applied for 4-5 min for drying. The filter papers were moved into 0.5 ml centrifuge tubes, and 500 μl of lipid-soluble scintillation solution was added. The tubes were allowed to stand still for over 30 min in dark, and the intensities of radioactivity were measured by a counter. The percentage inhibition rates of each compound against the binding of isotope ligands were calculated according to the following formula:

Inhibition rate ( I %)=(Total binding tube cpm−compound cpm)/(Total binding tube cpm−non-specific binding tube cpm)×100%

The results are listed in Table 2.

5HT 2 Receptor Assay

Preparation of 5HT 2C Membrane

Rats were sacrificed by cervical dislocation on ice. Cerebral cortex was rapidly taken, and 2 cerebral cortexes were combined into a centrifuge tube, to which 3 ml of buffer (0.05 M Tris-HCl buffer, 6.05 g of Tris was dissolved in 1000 ml of double-distilled water, and concentrated HCl was used to adjust to pH 7.5) was added, homogenization was conducted for 3-4 s at level 4 for four times, and then 5 ml of buffer was added. Incubation at 37° C. was conducted for 10 min, the weight of the tubes were adjusted using a balance after the incubation. Centrifugation was conducted at 12000 r, 4° C. for 20 min, the supernatant was discarded, and 3 ml of A solution was added. Vortex mixer was used for blending, and then 5 ml of buffer was added. Centrifugation was conducted (repeated 3 times). After the centrifugations, the supernatant was discarded, and the pellets were stored at −80° C. for future use.

Materials for the Receptor Binding Assay

Isotope ligand [ 3 H]-mesulergine (67.0 Ci/mmol) was purchased from PerkinElmer Company; mianserin was purchased from RBI Company; GF/C glass fiber filter paper was purchased from Whatman Company; Tris was imported and divided into aliquots; PPO, POPOP were purchased from Shanghai No. 1 Reagent Factory; lipid-soluble scintillation solution. Beckman LS-6500 Multi-function Liquid Scintillation Counter was used.

Procedures

(1) The prepared membrane was first applied with appropriate amount of homogenized liquid, and homogenizer was used for evenly dispersing. 15 tubes were mixed into a 100 ml container, and appropriate amount of homogenized liquid was added to give 50 ml of membrane suspension, which was reserved for future use.

(2) 100 μL of membrane preparation and 100 μL of buffer were added into each reaction tube.

(3) 100 μL of homogenized liquid was added into the total binding tube (TB), 100 μL of mianserin (final concentration 10 −5 M) was added into the non-specific binding tube (NB), and 100 μL of the test compound (final concentration 10 −5 M) was added into the specific binding tube (SB) for each compound.

(4) 10 μL of radioactive ligand [ 3 H]-mesulergine was respectively added into each reaction tube (2 parallel tubes were used for each reaction tube, and each of them was placed on ice when adding sample).

(5) Each of the reaction tubes was incubated at 37° C. for 15 min. After the reaction was completed, the bound ligands were rapidly filtered under reduced pressure, and the ice-chilled assay buffer was used for adequate washing. The filter was taken out and put into a 3 ml scintillation vial, and 2 ml of toluene scintillation solution was added and blended.

(6) The scintillation vials were put into Liquid Scintillation Counter for counting.

Inhibition rate ( I %)=(Total binding tube cpm−compound cpm)/(Total binding tube cpm−non-specific binding tube cpm)×100%

Each assay for the compounds was conducted in duplicate.

The results are listed in Table 2.

The results of in vitro assay indicate that compound 7 has relatively stronger affinities for four receptors (D 2 , D 3 , 5-HT 1A and 5-HT 2A ), while lower affinity for 5HT 2C .

MK-801 Induced High Activity—the In Vivo Anti-Schizophrenia Activity of the Compounds

Animals and Reagents

Healthy mice of Kunming breed (with half male and half female, (20±2) g) were provided by Qinglongshan Animal Cultivation Center, Nanjing.

Ascorbic acid was provided by Sinopharm Chemical Reagent Co. Ltd.

MK-801 was produced by Sigma Company, USA; the formulation method: 0.1% vitamin C was used to formulate a 1 mg/ml solution.

Test positive drugs: haloperidol, clozapinc, risperidone, olanzapine, aripiprazole, ziprasidone, quetiapine.

Tween 80, with the concentration of 10%.

Procedures

Mice with qualified body weight were selected, and randomly divided into blank group, model group, positive control group (risperidone group) and drug group. 10% Tween was administered intragastrically to the blank group and the model group at 0.1 ml/10 g; risperidone was administered intragastrically to the positive control group at 0.1 mg/kg; and corresponding amounts of drugs were administered intragastrically to the drug groups, respectively. 1 h after the administration, 0.1% of ascorbic acid was intraperitoneally injected to the blank group at 0.1 ml/10 g; and the model group, the positive control group (30 min) and the drug groups were intraperitoneally injected the MK-801 solution at 0.1 mg/kg. Subsequently, the spontaneous activities of the mice of each group in 90 min were measured.

The results are listed in Table 3.

The results of this assay indicate that, when compared to the model group, risperidone, compound 7 can not only significantly improve the MK-801 induced high activity, but also effectively improve the apomorphine induced clambering symptoms, and they did not cause EPS at effective dosage, indicating that they have notable anti-schizophrenia effects.

Apomorphine Induced Clambering Assay of Mice

Animals

Healthy KM mice (male, with body weight of 18-22 g) were provided by Qinglongshan Animal Cultivation Center, Nanjing.

Main Reagents

Test positive drugs: haloperidol, clozapine, risperidone, olanzapine, aripiprazole, ziprasidone, quetiapine.

Apomorphine provided by Sigma Company was dissolved in 0.9% NaCl (containing 0.1% vitamin C) before use, and was freshly formulated before use.

Vitamin C, F20061113, was provided by Sinopharm Chemical Reagent Co. Ltd.

Sodium chloride injection, H32026305, was provided by Xuzhou No. 5 Pharmaceutical Factory Co. Ltd.

Instruments: self-made clambering cage, chronograph.

Procedures: apomorphine induced clambering assay of mice

KM mice (male, with body weight of 18-22 g) were randomly divided into negative control group, model group, positive drug groups for each dosage (risperidone, aripiprazole, ziprasidone, quetiapine, olanzapine, haloperidol, clozapine), and compound groups for each dosage (the specific dosages are listed in Table 4), with 10 mice in each group. Corresponding solvent double-distilled water was administered intragastrically to the negative control group and the model group, corresponding positive drugs were administered intragastrically to the positive drug groups (a small amount of acetic acid was first added and then double-distilled water was added when dissolving), and corresponding dosages of compounds were administered intragastrically to the compound groups for each dosage, with the volume for intragastric administration as 0.1 ml/10 g. 1 hour after the intragastric administration, apomorphine was subcutaneously injected (1 mg/kg), with the volume as 0.1 ml/10 g. After the injection of apomorphine, the mice were immediately put into the clambering cages. After 5 min of adaptation, the behaviors of the mice at 10-11, 20-21, and 30-31 min after the injection of apomorphine were observed and scored. Scoring criteria: 4 paws on the floor was scored as 0; 2 forepaws on the cage was scored as 1; and 4 paws on the cage was scored as 2.

Catalepsy Assay

Animals

Healthy mice of Kunming breed (with half male and half female, (22±2) g) were provided by Qinglongshan Animal Cultivation Center, Nanjing.

Main reagents: the test drugs, haloperidol, clozapine, risperidone, olanzapine, aripiprazole, ziprasidone.

Instruments: self-made bar-grabbing apparatus: stainless steel bar in mice box, which was 0.3 cm in diameter and 5 cm above the bench.

Procedures

KM mice (half male and half female, with body weight of 20-24 g) were randomly divided into negative control group, model group, positive drug groups for each dosage (risperidone, aripiprazole, ziprasidone, quetiapine, olanzapine, haloperidol, clozapine), and compound groups for each dosage, with 10 mice in each group. Corresponding solvent double-distilled water was administered intragastrically to the negative control group and the model group, corresponding positive drugs were administered intragastrically to the positive drug groups (a small amount of acetic acid was first added and then double-distilled water was added when dissolving), and corresponding dosages of compounds were administered intragastrically to the compound groups for each dosage, with the volume for intragastric administration as 0.1 ml/10 g. At 30 min, 60 min, 90 min after the intragastric administration, the two forepaws of the mice were gently placed on the bars (which were 20 cm in length, 0.3 cm in diameter, and 5.5 cm above the bench), and the hindpaws of the animals were placed on the bottom of the box. The durations for the mice to maintain the posture with the two forepaws on the bars were recorded, and 30 s of spasticity without moving was considered as the positive response. In the case the forepaws of the mice were not put down persistently, the observation was terminated at 60 s. The numbers of animals with positive response in each of the compound dosage groups were counted.

Acute Toxicity Study

Limit Test of Sequential Assay

KM mice (half male and half female) were randomly divided into several groups (with 2-5 mice in each group), which were respectively the 2000 mg/kg groups for each compound, and the solvent group. 0.2 ml/10 g were administered intragastrically. The death of the animals in 3 days were observed. (In the case 3 or more animals survived in 3 days without notable abnormity in their life states, the observation was continued until the assay was completed in 7 days. In the case 3 or more animals died in 3 days, the median lethal dose method was used to determine the LD 50 ).

Pre-Assay for the Median Lethal Dose Method

KM mice (half male and half female) were randomly divided into several groups (with 4 mice in each group), which were respectively the 1500 mg/kg, 1000 mg/kg, 500 mg/kg groups for each compound, and the solvent group. 0.2 ml/10 g were administered intragastrically, and the death of the animals in 1-3 days were observed.

Results

The LD 50 of single intragastric administration of compound 7 in mice was greater than 2000 mg/kg, which was comparable to ziprasidone (>2000 mg/kg), and was far greater than risperidone (82.1 mg/kg) and aripiprazole (93 mg/kg), indicating a relatively low acute toxicity.

Tablet

The raw excipients were sieved with 80 mesh for use. The prescription doses of active ingredient, microcrystalline cellulose, lactose, Povidone K30 were weighed and introduced into a high speed mixing granulator, whereby they were mixed uniformly at low speed. A suitable amount of purified water was added, the stirring was performed at low speed, and high speed shear granulation was carried out. The wet granules were dried at 60° C. for 3 h, and sieved with 24 mesh. The prescription doses of carboxymethyl starch sodium, silica and magnesium stearate were added for mixing totally. The compression was performed in a rotary tablet press.

Capsule

The raw excipients were sieved with 80 mesh for use. The prescription doses of active ingredient, lactose, starch, Povidone K30 were weighed and introduced into a high speed mixing granulator, whereby they were mixed uniformly at low speed. A suitable amount of purified water was added, the stirring was performed at low speed, and high speed shear granulation was carried out. The wet granules were dried at 60° C. for 3 h, and sieved with 24 mesh. The prescription doses of silica and magnesium stearate were added for mixing totally. The capsules were filled in a capsule filling machine.

While this invention has been described in conjunction with the specific embodiments outlined above, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the preferred embodiments of the invention as set forth above are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the inventions as defined in the following claims.