2-arylmethyloxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivatives, process for their synthesis and use

Abstract

This invention discloses 2-arylmethoxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivatives, their synthesis methods, and applications. The general structural formula of the 2-arylmethoxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivatives is shown in Formula I. Through experimental verification in the embodiments of this invention, the 2-arylmethoxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivatives provided by this invention can serve as a good small molecule inhibitor of PD-1 / PD-L1. They have a novel structure and have achieved good results in protein, cell, and animal level evaluations, demonstrating good drug-like properties and development potential.

Description

Technical Field

[0001] This invention relates to 2-arylmethoxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivatives, their synthesis methods, and applications. Background Technology

[0002] The tumor microenvironment can protect tumor cells from being recognized and killed by the collective immune system, i.e., immune escape occurs. Immune escape by tumor cells plays a crucial role in tumor development and progression. The activation or inhibition of the body's immune cells is regulated through positive and negative signals. Programmed death protein 1 (PD-1) and its ligand (PD-L1) form a pair of negative immunomodulatory signals, inhibiting T cell immune activity and mediating tumor cell immune escape.

[0003] The ability of tumor cells to evade the immune system is achieved through the binding of programmed death ligand (PD-L1) expressed on the surface of tumor cells to the PD-1 protein expressed on the surface of T cells. The tumor microenvironment in the body induces infiltrating T cells to overexpress PD-1 protein, and tumor cells overexpress PD-1 ligands PD-L1 and PD-L2, leading to the continuous activation of the PD-1 / PD-L1 pathway in the tumor microenvironment. This suppresses T cell function, preventing them from detecting tumors and thus from sending signals to the immune system to attack and kill tumor cells. Therefore, to activate the function of T cells in killing tumor cells, a monoclonal antibody strategy can be used. Specifically, PD-1 or PD-L1 monoclonal antibodies are antibody proteins that target and bind to PD-1 or PD-L1, respectively, preventing the two proteins from binding and thus blocking this pathway, restoring T cell function, and allowing them to continue killing tumor cells.

[0004] Tumor immunotherapy based on PD-1 / PD-L1 immune checkpoint inhibitors is a promising new generation of immunotherapy for cancer. It aims to utilize the body's own immune system to kill tumor cells by inducing apoptosis through blocking the PD-1 / PD-L1 signaling pathway, and has shown efficacy in treating various types of tumors. On September 4, 2014, Merck & Co., Ltd. developed the monoclonal antibody drug pembrolizumab (trade name...). PD-1 monoclonal antibodies are used to treat patients with advanced or unresectable melanoma who have not responded to other drug therapies. To date, more than a dozen PD-1 and PD-L1 monoclonal antibodies have been marketed. Tumor immunotherapy is considered a revolution in cancer treatment after targeted therapy. However, monoclonal antibody therapies have their own drawbacks: they are easily broken down by digestive juices, unstable in vivo, and cannot be taken orally; they are prone to immune cross-reactions; product quality is difficult to control, and manufacturing technology is highly demanding; large-scale preparation and purification are difficult, resulting in high production costs; and they are inconvenient to use, requiring only intravenous injection or infusion, with an average response rate of less than 30%. Therefore, developing tumor immunotherapy drugs that inhibit the interaction between PD-1 and PD-L1 through small molecules has significant practical implications. Summary of the Invention

[0005] The present invention aims to provide a small molecule 2-arylmethoxy-4-(3-aryl-2-fluoromethylbenzyloxy)benzylamine derivative (general structural formula shown in Formula I) that inhibits PD-1 / PD-L1 interaction, as well as its stereoisomers and pharmaceutically acceptable salts, its preparation method and its use in the preparation of drugs for the prevention or treatment of diseases related to the PD-1 / PD-L1 signaling pathway.

[0006]

[0007] R1 is one of the following: Where R 11 and R 13 Each is independently selected from one of the following:

[0008] Where n = 1, 2, or 3; where R 14 It can be hydrogen, C1-C4 alkyl, C1-C4 alkylamide C1-C4 alkyl, C1-C4 alkylamide C1-C4 alkyl, amide C1-C4 alkyl, amino C1-C4 alkyl, di(C1-C4 alkyl)amide C1-C4 alkyl, di(C1-C4 alkyl)amino C1-C3 alkyl, hydroxy C1-C4 alkyl, phenyl, or methoxy-substituted phenyl; R 12 It can be hydrogen, C1-C3 alkyl, cyano, halogen, or halogenated C1-C3 alkyl;

[0009] R2 can be:

[0010] X can be one of the following: hydrogen, chlorine, bromine, iodine, C1-4 alkyl, methoxy;

[0011] R3 can be one of the following: R4 can be one of the following: a substituted C1-8 saturated alkylamino group, a substituted C2-6 unsaturated alkylamino group, or a substituted C2-6 aza-1-yl group. The substituent can be hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, C1-5 alkyl, C1-5 alkoxy, amino, C1-6 alkylamino, acetamido, cyano, ureoyl, guanidinyl, ureaamino, guanidinylamino, sulfonylamino, aminosulfonyl, fluorosulfonyl, fluorosulfonylamino, fluorosulfonate, methanesulfonylamino, hydroxyformyl, C1-8 alkoxyformyl, mercapto, imidazole, thiazolyl, oxazolyl, or tetrazolyl.

[0012] Furthermore, R4 can be one of the following:

[0013]

[0014] R = methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl.

[0015] The most preferred compound could be:

[0016] N-hydroxyethyl-[2-(5-cyanopyridine-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)]benzylamine

[0017] N-[2-(3-cyanobenzyloxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]L-serine

[0018] N-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]piperidine-2-carboxylic acid

[0019] N-[2-(5-cyanopyridine-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]glycine

[0020] N-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]L-proline

[0021] N-[2-(5-cyanopyridine-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]pyrrolidine-3-carboxylic acid

[0022] N-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]L-serine

[0023] N-[2-(5-cyanopyridine-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]D-serine

[0024] N-{2-(5-cyanopyridine-3-methoxy)-4-[3-(2,5-bisfluorophenyl)-2-monofluoromethyl-benzyloxy]benzyl}L-serine

[0025] N-hydroxyethyl-{2-(3-cyanobenzyloxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-monofluoromethyl-benzyloxy]}benzylamine

[0026] N-{2-(5-cyanopyridine-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-monofluoromethyl-benzyloxy]benzyl}-L-serine

[0027]

[0028] N-hydroxyethyl-{2-(5-cyanopyridine-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-monofluoromethyl-benzyloxy]}benzylamine

[0029]

[0030] N-[2-(5-cyanopyridine-3-methoxy)-4-(3-o-fluorophenyl-2-bisfluoromethyl-benzyloxy)-benzyl]L-serine

[0031]

[0032] N-hydroxyethyl-{2-(5-cyanopyridine-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-bisfluoromethyl-benzyloxy]}benzylamine

[0033]

[0034] Furthermore, the starting materials and intermediates in the above reactions are relatively easy to obtain. Pharmaceutically acceptable salts of Formula I include salts formed from various acids, including: hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, citric acid, maleic acid, tartaric acid, fumaric acid, citric acid, or lactic acid. Pharmaceutically acceptable salts of Formula I also include various alkali metal salts (lithium, sodium, potassium), alkaline earth metal salts (calcium, magnesium), and ammonium salts. All these salts within the scope of this invention can be prepared using conventional methods. During the preparation of the compounds of Formula I, their solvates, and their salts, polycrystalline or eutectic crystals may occur under different crystallization conditions.

[0035] The second aspect of the present invention provides a method for preparing the compound described in the first aspect: To prepare the compound of general formula IA-1 according to the structure of general formula IA-1, the preparation of the compound of general formula IA-1 according to the present invention will be divided into eight steps (Route 1):

[0036]

[0037] (a) Using methyl 3-bromo-2-methylbenzoate as the starting material, and with azobisisobutyronitrile (AIBN) as a free radical initiator, brominate one hydrogen atom of the methyl group at the 2-position of the benzene ring of the compound of Formula 1 to obtain intermediate 2.

[0038] (b) Using tetrabutylammonium fluoride as a fluorination agent, the monobromomethyl group at the 2-position of the benzene ring of intermediate 2 is converted into a monofluoromethyl group to obtain intermediate 3.

[0039] (c) Using intermediate 3 and various substituted aromatic boric acids R1-B(OH)2 as basic raw materials, intermediate 4 was obtained by Suzuki coupling reaction;

[0040] (d) The ester group of intermediate 4 was reduced with lithium aluminum hydride to obtain benzyl alcohol intermediate 5;

[0041] (e) Using intermediate 5 as a raw material, benzyl alcohol is brominated with carbon tetrabromide to obtain brominated intermediate 6;

[0042] (f) Using intermediate 6 as a raw material, react with intermediate 5-X-2,4-dihydroxybenzaldehyde under alkaline conditions to obtain benzyl aryl ether intermediate 7;

[0043] (g) Using intermediate 7 as a raw material, react with various substituted aryl halides R3-Cl under alkaline conditions to obtain intermediate 8;

[0044] (h) Using aldehyde-containing intermediate 8 as a raw material, the target compound IA-1 is obtained by reductive amination reaction with various amino or imino-containing HR4 compounds; the substituents R1, R3, R4, and X in the raw material compounds added during the reaction are defined as in the target compound.

[0045] To prepare the compound of general formula IA-2 according to the structure of general formula IA-2, the preparation of the compound of general formula IA-2 according to the present invention will be divided into eight steps (route two):

[0046] (a) Using 2-difluoromethyl-3-bromobenzyl alcohol as shown in Formula 9 and various substituted aromatic boric acids R1-B(OH)2 as basic raw materials, intermediate 10 was obtained by Suzuki coupling reaction;

[0047] (b) Using intermediate 10 as a raw material, substituted benzyl alcohol is brominated with carbon tetrabromide to obtain brominated intermediate 11;

[0048] (c) Using intermediate 11 as a raw material, react with 5-X-2,4-dihydroxybenzaldehyde under alkaline conditions to obtain benzyl aryl ether intermediate 12;

[0049] (d) Using intermediate 12 as a raw material, it was reacted with various substituted aryl halides R3-Cl under alkaline conditions to obtain intermediate 13;

[0050] (e) Using aldehyde-containing intermediate 13 as a starting material, a reductive amination reaction was carried out with various amino or imino-containing HR4 compounds to obtain the target compound IA-2; the substituents R1, R3, R4, and X in the starting material compounds added during the reaction were the same as those in the target compound. The reaction formulas for the synthetic route are as follows:

[0051]

[0052] The beneficial effects achieved by this invention are: a novel type of PD-1 / PD-L1 small molecule inhibitor has been obtained, and it has achieved good results in protein, cell and animal level evaluations, showing good drug-like properties and development potential. Attached Figure Description

[0053] Figure 1 The compound helps restore the ability of T cells to secrete interferon-γ levels;

[0054] Figure 2 This enhances the ability of compounds to induce T cells to kill tumor cells.

[0055] Figure 3 To demonstrate the antitumor activity of the compound in a homologous mouse subcutaneous tumor-bearing model;

[0056] Figure 4Antitumor activity of the compound in a PD-1 / PD-L1 humanized mouse subcutaneous tumor model and immunocellular typing in tumor tissue;

[0057] Figure 5 Immunohistochemical staining results of tumor tissue in a homologous mouse subcutaneous tumor-bearing model;

[0058] Figure 6 The results of eosin hematoxylin staining of tissues and organs in a homologous mouse subcutaneous tumor model. Detailed Implementation

[0059] The invention will be further described below with reference to embodiments, but this does not limit the scope of the invention.

[0060] (I) Preparation of Compounds

[0061] Instruments used for determination: Nuclear magnetic resonance (NMR) spectroscopy of the compounds was performed using a Bruker AV-400 or Bruker AV-600 NMR spectrometer. Mass spectrometry of the compounds was performed using a ZAD-2F and a VG300 mass spectrometer.

[0062] Example 1: N-hydroxyethyl-[2-(3-cyanobenzoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzoxy)]benzylamine

[0063]

[0064] (1) Methyl 3-bromo-2-bromomethylbenzoate

[0065] In a 100 mL round-bottom flask, methyl 3-bromo-2-methylbenzoate (9.83 g, 42.9 mmol, 1.0 eq), Br2 (8.24 g, 51.5 mmol, 1.2 eq), and acetonitrile (60 mL) were added. After stirring and dissolving, AIBN catalyst (705 mg, 4.3 mmol, 0.1 eq) was added. After the addition was complete, the mixture was slowly heated to reflux (heated to below the boiling point of acetonitrile) and reacted overnight. After TLC analysis showed that the reactants had essentially reacted completely, the system was cooled to room temperature. After removing the solvent under reduced pressure, DCM and water were added, and the mixture was stirred to separate the layers. The organic phase was collected. The organic phase was washed again with water, dried over anhydrous sodium sulfate, concentrated to dryness, and the residue was purified by column chromatography using petroleum ether (PE):ethyl acetate (EA) at a volume ratio of 40:1, yielding 12 g of a white solid, with a yield of 90%.

[0066] (2) Methyl 3-bromo-2-monofluoromethyl benzoate

[0067] In a 100 mL round-bottom flask, add methyl 3-bromo-2-bromomethylbenzoate (12.0 g, 39 mmol, 1.0 eq), Bu4N + F- (20.4 g, 78.0 mmol, 2.0 eq), acetonitrile (60 mL), stirred overnight at room temperature. After the reaction of the starting material was confirmed to be complete by TLC, the solvent was removed by vacuum distillation, EA and water were added, and the mixture was stirred to separate the layers. The aqueous phase was extracted once with EA, and the organic phases were combined. The organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain a white solid. The solid was purified by column chromatography with PE:EA = 40:1 (v / v) to give 6.60 g of white solid, yield: 68.0%.

[0068] (3) Methyl 3-o-fluorophenyl-2-monofluoromethyl benzoate

[0069] In a 100 mL round-bottom flask, methyl 3-bromo-2-monofluoromethyl benzoate (2.02 g, 8.20 mmol, 1.0 eq), o-fluorophenylboronic acid (1.30 g, 9.30 mmol, 1.1 eq), Cs₂CO₃ (6.50 g, 20.5 mmol, 2.5 eq), and toluene (25 mL) were added. After purging with nitrogen three times, the mixture was stirred for 20 min, and then Pd(pph₃)₄ (284.3 mg, 0.24 mmol, 0.03 eq) was added. A condenser was fitted to the round-bottom flask, and the reaction mixture was purged with nitrogen three more times. The mixture was heated to 80 °C and stirred for 18 h. After the reaction was complete as detected by TLC, heating was stopped, the reaction mixture was cooled to room temperature, and filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure and extracted three times with water and ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to dryness to obtain crude product, and purified by column chromatography with eluent PE:EA = 40:1 (v / v) to obtain product: 1.60 g, yield: 81.0%. 1 HNMR (500MHz, Chloroform-d): δ7.95(ddd,J=7.6,1.7,1.0Hz,1H),7.55–7.46(m,2H),7.42(dddd,J=8.3,7.2,5.2,1.9Hz,1H),7.33(td,J=7 .6, 1.9Hz, 1H), 7.25 (td, J = 7.5, 1.2Hz, 1H), 7.18 (ddd, J = 9.6, 8.3, 1.2Hz, 1H), 5.80 (d, J = 47.6Hz, 1H), 5.37 (d, J = 45.3Hz, 1H), 3.97 (s, 3H).

[0070] (4) 3-o-fluorophenyl-2-monofluoromethyl-benzyl alcohol

[0071] In a 100 mL three-necked flask, lithium aluminum hydride (490 mg, 13.0 mmol, 2.0 eq) was added, followed by purging with nitrogen twice. Under ice bath conditions, 15 mL of THF was added to the sealed system using a syringe, resulting in bubble formation. After thorough stirring, a THF solution of methyl 3-o-fluorophenyl-2-monofluoromethyl benzoate (1.60 g, 6.50 mmol, 1.0 eq) was added dropwise using a syringe. After the addition was complete, the mixture was slowly brought to room temperature and reacted overnight. TLC analysis showed that the reactants were essentially completely converted, and the reaction was quenched with saturated sodium sulfate solution. The reaction system was extracted three times with EA, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to dryness to obtain a white solid: 1.50 g of crude product was directly added to the next step. 1 H NMR (500MHz, Chloroform-d): δ7.59 (dt, J=7.7, 1.3Hz, 1H), 7.49 (td, J=7.6, 2.4Hz, 1H), 7.41 (dddd, J=8.3, 7.2, 5.2, 1. 9Hz,1H),7.35–7.29(m,2H),7.25(td,J=7.4,1.2Hz,1H),7.21–7.14(m,1H),5.42(dd,J=98.5,47.9Hz,2H),4.91(s,2H).

[0072] (5) 3-o-fluorophenyl-2-monofluoromethyl-benzyl bromide

[0073] In a 100 mL round-bottom flask, 3-o-fluorophenyl-2-monofluoromethylbenzyl alcohol (1.50 g, 6.40 mmol, 1.0 eq), triphenylphosphine (2.50 g, 9.60 mmol, 1.5 eq), and dichloromethane (30 mL) were added. After stirring to dissolve, carbon tetrabromide (3.18 g, 9.60 mmol, 1.0 eq) was added while cooling in an ice bath. After the addition was complete, the mixture was slowly brought to room temperature and reacted for 4 h. After the reaction was confirmed to be complete by TLC, three times the amount of silica gel was added to the reaction solution, and the mixture was concentrated to dryness. The solution was then purified by column chromatography with PE:EA = 40:1 (v / v) to give a colorless oily substance: 890 mg, yield: 47.0%.

[0074] (6) 2-Hydroxy-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzaldehyde

[0075] In a 50 mL round-bottom flask, 3-o-fluorophenyl-2-monofluoromethyl-benzyl bromide (890 mg, 3.00 mmol, 1.0 eq), 2,4-dihydroxybenzaldehyde (496 mg, 3.60 mmol, 1.2 eq), Cs₂CO₃ (1.27 mg, 3.90 mmol, 1.3 eq), sodium iodide (225 mg, 1.50 mmol, 0.5 eq), and DMF (7 mL) were added. After the addition was complete, the mixture was heated to 60 °C and reacted for 2 h. After the reaction solution cooled to room temperature, water and ethyl acetate were added, and the mixture was stirred for 5 min until it separated into layers. The aqueous phase was extracted three times with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure to dryness, and purified by column chromatography with eluent PE:EA = 10:1 (v / v) to give a white solid: 393 mg, yield: 38.0%.

[0076] (7) 2-(3-cyanobenzyloxy)-4-(3-o-fluorophenyl-2-monofluoromethylbenzyloxy)benzaldehyde

[0077] In a 50 mL round-bottom flask, 2-hydroxy-4-(3-o-fluorophenyl-2-monofluoromethylbenzyloxy)benzaldehyde (393 mg, 1.10 mmol, 1.0 eq), m-cyanobenzyl chloride (185 mg, 1.20 mmol, 1.1 eq), Cs₂CO₃ (465 mg, 1.40 mmol, 1.3 eq), and DMF (7 mL) were added. After the addition was complete, the mixture was heated to 60 °C and reacted for 2 h. After the reaction solution cooled to room temperature, water and ethyl acetate were added, and the mixture was stirred for 5 min until it separated into layers. The aqueous phase was extracted three times with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to dryness to obtain a crude product of 520 mg, which was then directly used for the next step.

[0078] (8) N-hydroxyethyl-[2-(3-cyanobenzyloxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)]benzylamine

[0079] 2-(3-cyanobenzyloxy)-4-(3-o-fluorophenyl-2-monofluoromethylbenzyloxy)benzaldehyde (520 mg, 1.10 mmol, 1.0 eq) was dissolved in 4 mL of DMF, and 2-aminoethanol (134 mg, 2.20 mmol, 2.0 eq) and three drops of glacial acetic acid were added to the reaction system. After stirring at room temperature for 20 min, sodium cyanoborohydride (138 mg, 2.20 mmol, 2.0 eq) was added, and the mixture was stirred overnight at room temperature. The reaction was stopped, and the mixture was extracted with water and ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, evaporated to dryness under reduced pressure, and purified by column chromatography with DCM:MeOH = 10:1 (v / v) to give a white solid: 171 mg, yield: 30.0%, HPLC purity: 96.12%, melting point: 65-67℃.1 H NMR (500MHz, Chloroform-d): δ7.75 (d, J=1.8Hz, 1H), 7.70 (dt, J=7.9, 1.5Hz, 1H), 7. 62–7.54(m,2H),7.52–7.44(m,2H),7.41(ddd,J=5.4,2.0,1.1Hz,1H),7.34–7.29(m,3 H),7.25(dd,J=7.5,1.2Hz,1H),7.17(ddd,J=9.6,8.3,1.2Hz,1H),6.67–6.62(m,1H), 6.61(d,J=2.3Hz,1H), 5.21(d,J=28.4Hz,6H), 4.14(s,2H), 3.76(s,2H), 3.01(s,2H).

[0080] The experimental procedures of Examples 2-29 of this invention are repeated in Example 1, except that at least one of the various substituted aromatic boric acid compounds R1-B(OH)2, 5-X-2,4-dihydroxybenzaldehyde, or various substituted aryl halides R3-Cl is replaced in equimolar amounts with a starting material compound with a different substituent structure. The definitions of substituents R1, R3, R4, and X in the starting material compound added during the reaction are the same as those in the target compound. The experimental results are shown in Examples 2-29.

[0081] Example 2: N-[2-(3-cyanobenzyloxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]L-serine

[0082]

[0083] Replacing an equimolar amount of 2-aminoethanol with L-serine, and performing the remaining procedures as in Example 1, yielded a white solid: 190 mg, yield: 24.0%. HPLC purity: 96.42%, melting point: 153-156 °C. 1H NMR (500MHz, Chloroform-d): δ7.75(d,J=7.4Hz,2H),7.64(d,J=7.7Hz,1H),7.55(q,J=8.1Hz,2H) ,7.48(td,J=7.7,2.2Hz,1H),7.42(tdd,J=7.5,5.1,1.9Hz,1H),7.34–7.28(m,3H),7.25(td,J=7. 5,1.2Hz,1H),7.21–7.14(m,1H),6.68(dd,J=8.3,2.3Hz,1H),6.63(d,J=2.3Hz,1H),5.27(d,J=12 .1Hz,4H),5.18(d,J=3.6Hz,2H),4.23(t,J=10.4Hz,2H),3.94–3.81(m,2H),3.46(d,J=6.0Hz,1H).

[0084] Example 3: N-{2-[2-(3-cyanobenzyloxy)-4-(2'-fluoro-2-fluoromethyl-biphenyl-3-ylmethoxy)-benzylamine]-ethyl}-acetamide

[0085]

[0086] Replacing an equal molar amount of 2-aminoethanol with N-(2-amino-ethyl)-acetamide, and performing the remaining operations as in Example 1, the reaction of N-(2-amino-ethyl)-acetamide yielded an off-white solid of 24.0 mg, yield: 43.0%. HPLC purity: 95.77%, melting point: 72-75℃. 1 H NMR(400MHz,Chloroform-d)δ7.79(s,1H),7.71(d,J=7.9Hz,1H),7.63(d,J=7.7Hz,1H),7.59–7.23(m,8H),7.18(t,J=9.1Hz,1H),6.66(dd ,J=8.3,2.6Hz,1H),6.60(d,J=2.4Hz,1H),5.56–5.07(m,6H),4.09(s,2H),3.46(q,J=5.3,4.6Hz,2H),3.08(t,J=4.9Hz,2H),1.93(s,3H).

[0087] Example 4: N-[2-(3-cyanobenzoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzoxy)benzyl]L-proline

[0088]

[0089] Replacing an equimolar amount of 2-aminoethanol with L-proline, and performing the remaining procedures as in Example 1, yielded an off-white solid: 22.0 mg, yield: 39.0%. HPLC purity: 97.43%, melting point: 136-139 °C. 1 H NMR (400MHz, CDCl3-d) δ7.78–7.71(m,2H),7.62(d,J=7.7Hz,1H),7.58–7.38(m,4H ),7.33(q,J=8.5,7.7Hz,3H),7.24(t,J=7.5Hz,1H),7.17(t,J=9.1Hz,1H),6.66–6 .58(m,2H),5.20(d,J=23.0Hz,6H),4.34–4.21(m,2H),3.90(t,J=6.9Hz,1H),3.57 (dt,J=11.3,5.4Hz,1H),2.92(m,1H),2.30(t,J=6.8Hz,2H),1.95(t,J=7.1Hz,2H).

[0090] Example 5: N-hydroxyethyl-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)]benzylamine

[0091]

[0092] Yield: 28.0%. HPLC purity: 95.43%. Melting point: 151-153℃. 1 H NMR (500MHz, CDCl3-d): δ8.89 (dd, J=6.8, 2.1Hz, 1H), 8.78 (dd, J=4.3, 2.0Hz, 1H), 8.16 (t, J= 2.1Hz,1H),7.57(d,J=7.7Hz,1H),7.49–7.43(m,1H),7.43–7.36(m,1H),7.31(ddd,J=7.3,4. 9,1.9Hz,3H),7.23(td,J=7.5,1.2Hz,1H),7.16(ddd,J=9.5,8.3,1.2Hz,1H),6.71–6.61(m,2 H), 5.48-5.13 (m, J = 32.7Hz, 6H), 4.11 (d, J = 15.1Hz, 2H), 3.75 (t, J = 4.9Hz, 2H), 3.00 (s, 2H).

[0093] Example 6: N-acetamide ethyl-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)]benzylamine

[0094]

[0095] Yield: 25.0%. HPLC purity: 98.43%. Melting point: 81-84℃. 1 H NMR (500MHz, CDCl3-d): δ8.92(s,1H),8.82(s,1H),8.20(d,J=2.0Hz,1H),7.57(d,J=7.6Hz,1H) ,7.47(td,J=7.7,2.3Hz,1H),7.40(dddd,J=8.2,7.2,5.2,1.9Hz,1H),7.32(dd,J=7.5,1.7Hz,3H ),7.24(td,J=7.5,1.2Hz,1H),7.16(ddd,J=9.5,8.3,1.1Hz,1H),6.67(dd,J=8.4,2.2Hz,1H),6. 62(d,J=2.2Hz,1H),5.24(d,J=33.0Hz,6H),4.10(s,2H),3.46(s,2H),3.11(s,2H),1.93(s,3H).

[0096] Example 7: N-[2-(5-cyanopyridine-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]-3-hydroxypyrrolidine

[0097]

[0098] Yield: 31.0%. HPLC purity: 97.32%. Melting point: 155-157℃. 1 H NMR (500MHz, CDCl3-d): δ8.92(s,1H),8.83(s,1H),8.18(t,J=2.0Hz,1H),7.59(d,J=7.7Hz,1H),7.49(td ,J=7.7,2.2Hz,1H),7.46–7.37(m,2H),7.33(td,J=7.6,1.9Hz,2H),7.24(d,J=1.2Hz,1H),7.17(ddd,J=9 .6,8.2,1.1Hz,1H),6.71(dd,J=8.4,2.2Hz,1H),6.64(d,J=2.3Hz,1H),5.61–5.10(m,6H),4.59(s,1H),4 .25(t,J=8.0Hz,2H),3.48–3.41(m,1H),3.31(d,J=17.8Hz,3H),2.28–2.22(m,1H),2.11(d,J=7.3Hz,1H).

[0099] Example 8: N-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]piperidine-2-carboxylic acid

[0100]

[0101] Yield: 21.0%. HPLC purity: 98.22%. Melting point: 174-176℃. 1 H NMR (500MHz, CDCl3-d): δ8.90–8.81(m,2H),8.20(s,1H),7.55(d,J=7.7Hz,1H),7.48(ddt,J=9.9,7. 7,3.3Hz,2H),7.44–7.38(m,1H),7.32(td,J=7.6,1.8Hz,2H),7.24(td,J=7.5,1.2Hz,1H),7.17(ddd, J=9.6,8.3,1.1Hz,1H),6.70(d,J=8.2Hz,1H),6.59(d,J=2.2Hz,1H),5.55–5.05(m,6H),4.53(d,J=1 2.9Hz, 2H), 3.56 (s, 1H), 3.43 (s, 2H), 2.66 (s, 1H), 2.25 (d, J = 13.7Hz, 1H), 1.93 (s, 2H), 1.75 (s, 2H).

[0102] Example 9: N-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]glycine

[0103]

[0104] Yield: 39.0%. HPLC purity: 97.50%. Melting point: 162-165℃. 1 H NMR (500MHz, CDCl3-d): δ8.83(s,1H),8.59(s,1H),7.49(d,J=7.2Hz,1H),7.42–7.30(m,4H),7.28-7.25(m,2H),7.23– 7.18(m,1H),7.13(t,J=9.0Hz,1H),6.55(d,J=11.9Hz,2H),5.55–4.88(m,6H),4.10(s,2H),3.42(s,2H),2.10(s,1H).

[0105] Example 10: N-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]L-proline

[0106]

[0107] Yield: 28.0%. HPLC purity: 97.88%. Melting point: 137-139℃. 1 H NMR (500MHz, CDCl3-d): δ8.91(d,J=2.1Hz,1H),8.86(d,J=1.9Hz,1H),8.20(t,J=2.1Hz,1H),7.58(d,J=7.7Hz,1H), 7.49(td,J=7.7,2.2Hz,1H),7.42(dddd,J=8.3,7.2,5.2,1.9Hz,1H),7.37–7.28(m,3H),7.26(dd,J=7.5,1.1Hz,1H), 7.18(ddd,J=9.5,8.3,1.1Hz,1H),6.66(dd,J=8.3,2.2Hz,1H),6.62(d,J=2.2Hz,1H),5.62–5.08(m,6H),4.27–4.18( m,2H),3.79(dd,J=9.1,4.9Hz,1H),3.56–3.47(m,1H),2.94–2.85(m,1H),2.33–2.24(m,2H),1.97(d,J=12.6Hz,2H).

[0108] Example 11: N-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]pyrrolidine-3-carboxylic acid

[0109]

[0110] Yield: 22.0%. HPLC purity: 95.88%. Melting point: 82-85℃. 1 H NMR (500MHz, CDCl3-d): δ8.87(d,J=10.9Hz,2H),8.10(s,1H),7.59(d,J=7.7Hz,1H), 7.53–7.38(m,3H),7.34(td,J=7.5,1.8Hz,2H),7.29–7.22(m,1H),7.18(dd,J=9.8,8 .2Hz,1H),6.72–6.67(m,1H),6.60(d,J=2.2Hz,1H),5.58–5.21(m,4H),5.15(s,2H), 4.25–4.04(m,2H),3.71(s,2H),3.30(s,2H),3.06(s,1H),2.35(s,1H),2.22(s,1H).

[0111] Example 12: N-[2-(5-cyanopyridine-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]acetidine-3-carboxylic acid

[0112]

[0113] Yield: 19.0%. HPLC purity: 96.44%. Melting point: 148-152℃. 1 H NMR (500MHz, CDCl3-d): δ8.84(d,J=16.3Hz,2H),8.18(s,1H),7.58(t,J=7.5Hz,1 H),7.48(t,J=6.9Hz,1H),7.45–7.36(m,2H),7.33(dd,J=7.5,5.3Hz,2H),7.27–7. 23(m,1H),7.18(t,J=9.0Hz,1H),6.69(s,1H),6.59(d,J=9.6Hz,1H),5.57–5.21( m,4H),5.15(s,2H),4.29(s,2H),4.17(s,2H),3.97(d,J=8.5Hz,2H),3.30(s,1H).

[0114] Example 13: N-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]L-serine

[0115]

[0116] Yield: 26.0%. HPLC purity: 97.35%. Melting point: 137-140℃. 1 H NMR (500MHz, CDCl3-d): δ8.85(s,1H),8.62(s,1H),8.11(s,1H),7.47(d,J=6.4Hz,1H),7.37(d,J=7.8Hz,3H),7.28(s,2 H),7.20(t,J=7.4Hz,1H),7.13(t,J=9.0Hz,1H),6.55(s,2H),5.10(s,6H),4.17(s,2H),4.02–3.73(m,2H),3.48(s,1H).

[0117] Example 14: N-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]D-serine

[0118]

[0119] Yield: 24.0%. HPLC purity: 96.54%. Melting point: 139-142℃. 1 HNMR(500MHz, CDCl3-d): δ8.88–8.85(m,1H),8.67(s,1H),8.15(s,1H),7.5 2(d,J=7.8Hz,1H),7.39(dddd,J=17.6,15.5,7.7,4.4Hz,3H),7.31-7.28(m, 2H),7.22(td,J=7.5,1.2Hz,1H),7.17–7.13(m,1H),6.63–6.55(m,2H),5.6 5–5.01(m,6H),4.24(d,J=13.3Hz,2H),3.88(d,J=13.6Hz,2H),3.47(s,1H).

[0120] Example 15: N-{2-(3-cyanobenzyloxy)-4-[3-(2,5-bisfluorophenyl)-2-monofluoromethyl-benzyloxy]benzyl}L-serine

[0121] Yield: 13.1%. HPLC purity: 97.69%. Melting point: 125-127℃. 1 H NMR(500MHz,Chloroform-d)δ7.73–7.30(m,6H),7.26(s,3H),7.22–6.94(m,3H),6.48(t,J=2 4.7Hz,2H),5.70–5.22(m,2H),5.04(d,J=16.6Hz,4H),4.05(d,J=143.6Hz,4H),3.44(s,1H).

[0122] Example 16: N-{2-(5-cyanopyridine-3-methoxy)-4-[3-(2,5-bisfluorophenyl)-2-monofluoromethyl-benzyloxy]benzyl}L-serine

[0123]

[0124] Yield: 19.0%. HPLC purity: 96.77%. Melting point: 102-106℃. 1H NMR (500MHz, CDCl3-d): δ8.83(s,1H),8.62(s,1H),8.09(s,1H),7.55(dd,J=19.3,7.9Hz,1H),7.46–7.30(m,2H),7.28(s,1H),7.19(t, J=7.7Hz,1H),7.09(dd,J=4.6,2.4Hz,2H),6.54(d,J=20.2Hz,2H),5.65–5.01(m,6H),4.20(s,2H),3.87(s,2H),3.48(d,J=6.6Hz,1H).

[0125] Example 17: N-acetamide ethyl-{2-(3-cyanobenzoxy)-4-[3-(2,5-bisfluorophenyl)-2-monofluoromethyl-benzoxy]}benzylamine

[0126]

[0127] Yield: 35.3%. HPLC purity: 96.98%. Melting point: 128-130℃. 1 H NMR(600MHz,Chloroform-d)δ7.80–7.75(m,1H),7.72(t,J=8.9Hz,1H),7.64–7.57(m,2H),7.53 (t,J=7.8Hz,1H),7.49(td,J=7.7,2.2Hz,1H),7.32(dt,J=15.4,7.8Hz,3H),7.18–7.07(m,2H),7 .04(ddd,J=8.7,5.8,3.1Hz,1H),6.65(dd,J=8.4,2.3Hz,1H),6.60(d,J=2.3Hz,1H),5.24(s,2H) ,5.18(d,J=14.3Hz,2H),4.13(s,2H),3.48(q,J=5.2Hz,2H),3.13(t,J=5.0Hz,2H),1.93(s,3H).

[0128] Example 18: N-(1,2-dihydroxyethyl)-{2-(3-cyanobenzyloxy)-4-[3-(2,5-bisfluorophenyl)-2-monofluoromethyl-benzyloxy]}benzylamine

[0129] Yield: 30.5%. HPLC purity: 98.22%. Melting point: 145-147℃. 1H NMR(600MHz,Chloroform-d)δ7.73(d,J=9.0Hz,1H),7.62(dd,J=36.7,7.8Hz,3H),7.53–7.39(m,2H),7.34–7.19(m,2H),7.17–6.97(m,2H),6.68 –6.46(m,2H),5.51(dd,J=191.1,41.9Hz,2H),5.27–5.02(m,4H),3.95–3.86(m,2H),3.64(dd,J=11.6,3.8Hz,1H),3.52(dd,J=11.6,4.8Hz,1H).

[0130] Example 19: N-hydroxyethyl-{2-(3-cyanobenzoxy)-4-[3-(2,5-bisfluorophenyl)-2-monofluoromethyl-benzoxy]}benzylamine

[0131]

[0132] Yield: 27.5%. HPLC purity: 96.14%. Melting point: 137-140℃. 1 H NMR(600MHz,Chloroform-d)δ8.03(s,1H),7.78(s,1H),7.74(d,J=8.0Hz,1H),7.63(d,J=7.7 Hz,1H),7.60(d,J=7.7Hz,1H),7.56–7.47(m,2H),7.34(dd,J=15.4,7.9Hz,2H),7.29(s,1H),7 .14(ddq,J=14.7,7.3,3.9,3.3Hz,2H),7.06(ddd,J=8.6,5.7,3.0Hz,1H),6.67(dd,J=8.4,2.3 Hz,1H),6.63(d,J=2.2Hz,1H),5.23(d,J=24.9Hz,5H),4.19(s,2H),3.80(s,2H),3.07(s,2H).

[0133] Example 20: N-acetamide ethyl-{2-(5-cyanopyridine-3-methoxy)-4-[3-(2,5-bisfluorophenyl)-2-monofluoromethyl-benzyloxy]}benzylamine

[0134]

[0135] Yield: 27.3%. HPLC purity: 97.21%. Melting point: 98-101℃. 1H NMR(500MHz,Chloroform-d)δ8.94–8.80(m,2H),8.18(dt,J=7.6,2.2Hz,1H),7.61 (dd,J=16.0,7.8Hz,1H),7.42(dd,J=24.8,5.8Hz,2H),7.36–7.28(m,2H),7.18–7. 00(m,1H),6.67–6.48(m,2H),5.68(d,J=47.7Hz,1H),5.17(d,J=7.0Hz,4H),3.96( d,J=2.9Hz,2H),3.38(d,J=5.5Hz,2H),2.97–2.86(m,3H),1.95(d,J=14.4Hz,4H).

[0136] Example 21: N-hydroxyethyl-{2-(5-cyanopyridine-3-methoxy)-4-[3-(2,5-bisfluorophenyl)-2-monofluoromethyl-benzyloxy]}benzylamine

[0137]

[0138] Yield: 17.0%. HPLC purity: 95.84%. Melting point: 145-147℃. 1 H NMR (500MHz, CDCl3-d): δ8.89(s,1H),8.77(s,1H),8.15(dt,J=5.6,2.4Hz,1H),7.61(dd,J=7.7,2.6Hz,1H),7.48(dt,J=10. 2,7.7Hz,1H),7.28(s,3H),7.20–6.96(m,2H),6.69–6.59(m,2H),5.52–4.98(m,6H),4.14(s,2H),3.76(s,2H),3.02(s,2H).

[0139] Example 22: N-hydroxyethyl-{2-(3-cyanobenzoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-monofluoromethyl-benzoxy]}benzylamine

[0140]

[0141] Yield: 20.0%. HPLC purity: 95.66%. Melting point: 139-143℃. 1H NMR (500MHz, CDCl3-d): δ7.77(t,J=1.6Hz,1H),7.74–7.69(m,1H),7.69–7.62(m,1H),7.55–7.47(m,2H), 7.43(td,J=7.6,2.3Hz,1H),7.38–7.30(m,2H),6.94(d,J=8.2Hz,1H),6.91(d,J=2.2Hz,1H),6.88–6.81(m ,1H),6.65(dd,J=8.3,2.4Hz,1H),6.59(d,J=2.3Hz,1H),5.46(s,1H),5.37(s,1H),5.23(d,J=1.5Hz,2H), 5.16(d,J=3.4Hz,2H),4.33(s,4H),3.98(s,2H),3.67(dd,J=6.1,3.7Hz,2H),2.89(dd,J=5.9,3.3Hz,2H).

[0142] Example 23: N-hydroxyethyl-{2-(5-cyanopyridine-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-monofluoromethyl-benzyloxy]}benzylamine

[0143]

[0144] Yield: 24.1%. HPLC purity: 98.67%. Melting point: 133-136℃. 1 H NMR(500MHz,Chloroform-d)δ8.91(d,J=2.2Hz,1H),8.83(d,J=2.0Hz,1H),8.22(t,J=2.1Hz,1H),7.49(d,J =7.7Hz,1H),7.43(td,J=7.6,2.3Hz,1H),7.33(q,J=8.2,7.4Hz,2H),6.96–6.88(m,3H),6.87–6.80(m,1H), 6.67(dd,J=8.4,2.3Hz,1H),6.61(d,J=2.3Hz,1H),5.47(d,J=9.5Hz,1H),5.37(d,J=9.6Hz,2H),5.25(d,J= 10.5Hz,2H),5.20(s,2H),4.32(d,J=2.6Hz,6H),4.06(s,2H),3.65(d,J=4.9Hz,2H),2.95(d,J=8.9Hz,3H).

[0145] Example 24: N-{2-(5-cyanopyridin-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-monofluoromethyl-benzyloxy]benzyl}-L-serine

[0146]

[0147] Yield: 35.4%. HPLC purity: 98.46%. Melting point: 110-113℃. 1 H NMR(600MHz,DMSO-d6)δ9.01(dd,J=14.7,2.1Hz,2H),8.51(t,J=2.0Hz,1H),7.56(d,J=7.6Hz,1H) ,7.49(td,J=7.6,2.3Hz,1H),7.34(dd,J=20.4,8.0Hz,2H),6.96(d,J=8.2Hz,1H),6.84(ddd,J=30 .0,7.5,2.2Hz,3H),6.73(dd,J=8.5,2.3Hz,1H),5.40(d,J=48.1Hz,2H),5.34–5.21(m,4H),4.30( s,4H),4.16–4.01(m,2H),3.75(dd,J=11.7,4.2Hz,2H),3.64(dd,J=11.4,6.8Hz,2H),3.58(s,1H).

[0148] Example 25: N-acetamide ethyl-{2-(5-cyanopyridin-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-monofluoromethyl-benzyloxy]}benzylamine

[0149]

[0150] Yield: 32.2%. HPLC purity: 98.89%, melting point: 105-107℃. 1H NMR(400MHz,Chloroform-d)δ8.93(s,1H),8.81(s,1H),8.22(s,1H),7.47(d,J=7.6Hz,1H),7.44– 7.35(m,2H),7.32(d,J=7.6Hz,1H),7.24(d,J=20.5Hz,1H),6.95–6.86(m,2H),6.82(dd,J=8.5,2. 1Hz,1H),6.65(d,J=8.3Hz,1H),6.59(d,J=2.2Hz,1H),5.45(s,2H),5.33(s,2H),5.20(d,J=3.8Hz ,5H),4.30(s,5H),4.09(s,2H),3.50–3.40(m,3H),3.06(d,J=6.6Hz,2H),1.95(d,J=19.1Hz,4H).

[0151] Example 26: N-Dimethylaminoethyl-{2-(5-cyanopyridin-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-monofluoromethyl-benzyloxy]}benzylamine

[0152]

[0153] Yield: 35.5%. HPLC purity: 96.33%, melting point: 127-129℃. 1 H NMR(4+00MHz,Chloroform-d)δ8.89(d,J=8.4Hz,2H),8.02(dd,J=5.8,3.8Hz,2H),7.55–7.43( m,2H),7.37(dt,J=7.6,1.5Hz,1H),7.31(d,J=8.4Hz,2H),6.98–6.90(m,2H),6.86(dd,J=8.3, 2.1Hz,1H),6.77(dd,J=8.5,2.3Hz,1H),6.65(d,J=2.3Hz,1H),5.45(d,J=48.1Hz,2H),5.31(t ,J=1.8Hz,2H),5.17(s,2H),4.34(s,4H),4.12(s,2H),2.98(s,2H),2.90(s,2H),2.57(s,6H).

[0154] Example 27: N-{2-(5-cyanopyridin-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-monofluoromethyl-benzyloxy]benzyl}-pyrrolidine-3-carboxylic acid

[0155]

[0156] Yield: 31.8%. HPLC purity: 96.38%. Melting point: 145-128℃. 1 H NMR(400MHz,Chloroform-d)δ8.92–8.79(m,2H),8.06(d,J=33.4Hz,1H),7.56 –7.28(m,4H),6.96–6.79(m,3H),6.69(d,J=7.6Hz,1H),6.58(d,J=2.1Hz,1H) ,5.47(s,1H),5.35(s,1H),5.25(s,2H),5.15(s,2H),4.31(s,4H),4.27–4.06 (m,2H),3.73(s,1H),3.30(s,2H),3.14–2.76(m,3H),2.28(d,J=49.7Hz,2H).

[0157] Example 28: N-{2-(5-cyanopyridin-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-monofluoromethyl-benzyloxy]benzyl}-glycine

[0158]

[0159] Yield: 23.0%. HPLC purity: 96.22%. Melting point: 162-164℃. 1 H NMR (400MHz, CDCl3-d): δ8.90(s,1H),8.69(s,1H),8.20(s,1H),8.11(d,J=3.9Hz,1H),7.46(d,J=7.8Hz,1H),7.43–7.35(m,2H),7.31(d ,J=7.7Hz,1H),7.00–6.79(m,3H),6.57(s,2H),5.44(s,1H),5.32(s,1H),5.17(d,J=13.6Hz,4H),4.31(s,4H),4.20(s,2H),3.53(s,2H).

[0160] Example 29: N-{2-(5-cyanopyridin-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-monofluoromethyl-benzyloxy]benzyl}piperidine-2-carboxylic acid

[0161]

[0162] Yield: 21.0%. HPLC purity: 97.34%. Melting point: 140-142℃.1 HNMR (400MHz, CDCl3-d): δ8.90–8.84(m,2H),8.15(s,1H),7.47(dtd,J=14.9,7.5,1.9Hz,3H) ,7.39–7.33(m,1H),6.98–6.82(m,3H),6.75–6.69(m,1H),6.60(d,J=2.1Hz,1H),5.48(s,1H) ,5.36(s,1H),5.23(d,J=24.4Hz,4H),4.46(s,2H),4.33(s,4H),3.59(d,J=10.2Hz,1H),3.50 (d,J=12.0Hz,1H),2.74(s,1H),2.29(d,J=13.8Hz,1H),1.84(d,J=34.0Hz,4H),1.44(s,1H).

[0163] Example 30: N-hydroxyethyl-[2-(3-cyanobenzoxy)-4-(3-o-fluorophenyl-2-bisfluoromethyl-benzoxy)]benzylamine

[0164]

[0165] (1) 3-o-fluorophenyl-2-difluoromethylbenzyl alcohol

[0166] In a 100 mL round-bottom flask, 3-bromo-2-difluoromethylbenzyl alcohol (580 mg, 2.40 mmol, 1.0 eq), o-fluorophenylboronic acid (364 mg, 2.60 mmol, 1.1 eq), Cs₂CO₃ (1954 mg, 6.00 mmol, 2.5 eq), and toluene (20 mL) were added. After purging with nitrogen three times, the mixture was stirred for 20 min, and then PdCl₂ (dppf) (88.0 mg, 0.120 mmol, 0.05 eq) was added. A condenser was fitted to the round-bottom flask, and the reaction mixture was purged with nitrogen three more times. The mixture was heated to 80 °C and stirred for 18 h. After the reaction was complete as detected by TLC, heating was stopped, the reaction mixture was cooled to room temperature, and filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure and extracted three times with water and ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to dryness to obtain crude product, and purified by column chromatography with eluent PE:EA = 10:1 (v / v) to obtain product: 500 mg, yield: 82.6%. 1HNMR (500MHz, CDCl3-d): δ7.75 (d, J=7.7Hz, 1H), 7.60–7.54 (m, 1H), 7.47–7.39 (m, 1H), 7. 31–7.23(m,3H),7.22–7.17(m,1H),6.57(d,J=1.2Hz,1H),5.06–5.02(m,2H),2.02(s,1H).

[0167] (2) 3-o-fluorophenyl-2-difluoromethyl-benzyl bromide

[0168] In a 100 mL round-bottom flask, 3-o-fluorophenyl-2-difluoromethylbenzyl alcohol (500 mg, 2.00 mmol, 1.0 eq), triphenylphosphine (787 mg, 3.00 mmol, 1.5 eq), and dichloromethane (15 mL) were added. After stirring to dissolve, carbon tetrabromide (995 mg, 3.00 mmol, 1.5 eq) was added while cooling in an ice bath. After the addition was complete, the mixture was slowly brought to room temperature and reacted for 4 h. After the reaction was confirmed to be complete by TLC, three times the amount of silica gel was added to the reaction solution, and the mixture was concentrated to dryness. The solution was then purified by column chromatography with PE:EA = 40:1 (v / v) to give a colorless oily substance: 470 mg, yield: 74.6%. 1 H NMR (600MHz, CDCl3-d): δ7.68 (dd, J=7.8, 1.2Hz, 1H), 7.55 (tt, J=7.7, 1.2Hz, 1H), 7.48–7.42 (m, 1H), 7.32–7.25(m,3H),7.21(ddd,J=9.5,8.3,1.1Hz,1H),6.60(td,J=53.5,1.3Hz,1H),4.93–4.81(m,2H).

[0169] (3) 2-Hydroxy-4-(3-o-fluorophenyl-2-bisfluoromethyl-benzyloxy)benzaldehyde

[0170] In a 50 mL round-bottom flask, 3-o-fluorophenyl-2-difluoromethylbenzyl bromide (158 mg, 0.500 mmol, 1.0 eq), 2,4-dihydroxybenzaldehyde (83 mg, 0.6 mmol, 1.2 eq), Cs₂CO₃ (211.8 mg, 0.650 mmol, 1.3 eq), sodium iodide (37.0 mg, 0.250 mmol, 0.5 eq), and DMF (5 mL) were added. After the addition was complete, the mixture was heated to 60 °C and reacted for 2 h. After the reaction solution cooled to room temperature, water and ethyl acetate were added, and the mixture was stirred for 5 min until it separated into layers. The aqueous phase was extracted three times with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure to dryness, and purified by column chromatography with eluent PE:EA = 10:1 (v / v) to give 100 mg of white solid, yield: 53.7%.

[0171] (4) 2-(3-cyanobenzyloxy)-4-(3-o-fluorophenyl-2-bisfluoromethyl-benzyloxy)benzaldehyde

[0172] In a 50 mL round-bottom flask, 2-hydroxy-4-(3-o-fluorophenyl-2-difluoromethylbenzyloxy)benzaldehyde (100 mg, 0.270 mmol, 1.0 eq), m-cyanobenzyl chloride (45.5 mg, 0.300 mmol, 1.1 eq), and Cs₂CO₃ (114 mg, 0.350 mmol, 1.3 eq) were added. After the addition was complete, the mixture was heated to 60 °C and reacted for 2 h. After the reaction solution cooled to room temperature, water and ethyl acetate were added, and the mixture was stirred for 5 min until it separated into layers. The aqueous phase was extracted three times with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to dryness to obtain a crude product of 130 mg, which was then directly used for the next step.

[0173] (5) N-hydroxyethyl-[2-(3-cyanobenzyloxy)-4-(3-o-fluorophenyl-2-bisfluoromethyl-benzyloxy)]benzylamine

[0174] 2-(3-cyanobenzyloxy)-4-(3-o-fluorophenyl-2-difluoromethylbenzyloxy)benzaldehyde (73.1 mg, 0.150 mmol, 1.0 eq) was dissolved in 2 mL of DMF, and 2-aminoethanol (18.3 mg, 0.300 mmol, 2.0 eq) and three drops of glacial acetic acid were added to the reaction system. After stirring at room temperature for 20 min, sodium cyanoborohydride (18.8 mg, 0.300 mmol, 2.0 eq) was added, and the mixture was stirred overnight at room temperature. The reaction was stopped, and the mixture was extracted with water and ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, evaporated to dryness under reduced pressure, and purified by column chromatography with DCM:MeOH = 10:1 (v / v) to give a white solid: 34.0 mg, yield: 42.5%, HPLC purity: 96.51%, melting point: 71-75℃.1 H NMR (500MHz, CDCl3-d): δ7.75–7.66(m,3H),7.60(dt,J=7.7,1.4Hz,1H),7.50(t,J=7.9Hz,2H),7.48–7.39(m,1H),7.31–7.2 4(m,4H),7.20(s,1H),6.71–6.41(m,3H),5.41(s,2H),5.19–5.10(m,2H),4.03(s,2H),3.75–3.69(m,2H),2.95–2.89(m,2H).

[0175] The experimental procedures of Examples 31-48 of this invention are repeated in Example 30, except that at least one of the various substituted aromatic boric acid compounds R1-B(OH)2, 5-X-2,4-dihydroxybenzaldehyde or various substituted aryl halides R3-Cl is replaced in equimolar amounts with a starting compound with a different substituent structure. The definitions of substituents R1, R3, R4, and X in the starting compound added during the reaction are the same as those in the target compound. The experimental results are shown in Examples 31-48.

[0176] Example 31: N-[2-(3-cyanobenzyloxy)-4-(3-o-fluorophenyl-2-bisfluoromethyl-benzyloxy)-benzyl]L-serine

[0177] Replacing an equimolar amount of 2-aminoethanol with L-serine, and performing the remaining procedures as in Example 30, yielded a white solid: 38.0 mg, yield: 45.0%. HPLC purity: 97.31%, melting point: 122-125 °C. 1 H NMR (500MHz, CDCl3-d): δ7.65(d,J=8.6Hz,3H),7.50–7.38(m,4H),7.27(m,1H),7.26–7.21(m,3H),7.18(dd,J=9.7,8.1 Hz,1H),6.69–6.39(m,3H),5.32(d,J=2.3Hz,2H),5.16–4.94(m,2H),4.21(d,J=26.6Hz,2H),3.93(s,2H),3.45(s,1H).

[0178] Example 32: N-hydroxyethyl-[2-(5-cyanopyridine-3-methoxy)-4-(3-o-fluorophenyl-2-bisfluoromethyl-benzyloxy]benzylamine

[0179]

[0180] Yield: 31.0%. HPLC purity: 95.81%. Melting point: 92-95℃. 1 H NMR (500MHz, CDCl3-d): δ8.92–8.88(m,1H),8.79(d,J=1.9Hz,1H),8.16(t,J= 2.0Hz,1H),7.71(d,J=7.8Hz,1H),7.53(t,J=7.8Hz,1H),7.44(dddd,J=8.5,7 .0,5.2,2.3Hz,1H),7.32–7.23(m,4H),7.23–7.16(m,1H),6.73–6.42(m,3H), 5.50–5.34(m,2H),5.24–5.14(m,2H),4.12(s,2H),3.76(s,2H),3.00(s,2H).

[0181] Example 33: N-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-bisfluoromethyl-benzyloxy)-benzyl]L-serine

[0182]

[0183] Yield: 30.0%. HPLC purity: 96.11%. Melting point: 149-152℃. 1 H NMR (500MHz, CDCl3-d): δ8.85(s,1H),8.64(s,1H),8.08(s,1H),7.64(d,J=7.8Hz,1H),7.42(dq,J=14.2,7.1,6.6Hz,2H),7.23( q,J=9.6,8.3Hz,4H),7.16(t,J=8.9Hz,1H),6.65–6.36(m,3H),5.30(s,2H),5.09(s,2H),4.21(s,2H),3.92(s,2H),3.50(s,1H).

[0184] Example 34: N-{2-[2-(5-cyano-pyridin-3-yl-methoxy)-4-(2-bisfluoromethyl-2'-fluoro-biphenyl-3-yl-methoxy)-benzylamine]-ethyl}-

[0185]

[0186] Yield: 36.0%. HPLC purity: 96.83%. Melting point: 98-102℃. 1H NMR (400MHz, CDCl3-d) δ8.92 (s, 1H), 8.84 (s, 1H), 8.21 (d, J = 3.0Hz, 1H), 8.02 (s, 1H) ,7.72(d,J=7.8Hz,1H),7.59–7.50(m,2H),7.48–7.42(m,1H),7.34(d,J=8.5Hz,1H),7 .27(s,1H),7.20(t,J=8.9Hz,1H),6.80–6.52(m,3H),5.44(s,2H),5.20(d,J=4.0Hz, 2H), 4.10 (s, 2H), 3.48 (d, J = 5.6Hz, 2H), 3.11 (d, J = 5.3Hz, 2H), 1.94 (d, J = 2.8Hz, 3H).

[0187] Example 35: N-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-bisfluoromethyl-benzyloxy)-benzyl]L-proline

[0188]

[0189] Yield: 51.0%. HPLC purity: 97.32%. Melting point: 156-158℃. 1 H NMR (400MHz, CDCl3-d) δ8.90(s,1H),8.83(s,1H),8.20(s,1H),7.71(d,J=7.8Hz,1H),7.54( t,J=7.9Hz,1H),7.45(q,J=7.0,6.1Hz,1H),7.28(d,J=6.9Hz,4H),7.20(t,J=9.3Hz,1H),6. 74–6.52(m,3H),5.45(s,2H),5.21(d,J=4.8Hz,2H),4.29(s,2H),3.86(t,J=7.2Hz,1H),3.5 8(dd,J=11.0,5.5Hz,1H),2.91(d,J=16.6Hz,1H),2.29(q,J=7.3Hz,2H),1.97–1.92(m,2H).

[0190] Example 36: N-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-bisfluoromethyl-benzyloxy)-benzyl]L-glycine

[0191]

[0192] Yield: 15.0%. HPLC grade: 95.11%, melting point: 171-174℃. 1H NMR(400MHz, CDCl-d)δ8.86(s,1H),8.68(s,1H),8.17(s,1H),7.68(d,J=8.0Hz,1H),7.53–7.42( m,2H),7.31–7.14(m,5H),6.79–6.36(m,3H),5.36(s,2H),5.11(s,2H),4.16(s,2H),3.42(s,2H).

[0193] Example 37: N-hydroxyethyl-[2-(5-cyanopyridine-3-methoxy)-4-(3-(2,5-difluorophenyl)-2-bisfluoromethyl-benzyloxy]benzylamine

[0194]

[0195] Yield: 21.1%. HPLC purity: 97.99%. 1 H NMR(400MHz,Chloroform-d)δ8.90(d,J=20.1Hz,4H),8.14(d,J=48.8Hz,2H),7.55–7.29(m,3H),6.60(d,J=1 0.2Hz,2H),5.32(d,J=19.1Hz,1H),5.17(d,J=25.5Hz,5H),3.99(s,1H),3.70(s,3H),2.92(t,J=14.9Hz,2H).

[0196] Example 38: N-hydroxyethyl-{2-(3-cyanobenzoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-bisfluoromethyl-benzoxy]}benzylamine

[0197]

[0198] Yield: 25.4%. HPLC purity: 99.29%. 1 H NMR(400MHz,Chloroform-d)δ7.79–7.57(m,3H),7.47(dt,J=20.1,8.0Hz,2H),7.37–7.17(m,2H),6.93(d,J=7.9Hz,1H),6.87–6.66( m,3H),6.65–6.46(m,2H),5.42(d,J=33.0Hz,2H),5.17(s,1H),4.31(s,4H),4.10(d,J=27.0Hz,2H),3.79–3.60(m,3H),2.94(s,1H).

[0199] Example 39: N-{2-(3-cyanobenzyloxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-bisfluoromethyl-benzyloxy]-benzylamine]}-ethyl-acetamide

[0200]

[0201] Yield: 32.3%. HPLC purity: 96.88%, melting point: 105-107℃. 1 H NMR(400MHz,Chloroform-d)δ7.82–7.67(m,2H),7.61(t,J=9.2Hz,3H),7.48(dt ,J=21.9,7.9Hz,2H),7.36(d,J=8.0Hz,1H),7.25(d,J=9.4Hz,2H),6.93(d,J=8. 1Hz,1H),6.88–6.68(m,3H),6.66–6.46(m,2H),5.34(s,2H),5.20–4.94(m,2H), 4.32(s,4H),4.01(s,3H),3.42(d,J=5.7Hz,3H),3.08–2.89(m,2H),1.91(s,3H).

[0202] Example 40: N-hydroxyethyl-{2-(5-cyanopyridine-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-bisfluoromethyl-benzyloxy]}benzylamine

[0203]

[0204] Yield: 35.3%. HPLC purity: 96.77%, melting point: 127-129℃. 1 H NMR(400MHz,Chloroform-d)δ9.09–8.55(m,2H),8.16(t,J=2.0Hz,1H),7.63(d, J=7.7Hz,1H),7.45(t,J=7.8Hz,1H),7.29(d,J=8.9Hz,1H),7.25(d,J=7.7Hz,1H ),6.93(d,J=8.2Hz,1H),6.87–6.80(m,1H),6.79–6.70(m,2H),6.67–6.55(m,2H ),5.40(s,2H),5.23(s,2H),4.31(s,5H),3.78(s,2H),3.67(s,7H),3.05(s,2H).

[0205] Example 41: N-hydroxyethyl-{2-(pyridin-4-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-bisfluoromethyl-benzyloxy]}benzylamine

[0206]

[0207] Yield: 19.8%. HPLC purity: 97.29%, melting point: 105-107℃. ¹H NMR (600MHz, Chloroform-d): δ 8.51 (d, J = 17.5Hz, 3H), 7.62 (d, J = 7.8Hz, 1H), 7.45 (t, J = 7.8Hz, 1H), 7.40–7.19 (m, 4H), 6.93 (d, J = 8.2Hz, 1H), 6.87–6.69 (m, 3H), 6.67–6.54 (m, 2H), 5.39 (s, 2H), 5.14 (s, 2H), 4.31 (s, 4H), 4.20 (s, 2H), 3.86–3.62 (m, 3H), 3.14–2.93 (m, 2H).

[0208] Example 42: N-{2-(5-cyanopyridin-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-bisfluoromethyl-benzyloxy]-benzyl}D-proline

[0209]

[0210] Yield: 33.8%. HPLC purity: 98.98%. 1 H NMR(400MHz,Chloroform-d)δ8.88(dd,J=22.6,2.0Hz,2H),8.24–8.11(m,1H),7.62(d,J=7.8Hz ,1H),7.47(t,J=7.7Hz,1H),7.33–7.22(m,3H),6.94(d,J=8.2Hz,1H),6.89–6.81(m,1H),6.81–6 .69(m,2H),6.68–6.53(m,2H),5.42(s,2H),5.23(s,2H),4.32(s,4H),3.67(s,3H),3.51(dt,J= 14.2,7.4Hz,1H),2.30(dd,J=8.9,6.2Hz,4H),1.95(q,J=8.1,7.6Hz,2H),1.62(q,J=7.3Hz,2H).

[0211] Example 43: N-{2-(5-cyanopyridine-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-bisfluoromethyl-benzyloxy]-benzylamine}-ethyl-acetamide

[0212]

[0213] Yield: 27.5%. HPLC purity: 98.44%. Melting point: 118-120℃. 1 H NMR(600MHz,Chloroform-d)δ8.86(d,J=50.0Hz,2H),8.18(d,J=2.2Hz,1H),7.6 2(d,J=7.8Hz,1H),7.46(t,J=7.8Hz,1H),7.27(dd,J=23.5,7.9Hz,3H),6.93(d, J=8.2Hz,1H),6.85–6.70(m,3H),6.67–6.58(m,2H),5.41(s,2H),5.21(s,2H),4 .31(s,5H),4.07(s,2H),3.50–3.38(m,2H),3.09(t,J=4.9Hz,2H),1.93(s,4H).

[0214] Example 44: N-Dimethylaminoethyl-{2-(5-cyanopyridin-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-difluoromethyl-benzyloxy]}benzylamine

[0215]

[0216] Yield: 29.8%. HPLC purity: 98.77%, melting point: 109-111℃. 1 H NMR(600MHz,Chloroform-d)δ8.95–8.79(m,2H),8.12(q,J=4.8,3.4Hz,1H),7.64(d,J=7.8Hz,1H),7.47(t,J=7.7Hz,1H),7.29(dd,J=25.4,8.0Hz,3H),6.9 5(d,J=8.2Hz,1H),6.86–6.71(m,3H),6.69–6.59(m,2H),5.42(s,2H),5.24(s ,2H),4.33(s,5H),4.09(s,2H),3.01(s,2H),2.78–2.69(m,2H),2.33(s,6H).

[0217] Example 45: N-(1,2-dihydroxyethyl)-{2-(5-cyanopyridin-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-bisfluoromethyl-benzyloxy]}benzylamine

[0218]

[0219] Yield: 24.6%. HPLC purity: 98.71%, melting point: 100-102℃. 1 H NMR(600MHz,Chloroform-d)δ9.07–8.56(m,2H),8.15(s,1H),7.63(d,J=7.8Hz,1H),7.44(t,J=7.8Hz,1H),7.36–7.28(m,1H),7.24(d,J=7.7Hz,1H), 6.93(d,J=8.2Hz,1H),6.87–6.68(m,3H),6.63(s,2H),5.35(d,J=37.0Hz, 4H),4.31(s,4H),4.23–3.95(m,2H),3.71–3.40(m,2H),3.20–2.79(m,2H).

[0220] Example 46: N-Dimethylaminoethyl-{2-(pyridin-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-difluoromethyl-benzyloxy]}benzylamine

[0221]

[0222] Yield: 25.6%. HPLC purity: 97.44%, melting point: 110-112℃. 1 H NMR(600MHz,Chloroform-d)δ8.65(d,J=57.8Hz,2H),7.83(d,J=7.9Hz,1H),7. 65(d,J=7.9Hz,1H),7.44(dt,J=29.9,4.6Hz,2H),7.26(d,J=7.1Hz,3H),6.94( d,J=8.2Hz,1H),6.86–6.60(m,5H),5.44(s,2H),5.14(s,2H),4.31(d,J=5.1Hz ,4H),4.04(d,J=48.5Hz,2H),2.98(s,2H),2.67(t,J=5.7Hz,2H),2.20(s,5H).

[0223] Example 47A25: N-Dimethylaminoethyl-{2-(pyridin-4-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-difluoromethyl-benzyloxy]}benzylamine

[0224]

[0225] Yield: 22.2%. HPLC purity: 99.11%, melting point: 132-135℃. 1H NMR(600MHz,Chloroform-d)δ8.62(d,J=4.7Hz,2H),7.61(d,J=7.8Hz,1H),7.45( t,J=7.7Hz,1H),7.32(d,J=4.7Hz,2H),7.28–7.22(m,2H),6.93(d,J=8.2Hz,1H),6 .86–6.71(m,3H),6.68–6.55(m,2H),5.41(s,2H),5.14(s,2H),4.31(s,5H),4.12 (s,2H),3.68(d,J=5.5Hz,1H),3.04–2.88(m,2H),2.70–2.53(m,2H),2.18(s,6H).

[0226] Example 48: N-(1,2-dihydroxyethyl)-{2-(pyridin-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-difluoromethyl-benzyloxy]}benzylamine

[0227]

[0228] Yield: 33.6%. HPLC purity: 97.34%, melting point: 129-132℃. 1 H NMR(600MHz,Chloroform-d)δ8.71(s,1H),8.37(s,1H),7.91–7.59(m,2H),7.57–7.15(m,4H),7.08–6.41(m ,6H),5.26(d,J=150.7Hz,4H),4.31(s,4H),4.18(s,2H),3.65(s,1H),3.48(s,1H),2.96(t,J=34.8Hz,2H).

[0229] (II) Pharmacological activity of the compound

[0230] Example 49: Inhibitory activity of the compound at the in vitro molecular level against the interaction between PD-1 and PD-L1

[0231] In vitro protein levels were detected using the Cisbio PD-1 / PD-L1 binding assay kit. The screening principles and methods for PD-1 / PD-L1 small molecule inhibitors are as follows:

[0232] (1) Principle: The PD-1 protein carries an HIS tag, and its ligand PD-L1 carries an hFc tag. Eu-labeled anti-hFc antibody and XL665-labeled anti-HIS antibody bind to these two tagged proteins, respectively. Upon laser excitation, energy is transferred from the donor Eu to the receptor XL665, causing XL665 to luminesce. Adding an inhibitor (compound or antibody) blocks the binding of PD-1 to PD-L1.

[0233] The combination of L1 results in a large distance between Eu and XL665, preventing energy transfer and thus XL665 does not emit light.

[0234] (2) Experimental Methods: Specific methods can be found in the Cisbio PD-1 / PD-L1 kit (catalog number 64ICP01PEG). A brief description is as follows: In a 384-well white microplate, add 4 μL of PD-1 protein and 4 μL of PD-L1 protein to each well. Then add 2 μL of diluent or the target compound diluted with diluent to each well. Incubate at room temperature for 15 min. Finally, add 10 μL of anti-Tag 1-

[0235] A mixture of Eu3+ and anti-Tag2-XL665 was incubated at room temperature for 1-4 hours, followed by detection of fluorescence signals at 665 nm and 620 nm using a multi-mode microplate reader. HTRF rate = (665 nm / 620 nm) * 10 4 Eight to ten concentrations were measured for each compound, and the IC50 was calculated using Graphpad software. 50 .

[0236] (3) The screening results are shown in Table 1:

[0237] Table 1. Screening results of the inhibitory activity evaluation of the compounds in the examples against the PD-1 and PD-L1 interaction at the molecular level.

[0238]

[0239] Example 50: The ability of the compound to restore the level of interferon-γ secreted by T cells

[0240] (1) Principle: The compound blocks the binding of PD-1 / PD-L and restores the level of interferon-γ secreted by T cells. The concentration of interferon-γ in the cell supernatant is detected by an ELISA kit.

[0241] (2) Experimental methods: PBMCs were isolated from the blood of healthy donors using Ficoll Paque. PBMCs were obtained by density gradient centrifugation. PBMCs were co-stimulated with CD3 antibody (2 μg / m) and CD28 antibody (2 μg / m), and IL-2 (10 ng / m) was added simultaneously for 3–4 days. Subsequently, 1, 2, or 4 μM of the compound and 5 pg / mL of recombinant PD-L1 protein (i.e., hPD-L1 protein) were added to the co-culture system. The cells were co-cultured at 37°C in a 5% CO2 cell culture incubator for 48 h. The supernatant was collected, and the IFN-γ secretion level was detected by ELISAkt.

[0242] (3) Experimental results are as follows Figure 1 As shown (all data are expressed as mean ± SEM (n=3); *p<0.05, **p<0.01, ***p

[0243] <0.001).

[0244] Example 51: The ability of compounds to induce T cells to kill tumor cells

[0245] (1) Principle: The compound blocks the binding of PD-1 / PD-L1, which can restore T cell function and kill tumor cells in the co-culture environment. The cell survival rate in the co-culture system is detected by the CCK-8 kit.

[0246] (2) Experimental Methods: Jurkat T cells were stimulated with 50 ng / mL PMA for no more than 5 h to stably express human PD-1 (hPD-1). Tumor cells cultured to the logarithmic growth phase were digested and seeded into 96-well plates 12 h in advance, 2000 cells per well. Activated Jurkat T cells were seeded into the wells. Jurkat T cells and different tumor cells were cultured in DMEM medium at a ratio of 5:1. Then, different concentrations of compounds were added, and the cells were co-cultured at 37°C and 5% CO2 for 48 h. The CCK8 assay was used to detect T cell-mediated tumor cell mortality. All concentrations of compounds used in the co-culture system were non-toxic to T cells and tumor cells. The formula is as follows: Cell viability (%) = (OD experimental group - OD blank group) × 100% / (OD control group -

[0247] (OD blank group). The OD blank group corresponds to the experimental results without the addition of any compound.

[0248] (3) The results are as follows Figure 2 As shown (all data are expressed as mean ± SEM (n=3); *p<0.05, **p<0.01, ***p<

[0249] 0.001).

[0250] Example 52: Antitumor activity of the compound in a homologous mouse subcutaneous tumor-bearing model

[0251] (1) Experimental method: MC38 cells (5×10⁻⁶) were used to prepare the cells. 5 A colon tumor model was established by subcutaneously inoculating female C57BL / 6 mice with 100 μL of the compound in PBS. Mice were randomly divided into 6 groups. Throughout the in vivo experiments, the compound was dissolved in physiological saline containing 2% HS15. Mice in the solvent control group were administered 200 μL of the solvent (physiological saline containing 2% HS15) by gavage. Anti-mouse PD-L1k antibody was injected via tail vein twice weekly at 5 mg / kg. The solvent control group, compounds from Example 24 (25, 50 mg / kg), and compounds from Example 27 (25, 50 mg / kg) were administered by gavage once daily for 12 consecutive days. Tumor volume was measured daily using calipers (0.5 × length × width × width). Furthermore, the potential toxicity of the compound was assessed by measuring body weight. Mice were sacrificed after treatment, and the tumor and organ weights were measured. The tumor growth inhibition rate was calculated using TGI (%) = [1 - Vt / Vv] × 100%, where Vt and Vv are the mean tumor volumes of the treatment group and solvent control group after 12 days of administration, respectively.

[0252] (2) Experimental results are as follows Figure 3 As shown (all data are expressed as mean ± SEM (n=3); *p<0.05, **p<0.01),

[0253] ***p<0.001).

[0254] Example 53: Antitumor activity of the compound in a PD-1 / PD-L1 humanized mouse subcutaneous tumor model and immunocellular typing in tumor tissue

[0255] (1) Experimental Methods: hPD-L1 MC38 cells were seeded into the right axilla of PD-1 / PD-L1 humanized C57BL / 6 mice to establish a subcutaneous tumor-bearing model in humanized mice. When the tumor size reached 50 mm... 3 Mice were randomly divided into two groups (n=6). The experimental group was administered compound 50 mg / kg of Example 27 by gavage daily, while the control group received physiological saline (2% HS15) as a solvent. Tumor volume and body weight were measured daily. Tumor volume was calculated using the same formula as before. After the experiment, tumor tissue was obtained, digested with collagenase into single cells, and immunocellular typing of the tumor tissue was detected by fluorescent dye staining.

[0256] (2) Experimental results are as follows Figure 4 As shown (all data are expressed as mean ± SEM (n=3); *p<0.05, **p<0.01),

[0257] ***p<0.001).

[0258] Example 54: Immunohistochemical staining of tumor tissue in a homologous mouse subcutaneous tumor-bearing model

[0259] (1) Experimental Methods: Mouse tumor tissue obtained from the mouse model was fixed in formalin for 24 hours and dehydrated using the method described in Example 53, then embedded in paraffin. The tissue was transferred to a glass slide and dewaxed. Anti-granzyme B antibody (1:100 dilution) and anti-perforin antibody (1:100 dilution) were added to the slide and incubated overnight at 4°C. The slide was washed three times with PBS, and the secondary antibody was added to the slide. DAB substrate solution was spread on the slide to show the antibody staining color. Then, it was counterstained with hematoxylin. Immunohistochemical images were obtained under a microscope. Figure 5 In the control group, the levels of granzyme B and perforin in the tumor tissue were extremely low. In mice that were injected with monoclonal antibodies or administered compounds 24 and 27 by gavage for 12 days, the levels of granzyme B and perforin in the tumor tissue were significantly increased, and the levels of both increased in a concentration-dependent manner, especially compound 27.

[0260] (2) Results Figure 5 As shown (scalebars, 200μm).

[0261] Example 55: Eosin hematoxylin staining of tissues and organs in a homologous mouse subcutaneous tumor-bearing model

[0262] (1) Experimental Methods: Mouse tumor organs obtained using the method described in Example 53 were embedded in OCT embedding medium (SAKURA 4583) and then rapidly frozen in liquid nitrogen. Sections were prepared using a cryostat and mounted onto positive ion adsorption slides. The slides were dried in a 37°C oven for approximately 1–2 hours, and the OCT on the slides was removed by rinsing with running water. The slides were then stained with hematoxylin for 1 minute, rinsed with running water for 5 minutes, then stained with eosin dye and rinsed with running water for 5 minutes. Following this, the slides were subjected to immersion in 70%, 90%, and 100% ethanol for differentiation. The slides were then immersed in xylene solution for 10 minutes, replaced with fresh xylene solution, and immersed again for 10 minutes. Residual xylene was blotted from the tissue edges with lens paper, and the slides were mounted with neutral resin before the edges curled up. The mounted slides were then observed and photographed under a microscope.

[0263] (2) Results Figure 6 As shown, no significant tissue morphological changes were observed in the heart, liver, spleen, lungs, and kidneys of mice, indicating that compounds 24 and 27 administered orally in Examples did not cause damage to normal tissues.

Claims

1. 2-arylmethoxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivatives as shown in general formula I, their stereoisomers, or pharmaceutically acceptable salts thereof. ； R1 is one of the following: , , , , , , ; Where R 11 and R 13 Each is independently selected from hydrogen or one of the following: ； in, n=1, 2, or 3; R 14 It is hydrogen, C1-C4 alkyl, C1-C4 alkylamino-substituted C1-C4 alkyl, C1-C4 alkylamino-substituted C1-C4 alkyl, amide C1-C4 alkyl, amino C1-C4 alkyl, di(C1-C4 alkyl)amide C1-C4 alkyl, di(C1-C4 alkyl)amino C1-C3 alkyl, hydroxy C1-C4 alkyl, pyridyl, phenyl, or methoxy-substituted phenyl; R 12 It is hydrogen, C1-C3 alkyl, cyano, halogen, or halo-C1-C3 alkyl; R2 is: or ; X is one of the following: hydrogen, chlorine, bromine, iodine, C1-C4 alkyl, methoxy; R3 is one of the following: ; R4 is one of the following: a substituted C1-8 saturated alkylamino group, a substituted C2-6 unsaturated alkylamino group, or a substituted C2-6 aza-1-yl group, wherein the substituent is hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, C1-5 alkyl, C1-5 alkoxy, amino, C1-6 alkylamino, acetamido, cyano, ureoyl, guanidinyl, ureaamino, guanidinylamino, sulfonylamino, aminosulfonyl, fluorosulfonyl, fluorosulfonylamino, fluorosulfonate, methanesulfonylamino, hydroxyformyl, C1-8 alkoxyformyl, mercapto, imidazole, thiazolyl, oxazolyl, or tetrazolyl.

2. The 2-arylmethoxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivatives and their stereoisomers or pharmaceutically acceptable salts as described in claim 1, characterized in that... The compound structures of the 2-arylmethoxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivatives are shown in formula (IA-1): ； R1 is one of the following: ; R3 and R4 are the same as in Equation I.

3. The 2-arylmethoxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivatives and their stereoisomers or pharmaceutically acceptable salts as described in claim 1, characterized in that... The compound structures of the 2-arylmethoxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivatives are shown in formula (IA-2): ； R1, R3, and R4 are the same as in Equation I.

4. The 2-arylmethoxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivatives and their stereoisomers or pharmaceutically acceptable salts as described in claim 1, characterized in that... R4 is one of the following: ； R = methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl.

5. The 2-arylmethoxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivatives and their stereoisomers or pharmaceutically acceptable salts as described in claim 1, characterized in that... The 2-arylmethoxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivative is one of the following: N-hydroxyethyl-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)]benzylamine ； N-[2-(3-cyanobenzyloxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]L-serine ； N-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]piperidine-2-carboxylic acid ； N-[2-(5-cyanopyridine-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]glycine ； N-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]L-proline ； N-[2-(5-cyanopyridine-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]pyrrolidine-3-carboxylic acid ； N-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]L-serine ； N-[2-(5-cyanopyridin-3-methoxy)-4-(3-o-fluorophenyl-2-monofluoromethyl-benzyloxy)benzyl]D-serine ； N-{2-(5-cyanopyridine-3-methoxy)-4-[3-(2,5-bisfluorophenyl)-2-monofluoromethyl-benzyloxy]benzyl}L-serine ； N-hydroxyethyl-{2-(3-cyanobenzoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-monofluoromethyl-benzyloxy]} benzylamine ； N-{2-(5-cyanopyridine-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-monofluoromethyl-benzyloxy]benzyl}-L-serine ； N-hydroxyethyl-{2-(5-cyanopyridine-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-monofluoromethyl-benzyloxy]}benzylamine ； N-[2-(5-cyanopyridine-3-methoxy)-4-(3-o-fluorophenyl-2-bisfluoromethyl-benzyloxy)-benzyl]L-serine ； N-hydroxyethyl-{2-(5-cyanopyridine-3-methoxy)-4-[3-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-2-bisfluoromethyl-benzyloxy]}benzylamine 。 6. The 2-arylmethoxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivatives and their stereoisomers or pharmaceutically acceptable salts as described in claim 1, characterized in that... The medicinal salts include salts formed by combining 2-arylmethoxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivatives with inorganic acids, organic acids, alkali metal ions, alkaline earth metal ions, or organic bases that can provide physiologically acceptable cations, as well as ammonium salts. The inorganic acid is hydrochloric acid, hydrobromic acid, phosphoric acid, or sulfuric acid; the organic acid is methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, citric acid, maleic acid, tartaric acid, fumaric acid, citric acid, or lactic acid; the alkali metal ion is lithium ion, sodium ion, or potassium ion; the alkaline earth metal ion is calcium ion or magnesium ion; the organic base that can provide physiologically acceptable cations is methylamine, dimethylamine, trimethylamine, piperidine, or morpholine.

7. The synthesis of the 2-arylmethoxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivative of claim 2, characterized in that... Its synthetic route is as follows: ； (a) Using methyl 3-bromo-2-methylbenzoate as the starting material, and with azobisisobutyronitrile (AIBN) as a free radical initiator, bromination of one hydrogen atom of the methyl group at the 2-position of the benzene ring of the compound of Formula 1 with bromine is carried out to obtain intermediate 2. (b) Using tetrabutylammonium fluoride as a fluorination agent, the monobromomethyl group at the 2-position of the benzene ring of intermediate 2 is converted into a monofluoromethyl group to obtain intermediate 3; (c) Using intermediate 3 and various substituted aromatic boric acid compounds R1-B(OH)2 as basic raw materials, intermediate 4 was obtained by Suzuki coupling reaction; (d) The ester group of intermediate 4 was reduced with lithium aluminum hydride to give benzyl alcohol intermediate 5; (e) Using intermediate 5 as a raw material, benzyl alcohol is brominated with carbon tetrabromide to obtain brominated intermediate 6; (f) Using intermediate 6 as a raw material, react with 5-X-2,4-dihydroxybenzaldehyde under alkaline conditions to obtain benzyl aryl ether intermediate 7; (g) Using intermediate 7 as a raw material, react with various substituted aryl halides R3-Cl under alkaline conditions to obtain intermediate 8; (h) Using aldehyde-containing intermediate 8 as a raw material, the target compound IA-1 is obtained by reductive amination reaction with various amino or imino-containing HR4 compounds; the substituents R1, R3, R4, and X in the raw material compounds added during the reaction are defined as in the target compound.

8. The synthesis of the 2-arylmethoxy-4-(3-aryl-2-fluoromethyl-benzyloxy)benzylamine derivative of claim 3, characterized in that... Its synthetic route is as follows: ； (a) Using 2-difluoromethyl-3-bromobenzyl alcohol as shown in Formula 9 and various substituted aromatic boric acid compounds R1-B(OH)2 as basic raw materials, intermediate 10 was obtained by Suzuki coupling reaction; (b) Using intermediate 10 as a raw material, substituted benzyl alcohol is brominated by carbon tetrabromide to obtain brominated intermediate 11; (c) Using intermediate 11 as a raw material, react with 5-X-2,4-dihydroxybenzaldehyde under alkaline conditions to obtain benzyl aryl ether intermediate 12; (d) Using intermediate 12 as a raw material, it was reacted with various substituted aryl halides R3-Cl under alkaline conditions to obtain intermediate 13; (e) Using aldehyde-containing intermediate 13 as a raw material, the target compound IA-2 is obtained by reductive amination reaction with various amino or imino-containing HR4 compounds; the substituents R1, R3, R4, and X in the raw material compounds added during the reaction are the same as those in the target compound.

9. The use of the 3-aryl-2-fluoromethyl-benzylresorcinol ether derivatives and their stereoisomers, as well as their pharmaceutically acceptable salts, according to any one of claims 1-8, in the preparation of medicaments for the prevention and / or treatment of diseases related to the PD-1 / PD-L1 signaling pathway.

10. The application according to claim 9, characterized in that, The diseases related to the PD-1 / PD-L1 signaling pathway are selected from cancer, infectious diseases, or autoimmune diseases. The cancers include skin cancer, lung cancer, urinary tract tumors, hematological malignancies, breast cancer, glioma, digestive system tumors, reproductive system tumors, lymphoma, nervous system tumors, brain tumors, or head and neck cancer. The infectious diseases are bacterial or viral infections. The autoimmune diseases are organ-specific autoimmune diseases or systemic autoimmune diseases. Organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage-nephritis syndrome, primary biliary cirrhosis, multiple cerebral sclerosis, or acute idiopathic polyneuritis. Systemic autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, systemic vasculitis, scleroderma, or autoimmune hemolytic anemia.

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