AROMATIC RING COMPOUND

Abstract

A novel aromatic ring compound is represented by general formula (I). This compound, isomer, prodrug, solvate, pharmaceutically acceptable salt and pharmaceutical composition thereof are useful in preparing medicaments for treating depression and related symptoms.

##STR00001##

Claims

1. An aromatic ring compound of formula I, an isomer, a prodrug, a solvate, a pharmaceutically acceptable salt or an isotopically labeled compound thereof, ##STR00016## wherein, R.sub.1 and R.sub.2 each independently represent H or a saccharide unit, and at least one of R.sub.1 and R.sub.2 is a saccharide unit; the saccharide unit may be selected from C.sub.4-6 monosaccharides such as glucose, mannose, allose, galactose, arabinose and xylose, or may be selected from disaccharides and higher-order oligosaccharides such as sucrose, lactose, cellobiose and maltose, wherein carbon and oxygen atoms on the saccharide unit may be optionally substituted with sulfur, nitrogen or carbon; when R.sub.1 and R.sub.2 each independently represent H, the —X.sub.1— and —X.sub.2— to which they are connected respectively represent —O—, —S— or a bond; when R.sub.1 and R.sub.2 each independently represent a saccharide unit, the —X.sub.1— and —X.sub.2— to which they are connected respectively represent glycosidic bond formed by the saccharide unit and a non-saccharide unit (aromatic aglycon) and each independently represent —O—, —O—, —N— or a bond (i.e., O-glycosidic bond, S-glycosidic bond, N-glycosidic bond, or C-glycosidic bond is formed); or —X.sub.1— and —X.sub.2— are —CH.sub.2—; Y and Z each independently represent C, O, N, S, P or Si; R.sub.3 represents hydrogen, hydroxyl, or a substituted or unsubstituted C.sub.1-C.sub.20 aliphatic hydrocarbyl; n is selected from 1, 2, 3, 4 and 5; the aromatic ring may be ##STR00017## (with absence of ring A) or ##STR00018## ring A may be a C.sub.6-10 aryl, a C.sub.3-8 cycloalkyl, a 3-10 membered heterocycloalkyl, or a 5-12 membered heteroaryl.

2. The aromatic ring compound, the isomer, the prodrug, the solvate, the pharmaceutically acceptable salt or the isotopically labeled compound thereof according to claim 1, wherein ring A may be phenyl ring, a 5-6 membered heteroaryl, a C.sub.5-6 cycloalkyl or a 5-6 membered heterocycloalkyl; in ring A, a heteroatom, if present, may be O, S or N; ring A may be, for example, phenyl ring, cyclopentane, cyclohexane, or a nitrogen- or oxygen-containing 5-6-membered heterocyclic ring; and/or, the C.sub.1-C.sub.20 aliphatic hydrocarbyl may be a saturated hydrocarbyl or an unsaturated hydrocarbyl, for example, selected from a C.sub.1-C.sub.20 alkyl, a C.sub.2-C.sub.20 alkenyl and a C.sub.2-C.sub.20 alkynyl, and specifically, selected from a (C.sub.1-C.sub.6) alkyl, a (C.sub.2-C.sub.6) alkenyl and a (C.sub.2-C.sub.6) alkynyl; and/or, the substituted C.sub.1-C.sub.20 aliphatic hydrocarbyl may be a C.sub.1-C.sub.20 aliphatic hydrocarbyl containing one, two or more halogen and/or oxygen, sulfur, nitrogen, phosphorus atoms; for example, a halogenated (C.sub.1-C.sub.6) alkyl, a halogenated (C.sub.1-C.sub.6) alkoxy, or a (C.sub.1-C.sub.6) alkoxy, and specifically, CF.sub.3, CHF.sub.2, and OCH.sub.3; for example, a C.sub.1-C.sub.20 aliphatic hydrocarbyl substituted with hydroxyl, amino, carboxyl, fluorine, trifluoromethyl, difluoromethyl, formyl, or phosphate, sulfate, phosphate or sulfonate group; the halogen is selected from F, Cl, Br and I; and/or, the saccharide unit is preferably glucose, mannose, allose, galactose, arabinose or xylose; and/or, the saccharide unit may be in the D configuration or L configuration; and/or the configurations of the glycosidic bonds formed by the saccharide unit and the aromatic aglycon are independently selected from an a configuration and a β configuration, preferably a β configuration; and/or, the glycosidic bond may be formed by connecting the aglycon to the C1 position of the ring moiety of the saccharide unit; and/or, the aromatic ring may be phenyl ring, ##STR00019## and/or, in the isotopically labeled compound, the isotopically labeled atoms include, but are not limited to, hydrogen, carbon, nitrogen, oxygen and phosphorus, as they can be substituted by isotopically labeled atoms .sup.2H, .sup.3H, .sup.11C, .sup.13C, .sup.14C, .sup.15N, .sup.31P, .sup.32P and .sup.35S.

3. The aromatic ring compound, the isomer, the prodrug, the solvate, the pharmaceutically acceptable salt or the isotopically labeled compound thereof according to claim 1, wherein when R.sub.3 is a C.sub.1-C.sub.20 aliphatic hydrocarbyl containing amino functional group or the aromatic ring is a nitrogen-containing heterocyclic ring, the compound can form the pharmaceutically acceptable salt with an acid; preferably, the acid is selected from sulfuric acid, phosphoric acid, methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, citric acid, oxalic acid, lactic acid, acetic acid, succinic acid, any one of the 20 natural L-amino acids and corresponding D-amino acids thereof, and an oxygen-free acid; the oxygen-free acid may be HCl, HBr, HI or HF.

4. The aromatic ring compound, or the isomer, the prodrug, the solvate, the pharmaceutically acceptable salt or the isotopically labeled compound thereof according to claim 1, wherein the aromatic ring compound is selected from: ##STR00020## ##STR00021## ##STR00022## ##STR00023##

5. A pharmaceutical composition comprising the aromatic ring compound, the isomer, the prodrug, the solvate, the pharmaceutically acceptable salt or the isotopically labeled compound thereof according to claim 1, and a pharmaceutically acceptable carrier.

6. Use of the aromatic ring compound, the isomer, the prodrug, the solvate, the pharmaceutically acceptable salt or the isotopically labeled compound thereof according to claim 1, in preparing a medicament for treating depressive disorders.

7. The use according to claim 6, wherein the aromatic ring compound, the isomer, the prodrug, the solvate, the pharmaceutically acceptable salt or the isotopically labeled compound thereof is used alone or in combination with other therapeutic agents for treating nerve damage and depressive disorders.

8. A pharmaceutical formulation comprising the aromatic ring compound, the isomer, the prodrug, the solvate, the pharmaceutically acceptable salt or the isotopically labeled compound thereof according to claim 1; preferably, the formulation is selected from an injection, an oral capsule and tablet, and other conventional dosage forms.

9. A compound of the following formula: ##STR00024##

10. A method for preparing the aromatic ring compound, or the isomer, the prodrug, the solvate, the pharmaceutically acceptable salt or the isotopically labeled compound thereof according to claim 1, comprising: condensing a hydroxyl-protected saccharide starting material with an aglycon, followed by deprotecting to give a product; wherein, the method further may comprise a post-treatment procedure; preferably, the reaction scheme is as follows: ##STR00025## wherein R.sub.4 is selected from hydrogen and an isotopically-labeled atom thereof; the preparation method comprises the following procedures: 1) subjecting a compound of formula 1-1 and compound a to Mitsunobu reaction to give a compound of formula 1-2; and 2) subjecting the compound of formula 1-2 to catalytic hydrogenolysis reaction to give a compound of formula 1.

11. A method for treatment of depressive disorders, comprising administering a therapeutically effective amount of the aromatic ring compound, the isomer, the prodrug, the solvate, the pharmaceutically acceptable salt or the isotopically labeled compound thereof according to claim 1.

12. The method of claim 11, wherein the aromatic ring compound, the isomer, the prodrug, the solvate, the pharmaceutically acceptable salt or the isotopically labeled compound thereof according claim 1 is administered alone or in combination with other therapeutic agents.

Description

DETAILED DESCRIPTION

[0095] The present invention is further illustrated by the following examples; however, these examples should not be construed as limiting the present invention. Experimental procedures without specified conditions in the following examples can be selected in accordance with conventional procedures and conditions. In the following examples, all the starting/auxiliary materials or reagents are commercially available unless otherwise specified.

[0096] The “nitrogen atmosphere” condition of the invention can be replaced by other inert gas atmosphere, such as “argon atmosphere”.

[0097] The structure of the compounds of the present invention can be identified by nuclear magnetic resonance (.sup.1H NMR) and/or mass spectrometry (MS).

[0098] .sup.1H NMR chemical shifts (6) were recorded in PPM (10.sup.−6). NMR was performed on a Bruker AVANCE-400 spectrometer. Suitable solvents are deuterated chloroform (CDCl.sub.3), deuterated methanol (MeOD-d.sub.4), deuterated dimethyl sulfoxide (DMSO-d.sub.6) and tetramethylsilane (TMS) as internal standard. Liquid chromatography-mass spectrometry (LCMS) was conducted by an Agilent 1200HPLC/6120 mass spectrometer using XBridge C18, 4.6×50 mm, 3.5 μm. Gradient elution conditions one: 80-5% solvent A.sub.1 and 20-95% solvent B.sub.1 (1.8 minutes), followed by 95% solvent B.sub.1 and 5% solvent A.sub.1 (more than 3 minutes), the percentages being the volume percentage of a certain solvent in the total solvent volume. Solvent A.sub.1: 0.01% trifluoroacetic acid (TFA) in water; solvent B.sub.1: 0.01% trifluoroacetic acid in acetonitrile; the percentages being the volume percent of solute in solution. Gradient elution conditions two: 80-5% solvent A.sub.2 and 20-95% solvent B.sub.2 (1.5 minutes), followed by 95% solvent B.sub.2 and 5% solvent A.sub.2 (more than 2 minutes), the percentages being the volume percentage of a certain solvent in the total solvent volume. Solvent A.sub.2: 10 mM aqueous ammonium bicarbonate; solvent B.sub.2: acetonitrile.

[0099] The compounds of the present invention can be separated and purified by using conventional column chromatography, flash separator or high-performance liquid chromatography, and the elution system can be an ethyl acetate/petroleum ether system or a dichloromethane/methanol system.

[0100] The fast separator (flash column chromatography; flash system/Cheetah™) was equipped with Agela Technologies MP200, and the flash chromatographic column was Flash column Silica-CS (80 g; Cat No. CS140080-0).

[0101] The preparative high-performance liquid chromatograph (prep-HPLC) was equipped with Shimadzu LC-20, and the column was Waters Xbridge Pre C18, 10 μm, 19 mm×250 mm. Mobile phase A: 0.05% trifluoroacetic acid in water (percentage being a volume percent), mobile phase B: acetonitrile; detection wavelength: 214 nm & 254 nm; flow rate: 15.0 mL/min.

[0102] The column chromatography generally used 200-mesh and 300-mesh silica gel (Huanghai, Yantai) as the resin. The thin layer chromatograph (TLC) was equipped with HSGF254 (Huanghai, Yantai) or GF254 (Qingdao) silica gel plate.

EXAMPLES 1-3

Preparation of compound of formula 1-2

EXAMPLE 1

[0103] ##STR00014##

[0104] A solution of triphenylphosphine (52.82 g), compound a (10 g, 1 eq.) and a compound of formula 1-1 (91.46 g) in anhydrous tetrahydrofuran (TRF; 1000 mL) was cooled to −15° C. in an ice bath. DIAD (diisopropylazodicarboxylate; 40.72 g, 2.5 eq.) was slowly and dropwise added to the solution, and the solution gradually turned yellow. After 30 minutes of stirring at 0° C., the ice bath was removed. The reaction system was warmed to room temperature (25° C.) and continuously stirred until the starting material disappeared (TLC monitoring, about 6 hours).

[0105] After the reaction was completed, the solution was concentrated in vacuum. The product was separated and purified by silica gel column chromatography to give a white foamy solid (53% yield). Rf 0.37 (EtOAc/hexane =1/5); 1H NMR (600 MHz, CDCl.sub.3,) δ 7.25-7.05 (m, 40H), 6.98-6.95 (m, 1H) 6.78-6.74 (m, 1H) 6.65-6.55 (m, 1H), 5.05-4.89 (m, 4H), 4.85-4.62 (m, 8H), 4.55-4.40 (m, 6H), 4.71-4.45 (m, 12H) 2.16 (s, 3H).

EXAMPLE 2

[0106] A solution of tributylphosphine (40.75 g), compound a (10 g, 1 eq.) and a compound of formula 1-1 (91.46 g) in dichloromethane (800 mL) was cooled to −10° C. in an ice bath. Dimethyl azodicarboxylate (DMAD, 61.79 g, 2.5 eq.) was slowly and dropwise added to the solution, and the solution gradually turned yellow. After 40 minutes of stirring at 0° C., the ice bath was removed. The reaction system was warmed to 50° C. and continuously stirred until the starting material disappeared (TLC monitoring, about 5 hours).

[0107] After the reaction was completed, the solution was concentrated in vacuum. The product was separated and purified by silica gel column chromatography to give a white foamy solid (56% yield). Rf 0.37 (EtOAc/hexane=1/5); 1H NMR (600 MHz, CDCl.sub.3,) δ7.25-7.05 (m, 40H), 6.98-6.95 (m, 1H) 6.78-6.74 (m, 1H) 6.65-6.55 (m, 1H), 5.05-4.89 (m, 4H), 4.85-4.62(m, 8H), 4.55-4.40 (m, 6H), 4.71-4.45 (m, 12H) 2.16 (s, 3H).

EXAMPLE 3

[0108] A solution of trimethylphosphine (15.32 g), compound a (10 g, 1 eq.) and a compound of formula 1-1 (91.46 g) in toluene (800 mL) was cooled to 0° C. in an ice bath. Diethyl azodicarboxylate (DEAD, 73.65g, 2.5 eq.) was slowly & dropwise added to the solution, and the solution gradually turned yellow. After 20 minutes of stirring at 0° C., the ice bath was removed. The reaction system was warmed to 20° C. and continuously stirred until the starting material disappeared (TLC monitoring, about 6 hours). After the reaction was completed, the solution was concentrated in vacuum. The product was separated and purified by silica gel column chromatography to give a white foamy solid (50% yield).

[0109] Rf 0.37 (EtOAc/hexane=1/5); 1H NMR (600 MHz, CDCl.sub.3,) δ7.25-7.05 (m, 40H), 6.98-6.95 (m, 1H) 6.78-6.74 (m, 1H) 6.65-6.55 (m, 1H), 5.05-4.89 (m, 4H), 4.85-4.62 (m, 8H), 4.55-4.40 (m, 6H), 4.71-4.45 (m, 12H) 2.16 (s, 3H).

EXAMPLES 4-6

Preparation of compound of formula 1

EXAMPLE 4

[0110] ##STR00015##

[0111] The compound of formula 1-2 (47 g) was dissolved in methanol (1000 mL). A palladium hydroxide on activated carbon catalyst (22.58 g, containing 20 wt % of palladium hydroxide) was added in nitrogen atmosphere and the reaction system was stirred at 45° C. in hydrogen atmosphere at one standard atmosphere pressure until complete conversion of the starting materials (TLC monitoring, about 12 hours). The reaction solution was filtered under reduced pressure. The filter cake was washed with methanol (3×200 mL). The filtrates were combined and the solvent was removed under reduced pressure to give a product in the form of a white powder (16 g, 88% yield).

[0112] Rf 0.37 (MeOH/CH.sub.2Cl.sub.2=1/5);.sup.1H NMR (600 MHz, CDCl.sub.3,) δ7.14-7.13 (d, 1H), 6.80 (s, 1H) 6.73-6.71 (d, 1H) 5.00-4.98 (m, 2H),3.88-3.85 (m, 2H), 3.68-3.60 (m, 2H), 3.60-3.40 (m, 8H), 2.13 (s, 3H); .sup.13C NMR (600 MHz, CDCl.sub.3,) δ155.5, 155.1, 131.4, 122.3, 110.6, 103.8, 100.2, 76.2, 75.5, 72.8, 69.5, 60.7, 48.8, 14.6;IR (thin film,cm-1): 3257, 2928, 1612, 1592, 1503, 1395, 1264, 1169, 1058, 922, 896;HRMS (ESI-TOF) m/z Calcd.for C.sub.19H.sub.28O.sub.12: [M+Na].sup.+471.1478, found 471.1484;[α].sup.23.sub.D=−74.0° (c 1, H.sub.2O).

EXAMPLE 5

[0113] The compound of formula 1-2 (47 g) was dissolved in ethanol (1000 mL). A palladium on carbon catalyst (5.0 g, containing 10 wt % of Pd) was added in nitrogen atmosphere and the reaction system was stirred at 20° C. in hydrogen atmosphere at one standard atmosphere pressure until complete conversion of the starting materials (TLC monitoring, about 13 hours). The reaction solution was filtered under reduced pressure. The filter cake was washed with methanol (3×200 mL). The filtrates were combined and the solvent was removed under reduced pressure to give a product in the form of a white powder (15.5 g, 85.2% yield).

[0114] Rf 0.37 (MeOH/CH.sub.2Cl.sub.2=1/5);.sup.1H NMR (600 MHz, CDCl.sub.3,) δ7.14-7.13 (d, 1H), 6.80 (s, 1H) 6.73-6.71 (d, 1H) 5.00-4.98 (m, 2H),3.88-3.85 (m, 2H), 3.68-3.60 (m, 2H), 3.60-3.40 (m, 8H), 2.13 (s, 3H);.sup.13C NMR (600 MHz, CDCl.sub.3,) δ155.5, 155.1, 131.4, 122.3, 110.6, 103.8, 100.2, 76.2, 75.5, 72.8, 69.5, 60.7, 48.8, 14.6;IR (thin film,cm-1): 3257, 2928, 1612, 1592, 1503, 1395, 1264, 1169, 1058, 922, 896;HRMS (ESI-TOF) m/z Calcd. for C.sub.19H.sub.28O.sub.12: [M+Na].sup.+471.1478, found 471.1484;[α].sup.23.sub.D=−74.0° (c 1, H.sub.2O).

EXAMPLE 6

[0115] The compound of formula 1-2 (47g) was dissolved in isobutanol (1000 mL). A palladium hydroxide on activated carbon catalyst (22.58 g, containing 20 wt % of palladium hydroxide) was added in nitrogen atmosphere and the reaction system was stirred at 50° C. in hydrogen atmosphere at one standard atmosphere pressure until complete conversion of the starting materials (TLC monitoring, about 12 hours). The reaction solution was filtered under reduced pressure. The filter cake was washed with methanol (3×200 mL). The filtrates were combined and the solvent was removed under reduced pressure to give a product in the form of a white powder (90% yield).

[0116] Rf 0.37 (MeOH/CH.sub.2Cl.sub.2=1/5);.sup.1H NMR (600 MHz, CDCl.sub.3,) δ7.14-7.13 (d, 1H), 6.80 (s, 1H) 6.73-6.71 (d, 1H) 5.00-4.98 (m, 2H),3.88-3.85 (m, 2H), 3.68-3.60 (m, 2H), 3.60-3.40 (m, 8H), 2.13 (s, 3H);.sup.13C NMR (600 MHz, CDCl.sub.3,) δ155.5, 155.1, 131.4, 122.3, 110.6, 103.8, 100.2, 76.2, 75.5, 72.8, 69.5, 60.7, 48.8, 14.6;IR (thin film,cm-1): 3257, 2928, 1612, 1592, 1503, 1395, 1264, 1169, 1058, 922, 896;HRMS (ESI-TOF) m/z Calcd. for C.sub.19H.sub.28O.sub.12: [M+Na].sup.+471.1478, found 471.1484;[α].sup.23.sub.D=−74.0° (c 1, H.sub.2O).

EXAMPLE 7

Efficacy of Oral Administration for Compound of Formula 1 in Treating Depression

[0117] (1) Experimental Animals

[0118] The experimental animals were male SPF grade KM mice purchased from Kunming Medical University, weighed 21-24 g, certificate no. SCXK(Dian)K2015-0002. The experimental animals were bred in individually ventilated cages (IVCs) in animal room in Dianqing Biotechnology, Ltd., Yunnan (facility no. 13-11-078 and 13-11-079; manufacture date: Nov. 24, 2013). The room temperature was controlled at 22-24° C. with humidity at 40-70% and 12-hour light/dark cycle (7:00 am/19:00 pm). The cages and padding were changed twice a week. The raising method was group raising and there were 10 mice accommodated in each cage. The feed was sterilized feed from Jiangsu Xietong Medical Bioengineering, Ltd., certificate no.: (2014)01008. Feed was supplied once daily with free access . Tap water was supplied in boxes with free access.

[0119] (2) Test Compound

[0120] The test compound was the compound of formula 1 (product in Example 4), with a molecular weight of 448.16, which is easily soluble in water and was sealed at 4° C.

[0121] (3) Experimental Procedures

[0122] Grouping:

[0123] A. Behavioral testing was performed in different treatment groups (1.0 mg/kg, 5.0 mg/kg, 10.0 mg/kg, 15.0 mg/kg, 30.0 mg/kg) 1 h after intragastric (i.g.) administration of the compound of formula 1;

[0124] B. In the negative control group receiving normal saline (NS), the behavioral testing was conducted at the same time;

[0125] C. In the positive control group receiving imipramine (IMI; 15 mg/kg), the behavioral testing was conducted at the same time;

[0126] Route of administration: The test compound of formula 1, normal saline or imipramine was administered by intragatric administration according to the experimental design.

[0127] Time of administration: The animals were accommodated to the experimental environment for 1 hour, administrated by intragatric administration, and subjected to tail suspension test 1 hour after the administration.

[0128] Test criterion: The mice were subjected to a 6-minute tail suspension test, and the cumulative time of immobility in the first 2 minutes and the last 4 minutes was recorded. The criterion for immobility is that the mouse stops struggling and remains steady.

[0129] (4) Procedures

[0130] A depression model of behavioral despair, ie. tail suspension model, was used. Animals were accommodated to the company's breeding environment for 1 day. During the adaptation, animals with non-smooth and unclean hair, high alertness or aggressivity were excluded.

[0131] Animals were accommodated to the experimental environment for 1 hour, weighed, and randomized according to the body weight into a normal saline control group, an imipramine control group and treatment groups with different doses.

[0132] In the tail suspension test, animals in all groups were administered with corresponding drugs by a single intragastric administration. 1 h after the administration, tails of mice were fixed with medical tape at about 1-2 cm from the end, such that the mice were hanged in the tail suspension box with the heads about 10 cm above the bottom of the box. The observation started immediately after hanging. In the 6-minute observation, the cumulative time of immobility in the first 2 minutes and the last 4 minutes was recorded. A video was recorded with obviously contrasted background with the hair color of the mice, for example, black background was used for white mice.

[0133] (5) Statistics

[0134] The cumulative time of immobility within the last 4 minutes was compared between the test compound groups and the normal saline group using SPSS 11.0 software. One-way analysis of variance (ANOVA) was used for comparison among multiple groups, and independent sample T test was performed for comparisons in pairs. P <0.05 indicates a statistically significant difference. All statistical diagrams were plotted in mean ±SEM using Origin 8.0 software.

[0135] (6) Results

[0136] Mice were administered with different doses of the compound of formula 1 by intragastric administration, and subjected to a 6-minute tail suspension test 1 hours after the administration. The results show that: the time of immobility in each treatment group (1.0 mg/kg, 5.0 mg/kg, 10.0 mg/kg, 15.0 mg/kg, 30.0 mg/kg) of the compound of formula 1 was lower than that of the control group (100%). Among these, the time of immobility in 5.0 mg/kg, 10.0 mg/kg, 15.0 mg/kg treatment groups was very significantly different as compared to the normal saline group (**P <0.01); the time of immobility in the 30.0 mg/kg group was significantly different as compared to the normal saline group (*P <0.05). The compound of formula 1 is capable of significantly reducing the time of immobility in mice and has significant dose-response relationship. The detailed results are shown in Table 1.

TABLE-US-00001 TABLE 1 Efficacy of oral administration for compound of formula 1 in treating depression Grouping Dose (mg/kg, i.g.) N Time of immobility (% of NS) NS — 69 100.00 ± 5.08     IMI 15 71 58.64 ± 5.42*** Compounds  1 30 87.14 ± 11.27   of formula 1  5 29 72.67 ± 7.89**  10 25 66.86 ± 8.80**  15 31 68.33 ± 7.17**  30 29 77.38 ± 6.00*   *P < 0.05; **P < 0.01; ***P < 0.001 (compared to NS), least significant difference test after one-way ANOVA.

[0137] Conclusion: the compound of formula 1 can significantly reduce the time of immobility in mice through oral administration, suggesting that the compound has anti-depression efficacy and significant dose-response relationship.

EXAMPLE 8

Efficacy of Intraperitoneal Injection for Compound of Formula 1 in Treating Depression

[0138] (1) Experimental Animals

[0139] The experimental animals were male SPF grade KM mice purchased from Kunming Medical University, weighed 21-24 g, certificate no. SCXK(Dian)K2015-0002. The experimental animals were bred in individually ventilated cages (IVCs) in animal room in Dianqing Biotechnology, Ltd., Yunnan (facility no. 13-11-078 and 13-11-079; manufacture date: Nov. 24, 2013). The room temperature was controlled at 22-24° C. with humidity at 40-70% and 12-hour light/dark cycle (7:00 am/19:00 pm). The cages and padding were changed twice a week. The raising method was group raising and there were 10 mice accommodated in each cage. The feed was sterilized feed from Jiangsu Xietong Medical Bioengineering, Ltd., certificate no.: (2014)01008. Feed was supplied once daily with free access. Tap water was supplied in boxes with free access.

[0140] (2) Test Compound

[0141] The test compound was the compound of formula 1 (product in Example 4), with a molecular weight of 448.16, which is easily soluble in water and was sealed at 4° C.

[0142] (3) Experimental Procedures

[0143] Grouping:

[0144] A. Behavioral testing was performed in different treatment groups (1.0 mg/kg, 5.0 mg/kg, 10.0 mg/kg, 20.0 mg/kg) 0.5 h after intraperitoneal (i.p.) injection of the compound of formula 1;

[0145] B. In the control group receiving normal saline (NS), the behavioral testing was conducted at the same time;

[0146] C. In the control group receiving imipramine (IMI; 15 mg/kg), the behavioral testing was conducted at the same time;

[0147] Route of administration: The test compound of formula 1, normal saline or imipramine was administered by intraperitoneal injection according to the experimental design.

[0148] Time of administration: The animals were accommodated to the experimental environment for 1 hour, administrated by intraperitoneal injection and subjected to tail suspension test 0.5 hour after the administration.

[0149] Test criterion: The mice were subjected to a 6-minute tail suspension test, and the cumulative time of immobility in the first 2 minutes and the last 4 minutes was recorded. The criterion for immobility was that the mouse stops struggling and remains steady.

[0150] (4) Procedures

[0151] A depression model of behavioral despair, ie. tail suspension model, was used. Animals were accommodated to the company's breeding environment for 1 day. During the adaptation, animals with non-smooth and unclean hair, high alertness or aggressivity were excluded.

[0152] Animals were accommodated to the experimental environment for 1 hour, weighed, and randomized according to the body weight into a normal saline control group, an imipramine control group and treatment groups with different doses.

[0153] In the tail suspension test, animals in all groups for tail suspension test were administered with corresponding drugs by a single intraperitoneal injection. 0.5 h after the administration, tails of mice were fixed with medical tape at about 1-2 cm from the end, such that the mice were hanged in the tail suspension box with the heads about 10 cm above the bottom of the box. The observation started immediately after hanging. In the 6-minute observation, the cumulative time of immobility in the first 2 minutes and the last 4 minutes was recorded. A video was recorded with obviously contrasted background with the hair color of the mice, for example, black background was used for white mice.

[0154] (5) Statistics

[0155] The cumulative time of immobility within the last 4 minutes was compared between the test compound groups and the normal saline group using SPSS 11.0 software. One-way analysis of variance (ANOVA) was used for comparison among multiple groups, and independent sample T test was performed for comparisons in pairs. P <0.05 indicates a statistically significant difference. All statistical diagrams were plotted in mean ±SEM using Origin 8.0 software.

[0156] (6) Results

[0157] Mice were administered with different doses of the compound of formula 1 by intraperitoneal injection, and subjected to a 6-minute tail suspension test 30 minutes after the administration. The results show that: the time of immobility in 5.0 mg/kg and 10.0 mg/kg treatment groups (1.0 mg/kg, 5.0 mg/kg, 10.0 mg/kg, 20.0 mg/kg) of the compound of formula 1 was significantly lower than that of the control group. Among these, the time of immobility in 5.0 mg/kg treatment group was significantly different as compared to the normal saline group (**P <0.05); the time of immobility in the 10.0 mg/kg group was very significantly different as compared to the normal saline group (*P <0.01). The intraperitoneal injection of the compound of formula 1 is capable of significantly reducing the time of immobility in mice and has significant dose-response relationship. The detailed results are shown in Table 2.

TABLE-US-00002 TABLE 2 Efficacy of intraperitoneal injection for compound of formula 1 in treating depression Grouping Dose (mg/kg, i.p.) N Time of immobility (% of NS) NS — 28 100.00 ± 5.95     IMI 15 31 58.12 ± 5.66*** Compounds  1 14 104.02 ± 16.80    of formula 1  5 24 72.12 ± 8.57*   10 24 60.44 ± 7.53**  20 14 104.66 ± 14.75    *P < 0.05; **P < 0.01; ***P < 0.001 (compared to NS), least significant difference test after one-way ANOVA.

[0158] Conclusion: the compound of formula 1 can significantly reduce the time of immobility in mice through intraperitoneal injection, suggesting that the compound has significant dose-response relationship.

EXAMPLE 9

Positive Regulation of NMDA Receptor Current by Compound of Formula 1

[0159] (1) Experimental Animals

[0160] Male C57BL/6 mice, aged 3-9 weeks, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The animals were bred at 23 ±1° C. The room temperature was controlled at 22-24° C. with humidity at 40-70% and 12-hour light/dark cycle (7:00 am/19:00 pm).

[0161] The cages and padding were changed twice a week. The raising method was group raising and there were 3-5 mice accommodated in each cage. Feed was supplied once daily with free access. Tap water was supplied in boxes with free access.

[0162] (2) Test Compound

[0163] The test compound was the compound of formula 1 (product in Example 4), with a molecular weight of 448.16, which is easily soluble in water and was sealed at 4° C.

[0164] (3) Experimental Procedures

[0165] Grouping:

[0166] A. The baseline whole-cell NMDAR current was recorded in 10 minutes before the compound of formula 1 (0.76, 3.8 mM/L) was dissolved in artificial cerebrospinal fluid (vehicle) and added with the brain slice circulation fluid.

[0167] B. An identical volume of artificial cerebrospinal fluid (vehicle) was added with the brain slice circulation fluid.

[0168] Treatment: The brain slice circulation fluid was directly added.

[0169] Time of treatment: 10 minutes after baseline recording.

[0170] Test item: depolarization voltage-induced whole-cell NMDAR receptor currents.

[0171] Hippocampal brain slices: The animals were anesthetized with isoflurane and the brains were collected quickly. Brain slices with a thickness of 350 micron were prepared with a vibrating blade microtome (Leica VT1000S, Leica Microsystems, Germany) and incubated in artificial cerebrospinal fluid with saturated oxygen (95% 02/5% CO.sub.2) on ice: 206 mM sucrose, 2.5 mM KCl, 1.25 mM NaH2PO.sub.4, 26 mM NaHCO.sub.3, 10 mM D-glucose, 2 mM MgSO.sub.4.7H.sub.2O and 2 mM CaCl.sub.2.H.sub.2O (pH 7.2-7.4, 290-300 mOsm). Subsequently, the brain slices were incubated at 32° C. for another 45 minutes in artificial cerebrospinal fluid with saturated oxygen (95% 02/5% CO.sub.2). Finally, the brain slices were transferred to a recording tank containing continuously circulating artificial cerebrospinal fluid with saturated oxygen.

[0172] Whole-cell NMDAR current recording: NMDAR whole cell EPSC recording was performed in hippocampal CA1 pyramidal cells. Recording electrodes were made with a micropipette puller (P-1000, Sutter, USA) with an input resistance of about 5-7 MQ. The filling electrode solution: 130 mM Cs-methanesulfonate, 0.15 mM CaCl.sub.2.2H.sub.2O, 2.0 mM MgC12, 2.0 mM EGTA, 10 mM HEPES, 2 mM Mg-ATP, 0.3 mM Na-GTP, and 10 mM QX-314 with osmolarity adjusted to 285-290 mOsm/kg and pH adjusted to 7.2 with CsOH. Hippocampal pyramidal cells were clearly visible under a 40-fold water microscope and near infrared visual system (Olympus, BX51WI, Japan). The electrical signal in whole cells was recorded using a Clampfit 10.3 software (Axon Instruments) equipped with an Axopatch-700B amplifier (Axon Instruments, Foster City, Calif.) and a Digiclata 1440A digital-to-analog converter, with filtering set at 2.8 kHz and sampling at 10 kHz. Hippocampal CA1 pyramidal cells were clamped at a membrane potential of +40 mV. Schaffer collaterals were stimulated using a white iraurita electrode, triggering glutamate release resulting in whole cell NMDAR-mediated EPSC. The NIVIDAR-EPSC were validated with antagonist AP-5 of NMDARs. After 10 minutes of NMDAR-EPSC recording (once every 20 seconds), vehicle or the compound of formula 1 was added to the circulating artificial cerebrospinal fluid for another 20 minutes, and the NMDAR-EPSC current intensity (pA) was measured for the amplitude of the last 10 minutes of recorded EPSC and the amplitude of baseline.

[0173] (4) Statistics

[0174] The data were compared between the test compound groups and the vehicle group using SPSS 11.0 software. One-way analysis of variance (ANOVA) was used for comparison among multiple groups, and independent sample T test was performed for comparisons in pairs. P <0.05 indicates a statistically significant difference. All statistical diagrams were plotted in mean ±SEM using Origin 8.0 software.

[0175] (5) Results

[0176] Addition of vehicle to oxygenated circulating cerebrospinal fluid had no effect on NMDAR-EPSC, giving a result of 98.31 ±3.28% compared to the baseline. However, the addition of 0.76 mM/L of the compound of formula 1 slightly increased NMDAR-EPSC to 108.80 ±5.86% compared to baseline, which, however, is statistically insignificant. However, the addition of 3.8 mM/L of the compound of formula 1 significantly increased NMDAR-EPSC to 155.53 ±20.85% compared to baseline, which is statistically significant (**P <0.01). The detailed results are shown in Table 3.

TABLE-US-00003 TABLE 3 Positive regulation of NMDA receptor current by compound of formula 1 NMDAR current Grouping Dose (mM/L) N (% of Baseline) Vehicle — 5 98.31 ± 3.28   Compounds of 0.76 6 108.80 ± 5.86    formula 1 3.8  4 155.53 ± 20.85** **P < 0.01 (compared to Vehicle), least significant difference test after one-way ANOVA.

[0177] Conclusion: The compound of formula 1 can directly up-regulate NMDA receptor function and has a dose-response relationship.