Lat1 inhibitors, methods of making, and uses thereof

Abstract

The application discloses a kind of LAT1 inhibitor and its preparation and application;The inhibitor is as shown in general formula I, the inhibitor or its pharmaceutically acceptable salt, isomer, metabolite, solvate, is applied in LAT1-mediated disease, with excellent LAT1 affinity and selectivity, and with good metabolic stability and oral bioavailability, widen the skeleton structure and indication of LAT1 inhibitor, provide more choices for the research and application of LAT1 inhibitor.

Description

TECHNICAL FIELD

[0001] The present application relates to a LAT1 inhibitor, and also relates to the preparation and use of the above-mentioned inhibitor. BACKGROUND

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial hyperplasia and progressive small joint destruction. Multiple cell types, including T cells, B cells, macrophages and fibroblast-like synoviocytes, are involved in the complex pathogenesis of RA. At present, the drugs for treating RA in clinic are mainly large molecule antibodies and small molecule kinase inhibitors. Upadacitinib is the first FDA-approved selective JAK1 small molecule inhibitor, which shows excellent therapeutic effect and better safety in the treatment of rheumatoid arthritis due to its good JAK1 selectivity. However, it still has potential hematologic toxicity risk and has been issued a black box warning by FDA.

[0003] L-type amino acid transporter 1 (LAT1) belongs to the solute carrier protein SLC7A family and mainly mediates the transport of large neutral amino acids including branched-chain amino acids and aromatic amino acids in a sodium ion-independent manner. The expression level of LAT1 in tumor cells and activated T cells and related inflammatory cells is significantly higher than that in normal cells, in order to meet the large intake of amino acids in the rapid proliferation and metabolism process of these cells. LAT1 is closely related to energy metabolism and signal transduction pathways in the production and development of inflammation. Among many inflammatory diseases, RA has a high correlation with LAT1, and LAT1 plays an important regulatory role in the activation, development, migration and invasion of RA through mediating the uptake of amino acids.

[0004] At present, there are few studies on LAT1 inhibitors, and only two have entered clinical research and development, and mainly focus on tumor treatment. And the current LAT1 small molecule inhibitors are mostly limited to phenylalanine substrate structure, and the skeleton structure is relatively single. Among them, JPH203 is the best LAT1 inhibitor reported so far, with an IC50 of 60 nM for leucine uptake inhibition in HT-29 cells, an IC50 of 4.1 μM for cell proliferation inhibition, showing good in vitro activity and good LAT1 selectivity. It is used for the treatment of advanced biliary tract cancer in clinic, and entered clinical phase II trial in 2018 and clinical phase II / III trial in 2022. However, it is prone to phase II metabolism in vivo, and the exposed amino group is acetylated, resulting in a decrease in activity and rapid clearance, so its clinical application is limited. In addition, it also has problems such as poor water solubility, poor membrane permeability, and limited indications. Therefore, it is of great significance to develop it. 50 For 4.1 μM, showing good in vitro activity and good LAT1 selectivity. It is used for the treatment of advanced biliary tract cancer in clinic, and entered clinical phase II trial in 2018 and clinical phase II / III trial in 2022. However, it is prone to phase II metabolism in vivo, and the exposed amino group is acetylated, resulting in a decrease in activity and rapid clearance, so its clinical application is limited. In addition, it also has problems such as poor water solubility, poor membrane permeability, and limited indications. Therefore, it is of great significance to develop it. SUMMARY

[0005] The application aims to provide a LAT1 small molecule inhibitor with good metabolic stability and water solubility, and a preparation method and application of the inhibitor.

[0006] Technical scheme: The LAT1 inhibitor of the application is shown in the general formula I:

[0007]

[0008] wherein, Q 1 , Q 2 is a single bond, R a is hydrogen or methyl, or multiple R a form a ring with the carbon atom connected thereto;

[0009] R 1 is hydrogen, halogen, amino, hydroxyl, cyano, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy or C1-C4 haloalkoxy;

[0010] L 1 is -[C(R b R c )] 0-5 -, -[C(R b R c )] 0-2 -C(O)-[C(R b R c )] 0-2 -, -[C(R b R c )] 0-2 -O-[C(R b R c )] 0-2 -, -[C(R b R c )] 0-2 -C(O)NH-[C(R b R c )] 0-2 -, -[C(R b R c )] 0-2 -S(O)2NH-[C(R b R c )] 0-2 -, -[C(R b R c )] 0-2 -S(O)2-[C(R b R c )] 0-2 -.

[0011] X is a single bond, 6-10 membered aryl, 5-10 membered heteroaryl, 3-10 membered cycloalkyl or C1-C6 branched alkyl;

[0012] R 2 is hydrogen, halogen, amino, hydroxyl, cyano, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy or C1-C4 haloalkoxy, 1-4 hydrogens of which are replaced by R 2a substituted 6-10 membered aryl or 5-10 membered heteroaryl, or R 2b -L 2 ;

[0013] R 2b -L 2 is R 2b -[C(R b R c )] 0-5 -, R 2b -[C(R b R c )] 0-2 -O-[C(R b R c )] 0-2 -, R 2b -[C(R b R c )] 0-2 -C(O)-[C(R b R c )] 0-2 -, R 2b -[C(R b R c )] 0-2 -C(O)NH-[C(R b R c )] 0-2 -, R 2b -[C(R b R c )] 0-2 -NHC(O)-[C(R b R c )] 0-2 -, R 2b -[C(R b R c )] 0-2 -S(O)2NH-[C(R b R c )] 0-2 -, R 2b -[C(R b R c )] 0-2 -NHS(O)2-[C(R b Rc 0-2 - or R 2b - [C(R b R c 0-2 - S(O)2- [C(R b R c 0-2 - ;

[0014] R 2b is 6-10 membered aryl or 5-10 membered heteroaryl, each of which is substituted with 1-4 hydrogen by R 2c ;

[0015] R 2a and R 2c are hydrogen, halogen, amino, hydroxyl, cyano, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, or C1-C4 haloalkoxy;

[0016] R b and R c are hydrogen, halogen, C1-C3 alkyl, or C3-C6 cycloalkyl, or R b , R c form a cyclopropyl ring with the carbon atom to which they are attached;

[0017] m is 0, 1, 2, 3, or 4;

[0018] n is 0, 1, 2, 3, or 4;

[0019] p is 1, 2, or 3;

[0020] z is 0, 1, or 2;

[0021] the heteroatoms in the 5-10 membered heteroaryl are N, O, or S, and the number of heteroatoms is 1, 2, 3, or 4.

[0022] Preferably, the inhibitors have the structure of Formula Ia, Formula Ib, or Formula Ic:

[0023]

[0024] wherein R 1 , R 2 , Q 1 , Q 2 , L 1 , X, and p are as defined in claim 1.

[0025] Preferably, the inhibitors have the structure of Formula Ia, Formula Ib, or Formula Ic:

[0026] Q 1 , Q 2 are ​​​

[0027] R a It can be hydrogen or methyl, or two Rs a Form with the attached carbon atom

[0028] R 1 It can be hydrogen, fluorine, chlorine, amino, hydroxyl, cyano, methyl, isopropyl, tert-butyl, trifluoromethyl, methoxy, trifluoromethoxy, ethoxy, or isopropoxy;

[0029] L 1 It is -CH2O-, -OCH2-, -C(O)NH-, -NHC(O)-, -S(O)2NH- or -NHS(O)2-;

[0030] X is either phenyl or pyridinyl;

[0031] R 2 For 1-4 hydrogens R 2a Substituted phenyl, pyridyl, naphthyl, or R 2b -L 2 ;

[0032] R 2b -L 2 For R 2b -CH2O-、R 2b -OCH2-、R 2b -C(O)NH-, R 2b -NHC(O)-、R 2b -S(O)2NH- or -R 2b -NHS(O)2-;

[0033] R 2b For 1-4 hydrogens R 2c Substituted phenyl;

[0034] R 2a and R 2c It can be hydrogen, fluorine, chlorine, methyl, trifluoromethyl, methoxy, or trifluoromethoxy;

[0035] m can be 0, 1, 2, 3, or 4;

[0036] n is 0, 1, 2, 3 or 4;

[0037] p is 1, 2, or 3;

[0038] z can be 0, 1, or 2.

[0039] Preferably, the structure of the inhibitor includes:

[0040] Q 1 for

[0041] R a is hydrogen or methyl, or the two R a form together with the carbon atom to which they are attached

[0042] Q 2 is a single bond;

[0043] R 1 is hydrogen, fluorine, chlorine, amino, hydroxy, cyano, methyl, isopropyl, tert-butyl, trifluoromethyl, methoxy, trifluoromethoxy, ethoxy or isopropoxy;

[0044] L 1 is -CH2O-, -OCH2-, -C(O)NH-, -NHC(O)-, -S(O)2NH- or -NHS(O)2-;

[0045] X is a single bond;

[0046] R 2 is hydrogen;

[0047] m is 0, 1, 2, 3 or 4;

[0048] p is 1, 2 or 3;

[0049] z is 0, 1 or 2.

[0050] Preferably, the structure of the inhibitor is:

[0051] Q 1 , Q 2 is

[0052] R a is hydrogen or methyl, or the two R a form together with the carbon atom to which they are attached

[0053] R 1 is hydrogen, fluorine, chlorine, amino, hydroxy, cyano, methyl, isopropyl, tert-butyl, trifluoromethyl, methoxy, trifluoromethoxy, ethoxy or isopropoxy;

[0054] L 1 is -CH2O-, -OCH2-, -C(O)NH-, -NHC(O)-, -S(O)2NH- or -NHS(O)2-;

[0055] X is hydrogen, methyl, isopropyl, tert-butyl, isoamyl, phenyl, furanyl, pyridinyl, cyclopropyl, cyclobutyl, cyclohexyl, piperidinyl, piperazinyl or adamantyl;

[0056] R 2hydrogen, fluorine, chlorine, amino, hydroxyl, cyano, methyl, isopropyl, tert-butyl, trifluoromethyl, methoxy, trifluoromethoxy, ethoxy or isopropoxy;

[0057] m is 0, 1, 2, 3 or 4;

[0058] n is 0, 1, 2, 3 or 4;

[0059] p is 1, 2 or 3;

[0060] z is 0, 1 or 2.

[0061] Preferably, the inhibitor is selected from any of the following structures:

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] Preferably, the inhibitor further comprises an isomer, a metabolite, a solvate, a pharmaceutically acceptable salt or a mixture thereof; the pharmaceutically acceptable salt is a salt of the compound with an acid or a base, the acid is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid, the base is an inorganic base containing an alkali metal cation, an alkaline earth metal cation or an ammonium cation salt.

[0068] Preferably, the inhibitor and the pharmaceutically acceptable carrier form a pharmaceutical composition, which is prepared into a common pharmaceutical preparation, such as a tablet, a capsule, a syrup, a suspension or an injection, and the preparation can be added with a flavoring agent, a sweetener, a liquid / solid filler, a diluent and other common pharmaceutical excipients.

[0069] The present application also provides a preparation method of the above-mentioned compound, which comprises acylating a raw material containing an amino benzaldehyde or a hydroxyl benzaldehyde with a raw material containing a substituted benzoyl chloride or a substituted benzene sulfonyl chloride under alkaline conditions to obtain a reaction product, then subjecting the reaction product to a reductive amination reaction to obtain a Boc-protected product, and finally removing the Boc under acidic conditions to obtain the LAT1 inhibitor as described in the general formula I.

[0070] Specifically, when the LAT1 inhibitor is a compound of general formula I-1, the compound of general formula 3 is prepared from the compound of general formula 1 and compound 2 by acylation reaction under alkaline conditions, the Boc-protected compound 5 is prepared from compound 3 and compound 4 by reductive amination reaction, and the compound of general formula (I-1) is finally prepared from compound 5 by deprotection of Boc under acidic conditions, and the synthetic route is as follows:

[0071]

[0072] wherein, R 1 , R a and p are defined as above;

[0073] Further, R 1 is hydrogen, fluorine, chlorine, amino, hydroxyl, cyano, methyl, isopropyl, tert-butyl, trifluoromethyl, methoxy, trifluoromethoxy, ethoxy or isopropoxy;

[0074] Further, in the process of preparing compound 3 from compound 1, the base required for the reaction can be potassium carbonate, triethylamine, and the reaction solvent can be tetrahydrofuran, dichloromethane and acetone.

[0075] When the LAT1 inhibitor is a compound of general formula I-2, the compound of general formula 8 is prepared from the compound of general formula 6 and compound 7 by substitution reaction under alkaline conditions, the Boc-protected compound 9 is prepared from compound 8 and compound 4 by reductive amination reaction, and the compound of general formula (I-2) is finally prepared from compound 9 by deprotection of Boc under acidic conditions, and the synthetic route is as follows:

[0076]

[0077] wherein, R 1 , R a and p are defined as above;

[0078] Further, R 1 is hydrogen, fluorine, chlorine, amino, hydroxyl, cyano, methyl, isopropyl, tert-butyl, trifluoromethyl, methoxy, trifluoromethoxy, ethoxy or isopropoxy;

[0079] Further, in the process of preparing compound 7 from compound 6, the base required for the reaction can be potassium carbonate, cesium carbonate and sodium carbonate, and the reaction solvent can be tetrahydrofuran, DMF and acetone.

[0080] Further, in the process of preparing compound 9 from compound 8, the reagent required for the reaction can be sodium cyanoborohydride and triacetoxyborohydride, and the reaction solvent can be tetrahydrofuran, methanol and ethanol.

[0081] Further, in the process of preparing compound I-2 from compound 9, the reaction reagent can be hydrochloric acid and trifluoroacetic acid, and the reaction solvent can be ethyl acetate and dioxane.

[0082] When the LAT1 inhibitor is a compound of general formula I-3, the Boc-protected compound 10 is prepared from compound 3 via reductive amination reaction with compound 4, and the compound of general formula (I-3) is finally prepared by removing Boc under acidic conditions, and the synthetic route is as follows:

[0083]

[0084] wherein, R 1 , R a and p are as defined above;

[0085] Further, R 1 is hydrogen, fluorine, chlorine, amino, hydroxyl, cyano, methyl, isopropyl, tert-butyl, trifluoromethyl, methoxy, trifluoromethoxy, ethoxy or isopropoxy;

[0086] Further, in the process of preparing compound 10 from compound 3, the reaction reagent can be sodium cyanoborohydride and sodium triacetoxyborohydride, and the reaction solvent can be tetrahydrofuran, methanol and ethanol.

[0087] Further, in the process of preparing compound I-3 from compound 10, the reaction reagent can be hydrochloric acid and trifluoroacetic acid, and the reaction solvent can be ethyl acetate and dioxane.

[0088] When the LAT1 inhibitor is a compound of general formula I-4, the Boc-protected compound 12 is prepared from compound 9 via reductive amination reaction with compound 11, and the compound of general formula (I-4) is finally prepared by removing Boc under acidic conditions, and the synthetic route is as follows:

[0089]

[0090] wherein, R 1 , R 2 , R a , X and p are as defined above;

[0091] Further, R 1 is hydrogen, fluorine, chlorine, amino, hydroxyl, cyano, methyl, isopropyl, tert-butyl, trifluoromethyl, methoxy, trifluoromethoxy, ethoxy or isopropoxy;

[0092] Further, X is hydrogen, methyl, isopropyl, tert-butyl, isoamyl, phenyl, furanyl, pyridyl, cyclopropyl, cyclobutyl, cyclohexyl, piperidyl, piperazinyl or adamantyl;

[0093] Further, R2 hydrogen, fluorine, chlorine, amino, hydroxyl, cyano, methyl, isopropyl, tert-butyl, trifluoromethyl, methoxy, trifluoromethoxy, ethoxy or isopropoxy;

[0094] Further, in the process for preparing compound 12 from compound 9, the reaction reagent can be sodium cyanoborohydride and sodium triacetoxyborohydride, and the reaction solvent can be tetrahydrofuran, methanol and ethanol.

[0095] Further, in the process for preparing compound I-4 from compound 12, the reaction reagent can be hydrochloric acid and trifluoroacetic acid, and the reaction solvent can be ethyl acetate and dioxane.

[0096] When the LAT1 inhibitor is a compound of general formula I-5, the Boc-protected compound 13 is prepared from compound 10 and compound 11 by reductive amination, and the compound of general formula (I-5) is finally prepared by removing Boc under acidic conditions, and the synthetic route is as follows:

[0097]

[0098] wherein, R 1 , R 2 , R a , X and p are as defined above;

[0099] Further, R 1 is hydrogen, fluorine, chlorine, amino, hydroxyl, cyano, methyl, isopropyl, tert-butyl, trifluoromethyl, methoxy, trifluoromethoxy, ethoxy or isopropoxy;

[0100] Further, X is hydrogen, methyl, isopropyl, tert-butyl, isoamyl, phenyl, furanyl, pyridyl, cyclopropyl, cyclobutyl, cyclohexyl, piperidyl, piperazinyl or adamantyl;

[0101] Further, R 2 is hydrogen, fluorine, chlorine, amino, hydroxyl, cyano, methyl, isopropyl, tert-butyl, trifluoromethyl, methoxy, trifluoromethoxy, ethoxy or isopropoxy;

[0102] Further, in the process for preparing compound 13 from compound 10, the reaction reagent can be sodium cyanoborohydride and sodium triacetoxyborohydride, and the reaction solvent can be tetrahydrofuran, methanol and ethanol.

[0103] Further, in the process for preparing compound I-5 from compound 13, the reaction reagent can be hydrochloric acid and trifluoroacetic acid, and the reaction solvent can be ethyl acetate and dioxane.

[0104] The application further discloses a pharmaceutical composition containing the inhibitor or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer of the inhibitor and a pharmaceutically acceptable carrier or excipient.

[0105] The inhibitor or the pharmaceutical composition is used for preparing a medicine for treating inflammatory immune diseases or cancers.

[0106] Preferably, the inflammatory immune diseases are rheumatoid arthritis, dermatitis and the like.

[0107] Principle: The LAT1 inhibitor of the non-phenylalanine analogue skeleton of the application breaks through the limitation of the prior art phenylalanine analogue. The LAT1 inhibitor of the application takes 2,4-diaminobutyric acid as a mother nucleus, as a kind of leucine analogue, and the amino acid structure is conducive to forming hydrogen bond force with the substrate binding pocket of the LAT1 transport protein, so that it has good LAT1 affinity. According to the different accommodation volume and shape of the substrate binding pocket of different amino acid transport proteins, the good selectivity of the compound to LAT1 is realized through the limitation of molecular conformation and the modification of hydrophobic fragments. The LAT1 inhibitor of the application contains a tertiary amine structure, which can form different forms of organic salts, so that the water solubility of the compound is significantly improved, and the poor solubility problem of the benzene amino acid compound is greatly improved. In addition, through structure modification, group replacement and the like of the metabolizable site by structure-activity relationship study, the advantage compound with greatly improved in-vivo metabolic stability is finally screened out. The LAT1 inhibitor of the application realizes the metabolic regulation of tumor cells or inflammatory cells by inhibiting the transport activity of LAT1 and affecting the uptake and utilization of amino acids by the tumor cells or inflammatory cells, and is suitable for application in the treatment of inflammatory immune diseases or cancers; and is especially suitable for application in the treatment of rheumatoid arthritis.

[0108] Rheumatoid arthritis is a disease caused by the disorder of immune cells, and the pathogenesis mechanism includes the stimulation, proliferation and activation of T cells, the invasion and migration of other inflammatory cells such as FLS and the like. LAT1 plays an important role in the development of rheumatoid arthritis, and the LAT1 inhibitor of the application realizes the treatment of rheumatoid arthritis from multiple aspects by inhibiting the transport activity of LAT1 and affecting the uptake of amino acids by cells, on the one hand, the energy and nutrient sources required for the proliferation and differentiation of inflammatory cells can be blocked, and on the other hand, the amino acids taken by LAT1 are often used as signal molecules in intracellular signal pathways, and the inhibition of LAT1 will cause the blockage of amino acid transport, and thus the activation of the signal pathway related to cell proliferation is inhibited. The LAT1 inhibitor of the application brings new application through a new skeleton structure, and provides more choices for the research and use of the LAT1 inhibitor.

[0109] Advantages: Compared with the prior art, the application has the following obvious advantages:

[0110] (1) The LAT1 inhibitor compound of the present application has significantly higher inhibitory activity on LAT1 than the inhibitor BCH, and the inhibitory level is comparable to that of the lead compound V9302, which solves the problem of short half-life of V9302 while retaining good LAT1 target activity;

[0111] (2) The metabolic stability of the compound of the present application is extremely high, and the liver microsomal half-life is significantly higher than that of the lead compound V9302, and the metabolic stability of the LAT1 inhibitor is significantly improved compared with the current clinical LAT1 inhibitor JPH203; wherein the in vitro rat liver microsomal metabolic half-life of compound L-31 is about 5-10 times better than that of the clinical drug JPH203; in the pharmacokinetic study, the in vivo clearance of compound L-31 through intravenous injection is much smaller than that of the clinical drug JPH203, and the half-life is also significantly improved, and it has good metabolic stability in vivo;

[0112] (3) The oral bioavailability is extremely high, and the initial blood drug concentration and plasma drug exposure of compound L-31 have a great advantage compared with JPH203, and it has good in vivo absorption;

[0113] (4) It has a significant effect on rheumatoid arthritis, and the in vivo pharmacodynamic test results of the rat AIA model show that after 20 days of drug treatment, the rheumatoid arthritis of the high and low dose groups of rats is obviously reduced, and the parameters such as foot swelling volume, foot palm thickness and ankle joint thickness are all reduced to the level before modeling, which is consistent with the level of the blank normal group. BRIEF DESCRIPTION OF DRAWINGS

[0114] Figure 1 is the general formula of the LAT1 inhibitor of the present application;

[0115] Figure 2 is a line graph of the pharmacodynamic test results of the rat AIA model, wherein Figure A is a line graph of the body weight of rats in each group during the drug administration period, and Figure B is a line graph of the foot palm thickness of rats in each group during the drug administration period;

[0116] Figure 3 is a column graph of the liver and spleen weight index and the expression amount of inflammatory factors in the spleen of rats, wherein Figure A is a column graph of the liver weight index of rats in each group, Figure B is a column graph of the spleen weight index of rats in each group, Figure C is a column graph of the IL-1β mRNA expression amount in the spleen of rats in each group, and Figure D is a column graph of the INFα mRNA expression amount in the spleen of rats in each group. DETAILED DESCRIPTION

[0117] The technical solutions of the present application are further described below through specific examples; the raw materials and equipment used are known products, which are obtained by purchasing commercially available products, deuterated chloroform CDCl3, N,N-dimethylformamide DMF, dimethyl sulfoxide DMSO, thin layer chromatography TLC, proton nuclear magnetic resonance spectrum 1 H NMR, 13 C nuclear magnetic resonance spectrum 13 C NMR, high resolution mass spectrum HRMS.

[0118] Example 1

[0119] Preparation of (S)-2-amino-4-(bis(2-(3-methoxybenzamido)benzyl)amino)butyric acid (L-1):

[0120]

[0121] Step 1: Preparation of N-(2-formylphenyl)-3-methoxybenzamide (I-3): 2-aminobenzaldehyde (I-1) (1.18 g, 9.8 mmol) and 30 ml of dichloromethane were added to a 100 mL reaction bottle, cooled to 0°C, and then triethylamine (1.98 g, 19.6 mmol) and 3-methoxybenzoyl chloride (I-2) (2.50 g, 14.7 mmol) were added dropwise. After the dropwise addition was completed, the reaction was warmed to room temperature for 3 h. After TLC detection showed that the raw material was completely reacted, 50 mL of water and 100 mL of dichloromethane were added to the reaction liquid, and the water layer was adjusted to pH 4-5 with 1N HCl. The organic layer was washed with water, saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. Isopropyl ether (150 mL) was added to the crude product and stirred at room temperature for 30 min. The filter cake was washed and dried to obtain the intermediate I-3 1.89 g, white solid, yield 76%. 1 H NMR (300 MHz, DMSO-d6): δ = 10.36 (s, 1H), 7.98 (s, 1H), 7.74-7.68 (m, 3H), 7.57 (t, J = 12 Hz, 1H), 7.49-7.43 (m, 3H), 7.21-7.18 (d, J = 9 Hz, 1H), 3.87 (s, 3H) ppm. HRMS (ESI + ): cacld for C 15 H 14 NO3 + (M+H) + , 256.0974; found 256.0969.

[0122] Step 2: Preparation of (S)-4-(bis(2-(3-methylbenzamido)benzyl)amino)-2- ((tert-butoxycarbonyl)amino)butanoic acid (1-5): N-(2-formylphenyl)-3- methoxybenzamide (1-3) (6.58 g, 27.5 mmol), (S)-4-amino-2-((tert- butoxycarbonyl)amino)butanoic acid (1-4) (2.00 g, 9.2 mmol) and 50 mL of methanol were added into a 100 mL reaction flask, reacted at room temperature for 1 h, sodium cyanoborohydride (2.31 g, 36.7 mmol) was added in 5 batches, reacted at room temperature for 12 h, the solvent was removed under reduced pressure, and the intermediate 1-5 was obtained by column chromatography (dichloromethane:methanol = 30:1) 2.97 g, white solid, yield 49%. 1 HNMR (300 MHz, DMSO-d6): δ = 7.87-7.80 (m, 4H), 7.38-7.28 (m, 8H), 6.99-6.91 (m, 4H), 4.12 (s, 4H), 4.05 (t, J = 12.0 Hz, 1H), 3.89 (s, 6H), 2.68-2.62 (m, 2H), 1.95-1.86 (m, 2H), 1.35 (s, 9H) ppm. HRMS (ESI + ): cacld for C 39 H 45 N4O8 + (M+H) + ,697.3237; found 697.3240.

[0123] Step 3: Preparation of (S)-2-amino-4-(bis(2-(3-methoxybenzamido)benzyl)amino)butanoic acid (L-1): (S)-4-(bis(2-(3-methylbenzamido)benzyl)amino)-2-((tert- butoxycarbonyl)amino)butanoic acid (1-5) (0.34 g, 0.5 mmol) was dissolved in 10 mL of ethyl acetate, 4N hydrogen chloride ethyl acetate solution 5 mL was added, reacted at room temperature for 2 h, the solvent was removed under reduced pressure, the obtained solid was slurried with ethyl acetate, suction filtered, and the filter cake was washed with ethyl acetate and dried to obtain compound L-1 0.27 g, white solid, yield 91%. 1 H NMR (300 MHz, DMSO-d6): δ = 7.88-7.82 (m, 4H), 7.36-7.27 (m, 8H), 7.01-6.93 (m, 4H), 4.27 (s, 4H), 3.99 (t, J = 11.7 Hz, 1H), 3.94 (s, 6H), 2.64-2.60 (m, 2H), 1.96-1.90 (m, 2H) ppm. HRMS (ESI + ): cacld for C 34 H37 N4O6 + (M+H) + ,597.2713; found 597.2721.

[0124] The compounds described in Examples 2-6 were prepared according to the methods and preparation routes described in Example 1.

[0125] Example 2

[0126] Preparation of (S)-2-amino-4-(bis(2-(3-methylbenzamido)benzyl)amino)butanoic acid (L-2)

[0127]

[0128] 1 H NMR (300 MHz, DMSO-d6): δ = 7.91 - 7.80 (m, 4H), 7.33 - 7.22 (m, 8H), 7.05 - 6.99 (m, 4H), 4.26 (t, J = 12.0 Hz, 1H), 4.11 (s, 4H), 4.26 (t, J = 12.0 Hz, 1H), 2.77 - 2.71 (m, 2H), 2.34 (s, 6H), 1.84 - 1.80 (m, 2H) ppm. HRMS (ESI + ): calcd for C 34 H 37 N4O4 + (M+H) + ,565.2815; found 565.2809.

[0129] Example 3

[0130] Preparation of (S)-2-amino-4-(bis(2-(3-methylbenzamido)benzyl)amino)butanoic acid (L-2)

[0131]

[0132] 1 H NMR (300 MHz, DMSO-d6): δ = 7.93 - 7.85 (m, 4H), 7.30 - 7.23 (m, 8H), 7.15 - 7.04 (m, 4H), 4.16 (s, 4H), 3.94 (t, J = 11.4 Hz, 1H), 2.81 - 2.73 (m, 2H), 1.90 - 1.83 (m, 2H) ppm. HRMS (ESI + ): calcd for C 32 H 31 Cl2N4O4 + (M+H)+ , 605.1722; found 605.1731.

[0133] Example 4

[0134] Preparation of (S)-2-amino-4-(bis(2-(3-(trifluoromethyl)benzamido)benzyl)amino)butanoic acid (L-4)

[0135]

[0136] 1 H NMR (300 MHz, DMSO-d6): δ = 7.89 - 7.80 (m, 4H), 7.34 - 7.25 (m, 8H), 7.19 - 7.03 (m, 4H), 4.20 (s, 4H), 4.11 (t, J = 12.3 Hz, 1H), 2.85 - 2.74 (m, 2H), 1.92 - 1.87 (m, 2H) ppm. HRMS (ESI + ): calcd for C 34 H 31 F6N4O4 + (M+H) + , 673.2249; found 673.2240.

[0137] Example 5

[0138] Preparation of (S)-2-amino-4-(bis(2-(3-(trifluoromethyl)benzamido)benzyl)amino)butanoic acid (L-4)

[0139]

[0140] 1 H NMR (300 MHz, DMSO-d6): δ = 7.76 - 7.68 (m, 6H), 7.26 - 7.21 (m, 8H), 7.19 - 7.03 (m, 4H), 4.09 (s, 4H), 3.88 (t, J = 12.0 Hz, 1H), 2.86 - 2.78 (m, 2H), 1.95 - 1.86 (m, 2H) ppm. HRMS (ESI + ): calcd for C 32 H 33 N4O4 + (M+H) + , 537.2502; found 537.2513.

[0141] Example 6

[0142] Preparation of (S)-2-amino-4-(bis(2-(3-(trifluoromethoxy)benzamido)benzyl)amino)butanoic acid (L-6)

[0143]

[0144] 1 H NMR (300 MHz, DMSO-d6): δ = 7.95-7.86 (m, 4H), 7.37-7.29 (m, 8H), 7.24-7.16 (m, 4H), 4.23 (s, 4H), 4.17 (t, J = 11.7 Hz, 1H), 2.81-2.72 (m, 2H), 1.87-1.83 (m, 2H) ppm. HRMS (ESI + ): calcd for C 34 H 31 F6N4O6 + (M+H) + , 705.2148; found 705.2156.

[0145] Example 7

[0146] Preparation of (S)-2-amino-4-((2-((3-methoxybenzyl)oxy)benzyl)amino)butanoic acid (L-7)

[0147]

[0148] Step 1: Preparation of 2-((3-methoxybenzyl)oxy)benzaldehyde (II-3): Salicylaldehyde (II-1) (1.22 g, 10.0 mmol) and 50 mL of acetone were added to a 100 mL reaction flask, 1-(bromomethyl)-3-methoxybenzene (II-2) (2.40 g, 12.0 mmol) and potassium carbonate (2.07 g, 15.0 mmol) were added, and the reaction was heated to reflux for 3 h. After TLC detection that the raw material was completely reacted, the reaction liquid was cooled to room temperature, the potassium carbonate was removed by suction filtration, the filtrate was concentrated under reduced pressure, and the filter cake was obtained after isopropyl ether was pulped and suction filtered. 2.1 g of white solid was obtained as intermediate (II-3), with a yield of 87%. 1 H NMR (300 MHz, DMSO-d6): δ = 10.48 (s, 1H), 7.83-7.80 (d, J = 9.0 Hz, 1H), 7.63 (t, J = 11.4 Hz, 1H), 7.26-7.03 (m, 4H), 6.97-6.91 (m, 2H), 5.61 (s, 2H), 3.70 (s, 3H) ppm. HRMS (ESI + ): calcd for C 15 H 15 O3 +(M+H) + ,243.1021; found 243.1029.

[0149] Step 2: Preparation of (S)-2-((tert-butoxycarbonyl)amino)-4-((2-((3- methoxybenzyl)oxy)benzyl)amino)butanoic acid (II-5): 2-((3-methoxybenzyl)oxy)benzaldehyde (II-3) (4.36 g, 18.0 mmol), (S)-4-amino-2-((tert- butoxycarbonyl)amino)butanoic acid (II-4) (3.27 g, 15.0 mmol) and 30 mL of methanol were added into a 100 mL reaction flask, and the reaction was carried out at room temperature for 1 h, sodium cyanoborohydride (1.88 g, 30 mmol) was added in 5 batches, and the reaction was carried out at room temperature for 12 h, the solvent was removed under reduced pressure, and the intermediate II-5 was obtained by column chromatography (dichloromethane:methanol = 10:1), 4.59 g, white solid, yield 69%. 1 HNMR (300 MHz, DMSO-d6): δ = 7.85-7.82 (d, J = 8.7 Hz, 1H), 7.66 (t, J = 11.7 Hz, 1H), 7.28-7.13 (m, 4H), 7.02-6.92 (m, 2H), 5.48 (s, 2H), 4.26 (s, 2H), 4.05-3.97 (m, 1H), 3.89 (s, 3H), 2.78-2.72 (m, 2H), 1.84-1.78 (m, 2H), 1.33 (s, 9H) ppm. HRMS (ESI + ): cacld for C 24 H 33 N2O6 + (M+H) + ,445.2339; found 445.2328.

[0150] Step 3: Preparation of (S)-2-amino-4-((2-((3-methoxybenzyl)oxy)benzyl)amino)butanoic acid (L-7): (S)-2-((tert-butoxycarbonyl)amino)-4-((2-((3- methoxybenzyl)oxy)benzyl)amino)butanoic acid (II-5) (0.27 g, 0.6 mmol) was dissolved in 10 mL of ethyl acetate, 5 mL of 4N hydrogen chloride ethyl acetate solution was added, and the reaction was carried out at room temperature for 2 h, the solvent was removed under reduced pressure, the obtained solid was slurried with ethyl acetate, and the filter cake was washed with ethyl acetate and dried to obtain compound L-7 0.19 g, white solid, yield 93%. 1H NMR (300 MHz, DMSO-d6): δ = 7.87-7.84 (d, J = 8.7 Hz, 1H), 7.71 (t, J = 12.0 Hz, 1H), 7.26-7.15 (m, 4H), 7.07-6.95 (m, 2H), 5.53 (s, 2H), 4.28 (s, 2H), 4.11-4.06 (m, 1H), 3.91 (s, 3H), 2.76-2.73 (m, 2H), 1.83-1.78 (m, 2H) ppm. HRMS (ESI + ): calcd for C 19 H 25 N2O4 + (M+H) + , 345.1814; found 345.1806.

[0151] Example 8

[0152] Preparation of (S)-2-amino-4-((2-((3-methylbenzyl)oxy)benzyl)amino)butanoic acid (L-8)

[0153]

[0154] Prepared according to the method and preparation route described in Reference Example 7. 1 H NMR (300 MHz, DMSO-d6): δ = 7.86-7.83 (d, J = 9.3 Hz, 1H), 7.73 (t, J = 11.7 Hz, 1H), 7.27-7.16 (m, 4H), 7.09-6.93 (m, 2H), 5.59 (s, 2H), 4.17 (s, 2H), 3.94-3.89 (m, 1H), 2.76-2.73 (m, 2H), 2.34 (s, 3H), 1.83-1.78 (m, 2H) ppm. HRMS (ESI + ): calcd for C 19 H 25 N2O3 + (M+H) + , 329.1865; found 329.1851.

[0155] Example 9

[0156] Preparation of (S)-2-amino-4-((2-(3-methoxybenzamido)benzyl)amino)butanoic acid (L-9)

[0157]

[0158] Intermediate III-1 was prepared according to the procedure described in Example 1 and the preparation route

[0159] Step 1: Preparation of (S)-2-((tert-butoxycarbonyl)amino)-4-((2-(3- methoxybenzamido)benzyl)amino)butanoic acid (III-3): N-(2- formylphenyl)benzamide (III-1) (9.11 g, 35.8 mmol), (S)-4-amino-2- ((tert-butoxycarbonyl)amino)butanoic acid (III-2) (6.00 g, 27.5 mmol) and 100 mL of methanol were added into a 250 mL reaction flask, and the reaction was carried out at room temperature for 1 h. Sodium cyanoborohydride (3.45 g, 55 mmol) was added in 5 batches, and the reaction was carried out at room temperature for 12 h. The solvent was removed under reduced pressure, and the intermediate III-3 was obtained by column chromatography (dichloromethane:methanol = 10:1) as a white solid with a yield of 63%. 1 H NMR (300 MHz, DMSO-d6): δ = 7.94-7.92 (d, J = 5.7 Hz, 1H), 7.55-7.42 (m, 3H), 7.38-7.31 (m, 2H), 7.17-7.15 (d, J = 6.0 Hz, 2H), 3.92 (s, 2H), 3.85 (s, 3H), 3.92-3.81 (m, 1H), 2.72 (t, J = 9.0 Hz, 2H), 1.86-1.80 (m, 2H), 1.35 (s, 9H) ppm. HRMS (ESI + ): cacld for C 24 H 32 N3O6 + (M+H) + ,458.2291; found 458.2283.

[0160] Step 2: Preparation of (S)-2-amino-4-((2-(3-methoxybenzamido)benzyl)amino)butanoic acid (L-9): ((S)-2-((tert-butoxycarbonyl)amino)-4-((2-(3- methoxybenzamido)benzyl)amino)butanoic acid (III-3) (0.23 g, 0.5 mmol) was dissolved in 10 mL of ethyl acetate, and 5 mL of 4N hydrogen chloride ethyl acetate solution was added. The reaction was carried out at room temperature for 2 h. The solvent was removed under reduced pressure, and the obtained solid was slurried with ethyl acetate. The filter cake was washed with ethyl acetate and dried to obtain compound L-9 0.18 g as a white solid with a yield of 93%. 1H NMR (300 MHz, DMSO-d6): δ = 7.96 - 7.94 (d, J = 6.0 Hz 1H), 7.58 - 7.44 (m, 3H), 7.40 - 7.35 (m, 2H), 7.16 - 7.14 (d, J = 6.0 Hz, 2H), 3.97 (s, 2H), 3.91 (s, 3H), 3.89 - 3.85 (m, 1H), 2.74 (t, J = 9.3 Hz, 2H), 1.85 - 1.81 (m, 2H) ppm. HRMS (ESI + ): calcd for C 19 H 24 N3O4 + (M+H) + , 358.1767; found 358.1761.

[0161] The compounds described in Examples 10-14 were prepared according to the methods and routes described in Example 9.

[0162] Example 10

[0163] Preparation of (S)-2-amino-4-((2-(3-fluorobenzamido)benzyl)amino)butanoic acid (L-10)

[0164]

[0165] 1 H NMR (300 MHz, DMSO-d6): δ = 8.01 - 7.99 (d, J = 5.7 Hz, 1H), 7.55 - 7.41 (m, 3H), 7.43 - 7.32 (m, 2H), 7.19 - 7.17 (d, J = 6.0 Hz, 2H), 4.07 (s, 2H), 3.98 - 3.92 (m, 1H), 2.78 - 2.72 (m, 2H), 1.87 - 1.83 (m, 2H) ppm. HRMS (ESI + ): calcd for C 18 H 21 FN3O3 + (M+H) + , 346.1567; found 346.1555.

[0166] Example 11

[0167] Preparation of (S)-2-amino-3-((2-(3-methoxybenzamido)benzyl)amino)propanoic acid (L-11)

[0168]

[0169] 1 H NMR (300 MHz, DMSO-d6): δ = 7.92-7.90 (d, J = 6.3 Hz, 1H), 7.51-7.43 (m, 3H), 7.39-7.31 (m, 2H), 7.16-7.14 (d, J = 5.7 Hz, 2H), 4.11 (s, 2H), 4.03-3.97 (m, 1H), 3.81 (s, 3H), 2.92-2.85 (m, 2H) ppm. HRMS (ESI + ): calcd for C 18 H 22 N3O4 + (M+H) + , 344.1610; found 344.1623.

[0170] Example 12

[0171] Preparation of (S)-2-amino-5-((2-(3-methoxybenzamido)benzyl)amino)pentanoic acid (L-12)

[0172]

[0173] 1 H NMR (300 MHz, DMSO-d6): δ = 7.95-7.93 (d, J = 5.7 Hz, 1H), 7.49-7.44 (m, 3H), 7.37-7.32 (m, 2H), 7.15-7.13 (d, J = 6.0 Hz, 2H), 4.14 (s, 2H), 3.97-3.93 (m, 1H), 3.85 (s, 3H), 2.97-2.88 (m, 2H), 1.86-1.81 (m, 2H), 1.51-1.39 (m, 2H) ppm. HRMS (ESI + ): calcd for C 20 H 26 N3O4 + (M+H) + , 372.1923; found 372.1918.

[0174] Example 13

[0175] Preparation of (S)-2-amino-4-((2-((3-chlorophenyl)sulfonamido)benzyl)amino)butanoic acid (L-13)

[0176]

[0177] 1H NMR (300 MHz, DMSO-d6): δ = 7.81 - 7.77 (m, 1H), 7.73 - 7.64 (m, 4H), 7.41 - 7.29 (m, 2H), 6.76 - 6.74 (d, J = 7.1 Hz, 2H), 4.19 (s, 2H), 4.00 - 3.91 (m, 1H), 3.26 - 3.17 (m, 2H), 2.31 - 2.15 (m, 2H) ppm. HRMS (ESI + ): calcd for C 17 H 21 ClN3O4S + (M+H) + , 398.0941; found 398.0933.

[0178] Example 14

[0179] Preparation of (S)-2-amino-4-((2-((4-methoxyphenyl)sulfonamido)benzyl)amino)butanoic acid (L-14)

[0180]

[0181] 1 H NMR (300 MHz, DMSO-d6): δ = 7.85 - 7.79 (m, 1H), 7.76 - 7.65 (m, 4H), 7.43 - 7.33 (m, 2H), 6.78 - 6.76 (d, J = 6.3 Hz, 2H), 4.24 (s, 2H), 4.06 - 4.02 (m, 1H), 3.93 (s, 3H), 3.17 - 3.09 (m, 2H), 2.28 - 2.17 (m, 2H) ppm. HRMS (ESI + ): calcd for C 18 H 24 N3O5S + (M+H) + , 394.1437; found 394.1445.

[0182] Example 15

[0183] Preparation of (S)-2-amino-4-(benzyl(2-(3-methoxybenzamido)benzyl)amino)butanoic acid (L-15)

[0184]

[0185] Preparation of Intermediate IV-1 can be prepared according to the method and preparation route described in Example 9

[0186] Step 1: Preparation of (S)-4-(benzyl(2-(3-methoxybenzamido)benzyl)amino)-2- ((tert-butoxycarbonyl)amino)butanoic acid (IV-3): (S)-2-((tert- butoxycarbonyl)amino)-4-((2-(3-methoxybenzamido)benzyl)amino)butanoic acid (IV-1) (0.38 g, 0.7 mmol), benzaldehyde (IV-2) (0.14 g, 1.4 mmol) and 8 mL of methanol were added into a 50 mL reaction flask, reacted at room temperature for 1 h, sodium cyanoborohydride (0.09 g, 1.4 mmol) was added in 5 batches, reacted at room temperature for 12 h, the solvent was removed under reduced pressure, and the intermediate IV-3 was obtained by column chromatography (dichloromethane:methanol = 20:1) 0.37 g, white solid, yield 81%. 1 H NMR (300 MHz, DMSO-d6): δ = 7.72-7.69 (d, J = 9 Hz, 1H), 7.55-7.38 (m, 8H), 7.25-7.18 (m, 4H), 4.36-4.26 (m, 2H), 4.16 (s, 2H), 3.96-3.92 (m, 1H), 3.85 (s, 3H), 3.27-3.18 (m, 2H), 2.39-2.32 (m, 2H), 1.35 (s, 9H) ppm. HRMS (ESI + ): cacld for C 31 H 38 N3O6 + (M+H) + ,548.2761; found 548.2749.

[0187] Step 2: Preparation of (S)-2-amino-4-(benzyl(2-(3-methoxybenzamido)benzyl)amino)butanoic acid (L-15): (S)-4-(benzyl(2-(3-methoxybenzamido)benzyl)amino)-2- ((tert-butoxycarbonyl)amino)butanoic acid (IV-3) (0.22 g, 0.5 mmol) was dissolved in 10 mL of ethyl acetate, 5 mL of 4N hydrogen chloride ethyl acetate solution was added, and reacted at room temperature for 2 h. The solvent was removed under reduced pressure, the obtained solid was slurried with ethyl acetate, and filtered. The filter cake was washed with ethyl acetate and dried to obtain compound L-15 0.17 g, white solid, yield 89%. 1H NMR (300 MHz, DMSO-d6): δ = 7.72-7.69 (d, J = 7.4 Hz, 1H), 7.55-7.38 (m, 8H), 7.25-7.19 (m, 4H), 4.34-4.28 (m, 2H), 4.16 (s, 2H), 3.97-3.92 (m, 1H), 3.85 (s, 3H), 3.28-3.16 (m, 2H), 2.39-2.32 (m, 2H) ppm. HRMS (ESI + ): calcd for C 26 H 30 N3O4 + (M+H) + , 448.2236; found 448.2243.

[0188] The compounds described in Examples 16-47 were prepared according to the methods and routes described in Example 15.

[0189] Example 16

[0190] Preparation of (S)-2-amino-4-((4-(tert-butyl)benzyl)(2-(3-methoxybenzamido)benzyl)amino)butanoic acid (L-16)

[0191]

[0192] 1 H NMR (300 MHz, DMSO-d6): δ = 7.66-7.63 (d, J = 7.9 Hz, 1H), 7.57-7.44 (m, 5H), 7.39-7.34 (m, 3H), 7.23-7.16 (m, 3H), 4.21-4.10 (m, 2H), 4.02 (s, 2H), 3.91 (t, J = 11.4 Hz, 1H), 3.84 (s, 3H), 3.22-3.06 (m, 2H), 2.32-2.23 (m, 2H), 1.15 (s, 9H) ppm. HRMS (ESI + ): calcd for C 30 H 38 N3O4 + (M+H) + , 504.2862; found 504.2850.

[0193] Example 17

[0194] Preparation of (S)-2-amino-4-((2-(3-methoxybenzamido)benzyl)(4-methoxybenzyl)amino)butanoic acid (L-17)

[0195]

[0196] 1 H NMR (300 MHz, DMSO-d6): δ = 7.65-7.62 (d, J = 7.6 Hz, 1H), 7.55-7.50 (m, 1H), 7.42-7.30 (m, 7H), 7.20-7.18 (m, 1H), 6.68-6.65 (d, J = 8.5 Hz, 2H), 4.28-4.20 (m, 2H), 4.15-4.10 (m, 2H), 3.81 (s, 3H), 3.79-3.75 (m, 1H), 3.59 (s, 3H), 3.26-3.13 (m, 2H), 2.27-2.16 (m, 2H) ppm. HRMS (ESI + ): calcd for C 27 H 32 N3O5 + (M+H) + , 478.2342; found 478.2352.

[0197] Example 18

[0198] Preparation of (S)-2-amino-4-((2-(3-methoxybenzamido)benzyl)(4- (trifluoromethyl)benzyl)amino)butanoic acid (L-18)

[0199]

[0200] 1 H NMR (300 MHz, DMSO-d6): δ = 7.59-7.56 (m, 3H), 7.53-7.47 (m, 1H), 7.44-7.32 (m, 7H), 7.20-7.17 (d, J = 9.0 Hz, 1H), 4.37-4.29 (m, 2H), 4.08 (s, 2H), 3.78 (s, 3H), 3.78-3.75 (m, 1H), 3.30-3.16 (m, 2H), 2.25-2.15 (m, 2H) ppm. HRMS (ESI + ): calcd for C 27 H 29 F3N3O4 + (M+H) + , 516.2110; found 516.2105.

[0201] Example 19

[0202] Preparation of (S)-2-amino-4-((2-(3-methoxybenzamido)benzyl)(3- (trifluoromethyl)benzyl)amino)butanoic acid (L-19)

[0203]

[0204] 1 H NMR (300 MHz, DMSO-d6): δ = 7.61 - 7.57 (m, 3H), 7.51 - 7.48 (m, 1H), 7.45 - 7.38 (m, 7H), 7.22 - 7.19 (d, J = 9.0 Hz, 1H), 4.33 - 4.26 (m, 2H), 4.11 (s, 2H), 3.86 (s, 3H), 3.81 - 3.74 (m, 1H), 3.27 - 3.18 (m, 2H), 2.28 - 2.19 (m, 2H) ppm. HRMS (ESI + ): calcd for C 27 H 29 F3N3O4 + (M+H) + , 516.2110; found 516.2117.

[0205] Example 20

[0206] Preparation of (S)-2-amino-4-((2-(3-methoxybenzamido)benzyl)(2- (trifluoromethyl)benzyl)amino)butanoic acid (L-20)

[0207]

[0208] 1 H NMR (300 MHz, DMSO-d6): δ = 7.68 - 7.59 (m, 3H), 7.55 - 7.50 (m, 1H), 7.47 - 7.41 (m, 7H), 7.31 - 7.29 (d, J = 9.0 Hz, 1H), 4.38 - 4.29 (m, 2H), 4.16 (s, 2H), 3.89 (s, 3H), 3.88 - 3.75 (m, 1H), 3.26 - 3.15 (m, 2H), 2.29 - 2.16 (m, 2H) ppm. HRMS (ESI + ): calcd for C 27 H 29 F3N3O4 + (M+H) + , 516.2110; found 516.2101.

[0209] Example 21

[0210] Preparation of (S)-2-amino-4-((2-(3-methoxybenzamido)benzyl)(pyridin-4- ylmethyl)amino)butanoic acid (L-21)

[0211]

[0212] 1 H NMR (300 MHz, DMSO-d6): δ = 7.78-7.75 (d, J = 9.3 Hz, 1H), 7.66-7.59 (m, 7H), 7.38-7.24 (m, 4H), 4.43-4.37 (m, 2H), 4.32 (s, 2H), 4.05-3.98 (m, 1H), 3.91 (s, 3H), 3.37-3.23 (m, 2H), 2.37-2.31 (m, 2H) ppm. HRMS (ESI + ): calcd for C 25 H 29 N4O4 + (M+H) + , 449.2189; found 449.2195.

[0213] Example 22

[0214] Preparation of (S)-2-amino-4-((furan-3-ylmethyl)(2-(3-methoxybenzamido)benzyl)amino)butanoic acid (L-22)

[0215]

[0216] 1 H NMR (300 MHz, DMSO-d6): δ = 7.77 (s, 1H), 7.66-7.64 (d, J = 7.2 Hz, 1H), 7.54-7.45 (m, 7H), 7.20-7.18 (d, J = 6.9 Hz, 1H), 6.54 (s, 1H), 4.19-4.14 (m, 4H), 3.82 (s, 4H), 3.27-3.08 (m, 2H), 2.29-2.14 (m, 2H) ppm. HRMS (ESI + ): calcd for C 24 H 28 N3O5 + (M+H) + , 438.2029; found 438.2020.

[0217] Example 23

[0218] Preparation of (S)-2-amino-4-(((3'-methoxy-[l,l'-biphenyl]-2-yl)methyl)(2-(3- methoxybenzamido)benzyl)amino)butanoic acid (L-23)

[0219]

[0220] 1 H NMR (300 MHz, DMSO-d6): δ = 7.80 - 7.63 (m, 3H), 7.47 - 7.32 (m, 5H), 7.29 - 7.19 (m, 4H), 7.04 - 6.91 (m, 3H), 6.77 (s, 1H), 4.32 (s, 2H), 4.20 - 4.11 (m, 2H), 4.09 - 4.02 (m, 1H), 3.97 (s, 3H), 3.84 (s, 3H), 3.34 - 3.11 (m, 2H), 2.33 - 2.17 (m, 2H) ppm. HRMS (ESI + ): calcd for C 33 H 36 N3O5 + (M+H) + ,554.2655; found 554.2643.

[0221] Example 24

[0222] Preparation of (S)-4-(([l,l'-biphenyl]-2-ylmethyl)(2-(3-methoxybenzamido)benzyl)amino)-2- aminobutanoic acid (L-24)

[0223]

[0224] 1 H NMR (300 MHz, DMSO-d6): δ = 7.76 - 7.64 (m, 4H), 7.44 - 7.30 (m, 5H), 7.27 - 7.21 (m, 4H), 7.08 - 6.93 (m, 3H), 6.79 (s, 1H), 4.36 - 4.30 (m, 2H), 4.22 - 4.16 (m, 2H), 4.13 - 4.07 (m, 1H), 3.81 (s, 3H), 3.31 - 3.15 (m, 2H), 2.35 - 2.19 (m, 2H) ppm. HRMS (ESI + ): calcd for C 32 H 34 N3O4 + (M+H) + ,524.2549; found 524.2540.

[0225] Example 25

[0226] Preparation of (S)-2-amino-4-((2-(3-methoxybenzamido)benzyl)(2- (pyridin-4-yl)benzyl)amino)butanoic acid (L-24)

[0227]

[0228] 1 H NMR (300 MHz, DMSO-d6): δ = 7.81 - 7.69 (m, 4H), 7.47 - 7.36 (m, 4H), 7.28 - 7.24 (m, 4H), 7.11 - 6.95 (m, 3H), 6.77 (s, 1H), 4.29 - 4.23 (m, 2H), 4.21 - 4.16 (m, 2H), 4.15 - 4.08 (m, 1H), 3.93 (s, 3H), 3.33 - 3.19 (m, 2H), 2.32 - 2.18 (m, 2H) ppm. HRMS (ESI + ): calcd for C 31 H 33 N4O4 + (M+H) + , 525.2502; found 525.2513.

[0229] Example 26

[0230] Preparation of (S)-2-amino-4-((2-(3-methoxybenzamido)benzyl)(2- (pyridin-4-yl)benzyl)amino)butanoic acid (L-24)

[0231]

[0232] 1 H NMR (300 MHz, DMSO-d6): δ = 7.81 - 7.69 (m, 4H), 7.47 - 7.36 (m, 4H), 7.28 - 7.24 (m, 4H), 7.11 - 6.95 (m, 3H), 6.77 (s, 1H), 4.29 - 4.23 (m, 2H), 4.21 - 4.16 (m, 2H), 4.15 - 4.08 (m, 1H), 3.93 (s, 3H), 3.33 - 3.19 (m, 2H), 2.32 - 2.18 (m, 2H) ppm. HRMS (ESI + ): calcd for C 31 H 33 N4O4 + (M+H) + , 525.2502; found 525.2514.

[0233] Example 27

[0234] Preparation of (S)-2-amino-4-((2-(3-methoxybenzamido)benzyl)(piperidin-4- ylmethyl)amino)butanoic acid (L-27)

[0235]

[0236] 1 H NMR (300 MHz, DMSO-d6): δ = 7.76-7.73 (d, J = 8.7 Hz, 1H), 7.57-7.42 (m, 3H), 7.28-7.20 (m, 4H), 4.16-4.11 (m, 2H), 4.07-4.02 (m, 1H), 3.90 (s, 3H), 3.43-3.31 (m, 4H), 2.97-2.84 (m, 2H), 2.48-2.35 (m, 2H), 2.26-2.19 (m, 2H), 1.56-1.31 (m, 5H) ppm. HRMS (ESI + ): calcd for C 25 H 35 N4O4 + (M+H) + , 455.2658; found 455.2666.

[0237] Example 28

[0238] Preparation of (S)-4-((((3S,5S,7S)-adamantan-l-yl)methyl)(2-(3- methoxybenzamido)benzyl)amino)-2-aminobutanoic acid (L-28)

[0239]

[0240] 1 H NMR (300 MHz, DMSO-d6): δ = 7.69-7.66 (d, J = 9.3 Hz, 1H), 7.52-7.40 (m, 3H), 7.25-7.18 (m, 4H), 4.14-4.09 (m, 2H), 4.04-3.98 (m, 1H), 3.83 (s, 3H), 2.99-2.86 (m, 2H), 2.45-2.32 (m, 2H), 2.24-2.17 (m, 2H), 1.91-1.65 (m, 15H) ppm. HRMS (ESI + ): calcd for C 30 H 40 N3O4 + (M+H) +, 506.3019; found 506.3027.

[0241] Example 29

[0242] Preparation of ((S)-2-amino-4-((cyclohexylmethyl)(2-(3- methoxybenzamido)benzyl)amino)butanoic acid (L-29)

[0243]

[0244] 1 H NMR (300 MHz, DMSO-d6): δ = 7.67-7.57 (m, 4H), 7.52-7.41 (m, 3H), 7.24-7.21 (d, J = 7.9 Hz, 1H), 4.37-4.25 (m, 2H), 3.88 (t, J = 12.0 Hz, 1H), 3.88 (s, 3H), 3.40-3.22 (m, 2H), 2.94-2.90 (m, 2H), 2.28-2.14 (m, 2H), 1.68-1.56 (m, 3H), 1.51-1.42 (m, 3H), 1.00-0.90 (m, 3H), 0.84-0.72 (m, 2H) ppm. HRMS (ESI + ): calcd for C 26 H 36 N3O4 + (M+H) + , 454.2706; found 454.2695.

[0245] Example 30

[0246] Preparation of ((S)-2-amino-4-((cyclobutylmethyl)(2-(3- methoxybenzamido)benzyl)amino)butanoic acid (L-30)

[0247]

[0248] 1 H NMR (300 MHz, DMSO-d6): δ = 7.82-7.73 (m, 3H), 7.61-7.55 (m, 1H), 7.48-7.40 (m, 3H), 7.22-7.19 (d, J = 9.0 Hz, 1H), 4.37-4.33 (m, 2H), 4.05-3.98 (m, 1H), 3.88 (s, 3H), 3.41-3.30 (m, 2H), 3.08-3.06 (d, J = 5.7 Hz, 2H), 2.32-2.23 (m, 3H), 1.72-1.47 (m, 6H) ppm. HRMS (ESI +): calcd for C 24 H 32 N3O4 + (M+H) + ,426.2393; found 426.2404.

[0249] Example 31

[0250] Preparation of (S)-2-amino-4-((cyclopropylmethyl)(2-(3- methoxybenzamido)benzyl)amino)butanoic acid (L-31)

[0251]

[0252] 1 H NMR (300 MHz, DMSO-d6): δ = 7.76 - 7.67 (m, 3H), 7.58 - 7.53 (m, 1H), 7.49 - 7.43 (m, 3H), 7.20 - 7.17 (d, J = 9.0 Hz, 1H), 4.45 - 4.32 (m, 2H), 4.03 - 3.98 (m, 1H), 3.85 (s, 3H), 3.44 - 3.28 (m, 2H), 3.05 - 3.03 (d, J = 6.3 Hz, 2H), 2.37 - 2.26 (m, 2H), 1.14 - 1.07 (m, 1H), 0.50 - 0.47 (d, J = 7.2 Hz, 2H), 0.35 - 0.33 (d, J = 7.2 Hz, 2H) ppm. HRMS (ESI + ): calcd for C 23 H 30 N3O4 + (M+H) + ,412.2236; found 412.2243.

[0253] Example 32

[0254] Preparation of (S)-2-amino-4-((2-(3-methoxybenzamido)benzyl)(methyl)amino)butanoic acid (L-32)

[0255]

[0256] 1H NMR (300 MHz, DMSO-d6): δ = 7.85-7.73 (m, 3H), 7.66-7.57 (m, 1H), 7.51-7.45 (m, 3H), 7.24-7.18 (m, 1H), 4.39-4.31 (m, 2H), 4.06-3.99 (m, 1H), 3.82 (s, 3H), 3.23-3.15 (m, 2H), 2.56 (s, 3H), 2.37-2.26 (m, 2H) ppm. HRMS (ESI + ): calcd for C 20 H 26 N3O4 + (M+H) + , 372.1923; found 372.1916.

[0257] Example 33

[0258] Preparation of (S)-2-amino-4-((2-(3-methoxybenzamido)benzyl)(neopentyl)amino)butanoic acid (L-33)

[0259]

[0260] 1 H NMR (300 MHz, DMSO-d6): δ = 7.80-7.74 (m, 3H), 7.66-7.52 (m, 1H), 7.51-7.43 (m, 3H), 7.25-7.22 (d, J = 8.7 Hz, 1H), 4.28-4.20 (m, 2H), 4.11-4.04 (m, 1H), 3.94 (s, 3H), 3.09-3.03 (m, 2H), 2.64-2.59 (m, 2H), 2.26-2.18 (m, 2H), 1.05 (s, 9H) ppm. HRMS (ESI + ): calcd for C 24 H 34 N3O4 + (M+H) + , 428.2549; found 428.2556.

[0261] Example 34

[0262] Preparation of (S)-2-amino-4-((2-ethylbutyl)(2-(3-methoxybenzamido)benzyl)amino)butanoic acid (L-34)

[0263]

[0264] 1H NMR (300 MHz, DMSO-d6): δ = 7.74-7.71 (m, 3H), 7.65-7.56 (m, 1H), 7.47-7.41 (m, 3H), 7.26-7.24 (d, J = 9.0 Hz, 1H), 4.27-4.24 (m, 2H), 4.13-4.06 (m, 1H), 3.91 (s, 3H), 3.18-3.12 (m, 2H), 2.62-2.55 (m, 2H), 2.29-2.17 (m, 2H), 1.45-1.41 (m, 5H), 1.14 (t, J = 11.7 Hz, 6H) ppm. HRMS (ESI + ): calcd for C 25 H 36 N3O4 + (M+H) + , 442.2706; found 442.2718.

[0265] Example 35

[0266] Preparation of (2S)-2-amino-4-(((2-fluorocyclopropyl)methyl)(2-(3- methoxybenzamido)benzyl)amino)butanoic acid (L-35)

[0267]

[0268] 1 H NMR (300 MHz, DMSO-d6): δ = 7.78-7.67 (m, 3H), 7.59-7.52 (m, 1H), 7.50-7.44 (m, 3H), 7.21-7.18 (d, J = 9.3 Hz, 1H), 4.42-4.35 (m, 2H), 4.31-4.27 (m, 1H), 4.07-3.99 (m, 1H), 3.88 (s, 3H), 3.41-3.26 (m, 2H), 3.08-3.06 (d, J = 6.0 Hz, 2H), 2.36-2.23 (m, 2H), 1.19-1.12 (m, 1H), 0.68-0.49 (m, 1H), 0.35-0.33 (m, 1H) ppm. HRMS (ESI + ): calcd for C 23 H 29 FN3O4 + (M+H) + , 430.2142; found 430.2135.

[0269] Example 36

[0270] Preparation of (2S)-2-amino-4-(((2,2-difluorocyclopropyl)methyl)(2-(3- methoxybenzamido)benzyl)amino)butanoic acid (L-36)

[0271]

[0272] 1 H NMR (300 MHz, DMSO-d6): δ = 7.83 - 7.72 (m, 3H), 7.63 - 7.55 (m, 1H), 7.46 - 7.41 (m, 3H), 7.22 - 7.19 (d, J = 8.7 Hz, 1H), 4.36 - 4.28 (m, 2H), 4.14 - 4.02 (m, 1H), 3.90 (s, 3H), 3.39 - 3.23 (m, 2H), 3.10 - 3.08 (d, J = 6.5 Hz, 2H), 2.35 - 2.24 (m, 2H), 1.47 - 1.41 (m, 1H), 0.83 - 0.74 (m, 1H), 0.59 - 0.50 (m, 1H) ppm. HRMS (ESI + ): calcd for C 23 H 28 F2N3O4 + (M+H) + , 448.2048; found 448.2040.

[0273] Example 37

[0274] Preparation of (S)-2-amino-4-(((cyclopropyl-2,2,3,3-d4)methyl)(2-(3- methoxybenzamido)benzyl)amino)butanoic acid (L-37)

[0275]

[0276] 1 H NMR (300 MHz, DMSO-d6): δ = 7.84 - 7.72 (m, 3H), 7.60 - 7.54 (m, 1H), 7.53 - 7.45 (m, 3H), 7.27 - 7.14 (d, J = 9.4 Hz, 1H), 4.29 - 4.16 (m, 2H), 4.08 - 4.00 (m, 1H), 3.84 (s, 3H), 3.24 - 3.17 (m, 2H), 3.09 - 3.07 (d, J = 6.4 Hz, 2H), 2.31 - 2.23 (m, 2H), 1.13 - 1.06 (m, 1H) ppm. HRMS (ESI + ): calcd for C 23 H 26 D4N3O4 +(M+H) + ,416.2487; found 416.2493.

[0277] Example 38

[0278] Preparation of (S)-2-amino-4-((cyclopropylmethyl-d2)(2-(3- methoxybenzamido)benzyl)amino)butanoic acid (L-38)

[0279]

[0280] 1 H NMR (300 MHz, DMSO-d6): δ = 7.80-7.69 (m, 3H), 7.64-7.58 (m, 1H), 7.43-7.37 (m, 3H), 7.25-7.23 (d, J = 8.6 Hz, 1H), 4.42-4.28 (m, 2H), 3.97-3.94 (m, 1H), 3.82 (s, 3H), 3.13-3.06 (m, 2H), 2.25-2.12 (m, 2H), 1.08-1.02 (m, 1H), 0.53-0.49 (d, J = 11.6 Hz, 2H), 0.41-0.36 (d, J = 7.2 Hz, 2H) ppm. HRMS (ESI + ): calcd for C 23 H 28 D2N3O4 + (M+H) + ,414.2362; found 414.2353.

[0281] Example 39

[0282] Preparation of (S)-2-amino-4-((cyclopropylmethyl)(2-(4- methoxybenzamido)benzyl)amino)butanoic acid (L-39)

[0283]

[0284] 1H NMR (300 MHz, DMSO-d6): δ = 7.99-7.96 (d, J = 9.3 Hz, 2H), 7.32-7.27 (m, 2H), 7.11-7.08 (d, J = 8.6 Hz, 2H), 6.96-6.91 (m, 2H), 4.27-4.16 (m, 2H), 4.05-4.00 (m, 1H), 3.89 (s, 3H), 3.37-3.25 (m, 2H), 3.08-3.06 (d, J = 5.8 Hz, 2H), 2.34-2.23 (m, 2H), 1.13-1.06 (m, 1H), 0.52-0.44 (d, J = 7.2 Hz, 2H), 0.34-0.32 (d, J = 7.2 Hz, 2H) ppm. HRMS (ESI + ): calcd for C 23 H 30 N3O4 + (M+H) + , 412.2236; found 412.2243.

[0285] Example 40

[0286] Preparation of (S)-2-amino-4-((cyclopropylmethyl)(2-(2- methoxybenzamido)benzyl)amino)butanoic acid (L-40)

[0287]

[0288] 1 H NMR (300 MHz, DMSO-d6): δ = 7.99-7.96 (d, J = 9.3 Hz, 2H), 7.32-7.27 (m, 2H), 7.11-7.08 (d, J = 8.6 Hz, 2H), 6.96-6.91 (m, 2H), 4.27-4.16 (m, 2H), 4.05-4.00 (m, 1H), 3.89 (s, 3H), 3.37-3.25 (m, 2H), 3.08-3.06 (d, J = 5.8 Hz, 2H), 2.34-2.23 (m, 2H), 1.13-1.06 (m, 1H), 0.52-0.44 (d, J = 7.2 Hz, 2H), 0.34-0.32 (d, J = 7.2 Hz, 2H) ppm. HRMS (ESI + ): calcd for C 23 H 30 N3O4 + (M+H) + , 412.2236; found 412.2243.

[0289] Example 41

[0290] Preparation of (S)-2-amino-4-((cyclopropylmethyl)(2-(3- methoxybenzamido)benzyl)amino)butanoic acid (L-40)

[0291]

[0292] 1 H NMR (300 MHz, DMSO-d6): δ = 7.32-7.19 (m, 6H), 6.98-6.92 (m, 2H), 4.43-4.34 (m, 2H), 4.07-4.02 (m, 1H), 3.39-3.32 (s, 6H), 3.26-3.17 (m, 2H), 3.06-3.04 (d, J = 5.8 Hz, 2H), 2.41-2.35 (m, 2H), 1.16-1.03 (m, 1H), 0.55-0.53 (d, J = 7.3 Hz, 2H), 0.36-0.34 (d, J = 7.2 Hz, 2H) ppm. HRMS (ESI + ): calcd for C 24 H 33 N4O3 + (M+H) + , 425.2553; found 425.2566.

[0293] Example 42

[0294] Preparation of (S)-2-amino-4-((cyclopropylmethyl)(2-(3-methylbenzamido)benzyl)amino)butanoic acid (L-42)

[0295]

[0296] 1 H NMR (300 MHz, DMSO-d6): δ = 7.85-7.81 (m, 2H), 7.37-7.28 (m, 4H), 7.02-6.94 (m, 2H), 4.36-4.21 (m, 2H), 4.14-4.06 (m, 1H), 3.33-3.22 (m, 2H), 3.08-3.06 (d, J = 6.2 Hz, 2H), 2.40 (s, 3H), 2.35-2.27 (m, 2H), 1.14-1.07 (m, 1H), 0.59-0.57 (d, J = 6.9 Hz, 2H), 0.38-0.36 (d, J = 6.9 Hz, 2H) ppm. HRMS (ESI + ): calcd for C 23 H 30 N3O3 + (M+H) +, 396.2287; found 396.2275.

[0297] Example 43

[0298] Preparation of (S)-2-amino-4-((cyclopropylmethyl)(2-(3-(trifluoromethyl)benzamido)benzyl)amino)butanoic acid (L-43)

[0299]

[0300] 1 H NMR (300 MHz, DMSO-d6): δ = 7.71 - 7.63 (m, 2H), 7.32 - 7.19 (m, 3H), 6.99 - 6.78 (m, 3H), 4.34 - 4.28 (m, 2H), 4.10 - 4.05 (m, 1H), 3.26 - 3.20 (m, 2H), 3.15 - 3.13 (d, J = 6.7 Hz, 2H), 2.27 - 2.21 (m, 2H), 1.18 - 1.13 (m, 1H), 0.58 - 0.56 (d, J = 6.5 Hz, 2H), 0.43 - 0.41 (d, J = 6.5 Hz, 2H) ppm. HRMS (ESI + ): calcd for C 23 H 27 F3N3O3 + (M+H) + , 450.2005; found 450.1996.

[0301] Example 44

[0302] Preparation of (S)-2-amino-4-((cyclopropylmethyl)(2-(3-(trifluoromethoxy)benzamido)benzyl)amino)butanoic acid (L-44)

[0303]

[0304] 1 H NMR (300 MHz, DMSO-d6): δ = 7.71 - 7.63 (m, 2H), 7.32 - 7.19 (m, 3H), 6.99 - 6.78 (m, 3H), 4.34 - 4.28 (m, 2H), 4.10 - 4.05 (m, 1H), 3.26 - 3.20 (m, 2H), 3.15 - 3.13 (d, J = 6.7 Hz, 2H), 2.27 - 2.21 (m, 2H), 1.18 - 1.13 (m, 1H), 0.58 - 0.56 (d, J = 6.5 Hz, 2H), 0.43 - 0.41 (d, J = 6.5 Hz, 2H) ppm. HRMS (ESI +): calcd for C 23 H 27 F3N3O4 + (M+H) + ,466.1954; found 466.1960.

[0305] Example 45

[0306] Preparation of (S)-2-amino-4-((cyclopropylmethyl)(2-((3- methoxyphenyl)aminocarbonyl)benzyl)amino)butanoic acid (L-46)

[0307]

[0308] 1 H NMR (300 MHz, DMSO-d6): δ = 7.72-7.64 (m, 2H), 7.54-7.40 (m, 2H), 7.32-7.27 (m, 2H), 7.03-6.92 (m, 2H), 4.31-4.25 (m, 2H), 4.13-4.08 (m, 1H), 3.25-3.19 (m, 2H), 3.17-3.15 (d, J = 6.4 Hz, 2H), 2.33-2.24 (m, 2H), 1.08-1.02 (m, 1H), 0.62-0.60 (d, J = 5.9 Hz, 2H), 0.47-0.45 (d, J = 5.9 Hz, 2H) ppm. HRMS (ESI + ): calcd for C 22 H 27 FN3O3 + (M+H) + ,400.2036; found 400.2023.

[0309] Example 46

[0310] Preparation of (S)-2-amino-4-((cyclopropylmethyl)(2-((3- methoxyphenyl)aminocarbonyl)benzyl)amino)butanoic acid (L-46)

[0311]

[0312] 1H NMR (300 MHz, DMSO-d6): δ = 7.84-7.81 (d, J = 9.3 Hz, 1H), 7.58-7.46 (m, 3H), 7.21-7.18 (m, 3H), 6.67-6.64 (d, J = 8.5 Hz, 1H), 4.32-4.27 (m, 2H), 4.09-4.02 (m, 1H), 3.82 (s, 3H), 3.36-3.27 (m, 2H), 3.06-3.04 (d, J = 6.5 Hz, 2H), 2.34-2.25 (m, 2H), 1.11-1.06 (m, 1H), 0.52-0.50 (d, J = 7.4 Hz, 2H), 0.41-0.39 (d, J = 7.4 Hz, 2H) ppm. HRMS (ESI + ): calcd for C 23 H 30 N3O4 + (M+H) + , 412.2236; found 412.2243.

[0313] Example 47

[0314] Preparation of (S)-2-amino-4-((cyclohexylmethyl)(2-((3- methoxyphenyl)sulfonamido)benzyl)amino)butanoic acid (L-47)

[0315]

[0316] 1 H NMR (300 MHz, DMSO-d6): δ = 7.85-7.79 (m, 1H), 7.59-7.54 (m, 3H), 7.34-7.24 (m, 2H), 7.07-7.05 (d, J = 8.3 Hz, 2H), 6.72-6.66 (m, 1H), 3.79 (s, 3H), 3.67-3.65 (m, 1H), 3.28-3.11 (m, 4H), 2.75-2.69 (m, 2H), 2.22-2.01 (m, 2H), 1.75-1.52 (m, 6H), 1.25-1.07 (m, 5H) ppm. HRMS (ESI + ): calcd for C 25 H 36 N3O5S + (M+H) + , 490.2376; found 490.2365.

[0317] Example 48

[0318] Preparation of (S)-2-amino-4-((cyclopropylmethyl)(2-((3- methoxyphenyl)sulfonamido)benzyl)amino)butanoic acid (L-48)

[0319]

[0320] 1 H NMR (300 MHz, DMSO-d6): δ = 7.88-7.82 (m, 1H), 7.62-7.55 (m, 3H), 7.37-7.26 (m, 2H), 7.08-7.06 (d, J = 8.1 Hz 2H), 6.74-6.63 (m, 1H), 3.88 (s, 3H), 3.79-3.67 (m, 1H), 3.32-3.16 (m, 4H), 2.75-2.68 (m, 2H), 2.24-2.06 (m, 2H), 1.14-1.07 (m, 1H), 0.50-0.47 (d, J = 7.2 Hz, 2H), 0.35-0.33 (d, J = 7.2 Hz, 2H) ppm. HRMS (ESI + ): calcd for C 22 H 30 N3O5S + (M+H) + , 448.1906; found 448.1917.

[0321] Example 49

[0322] Preparation of (S)-2-amino-3-((cyclohexylmethyl)(2-(3- methoxybenzamido)benzyl)amino)propanoic acid (L-49)

[0323]

[0324] 1 H NMR (300 MHz, DMSO-d6): δ = 7.63-7.56 (m, 3H), 7.51-7.45 (m, 2H), 7.44-7.37 (m, 2H), 7.22-7.17 (m, 1H), 4.30-4.24 (m, 1H), 4.14-4.08 (m, 2H), 3.82 (s, 3H), 3.36-3.20 (m, 2H), 2.82-2.72 (m, 2H), 1.65-1.38 (m, 6H), 1.01-0.89 (m, 2H), 0.82-0.66 (m, 3H) ppm. HRMS (ESI + ): calcd for C 25 H 34 N3O4 + (M+H)+ ,440.2549; found 440.2556.

[0325] Example 50

[0326] Preparation of (S)-2-amino-3-((cyclopropylmethyl)(2-(3- methoxybenzamido)benzyl)amino)propanoic acid (L-50)

[0327]

[0328] 1 H NMR (300 MHz, DMSO-d6): δ = 7.86-7.83 (d, J = 7.0 Hz, 1H), 7.64 (s, 2H), 7.48-7.37 (m, 4H), 7.16-7.14 (d, J = 7.7 Hz, 1H), 4.66-4.55 (m, 2H), 4.40-4.35 (m, 1H), 3.83 (s, 3H), 3.76-3.72 (m, 2H), 3.12-2.97 (m, 2H), 1.11-1.03 (m, 1H), 0.51-0.42 (m, 2H), 0.34-0.24 (m, 2H) ppm. HRMS (ESI + ): calcd for C 22 H 28 N3O4 + (M+H) + ,398.2080; found 398.2074.

[0329] Example 51

[0330] Preparation of (S)-2-amino-5-((cyclohexylmethyl)(2-(3- methoxybenzamido)benzyl)amino)pentanoic acid (L-51)

[0331]

[0332] 1 H NMR (300 MHz, DMSO-d6): δ = 7.62-7.49 (m, 4H), 7.47-7.33 (m, 3H), 7.19-7.16 (d, J = 8.6 Hz, 1H), 4.27-4.21 (m, 2H), 3.77 (s, 4H), 3.07-2.98 (m, 2H), 2.90-2.83 (m, 2H), 1.81-1.67 (m, 4H), 1.61-1.51 (m, 3H), 1.46-1.38 (m, 3H), 0.97-0.68 (m, 5H) ppm. HRMS (ESI + ): calcd for C27 H 38 N3O4 + (M+H) + ,468.2862;found 468.2854.

[0333] Example 52

[0334] Preparation of (S)-2-amino-5-((cyclopropylmethyl)(2-(3- methoxybenzamido)benzyl)amino)pentanoic acid (L-52)

[0335]

[0336] 1 H NMR (300 MHz, DMSO-d6): δ = 7.74-7.72 (d, J = 6.9 Hz, 1H), 7.68-7.63 (m, 2H), 7.57-7.51 (m, 1H), 7.48-7.39 (m, 3H), 7.19-7.17 (d, J = 7.5 Hz, 1H), 4.35 (s, 2H), 3.83 (s, 4H), 3.12-3.04 (m, 2H), 3.01-2.96 (m, 2H), 1.89-1.72 (m, 4H), 1.13-1.04 (m, 1H), 0.49-0.46 (m, 2H), 0.33-0.29 (m, 2H) ppm. HRMS (ESI + ): calcd for C 24 H 32 N3O4 + (M+H) + ,426.2393;found426.2385.

[0337] Example 53

[0338] In vitro activity experiment of the compound of the present application

[0339] 1. Leucine transport experiment:

[0340] (1) Cell culture

[0341] (2) Plating: 40000 / well in 24-well plate, 37°C, 5% CO2 culture for 24h;

[0342] (3) When the confluence of HEK-293T cells reached 60%, the SLC7A5 plasmid was transfected into the cells according to the instructions of jetprime transfection reagent. After 48h of transfection, the 24-well plate was taken out from the incubator;

[0343] (4) Discard the medium in the plate, wash 3 times with 37℃ preheated Na+-free Hank's solution (125mM choline chloride, 25mM Hepes, 4.8mM KCl, 1.2mM KH2PO4, 1.2mM MgSO4·7H2O, 1.3mM CaCl2, 5.6mM D-glucose. pH = 7.4) and pre-warm incubation at 37℃ for 7min;

[0344] (5) Add 250μL of different concentrations of inhibitor solution (10mM compound stock solution diluted to 20μM, 2μM with 5μM 2H-Leu in Na+-free Hank's solution), 37℃ water bath for 15min;

[0345] (6) After incubation, aspirate the liquid, wash the cells with cold Na+-free Hank's solution three times. Add 200μL of ultrapure water. Freeze-thaw three times at -80℃, scrape the cells into 1.5mL EP tubes, ultrasonic 15min to break the cell samples, take 30μL into 1.5mL EP tubes, add 30μL of diluent, 60μL of internal standard solution, vortex for 2min, centrifuge at 4℃, 12,000rpm for 10min. Take 60μL of supernatant into the sample vial, and detect by LC-MS / MS 2 H-Leu content.

[0346] 2. Data analysis:

[0347] Leucine transport experiment calculation formula %Inhibition = [1-(A_sample / A_max)] Wherein: A_sample represents the content of H-Leu in the sample 2 H-Leu, A_max represents the content of H-Leu in the blank sample 2 H-Leu; the inhibition of leucine uptake transport is shown in Table 1.

[0348] Table 1, the inhibition rate of compound in vitro leucine transport

[0349]

[0350]

[0351] Note: A: 70-90%, B: 40-70%, C: 10-40%

[0352] As shown in Table 1, all the test compounds have inhibitory effect on LAT1 leucine transport, wherein the LAT1 leucine transport inhibition rates of compounds L-29, L-31, L-35, L-36, L-39, L-40, L-42 to L-45 at 2μM concentration are all greater than 70%, which have good inhibitory activity.

[0353] The above-mentioned compound has better leucine uptake inhibitory activity, which proves that the LAT1 inhibitor has better LAT1 inhibitory activity, and the inhibition of LAT1 activity has been proved to play a major role in the treatment of rheumatoid arthritis.

[0354] Example 54

[0355] Pharmacodynamic study of the compound of the present application in the AIA model of rats in vivo

[0356] 1. Establishment of AIA rat model

[0357] 100 μL of complete Freund's adjuvant (CFA, Chondrex, 10 mg / ml) was subcutaneously injected into the right plantar region and tail root of SD rats, respectively, and the control group was given the same amount of blank solvent (day 0). After 21 days, the modeling condition was evaluated, and the rats were randomly divided into 5 groups according to the foot volume and body weight: control group, model group, compound L-31 low dose (50 mg / kg) group, and high dose (100 mg / kg) group. The rats were orally administered with the drug at a fixed time every afternoon, and the administration was continuously performed for 16 days.

[0358] 2. Measurement of rat foot thickness

[0359] The left feet of rats in each group were marked at the same position, and the thickness of rat footpads was measured every 2 days using a vernier caliper.

[0360] 3. Measurement of rat liver and spleen weights

[0361] After the administration was completed, the rats were sacrificed, and the rat liver and spleen were obtained by dissection, weighed, and then preserved.

[0362] 4. Rat IL-1β High Sensitivity ELISA Kit detection

[0363] The spleen tissue of rats in each group was weighed (50 mg-100 mg), 10 times the volume of PBS was added, and the homogenate was frozen for 10 min at 12500 rpm / min, centrifuged for 15 min, and the supernatant was subjected to ELASA detection according to the instructions of Rat IL-1β High Sensitivity ELISA Kit detection kit (MULTI SCIENCE, EK301BHS).

[0364] 5. Data analysis

[0365] From Figure 2 and Figure 3As can be seen, during the administration of compound L-31, the body weight of the rats remained steadily increasing, the foot volume of the rats in the high and low dose administration groups decreased significantly, and was basically consistent with the treatment trend of the marketed drug upatin. After the administration ended, the rat foot thickness of the L-31 administration group basically returned to the normal level before modeling, that is, close to the blank healthy group, which showed that the compound L-31 had good effect on treating rheumatoid arthritis. The weight of the liver and spleen often increases during inflammation, and can effectively reflect the condition of inflammation. The results of the liver and spleen weight of the rats showed that the liver and spleen weight of the rats in the high and low dose administration groups of compound L-31 was lower than that of the modeling group, which was consistent with the level after upatin treatment, indicating that compound L-31 could effectively alleviate the inflammation. In addition, the expression amount of IL-1β and INF-α in the spleen was detected, and the results showed that the administration of compound L-31 could effectively reduce the expression amount of IL-1β and INF-α and other inflammatory factors in the spleen of the AIA model rats and return to the normal level, proving the anti-inflammatory effect of compound L-31.

[0366] Example 55

[0367] Metabolic stability of the compound of the present application in human liver microsomes in vitro

[0368] The DMSO mother liquor of the test compound was diluted and vortexed, and the final concentration of DMSO was controlled at 0.01%; the compound was incubated with the liver microsome system (PBS, MgCl2, HLM, NADPH), and sampled at different time points (0 min, 5 min, 15 min, 30 min, 60 min, 120 min), and after termination, the supernatant was collected and stored in a-80℃ ultra-low temperature refrigerator until LC-MS / MS analysis.

[0369] Table 2, metabolic stability of the compound in human liver microsomes in vitro

[0370] Compound HLM T 1 / 2 (min) L-15 47.89 L-29 63.25 L-31 168.72 L-47 83.42 V9302 5.12

[0371] As shown in Table 2, the half-lives of compounds L-29, L-31 and L-47 were significantly improved compared with the lead compound V9302, and L-31 had obvious advantages and excellent metabolic stability in rat liver microsomes in vitro.

Claims

1. A LAT1 inhibitor, characterized in that, The inhibitor is selected from any one of the following structures:

2. The inhibitor of claim 1, wherein The inhibitor further comprises a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer, a pharmaceutically acceptable salt or a mixture thereof; the pharmaceutically acceptable salt is a salt of the inhibitor compound with an acid or a base, the acid is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid, the base is an inorganic base containing an alkali metal cation, an alkaline earth metal cation or an ammonium cation salt.

3. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the inhibitor of claim 1 or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer and a pharmaceutically acceptable carrier or excipient thereof.

4. Use of the inhibitor of claim 1 or the pharmaceutical composition of claim 3 in the preparation of a medicament for treating inflammatory immune diseases.

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