Novel acyl phenylalanine derivative, preparation method thereof and application of novel acyl phenylalanine derivative as medicine

Abstract

The invention relates to an effective amount of novel acyl phenylalanine derivative as shown in a general formula (I), a preparation method thereof and application of a pharmaceutical composition containing the derivative as a medicine, and compared with the prior art, the novel acyl phenylalanine derivative has a wider application prospect. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a preparation method and application of a novel acylphenylalanine derivative, belonging to the field of pharmaceutical chemistry. The structure of the related acylphenylalanine derivative in the present invention is unique and novel in the field. Background Art

[0002] The prevalence of obesity continues to rise worldwide. Rapid modernization, urbanization and accelerated socioeconomic growth have helped improve living standards, but in the past two decades, stressful, sedentary lifestyles and unhealthy eating habits have made obesity a global epidemic that affects almost every organ system and threatens people's lives. Currently, obesity has become one of the most common non-communicable diseases and a serious global public health problem.

[0003] The main cause of obesity is a chronic imbalance between calorie intake and expenditure. Subcutaneous fat is the main depot for storing energy. It is associated with improved metabolism and insulin sensitivity because it includes brown adipocytes containing a large number of mitochondria that perform thermogenic functions. However, when energy intake is excessive, when the storage capacity of subcutaneous fat is exhausted, fat accumulates around abdominal visceral organs, such as the liver, forming non-alcoholic fatty liver disease (NAFLD), in the vascular system causing atherosclerotic cardiovascular disease (ASCVD) or in muscles causing insulin resistance.

[0004] Given these complex connections in the pathogenesis of obesity, the current treatment options for managing and treating obesity and type 2 diabetes share some similarities, including lifestyle interventions, drug therapy, various newly developed medical devices, and bariatric surgery, which are becoming increasingly popular and technologically advanced. Lifestyle intervention is the foundation and first-line treatment for obesity and type 2 diabetes, and effective management of obesity is of paramount importance. Surgical procedures, whether endoscopic or non-endoscopic, have been recognized in patients who are suitable for different methods, but the inevitable risks and complications of surgery still remind us that these technologies are far from perfect. Therefore, for obese people who cannot be managed through lifestyle interventions, drug control is a relatively low-risk option, and anti-obesity drugs have anti-diabetic effects to a certain extent. At present, the most popular direction of weight loss drug research is peptide drugs, such as glucagon-like 1 peptide (GLP-1), which is a hot topic in the field of weight loss research of major pharmaceutical companies. It can not only slow down gastric emptying, increase satiety, and reduce appetite, but also promote insulin secretion in a glucose-dependent manner. However, since it is a peptide drug, it can only be used by injection to exert its efficacy, and there is currently a lack of safe and effective oral weight loss drugs.

[0005] In recent years, studies have shown that the dipeptide molecule N-lactic acid-phenylalanine (Lac-Phe) synthesized from lactic acid and phenylalanine can inhibit eating and reduce obesity. N-lactic acid-phenylalanine is a new metabolite, and there are few studies on it. It has been reported that exercise can increase Lac-Phe levels, and the in vitro cytoplasmic enzyme CNDP2 can catalyze Lac-Phe synthesis through the condensation of lactic acid and phenylalanine. Studies have found that injection of Lac-Phe in obese mice fed with a high-fat diet can significantly inhibit the eating of mice, reduce obesity and improve glucose homeostasis (Nature, 2022, 606, 785-790). However, the above studies are limited to injection, and oral administration is ineffective. Therefore, the development of oral Lac-Phe derivatives is very promising and is expected to change the current situation of lack of safe and effective oral weight loss drugs on the market. In addition, in-depth research on other medicinal uses of such derivatives is expected to expand their indications and lay the foundation for the development of safer and more effective drugs or health products. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide for the first time a method for preparing a novel acylphenylalanine derivative, which provides possibilities for the treatment and prevention of obesity, hyperlipidemia, non-alcoholic fatty liver disease, drug-induced liver injury, diabetes and other related diseases.

[0007] The novel acylphenylalanine derivative of the present invention contains an effective amount of a compound represented by the general formula (I):

[0008]

[0009] Wherein, R1 is selected from the following structures:

[0010] Wherein, R is selected from hydrogen, alkyl, alkoxy, halogen, cycloalkyl, trifluoromethyl;

[0011] R2 is selected from the following structures:

[0012]

[0013] Preferably, the compound of the present invention has the general formula (I):

[0014] Wherein, R1 is selected from the following structures:

[0015] Wherein, R is optionally selected from hydrogen, C1-C6 alkyl, alkoxy, chlorine, fluorine, C3-C6 cycloalkyl, trifluoromethyl;

[0016] R2 is selected from the following structures:

[0017]

[0018] More preferred compounds of the present invention include, but are not limited to:

[0019] ((S)-2-Hydroxybutyryl)-L-phenylalanine (4),

[0020] ((S)-2-Hydroxy-3-phenylpropionyl)-L-phenylalanine (7),

[0021] ((S)-2-Hydroxypropionyl)-D-phenylalanine (8),

[0022] (S)-3-(4-Fluorophenyl)-2-((S)-2-hydroxy-3-methylbutyramide)propanoic acid (9),

[0023] (S)-3-(4-(Benzyloxy)phenyl)2-propionamidopropionic acid (13),

[0024] (S)-3-(4-(Benzyloxy)phenyl)-2-((S)-2-hydroxypropionamido)propanoic acid (14),

[0025] (R)-3-(4-(Benzyloxy)phenyl)-2-((R)-2-hydroxy-3-methylbutyramido)propanoic acid (15),

[0026] (R)-3-(4-(Benzyloxy)phenyl)-2-propionamidopropionic acid (16),

[0027] (R)-3-(4-(Benzyloxy)phenyl)-2-((S)-2-hydroxypropionamido)propanoic acid (17),

[0028] (R)-3-(4-(Benzyloxy)phenyl)-2-(cyclopropanecarboxamido)propanoic acid (18),

[0029] (R)-3-(4-((4-fluorobenzyl)oxy)phenyl)-2-((S)-2-hydroxypropionamido)propanoic acid (19),

[0030] (R)-3-(4-(cyclopropylmethoxy)phenyl)-2-((S)-2-hydroxypropionamido)propanoic acid (20),

[0031] (R)-2-((S)-2-Hydroxypropionamido)-3-(4-methoxyphenyl)propanoic acid (21).

[0032] Another aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective dose of the compound and a suitable carrier, diluent or excipient.

[0033] The present invention also relates to the use of the compound or its pharmaceutical composition in the preparation of a drug for preventing and / or treating at least one of obesity, hyperlipidemia, non-alcoholic fatty liver, alcoholism, alcoholic steatohepatitis, drug-induced liver injury, dyslipidemia, organ fibrosis, cirrhosis, liver failure, diabetes, diabetic complications, hyperuricemia, gout, altitude sickness, cerebral hypoxia, cerebral ischemia, and myocardial infarction.

[0034] Detailed description of the invention

[0035] Unless otherwise stated, the following terms used in the specification and claims have the following meanings.

[0036] Any general formula or structure shown herein, including compounds of general formula (I), is also intended to represent unlabeled and isotopically labeled forms of the compounds. Examples of isotopes that may be included in the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, and in the compounds of the present invention, any atom that is not explicitly indicated as a specific isotope is intended to represent any stable isotope of the atom. Unless otherwise stated, when a position is explicitly indicated with "H" or "hydrogen", it is understood that the position is hydrogen of its isotopic composition. Therefore, in the compounds of the present invention, any atom explicitly indicated as deuterium (D) is intended to represent deuterium.

[0037] "Pharmaceutical composition" means a mixture containing one or more compounds of the present invention and other chemical components, such as pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to promote the absorption of the active ingredients by the organism and facilitate the biological activity in the organism. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 :Compound 18 improves the histological characteristics of DIO mice: (A) Mouse body weight; (B) Mouse liver mass, (C) Mouse white fat mass (D) HE-stained liver section, scale bar: 50 μm. *P<0.05, **P<0.01, ***P<0.001, compared with the model group.

[0039] Figure 2 Effects of compound 18 on lipid metabolism in DIO mice: (A) liver TC level; (B) liver TG level, (C) serum TG level. One-way ANOVA and Tukey's multiple-comparison post hoc test were used for analysis (n=6), *P<0.05, **P<0.01, ***P<0.001, compared with the model group.

[0040] Figure 3:Compound 18 improves the histological characteristics of NASH mice: (A) NAS score; (B) HE-stained liver sections, scale bar: 100μn. One-way ANOVA and Tukey's multiple-comparison post hoc test were used for analysis (n=6), *P<0.05, **P<0.01, ***P<0.001, compared with the model group.

[0041] Figure 4 Effects of compound 18 on lipid metabolism and liver toxicity in NASH mice: (A) liver TC level; (B) liver TG level, (C) serum TG level; (D) serum ALT level; (E) serum AST level. One-way ANOVA and Tukey's multiple-comparison post hoc test were used for analysis (n=6), *P<0.05, **P<0.01, ***P<0.001, compared with the model group.

[0042] Figure 5 Effects of Compound 1, Compound 18, and Compound 19 on liver toxicity in mice with APAP-induced liver injury: (A) serum AST level; (B) serum ALT level, (C) HE-stained liver sections. One-way ANOVA and Tukey's multiple-comparison post ho ctest were used for statistical analysis (n=6), *P<0.05, **P<0.01, ***P<0.001, compared with the model group. DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with the examples. It should be noted that the following examples are only for illustration, and are not intended to limit the present invention. Various changes made by those skilled in the art based on the teachings of the present invention should be within the scope of protection of the claims of this application.

[0044] Synthesis and preparation of the compounds of the present invention:

[0045] Synthetic route 1:

[0046]

[0047] Reagents and conditions: (i) ia: HBTU, DIPEA, DCM, rt, 10 hours, 60%; ib: pyridine, DCM, 0°C, 3h, 80%; (ii) LiOH·H2O, HF / MeOH / H2O, rt, 4 hours, 98%.

[0048] (ia): To a solution of the selected carboxylic acid in dichloromethane (10 mL) were added HBTU (1.2 equiv), DIPEA (3 equiv) and the desired amine (1.2 equiv. After stirring at room temperature for 12 h, the reaction mixture was concentrated, washed sequentially with saturated sodium bicarbonate solution and 1N HCl, and extracted with ethyl acetate. It was then purified by silica gel column chromatography using petroleum / ethyl acetate (3:1; v / v) to give the product.

[0049] (ib): A solution of the acid chloride (1 equiv) in dichloromethane (5 mL) was slowly added to a stirred solution of phenylalanine methyl ester (1.2 equiv) in dichloromethane (5 mL) and pyridine (0.5 mL) at 0°C. After stirring for another 3 hours at room temperature, 4 ml of water was added to quench the reaction. After the organic layer was concentrated under vacuum, the mixture was diluted with water. The aqueous layer was adjusted to pH 4-5 with 1N HCl and extracted with ethyl acetate. The organic layers were combined, washed with brine, dried over anhydrous sodium sulfate, concentrated in vacuo, and then purified by silica gel column chromatography using petroleum / ethyl acetate (3:1; v / v) to give the intermediate product.

[0050] (ii): The obtained intermediate was dissolved in 3:3:1 THF / MeOH / H2O (7 mL) solution, and LiOH·H2O (3 equiv) was added. After stirring at room temperature for 4 hours, the solution was acidified with hydrochloric acid solution, filtered, and the filter cake was washed with cold water to obtain the compound.

[0051] Synthesis route 2:

[0052]

[0053] Reagents and conditions: (i) K2CO3, MeCN, reflux, 3 hours, yield 87-89%; (ii) TFA, DCM, 0°C, 3h, yield 88%; (iii) ① HBTU, DIPEA, DCM, rt, 12 hours, 60%; ② LiOH·H2O, THF / MeOH / H2O, room temperature, 4 hours, 98%. (i): Potassium carbonate (3.00 equiv) and ethyl 8-bromooctanoate (1.10 equiv) were added to MeCN (10 mL) of butoxycarbonyl-D-tyrosine-methoxy ester (1.00 equiv), and the mixture was stirred at 70°C for 3 hours. After the reaction was completed, it was cooled to room temperature, filtered under reduced pressure, and the filtrate was spin-dried and then purified by silica gel column chromatography using petroleum / ethyl acetate (5:1; v / v) to obtain the intermediate product.

[0054] (ii): TFA (3 equiv) was slowly added dropwise to a solution of the selected amide in dichloromethane (10 mL) at 0°C. After stirring at room temperature for 4 hours, the reaction mixture was concentrated and extracted with saturated sodium bicarbonate solution and ethyl acetate (20 mL x 3). The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo to obtain the product.

[0055] (iii): To a solution of the selected carboxylic acid in dichloromethane (10 mL) were added HBTU (1.2 equiv), DIPEA (3 equiv) and the desired amine (1.2 equiv). After stirring at room temperature for 16 hours, the reaction mixture was concentrated, washed with saturated sodium bicarbonate solution and 1N HCl in sequence, and extracted with ethyl acetate. It was then purified by silica gel column chromatography using petroleum / ethyl acetate (5:1; v / v) to obtain the intermediate. The obtained intermediate was dissolved in a 3:3:1 THF / MeOH / H2O (7 mL) solution, and LiOH·H2O (3 equiv) was added. After stirring at room temperature for 3 hours, the solution was acidified with hydrochloric acid solution, filtered, and the filter cake was washed with cold water to obtain the compound.

[0056] Example 1

[0057] ((S)-2-Hydroxypropionyl)-L-phenylalanine (1)

[0058]

[0059] (S)-2-amino-3-phenylpropionic acid methyl ester hydrochloride (1.0 equiv), sodium propionate (1.2 equiv), HBTU (1.2 equiv) and DIPEA (3 equiv) were dissolved in dichloromethane and stirred at room temperature for 10 h. After the reaction, the reaction mixture was concentrated, washed with saturated sodium bicarbonate solution and 1N HCl in sequence, and extracted with ethyl acetate. Then, the target compound was purified by silica gel column chromatography using petroleum / ethyl acetate (3:1; v / v). Yield: 51%, white powder. 1 H NMR (400MHz, DMSO-d6) δ7.61 (d, J=8.2Hz, 1H), 7.30-7.24 (m, 2H), 7.23-7.19 (m, 1H), 7.19-7.15 (m, 2H), 4.51 (tdJ=8.0 , 5.2Hz, 1H), 3.93 (q, J=6.7Hz, 1H), 3.10 (dd, J=13.7, 5.1Hz, 1H), 3.01 (dd, J=13.7, 8.0Hz, 1H), 1.10 (d, J=6.8Hz, 3H). 13C NMR (101MHz, DMSO-d6) δ174.17, 172.74, 137.20, 129.28, 128.17, 126.52, 67.11, 52.36, 36.62, 21.02.ESI-MS m / z: [MH] - calcd.for C 12 H 14 NO4 - , 236.0928; Found, 236.0920.

[0060] Acetyl-L-phenylalanine (2)

[0061]

[0062] The synthesis method is the same as compound 1. Yield: 88%, white powder. 1 H NMR (400MHz, DMSO-d6) δ12.64 (s, 1H), 8.18 (d, J=8.1Hz, 1H), 7.31-7.25 (m, 2H), 7.25-7.17 (m, 3H), 4 .40 (td, J=9.1, 5.0Hz, 1H), 3.04 (dd, J=13.8, 4.9Hz, 1H), 2.83 (dd, J=13.8, 9.6Hz, 1H), 1.78 (s, 3H). 13 CNMR (101MHz, DMSO-d6) δ173.20, 169.26, 137.75, 129.07, 128.20, 126.42, 53.52, 36.79, 22.35.ESI-MS m / z: [MH] - calcd.for C 11 H 12 NO3 - , 206.0823; Found, 206.0821.

[0063] Propionyl-L-phenylalanine (3)

[0064]

[0065] The synthesis method is the same as compound 1. Yield: 62%, white powder. 11H NMR (400MHz, DMSO-d6) δ12.63 (s, 1H), 8.08 (d, J = 8.1Hz, 1H), 7.30-7.24 (m, 2H), 7.24-7.16 (m, 3H), 4.41 (td, J = 9.1, 4.9Hz, 1H), 3.05 (dd, J=13.8, 4.8Hz, 1H), 2.84 (dd, J=13.7, 9.7Hz, 1H), 2.05 (q, J=7.5Hz, 2H), 0.90 (t, J=7.6Hz, 3H). 13 C NMR (101MHz, DMSO-d6) δ173.25, 172.93, 137.77, 129.10, 128.14, 126.38, 53.33, 36.80, 28.22, 9.79.ESI-MS m / z: [MH] - calcd.forC 12 H 14 NO3 - , 220.0979; Found, 220.0971.

[0066] ((S)-2-Hydroxybutyryl)-L-phenylalanine (4)

[0067]

[0068] The synthesis method is the same as compound 1. Yield: 58%, white powder. 1 H NMR (400MHz, DMSO-d6) δ12.8.2 (s, 1H), 7.63 (d, J=8.2Hz, 1H), 7.30-7.23 (m, 2H), 7.22-7.14 (m, 3H), 5.51 (s, 1H), 4.54 (td, J=8.2, 5.1Hz, 1H), 3.78 (s, 1H), 3.09 (dd, J=13.8, 4.9Hz, 1H), 3.00 (dd, J=13.8, 8.2Hz, 1H), 1.59-1.49 (m, 1H), 1.43-1.33 (m, 1H), 0.72 (t, J=7.4Hz, 3H). 13 C NMR (101MHz, DMSO-d6) δ173.44, 172.79, 137.26, 129.20, 128.16, 126.48, 71.80, 52.39, 36.70, 27.31, 9.14.ESI-MS m / z: [MH] - calcd.for C 13 H 16 NO4 -, 250.1085; Found, 250.1081.

[0069] ((S)-2-Hydroxy-3-methylbutyryl)-L-phenylalanine (5)

[0070]

[0071] The synthesis method is the same as compound 1. Yield: 55%, white powder. 1 H NMR (400MHz, DMSO-d6) δ12.78 (s, 1H), 7.65 (d, J=8.3Hz, 1H), 7.28-7.23 (m, 2H), 7.22-7.15 (m, 3H), 4.56 (td, J=8.4, 5.1Hz, 1H), 3.65 (d, J =3.7Hz, 1H), 3.08 (dd, J=13.9, 5.0Hz, 1H), 3.00 (dd, J=13.9, 8.6Hz, 1H), 1.94-1.80 (m, 1H), 0.79 (d, J=6.9Hz, 3H), 0.57 (d, J=6.8Hz, 3H). 13 C NMR (101MHz, DMSO-d6) δ173.08, 172.84, 137.34, 129.15, 128.19, 126.46, 75.07, 52.43, 36.80, 31.23, 19.12, 15.94.ESI-MS m / z: [MH] - calcd.for C 14 H 18 NO4 - , 264.1241; Found, 264.1238.

[0072] ((S)-2-Hydroxy-2-phenylacetyl)-L-phenylalanine (6)

[0073]

[0074] The synthesis method is the same as compound 1. Yield: 55%, white powder. 1H NMR (400MHz, DMSO-d6) δ12.87 (s, 1H), 8.06 (d, J=8.1Hz, 1H), 7.38-7.30 (m, 2H), 7.30-7.21 (m, 5H), 7.20-7.13 (m, 2H), 7.13-7.07 (m, 1H), 6.22-6.08 (m, 1H), 4.93 (s, 1H), 4.47 (td, J=8.4, 4.2Hz, 1H), 3.09 (dd, J=13.8, 4.3Hz, 1H), 3.01 (dd, J=13.6, 8.4Hz, 1H). 13 C NMR (101MHz, DMSO-d6) δ172.66, 171.83, 141.15, 137.31, 129.19, 128.21, 127.85, 127.34, 126.80, 126.47, 73.05, 52.90, 36.50.ESI-MS m / z: [MH] - calcd.for C 17 H 16 NO4 - , 298.1085; Found, 298.1081.

[0075] ((S)-2-Hydroxy-3-phenylpropionyl)-L-phenylalanine (7)

[0076]

[0077] The synthesis method is the same as compound 1. Yield: 61%, white powder. 1 H NMR (400MHz, DMSO-d6) δ12.92 (s, 1H), 7.66 (d, J=8.2Hz, 1H), 7.30-7.22 (m, 4H), 7.22-7.17 (m, 4H), 7.06 (d, J=6.8Hz, 2H), 5.72 (d, J=5.3H z, 1H), 4.55 (td, J=8.2, 3.3Hz, 1H), 4.12-4.03 (m, 1H), 2.99 (d, J=6.3Hz, 2H), 2.88 (dd, J=13.8, 3.4Hz, 1H), 2.61 (dd, J=13.8, 8.3Hz, 1H). 13 C NMR (101MHz, DMSO-d6) δ173.34, 173.05, 138.84, 137.46, 130.00, 129.70, 128.62, 128.39, 126.96, 126.51, 72.39, 52.81, 40.79, 37.23.ESI-MS m / z:[MH] -calcd.for C 18 H 18 NO4 - , 312.1241; Found, 312.1237.

[0078] ((S)-2-Hydroxypropionyl)-D-phenylalanine (8)

[0079]

[0080] The synthesis method is the same as compound 1. Yield: 56%, white powder. 1 H NMR (400MHz, DMSO-d6) δ12.75 (s, 1H), 7.68 (d, J=8.2Hz, 1H), 7.30-7.23 (m, 2H), 7.23-7.15 (m, 3H), 5.49 (s, 1H), 4.47 (td, J =8.2, 5.0Hz, 1H), 3.94 (q, J=6.7Hz, 1H), 3.08 (dd, J=13.8, 5.0Hz, 1H), 3.00 (dd, J=13.8, 8.3Hz, 1H), 1.13 (d, J=6.8Hz, 3H). 13 C NMR (101MHz, DMSO-d6) δ174.25, 172.73, 137.35, 129.19, 128.21, 126.50, 66.94, 52.66, 36.58, 20.96.ESI-MS m / z: [MH] - calcd.for C 12 H 14 NO4 - , 236.0928; Found, 236.0924.

[0081] (S)-3-(4-Fluorophenyl)-2-((S)-2-hydroxy-3-methylbutyramide)propanoic acid (9)

[0082]

[0083] The synthesis method is the same as compound 1. Yield: 55%, white powder. 1H NMR (400MHz, DMSO-d6) δ12.86 (s, 1H), 7.70 (d, J=8.4Hz, 1H), 7.29-7.20 (m, 2H), 7.07 (t, J=8.8Hz, 2H), 5.45 (s, 1H), 4.54 (td, J=8.7, 5.0Hz, 1H), 3 .64 (d, J=3.4Hz, 1H), 3.07 (dd, J=13.8, 4.8Hz, 1H), 2.99 (dd, J=14.0, 8.9Hz, 1H), 1.94-1.78 (m, 1H), 0.78 (d, J=6.9Hz, 3H), 0.54 (d, J=6.8Hz, 3H). 13 C NMR (101MHz, DMSO-d6)) δ173.10, 172.79, 161.08 (d, J=243.41Hz), 133.61 (d, J=3.03Hz), 130 .98 (d, J=8.08Hz), 114.85 (d, J=21.21Hz), 75.10, 52.44, 35.86, 31.23, 19.06, 15.91.ESI-MS m / z:[MH] - calcd.for C 14 H 17 FNO4 - , 282.1147; Found, 282.1143.

[0084] Propionyl-D-phenylalanine (10)

[0085]

[0086] The synthesis method is the same as compound 1. Yield: 59%, white powder. 1 H NMR (400MHz, DMSO-d6) δ12.66 (s, 1H), 8.09 (d, J=8.1Hz, 1H), 7.31-7.24 (m, 2H), 7.24-7.15 (m, 3H), 4.41 (td, J=9.4, 4.9Hz, 1H), 3.04 (dd, J=13.7, 4.8Hz, 1H), 2.84 (dd, J=13.7, 9.8Hz, 1H), 2.05 (q, J=7.4Hz, 2H), 0.90 (t, J=7.6Hz, 3H). 13 C NMR (101MHz, DMSO-d6) δ173.25, 172.93, 137.78, 129.10, 128.14, 126.38, 53.34, 36.81, 28.22, 9.79.ESI-MS m / z: [MH] -calcd.forC 12 H 14 NO3 - , 220.0979; Found, 220.0972.

[0087] ((S)-2-Hydroxy-3-methylbutyryl)-D-phenylalanine (11)

[0088]

[0089] The synthesis method is the same as compound 1. Yield: 58%, white powder. 1 H NMR (400MHz, DMSO-d6) δ12.78 (s, 1H), 7.73 (d, J=8.2Hz, 1H), 7.30-7.24 (m, 2H), 7.23-7.15 (m, 3H), 5.33 (s, 1H), 4.50 (td, J=8.4, 4.9Hz, 1H ), 3.65 (s, 1H), 3.09 (dd, J=13.8, 4.9Hz, 1H), 2.99 (dd, J=13.9, 8.6Hz, 1H), 1.95-1.86 (m, 1H), 0.83 (d, J=6.9Hz, 3H), 0.67 (d, J=6.8Hz, 3H). 13 C NMR (101MHz, DMSO-d6) δ173.27, 172.82, 137.43, 129.15, 128.24, 126.48, 74.97, 52.69, 36.63, 31.35, 19.21, 15.87.ESI-MS m / z: [MH]-calcd.for C 14 H 18 NO4 - , 264.1241; Found, 264.1236.

[0090] (Cyclopropanecarbonyl)-D-phenylalanine (12)

[0091]

[0092] The synthesis method is the same as compound 1. Yield: 67%, white powder. 1H NMR (400MHz, DMSO-d6) δ12.64 (s, 1H), 8.40 (d, J=8.0Hz, 1H), 7.32-7.25 (m, 2H), 7.25-7.16 (m, 3H), 4.42 (dt, J=9 .2, 5.0Hz, 1H), 3.04 (dd, J=13.8, 5.0Hz, 1H), 2.86 (dd, J=13.8, 9.5Hz, 1H), 1.66-1.54 (m, 1H), 0.69-0.49 (m, 4H). 13 C NMR (101MHz, DMSO-d6) δ173.26, 172.74, 137.73, 129.10, 128.21, 126.43, 53.64, 36.91, 13.35, 6.46, 6.35.ESI-MS m / z: [MH] - calcd.forC 13 H 14 NO3 - , 232.0979; Found, 232.0971.

[0093] (S)-3-(4-(Benzyloxy)phenyl)-2-propionamidopropionic acid (13)

[0094]

[0095] The synthesis method is the same as compound 1. Yield: 54%, white powder. 1H NMR (400MHz, DMSO-d6) δ12.63 (s, 1H), 8.05 (d, J = 8.1 Hz, 1H), 7.43 (d, J = 7.1 Hz, 2H), 7.38 (t, J = 7.3 Hz, 2H), 7.32 (t, J = 7.1 Hz, 1H), 7.14 (d, J = 8.5 Hz, 2H), 6.91 (d, J=8.6Hz, 2H), 5.05 (s, 2H), 4.36 (td, J=9.2, 5.0Hz, 1H), 2.97 (dd, J=13.8, 4.8Hz , 1H), 2.77 (dd, J=13.8, 9.6Hz, 1H), 2.06 (q, J=7.5Hz, 2H), 0.91 (t, J=7.6Hz, 3H). 13 C NMR (101MHz, DMSO-d6) δ173.33, 172.92, 157.00, 137.21, 130.15, 129.86, 128.43, 127.80, 127.69, 114.45, 69.12, 53.59, 35.98, 28.22, 9.82.ESI-MS m / z:[MH] -calcd.for C 19 H 20 NO4 - , 326.1398; Found, 326.1394.

[0096] (S)-3-(4-(Benzyloxy)phenyl)-2-((S)-2-hydroxypropionamido)propanoic acid (14)

[0097]

[0098] The synthesis method is the same as compound 1. Yield: 50%, white powder. 1 H NMR (400MHz, DMSO-d6) δ12.88 (s, 1H), 7.58 (d, J=8.1Hz, 1H), 7.47-7.41 (m, 2H), 7. 41-7.35 (m, 2H), 7.35-7.29 (m, 1H), 7.09 (d, J = 8.6Hz, 2H), 6.91 (d, J = 8.7Hz, 2H), 5 .72-5.51 (m, 1H), 5.05 (s, 2H), 4.45 (td, J=7.8, 5.2Hz, 1H), 3.94 (q, J=6.7Hz, 1H), 3.03 (dd, J=13.8, 5.1Hz, 1H), 2.94 (dd, J=13.8, 7.7Hz, 1H), 1.12 (d, J=6.8Hz, 3H). 13 C NMR (101MHz, DMSO-d6) δ174.12, 172.77, 157.11, 137.18, 130.29, 129.21, 1 28.42, 127.80, 127.69, 114.47, 69.14, 67.11, 52.55, 35.78, 21.04.ESI-MS m / z:[MH] - calcd.for C 19 H 20 NO5 - , 342.1347; Found, 342.1343.

[0099]

[0100] (R)-3-(4-(Benzyloxy)phenyl)-2-((R)-2-hydroxy-3-methylbutyramido)propanoic acid (15) Synthesis method is the same as compound 1. Yield: 58%, white powder. 1H NMR (400MHz, DMSO-d6) δ12.76 (s, 1H), 7.61 (d, J=8.3Hz, 1H), 7.43 (d, J=7.0Hz, 2H), 7.38 (t, J=7 .3Hz, 2H), 7.31 (t, J=7.1Hz, 1H), 7.11 (d, J=8.6Hz, 2H), 6.90 (d, J=8.6Hz, 2H), 5.45 (d, J=5.6Hz, 1H), 5.05 (s, 2H), 4.51 (td, J=8.1, 5.3Hz, 1H), 3.66 (t, J=4.3Hz, 1H), 3.00 (dd, J=14.1, 5.3Hz, 1H ), 2.94 (dd, J=14.0, 8.2Hz, 1H), 1.93-1.83 (m, 1H), 0.81 (d, J=6.9Hz, 3H), 0.60 (d, J=6.8Hz, 3H). 13 C NMR (101MHz, DMSO-d6) δ173.48, 173.33, 157.55, 137.66, 130.63, 129.80, 128.87, 128.22, 128.08, 114.97, 75.52, 69.59, 53.03, 36.48, 31.70, 19.58, 16.42.ESI-MS m / z:[MH] - calcd.for C 21 H 24 NO5 - , 370.1660; Found, 370.1655.

[0101] (R)-3-(4-(Benzyloxy)phenyl)-2-propionamidopropionic acid (16)

[0102]

[0103] Potassium carbonate (3.00 equiv) and ethyl 8-bromooctanoate (1.10 equiv) were added to butoxycarbonyl-D-tyrosine-methoxy ester (1.00 equiv) in MeCN (10 mL), and the mixture was stirred at 70 ° C for 3 hours. After the reaction was completed, it was cooled to room temperature, filtered under reduced pressure, and the filtrate was spin-dried and then purified by silica gel column chromatography using petroleum / ethyl acetate (5:1; v / v) to obtain intermediate 16a. TFA (3 equiv) was slowly added dropwise to a solution of 16a in dichloromethane (10 mL) at 0 ° C. After stirring at room temperature for 4 hours, the reaction mixture was concentrated and extracted with saturated sodium bicarbonate solution and ethyl acetate (20 mL×3). The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo to obtain intermediate 16b. HBTU (1.2 equiv), DIPEA (3 equiv) and propionic acid (1.2 equiv) were added to a solution of 16b in dichloromethane (10 mL). After stirring at room temperature for 16 hours, the reaction mixture was concentrated, washed with saturated sodium bicarbonate solution and 1N HCl, and extracted with ethyl acetate. Then, the product 16 was purified by silica gel column chromatography using petroleum / ethyl acetate (10:7; v / v). Yield: 59%, white powder. 1H NMR (400 MHz, DMSO-d6) δ 12.62 (s, 1H), 8.05 (d, J = 8.1 Hz, 1H), 7.43 (d, J = 7.0 Hz, 2H), 7.38 (t, J = 7.3 Hz, 2H), 7.32 (t, J = 7.1 Hz, 1H), 7.14 (d, J = 8.6 Hz, 2H), 6.91 (d, J=8.6Hz, 2H), 5.05 (s, 2H), 4.36 (td, J=9.2, 4.9Hz, 1H), 2.97 (dd, J=13.8, 4.9Hz , 1H), 2.78 (dd, J=13.8, 9.6Hz, 1H), 2.06 (q, J=7.6Hz, 2H), 0.91 (t, J=7.6Hz, 3H). 13 C NMR (101MHz, DMSO-d6) δ173.33, 172.92, 157.00, 137.21, 130.15, 129.87, 128.43, 127.80, 127.69, 114.45, 69.12, 53.60, 35.99, 28.22, 9.82.ESI-MS m / z:[MH] - calcd.for C 19 H 20 NO4 - , 326.1398; Found, 326.1391.

[0104] (R)-3-(4-(Benzyloxy)phenyl)-2-((S)-2-hydroxypropionamido)propanoic acid (17)

[0105]

[0106] The synthesis method is the same as compound 16. Yield: 56%, white powder. 1 H NMR (400MHz, DMSO-d6) δ12.88 (s, 1H), 7.58 (d, J=8.1Hz, 1H), 7.47-7.41 (m, 2H), 7. 41-7.35 (m, 2H), 7.35-7.29 (m, 1H), 7.09 (d, J = 8.6Hz, 2H), 6.91 (d, J = 8.7Hz, 2H), 5 .72-5.51 (m, 1H), 5.05 (s, 2H), 4.45 (td, J=7.8, 5.2Hz, 1H), 3.94 (q, J=6.7Hz, 1H), 3.03 (dd, J=13.8, 5.1Hz, 1H), 2.94 (dd, J=13.8, 7.7Hz, 1H), 1.12 (d, J=6.8Hz, 3H). 13 C NMR (101MHz, DMSO-d6) δ174.23, 172.79, 157.11, 137.19, 130.25, 129.37, 1 28.82, 127.82, 127.73, 114.51, 69.15, 66.97, 52.85, 35.78, 20.97.ESI-MS m / z:[MH] - calcd.for C 19 H 20 NO5 - , 342.1347; Found, 342.1345.

[0107] (R)-3-(4-(Benzyloxy)phenyl)-2-(cyclopropanecarboxamido)propanoic acid (18)

[0108]

[0109] The synthesis method is the same as compound 16. Yield: 61%, white powder. 1H NMR (400MHz, DMSO-d6) δ12.62 (s, 1H), 8.36 (d, J = 6.7Hz, 1H), 7.53-7.41 (m, 2H), 7.41-7.35 (m, 2H), 7.35-7.27 (m, 1H), 7.15 (d, J = 6.9Hz, 2 H), 6.92 (d, J=6.8Hz, 2H), 5.06 (s, 2H), 4.51-4.24 (m, 1H), 2.97 (d, J=13.0Hz, 1H), 2.82 (d, J=9.6Hz, 1H), 1.74-1.51 (m, 1H), 0.61 (s, 4H). 13 C NMR (101MHz, DMSO-d6) δ173.33, 172.73, 157.04, 137.22, 130.14, 129.81, 128.44, 127.81, 127.71, 114.49, 69.14, 53.90, 36.07, 13.35, 6.49.ESI-MS m / z:[MH] - calcd.forC 20 H 20 NO4 - , 338.1398; Found, 338.1393.

[0110] (R)-3-(4-((4-Fluorobenzyl)oxy)phenyl)-2-((S)-2-hydroxypropionamido)propanoic acid (19)

[0111]

[0112] The synthesis method is the same as compound 16. Yield: 53%, white powder. 1 H NMR (400MHz, DMSO-d6). δ12.78 (s, 1H), 7.64 (d, J = 8.2Hz, 1H), 7.50 (d, J = 5.7Hz, 1H) , 7.47 (d, J=5.6Hz, 1H), 7.21 (t, J=8.9Hz, 2H), 7.11 (d, J=8.6Hz, 2H), 6.91 (d, J=8.6 Hz, 2H), 5.50 (s, 1H), 5.03 (s, 2H), 4.43 (td, J=8.0, 5.1Hz, 1H), 3.95 (d, J=5.5Hz, 1H ), 3.01 (dd, J=13.8, 5.1Hz, 1H), 2.94 (dd, J=13.9, 8.0Hz, 1H), 1.15 (d, J=6.8Hz, 3H). 13C NMR (101MHz, DMSO-d6) δ174.66, 173.23, 162.21 (d, J=244.42Hz), 157.44, 133.84 (d, J=3.03Hz), 130.69, 130.41 (d, J=9.09Hz), 129.89, 115.68 (d, J=21.21Hz), 114.96, 68.87, 67.41, 53.28, 36.22, 21.41.ESI-MS m / z:[MH] - calcd.for C 19 H 19 FNO5 - , 360.1253; Found, 360.1250.

[0113] (R)-3-(4-(Cyclopropylmethoxy)phenyl)-2-((S)-2-hydroxypropionamido)propanoic acid (20)

[0114]

[0115] The preparation method is the same as compound 16. Yield: 56%, white powder. 1 H NMR (400MHz, DMSO-d6) δ12.78 (s, 1H), 7.62 (d, J = 8.1Hz, 1H), 7.07 (d, J = 8.5Hz, 2H) , 6.81 (d, J = 8.5Hz, 2H), 5.50 (s, 1H), 4.42 (dt, J = 7.9, 5.1Hz, 1H), 3.94 (d, J = 6.5Hz, 1H), 3.75 (d, J=7.0Hz, 2H), 3.00 (dd, J=13.8, 5.2Hz, 1H), 2.92 (dd, J=13.8, 7.9Hz, 1 H), 1.21-1.16 (m, 1H), 1.15 (d, J=6.7Hz, 3H), 0.61-0.47 (m, 2H), 0.35-0.25 (m, 2H). 13 C NMR (101MHz, DMSO-d6) δ174.20, 172.80, 157.37, 130.19, 128.91, 114.17, 71.89, 66.97, 52.86, 35.78, 20.97, 10.22, 3.11.ESI-MS m / z: [MH] - calcd.for C 16 H 20 NO5 - , 306.1347; Found, 306.1340.

[0116] (R)-2-((S)-2-Hydroxypropionamido)-3-(4-methoxyphenyl)propanoic acid (21)

[0117]

[0118] The preparation method is the same as compound 16. Yield: 58%, white powder. 1 H NMR (400MHz, DMSO-d6) δ12.78 (s, 1H), 7.63 (d, J = 7.6Hz, 1H), 7.10 (d, J = 7.7Hz, 2H), 6.83 (d, J = 7.6Hz, 2H), 5.50 (s, 1H), 4.53-4.33 (m, 1H), 4.00-3.90 (m, 1H), 3.71 (s, 3H), 2.99 (d, J=7.7Hz, 1H), 2.95 (d, J=7.7Hz, 1H), 1.15 (d, J=6.4Hz, 3H). 13 C NMR (101MHz, DMSO-d6) δ174.22, 172.80, 157.98, 130.23, 129.09, 113.65, 66.97, 54.97, 52.88, 35.77, 20.97.ESI-MS m / z: [MH] - calcd.forC 13 H 16 NO5 - ,266.1034;Found,266.1030.0

[0119] Example 2 In vivo appetite suppressant activity study

[0120] Five-week-old male C57BL / 6 mice weighing 16-20 g were purchased from the Guangdong Medical Laboratory Animal Center (license number: SCXK(Y)2022-0002) and housed in an SPF barrier environment. During the entire experiment, the temperature and relative humidity of the animal room were controlled at 23±2°C, 50±10%, and the light / dark cycle was constant at 12 hours. All mice were adapted for one week before the start of the animal experiment. Unless otherwise stated, mice were housed under specific pathogen-free conditions with free access to feed and water. The Ethics Committee of Guangdong Pharmaceutical University approved all animal-related procedures and was carried out in accordance with the Regulations on Laboratory Animal Management of China and the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health of the United States (NIH Publication No. 85-23, revised in 2011).

[0121] Mice were randomly divided into a control group and a test compound administration group, with 6 mice in each group. Both the control group and the test compound administration group were fed with a high-fat diet (Dyets, D12492, containing 60% fat) and normal drinking water. After the adaptation to feeding, the mice were intraperitoneally injected daily, and the control group was given water for injection and the test compound group (50 mg / kg) for 7 consecutive days, and the cumulative food intake inhibition rate was calculated. The results of the in vivo appetite suppression activity are shown in Table 1. The results show that the compounds of the present invention have good appetite suppression activity, indicating that the compounds of the present invention have weight loss potential.

[0122] Table 1: In vivo appetite suppressant activity

[0123]

[0124] Example 3 Study on the anti-obesity activity of oral administration in vivo

[0125] C57BL / 6 mice were randomly divided into normal group, model group, compound 1 (150 mg / kg) group, compound 18 (50 mg / kg) group, compound 18 (150 mg / kg) group, and compound 18 (300 mg / kg) group, with 6 mice in each group. The normal group was given normal basal feed, and the other groups were fed with high-fat feed (Dyets, D12492, containing 60% fat) and normal drinking water. Starting from the 17th week, daily gavage was performed, and the normal group and the model group were given 0.5% sodium carboxymethylcellulose (CMC-Na), compound 1 (150 mg / kg), compound 18 (50 mg / kg), compound 18 (150 mg / kg), and compound 18 (300 mg / kg) for 4 weeks. Before sampling, the mice were fasted for 12 hours (free access to water), and anesthetized by intraperitoneal injection of 2% sodium pentobarbital (50 mg / kg). Blood was then collected through the fundus venous plexus. The mice were killed by cervical dislocation and fixed on a mouse stand. The skin, subcutaneous tissue, and peritoneum were cut along the midline of the abdomen to expose the abdominal cavity. The liver tissue was removed, washed with saline, and the liver tissue of the left lobe of appropriate size was cut and fixed in tissue fixative. (1×1×0.5cm was taken from the right lobe of the liver 5mm away from the edge. 3 The liver tissues were fixed in 4% paraformaldehyde for paraffin embedding and HE staining. The remaining tissues were placed in cryopreservation tubes sealed with liquid nitrogen and stored in a -80℃ refrigerator for later use. Figure 1 and 2 .

[0126] The experimental results showed that compound 1 and compound 18 could significantly reduce the body weight, liver weight and white fat weight of diet-induced obese (DIO) mice. HE pathological sections showed that the model induced liver fatty degeneration and lobular inflammation. Compared with the model group, compound 1 had fewer lipid vacuoles and no inflammatory cell infiltration. After treatment with compound 18, lipid vacuoles were significantly reduced, the hepatocyte morphology was normal, there was no inflammatory cell infiltration, and the therapeutic effect was better than that of the control compound 1 (see Appendix). Figure 1 ).

[0127] In addition, compared with the normal group, the levels of total triglyceride (TG), total cholesterol (TC) and serum TG in the HFD-induced DIO mouse model were significantly increased, while compound 1 could significantly reduce the total TC in the liver. Compound 18 significantly reduced the levels of total TG, total TC and serum TG in the liver after treatment, and the effect of compound 18 was stronger than that of compound 1. This indicates that oral administration of compound 18 has a good improvement effect on obesity, and its oral efficacy is better than that of the control compound 1 (see Appendix Figure 2 ).

[0128] Example 4 In vivo study of the anti-nonalcoholic steatohepatitis (NASH) activity of oral administration

[0129] C57BL / 6 mice were randomly divided into normal group, model group, obeticholic acid (OCA) administration group, compound 18 (50 mg / kg) group, compound 18 (150 mg / kg) group, and compound 18 (300 mg / kg) group, with 6 mice in each group. The normal group was given normal basal feed and drinking water, and the other groups were fed with Western diet feed (Dyets, D18061501, containing 21.1% fat, 41% fructose, and 1.25% cholesterol) and prepared with sugar water (23.1 g / L fructose: 18.9 g / L glucose). At the same time, except for the normal group, the other groups were intraperitoneally injected with 0.3 μL / g CCl4 oil solution once a week, and the model was continuously established for 12 weeks to induce NASH model. Starting from the 9th week, the mice were given oral administration every day. The normal group and the model group were given 0.5% sodium carboxymethylcellulose (CMC-Na), OCA group (20 mg / kg), compound 18 (50 mg / kg), compound 18 (150 mg / kg), and compound 18 (300 mg / kg) for 4 weeks. Half an hour after the last administration, 0.3 μL / g of CCl4 oil solution was injected. The mice were fasted for 12 hours before sampling (free drinking water), and 2% sodium pentobarbital (50 mg / kg) was given for intraperitoneal anesthesia. Then, blood was collected through the fundus venous plexus. The mice were killed by cervical dislocation and fixed on the mouse stand. The skin, subcutaneous tissue, and peritoneum were cut along the midline of the abdomen in turn to expose the abdominal cavity. The liver tissue was taken out, washed with physiological saline, and the liver tissue of the left lobe of appropriate size was uniformly cut and soaked in tissue fixative for fixation. (1×1×0.5cm was taken from the right lobe of the liver 5mm away from the edge)3 The liver tissues were fixed in 4% paraformaldehyde for paraffin embedding and HE staining. The remaining tissues were placed in cryopreservation tubes sealed with liquid nitrogen and stored in a -80℃ refrigerator for later use. Figure 3 and 4 .

[0130] The experimental results showed that the model induced typical histological features of NASH, including fatty degeneration, lobular inflammation and hepatocyte ballooning. Compared with the model group, the OCA treatment group had significantly fewer lipid vacuoles and no dense inflammatory cell infiltration. After treatment with compound 18, inflammation was significantly alleviated, the hepatocyte morphology was normal, there was no ballooning, the cells were arranged more neatly, and only some slight lipid vacuoles were observed. The NAS score results showed that the therapeutic effect of compound 18 was comparable to that of OCA (see Appendix). Figure 3 ).

[0131] In addition, compared with the normal group, the levels of total triglyceride (TG), total cholesterol (TC) and serum TG in the liver of the NASH mouse model were significantly increased, while these indicators were significantly reduced after treatment with OCA and compound 18, and the effects of the two were comparable. Since the interference of the fibrosis promoter CCl4 caused a certain degree of damage to the mouse liver, the levels of serum AST and ALT in the model group were significantly increased compared with the normal group. The AST and ALT in the drug-treated group were significantly reduced, indicating that OCA and compound 18 have a good protective effect on liver function and can be used to prevent and treat liver fibrosis (see Appendix Figure 4 ), indicating that the oral anti-NASH efficacy of compound 18 is comparable to that of the marketed drug OCA, and has broad application prospects.

[0132] Example 5 In vivo oral administration anti-diabetic activity study

[0133] C57BL / 6 mice were given normal basal feed and drinking water, and diabetes analysis was performed after 1 week of adaptive feeding. STZ was injected intraperitoneally on an empty stomach at a dose of 50 mg / kg. Blood glucose levels were measured after three consecutive injections. Mice with fasting blood glucose greater than 11.1 mmol / L and random blood glucose greater than 16.7 mmol / L were randomly divided into a normal group, a model group, an empagliflozin group, and a compound 18 (150 mg / kg) group, with 6 mice in each group. After grouping, 0.5% sodium carboxymethylcellulose (CMC-Na), empagliflozin (20 mg / kg), and compound 18 (150 mg / kg) were administered orally for 2 weeks. The results of the blood glucose improvement effect are shown in Table 2. The results show that compound 18 of the present invention has a good blood glucose improvement effect, and its efficacy is even better than that of the marketed drug empagliflozin.

[0134] Table 2: Blood glucose levels in mice

[0135]

[0136] Example 6 Study on the ability to resist hypoxia in normal pressure hypoxia environment

[0137] Five-week-old male Kunming mice weighing 18-22 g were randomly divided into a model group, a test compound 1 group, a test compound 8 group, and a test compound 14 group, with 6 mice in each group. The model group was given 0.5% sodium carboxymethylcellulose (CMC-Na) by gavage, and the other test compounds were given compound 1 (150 mg / kg), compound 8 (150 mg / kg) and compound 14 (150 mg / kg) by gavage, respectively. After three consecutive days of administration, the mice were placed in a 125 ml wide-mouth bottle, sealed with a rubber stopper, and the asphyxiation time of the animals was closely observed and recorded. The results are shown in Table 3. After the experiment was completed, the mice were anesthetized and killed, and the brain tissue of the mice was immediately dissected and placed in a cryopreservation tube, sealed with liquid nitrogen, and placed in a -80°C refrigerator. The content of MDA, SOD, and GSH-Px in the brain tissue was subsequently detected using a kit. The experimental results are shown in Table 4.

[0138] Table 3: Duration of animal suffocation

[0139]

[0140]

[0141] Table 4: Contents of MDA, SOD, and GSH-Px in brain tissue

[0142]

[0143] The experimental results showed that compound 14 could significantly increase the asphyxiation time of mice in a closed hypoxic environment, compound 1, compound 8, and compound 14 could significantly increase the content of SOD and GSH-Px, and compound 1 and compound 14 could significantly reduce the content of MDA in brain tissue. This indicates that compound 14 can enhance the hypoxia tolerance of mice in a normal pressure hypoxia environment, indicating its potential for use in cerebral ischemia, myocardial infarction, altitude sickness, etc.

[0144] Example 7 Study on the Ability to Tolerate Hypoxia in a Low-Pressure Hypoxic Environment (Simulating a Plateau Low-Pressure, Hypoxic Environment)

[0145] Kunming mice were randomly divided into model group, test compound 1 group, test compound 10 group, and test compound 14 group, with 6 mice in each group. The model group was given 0.5% sodium carboxymethyl cellulose (CMC-Na) by gavage, and the other test compounds were given compound 1 (150 mg / kg), compound 10 (150 mg / kg) and compound 14 (150 mg / kg) by gavage, respectively. After 3 consecutive days of administration, the mice were placed in a 125ml wide-mouth bottle containing about 5g of soda lime, sealed with a rubber stopper, and the asphyxiation time of the animals was closely observed and recorded. The results are shown in Table 5. After the experiment was completed, the mice were anesthetized and killed, and the mouse brain tissue was immediately dissected and placed in a cryopreservation tube, sealed with liquid nitrogen, and placed in a -80°C refrigerator. The content of MDA, SOD, and GSH-Px in the brain tissue was subsequently detected using a kit. The experimental results are shown in Table 6.

[0146] Table 5: Duration of animal suffocation

[0147]

[0148] Table 6: Contents of MDA, SOD, and GSH-Px in brain tissue

[0149]

[0150] The experimental results showed that compound 14 could significantly increase the survival time of mice in a closed low-pressure hypoxic environment, and compound 1 and compound 14 could significantly increase the content of SOD and GSH-Px and significantly reduce the content of MDA in brain tissue. This indicates that compound 1 and compound 14 can enhance the hypoxia tolerance of mice in a low-pressure hypoxic environment and have the potential to prevent and treat altitude sickness.

[0151] Example 8 In vivo anti-hyperuricemia activity study

[0152] Kunming mice were randomly divided into model group, test compound 1 group, test compound 8 group, test compound 18 group, test compound 19 group, 6 mice in each group. The animals were given 0.5% sodium carboxymethylcellulose (CMC-Na), compound 1 (150 mg / kg), compound 8 (150 mg / kg), compound 18 (150 mg / kg) and compound 19 (150 mg / kg) by gavage at -1h, hypoxanthine 200 mg / kg was injected intraperitoneally at 0min, potassium oxonate 120 mg / kg was injected subcutaneously to establish the model, and blood uric acid levels were measured at the tip of the tail at 1h, 2h, and 4h after modeling. The results are shown in Table 7.

[0153] Table 7: Blood uric acid levels in mice

[0154]

[0155] The experimental results show that compound 1, compound 8 and compound 19 have good anti-hyperuricemia activity.

[0156] Example 9 Study on the activity against acute alcohol intoxication in vivo

[0157] Kunming mice were randomly divided into model group, test compound 1 group, test compound 8 group, test compound 18 group, test compound 19 group, 10 mice in each group. The animals were given 0.5% sodium carboxymethylcellulose (CMC-Na), compound 1 (150 mg / kg), compound 8 (150 mg / kg), compound 18 (150 mg / kg) and compound 9 (150 mg / kg) by gavage at -1h, and 0.025ml / g of 52% liquor was given to each mouse at 0min. The disappearance of the "righting reflex" of mice was used as the judgment index of drunkenness, and the number of drunken and dead mice was recorded, and the drunkenness rate and mortality rate were calculated. The results are shown in Table 8.

[0158] Table 8: Intoxication rate and mortality rate of mice

[0159]

[0160] The experimental results show that compound 1, compound 18 and compound 19 have good anti-acute alcohol intoxication activity.

[0161] Example 10 In vivo study of the activity against acetaminophen-induced liver injury

[0162] Kunming mice were randomly divided into normal group, model group, test compound 1 group, test compound 18 group, and test compound 19 group, with 6 mice in each group. The normal group and model group were given 0.5% sodium carboxymethyl cellulose (CMC-Na) by gavage, and the other test compound groups were given compound 1 (150 mg / kg), compound 18 (150 mg / kg), and compound 19 (150 mg / kg) by gavage for 3 days. On the third day, they were fasted for 12 hours and had normal drinking water. After fasting, except for the normal group, each mouse was intraperitoneally injected with 300 mg / kg APAP solution. After 10 hours, 2% sodium pentobarbital (50 mg / kg) was given for intraperitoneal anesthesia. Then blood was collected by orbital bleeding. The mice were killed by cervical dislocation and fixed on a mouse stand. The skin, subcutaneous tissue, and peritoneum were cut along the midline of the abdomen to expose the abdominal cavity. The liver tissue was removed and washed with saline. The liver tissue of the left lobe of appropriate size was uniformly cut and soaked in tissue fixative for fixation. (1×1×0.5cm was taken from the right lobe of the liver 5mm away from the edge. 3 The liver tissues were fixed in 4% paraformaldehyde for paraffin embedding and HE staining. The remaining tissues were placed in cryopreservation tubes sealed with liquid nitrogen and stored in a -80℃ refrigerator for later use. Figure 5 .

[0163] The experimental results showed that: HE pathological sections showed that the model induced liver damage in mice. After treatment with compound 1, compound 18, and compound 19, liver damage was significantly improved, and compared with the model group, the serum AST and ALT of mice were significantly decreased, indicating that compound 1, compound 18, and compound 19 had a good liver protection effect (Appendix Figure 5 ).

Claims

1. A compound represented by the general formula (I) containing an effective amount: in, R1 is selected from the following structures: Wherein, R is selected from hydrogen, alkyl, alkoxy, halogen, cycloalkyl, trifluoromethyl; R2 is selected from the following structures:

2. A compound of the general formula (I) as defined in claim 1: in, R1 is selected from the following structures: Wherein, R is optionally selected from hydrogen, C1-C6 alkyl, alkoxy, chlorine, fluorine, C3-C6 cycloalkyl, trifluoromethyl; R2 is selected from the following structures:

3. The compound of general formula (I) as defined in any one of claims 1 to 2 is selected from: ((S)-2-Hydroxybutyryl)-L-phenylalanine; ((S)-2-hydroxy-3-phenylpropionyl)-L-phenylalanine; ((S)-2-Hydroxypropionyl)-D-phenylalanine; (S)-3-(4-Fluorophenyl)-2-((S)-2-hydroxy-3-methylbutanamide)propanoic acid; (S)-3-(4-(Benzyloxy)phenyl)-2-propionamidopropionic acid; (S)-3-(4-(Benzyloxy)phenyl)-2-((S)-2-hydroxypropionamido)propanoic acid; (R)-3-(4-(Benzyloxy)phenyl)-2-((R)-2-hydroxy-3-methylbutyramido)propanoic acid; (R)-3-(4-(Benzyloxy)phenyl)-2-propionamidopropionic acid; (R)-3-(4-(Benzyloxy)phenyl)-2-((S)-2-hydroxypropionamido)propanoic acid; (R)-3-(4-(Benzyloxy)phenyl)-2-(cyclopropanecarboxamido)propanoic acid; (R)-3-(4-((4-fluorobenzyl)oxy)phenyl)-2-((S)-2-hydroxypropionamido)propanoic acid; (R)-3-(4-(cyclopropylmethoxy)phenyl)-2-((S)-2-hydroxypropionamido)propanoic acid; (R)-2-((S)-2-Hydroxypropionamido)-3-(4-methoxyphenyl)propanoic acid.

4. A pharmaceutical composition comprising the compound according to any one of claims 1 to 3 and a suitable carrier or excipient.

5. Use of the compound defined in any one of claims 1 to 3, or the pharmaceutical composition according to claim 4, in the preparation of a medicament for preventing and / or treating at least one of altitude sickness, cerebral hypoxia, cerebral ischemia and myocardial infarction.

6. Use of the compound defined in any one of claims 1 to 3, or the pharmaceutical composition according to claim 4, in the preparation of a medicament for preventing and / or treating at least one of obesity, hyperlipidemia, non-alcoholic fatty liver disease, alcoholism, alcoholic steatohepatitis, drug-induced liver injury, dyslipidemia, organ fibrosis, cirrhosis, liver failure, diabetes, diabetic complications, hyperuricemia, and gout.

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