KR20200023236A - 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 containing the same as an active component. A compound represented by chemical formula 1, a stereoisomer thereof, a racemate thereof, or pharmaceutically acceptable salt thereof according to the present invention can have an effect of inhibiting the activation of a lysophosphatidic acid 1 (LPA1) receptor, thereby being able to be usefully used as the pharmaceutical composition and a health functional food composition for preventing or treating LPA1-related diseases. In addition, the pharmaceutical composition and the health functional food composition can prevent or treat not only autoimmune diseases, but also other immune-related diseases with similar mechanisms of occurrence.

Description

Benzoic acid derivatives and compositions for preventing or treating autoimmune diseases containing the same as active ingredients {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 abnormal immune responses by misidentifying the substance of the body as an antigen. Psoriasis, Sjogren's syndrome, inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, optic neuromyelitis, Guillain-Barré syndrome, Graves disease, Behcet's disease Etc.

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

Recently, focusing on efficient immunosuppression, researches on developing autoimmune diseases have been undertaken at the cellular level (inhibition of activation of cell types responsible for immune responses in lymphocytes and diseased tissues) and immunosuppression at the molecular level.

Lysophospholipids, represented by lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), have long been regarded as phospholipid metabolites of cell membrane components, but are now G protein-linked receptors. Lipophospholipid receptor activation, which is classified as a coupled receptor (GPCR), has been reported to be an important extracellular signal that modulates a variety of biological functions.

Among them, LPA1 is the first lysophospholipid receptor identified (1996) and is a key factor in regulating nervous system development. Recent studies have revealed the importance in various diseases. Immune response control is drawing attention. Signaling mechanisms of LPA1 and organic systems with tissues in vivo revealed through various studies have shown that the neuropsychiatric system, as well as autoimmune diseases such as Sjogren's syndrome, stroke, psoriasis, inflammatory bowel disease, diabetic nephropathy and fibrosis and systemic sclerosis It has become an important data for the treatment of various disorders, including disorders, neurological pain, infertility, cardiovascular disease, and cancer. (Refer to 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, Augu st 2010.)

Current Steroids and Cyclosporine, Rapamycin, Methotrexate, Cyclophosphamide, IV Immunoglobulin, Axathioprine, Infliximab, etc. Although it is treated with a combination therapy of various immunosuppressive drugs, most of them are persistent inflammation and frequent recurrence, and many systemic side effects are caused by the treatment.

In particular, while rare autoimmune diseases have a small number of patients, the cost of the therapeutic agents to be applied is very high, and the economic burden on patients is very high, and the cost burden is increasing due to the need for long-term treatment. Although the number of patients with rare autoimmune diseases is increasing in Korea, it is still economic and social problem because there is no clear treatment and low cure rate.

Korea Patent Registration No. 10-1217066 (2012.12.24. Registration)

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.

According to the present invention, the compound represented by the following Chemical 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, trilomethyl, benzyloxy, (C1 ~ C4) cyclo Hexyl, 2H-1,2,3-triazol-2-yl and (C1-C4) benzoyl can be any one selected from the group consisting of.

A pharmaceutical composition for preventing or treating 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 an inhibitory effect of LPA1 (Lysophosphatidic acid 1) receptor activation. It can have

 Health functional food composition for improving or preventing LPA1-related diseases according to the present invention contains the compound, its stereoisomer, racemate or pharmaceutically acceptable salt thereof as an active ingredient, LPA1 (Lysophosphatidic acid 1) receptor activation It 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 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.

LPA1-related diseases in accordance with the present invention can be effectively prevented and treated autoimmune diseases using a pharmaceutical composition and health functional food composition for preventing or treating. In addition, the autoimmune disease, as well as other immune-related diseases similar to the developmental mechanism can be applied in various ways.

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

Hereinafter, the present invention will be described in detail.

The present invention provides an in vivo disease model for an autoimmune disease such as sjogren's disease, psoriasis, multiple sclerosis, and abnormal immune response diseases such as diabetic nephropathy and cerebral ischemia. Previous studies using) validated LPA1 as a novel target molecule for the treatment of autoimmune diseases and completed the present invention by finding compounds with LPA1 antagonism.

The present invention provides a compound represented by 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, trilomethyl, benzyloxy, (C1 ~ C4) cyclo Hexyl, 2H-1,2,3-triazol-2-yl and (C1-C4) benzoyl can be any one selected from the group consisting of.

The compound may have the same or different R ¹ and R ² , respectively, hydrogen, (C 1 -C 2) alkyl, halogen, trifluoromethyl, benzyloxy, (C 3 -C 4) 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 -(4-methylbenzoyl) benzoyl) -4,5,6,7-tetrahydropyrazolo [1,5-a] pyrazin-2-yl) propanoic acid.

The present invention, the stereoisomers thereof, racemates thereof or pharmaceutically acceptable salts thereof as an active ingredient, characterized in that 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 further comprising a suitable carrier, excipient or diluent pharmaceutically acceptable according to the above formulation. The term "pharmaceutically acceptable" means that the cell or the human body exposed to the pharmaceutical composition exhibits characteristics that are not toxic.

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

The pharmaceutical compositions according to the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. have.

When the pharmaceutical composition according to the present invention is formulated in the above form, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid form preparations contain at least one excipient such as starch, calcium carbonate, sucrose in the compound. ) Or lactose, gelatin and the like can be mixed.

In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, 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 the disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level is determined by the patient's health condition and ulcer. Type, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and rate of release, duration of treatment, factors including the drug being used in combination or concurrently, and other factors well known in the medical arts. .

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

The pharmaceutical composition may be administered to various mammals such as mice, mice, livestock, humans, and the like. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrabronchial inhalation, intrauterine dural or intracere-broventricular injection.

The present invention contains the compound, its stereoisomers, racemates thereof, or pharmaceutically acceptable salts thereof as an active ingredient, and has an inhibitory effect on LPA1 (Lysophosphatidic acid 1) receptor activation. Or it provides a preventive dietary supplement composition.

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 dietary supplement according to the present invention may be provided in the form of powder, granules, tablets, capsules, syrups or beverages, and the dietary supplement is used with other foods or food additives in addition to the compound according to the present invention as an active ingredient, It can be suitably used according to a conventional method. The mixed amount of the active ingredient can be suitably determined according to the purpose of use thereof, for example, prophylactic, health or therapeutic treatment.

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

There is no particular limitation on the type of health functional food according to the present invention, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products, including ice cream, various soups, Beverages, teas, drinks, alcoholic beverages and vitamin complexes.

The compounds according to the invention can be used in stereoisomeric forms of the compounds. In the present invention, "stereoisomers" refer to isomers caused by different spatial arrangements of atoms or groups of atoms in a molecule, and include both optical and geometric isomers. Optical isomers are paired enantiomers of four atoms or groups of atoms bonded to an asymmetric carbon atom, and two asymmetric carbon atoms in the molecule become diastereomers. All isomers may be included. In addition, when unsaturated hydrocarbons are present, they become geometric isomers, and may also include all geometric isomers that may occur.

The compounds according to the invention can be used in the racemic form of the compounds. In the present invention, the "racemate" refers to a substance in which the right-handed optical isomer and the left-handed optical isomer of the chiral molecule are mixed in the same amount and thus do not exhibit optical activity.

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 either a pharmaceutically acceptable basic salt or an acid salt.

The basic salt may be used in the form of organic salt or inorganic salt, and may be sodium salt, potassium salt, calcium salt, lithium salt, magnesium salt, cesium salt, aluminum salt, ammonium salt, or triethyl. It may be selected from the group consisting of aluminum salts and pyridinium salts, but is not limited thereto.

Acid salts are useful acid addition salts formed by free acid. The inorganic acid and organic acid may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, etc. , Benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, glyconic 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 compounds according to the present invention may include not only pharmaceutically acceptable salts, but also all salts, hydrates, and solvates that can be prepared by conventional methods.

The addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound represented by Formula 1 to Formula 3 in a water miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile. It can be prepared by adding excess organic base or adding an aqueous base solution of inorganic base followed by precipitation or crystallization. Alternatively, the solvent or excess base may be evaporated and dried in this mixture to obtain an addition salt, or the precipitated salt may be prepared by suction filtration.

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

Synthesis Method

All reagents required for synthesis were purchased from TCI, Sigma Aldrich and Alfa Aesar with a purity of at least 97%. Before the synthesis was carried out, 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 It was purchased from gold and anhydrous solvent from Sigma Aldrich. In the TLC, the progress of the reaction was confirmed by using a UV lamp and a coloring reagent 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 are expressed in ppm, and coupling constants (J) are expressed in Hz.

<Example 1>

Synthesis of Mother Nuclear Tetrahydropyrazolopyrazine

Scheme 1

Scheme 1 is a process for synthesizing tetrahydropyrazolopyrazine, which is a core mother core according to the present invention, in the same manner as in Scheme 1, using propargylamine as a starting material according to the following method as tetrahydropyrazol Synthesis of pyrazine (Tetrahydropyrazolopyrazine) was performed

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

At 0 ° C., propargylamine (Propargylamine, 0.5ml, 7.81mmol) and triethylamine (Triethylamine; TEA 1.3ml, 9.37mmol) were added to dichloromethane (7.5ml) to produce a mixture. Para-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 stirring was complete, 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. Then purified by column chromatography to give compound 1 (1.517g, 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-in-1-yl) benzenesulfonamide [N- (2-chloroethyl) -4-methyl- N- (prop-2-yn-1-yl) benzenesulfonamide]

Compound 1 (1.517 g, 7.25 mmol) and potassium carbonate (Potassium Carbonate, 1.2 g, 8.7 mmol) were added to acetone (Acetone, 30 ml) to form a mixture. Thereafter, 1-bromo-2-chloroethane (1-bromo-2-chloroethane, 1.2 ml, 14.5 mmol) was added dropwise to the mixture, followed by reflux 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 purified by column chromatography to obtain compound 2 (1.29g, 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]

Toluene (Toluene, 26.3ml) was added Compound 2 (6.05g, 22.26mmol) and diazo acetate (Diazoacetate, 23.7ml, 27.83mmol) and stirred for 12 hours at 140 ℃. After stirring, cesium carbonate (Cesium Carbonate, 10.88 g, 33.4 mmol) and tetrahydrofuran (10 ml) were added to the mixture, and the reaction was continued for 30 minutes. After the reaction was completed, the mixture was cooled to room temperature and neutralized by adding 1 normal hydrochloric acid. The product was extracted through ethyl acetate and water was removed through magnesium sulfate. The filtrate was concentrated under reduced pressure, and then purified by column chromatography to obtain compound 3 (4.215g, 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. Thereafter, the mixture was stirred for 1 hour at room temperature. After completion of the reaction, 1 normal hydrochloric acid was added to neutralize and the product was extracted through dichloromethane. Magnesium sulfate was added to the extracted product to remove water, and then 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]

Dichloromethane (10 ml) was added dimethyl sulfoxide (Dimethyl sulfoxide, 4.43 ml, 61.8 mmol) to form a mixture. Thereafter, oxalyl chloride (Oxalyl chloride, 2.651 ml, 30.9 mmol) dissolved in dichloromethane (15 ml) was added dropwise to the mixture at -60 ° C. After 5 minutes, compound 4 (1.9 g, 6.18 mmol) was added to the mixture and the reaction continued for 1 hour. After completion of the reaction, the temperature of the mixture was brought to room temperature and the product was extracted through dichloromethane. Magnesium sulfate was added to the extracted product to remove water, and 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 (1.3 g, 3.46 mmol) and 10% Pd / C (0.1 g) were added to methanol (Methanol, 20 ml) to form a mixture, and the mixture was stirred at room temperature under hydrogen stream for 2 hours. After the reaction was completed, the mixture was filtered through celite. The filtrate was concentrated under reduced pressure and purified by column chromatography to give 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]

To methanol (6 ml) compound 6 (388 mg, 1.03 mmol) and magnesium powder (247 mg, 10.3 mmol) were added to form a mixture. The mixture is then allowed to react for 1 hour at 0 ° C. After the reaction is completed, 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%), a tetrahydropyrazolopyrazine, which is the core 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, 2 H), 2.67 (m, 2 H); 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 Mother Nucleus and Benzoic Acid Derivatives

Scheme 2

Reaction Scheme 2 is a process for synthesizing seven kinds of tetrahydropyrazolopyrazine and benzoic acid derivatives, which are the core mothers according to the present invention, and in the same manner as in Scheme 2, tetrahydropyrazolopyrazine of compound 7 and various substituted benzoic acids 7 Compounds 8a to 8g were synthesized by amide coupling reaction, and methyl ester was hydrolyzed to synthesize 7 species of compounds 9a to 9g.

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

To a mixture of dichloromethane (0.5 ml) was added compound 7 (50 mg, 0.224 mmol), acid (45 mg, 0.269 mmol), and triethylamine (0.13 ml, 0.896 mmol). Hydroxybenzotriazol (37 mg, 0.269 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, 52 mg , 0.269 mmol) was added and stirred at room temperature for 12 hours. After the reaction was completed, the product was extracted through dichloromethane. Magnesium sulfate was added to the extracted product to remove water, and the filtrate was concentrated under reduced pressure and separated and purified through 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 (Lithium hydroxide, 55mg, 1.3mmol) dissolved in water (1ml) was added and stirred at room temperature for 12 hours. After completion of the reaction, 1 normal hydrochloric acid was added to neutralize and the product was extracted through dichloromethane. Magnesium sulfate was added to the extracted product to remove water, and then the filtrate was concentrated under reduced pressure and purified by column chromatography to obtain Compound 9a (32 mg, 90%). In the same manner, compounds 9b to 9g were obtained from compounds 8b to 8g.

[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

LPA1 overexpressing cell lines (RH7777-human EDG1 cells) were seeded at 1 × 10 ⁵ / well in 96 well black plates (clear bottom) and incubated for one day. Thereafter, the cells were replaced with a serum-free culture solution containing 0.1% fatty acid-free BSA (fatty acid-free BSA), followed by further culture.

Next, pretreatment with a drug (compound 9a-9g or AM152 (LPA1 antagonist), 1 μM) for 30 minutes, followed by treatment with Fluo-3AM (5 μM), a fluorescent dye for calcium measurement, was incubated for 60 minutes. In addition, the fluorescence intensity was measured (absorption: 506 nm) 1 minute after treatment with 10 μM LPA. (Fluorescence intensity: 3 layers victor used)

1 is a graph showing the results of calcium analysis according to an experimental example of the present invention. The Ca ²⁺ response in the graph represents the fold increase relative to the vehicle.

Referring to Figure 1, compounds 9a ~ 9g was confirmed to exhibit an efficacy of 20 to 50% compared to the AM152 (1μM) efficacy.

Experimental Example 2 Toxicity Evaluation

Compounds 9a, 9c, 9d, and 9g were suspended in male Balb / c mice in 0.5% methylcellulose solution, respectively, and administered once orally at a dose of 0.5g / kg, 1g / kg and 2g / kg. Survival and body weight of the mice were examined daily.

After the administration, the mortality, clinical symptoms, and weight changes of the animals were observed. Hematological and hematological examinations were performed. The necropsy was performed to observe abdominal and thoracic organ abnormalities.

As a result, no significant clinical symptoms or dead animals were noted 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 do not show a toxic change in the mouse up to 2g / kg, therefore, oral administration medium lethal dose (LD50) was determined to be a safe substance of more than 2g / kg.

Hereinafter, the preparation examples of the composition comprising the compound 9a according to the present invention will be described, but the present invention is not intended to be limited thereto but merely to describe in detail.

<Prescription Example 1> Prescription example of pharmaceutical composition

<Prescription Example 1-1> Preparation of Powder

A powder was prepared by mixing 20 mg of compound 9a, 100 mg of lactose and 10 mg of talc and filling into an airtight bag.

<Prescription Example 1-2> Preparation of Tablet

Tablets were prepared by mixing 20 mg of compound 9a, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate, followed by compression according to a conventional method for preparing tablets.

<Prescription Example 1-3> Preparation of Capsule

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

<Prescription Example 1-4> Preparation of Injection

10 mg of a compound 9a, a sterile distilled water injection amount and a pH adjusting agent amount were mixed, and then prepared in the above-described ingredient content per ampoule (2 ml) according to a conventional injection method.

<Prescription Example 1-5> Preparation of Ointment

Compound 9a 10 mg, PEG-4000 250 mg, PEG-400 650 mg, white petrolatum 10 mg, paraoxybenzoic acid methyl 1.44 mg, 0.18 mg of paraoxybenzoic acid propyl and the remaining amount of purified water were prepared according to the conventional method for preparing an ointment. It was.

<Prescription 2> Health functional food

<Prescription Example 2-1> Preparation of Health Food

Compound 9a 1 mg, vitamin mixture (70 mg of vitamin A acetate, 1.0 mg of vitamin E, 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 mineral mixture (1.75 mg ferrous sulfate, 0.82 mg zinc oxide, 25.3 mg magnesium carbonate, 15 mg potassium monophosphate, calcium diphosphate dibasic) 55 mg, potassium citrate 90 mg, calcium carbonate 100 mg, magnesium chloride 24.8 mg) were mixed, granules were prepared, and health food was prepared according to a conventional method.

Prescription Example 2-2 Preparation of Health Beverage

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 were added to make it total 900 ml.The above ingredients were mixed according to a conventional healthy beverage preparation method, and then, about 1 After stirring and heating at 85 ° C. for an hour, the resulting solution was collected by filtration into a sterilized 2 L container, sealed, sterilized, and refrigerated.

Having described the specific part of the present invention in detail, it is apparent to those of ordinary skill in the art that such a specific description is merely a preferred embodiment, thereby not limiting the scope of the present invention. Do. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (7)

A compound represented by Formula 1, a stereoisomer thereof, a racemate thereof or a pharmaceutically acceptable salt thereof:
[Formula 1]

Wherein R ¹ and R ² may each be the same or different, hydrogen, (C 1 -C 4) alkyl, (C 1 -C 4) alkoxy, halogen, trifluoromethyl, benzyloxy, (C 1 -C 4) cyclohexyl, 2H-1,2,3-triazol-2-yl and (C1-C4) benzoyl. The method according to claim 1,
The compound may have the same or different R ¹ and R ² , respectively, hydrogen, (C 1 -C 2) alkyl, halogen, trifluoromethyl, benzyloxy, (C 3 -C 4) cyclohexyl, 2H-1,2,3 A compound, a stereoisomer thereof, a racemate thereof or a pharmaceutically acceptable salt thereof, which is selected from the group consisting of -triazol-2-yl and (C1-C2) benzoyl. The method according to claim 1,
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-propylcyclo) Hexyl) 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- Triazol-2-yl) benzoyl) -4,5,6,7-tetrahydropyrazolo [1,5-a] pyrazin-2-yl) propanoic acid and 3- (5- (2- (4-methyl Benzoyl) benzoyl) -4,5,6,7-tetrahydropyrazolo [1,5-a] pyrazin-2-yl) propanoic acid, compound, stereoisomer thereof, la Semi Or a pharmaceutically acceptable salt thereof. The compound according to any one of claims 1 to 3, 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. , Pharmaceutical composition for the prevention or treatment of LPA1-related diseases. The method according to claim 4,
The LPA1-related disease is selected from the group consisting of stroke, psoriasis, Sjogren's syndrome, inflammatory bowel disease, diabetic nephropathy and fibrosis, pharmaceutical composition for preventing or treating LPA1-related diseases. The compound according to any one of claims 1 to 3, 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. , LPA1-related disease improvement or preventive dietary supplement composition. The method according to claim 6,
The LPA1-related disease is selected from the group consisting of stroke, psoriasis, Sjogren's syndrome, inflammatory bowel disease, diabetic nephropathy and fibrosis, LPA1-related diseases improvement or preventive health functional food composition.

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