Use of phenylalanine amide derivatives as allosteric antagonists of beta2-adrenergic receptors

Abstract

The present invention belongs to the field of medicinal chemistry, and particularly relates to the use of a phenylalanine amide derivative as an allosteric antagonist of the β2-adrenergic receptor. The structural formula of the phenylalanine amide derivative is shown in Formula 1 and Formula 2. The pharmacological results show that most of the target compounds synthesized in the present invention have antagonistic activity against β2AR and can negatively allosterically regulate the functional activity of isoproterenol.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and particularly relates to the application of a phenylalanine amide derivative as an allosteric antagonist of the β2 - adrenergic receptor. Background Art

[0002] G protein - coupled receptors (GPCRs), also known as seven - transmembrane receptors, are a general term for a large class of membrane protein receptors, and all have seven transmembrane α - helices in their three - dimensional structures (Lagerstrom, M.C. et al. Nat Rev Drug Discov, 2008, 7:339 - 357). G protein - coupled receptors activate a series of intracellular signaling pathways by binding to these ligands in the cell's surrounding environment, causing changes in the cell state and participating in almost all physiological, pathological, and pharmacological processes in the human body (Venkatakrishnan, A.J. et al. Nature, 2013, 494:185 - 194). The various functions of GPCRs are related to a variety of diseases, such as cardiovascular diseases, neurological diseases, inflammatory diseases, metabolic diseases, cancers, etc. Currently, more than 40% of modern drugs directly or indirectly target GPCRs [7] , and these drugs include opioid drugs, antihistamine drugs, α - and β - receptor antagonists, β - receptor agonists, dopamine receptor antagonists, angiotensin receptor antagonists, angiotensin - converting enzyme inhibitors, and selective serotonin reuptake inhibitors, etc. During the period of 2011 - 2015, the total sales of GPCR drugs accounted for 27% of the globally marketed drugs. Well - known drugs such as Losartan, Diazepam, Cinacalcet, and Carvedilol all belong to GPCR drugs.

[0003] β - adrenergic receptor (β-AR) is a very typical member of the G-protein-coupled receptor superfamily and is highly representative among transmembrane receptors. As one of the most studied CPCRs, β2-AR is widely expressed in bronchial smooth muscle, the cardiovascular system, and the lungs. There are many currently marketed and used β2-AR agonists and antagonists. β2-AR agonists are mainly used to treat lung diseases and asthma, such as short-acting β2-AR agonists salbutamol and terbutaline. β2-AR antagonists are very widely used prescription drugs in modern medicine and play a very important role in the drug treatment of heart failure, hypertension, coronary heart disease, arrhythmia, angina, and other cardiovascular diseases, serving as the cornerstone of cardiovascular disease treatment. Long-acting β2-AR agonists include salmeterol and formoterol, and β2-AR antagonists such as propranolo and kalalol.

[0004] The above-mentioned β2-AR drugs are all orthosteric ligands for it, but these orthosteric ligands have poor subtype selectivity for the receptor and have more adverse reactions and contraindications in the nervous system. In order to explore and develop β2-AR drugs with high selectivity and few side effects, more and more research has been carried out on β2-AR allosteric modulators.

[0005] In 2017, our research group and the research group of Professor Robert Lefkowitz jointly reported the structure of the β2-adrenergic receptor allosteric antagonist Cmpd-15 (Ahn, S.; et al. PNAS, 2017, 114: 1708 - 1713; Liu, X. et al. Nature, 2017, 548: 480 - 484). This is the first reported β2-AR allosteric modulator, opening a new milestone in the development and research of β2-AR allosteric modulators. Orthosteric antagonists of the β2-adrenergic receptor are a class of extremely important prescription drugs in the treatment of various cardiovascular diseases. As the first reported β2-AR allosteric antagonist, Cmpd-15 has important guiding significance for the discovery of other β2-AR allosteric modulators and the biological research of β2-AR allosteric binding sites. And conducting structural biology research on Cmpd-15 provides the possibility for the development of new drugs for treating cardiovascular-related diseases regulated by the β2-AR receptor. Summary of the Invention

[0006] As described in the background art, compound Cmpd-15 is a phenylalanine amide derivative, and its structure is composed of three parts, M1, M2, and M3. In order to explore the active structural skeleton of Cmpd-15, as well as to simplify the structure and synthetic route, in the present invention, we selected some acids to replace the M3 region of Cmpd-15 through the principle of scaffold hopping and bioisosteres, while keeping the M1 and M2 regions unchanged. Maintaining the structure of the M3 region without polar substituents is crucial for its ability to regulate the activity of agonist-induced β2-AR.

[0007] To further study the active centers of different regions of Cmpd-15, we synthesized derivatives coupled with M1-M3, M2-M3, and M1-M2. We found that when M1-M2 are connected, there are both agonist activities and antagonist activities. Finally, to simplify the structure, we directly connected some acids to the M1 part and synthesized a series of new derivatives with greatly simplified structures, and found that they generally have allosteric antagonist activities against β2-adrenergic receptors, and even some compounds have activities equivalent to or better than Cmpd-15.

[0008]

[0009] The purpose of the present invention is to provide a new phenylalanine amide derivative to develop derivatives of compound Cmpd-15 with simple structures but high stability and high biological activities, as allosteric modulators of β2-AR, providing a solid foundation for the creation of new drugs for diseases such as cardiovascular diseases, asthma, and cancer.

[0010] A phenylalanine amide derivative, the structural formula of which is shown in Formula 1 or Formula 2:

[0011]

[0012] R1 is one of the following structures:

[0013] H

[0014] R2 is: 3-Br, 4-CONH2.

[0015] The synthetic route of the type 1 derivative of compound Cmpd-15 is as follows:

[0016]

[0017]

[0018] The synthetic method of the type 1 derivative (A1-8A) of compound Cmpd-15:

[0019] The specific synthesis steps are as follows:

[0020] (1) In a pressure-resistant tube, palladium acetate (0.05 eq), potassium carbonate (0.5 eq), silver carbonate (1 eq), N-acetylglycine (0.3 eq), 4-iodobenzonitrile (1.5 eq), and compound 1 (1 eq) were dissolved in 0.2 mol / L hexafluoroisopropanol, and the mixture was vigorously stirred at 100 °C for 48 h. After the reaction was completed, ethyl acetate was added to dilute the reaction solution. The reaction mixture was obtained by suction filtration through diatomaceous earth and then distilled under reduced pressure. After purification by column chromatography, compound 2 was obtained;

[0021] (2) Compound 2 (1 eq) was dissolved in a 1:1 mixed solution of 0.2 mmol / L DCM and absolute ethanol. Copper acetate (0.02 eq) and diethylhydroxylamine (2 eq) were added, and the mixture was stirred at 35 °C for 18 h. The crude product was obtained by concentration, and then purified by column chromatography. The purified product was washed with ethyl acetate and DCM to obtain compound 3;

[0022] (3) In a pressure-resistant tube, palladium acetate (0.05 eq), potassium carbonate (0.5 eq), silver carbonate (1 eq), N-acetylglycine (0.3 eq), iodobenzene with different substituents (1.5 eq), and compound 1 (1 eq) were dissolved in 0.2 mol / L hexafluoroisopropanol, and the mixture was vigorously stirred at 100 °C for 16 h. After the reaction was completed, ethyl acetate was added for dilution. The reaction mixture was obtained by suction filtration through diatomaceous earth and then distilled under reduced pressure. After purification by column chromatography, compound 4 was obtained;

[0023] (4) Compound 4 (1 eq) and HOAT (1 eq) were dissolved in 0.2 mol / L DMF, and the mixture was stirred at room temperature for 10 min. Then the temperature was lowered to 0 °C, methylamine hydrochloride (2 eq) was added, and stirring was continued for 10 min. Then NMM (0.7 eq) and EDCI (1 eq) were added, and the reaction was continued at 0 °C for 1.5 h. The temperature was raised to room temperature and the reaction was continued for 13 h. The reaction solution was concentrated, and ethyl acetate was added to dissolve the crude product. Then the organic phase was washed successively with 0.5 M sodium bisulfate solution, saturated sodium bicarbonate solution, and saturated sodium chloride aqueous solution. After concentration, purification was carried out by column chromatography to obtain compound 5;

[0024] (5) Compound 5 (1 eq) was dissolved in 0.2 mol / L absolute ethanol. Ethylenediamine (5 eq) was slowly added at 0 °C, and then the mixture was stirred for 30 min. The temperature was slowly raised to room temperature and stirring was continued for 18 h. After the reaction was completed, saturated sodium bicarbonate solution and saturated brine were added, and the mixture was continuously stirred at 0 °C for 30 min. Then it was extracted with ethyl acetate. After concentration, purification was carried out by column chromatography to obtain intermediate 6;

[0025] (6) Dissolve compound 3 (1 eq) and HOAT (1.2 eq) in 0.2 mol / L DMF, stir at room temperature for 10 min, then add a DMF solution of compound 6 (1.2 eq) at 0 °C, continue stirring for 10 min, then add NMM (0.8 eq) and EDCI (1 eq), react at 0 °C for 1.5 h, then slowly raise the temperature to room temperature and react for 13 h. Concentrate the reaction solution and dilute it with ethyl acetate, wash it successively with 0.5 M sodium bisulfate solution, saturated sodium bicarbonate solution, and saturated brine, concentrate it and purify it by column chromatography to obtain compound 7;

[0026] (7) Dissolve compound 7 (1 eq) in 0.2 mol / L ethanol, add ethylenediamine (5 eq) at 0 °C, slowly raise the temperature to room temperature, stir for 18 h, add absolute ethanol, saturated brine, and saturated sodium bicarbonate, stir at 0 °C for 30 min, extract with ethyl acetate, concentrate the organic phase, and wash the crude product with ethyl acetate to obtain compound 8;

[0027] (8) Dissolve different carboxylic acid compounds 9 (including the R1 group) (1 eq), HOBT (1 eq), and HBTU (1 eq) in 0.2 mol / L DMF, stir at room temperature for 10 min, add a DMF solution of compound 8 (0.5 eq) at 0 °C, continue stirring for 10 min, slowly add DIEA (1.5 eq), react at 0 °C for 1 h, then raise the temperature to room temperature and react for 10 h. Wash the concentrated crude product with ethyl acetate, filter by suction to obtain a white solid, and recrystallize with methanol to obtain type A compounds.

[0028] The synthetic route of the type 2 derivative (B1 - B15) of compound Cmpd - 15 is as follows:

[0029]

[0030] The specific synthesis method steps are as follows:

[0031] Dissolve different acid compounds 9 (including the R1 group) and the activator in a solvent, add compound 6 under an ice bath, then add the acid-binding agent and the amide coupling agent and continue stirring to room temperature to obtain. The solvent is N,N - dimethylformamide; the activator is 1 - hydroxy - 7 - azabenzotriazole (HOAT); the acid-binding agent is N - methylmorpholine (NMM); the amide coupling agent is 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide hydrochloride (EDCI). Dissolve compound 9 (1 eq) and HOAT (1.2 eq) in DMF, stir for 10 min, add compound 6, at 0 °C, add NMM (0.7 eq), stir for 10 min, add EDCI (1.2 eq), keep stirring at 0 °C for 1.5 h, then react at room temperature for 12 h. Post-treatment: Rotavap the DMF, extract, and column chromatograph to obtain compound B.

[0032] The present invention uses the cumulative experiment of GloSensor cAMP to test the functional activity of target compounds on the G protein-dependent signaling pathway for β2AR and to clarify whether the new compounds can allosterically regulate the functional activity of the endogenous ligand isoproterenol (ISO) of β2AR. The pharmacological results show that most derivatives of the synthesized compound Cmpd-15 have antagonistic activity against β2AR and can negatively allosterically regulate the functional activity of ISO.

[0033] The beneficial effects of the present invention are as follows:

[0034] The beneficial effects of the present invention are mainly manifested in the following aspects. First, the allosteric antagonistic activity of the newly synthesized derivatives against the β2-AR target is improved compared with the lead compound Cmpd-15. Second, the newly synthesized derivatives have a novel skeleton and a greatly simplified structure compared with the lead compound Cmpd-15, which simplifies the synthesis route, greatly reduces the synthesis cost and difficulty, and significantly improves the economic benefits of this type of compound. Finally, among the compounds we synthesized, we found that some compounds had an activity inversion and became agonists, which is a new discovery and will also provide a new direction for our subsequent work. Brief Description of the Drawings

[0035] Figure 1 It is the dose-response curve of ISO mediated by the Cmpd-15 derivative B8. Detailed Embodiments

[0036] Now, the present invention will be further described in conjunction with examples.

[0037] Example 1

[0038]

[0039] Preparation of 4-(S)-3-(S)-3-(3-bromophenyl)-1-methylamino-1-oxopropan-2-ylamino)-3-oxo-2-(2-phenylacetamido)propyl)benzamide (A1)

[0040] Step 1: Synthesis of (S)-3-(4-cyanophenyl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acid 2

[0041] In a 100 mL pressure-resistant tube, palladium acetate (88 mg, 0.4 mmol), potassium carbonate (276 mg, 2 mmol), silver carbonate (1104 mg, 4 mmol), N-acetylglycine (140 mg, 1.2 mmol), 4-iodobenzoyl cyanide (1374 mg, 6 mmol) and compound 1 (876 mg, 4 mmol) were dissolved in 40 mL of hexafluoroisopropanol, and the mixture was vigorously stirred at 100 °C for 48 h. After the reaction was completed, 40 mL of ethyl acetate was added to dilute the reaction solution. The reaction mixture was obtained by suction filtration through diatomaceous earth and then distilled under reduced pressure. After purification by column chromatography [eluent: V(DCM) / V(MeOH) / V(HCOOH) = 100 / 1 / 1], compound 3 (yellow oil, 512 mg, yield 40%, 98% ee) was obtained. 1 H NMR (500 MHz, CDCl3) δ 7.79 (dd, J = 5.5, 3.0 Hz, 2H), 7.72 (dd, J = 5.5, 3.0 Hz, 2H), 7.51 - 7.49 (m, 2H), 7.30 - 7.28 (m, 2H), 5.20 (dd, J = 8.7, 7.8 Hz, 1H), 3.63 (d, J = 8.3 Hz, 2H). 13 C NMR (75 MHz, CDCl3) δ 173.4, 167.5, 142.2, 134.6, 132.6, 131.3, 129.8, 123.9, 118.7, 111.2, 52.4, 34.7. HRMS (ESI, positive): Calcd. for C 18 H 12 N2O4Na [M+Na] + 343.0689, found: 343.0693.

[0042] Step 2: Synthesis of (S)-3-(4-carbamoylphenyl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acid 3

[0043] Compound 2 (977.4 mg, 3 mmol) was dissolved in a 1:1 mixed solution of 10 mL of dichloromethane and absolute ethanol. Copper acetate (12 mg, 0.06 mmol) and diethylhydroxylamine (913 μL, 6 mmol) were added, and the mixture was stirred at 35 °C for 18 h. The crude product was obtained by concentration and purified by column chromatography [eluent: V(DCM) / V(HCOOH) / V(MeOH) = 20 / 1 / 0.2]. The purified product was washed with ethyl acetate and DCM, and compound 3 (white solid, 639 mg, yield 63%, 98% ee) was obtained. 11H NMR (400 MHz, (CD3)2SO) δ 7.85 (s, 5H), 7.69 (d, J = 8.1 Hz, 2H), 7.28 - 7.24 (m, 3H), 5.16 (dd, J = 11.9, 4.6 Hz, 1H), 3.54 (dd, J = 14.1, 4.8 Hz, 1H), 3.41 (dd, J = 14.0, 11.7 Hz, 1H). 13 13C NMR (75 MHz, (CD3)2SO) δ 170.0, 167.5, 167.2, 140.9, 135.0, 132.5, 130.7, 128.6, 127.6, 123.4, 52.9, 33.9. HRMS (ESI, positive): Calcd. for C 18 H 14 N2O5Na [M+Na] + 361.0795, found: 361.0788.

[0044] Step 3: Synthesis of (S)-3-(3-bromophenyl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acid 4

[0045] In a 100 ml pressure tube, palladium acetate (44 mg, 0.2 mmol), potassium carbonate (276 mg, 2 mmol), silver carbonate (1104 mg, 4 mmol), N-acetylglycine (140 mg, 1.2 mmol), 1-bromo-3-iodobenzene (780 μL, 6 mmol) and compound 1 (876 mg, 4 mmol) were dissolved in 40 ml of hexafluoroisopropanol, and stirred vigorously at 100 °C for 16 h. After the reaction was completed, 40 ml of ethyl acetate was added for dilution, filtered through diatomaceous earth, and the crude product was obtained by distillation under reduced pressure. After purification by column chromatography [eluent: V(DCM) / V(HCOOH) = 100 / 1], compound 4 (yellow oil, 1.2 g, yield 80%, 95% ee) was obtained. 1 1H NMR (400 MHz, CDCl3): δ 7.81 (dd, J = 5.5, 3.0 Hz, 2H), 7.71 (dd, J = 5.5, 3.1 Hz, 2H), 7.33 - 7.27 (m, 2H), 7.12 - 7.05 (m, 2H), 5.18 (dd, J = 9.9, 6.5 Hz, 1H), 3.59 - 3.50 (m, 2H). 1313C NMR(75MHz,CDCl3):δ173.5,167.5,138.9,134.5,132.1,131.6,130.3,130.3,127.5,123.8,122.7,52.8,34.2.HRMS(ESI,positive):Calcd.for C 17 H 11 BrNO4[M - H] - 371.9877,found:371.9875.

[0046] Step 4: Synthesis of (S)-3-(3-bromophenyl)-2-(1,3-dioxoisoindolin-2-yl)-N-methylpropanamide 5

[0047] Dissolve compound 4 (374 mg, 1 mmol) and HOAT (136.1 mg, 1 mmol) in 10 mL of DMF, stir at room temperature for 10 min, then cool to 0 °C, add methylamine hydrochloride (135.0 mg, 2 mmol), continue stirring for 10 min, then add NMM (79 μL, 0.7 mmol) and EDCI (191.7 mg, 1 mmol), continue the reaction at 0 °C for 1.5 h, raise the temperature to room temperature and react for 13 h. Concentrate the reaction solution, add 30 mL of ethyl acetate to dissolve the crude product, and then wash the organic phase successively with 0.5 M sodium bisulfate solution (10 mL), saturated sodium bicarbonate solution (10 mL) and saturated sodium chloride aqueous solution (10 mL). After concentration, purify by column chromatography [eluent: V(DCM) / V(MeOH) = 50 / 1] to obtain compound 5 (white solid, 360.1 mg, yield 93%, 93% ee). 1 1H NMR(400MHz,CDCl3)δ7.80(dd,J = 5.4,3.1Hz,2H),7.72(dd,J = 5.5,3.0Hz,2H),7.28(d,J = 1.8Hz,1H),7.26 - 7.23(m,1H),7.11 - 7.09(m,1H),7.05(t,J = 7.7Hz,1H),6.16(s,1H),5.05(dd,J = 10.7,6.0Hz,1H),3.58 - 3.46(m,2H),2.83(d,J = 4.7Hz,3H). 13 13C NMR(126MHz,CDCl3)δ168.8,168.0,139.3,134.5,132.0,131.4,130.2,130.1,127.5,123.7,122.6,55.7,34.4,26.7.HRMS(ESI,positive):Calcd.for C 18 H 15BrN2O3Na[M+Na] + 409.0158, found: 409.0173.

[0048] Step Five: Synthesis of (S)-2-Amino-3-(3-bromophenyl)-N-methylpropanamide 6

[0049] Dissolve compound 5 (1132 mg, 2.9 mmol) in 12 mL of anhydrous ethanol. Slowly add ethylenediamine (981 μL, 14.5 mmol) at 0 °C, then stir for 30 min. Slowly warm up to room temperature and stir for 18 h. After the reaction is completed, add 25 mL of saturated sodium bicarbonate solution and 50 mL of saturated brine, stir continuously at 0 °C for 30 min, then extract with ethyl acetate. After concentration, purify by column chromatography [eluent: V(DCM) / V(MeOH) / V(ammonia water) = 20 / 1 / 0.2] to obtain intermediate 6 (pale yellow liquid, 693.4 mg, yield 93%). 1 H NMR (400 MHz, CDCl3) δ 7.37 (dd, J = 6.2, 1.6 Hz, 2H), 7.20 - 7.13 (m, 2H), 3.59 (dd, J = 9.5, 4.0 Hz, 1H), 3.24 (dd, J = 13.8, 4.0 Hz, 1H), 2.81 (d, J = 5.0 Hz, 3H), 2.67 (dd, J = 13.8, 9.2 Hz, 1H). 13 C NMR (75 MHz, CDCl3) δ 174.5, 140.5, 132.4, 130.4, 130.1, 128.0, 122.8, 56.4, 40.8, 26.0. HRMS (ESI, positive): Calcd. for C 10 H 14 BrN2O[M+H] + 257.0284, found: 257.0284.

[0050] Step Six: Synthesis of 4-((S)-3-((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(1,3-dioxoisoindolin-2-yl)-3-oxopropyl)benzamide 7

[0051] Compound 3 (135 mg, 0.4 mmol) and HOAT (64.8 mg, 0.48 mmol) were dissolved in 2 mL of DMF and stirred at room temperature for 10 min. Then, a solution of compound 6 (123 mg, 0.48 mmol) in DMF (1 mL) was added at 0 °C, and the mixture was stirred for another 10 min. Subsequently, NMM (32 μL, 0.32 mmol) and EDCI (76.7 mg, 0.4 mmol) were added, and the reaction was carried out at 0 °C for 1.5 h. Then, the temperature was slowly raised to room temperature, and the reaction was continued for 13 h. After concentration of the reaction solution, it was diluted with ethyl acetate and washed successively with 0.5 M sodium bisulfate solution (20 mL), saturated sodium bicarbonate solution (20 mL), and saturated brine (20 mL). After concentration, it was purified by column chromatography [eluent: V(DCM) / V(MeOH) = 50 / 1] to obtain compound 7 (white solid, 157.1 mg, yield 68%). 1 H NMR (400 MHz, CD3OD) δ 7.76 (s, 4H), 7.66 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 1.9 Hz, 1H), 7.30 - 7.27 (m, 1H), 7.22 (d, J = 8.1 Hz, 2H), 7.11 (dt, J = 15.3, 7.6 Hz, 2H), 5.16 (dd, J = 11.0, 5.6 Hz, 1H), 4.58 (dd, J = 9.3, 5.8 Hz, 1H), 3.54 - 3.43 (m, 2H), 3.09 (dd, J = 13.9, 5.8 Hz, 1H), 2.89 - 2.83 (m, 1H), 2.68 (s, 3H). 13 C NMR (75 MHz, CD3OD) δ 173.5, 171.9, 170.6, 168.9, 142.8, 141.0, 135.6, 133.4, 133.2, 132.7, 131.2, 130.8, 130.3, 129.0, 128.8, 124.4, 123.3, 56.3, 55.6, 38.1, 35.2, 26.3. HRMS (ESI, positive): Calcd. for C 28 H 25 BrN4O5Na [M+Na] + 599.0900, found: 599.0880.

[0052] Step 7: Synthesis of 4 - ((S)-2 - amino - 3 - ((S)-3 - (3 - bromophenyl)-1 - (methylamino)-1 - oxopropan - 2 - yl)amino)-3 - oxopropyl)benzamide 8

[0053] Compound 7 (231 mg, 0.4 mmol) was dissolved in 4 mL of ethanol. Ethylenediamine (135 μL, 2 mmol) was added at 0 °C, and the mixture was slowly warmed to room temperature and stirred for 18 h. Then, 2 mL of absolute ethanol, 4 mL of saturated brine, and 2 mL of saturated sodium bicarbonate were added, and the mixture was stirred at 0 °C for 30 min. It was extracted with ethyl acetate (70 mL × 3), and the organic phase was concentrated. The crude product was washed with ethyl acetate to obtain compound 8 (white solid, 164.6 mg, yield 92%). 1 H NMR (400 MHz, (CD3)2SO) δ 8.14 (d, J = 8.8 Hz, 1H), 7.93 (dd, J = 10.7, 6.0 Hz, 2H), 7.76 (d, J = 7.8 Hz, 2H), 7.38 (d, J = 6.5 Hz, 2H), 7.28 (s, 1H), 7.19 (dt, J = 15.0, 7.3 Hz, 4H), 4.46 (q, J = 6.8 Hz, 1H), 3.38 (dd, J = 8.6, 4.8 Hz, 2H), 2.95 (dd, J = 13.5, 5.1 Hz, 1H), 2.82 (dt, J = 13.0, 6.1 Hz, 2H), 2.57 (d, J = 4.5 Hz, 3H). 13 C NMR (126 MHz, (CD3)2SO) δ 174.0, 170.9, 167.8, 142.2, 140.7, 132.2, 132.0, 130.2, 129.2, 129.1, 128.4, 127.4, 121.3, 56.1, 53.2, 40.6, 37.6, 25.5. HRMS (ESI, positive): Calcd. for C 20 H 23 BrN4O3Na [M+Na] + 469.0846, found: 469.0843.

[0054] Step 8: Synthesis of 4-((S)-3-((((S)-3-(3-bromophenyl)-1-(methylaminooxyimino)amino)-3-oxo-2-(2-phenylacetamido)propyl)benzamide

[0055] In a 25 mL single-necked flask, phenylacetic acid (31.4 mg, 0.2 mmol) was dissolved in N,N-dimethylformamide (5 mL). 1-Hydroxybenzotriazole HOBT (37.3 mg, 0.3 mmol) and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate HBTU (104.7 mg, 0.3 mmol) were added. Then, under nitrogen protection, after 10 min, a DMF (2 mL) solution of compound 8 (102.6 mg, 0.2 mmol) was added. The reaction system was cooled to 0 °C, and diethyl acetate (114 μL, 0.7 mmol) was slowly added dropwise to the reaction system. It was slowly warmed to room temperature and stirred at room temperature for 12 h. After the reaction was complete, the crude product obtained by distillation under reduced pressure was added with a small amount of ethyl acetate, and white solid A1 (113.2 mg, yield 87%) was obtained by suction filtration. 1 H NMR (400 MHz, DMSO-d6): δ 5.12 (d, J = 3.2 Hz, 3H), 2.74 - 2.83 (m, 2H), 2.98 (t, J = 10.0 Hz, 2H), 3.31 (s, 2H), 4.44 (d, J = 5.3 Hz, 1H), 4.55 (s, 1H), 7.01 (d, J = 6.1 Hz, 2H), 7.17 - 7.30 (m, 8H), 7.40 (d, J = 15.5 Hz, 2H), 7.74 (d, J = 8.1 Hz, 2H), 7.84 (d, J = 3.4 Hz, 1H), 7.91 (s, 1H), 8.23 (d, J = 7.9 Hz, 2H); 13 C NMR (75 MHz, DMSO-d6): δ 26.0, 37.8, 42.5, 54.1, 54.3, 121.9, 126.6, 127.7, 128.5, 128.8, 129.4, 129.5, 129.7, 130.7, 132.3, 132.7, 136.6, 141.0, 141.6, 168.2, 170.4, 171.2, 171.3; HRMS (ESI, positive): Calcd. for C 28 H 29 BrN4O4 [M+Na] + 587.1264, 589.1244, found: 587.1267, 589.1252.

[0056] Example 2

[0057]

[0058] Preparation of 4-(S)-3-(S)-3-(3-bromophenyl)-1-methylamino-1-oxopropyl-2-amino)-2-(2,2-diphenylacetamido)-3-oxopropyl)benzamide (A2)

[0059] The preparation method was the same as that of Example 1, except that 2,2-diphenylacetic acid was used instead of phenylacetic acid in Step 8, and a white solid A2 was obtained with a yield of 80%. 1 H NMR(400MHz,DMSO-d6):δ2.55(s,3H),2.79(t,J=7Hz,2H),2.97(t,J=14.4Hz,1H),4.64(s,1H),5.00(s,1H),6.92(m,2H),7.06-7.34(m,15H),7.71(d,J=7.4Hz,2H),7.91(d,J=13.0Hz,2H),8.38-8.47(m,2H); 13 CNMR(75MHz,DMSO-d6):δ25.5,37.4,37.6,53.7,54.0,56.0,121.4,126.4,126.6,127.3,128.0,128.2,128.3,128.8,129.1,129.2,130.2,131.9,132.2,140.0,140.3,140.6,141.0;HRMS(ESI,positive):Calcd.forC 34 H 33 BrN4O4[M+Na] + 663.1577,665.1557,found:663.1582,665.1569.

[0060] Example 3

[0061]

[0062] Preparation of 4-(S)-2-(2-cyclohexylacetamido)-3-((S)-1-methylamino-1-oxo-3-phenylpropan-2-yl)amino)-3-oxopropyl)benzamide (A3)

[0063] The preparation method was the same as that of Example 1, except that cyclohexaneacetic acid was used instead of phenylacetic acid in Step 8, and a white solid A3 was obtained with a yield of 82%. 1H NMR (400 MHz, DMSO-d6): δ 0.62 - 0.77 (m, 2H), 0.98 - 1.08 (m, 3H), 1.17 - 1.23 (m, 1H), 1.40 - 1.51 (m, 5H), 1.85 (d, J = 13.0 Hz, 2H), 2.56 (d, J = 4.4 Hz, 3H), 2.68 - 2.74 (m, 1H), 2.78 - 2.83 (m, 1H), 4.42 (dd, J1 = 13.6 Hz, J2 = 4.4 Hz, 1H), 4.51 - 4.57 (m, 1H), 7.21 (d, J = 5.0 Hz, 2H), 7.26 (d, J = 7.0 Hz, 3H), 7.38 - 7.41 (m, 2H), 7.76 (d, J = 8.0 Hz, 2H), 7.89 (s, 2H), 7.97 (d, J = 8.4 Hz, 1H), 8.12 (d, J = 8.1 Hz, 1H); 13C NMR (75 MHz, DMSO-d6): δ 25.4, 25.5, 25.6, 25.8, 32.1, 32.4, 34.6, 37.2, 37.4, 43.2, 53.6, 53.8, 121.4, 127.2, 128.3, 129.0, 129.2, 130.2, 131.9, 132.1, 140.6, 141.4, 167.6, 170.8, 171.1, 171.3; HRMS (ESI, positive): Calcd. for C 28 H 35 BrN4O4[M+Na] + 593.1734, 595.1713, found: 593.1735, 595.1721.

[0064] Example 4

[0065]

[0066] Preparation of 4-(S)-3-(S)-3-(3-bromophenyl)-1-methylamino-1-oxopropan-2-ylamino)-2-isobutyramido-3-oxopropyl)benzamide (A4)

[0067] The preparation method was the same as that of Example 1, except that isobutyric acid was used instead of phenylacetic acid in Step 8 to obtain white solid A4 with a yield of 76%. 1H NMR (400 MHz, DMSO-d6): δ 0.79 (d, J = 4.0 Hz, 3H), 0.89 (d, J = 6.4 Hz, 3H), 2.33 (t, J = 6.6 Hz, 1H), 2.56 (d, J = 3.4 Hz, 3H), 2.73 - 2.83 (m, 2H), 2.97 (d, J = 13.1 Hz, 2H), 4.42 - 4.48 (m, 2H), 7.21 - 7.29 (m, 5H), 7.40 (d, J = 12.5 Hz, 2H), 7.75 (d, J = 7.5 Hz, 2H), 7.84 - 7.90 (m, 3H), 8.07 (d, J = 7.8 Hz, 1H); 13C NMR (75 MHz, DMSO-d6): δ 19.2, 19.6, 25.6, 33.7, 37.3, 37.4, 53.4, 53.8, 121.4, 127.2, 128.3, 129.0, 129.3, 130.3, 131.9, 132.2, 140.6, 141.4, 167.8, 170.8, 171.0, 176.1; HRMS (ESI, positive): Calcd. for C 24 H 29 BrN4O4 [M+Na] + 539.1264, 541.1244, found: 539.1269, 541.1253.

[0068] Example 5

[0069]

[0070] Preparation of N-(S)-1-(S)-3-(3-bromophenyl)-1-methylamino-1-oxopropan-2-yl)amino)-3-(4-carbamoylphenyl)-1-oxopropan-2-yl)-1H-indole-2-carboxamide (A5)

[0071] The preparation method was the same as that of Example 1, except that indole-2-carboxylic acid was used instead of phenylacetic acid in Step 8 to obtain white solid A5 with a yield of 72%. 11H NMR (400 MHz, DMSO-d6): δ 2.58 (d, J = 3.8 Hz, 3H), 2.81 - 2.88 (m, 1H), 2.99 - 3.02 (m, 2H), 3.08 - 3.11 (m, 1H), 4.48 (t, J = 6.5 Hz, 1H), 4.75 (s, 1H), 7.03 (t, J = 7.2 Hz, 1H), 7.12 - 7.17 (m, 3H), 7.24 (d, J = 8.0 Hz, 2H), 7.33 - 7.44 (m, 5H), 7.63 (d, J = 7.8 Hz, 1H), 7.74 (d, J = 7.7 Hz, 2H), 7.85 (s, 1H), 7.93 (d, J = 4.0 Hz, 1H), 8.30 (d, J = 8.0 Hz, 1H), 8.56 (d, J = 8.2 Hz, 1H), 11.51 (s, 1H); 13C NMR (75 MHz, DMSO-d6): δ 25.6, 37.0, 37.5, 53.9, 54.2, 103.3, 112.3, 119.8, 121.4, 121.6, 123.5, 127.0, 127.3, 128.4, 129.0, 129.3, 130.2, 131.1, 131.9, 132.3, 136.5, 140.6, 141.7, 161.0, 167.8, 170.8, 171.0; HRMS (ESI, positive): Calcd. for C 29 H 28 BrN5O4 [M + Na] + 612.1217, 614.1196, found: 612.1213, 614.1192.

[0072] Example 6

[0073]

[0074] Preparation of N-(S)-3-(4-carbamoylphenyl)-1-((S)-1-methylamino-1-oxo-3-phenylpropan-2-ylamino)-1-oxopropan-2-yl)-1,2,3,4-tetrahydronaphthalene-1-carboxamide (A6)

[0075] The preparation method was the same as that of Example 1, except that in Step 8, 1,2,3,4-tetrahydro-1-naphthoic acid was used instead of phenylacetic acid to obtain white solid A6 with a yield of 76%. 1H NMR (400 MHz, DMSO-d6): δ 1.50 - 1.66 (m, 2H), 1.75 - 1.88 (m, 2H), 2.58 (d, J = 3.9 Hz, 3H), 2.63 (s, 2H), 2.76 - 2.85 (m, 2H), 2.99 - 3.09 (m, 2H), 3.53 - 3.63 (m, 1H), 4.47 - 4.63 (m, 2H), 6.13 - 6.71 (m, 1H), 6.96 - 7.09 (m, 3H), 7.17 - 7.27 (m, 3H), 7.34 - 7.41 (m, 3H), 7.46 (s, 1H), 7.77 - 7.84 (m, 2H), 7.97 (d, J = 13.5 Hz, 2H), 8.21 (t, J = 7.6 Hz, 1H), 8.27 - 8.35 (m, 1H); 13C NMR (75 MHz, DMSO-d6): δ 20.6, 20.8, 25.5, 26.7, 27.1, 28.8, 37.5, 45.0, 53.7, 53.8, 121.4, 125.2, 125.9, 127.3, 127.4, 128.1, 128.4, 128.8, 129.0, 129.2, 130.2, 131.9, 132.2, 134.8, 136.8, 140.6, 141.2, 141.5, 167.7, 170.8, 170.9; HRMS (ESI, positive): Calcd. for C 31 H 33 BrN4O4 [M+Na] + 627.1577, 629.1557, found: 627.1572, 629.1557.

[0076] Example 7

[0077]

[0078] Preparation of 4-(S)-2-amino-3-((S)-3-(3-bromophenyl)-1-methylamino-1-oxopropyl)amino)-3-oxopropyl)benzamide (8A)

[0079] The preparation method was the same as that of Example 1, except that Step 8 was omitted to obtain a white solid with a yield of 87%. 11H NMR (300 MHz, DMSO-d6): δ 2.59 (d, J = 6.0 Hz, 3H), 2.73 - 2.81 (m, 1H), 2.95 (dd, J1 = 13.8 Hz, J2 = 4.2 Hz, 1H), 4.09 - 4.19 (m, 4H), 7.23 - 7.43 (m, 7H), 7.54 (s, 1H), 7.64 (t, J = 6.6 Hz, 2H), 7.70 (d, J = 8.7 Hz, 1H), 7.87 - 8.01 (m, 3H); 13 13C NMR (75 MHz, DMSO-d6): δ 26.0, 38.0, 44.6, 53.6, 56.6, 121.8, 126.9, 127.8, 128.8, 129.5, 129.7, 130.7, 132.5, 132.6, 141.1, 142.3, 142.6, 168.2, 171.4, 174.3; HRMS (ESI, positive): Calcd. for C 20 H 23 BrN4O3 [M + Na] + 469.084, 471.0825, found: 469.0841, 471.0825.

[0080] Example 8

[0081]

[0082] Preparation of 2S-3-(3-bromophenyl)-2-(2-cyclohexyl-2-phenylacetamido)-N-methylpropanamide (B1)

[0083] In a 25 mL single-necked flask, dissolve 2-cyclohexyl-2-phenylacetic acid (32.0 mg, 0.1 mmol) in DMF (3 mL), add 1-hydroxybenzotriazole HOBT (23.8 mg, 0.2 mmol), O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate HBTU (66.8 mg, 0.2 mmol). Then, under nitrogen protection, after 10 min, add a DMF (1.5 mL) solution of compound 6 (37.7 mg, 0.1 mmol) with an R2 group of bromine at the 3-position. Cool the reaction system to 0 °C, slowly dropwise add N,N-diisopropylethylamine DIEA (72 μL, 1.4 mmol) to the reaction system, slowly warm to room temperature, and stir at room temperature for 12 h. After the reaction is complete, add a small amount of ethyl acetate to the crude product obtained by distillation under reduced pressure, and filter by suction to obtain white solid B1 (48.3 mg, yield 72%). 11H NMR (400 MHz, DMSO-d6): δ 0.87 - 1.23 (m, 10H), 1.85 - 1.92 (m, 1H), 2.56 (d, J = 4.2 Hz, 3H), 2.64 - 2.70 (m, 1H), 2.78 (dd, J1 = 13.6 Hz, J2 = 4.9 Hz, 1H), 3.22 (d, J = 10.6 Hz, 1H), 4.47 (dd, J1 = 13.6 Hz, J2 = 8.2 Hz, 1H), 6.92 (t, J = 7.1 Hz, 2H), 7.16 (d, J = 6.3 Hz, 1H), 7.19 - 7.22 (m, 5H), 7.29 (s, 1H), 7.92 (d, J = 4.4 Hz, 1H), 8.26 (d, J = 8.2 Hz, 1H); 13 13C NMR (75 MHz, DMSO-d6): δ 25.4, 26.1, 30.2, 30.6, 31.1, 37.2, 53.5, 57.7, 121.2, 126.3, 127.9, 128.1, 128.4, 128.9, 129.8, 131.8, 139.5, 140.5, 171.2, 172.0; HRMS (ESI, positive): Calcd. for C 24 H 29 BrN2O2 [M + Na] + 479.1305, 481.1284, found: 479.1299, 481.1282.

[0084] Example 9

[0085]

[0086] Preparation of 3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl benzofuran-2-carboxamide (B2)

[0087] The preparation method was the same as that in Example 8, except that in Step 4, benzofuran-2-carboxylic acid was used instead of 2-cyclohexyl-2-phenylacetic acid, and a white solid B2 was obtained with a yield of 65%. 11H NMR (400 MHz, DMSO-d6) δ 8.87 (d, J = 8.6 Hz, 1H), 8.20 (q, J = 4.5 Hz, 1H), 7.77 (d, J = 7.5 Hz, 1H), 7.66 (d, J = 8.3 Hz, 1H), 7.60 (s, 1H), 7.56 (s, 1H), 7.48 - 7.44 (m, 1H), 7.36 - 7.30 (m, 3H), 7.20 (t, J = 7.8 Hz, 1H), 4.71 - 4.64 (m, 1H), 3.16 - 3.12 (m, 1H), 3.06 - 3.00 (m, 1H), 2.63 (d, J = 4.5 Hz, 3H). 13 13C NMR (101 MHz, DMSO-d6) δ 170.9, 158.0, 154.2, 148.6, 141.2, 131.9, 130.2, 129.2, 128.3, 127.1, 126., 123.7, 122.8, 121.4, 111.8, 109.8, 54.2, 36.8, 25.7. MS (ESI): m / z 401 [M+1] + .

[0088] Example 10

[0089]

[0090] (S)-N-(3-Bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)benzo[b]thiophene-2-carboxamide (B3) Preparation

[0091] The preparation method was the same as that of Example 8, except that in Step 4, benzo[b]thiophene-2-carboxylic acid was used instead of 2-cyclohexyl-2-phenylacetic acid to obtain white solid B3 with a yield of 65%. 1 1H NMR (400 MHz, DMSO-d6) δ 9.01 (d, J = 8.5 Hz, 1H), 8.21 (s, 1H), 8.15 (q, J = 4.5 Hz, 1H), 8.02 - 7.93 (m, 2H), 7.58 (t, J = 1.8 Hz, 1H), 7.48 - 7.40 (m, 2H), 7.38 - 7.33 (m, 2H), 7.22 (t, J = 7.8 Hz, 1H), 4.64 (ddd, J = 10.6, 8.5, 4.3 Hz, 1H), 3.12 (dd, J = 13.6, 4.3 Hz, 1H), 2.99 (dd, J = 13.6, 10.6 Hz, 1H), 2.64 (d, J = 4.5 Hz, 3H). 1313C NMR (101 MHz, DMSO-d6) δ 171.2, 161.5, 141.3, 140.3, 139.4, 139.1, 132.0, 130.3, 129.2, 128.3, 126.3, 125.3, 125.3, 125.0, 122.8, 121.4, 54.9, 36.9, 25.7. MS (ESI): m / z 417 [M+1] + .

[0092] Example 11

[0093]

[0094] Preparation of (S)-4-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl N-(bicyclo[2.2.2]octane-1-carboxylate) (B4)

[0095] The preparation method was the same as that of Example 8, except that in Step 4, 4-(methoxycarbonyl)bicyclo[2.2.2]octane-1-carboxylic acid was used instead of 2-cyclohexyl-2-phenylacetic acid to obtain white solid B4 with a yield of 65%. 1 1H NMR (400 MHz, DMSO-d6) δ 7.82 (q, J = 4.5 Hz, 1H), 7.43 (s, 1H), 7.41 - 7.32 (m, 2H), 7.23 - 7.17 (m, 2H), 4.46 - 4.40 (m, 1H), 3.56 (s, 3H), 2.98 - 2.94 (m, 1H), 2.85 - 2.79 (m, 1H), 2.59 (d, J = 4.5 Hz, 3H), 1.69 - 1.53 (m, 12H). 13 13C NMR (101 MHz, DMSO-d6) δ 176.9, 175.8, 171.2, 141.0, 131.9, 129.8, 128.8, 128.1, 121.0, 53.4, 51.3, 37.9, 37.8, 36.7, 27.4, 27.1, 25.4. MS (ESI): m / z 451 [M+1] + .

[0096] Example 12

[0097]

[0098] Preparation of 3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-4-methylbenzamide (B5)

[0099] The preparation method was the same as that of Example 8, except that in Step 4, 4-methylbenzoic acid was used instead of 2-cyclohexyl-2-phenylacetic acid to obtain white solid B5 with a yield of 65%. 1HNMR(400MHz, DMSO-d6) δ 8.54 (d, J=8.5Hz, 1H), 8.05 (q, J=4.5, 3.9Hz, 1H), 7.72 (d, J=8.3Hz, 2H), 7.56 (s, 1H), 7.34 (t, J=8.9Hz, 2H), 7.28 - 7.18 (m, 3H), 4.66 - 4.60 (m, 1H), 3.10 - 3.06 (m, 1H), 3.00 - 2.94 (m, 1H), 2.62 (d, J=4.6Hz, 3H), 2.33 (s, 3H). 13 C NMR(101MHz, DMSO-d6) δ 171.8, 166.4, 141.7, 141.4, 132.2, 131.4, 130.4, 129.3, 128.9, 128.5, 127.7, 121.6, 54.9, 37.1, 25.9, 21.2. MS(ESI): m / z 375 [M + 1] + .

[0100] Example 13

[0101]

[0102] Preparation of 3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl benzamide (B6)

[0103] The preparation method was the same as that of Example 8, except that benzoic acid was used instead of 2-cyclohexyl-2-phenylacetic acid in Step 4, and a white solid B6 was obtained with a yield of 65%. 1 H NMR(400MHz, DMSO-d6) δ 8.63 (d, J=8.5Hz, 1H), 8.07 (q, J=4.4Hz, 1H), 7.82 (s, 1H), 7.80 (d, J=1.4Hz, 1H), 7.57 (s, 1H), 7.51 (t, J=7.3Hz, 1H), 7.44 (t, J=7.4Hz, 2H), 7.35 (t, J=7.9Hz, 2H), 7.22 (t, J=7.8Hz, 1H), 4.67 - 4.62 (m, 1H), 3.14 - 2.93 (m, 2H), 2.63 (d, J=4.6Hz, 3H). 13 C NMR(101MHz, DMSO-d6) δ 171.7, 166.5, 141.7, 134.2, 132.2, 131.6, 130.4, 129.4, 128.5, 128.4, 127.7, 121.6, 55.0, 37.1, 25.9. MS(ESI): m / z 361 [M + 1] + .

[0104] Example 14

[0105]

[0106] Preparation of 3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-1H-benzoimidazole-2-carboxamide (B7)

[0107] The preparation method was the same as that in Example 8, except that 1H-benzoimidazole-2-carboxylic acid was used instead of 2-cyclohexyl-2-phenylacetic acid in Step 4, to obtain white solid B7 with a yield of 65%. 1 H NMR(400MHz,DMSO-d6)δ13.22(s,1H),8.80(d,J=8.8Hz,1H),8.12(d,J=4.8Hz,1H),7.75(d,J=7.9Hz,1H),7.51(d,J=6.9Hz,2H),7.36 - 7.25(m,4H),7.19(t,J=7.8Hz,1H),4.73(td,J=9.2,4.5Hz,1H),3.18 - 3.06(m,2H),2.64(d,J=4.5Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ170.7,158.7,145.3,141.1,132.2,130.5,129.5,128.5,124.4,122.8,121.6,120.2,54.3,37.2,25.9.MS(ESI):m / z 401[M + 1] + .

[0108] Example 15

[0109]

[0110] Preparation of 3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-5-methyl-1H-benzoimidazole-2-carboxamide (B8)

[0111] The preparation method was the same as that in Example 8, except that 5-methyl-1H-benzoimidazole-2-carboxylic acid was used instead of 2-cyclohexyl-2-phenylacetic acid in Step 4, to obtain white solid B8 with a yield of 65%. 11H NMR (400 MHz, DMSO-d6) δ 13.08 (s, 1H), 8.75 (d, J = 8.8 Hz, 1H), 8.14 - 8.10 (m, 1H), 7.60 (s, 1H), 7.51 (s, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 7.9 Hz, 1H), 7.19 (t, J = 7.8 Hz, 1H), 7.10 (d, J = 8.2 Hz, 1H), 4.74 - 4.68 (m, 1H), 3.16 - 3.05 (m, 2H), 2.63 (d, 3H), 2.42 (s, 3H). MS (ESI): m / z 415 [M + 1] + .

[0112] Example 16

[0113]

[0114] Preparation of 3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-5-methoxy-1H-benzimidazole-2-carboxamide (B9)

[0115] The preparation method was the same as that of Example 8, except that in Step 4, 5-methoxy-1H-benzimidazole-2-carboxylic acid was used instead of 2-cyclohexyl-2-phenylacetic acid to obtain white solid B9 with a yield of 65%. 1 1H NMR (400 MHz, DMSO-d6) δ 13.08 (s, 1H), 8.68 (d, J = 8.8 Hz, 1H), 8.14 - 8.11 (m, 1H), 7.62 (d, J = 8.7 Hz, 1H), 7.51 (s, 1H), 7..40 - 7.34 (m, 1H), 7.28 (d, J = 7.7 Hz, 1H), 7.21 - 7.17 (m, 1H), 6.97 - 6.88 (m, 2H), 4.75 - 4.68 (m, 1H), 3.78 (s, 3H), 3.16 - 3.04 (m, 2H), 2.64 (s, 3H). MS (ESI): m / z 431 [M + 1] + .

[0116] Example 17

[0117]

[0118] Preparation of 3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-5-fluoro-1H-benzimidazole-2-carboxamide (B10)

[0119] The preparation method was the same as that of Example 8, except that in Step 4, 5-fluoro-1H-benzimidazole-2-carboxylic acid was used instead of 2-cyclohexyl-2-phenylacetic acid to obtain white solid B10 with a yield of 65%.1 1H NMR (400 MHz, DMSO-d6) δ 13.34 (s, 1H), 8.82 (d, J = 8.6 Hz, 1H), 8.13 (d, J = 4.7 Hz, 1H), 7.51 (s, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.19 (t, J = 7.8 Hz, 2H), 4.73 - 4.67 (m, 1H), 3.17 - 3.04 (m, 2H), 2.63 (d, J = 4.5 Hz, 3H). MS (ESI): m / z 419 [M+1] + .

[0120] Example 18

[0121]

[0122] Preparation of 3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-5-chloro-1H-benzimidazole-2-carboxamide (B11)

[0123] The preparation method was the same as that of Example 8, except that 5-chloro-1H-benzimidazole-2-carboxylic acid was used instead of 2-cyclohexyl-2-phenylacetic acid in Step 4 to obtain white solid B11 with a yield of 65%. 1 1H NMR (400 MHz, DMSO-d6) δ 13.42 (s, 1H), 8.91 (t, J = 8.8 Hz, 1H), 8.13 (d, J = 4.7 Hz, 1H), 7.82 - 7.75 (m, 1H), 7.53 - 7.51 (m, 2H), 7.35 - 7.27 (m, 3H), 7.19 (t, J = 7.8 Hz, 1H), 4.71 (td, J = 9.3, 4.4 Hz, 1H), 3.20 - 3.02 (m, 2H), 2.63 (d, J = 4.5 Hz, 3H). MS (ESI): m / z 435 [M+1] + .

[0124] Example 19

[0125]

[0126] Preparation of 5-bromo-N-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-1H-benzimidazole-2-carboxamide (B12)

[0127] The preparation method was the same as that of Example 8, except that 5-bromo-1H-benzimidazole-2-carboxylic acid was used instead of 2-cyclohexyl-2-phenylacetic acid in Step 4 to obtain white solid B12 with a yield of 65%. 11H NMR (400 MHz, DMSO-d6) δ 8.88 (d, J = 8.8 Hz, 1H), 8.14 (q, J = 4.5 Hz, 1H), 7.81 (s, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.51 (s, 1H), 7.42 (dd, J = 8.6, 1.9 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 7.7 Hz, 1H), 7.18 (t, J = 7.8 Hz, 1H), 4.72 (td, J = 9.2, 4.4 Hz, 1H), 3.18 - 3.05 (m, 2H), 2.63 (d, J = 4.5 Hz, 3H). MS (ESI): m / z 481 [M+1] + .

[0128] Example 20

[0129]

[0130] Preparation of 3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-5-nitro-1H-benzimidazole-2-carboxamide (B13)

[0131] The preparation method was the same as that of Example 8, except that 5-nitro-1H-benzimidazole-2-carboxylic acid was used instead of 2-cyclohexyl-2-phenylacetic acid in Step 4 to obtain white solid B13 with a yield of 65%. 1 1H NMR (400 MHz, DMSO-d6) δ 13.77 (s, 1H), 9.07 (d, J = 8.7 Hz, 1H), 8.53 (s, 1H), 8.19 (d, J = 8.8 Hz, 1H), 8.14 (d, J = 4.7 Hz, 1H), 7.79 (s, 1H), 7.52 (s, 1H), 7.36 - 7.28 (m, 2H), 7.19 (t, J = 7.7 Hz, 1H), 4.75 - 4.68 (m, 1H), 3.18 - 3.06 (m, 2H), 2.63 (d, J = 4.5 Hz, 3H). MS (ESI): m / z 446 [M+1] + .

[0132] Example 21

[0133]

[0134] Preparation of 3-(3-bromophenyl)-1-(methylamino)-1-oxo-2-naphthamide (B14)

[0135] The preparation method was the same as that of Example 8, except that 2-naphthoic acid was used instead of 2-cyclohexyl-2-phenylacetic acid in Step 4 to obtain white solid B14 with a yield of 65%. 11H NMR (400 MHz, DMSO-d6) δ 8.81 (d, J = 8.5 Hz, 1H), 8.42 (s, 1H), 8.11 (q, J = 4.6 Hz, 1H), 8.00 (dd, J = 6.2, 3.0 Hz, 1H), 7.96 (d, J = 8.4 Hz, 2H), 7.88 (dd, J = 8.6, 1.8 Hz, 1H), 7.67 - 7.53 (m, 3H), 7.43 - 7.31 (m, 2H), 7.22 (t, J = 7.8 Hz, 1H), 4.77 - 4.66 (m, 1H), 3.18 - 2.98 (m, 2H), 2.64 (d, J = 4.6 Hz, 3H). 13 13C NMR (101 MHz, DMSO-d6) δ 171.7, 166.6, 141.7, 134.4, 132.3, 131.6, 130.5, 129.4, 129.0, 128.5, 128.1, 127.8, 127.0, 124.6, 121.6, 55.1, 37.2, 25.9. MS (ESI): m / z 411 [M+1] + .

[0136] Example 22

[0137]

[0138] Preparation of 4-(2S)-2-(2-cyclohexyl-2-phenylacetamido)-3-methylamino-3-oxopropylbenzamide (B15)

[0139] The preparation method is the same as Steps 1, 2, 5, and 8 of Example 1, except that compound 3 is used instead of compound 5 in Step 5, and 2-cyclohexyl-2-phenylacetic acid is used instead of phenylacetic acid in Step 8. After suction filtration, white solid B15 (39.3 mg, yield 68%) is obtained. 11H NMR (400 MHz, DMSO-d6): δ 0.44 - 0.56 (m, 1H), 0.60 - 0.68 (m, 1H), 1.02 - 1.11 (m, 3H), 1.14 - 1.23 (m, 3H), 1.55 (s, 2H), 1.86 - 1.99 (m, 1H), 2.56 (d, J = 3.4 Hz, 3H), 2.81 - 2.88 (m, 1H), 2.96 (dd, J1 = 13.4 Hz, J2 = 3.4 Hz, 1H), 3.23 (d, J = 10.7 Hz, 1H), 4.36 (dd, J1 = 13.6 Hz, J2 = 7.2 Hz, 1H), 7.01 (d, J = 7.6 Hz, 2H), 7.21 (s, 4H), 7.30 (s, 1H), 7.56 (d, J = 7.6 Hz, 2H), 7.80 (d, J = 7.5 Hz, 2H), 7.94 (d, J = 5.6 Hz, 1H), 8.21 (d, J = 7.8 Hz, 1H); 13 13C NMR (75 MHz, DMSO-d6): δ 25.2, 25.5, 26.0, 30.2, 31.2, 37.5, 53.6, 57.7, 126.3, 127.1, 127.9, 128.2, 128.8, 132.0, 139.5, 140.9, 167.6, 171.2, 172.0; HRMS (ESI, positive): Calcd. for C 25 H 31 N3O3 [M + Na]+ 444.2258, found: 444.2257.

[0140] Biological activity test

[0141] GloSensor TM cAMP Assay measures the cAMP content:

[0142] cAMP is a key signaling molecule for many G protein-coupled receptors. The cumulative level of cAMP is mainly measured using GloSensor - a bioluminescence-based biosensor that can directly detect intracellular cAMP (Promega). The principle is that a cAMP-binding domain is inserted at the N-terminus and C-terminus of firefly luciferase by genetic engineering technology, rendering the enzyme in an inactive state. When cAMP binds to the cAMP-binding domain, the enzyme is activated, thus oxidizing the substrate luciferin to produce bioluminescence. The cAMP accumulation experiment is used to test the functional activity of the target compound on β2AR and to clarify whether the new compound is a negative allosteric modulator (NAM) of β2AR. Briefly described as follows, HEK 293T cells are seeded into 6-well plates, with 4×10 5Cells. On the next day, β2AR and pGloSensor-22FcAMP plasmids were co-transfected into HEK 293T cells using FuGene transfection reagent (Promega). After 48 h, the transfected cells were washed with CO2-independent medium and then incubated with a balanced solution containing 2% v / v GloSensor cAMP reagent stock solution (dissolved in CO2-independent medium containing 10% FBS). After incubation at 37 °C for 1 h and then at room temperature for 1 h, the bioluminescence signal was detected using a multimode microplate reader until a steady-state baseline signal was obtained. Then, new derivatives and the control compound Cmpd-15 at different concentration gradients were added to the cells. After incubation at 37 °C for 30 min, the positive control ISO (final concentration 1 nM - 100 μM) was added. The change in bioluminescence was read using a microplate reader.

[0143]

[0144] Table 1. Statistical table of the bioactivity test results of Cmpd-15 derivatives

[0145]

[0146]

[0147] Note: Among them, "+" indicates having the corresponding activity, and "-" indicates having no corresponding activity

[0148] The test results showed (see Table 1) that most of the Cmpd-15 derivatives had no agonist activity on β2-adrenergic receptor (β2AR), and had antagonist effects on β2-adrenergic receptor at high concentrations. Among them, 8A had both agonist and antagonist activities, which provided a direction for the later work.

[0149] Allosteric antagonist activity screening

[0150] Using the GloSensor cAMP accumulation experiment, with isoproterenol (ISO) at different concentration gradients as the positive control (final concentration 1 nM - 100 μM) and compound Cmpd-15 (final concentration 50 μM) as the reference control, the allosteric antagonist activities of Cmpd-15 derivatives (final concentration 50 μM) were compared with those of the lead compound Cmpd-15. The test results in Table 2 showed that all the synthesized compounds had allosteric antagonist effects on β2-adrenergic receptor to varying degrees. The activities of B5, B6, B13, and B14 were also equivalent to or better than those of compound 15, but the M2 part in the original structure of Cmpd-15 was removed from their structures, which greatly simplified the structure and the synthesis process of the β2-adrenergic receptor allosteric antagonist. The simplified structure still has research significance, and it is expected to discover β2-adrenergic receptor allosteric antagonists with better activities.

[0151] Table 2. Statistical table of the comparison results of the allosteric activity and the activity of Cmpd-15 of Cmpd-15 derivatives

[0152]

[0153]

[0154] Note: a The value represents the blocking activity relative to Cmpd-15; "-" indicates that the compound has no allosteric antagonistic activity

[0155] Study on allosteric mechanism

[0156] The ability of this type of compound to allosterically regulate the functional activity of the endogenous ligand ISO of β2-AR was further studied through the GloSensor cAMP accumulation experiment to further confirm that this type of compound is a negative allosteric modulator (NAM) of β2-AR. Taking the B-type derivative B8 as an example ( Figure 1 ), when the concentration of compound B8 reached 30 μM, the curve of ISO showed a large downward shift and almost reached the lower limit of the maximum downward shift of the dose-effect regulation of ISO functional activity, indicating that the IC 50 value of compound B8 might be between 10 μM and 30 μM, specifically manifested as a sharp drop in the concentration curve. Then, when the concentration of B8 increased from 30.0 μM to 100.0 μM, the downward shift degree of the ISO concentration curve was very weak. This phenomenon explained that as the concentration of compound B8 increased, the concentration-dependent curve of ISO showed a limited downward shift, indicating that the Cmpd-15 derivative B8 of the compound could effectively negatively allosterically regulate the functional activity of the endogenous ligand ISO of β2-AR. This allosteric regulation phenomenon was consistent with the reported allosteric antagonistic regulation mechanism. That is, the newly synthesized Cmpd-15 derivatives of the compound are all negative allosteric modulators (NAM) of β2-AR.

Claims

1. A phenylalanine amide derivative, characterized in that: The structural formula of the phenylalanine amide derivative is shown in Formula 1 or Formula 2: 、 ； Among them, R1 is one of the following structural formulas: , R2 is: 3-Br, 4-CONH2.

2. A phenylalanine amide derivative, characterized in that: The structure of the phenylalanine amide derivative is as follows: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 3. Use of a phenylalanine amide derivative as claimed in claim 1 or 2 in the preparation of a drug as an allosteric antagonist of the β2-adrenergic receptor.

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