KR102381002B1 - Benzoic acid derivatives and composition for preventing or treating autoimmune diseases comprising the same - Google Patents

Abstract

The present invention provides a benzoic acid derivative and a pharmaceutical composition for preventing or treating autoimmune diseases and a health functional food composition containing the same as an active ingredient. The compound represented by Formula 1 according to the present invention, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof may have an LPA1 (Lysophosphatidic acid 1) receptor activation inhibitory effect, for preventing or treating LPA1-related diseases It can be usefully used as a pharmaceutical composition and a health functional food composition. In addition, the pharmaceutical composition and health functional food composition can effectively prevent or treat not only autoimmune diseases, but also other immune-related diseases with similar developmental mechanisms.

Description

Benzoic acid derivative and composition for preventing or treating autoimmune disease containing same as an active ingredient {BENZOIC ACID DERIVATIVES AND COMPOSITION FOR PREVENTING OR TREATING AUTOIMMUNE DISEASES COMPRISING THE SAME}

The present invention relates to a benzoic acid derivative and a composition for preventing or treating autoimmune diseases containing the same as an active ingredient.

Autoimmune disease is a disease that causes an abnormal immune response by mistaken for an antigen in the body's own substance. etc.

The main cause of autoimmune diseases is that lymphocytes migrate to tissues and abnormalities in the autoimmune system are triggered, but the clear pathogenesis of each disease is not yet known.

Recently, with a focus on efficient immunosuppression, research on the development of therapeutic agents for autoimmune diseases is being conducted focusing on immunosuppression at the cellular level (inhibition of activation of lymphocytes and cell types responsible for immune responses in diseased lesion tissues) and molecular level immunosuppression.

Lysophospholipid represented by lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) has long been considered a phospholipid metabolite of cell membrane components, but now G protein-coupled receptor (G protein- It has been reported as an important extracellular signaling material that regulates various biological functions through activation of lysophospholipid receptors classified as coupled receptors; GPCRs.

Among them, LPA1 is the first lysophospholipid receptor identified (identified in 1996) and is a key factor regulating the development of the nervous system. Immune response regulation is attracting attention. The signaling mechanism of LPA1 revealed through various research data and the organic system with in vivo tissues, including autoimmune diseases such as Sjogren's syndrome, stroke, psoriasis, inflammatory bowel disease, diabetic nephropathy and fibrosis, and systemic sclerosis, as well as the neuropsychiatric system It has become important data for the treatment of a variety of disorders, including disorders, neuropathic pain, infertility, cardiovascular disease, and cancer. (Park et al, “Inhibition of lysophosphatidic acid receptor ameliorates Sjogren's syndrome in NOD mice” Oncotarget. 2017 , 8(16), 27240-27251, Yung et al, “LPA receptor signaling: pharmacology, physiology, and pathophysiology” J Lipid Res. 2014, 55, 1192-1214, Choi et al. “LPA receptors: subtypes and biological actions “Annu Rev Pharmacol Toxicol. 2010;50:157-86. Debendra Pattanaik et al, “A Role for Lysophosphatidic Acid and Sphingosine 1-Phosphate in the Pathogenesis of Systemic Sclerosis” Discov Med 10(51):161-167, August 2010 .)

Current steroids include Cyclosporine, Rapamycin, Methotrexate, Cyclophosphamide, IV Immunoglobulin, Axathioprine, Infliximab, etc. Although it is being treated with a combination therapy of various immunosuppressive agents, most of them have continuous inflammation and frequent relapses, and systemic side effects from treatment are also frequently occurring.

In particular, in rare autoimmune diseases, the number of patients is small, but the cost of the applied treatment is very high, so the economic burden on patients is very high, and the cost burden is increasing because it requires long-term treatment. Although the number of patients with rare autoimmune diseases is increasing in Korea, there is no clear treatment and the cure rate is low, so it is still an economic and social problem.

Korean Patent Registration No. 10-1217066 (Registered on December 24, 2012)

In order to solve the above problems, the present invention provides a benzoic acid derivative.

The present invention provides a pharmaceutical composition for preventing or treating autoimmune diseases containing the benzoic acid derivative as an active ingredient.

The present invention provides a health functional food composition for improving or preventing autoimmune diseases containing the benzoic acid derivative as an active ingredient.

The compound represented by Formula 1 according to the present invention, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof,

[Formula 1]

In Formula 1, R ¹ and R ² may be the same or different, respectively, hydrogen, (C1-C4) alkyl, (C1-C4) alkoxy, halogen, trifluoromethyl, benzyloxy, (C1-C4) cyclo It may be any one selected from the group consisting of hexyl, 2H-1,2,3-triazol-2-yl and (C1-C4) benzoyl.

The pharmaceutical composition for preventing or treating an LPA1-related disease according to the present invention contains the compound, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, and has an inhibitory effect on LPA1 (Lysophosphatidic acid 1) receptor activation can have

The health functional food composition for improving or preventing LPA1-related diseases according to the present invention contains the compound, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, and LPA1 (Lysophosphatidic acid 1) receptor activation may have an inhibitory effect.

Since the benzoic acid derivative according to the present invention has an inhibitory effect on LPA1 receptor activation, it is useful as a pharmaceutical composition and a health functional food composition for preventing or treating LPA1-related diseases such as stroke, psoriasis, Sjogren's syndrome, inflammatory bowel disease, diabetic nephropathy and fibrosis can be used

Autoimmune diseases can be effectively prevented and treated by using the pharmaceutical composition and health functional food composition for preventing or treating LPA1-related diseases according to the present invention. In addition, it can be variously applied to the treatment of other immune-related diseases with similar developmental mechanisms as well as the autoimmune diseases.

1 is a graph showing a calcium analysis result according to an experimental example of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention relates to autoimmune diseases such as sjogren's disease, psoriasis, and multiple sclerosis, and abnormal immune response diseases such as diabetic nephropathy and stroke (cerebral ischemia) in an animal model (in vivo disease model) ), verified that LPA1 is useful as a new target molecule for the treatment of autoimmune diseases, and completed the present invention by discovering a compound with LPA1 antagonistic action.

The present invention provides a compound represented by the following formula (1), a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1, R ¹ and R ² may be the same or different, respectively, hydrogen, (C1-C4) alkyl, (C1-C4) alkoxy, halogen, trifluoromethyl, benzyloxy, (C1-C4) cyclo It may be any one selected from the group consisting of hexyl, 2H-1,2,3-triazol-2-yl and (C1-C4) benzoyl.

In the compound, R ¹ and R ² may each be the same or different, and hydrogen, (C1~C2)alkyl, halogen, trifluoromethyl, benzyloxy, (C3~C4)cyclohexyl, 2H-1,2,3 - It may be any one selected from the group consisting of -triazol-2-yl and (C1-C2) benzoyl.

More specifically, the compound is 3-(5-(4-bromobenzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid, 3 -(5-(2-fluoro-3-(trifluoromethyl)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid, 3 -(5-(3-(benzyloxy)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid, 3-(5-(4- (4-propylcyclohexyl)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid, 3-(5-(4-(4-ethyl) Cyclohexyl)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid, 3-(5-(5-methyl-2-(2H-1) ,2,3-triazol-2-yl)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid and 3-(5-(2) It may be any one selected from the group consisting of -(4-methylbenzoyl)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid.

The present invention contains the compound, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, and has an LPA1 (Lysophosphatidic acid 1) receptor activation inhibitory effect, LPA1-related disease prevention Or it provides a pharmaceutical composition for treatment.

The LPA1-related disease may be any one selected from the group consisting of stroke, psoriasis, Sjogren's syndrome, inflammatory bowel disease, diabetic nephropathy, and fibrosis, but is not limited thereto.

The pharmaceutical composition according to the present invention may be prepared by further including an appropriate pharmaceutically acceptable carrier, excipient or diluent according to the formulation. The "pharmaceutically acceptable" refers to exhibiting properties that are not toxic to cells or humans exposed to the pharmaceutical composition.

Carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. there is.

When the pharmaceutical composition according to the present invention is formulated in the above form, it can be prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, etc. usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient to the compound, for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc. can be mixed to prepare it.

In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be administered in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level depends on the patient's health condition, the Type, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment period, factors including drugs used in combination or concurrently, and other factors well known in the medical field. .

The amount of the compound used as the active ingredient of the pharmaceutical composition according to the present invention may vary depending on the patient's age, sex, weight, and disease, but is 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, once to several times a day. can be administered. In addition, the dosage of the compound according to the present invention may be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age, and the like. Accordingly, the above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, endobronchial inhalation, intrauterine dural or intracerebroventricular injection.

The present invention contains the compound, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, and has an LPA1 (Lysophosphatidic acid 1) receptor activation inhibitory effect, LPA1-related disease improvement Or it provides a health functional food composition for prevention.

The LPA1-related disease may be any one selected from the group consisting of stroke, psoriasis, Sjogren's syndrome, inflammatory bowel disease, diabetic nephropathy, and fibrosis, but is not limited thereto.

The health functional food according to the present invention may be provided in the form of powder, granule, tablet, capsule, syrup or beverage, and the health functional food is used together with other food or food additives in addition to the compound according to the present invention as an active ingredient, It may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined according to the purpose of its use, for example, prophylactic, health or therapeutic treatment.

The effective dose of the compound contained in the health functional food according to the present invention can be used according to the effective dose of the pharmaceutical composition, but in the case of long-term intake for health and hygiene or health control, the It may be below the range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

The type of health functional food according to the present invention is not particularly limited, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, teas, drinks, alcoholic beverages, and vitamin complexes.

The compound according to the present invention may be used in the form of a stereoisomer of the compound. In the present invention, “stereoisomer” refers to isomers resulting from different spatial arrangements of atoms or groups of atoms in a molecule, and includes both optical isomers and geometric isomers. In optical isomerism, four atoms or groups of atoms bonded to an asymmetric carbon atom form a pair of enantiomers according to the bonding method, and if there are two asymmetric carbon atoms in a molecule, it becomes a diastereomer. It may include all isomers. In addition, when unsaturated hydrocarbons exist, they become geometric isomers, and all possible geometric isomers can be included.

The compound according to the present invention may be used in the form of a racemate of the compound. In the present invention, "racemate" refers to a substance that does not exhibit optical activity because the right-handed optical isomer and the left-handed optical isomer of a chiral molecule are mixed in equal amounts.

The compound according to the present invention may be used in the form of a pharmaceutically acceptable salt, and the salt may be used in the form of any one of a pharmaceutically acceptable basic salt or an acid salt.

The basic salt can be used in the form of any one of an organic base salt, an inorganic base salt, sodium salt, potassium salt, calcium salt, lithium salt, magnesium salt, cesium salt, aminium salt, ammonium salt, triethyl It may be selected from the group consisting of an aminium salt and a pyridinium salt, but is not limited thereto.

As the acid salt, an acid addition salt formed by a free acid is useful. As free acids, inorganic acids and organic acids can be used. As inorganic acids, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfurous acid, phosphoric acid, etc. can be used. As organic acids, citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, and methanesulfonic acid can be used. , Benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid etc. can be used. Preferably, hydrochloric acid may be used as the inorganic acid, and methanesulfonic acid may be used as the organic acid.

In addition, the compound according to the present invention may include all salts, hydrates and solvates that can be prepared by conventional methods as well as pharmaceutically acceptable salts.

The addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compounds represented by Chemical Formulas 1 to 3 in a water-miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile. It can be prepared by adding an excess of an organic base or adding an aqueous base solution of an inorganic base, followed by precipitation or crystallization. Alternatively, an addition salt may be obtained by evaporating the solvent or excess base from the mixture and drying, or it may be prepared by suction filtration of the precipitated salt.

Hereinafter, examples will be described in detail to help the understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Synthesis method>

All reagents necessary for synthesis were purchased from TCI, Sigma Aldrich, and Alfa Aesar with a purity of 97% or higher. Before performing the synthesis, the glassware to be used for the reaction was sufficiently washed with acetone, dried in an oven at 60° C., and the synthesis was performed under a nitrogen stream. Dichloromethane, tetrahydrofuran, methanol, acetone, toluene, diethyl ether, ethyl acetate, hexane are highly purified It was purchased from Gold, and for anhydrous solvents it was purchased from Sigma Aldrich. In TLC, the progress of the reaction was confirmed by using a UV lamp and color developing reagents AA, CAM, potassium permanganate (KMnO ₄ ), and ninhydrin. H NMR and C NMR spectra were measured using a BRUKER Ascend 600 spectrometer and Varian VnmrS-400, and CDCl3 and DMSO-d6 were used as solvents for analysis. Chemical shift values were expressed in ppm units, and coupling constants (J) were expressed in Hz units.

<Example 1>

Synthesis of parent nucleus tetrahydropyrazolopyrazine

[Scheme 1]

Scheme 1 is a process for synthesizing tetrahydropyrazolopyrazine, which is a core parent nucleus, according to the present invention. In the same manner as in Scheme 1, propargylamine as a starting material and tetrahydropyrazol according to the following method The synthesis of pyrazine (Tetrahydropyrazolopyrazine) was carried out

<Example 1-1> 4-methyl-N- (prop-2-nin-1-yl) benzenesulfonamide [4-methyl-N- (prop-2-yn-1-yl) benzenesulfonamide]

Propargylamine (Propargylamine, 0.5ml, 7.81mmol) and triethylamine (Triethylamine; TEA 1.3ml, 9.37mmol) were added to dichloromethane (7.5ml) at 0° C. to form a mixture. Para-toluene sulfonyl chloride (p-Toluene Sulfonyl Chloride, 1.49 g, 7.81 mmol) dissolved in dichloromethane (22.5 ml) was added dropwise to the mixture, followed by stirring at room temperature for 5 hours. After completion of the stirring, water was added to terminate the reaction, and the product was extracted through dichloromethane. Water was removed using sodium sulfate, and the filtrate was concentrated under reduced pressure. Thereafter, separation and purification were performed by column chromatography to obtain compound 1 (1.517 g, 92.84%).

Compound 1: ¹ H NMR (600 MHz, Chloroform-d) δ 7.78 (d, J = 8.3 Hz, 2H), 7.32 (d, J = 8.2 Hz, 2H), 4.71 (s, 1H), 3.83 (dd, J = 6.1, 2.5 Hz, 2H), 2.44 (s, 3H), 2.11 (s, 1H); ¹³ C NMR (151 MHz, Chloroform-d) δ 143.88, 136.49, 129.72, 127.41, 77.95, 73.02, 32.89, 21.58.

<Example 1-2> N- (2-chloroethyl) -4-methyl-N- (prop-2-nin-1-yl) benzenesulfonamide [N- (2-chloroethyl) -4-methyl- N-(prop-2-yn-1-yl)benzenesulfonamide]

The compound 1 (1.517g, 7.25mmol) and potassium carbonate (Potassium Carbonate, 1.2g, 8.7mmol) were added to acetone (Acetone, 30ml) to form a mixture. Then, 1-bromo-2-chloroethane (1-bromo-2-chloroethane, 1.2ml, 14.5mmol) was added dropwise to the mixture, followed by refluxing for 3 hours. After cooling, the product was extracted through ethyl acetate and water was removed through magnesium sulfate. The filtrate was concentrated under reduced pressure, and then separated and purified through column chromatography to obtain compound 2 (1.29 g, 65.48%).

Compound 2: ¹ H NMR (600 MHz, Chloroform-d) δ 7.74 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 7.9 Hz, 2H), 4.19 (s, 2H), 3.70 (t, J = 7.0 Hz, 2H), 3.50 (t, J = 6.9 Hz, 2H), 2.43 (s, 3H), 2.11 (m, 1H); ¹³ C NMR (151 MHz, Chloroform-d) δ 143.99, 135.48, 129.69, 127.66, 76.64, 74.17, 48.30, 41.78, 38.16, 21.58.

<Example 1-3> Ethyl 5-tosyl-4,5,6,7-tetrahydropyrazolo [1,5-a] pyrazine-2-carboxylate [Ethyl 5-tosyl-4,5,6,7 -tetrahydropyrazolo[1,5-a]pyrazine-2-carboxylate]

Compound 2 (6.05 g, 22.26 mmol) and diazoacetate (Diazoacetate, 23.7 ml, 27.83 mmol) were added to toluene (26.3 ml) and stirred at 140° C. for 12 hours. After stirring, cesium carbonate (Cesium Carbonate, 10.88 g, 33.4 mmol) and tetrahydrofuran (Tetrahydrofuran, 10 ml) were added to the mixture, and the reaction was continued for 30 minutes. After completion of the reaction, the mixture was cooled to room temperature, and 1 n hydrochloric acid was added to neutralize the mixture. The product was extracted via ethyl acetate and water removed via magnesium sulfate. The filtrate was concentrated under reduced pressure, and purified by column chromatography to obtain compound 3 (4.215 g, 54.19%).

Compound 3: ¹ H NMR (600 MHz, DMSO-d6) δ 7.73 (d, J = 8.3 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H), 6.59 (s, 1H), 4.33 (s, 1H), 4.23 (d, J = 7.1 Hz, 1H), 4.18 (t, J = 5.6 Hz, 1H), 3.60 (t, J = 5.6 Hz, 1H), 3.32 (s, 2H), 2.50 (s, 1H), 2.40 (s, 2H), 1.25 (s, 1H); ¹³ C NMR (151 MHz, Chloroform-d) δ 144.03, 136.09, 129.94, 127.25, 108.44, 61.50, 49.56, 41.45, 21.56, 14.25.

<Example 1-4> (5-tosyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)methanol [(5-tosyl-4,5,6, 7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)methanol]

Compound 3 (1.13 g, 3.23 mmol) and lithium aluminum hydride (159 mg, 4.2 mmol) were added to tetrahydrofuran (10 ml) at 0° C. to form a mixture. Then, the mixture was stirred for one hour at room temperature. After completion of the reaction, 1 n hydrochloric acid was added to neutralize, and the product was extracted through dichloromethane. After adding magnesium sulfate to the extracted product to remove water, the filtrate was concentrated under reduced pressure and purified by column chromatography to obtain compound 4 (940 mg, 94%).

Compound 4: ¹ H NMR (600 MHz, Chloroform-d) δ 7.71 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 6.02 (s, 1H), 4.61 (m, 2H), 4.30 (d, J = 2.1 Hz, 2H), 4.18 (m, 2H), 3.56 (m, 2H), 2.44 (s, 3H); ¹³ C NMR (151 MHz, Chloroform-d) δ 144.52, 134.70, 132.74, 130.05, 127.70, 100.47, 100.45, 58.86, 58.81, 46.82, 43.76, 43.28, 30.95, 21.59.

<Example 1-5> Ethyl (E)-3-(5-tosyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)acrylate [Ethyl (E )-3-(5-tosyl-4,5,6,7-tetrahydropyrazolo[1,5-a] pyrazin-2-yl)acrylate]

Dimethyl sulfoxide (Dimethyl sulfoxide, 4.43 ml, 61.8 mmol) was added to dichloromethane (10 ml) to form a mixture. Then, oxalyl chloride (2.651ml, 30.9mmol) dissolved in dichloromethane (15ml) at -60°C was added dropwise to the mixture. After 5 minutes, compound 4 (1.9 g, 6.18 mmol) was added to the mixture, and the reaction was continued for 1 hour. After completion of the reaction, the temperature of the mixture was brought close to room temperature, and the product was extracted through dichloromethane. After removing water by adding magnesium sulfate to the extracted product, the filtrate was concentrated under reduced pressure and purified by column chromatography to obtain compound 5 (1.34 g, 60%).

Compound 5: ¹ H NMR (600 MHz, Chloroform-d) δ 7.71 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 16.1 Hz, 1H), 7.36 (d, J = 7.9 Hz, 2H) , 7.27 (s, 1H), 6.35 (d, J = 16.1 Hz, 1H), 6.25 (s, 1H), 4.33 (s, 2H), 4.23 (t, J = 6.6 Hz, 4H), 3.59 (t, J = 5.4 Hz, 2H), 2.44 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H); ¹³ C NMR (151 MHz, Chloroform-d) δ 166.80, 148.18, 144.62, 136.06, 135.13, 132.71, 130.09, 127.70, 119.60, 101.14, 60.49, 47.23, 43.66, 43.17, 21.59, 14.28.

<Example 1-6> Ethyl 3- (5-tosyl-4,5,6,7-tetrahydropyrazolo [1,5-a] pyrazin-2-yl) propanoate [Ethyl 3- (5- tosyl-4,5,6,7-tetrahydropyrazolo[1,5-a] pyrazin-2-yl)propanoate]

Compound 5 (1.3g, 3.46mmol) and 10% Pd/C (0.1g) were added to methanol (Methanol, 20ml) to form a mixture, and the mixture was stirred at room temperature under a hydrogen stream for 2 hours. After completion of the reaction, the mixture was filtered through celite. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain compound 6 (1 g, 76%).

Compound 6: ¹ H NMR (600 MHz, Chloroform-d) δ 7.70 (m, 2H), 7.35 (m, 2H), 5.84 (d, J = 1.0 Hz, 1H), 4.27 (s, 2H), 4.14 ( m, 2H), 3.54 (m, 2H), 2.89 (t, J = 7.6 Hz, 2H), 2.61 (dd, J = 8.0, 7.2 Hz, 2H), 2.44 (s, 3H), 1.24 (t, J) = 7.1 Hz, 3H); ¹³ C NMR (151 MHz, Chloroform-d) δ 172.89, 151.73, 134.20, 132.76, 129.99, 127.71, 100.67, 60.41, 46.63, 43.83, 43.28, 33.96, 23.51, 21.58, 14.23.

<Example 1-7> Methyl 3- (4,5,6,7-tetrahydropyrazolo [1,5-a] pyrazin-2-yl) propanoate [Methyl 3- (4,5,6, 7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl) propanoate]

Compound 6 (388mg, 1.03mmol) and magnesium powder (247mg, 10.3mmol) were added to methanol (6ml) to form a mixture. After that, the mixture was allowed to react at 0 °C for 1 hour. After completion of the reaction, the mixture is filtered through celite. The filtrate was concentrated under reduced pressure and separated and purified through column chromatography to obtain compound 7 (283 mg, 73%) and tetrahydropyrazolopyrazine, which is the core parent nucleus of the compound according to the present invention.

Compound 7: ¹ H NMR (600 MHz, Chloroform-d) δ 5.78 (s, 1H), 4.05 (s, 2H), 4.01 (s, 2H), 3.69 (s, 3H), 3.28 (s, 2H), 2.93 (m, 2H), 2.67 (m, 2H); ¹³ C NMR (151 MHz, Chloroform-d) δ 173.53, 150.64, 138.07, 99.55, 51.62, 47.61, 43.62, 43.01, 33.90, 23.59.

<Example 2> Synthesis of parent nucleus and benzoic acid derivative

[Scheme 2]

Scheme 2 is the process of synthesizing tetrahydropyrazolopyrazine and 7 benzoic acid derivatives, which are the core parent nucleus according to the present invention, and in the same manner as in Scheme 2, tetrahydropyrazolopyrazine of compound 7 and variously substituted benzoic acid 7 The species was subjected to an amide coupling reaction to synthesize compounds 8a to 8g, and methyl esters were hydrolyzed to synthesize 7 species of compounds 9a to 9g.

<Example 2-1> Amide coupling reaction (8a-8g)

Compound 7 (50 mg, 0.224 mmol), acid (45 mg, 0.269 mmol), and triethylamine (0.13 ml, 0.896 mmol) were added to dichloromethane (0.5 ml) to form a mixture. Hydroxybenzotriazole (37mg, 0.269mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 52mg in the resulting mixture , 0.269 mmol) was added and stirred at room temperature for 12 hours. After completion of the reaction, the product was extracted through dichloromethane. After adding magnesium sulfate to the extracted product to remove water, the filtrate was concentrated under reduced pressure and purified by column chromatography to obtain compound 8 imide (40 mg, 42%).

<Example 2-2> Hydrolysis (9a-9g)

Compound 8a (40 mg, 0.1 mmol) was added to tetrahydrofuran (3 ml) to form a mixture. Then, lithium hydroxide (55 mg, 1.3 mmol) dissolved in water (1 ml) was added and stirred at room temperature for 12 hours. After completion of the reaction, 1 n hydrochloric acid was added to neutralize, and the product was extracted through dichloromethane. After removing water by adding magnesium sulfate to the extracted product, the filtrate was concentrated under reduced pressure and purified by column chromatography to obtain compound 9a (32 mg, 90%). Compounds 9b to 9g were obtained from compounds 8b to 8g in the same manner.

[Compound 9a~9g]

1) Compound 9a: 3-(5-(4-bromobenzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid [3-(5) -(4-bromobenzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid]

¹ H NMR (600 MHz, Chloroform-d) δ 7.60 (m, 2H), 7.34 (m, 2H), 5.91 (s, 1H), 4.78 (d, J = 84.3 Hz, 2H), 4.22 (s, 4H) ), 2.94 (t, J = 7.2 Hz, 2H), 2.72 (t, J = 7.3 Hz, 2H).

2) Compound 9b: 3-(5-(2-fluoro-3-(trifluoromethyl)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-2- yl)propanoic acid [3-(5-(2-fluoro-3-(trifluoromethyl)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid]

¹ H NMR (600 MHz, Chloroform-d) δ 9.85 (s, 1H), 7.75 (ddd, J = 8.3, 4.7, 1.5 Hz, 1H), 7.66 (ddd, J = 7.7, 5.9, 1.7 Hz, 1H) , 7.39 (m, 1H), 5.92 (d, J = 112.0 Hz, 1H), 4.77 (d, J = 244.6 Hz, 2H), 4.10 (m, 4H), 2.94 (dt, J = 21.7, 7.4 Hz, 2H), 2.71 (dt, J = 17.5, 7.4 Hz, 2H).

3) compound 9c: 3-(5-(3-(benzyloxy)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid [3- (5-(3-(benzyloxy)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid]

¹ H NMR (600 MHz, Methanol-d4) δ 7.25 (m, 9H), 5.99 (m, 1H), 5.14 (s, 2H), 4.02 (m, 4H), 3.31 (s, 2H), 2.74 (d , J = 149.9 Hz, 4H), 2.15 (s, 2H), 1.35 (d, J = 61.9 Hz, 2H).

4) Compound 9d: 3-(5-(4-(4-propylcyclohexyl)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid [3-(5-(4-(4-propylcyclohexyl)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid]

¹ H NMR (600 MHz, Methanol-d4) δ 7.37 (m, 4H), 5.96 (d, J = 86.0 Hz, 1H), 4.15 (s, 4H), 2.69 (m, 4H), 2.55 (tt, J) = 12.2, 3.3 Hz, 1H), 1.89 (dq, J = 13.8, 3.4 Hz, 4H), 1.51 (qd, J = 13.2, 12.6, 3.7 Hz, 2H), 1.38 (m, 3H), 1.24 (d, J = 8.4 Hz, 2H), 1.09 (qd, J = 13.6, 13.0, 3.6 Hz, 2H), 0.92 (t, J = 7.3 Hz, 3H).

5) Compound 9e: 3-(5-(4-(4-ethylcyclohexyl)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid [3-(5-(4-(4-ethylcyclohexyl)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid]

¹ H NMR (600 MHz, Methanol-d4) δ 7.37 (m, 4H), 5.96 (d, J = 86.1 Hz, 1H), 4.84 (s, 2H), 4.14 (s, 4H), 2.72 (m, 4H) ), 2.55 (tt, J = 12.1, 3.3 Hz, 1H), 1.90 (m, 4H), 1.51 (m, 2H), 1.29 (m, 3H), 1.10 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H).

6) Compound 9f: 3-(5-(5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoyl)-4,5,6,7-tetrahydropyrazolo[1 ,5-a]pyrazin-2-yl)propanoic acid [3-(5-(5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoyl)-4,5,6, 7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid]

¹ H NMR (600 MHz, Methanol-d4) δ 8.08 (s, 1H), 7.91 (dd, J = 20.0, 8.3 Hz, 1H), 7.72 (d, J = 117.0 Hz, 2H), 7.47 (tdd, J) = 8.3, 2.0, 0.8 Hz, 1H), 7.32 (m, 1H), 5.87 (d, J = 243.3 Hz, 1H), 4.39 (m, 3H), 3.94 (m, 2H), 3.69 (m, 1H) , 2.84 (dt, J = 45.5, 7.5 Hz, 2H), 2.60 (m, 2H), 2.45 (d, J = 5.1 Hz, 3H).

7) Compound 9g: 3-(5-(2-(4-methylbenzoyl)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid [ 3-(5-(2-(4-methylbenzoyl)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid]

¹ H NMR (600 MHz, Methanol-d4) δ 8.07 (s, 1H), 7.61 (m, 6H), 6.94 (d, J = 7.7 Hz, 1H), 5.95 (d, J = 96.6 Hz, 1H), 4.74 (d, J = 33.9 Hz, 2H), 3.89 (dd, J = 189.8, 98.8 Hz, 4H), 2.86 (t, J = 7.5 Hz, 2H), 2.62 (t, J = 7.6 Hz, 2H), 2.37 (d, J = 42.7 Hz, 3H).

<Experimental Example 1> Calcium assay

The LPA1-overexpressing cell line (RH7777-human EDG1 cell) was seeded at 1×10 ⁵ /well in a 96-well black plate (clear bottom) and cultured for one day. After that, it was replaced with a serum-free culture medium containing 0.1% fatty acid-free BSA (fatty acid-free BSA) and cultured for an additional day.

Next, the drug (compound 9a-9g or AM152 (LPA1 antagonist), 1 μM) was pre-treated for 30 minutes, and then treated with Fluo-3AM (5 μM), a fluorescent dye for calcium measurement, and incubated for 60 minutes. After treatment with 10 μM LPA, the fluorescence intensity was measured 1 minute later (absorption: 506 nm). (Fluorescence intensity: using 3 layer victor)

1 is a graph showing a calcium analysis result according to an experimental example of the present invention. The 'Ca ²⁺ response' of the graph indicates a fold increase compared to the vehicle.

Referring to FIG. 1 , compounds 9a to 9g were found to exhibit 20-50% of efficacy compared to AM152 (1 μM).

<Experimental Example 2> Toxicity evaluation

To male Balb/c mice, Compound 9a, Compound 9c, Compound 9d, and Compound 9g were each suspended in 0.5% methylcellulose solution and administered orally once at a dose of 0.5 g/kg, 1 g/kg and 2 g/kg 7 The daily survival rate and body weight of mice were investigated.

After such administration, the animal's mortality, clinical symptoms, and weight change were observed, and hematological and blood biochemical tests were performed. After autopsy, abnormalities in the abdominal and thoracic organs were visually observed.

As a result, there were no remarkable clinical symptoms or mortality in all animals, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings.

As a result, the compounds of the present invention did not show a change in toxicity up to 2 g/kg in mice, and therefore, the median lethal dose (LD50) for oral administration was judged to be a safe substance of 2 g/kg or more.

Hereinafter, a formulation example of a composition containing compound 9a according to the present invention will be described, but the present invention is not intended to limit the present invention, but merely to describe it in detail.

<Prescription Example 1> Prescription Example of Pharmaceutical Composition

<Formulation Example 1-1> Preparation of powder

20 mg of compound 9a, 100 mg of lactose, and 10 mg of talc were mixed and filled in an airtight bag to prepare a powder.

<Formulation Example 1-2> Preparation of tablets

20 mg of compound 9a, 100 mg of cornstarch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed, and then tableted according to a conventional tablet preparation method to prepare tablets.

<Formulation Example 1-3> Preparation of capsules

After mixing 10 mg of compound 9a, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate, the above ingredients were mixed according to a conventional capsule preparation method and filled in a gelatin capsule to prepare a capsule.

<Formulation Example 1-4> Preparation of injection

After mixing 10 mg of compound 9a, an appropriate amount of sterile distilled water for injection, and an appropriate amount of a pH adjuster, the content of the above components per 1 ampoule (2 ml) was prepared according to a conventional injection preparation method.

<Formulation Example 1-5> Preparation of ointment

10 mg of compound 9a, 250 mg of PEG-4000, 650 mg of PEG-400, 10 mg of white petrolatum, 1.44 mg of methyl paraoxybenzoate, 0.18 mg of propyl paraoxybenzoate and the remaining amount of purified water were mixed, and then an ointment was prepared according to a conventional ointment preparation method. did

<Prescription Example 2> Health functional food

<Formulation Example 2-1> Preparation of health food

Compound 9a 1 mg, vitamin mixture appropriate amount (vitamin A acetate 70 µg, vitamin E 1.0 mg, vitamin B 1 0.13 mg, vitamin B 2 0.15 mg, vitamin B 6 0.5 mg, vitamin B 12 0.2 µg, vitamin C 10 mg, biotin 10 μg, nicotinic acid amide 1.7 mg, folic acid 50 μg, calcium pantothenate 0.5 mg) and an appropriate amount of a mineral mixture (ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, potassium monophosphate 15 mg, dibasic calcium phosphate 55 mg, potassium citrate 90 mg, calcium carbonate 100 mg, magnesium chloride 24.8 mg) were mixed, and then granules were prepared, and health food was prepared according to a conventional method.

<Formulation Example 2-2> Preparation of health drink

Add 1 mg of compound 9a, 1000 mg of citric acid, 100 g of oligosaccharide, 2 g of plum concentrate, 1 g of taurine and purified water to make a total of 900 ml. After stirring and heating at 85° C. for a period of time, the resulting solution was filtered and acquired in a sterilized 2 L container, sealed and sterilized, and then stored in a refrigerator.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. Do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (7)

delete delete 3-(5-(4-bromobenzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid, 3-(5-(2-flo Rho-3-(trifluoromethyl)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid, 3-(5-(3-( Benzyloxy)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid, 3-(5-(4-(4-propylcyclohexyl)benzoyl )-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid, 3-(5-(4-(4-ethylcyclohexyl)benzoyl)-4, 5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid, 3-(5-(5-methyl-2-(2H-1,2,3-triazole- 2-yl)benzoyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid and 3-(5-(2-(4-methylbenzoyl)benzoyl) )-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)propanoic acid, characterized in that selected from the group consisting of a compound, a stereoisomer thereof, a racemate thereof, or A pharmaceutically acceptable salt thereof. The compound of claim 3, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof, as an active ingredient, is characterized in that it has an inhibitory effect on LPA1 (Lysophosphatidic acid 1) receptor activation. Prevention or As a therapeutic pharmaceutical composition,
The LPA1-related disease is stroke, psoriasis, Sjogren's syndrome, inflammatory bowel disease, diabetic nephropathy and fibrosis, characterized in that selected from the group consisting of, LPA1-related disease prevention or treatment pharmaceutical composition. delete The compound of claim 3, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof, as an active ingredient, is characterized in that it has an LPA1 (Lysophosphatidic acid 1) receptor activation inhibitory effect, LPA1-related disease improvement or As a health functional food composition for prevention,
Wherein the LPA1-related disease is selected from the group consisting of stroke, psoriasis, Sjogren's syndrome, inflammatory bowel disease, diabetic nephropathy and fibrosis, a health functional food composition for improving or preventing LPA1-related diseases. delete

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