USE OF TRIAZOLE COMPOUND AS GHRELIN RECEPTOR AGONIST

Abstract

The present invention relates to a use of a triazole compound as a ghrelin receptor agonist and, more specifically, to a composition for preventing or treating diseases mediated by the ghrelin receptor, the composition being of a triazole compound which strongly binds to the ghrelin receptor with very high specificity. The compound provided by the present invention exhibits a strong binding force to the ghrelin receptor with very high specificity, and thus may be very usefully employed for preventing or developing a therapeutic agent for diseases mediated by the ghrelin receptor.

Claims

1. A pharmaceutical composition for preventing or treating diseases mediated by a ghrelin receptor, comprising a compound of the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: ##STR00015## wherein R.sub.1 is hydrogen; straight or branched chain alkyl having 1 to 15 carbon atoms; or substituted or unsubstituted alkylcarbonyl having 1 to 5 carbon atoms, and R.sub.2 is hydrogen; straight or branched chain alkyl having 1 to 15 carbon atoms; alkenyl having 2 to 10 carbon atoms; or alkynyl having 2 to 10 carbon atoms.

2. The pharmaceutical composition of claim 1, wherein R.sub.1 is hydrogen.

3. The pharmaceutical composition of claim 1, wherein R.sub.2 is straight-chain or branched-chain alkyl having 1 to 15 carbon atoms.

4. The pharmaceutical composition of claim 1, wherein the compound of Formula 1 is 2-amino-2-(1-hexyl-1H-1,2,3-triazol-4-yl)propane-1,3-diol, 2-amino-2-(1-heptyl)-1H-1,2,3-triazol-4-yl)propane-1,3-diol, 2-amino-2-(1-octyl-1H-1,2,3-triazol-4-yl)propane-1,3-diol, 2-amino-2-(1-nonyl-1H-1,2,3-triazol-4-yl)propane-1,3-diol, 2-amino-2-(1-decyl-1H-1,2,3-triazol-4-yl)propane-1,3-diol, 2-amino-2-(1-undecyl-1H-1,2,3-triazole-4-yl)propane-1,3-diol or 2-amino-2-(1-dodecyl-1H-1,2,3-triazol-4-yl)propane-1,3-diol.

5. The pharmaceutical composition of claim 1, wherein the compound of Formula 1 is a ghrelin receptor agonist.

6. The pharmaceutical composition of claim 1, wherein the diseases mediated by the ghrelin receptor are selected from the group consisting of eating disorders; cancer anorexia or cachexia; cachexia or anorexia due to anticancer drugs; hyperalgesia due to anticancer drugs; chronic obstructive pulmonary disease (COPD) or COPD cachexia; sarcopenia; eating disorders; weight loss; generalized weakness after surgery in cancer patients; chronic airway infection; inflammation; inflammatory bowel disease (IBD); functional dyspepsia (FD); constipation; diabetic gastroparesis; heart failure; myocardial infarction; diabetic neuropathy; growth hormone deficiency; defecation disorders in patients with spinal injuries; postoperative ileus; anoxia; and morphine-induced intestinal obstruction.

7. A food composition for preventing or improving diseases mediated by a ghrelin receptor, comprising a compound of the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: ##STR00016## wherein R.sub.1 is hydrogen; straight or branched chain alkyl having 1 to 15 carbon atoms; or substituted or unsubstituted alkylcarbonyl having 1 to 5 carbon atoms, and R.sub.2 is hydrogen; straight or branched chain alkyl having 1 to 15 carbon atoms; alkenyl having 2 to 10 carbon atoms; or alkynyl having 2 to 10 carbon atoms.

8. A compound of the following Formula 2 or a salt thereof: ##STR00017## wherein R.sub.1 is hydrogen; straight or branched chain alkyl having 1 to 15 carbon atoms; or a substituted or unsubstituted alkylcarbonyl having 1 to 5 carbon atoms.

9. The compound or salt thereof according to claim 8, wherein the compound of Formula 2 is 2-amino-2-(1-decyl-1H-1,2,3-triazol-4-yl)propane-1,3-diol.

10. Use of a compound of the following Formula 1 or a pharmaceutically acceptable salt thereof in preparing an agent for the treatment of ghrelin receptor mediated diseases: ##STR00018## wherein R.sub.1 is hydrogen; straight or branched chain alkyl having 1 to 15 carbon atoms; or substituted or unsubstituted alkylcarbonyl having 1 to 5 carbon atoms, and R.sub.2 is hydrogen; straight or branched chain alkyl having 1 to 15 carbon atoms; alkenyl having 2 to 10 carbon atoms; or alkynyl having 2 to 10 carbon atoms.

11. The use of claim 10, wherein R.sub.1 is hydrogen.

12. The use of claim 10, wherein R.sub.2 is straight-chain or branched-chain alkyl having 1 to 15 carbon atoms.

13. A method for treating diseases mediated by a ghrelin receptor, comprising administering an effective amount of a composition comprising a compound of the following Formula 1 or a pharmaceutically acceptable salt thereof to a subject in need thereof: ##STR00019## wherein R.sub.1 is hydrogen; straight or branched chain alkyl having 1 to 15 carbon atoms; or substituted or unsubstituted alkylcarbonyl having 1 to 5 carbon atoms, and R.sub.2 is hydrogen; straight or branched chain alkyl having 1 to 15 carbon atoms; alkenyl having 2 to 10 carbon atoms; or alkynyl having 2 to 10 carbon atoms.

14. The method of claim 13, wherein R.sub.1 is hydrogen.

15. The method of claim 13, wherein R.sub.2 is straight-chain or branched-chain alkyl having 1 to 15 carbon atoms.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0097] FIG. 1 is a diagram showing EC50 that may represent 50% intracellular calcium influx according to concentrations of ghrelin and triazole compounds (KARI001, KARI101 and KARI201) in human embryonic kidney 293 (HEK293) cells overexpressing a ghrelin receptor.

[0098] FIG. 2 is a diagram showing changes in ghrelin receptor subsignal factors by treatment with 10 mM of ghrelin and triazole compounds (KARI001, KARI101 and KARI201) in HEK293 cells overexpressing a ghrelin receptor (n=3, **p<0.01, ***p<0.001).

[0099] FIG. 3 is a diagram showing the intracellular influx efficacy of a ghrelin receptor by treatment with 10 mM of ghrelin and triazole compounds (KARI001, KARI101 and KARI201) in HEK293 cells overexpressing a ghrelin receptor (n=6, **p<0.01).

[0100] FIGS. 4A and 4B are diagrams showing the efficacy of a triazole compound (KARI201) as an agonist or antagonist for 170 G-protein coupled receptors comprising a ghrelin receptor.

[0101] FIGS. 5A and 5B are diagrams showing the effect of improving delayed gastric emptying, which may evaluate the motor function of the stomach of a postoperative ileus (POI) mouse model after oral administration of a triazole compound (KARI 201) at different concentrations (n=5-10, *p<0.05, **p<0.01).

[0102] FIGS. 6A and 6B are diagrams showing the effect of improving delayed gastric emptying, which may evaluate the motor function of the stomach of a postoperative ileus (POI) mouse model after oral administration of a triazole compound (KARI 101) at different concentrations (n=5, **p<0.01, ***p<0.001).

[0103] FIGS. 7A to 7D are diagrams showing the colon transit time of feces (FIG. 7A), number and weight of feces (FIG. 7B), feed intake (FIG. 7C) and body weight (FIG. 7D), which may evaluate the motor function of the colon of a postoperative ileus (POI) mouse model after oral administration of 30 mg/kg of a triazole compound (KARI 201) (n=4, ***p<0.001, ****p<0.0001).

[0104] FIGS. 8A to 8D are diagrams showing the colon transit time of feces (FIG. 8A), number and weight of feces (FIG. 8B), feed intake (FIG. 8C) and body weight (FIG. 8D), which may evaluate the motor function of the colon of a postoperative ileus (POI) mouse model after oral administration of 30 mg/kg of a triazole compound (KARI 101) (n=4, *p<0.05, **p<0.01, ***p<0.001).

[0105] FIGS. 9A to 9C are diagrams confirming the outline of experiments performed to investigate the efficacy of triazole compounds (KARI 101 and KARI 201) in a cancer cachexia mouse model (FIG. 9A) and the effect of improving muscle strength and function after oral administration of 10 mg/kg of such compounds (FIG. 9B) (n=7-8, *p<0.05, **p<0.01, ***p<0.001).

[0106] FIGS. 10A to 10D are diagrams showing the cancer size (FIG. 10A), body weight (FIG. 10B), feed intake (FIG. 100) and average daily feed intake (FIG. 10D) of a cancer cachexia mouse model for the duration of oral administration of 10 mg/kg of triazole compounds (KARI 101 and KARI 201) (n=9, **p<0.01, ****p<0.0001).

[0107] FIGS. 11A to 11D are diagrams showing the total fat weight (FIG. 11A), visceral fat weight (FIG. 11B), subcutaneous fat weight (FIG. 11C) and muscle weight (FIG. 11D) of a cancer cachexia mouse model after oral administration of 10 mg/kg of triazole compounds (KARI 101, KARI 201) (n=6-9, *p<0.05, **p<0.01, ****p<0.0001).

MODE FOR INVENTION

[0108] Hereinafter, the present invention will be described in detail by the following embodiments. However, the following embodiments are only for illustrating the present invention, and the present invention is not limited thereto.

[0109] <Test Materials and Test Methods>

[0110] 0. Synthesis of Compounds

[0111] Substances KARI001 and KARI201 were produced and synthesized in view of an existing reference (Korean Patent No. 2017324). A ghrelin protein used as a positive control was purchased from TORIS.

[0112] KARI001: [0113] 2-amino-2-(1-dodecyl-1H-1,2,3-triazol-4-yl)propane-1,3-diol

[0114] KARI201: [0115] 2-amino-2-(1-nonyl-1H-1,2,3-triazol-4-yl)propane-1,3-diol

[0116] Substance KARI101, compound 2-amino-2-(1-decyl-1H-1,2,3-triazol-4-yl) propane-1,3-diol, was produced through the following series of processes.

##STR00010##

[0117] To synthesize 1-azidodecane of Scheme 1, sodium azide (4.9 g, 75 mmol, 2 eq) was added to a solution of 1-bromodecane (9.9 g, 37 mmole) of Formula 1 in DMF (50 ml). The mixture was stirred at room temperature for 2 days and ice water (200 ml) was added thereto to extract with ether. The organic layer was dried over H.sub.2O, brine and MgSO.sub.4 and concentrated to obtain 1-azidodecane of Formula 2 (6.2 g, 91%).

[0118] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.28 (t, 2H), 1.62 (m, 2H,), 1.40-1.29 (m, 14H), 0.91 (t, 3H)

##STR00011##

[0119] To synthesize 2-amino-2-(hydroxymethyl)propane-1, 3-diol of Scheme 2, Boc.sub.2O (49.5 g, 1.1 eq) was added to the suspension of tris (hydroxymethyl) amino-methane of Formula 3 (25.0 g, 0.206 mol) in DMF (500 ml). After the mixture was stirred at room temperature for 2 hours, 2,2-dimethoxypropane (30.4 ml, 1.2 eq) and p-TsOH.Math.H.sub.2O (2.0 g, 0.05 eq) were added thereto. The mixture was stirred at room temperature for 18 hours and diluted with Et.sub.2O (500 ml). The organic layer was washed with saturated NaHCO.sub.3 solution (300 ml) and brine (200 ml). The organic layer was dried over MgSO.sub.4 and concentrated. The residue was crystallized with n-hexane to obtain tert-butyl 5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxan-5-ylcarbamate of Formula 4 as a white solid (32.0 g, 59.4%).

[0120] .sup.1H NMR (600 MHz, CDCl.sub.3): δ 5.32 (s, 1H), 3.86-3.80 (m, 4H), 3.73 (s, 2H), 3.68 (s, 1H), 1.46-1.44 (m, 15H)

##STR00012##

[0121] To synthesize tert-butyl 5-formyl-2,2-dimethyl-1,3-dioxan-5-ylcarbamate of Scheme 3, a solution of oxalyl chloride (33.4 ml, 3.17 eq) in dried MC (340 ml) was mixed with DMSO (43.7 ml, 5 eq). The mixture was stirred for 15 minutes and then mixed with tert-butyl 5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxan-5-ylcarbamate of Formula 4 (32.0 g, 0.123 mol) in anhydrous MC (340 ml). After the mixture was stirred for 2 hours, Et.sub.3N (171 ml, 10 eq) was added thereto. The mixture was stirred for 10 minutes, the cooling bath was then removed, and the mixture was left at room temperature. The light brown suspension was diluted with EA (300 ml) and washed with 10% NH.sub.4OH (1, 500 ml). The organic layer was concentrated and applied to SiO.sub.2 column chromatography eluting the residue by EA/n-hexane=1/10 to obtain tert-butyl 5-formyl-2,2-dimethyl-1,3-dioxane-5-ylcarbamate of Formula 5 (15.0 g, 47.2%) as a white solid.

[0122] .sup.1H NMR (400 MHz, C.sub.DCl3): δ 9.64 (s, 1H), 5.56 (s, 1H), 4.07 (d, 2H, J=12.0 Hz), 3.95 (d, 2H, J=12.0 Hz), 1.47 (s, 15H)

##STR00013##

[0123] To synthesize tert-butyl 5-ethynyl-2, 2-dimethyl-1,3-dioxan-5-ylcarbamate of Scheme 4, dimethyl-2-oxopropyl-phosphonate (1.6 g, 1.02 eq) was added to an acetonitrile (50 ml) suspension comprising K2003 (3.0 g, 2.25 eq) and p-toluenesulfonylazide (14% solution in toluene, 15.8 ml, 1.05 eq), and the mixture was vigorously stirred at room temperature for 2.5 hours. A solution of tert-butyl 5-formyl-2, 2-dimethyl-1,3-dioxan-5-ylcarbamate of Formula 5 (2.5 g, 9.64 mmol) contained in methanol (40 ml) was added to the first reaction mixture. After adding K2003 (2.7 g, 2.06 eq), the mixture was stirred for 1.5 hours and concentrated under a reduced pressure, and the residue was diluted with MC (200 ml) and H.sub.2O (200 ml). The organic layer was washed with H.sub.2O (200 ml), dried over MgSO.sub.4 and concentrated under a reduced pressure. The residue was eluted by EA/n-hexane=1/9 and applied to SiO.sub.2 column chromatography to obtain tert-butyl 5-ethynyl-2,2-dimethyl-1,3-dioxan-5-ylcarbarmate of Formula 6 (2.3 g, 93.4%) as a white solid.

[0124] .sup.1H NMR (400 MHz, C.sub.DCl3): δ 5.15 (s, 1H), 4 .05-3. 95 (m, 4H), 2.43 (s, 1H), 1.48-1.38 (m, 15H)

##STR00014##

[0125] To synthesize 2-amino-2-(1-decyl-1H-1,2,3-triazol-4-yl)propane-1,3-diol, which is KARI101 of Scheme 5, CuSO.sub.4.Math.5H.sub.2O (1.56 g, 6 mmol) was added to a solution of tert-butyl 5-ethynyl-2,2-dimethyl-1,3-dioxan-5-ylcarbamate (4.0 g, 15 mmol) , 1-azidodecane of Scheme 1 (3.16 g, 17 mmol) , sodium L (4.03 g, 20 mmol), t-BuOH (60 ml), H.sub.2O (128 ml) and MC (104 ml). The two-phase solution was stirred in air for 18 hours, and the aqueous layer was extracted with MC. The organic layer was dried over MgSO.sub.4 and concentrated under a reduced pressure. The residue was eluted by EA/n-hexane=1/6 and applied to SiO.sub.2 column chromatography to obtain a solid (6.5 g). The resulting solid was mixed with concentrated HCL (21 ml) and ethanol (210 ml) and stirred at room temperature for 6 hours. The reaction mixture was concentrated under a reduced pressure and recrystallized from acetone to obtain 2-amino-2-(1-decyl-1H-1,2,3-triazol-4-yl)propane-1,3-diol as a white solid (2.6 g, 52.4%, molecular weight 298.4).

[0126] .sup.1H NMR (500 MHz, methanol-d.sub.4): δ 8.06 (s, 1H), 4.41 (t, 2H), 3.95 (dd, J=20 Hz, 15 Hz, 4H), 1.91 (t, 2H), 1.29-1.33 (m, 14H), 0.89 (t, 3H)

[0127] .sup.13C NMR (500 MHz, methanol-d.sub.4): δ 144.9, 124.3, 63.7(2C), 60.8, 51.5, 33.0, 31.3, 30.6, 30.5, 30.4, 30.1, 27.5, 23.7, 14.4

[0128] 1. Cell Culture

[0129] Human embryonic kidney 293 (HEK293) cells were purchased from ATCC and cultured in a DMEM medium comprising 10% FBS at 37° C. and in 5% CO.sub.2. To overexpress a ghrelin receptor, HEK293 cells were infected with a human ghrelin receptor cDNA vector (hGHSRa-pcDNA3.1+, cDNA Resource Center) and cultured for 48 hours. Thereafter, each triazole compound synthesized in the cell line and ghrelin were treated to evaluate efficacy as a ghrelin receptor agonist in terms of intracellular calcium influx, ghrelin receptor sub-signal factors and ghrelin receptor intracellular influx.

[0130] 2. Intracellular Calcium Influx Experiment

[0131] HEK293 cells overexpressing a ghrelin receptor were treated with Fluo-2AM, a calcium-specific fluorescent factor, and, 1 hour later, washed twice with a wash buffer comprising 20 mM Hepes, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2 and 0.7 mg/mL probenicid. After treating the cells with 100 ul of a wash buffer, fluorescently labeled intracellular calcium was imaged at an excitation wavelength of 485 nM and an emission wavelength of 520 nM for 1 minute at a shutter interval of 10 seconds under a laser scanning confocal microscope equipped with a temperature and humidity control equipment. Thereafter, ghrelin and triazole compounds were added for each concentration and then imaged under the same conditions. Intracellular calcium fluorescence intensity by treatment with compounds was quantified by calculating same based on the fluorescence intensity when not treated with compounds.

[0132] 3. Immunofluorescence

[0133] HEK293 cells overexpressing a ghrelin receptor were treated with 10 mM each of ghrelin and a triazole compound, and, 1 hour later, the cells were fixed and cultured together with an anti-ghrelin receptor (rabbit, 1:500, abcam) for the ghrelin receptor. One day later, the cells were washed and cultured together with an anti-rabbit 488 antibody for 1 hour, and analyzed by using a laser scanning confocal microscope equipped with Fluoview SV1000 imaging software (Olympus FV1000, Japan). Metamorph software (Molecular Devices) was used to quantify the percentage of the area of the stained area of the ghrelin receptor introduced into the cells.

[0134] 4. Western Blot

[0135] Protein expressions of pAMPK, AMPK, pPI3K, PI3K, pERK, and ERK were analyzed by using Western blotting. Antibodies to pAMPK, AMPK, pPI3K, PI3K, pERK, ERK (cell signaling) and β-actin (Santa Cruz) were used, and densitometric quantification was performed by using ImageJ software (US National Institutes of Health).

[0136] 5. Analysis of a G Protein Coupled Receptor Agonist and Antagonist

[0137] The efficacy of triazole compounds (10 mM) as agonists and antagonists for a total of 170 types of G protein-coupled receptors was performed by Eurofins Discovery.

[0138] 6. Establishment of Postoperative Ileus Mouse Model

[0139] Mice aged 6-8 weeks were fasted for one day and then anesthetized with a mixture of ketamine and xylazine. The abdomen and peritoneum were then incised, and the small intestine was exposed on a sterile gauze pad. Manipulation was performed from the duodenum to the cecum for 5 minutes by using a sterile cotton swab. After surgery, the abdomen was sutured and the mice were allowed to recover for 4 hours in a cage maintained at 32 degrees.

[0140] 7. Evaluation of Delayed Gastric Emptying

[0141] Triazole compounds (10 mg/kg, 20 mg/kg and 30 mg/kg) were orally administered 20 hours after POI surgery. After 4 hours, 1.5% methylcellulose aqueous solution and 0.05% phenol red (Sigma) were orally administered. After 30 minutes, the stomach of the mouse was removed and put into a 0.1N NaOH 2 ml solution to homogenize same. 3 ml of 0.1N NaOH solution was added and centrifuged at 3000 rpm at 4 degrees for 10 minutes. After adding 100 ml of 20% trichloroacetic acid to the supernatant, the mixture was centrifuged at 3000 rpm at 4 degrees for 10 minutes. After adding 400 ml of 0.5N NaOH solution to 500 ml of the supernatant, absorbance was measured at 562 nm. For baseline control, the stomach was removed immediately after oral administration of 1.5% methylcellulose aqueous solution and 0.05% phenol red, and the absorbance was measured in the sample obtained through the above-identified process. The percentage of the gastric emptying delay was calculated by (1−(absorbance of test sample)/(baseline control absorbance)×100.

[0142] 8. Evaluation of Colon Transit Time

[0143] To evaluate the colon transit time of feces, 200 ul of trypan blue dye was injected into the proximal colon before suturing after POI surgery. A triazole compound (30 mg/kg) was orally administered 4 hours after POI surgery. The mouse was transferred to a metabolic cage, and the weighed feed was administered thereinto, and the time at which the first feces stained with trypan blue appeared was measured for 24 hours. At the 24th hour, the number and weight of feces were measured, and the weight of the consumed feed and the mouse's body weight were measured.

[0144] 9. Establishment of a Cancer Cachexia Model

[0145] To establish a cancer acinar model, CT26 (colon tumor 26 cell line) cells were purchased from ATCC (CRL-2638) and cultured in a RPMI1640 medium comprising 10% FBS at 37° C. and in 5% CO.sub.2. Cultured 1×10.sup.6 cells were subcutaneously injected into the right side of the abdomen of 10-week-old mice. The size of the cancer grown in the right abdomen, body weight, and feed intake were measured once every time after cell injection, and 10 mg/kg of a triazole compound was orally administered daily from the 9th day. On the 18th day of cancer cell injection, each of the grip test and Rota-rod test for evaluating the muscle function was performed for 1 day. On the 20th day, subcutaneous fat, visceral fat and muscle (femur, calf) of the mouse were separated and weighed.

[0146] 10. Grip Test and Rota-Rod Test for Evaluating Muscle Function

[0147] For the evaluation of muscle function, the grip test was performed by holding the mouse by having the mouse hold the metal grid attached to the grip strength meter, holding the tail of the mouse, and pulling it from behind horizontally. The force applied to the metal grid immediately before losing the grip was recorded in the measuring instrument as the maximum tension in g, and the entire process was performed 9 times per subject to record the average value. In the Rota-rod test (Ugo Basile, Comerio, VA, Italy), Rota-rod exercise was performed at least three times at a rotational speed of 4 rpm by using a machine equipped with a rod with the diameter of 3 cm appropriately processed to provide grip to measure the endurance time of the tested animals in seconds and record the average value. Each Rota-rod behavior test was not to exceed 5 minutes per time.

[0148] 11. Statistical Analysis

[0149] The repeated measures analysis of Tukey's HSD test and variance test were performed according to the SAS statistical package (release 9.1; SAS Institute Inc., Cary, NC) for comparison of multiple groups. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 were considered significant.

[0150] <Results of Experiment>

[0151] 1. Confirmation of Effects as a Ghrelin Receptor Agonist from Increase of Intracellular Calcium Influx

[0152] In order to confirm the efficacy of triazole compounds as ghrelin receptor agonists, changes in intracellular calcium influx, which is a major function of ghrelin, were first measured. HEK293 cells overexpressing a human ghrelin receptor were reacted with a calcium-labeled fluorescent factor for 1 hour and then treated with ghrelin and triazole compounds KARI001, KARI101, and KARI201 for each concentration. As a result, it was confirmed that intracellular calcium influx increased as the concentration of the three triazole compounds increased (see FIG. 1). The EC50, which is the concentration capable of inducing 50% intracellular calcium influx, was confirmed to have ghrelin=39.45 nM, KARI001=128.3 nM, KARI101=188.5 nM, and KARI201=272.9 nM.

[0153] That is, it was confirmed that the three triazole compounds could induce intracellular calcium influx as ghrelin receptor agonists.

[0154] 2. Effect as a Ghrelin Receptor Agonist Through the Regulation of G Protein-Dependent Subfactors of a Ghrelin Receptor

[0155] It is known in the art that a ghrelin receptor is a G protein-coupled receptor, and, when ghrelin binds to the ghrelin receptor, the activities of different sub-signal factors are regulated depending on three major types of G proteins. For example, Gag-dependent signaling increases intracellular calcium influx to increase AMPK factor phosphorylation, and Gai/o-dependent signaling increases PI3K factor phosphorylation. B-arrestin-dependent signaling increases ERK factor phosphorylation or induces ghrelin receptor intracellular influx (FASEB J. 2019;33(1):518-531).

[0156] First, it was checked whether or not the three triazole compounds could regulate the phosphorylation of G protein-dependent signaling subfactors of the ghrelin receptor. HEK293 cells overexpressing a human ghrelin receptor were treated with 10 mM of ghrelin and three triazole compounds, cell proteins were extracted 1 hour later, and it was checked whether or not sub-factors were phosphorylated by using Western blotting. As a result, it was observed that the three triazole compounds could increase phosphorylation of AMPK factors (see FIG. 2).

[0157] In addition, in order to confirm the effect of the ghrelin receptor intracellular influx, HEK293 cells overexpressing a human ghrelin receptor were treated with 10 mM ghrelin and three triazole compounds, a ghrelin receptor antibody was cultured 1 hour later, and it was checked whether or not an intracellular ghrelin receptor was expressed. As a result, it was observed that cells not treated with ghrelin and three triazoles expressed ghrelin receptors in the cell membrane, whereas cells treated with ghrelin and three types of triazoles expressed ghrelin receptors in the perinuclear cytoplasm (see FIG. 3).

[0158] That is, it was found that the three triazole compounds were effective as agonists capable of regulating Gag-dependent signaling and B-arrestin-dependent signaling of ghrelin receptors.

[0159] 3. Effect Of Triazole Compounds as Ghrelin Receptor-Specific Agonists

[0160] The family of G protein-coupled receptors comprises many types of receptors comprising a ghrelin receptor. It was checked whether or not the triazole compounds have efficacy as agonists or antagonists of different types of G protein-coupled receptors. Different cell lines expressing a total of 170 G protein-coupled receptors comprising the ghrelin receptor were treated with 10 mM of triazole compound KARI201 alone (agonist effect) or together with ligands for each receptor (antagonist effect). As a result, it was confirmed that KARI201 only exhibited 83.7% efficacy as a ghrelin receptor agonist (see FIGS. 4A and 4B).

[0161] That is, through this result, it was found that the KARI201 triazole compound was effective as a ghrelin receptor-specific agonist.

[0162] 4. Effect of Triazole Compounds of Improving Delayed Gastric Emptying

[0163] In order to confirm the gastric motility promoting effect, which is one of the representative physiological effects of ghrelin as a ghrelin receptor, postoperative intestinal obstruction (POI) mice were used. POI mice whose intestinal obstruction was induced showed a delayed gastric emptying effect compared to normal mice, while POI mice administered with KARI201 and KARI101 20 hours after POI surgery showed a significant improvement in delayed gastric emptying as the concentration increased (see FIGS. 5A, 5B, 6A and 6B).

[0164] That is, it was found that triazole compounds KARI201 and KARI101 are ghrelin receptor agonists and are effective in improving delayed gastric emptying by improving the gastric motor function.

[0165] 5. Improvement of Triazole Compounds of Colon Motor Function

[0166] Based on the effect of triazole compounds KARI201 and KARI101 of delaying gastric emptying, it was checked whether or not they could improve the colon transit time of feces in POI mice. The mice administered with PBS 4 hours after POI surgery showed a long colon transit time of feces, whereas the mice administered with 30 mg/kg of KARI201 or KARI101 showed a significantly reduced colon transit time of feces (see FIGS. 7A and 8A). The number and weight of feces also increased by the administration of KARI201 (see FIGS. 7B and 8B). Feed intake also increased as compared to that of POI mice administered with PBS, whereas there was no change in their body weights (see FIGS. 7C, 7D, 8C and 8D).

[0167] That is, it was found that triazole compounds KARI201 and KARI101 as ghrelin receptor agonists also improve colon motor function and thus are effective for improving the excretion time of feces. Therefore, it was found that the triazole compounds may be very useful as preventives or therapeutic agents for diseases mediated by a ghrelin receptor.

[0168] 6. Effect of Triazole Compounds of Improving Strength and Function

[0169] Cancer cachexia mice were used to confirm the increase in feed intake, which is another physiological effect of ghrelin as a ghrelin receptor, and consequent improvement in fat and muscle loss. To establish a cancer cachexia mouse model, CT26 cancer cells were subcutaneously injected into the right abdominal layer of 10-week-old mice. It was confirmed that cancer cells grew in the right abdomen on the 9th day after cell injection, and 10 mg/kg of KARI101 and KARI201 were orally administered once daily. On the 18th day, Grip test and Rota-rod test were performed to evaluate muscle strength function (see FIG. 9A). It was confirmed that muscle strength function decreased in cancer cachectic mice injected with Vehicle, whereas muscle strength function was significantly improved in cancer cachexic mice administered with KARI101 or KARI201 (see FIGS. 9B and 9C).

[0170] 7. Effect of Triazole Compound of Improving Feed Intake and Fat and Muscle Loss

[0171] Based on the effect of triazole compounds KARI101 and KARI201 of improving muscle strength and function in a cancer cachexia mouse model, cancer size, body weight and feed intake were evaluated. Administration of KARI101 and KARI201 did not affect the cancer size (see FIG. 10A) and body weight (see FIG. 10B) of cancer cachexia mice, but increased daily feed intake as compared to cancer cachexia mice administered with Vehicle (see FIGS. 100 and 10D). In addition, KARI101 and KARI201 administration increased reduced total fat (FIG. 11A), visceral (see FIG. 11B) and subcutaneous fat (FIG. 11C) and increased reduced muscle mass (see FIG. 11D), as compared to cancer cachexia mice administered with Vehicle.

[0172] That is, it was found that triazole compounds KARI201 and KARI101 as ghrelin receptor agonists can increase the reduced feed intake of cancer cachectic mice and, thus, are effective for improving the reduced fat and muscle mass and muscle strength function.

INDUSTRIAL APPLICABILITY

[0173] The compound provided by the present invention exhibits a very strong binding force with very high specificity to a ghrelin receptor and thus is very useful for preventing or developing a preventive or therapeutic agent for diseases mediated by a ghrelin receptor and has high industrial applicability.