A method of electrochemically mediated glycine residue alpha-c-h functionalization enabling late-stage nitrogen heteroarylation of peptides

Abstract

The application discloses an electrochemical mediation method for glycine residue α The application discloses a method for realizing post-stage azaheteroarylation of a peptide by C-H functionalization, which realizes direct azaheteroarylation of a C-H bond under electrochemical conditions by using a controllable anodic oxidation approach α The strategy does not need a transition metal catalyst, avoids metal residues, and is particularly suitable for post-stage modification of a drug peptide and a biologically active molecule; the electrochemical indirect oxidation strategy is used, reaction conditions are mild, and functional group tolerance is excellent; direct post-stage modification of a complex molecule containing a glycine residue is realized, a synthesis route is simplified, and step economy is improved. In addition, the method not only has innovative value in a peptide modification method, but also provides an effective tool for exploring a biologically active molecule with further research potential, and azaheteroarylation products obtained by using the method have an inhibitory effect on a cancer cell line.

Description

Technical Field

[0001] This invention relates to chemical synthesis, specifically to a method for achieving late-stage azaarylation of peptides through electrochemically mediated α-CH functionalization of glycine residues. Background Technology

[0002] The synthesis and modification of amino acids occupy a unique position in the field of drug development. Glycine, as an amine α-functionalized substrate with important synthetic value, is a direct route for preparing high-value-added compounds such as amino acid derivatives through structural modification.

[0003] Peptides, as an important class of biomolecules, are composed of amino acids linked by amide bonds and possess a wide range of biological activities, finding extensive applications in the pharmaceutical and biotechnology fields. Therefore, peptide synthesis and modification occupy a unique position in the pharmaceutical field. Currently, modifying specific amino acids using bioactive groups has become an effective alternative method for peptide modification, contributing to the acquisition of new peptide-based drugs, antibody-drug conjugates, and chemical probes for bioimaging, among other high-value-added compounds. Glycine, as an α-functionalized amine substrate with significant synthetic value, offers a direct pathway for modifying glycine derivatives and peptides through structural modification. Under traditional transition metal catalysis or photochemical conditions, the α-functionalization of glycine has been extensively studied, with significant progress particularly in the construction of C-C bonds. In recent years, researchers have successfully constructed C-C and CP bonds through electro-oxidation strategies; however, currently reported methods still mainly focus on C-C bond formation. Developing new C-C bond functionalization strategies has become an urgent direction for expansion in this field.

[0004] Azo-containing aromatic compounds are important and advantageous structures in natural products and synthetic drugs. For example, the presence of the pyrazole skeleton in the approved drugs celecoxib and rimonaban highlights its importance in new drug development (Bioorg. Chem., 2020, 97, 103470-103532.). Furthermore, nitrogen-containing heterocyclic frameworks, represented by azoles and benzo[a]heterocyclic compounds, have been successfully integrated into peptide-based drug design to improve target specificity, pharmacological activity, metabolic stability, and cell permeability (Eur. J. Med. Chem., 2025, 290, 117534-117508; Bioorg. Chem., 2018, 76, 113-129; Antibiot., 2023, 12, 532-620.). The azo aromatic skeleton also possesses a broad spectrum of pharmacological activities, such as anticonvulsant (Eur. J. Med. Chem., 2009, 44, 3480-3487.); anticancer (Bioorg. Med. Chem., 2013, 21, 3859–3865; Eur. J. Med. Chem., 2013 70, 740-749.); analgesic (Eur. J. Med. Chem., 2013, 62, 410-415.); anti-inflammatory (Eur. J. Med. Chem., 1994, 29, 707-711; Bioorg. Med. Chem., 2007, 15, 5775-5786.); and antituberculosis (Bioorg. Med. Chem. Lett., 2012, 22, 666-678; Bioorg. Med. Chem. Lett., 2012, 22, 5129-5133.) etc. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing an electrochemically mediated method for the functionalization of the α-CH bond of glycine residues, enabling the late-stage azaarylation of peptides. This method selectively functionalizes the α-CH bonds of peptides, natural products, and drug molecules containing glycine residues through a controlled anodic oxidation pathway under mild conditions without transition metal catalysts, thereby directly introducing azaarylation groups into these bioactive molecules. Furthermore, this method not only represents an innovation in peptide modification techniques but also provides an effective tool for exploring bioactive molecules with further research potential. The azaarylation products obtained using this method have shown inhibitory effects on cancer cell lines.

[0006] The technical solution to achieve the objective of this invention is: A method for achieving late-stage azaarylation of peptides through electrochemically mediated α-CH functionalization of glycine residues, wherein the general synthetic formula for achieving late-stage azaarylation of peptides through α-CH functionalization of glycine residues is: , in: R 1 = Aromatic group, amino acid, polypeptide, natural product, or drug molecule; R 2 = Aryl or alkyl, or natural product or drug molecule; R 3 = Aryl or alkyl or halogen; The additives are: 2,2,6,6-tetramethylpiperidine oxide or ferrocene or ferric trifluoromethanesulfonate or triethylenediamine; the electrolytes are: sodium iodide or cuprous chloride or sodium bromide or potassium iodide or tetrabutylammonium iodide; the solvents are: acetonitrile or a mixture of acetonitrile and methanol or a mixture of acetonitrile and water.

[0007] The synthetic process for achieving late-stage azaarylation of the peptide through α-CH functionalization of glycine residues is as follows: 0.3 mmol of glycine derivative, 0.6 mmol of nitrogen heterocyclic compound, 0.2 mmol of additive, and 0.6 mmol of electrolyte were added to 10 mL three-necked flasks, dissolved in 6 mL of solvent. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 20-40 °C with stirring for 3-6 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with 3 × 5 mL of ethyl acetate. The organic layer was dried with anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography to obtain the target product. During purification, the ratio of silica gel, ethyl acetate / petroleum ether was 1:1-40.

[0008] This technical solution employs a controllable anodic oxidation pathway under electrochemical conditions to achieve direct nitrogen-asterylation of glycine-containing natural products, drug molecules, and peptide α-CH bonds. This method also provides new possibilities for discovering potential bioactive molecules.

[0009] This electrochemically mediated α-CH functionalization method for late-stage azaarylation of peptides utilizes a controlled anodic oxidation pathway to directly azaarylate glycine-containing natural products, drug molecules, and peptides. This strategy eliminates the need for transition metal catalysts, avoiding metal residues, and is particularly suitable for the late-stage modification of drug peptides and bioactive molecules. Employing an indirect electrochemical oxidation strategy, the reaction conditions are mild, and the functional groups exhibit excellent tolerance. It enables direct late-stage modification of complex molecules containing glycine residues, simplifying the synthetic route and improving procedural economy. This method not only represents an innovation in peptide modification but also provides an effective tool for exploring bioactive molecules with further research potential. Some azaarylate products obtained using this method have shown inhibitory effects on cancer cell lines. Detailed Implementation

[0010] The present invention will be further described below with reference to embodiments, but this is not intended to limit the scope of the invention.

[0011] Example 1: Preparation method and product characterization of (E)-2-(phenylimino)-2-(1H-pyrazole-1-yl)ethyl acetate (3a):

[0012] 0.3 mmol of ethylphenylglycine ester, 0.6 mmol of pyrazole, 0.2 mmol of 2,2,6,6-tetramethylpiperidine oxide, and 0.6 mmol of potassium iodide were added to a 10 mL three-necked flask, dissolved in 6 mL of acetonitrile. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 6 mA and 20 °C with stirring for 3.0 h. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:35 elution) to obtain the target product 3a. The product was characterized as follows: Yellow oil (86%, 62.8 mg). 1 H NMR (500 MHz, DMSO-d6) δ 8.58-8.55 (m,1H), 7.93 (d, J = 1.6 Hz, 1H), 7.42-7.36 (m, 2H), 7.24-7.19 (m, 1H), 7.03-6.97 (m, 2H), 6.71-6.70 (m, 1H), 4.18 (q, J = 7.1 Hz, 2H), 1.00 (t, J = 7.1Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 159.39, 145.87, 145.41, 144.17, 129.21,127.96, 125.46, 120.43, 110.12, 62.32, 13.54. HRMS (m / z) (ESI): calcd forC 13 H 14 N3O2 [M+H] + 244.1081, found 244.1083.

[0013] Example 2: Preparation method and product characterization of ethyl (E)-2-(1H-pyrazol-1-yl)-2-(p-tolylimino)acetate (3b):

[0014] 0.3 mmol of p-tolylglycine ethyl ester, 0.6 mmol of pyrazole, 0.2 mmol of ferrocene, and 0.6 mmol of potassium iodide were added to a 10 mL three-necked flask, dissolved in 6 mL of acetonitrile. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 8 mA and 20 °C with stirring for 3.0 h. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:35 elution) to obtain the target product 3b. The product was characterized as follows: Yellow oil (85%, 65.6 mg). 1 H NMR (400 MHz, Chloroform-d) δ 8.35-8.34(m, 1H), 7.76 (d, J = 1.6 Hz, 1H), 7.16-7.10 (m, 2H), 6.97-6.90 (m, 2H),6.51-6.49 (m, 1H), 4.26 (q, J = 7.1 Hz, 2H), 2.33 (s, 3H), 1.10 (t, J = 7.2Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 160.42, 145.79, 143.67, 143.40,135.19, 129.71, 127.43, 120.68, 109.25, 62.59, 21.08, 13.87. HRMS (m / z)(ESI): calcd for C 14 H16 N3O2 [M+H] + 258.1237, found 258.1241.

[0015] Example 3: Preparation method and product characterization of (E)-2-((4-bromophenyl)imino)-2-(1H-pyrazole-1-yl)ethyl acetate (3c):

[0016] 0.3 mmol of p-bromophenylglycine ethyl ester, 0.6 mmol of pyrazole, 0.2 mmol of ferrocene, and 0.6 mmol of potassium iodide were added to a 10 mL three-necked flask, dissolved in 6 mL of acetonitrile. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 7 mA and 25 °C with stirring for 3.5 h. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:30 elution) to obtain the target product 3c. The product was characterized as follows: Yellow oil (80%, 77.3 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.57-8.55 (m,1H), 7.95-7.94 (m, 1H), 7.61-7.56 (m, 2H), 7.00-6.92 (m, 2H), 6.72-6.70 (m,1H), 4.21 (q, J = 7.1 Hz, 2H), 1.05 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 159.14, 146.12, 144.73, 144.40, 132.09, 128.11, 122.70, 117.86,110.31, 62.53, 13.57. HRMS (m / z) (ESI): calcd for C 13 H 12 BrNaN3O2 [M+Na] + 344.0005, found 344.0010.

[0017] Example 4: Preparation method and product characterization of (E)-2-(naphth-2-ylimino)-2-(1H-pyrazol-1-yl)ethyl acetate (3d):

[0018] 0.3 mmol of ethylnaphthalene-2-ylglycine ester, 0.6 mmol of pyrazole, 0.2 mmol of ferric trifluoromethanesulfonate, and 0.6 mmol of sodium iodide were added to a 10 mL three-necked flask, dissolved in 6 mL of acetonitrile. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 9 mA and 27 °C with stirring for 3.5 h. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:30 elution) to obtain the target product 3d. The product was characterized as follows: Yellow oil (77%, 67.8 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.63 (d, J =2.8 Hz, 1H), 7.98-7.95 (m, 2H), 7.93-7.87 (m, 2H), 7.55-7.44 (m, 3H), 7.26-7.23 (m, 1H), 6.746.73 (m, 1H), 4.16 (q, J = 7.1 Hz, 2H), 0.94 (t, J = 7.1Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 159.47, 146.06, 144.28, 143.19, 133.21,130.86, 129.13, 128.06, 127.69, 127.45, 126.84, 125.60, 121.04, 116.54,110.20, 62.33, 13.54. HRMS (m / z) (ESI): calcd for C 17 H 16 N3O2 [M+H] + 294.1237, found 294.1240.

[0019] Example 5: Preparation method and product characterization of (E)-2-(phenylimino)-2-(1H-pyrazole-1-yl)phenyl acetate (3e):

[0020] 0.3 mmol of phenylglycine ester, 0.6 mmol of pyrazole, 0.2 mmol of ferrocene, and 0.6 mmol of potassium iodide were added to a 10 mL three-necked flask, dissolved in 6 mL of acetonitrile. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 9 mA and 30 °C with stirring for 4.0 h. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:35 elution) to obtain the target product 3e. The product was characterized as follows: Yellow oil (63%, 55.1 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J =2.7 Hz, 1H), 7.91 (d, J = 1.5 Hz, 1H), 7.49-7.43 (m, 2H), 7.35-7.27 (m, 4H),7.13-7.07 (m, 4H), 6.70-6.69 (m, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 157.88,148.40, 145.88, 143.58, 137.40, 128.93, 128.91, 127.83, 124.82, 124.42,120.69, 119.58, 109.68. HRMS (m / z) (ESI): calcd for C 17 H 13 NaN3O2 [M+Na] + 314.0900, found 314.0908.

[0021] Example 6: Preparation method and product characterization of (E)-2-(1H-pyrazol-1-yl)-2-(p-tolylimino)acetic acid benzo[d][1,3]m-dioxanepenten-5-yl methyl ester (3f):

[0022] 0.3 mmol of benzo[d][1,3]dioxolane-5-ylmethyl-p-tolylglycine ester, 0.6 mmol of pyrazole, 0.2 mmol of triethylenediamine, and 0.6 mmol of tetrabutylammonium iodide were added to 10 mL three-necked flasks, and 6 mL of a mixture of acetonitrile and methanol (v:v = 5:1) was added to dissolve the flasks. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 10 mA and 30 °C with stirring for 4.0 h. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:30 elution) to obtain the target product 3f, which was characterized as follows: Yellow oil (62%, 67.6 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J =2.7 Hz, 1H), 7.92 (d, J = 1.6 Hz, 1H), 7.11-7.08 (m, 2H), 6.84-6.81 (m, 3H), 6.70-6.67 (m, 2H), 6.61-6.60 (m, 1H), 6.03 (s, 2H), 5.13 (s, 2H), 2.29 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 159.52, 147.44, 147.21, 145.13, 143.98,142.55, 134.79, 129.61, 127.87, 127.79, 122.82, 120.31, 109.94, 109.08,107.99, 101.18, 67.57, 20.47. HRMS (m / z) (ESI): calcd for C 19 H 18 N3O2 [M+H] + 320.1394, found 320.1390.

[0023] Example 7: Preparation method and product characterization of (E)-2-(1H-pyrazol-1-yl)-2-(p-tolylimino)acetic acid ((3aR, 5S, 5aS, 8aS, 8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]m-dioxacyclopentenano)[4,5-b:4', 5'-d]pyran-5-yl)methyl ester (3g):

[0024] 0.3 mmol of ((3aR,5S,5aS,8aS,8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxane)[4,5-b:4',5'-d]pyran-5-yl)methyl-p-tolylglycine ester, 0.6 mmol of pyrazole, 0.2 mmol of triethylenediamine, and 0.6 mmol of tetrabutylammonium iodide were added to 10 mL three-necked flasks, and 6 mL of an acetonitrile-water mixture (v:v = 5:1) were added to dissolve the esters. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 10 mA and 35 °C with stirring for 4.0 h. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The mixture was then subjected to column chromatography (silica gel, ethyl acetate / petroleum ether = 1:25). (Elution) The residue was purified to obtain 3g of the target product, which was characterized as follows: Yellow oil (56%, 79.2 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.57-8.55 (m,1H), 7.91-7.90 (m, 1H), 7.19 (d, J = 6.8 Hz, 2H), 6.93 (d, J = 8.3 Hz, 2H), 6.71-6.69 (m, 1H), 5.45 (d, J = 4.9 Hz, 1H), 4.53-4.50 (m, 1H), 4.37-4.34 (m,1H), 4.25-4.19 (m, 2H), 3.89-3.86 (m, 1H), 3.83-3.78 (m, 1H), 2.30 (s, 3H),1.40 (s, 3H), 1.33 (s, 3H), 1.28 (s, 3H), 1.25 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 159.59, 144.78, 144.05, 142.55, 134.94, 129.84, 127.90, 120.46, 110.03, 108.59, 108.08, 95.52, 69.87, 69.65, 69.63, 64.60, 64.15, 25.79,25.71, 24.82, 24.14, 20.55. HRMS (m / z) (ESI): calcd for C 24 H 30 N3O7 [M+H] +472.2078, found 472.2083.

[0025] Example 8: Preparation method and product characterization of (E)-2-(1H-pyrazol-1-yl)-2-(p-tolylimino)acetic acid 2-((1R, 5S)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)ethyl ester (3h):

[0026] 0.3 mmol of 2-((1R,5S)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)ethyl p-tolyl glycine ester, 0.6 mmol of pyrazole, 0.2 mmol of ferrocene, and 0.6 mmol of sodium bromide were added to 10 mL three-necked flasks, and 6 mL of a mixture of acetonitrile and methanol (v:v = 5:1) were added to dissolve them. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 6 mA and 35 °C with stirring for 5.5 h. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:40 elution) to obtain the target product. The product was characterized as follows: Yellow oil (62%, 70.2 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J =2.8 Hz, 1H), 7.90 (d, J = 1.6 Hz, 1H), 7.19 (d, J = 7.9 Hz, 2H), 6.90 (d, J =8.1 Hz, 2H), 6.70-6.66 (m, 1H), 5.12-5.10 (m, 1H), 4.20-4.06 (m, 2H), 2.29(s, 3H), 2.28-2.26 (m, 1H), 2.21-2.14 (m, 1H), 2.12-2.04 (m, 3H), 2.02-1.99(m, 1H), 1.95-1.91 (m, 1H), 1.21 (s, 3H), 0.99 (d, J = 8.5 Hz, 1H), 0.71 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 159.72, 145.25, 143.91, 143.14, 142.72,134.79, 129.68, 127.78, 120.45, 118.24, 109.90, 64.08, 44.77, 40.15, 37.50,34.74, 31.00, 30.81, 25.97, 20.84, 20.48. HRMS (m / z) (ESI): calcd forC 23 H 28 N3O2 [M+H] + 378.2176 found 378.2177.

[0027] Example 9: Preparation method and product characterization of ethyl(E)-2-((3-fluoro-4-morpholinophenyl)imino)-2-(1H-pyrazol-1-yl)(3i):

[0028] 0.3 mmol of ethyl (3-fluoro-4-morpholinylphenyl)glycine ester, 0.6 mmol of pyrazole, 0.2 mmol of ferrocene, and 0.6 mmol of sodium bromide were added to a 10 mL three-necked flask, and dissolved in 6 mL of a mixture of acetonitrile and methanol (v:v = 5:1). Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 15 mA and 40 °C with stirring for 3 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:15 elution) to obtain the target product 3i. The product was characterized as follows: Yellow oil (65%, 67.5 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.53 (d, J =2.7 Hz, 1H), 7.93 (d, J = 1.6 Hz, 1H), 7.08-7.03 (m, 1H), 6.91-6.86 (m, 1H),6.82-6.77 (m, 1H), 6.70-6.68 (m, 1H), 4.25 (q, J = 7.1 Hz, 2H), 3.76-3.71 (m,4H), 3.01-2.95 (m, 4H), 1.07 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ159.57, 154.68 (d, J = 244.7 Hz, 1 J CF ), 145.85, 144.20, 139.90 (d, J = 11.6Hz, 2 J CF ), 137.42 (d, J = 8.5 Hz, 3 J CF ), 127.91, 119.41 (d, J = 4.4 Hz, 3 J CF ), 116.89 (d, J = 3 Hz, 4 J CF ), 110.07, 109.40 (d, J = 22.8 Hz, 2 J CF ), 66.14, 62.43, 50.58, 50.54, 13.57. 19 F NMR (376 MHz, DMSO-d6) δ -123.42. HRMS (m / z)(ESI): calcd for C 17 H 20 FN4O3 [M+H] + 347.1514, found 347.1517.

[0029] Example 10: Preparation method and product characterization of ethyl (E)-2-(1H-benzo[d][1, 2, 3]triazol-1-yl)-2-(phenylimino)ethyl acetate (3j):

[0030] 0.3 mmol of ethylphenylglycine ester, 0.6 mmol of benzotriazole, 0.2 mmol of 2,2,6,6-tetramethylpiperidine oxide, and 0.6 mmol of potassium iodide were added to a 10 mL three-necked flask, dissolved in 6 mL of acetonitrile. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 15 mA and 35 °C with stirring for 4 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:35 elution) to obtain the target product 3j. The product was characterized as follows: Yellow oil (81%, 71.5 mg).1 H NMR (400 MHz, DMSO-d6) δ 8.38 (d, J =8.2 Hz, 1H), 8.31 (d, J = 8.3 Hz, 1H), 7.83 (t, J = 7.6 Hz, 1H), 7.68 (t, J =7.7 Hz, 1H), 7.47 (t, J = 7.6 Hz, 2H), 7.29 (t, J = 7.4 Hz, 1H), 7.17-7.12(m, 2H), 4.31 (q, J = 7.1 Hz, 2H), 1.06 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 158.59, 145.60, 145.10, 130.82, 130.08, 129.35, 126.93, 126.05, 120.43, 120.24, 114.31, 63.07, 13.53. HRMS (m / z) (ESI): calcd for C 16 H 15 N4O2 [M+H] + 295.1190, found 295.1197.

[0031] Example 11: Preparation method and product characterization of ethyl (E)-2-(5-methyl-1H-benzo[d][1,2,3]triazol-1-yl)-2-(phenylimino)acetate (3k):

[0032] 0.3 mmol of ethylphenylglycine ester, 0.6 mmol of 5-methyl-1H-benzo[d][1,2,3]triazole, 0.2 mmol of 2,2,6,6-tetramethylpiperidine oxide, and 0.6 mmol of sodium iodide were added to a 10 mL three-necked flask, dissolved in 6 mL of acetonitrile. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 10 mA and 30 °C with stirring for 5 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:35 elution) to obtain the target product 3K. The product was characterized as follows: Yellow oil (78%, 72.2 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.25-8.06 (m,2H), 7.67-7.49 (m, 1H), 7.48-7.44 (m, 2H), 7.31-7.26 (m, 1H), 7.16-7.10 (m,2H), 4.34-4.26 (m, 2H), 2.56-2.53 (m, 3H), 1.08-1.03 (m, 3H). 13 C NMR (101MHz, DMSO-d6) δ 158.63, 145.20, 144.22, 141.55, 132.49, 130.51, 129.31,128.76, 125.95, 120.42, 119.66, 113.45, 62.99, 21.61, 13.52. HRMS (m / z)(ESI): calcd for C 17 H 17 N4O2 [M+H] + 309.1346, found 309.1352.

[0033] Example 12: Preparation method and product characterization of ethyl (E)-2-(5-chloro-1H-benzo[d][1, 2, 3]triazol-1-yl)-2-(phenylimino)ethyl acetate (3l):

[0034] 0.3 mmol of ethylphenylglycine ester, 0.6 mmol of 5-chloro-1H-benzo[d][1,2,3]triazole, 0.2 mmol of 2,2,6,6-tetramethylpiperidine oxide, and 0.6 mmol of sodium iodide were added to a 10 mL three-necked flask, dissolved in 6 mL of acetonitrile. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 9 mA and 35 °C with stirring for 5 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:35 elution) to obtain the target product 3 μL. The product was characterized as follows: Yellow oil (81%, 79.9 mg). 1H NMR (500 MHz, Chloroform-d) δ 8.37 (d,J = 8.7 Hz, 1H), 8.14 (d, J = 1.9 Hz, 1H), 7.63-7.60 (m, 1H), 7.42-7.38 (m,2H), 7.26-7.22 (m, 1H), 7.13-7.09 (m, 2H), 4.34 (q, J = 7.1 Hz, 2H), 1.15 (t,J = 7.1 Hz, 3H). 13 C NMR (126 MHz, Chloroform-d) δ 158.96, 147.02, 145.50,145.45, 132.09, 130.78, 129.64, 129.31, 126.13, 120.63, 119.82, 115.67,63.25, 13.84. HRMS (m / z) (ESI): calcd for C 16 H 14 ClN4O2 [M+H] + 329.0800, found329.0809.

[0035] Example 13: Preparation method and product characterization of (E)-2-(4-iodo-1H-pyrazol-1-yl)-2-(p-tolylimino)acetic acid ((1s, 3s)-adamantane-1-yl)methyl ester (3m):

[0036] 0.3 mmol ((3r,5r,7r)-adamantane-1-yl)methyl-p-tolylglycine ester, 0.6 mmol 4-iodo-1H-pyrazole, 0.2 mmol 2,2,6,6-tetramethylpiperidine oxide, and 0.6 mmol sodium iodide were added to a 10 mL three-necked flask, dissolved in 6 mL acetonitrile. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 9 mA and 35 °C with stirring for 5 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:35 elution) to obtain the target product 3m. The product was characterized as follows: Yellow oil (68%, 102.7 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.75 (s, 1H), 8.01 (s, 1H), 7.19 (d, J = 7.9 Hz, 2H), 6.90-6.86 (m, 2H), 3.75 (s, 2H), 2.27(s, 3H), 1.81 (s, 3H), 1.62-1.58 (m, 3H), 1.50-1.46 (m, 3H), 1.23-1.21 (m,6H). 13 C NMR (126 MHz, DMSO-d6) δ 159.20, 148.46, 144.37, 142.75, 134.88,132.27, 129.77, 120.25, 75.05, 65.12, 37.93, 36.09, 32.69, 27.24, 20.47. HRMS(m / z) (ESI): calcd for C 23 H 27 IN3O2 [M+H] + 504.1142, found 504.1152.

[0037] Example 14: Preparation method and product characterization of ethyl (E)-2-(4-(2-chloroethyl)-1H-pyrazol-1-yl)-2-(phenylimino)ethyl acetate (3n):

[0038] 0.3 mmol of ethylphenylglycine ester, 0.6 mmol of 4-(2-chloroethyl)-1H-pyrazole, 0.2 mmol of triethylenediamine, and 0.6 mmol of sodium bromide were added to a 10 mL three-necked flask, dissolved in 6 mL of acetonitrile. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 6 mA and 35 °C with stirring for 6 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:40 elution) to obtain the target product 3n. The product was characterized as follows: Yellow oil (77%, 70.6 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.47-8.46 (m,1H), 7.88-7.87 (m, 1H), 7.41-7.36 (m, 2H), 7.23-7.18 (m, 1H), 7.01-6.96 (m,2H), 4.17 (q, J = 7.1 Hz, 2H), 3.87 (t, J = 6.8 Hz, 2H), 3.01-2.95 (m, 2H), 0.99 (t, J = 7.1 Hz, 3H). 13 HRMS (m / z) (ESI): calcd for C 15 H 17 ClN3O2 [M+H] + 306.1004, found 306.1010.

[0039] Example 15: Preparation method and product characterization of ethyl (E)-2-(3-phenyl-1H-pyrazol-1-yl)-2-(p-tolylimino)acetate (3o):

[0040] 0.3 mmol of p-tolylglycine ethyl ester, 0.6 mmol of 3-phenyl-1H-pyrazole, 0.2 mmol of 2,2,6,6-tetramethylpiperidine oxide, and 0.6 mmol of cuprous chloride were added to a 10 mL three-necked flask, dissolved in 6 mL of a mixture of acetonitrile and methanol (v:v = 5:1). Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 8 mA and 40 °C with stirring for 5 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:35 elution) to obtain the target product 3o. The product was characterized as follows: Yellow oil (62%, 62.0 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J =2.8 Hz, 1H), 7.91-7.86 (m, 2H), 7.51-7.40 (m, 3H), 7.25-7.19 (m, 3H), 6.96-6.91 (m, 2H), 4.29 (q, J = 7.1 Hz, 2H), 2.30 (s, 3H), 1.14 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 159.66, 154.41, 145.31, 142.85, 134.77,131.24, 129.69, 129.48, 129.16, 128.94, 125.85, 120.49, 107.68, 62.32, 20.50,13.71. HRMS (m / z) (ESI): calcd for C 20 H 20 N3O2 [M+H] + 334.1550, found 334.1557.

[0041] Example 16: Preparation method and product characterization of (E)-2-(1H-benzo[d]imidazol-1-yl)-2-(p-tolylimino)ethyl acetate (3p):

[0042] 0.3 mmol of p-tolylglycine ethyl ester, 0.6 mmol of benzimidazole, 0.2 mmol of ferrocene, and 0.6 mmol of potassium iodide were added to a 10 mL three-necked flask, dissolved in 6 mL of acetonitrile. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 9 mA and 25 °C with stirring for 6 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:40 elution) to obtain the target product 3p. The product was characterized as follows: Yellow oil (72%, 62.0 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.72 (s, 1H),8.28-8.20 (m, 1H), 7.86-7.78 (m, 1H), 7.47-7.40 (m, 2H), 7.20 (d, J = 7.8 Hz,2H), 6.91 (d, J = 7.9 Hz, 2H), 4.27 (q, J = 7.1 Hz, 2H), 2.30 (s, 3H), 1.02(t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 159.06, 143.82, 143.67,142.94, 134.46, 131.09, 129.58, 125.15, 124.80, 120.21, 120.04, 115.24,63.17, 20.46, 13.29. HRMS (m / z) (ESI): calcd for C 18 H 18 N3O2 [M+H] + 308.1394, found 308.1402.

[0043] Example 17: Preparation method and product characterization of methyl (S,E)-2-((tert-butoxycarbonyl)amino)-3-(4-((2-ethoxy-2-oxo-1-(1H-pyrazol-1-yl)ethylene)amino)phenyl)propionate (3q):

[0044] 0.3 mmol of p-methyl(S)-2-((tert-butyloxycarbonyl)amino)-3-(4-(((2-ethoxy-2-oxoethyl)amino)phenyl)propionate, 0.6 mmol of pyrazole, 0.2 mmol of ferric trifluoromethanesulfonate, and 0.6 mmol of tetrabutylammonium iodide were added to a 10 mL three-necked flask, dissolved in 6 mL of acetonitrile. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 15 mA and 25 °C with stirring for 6 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:5 elution) to obtain the target product 3q. The product was characterized as follows: Yellow oil (76%, 101.3 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J =2.8 Hz, 1H), 7.92 (d, J = 1.6 Hz, 1H), 7.31-7.24 (m, 3H), 6.92 (d, J = 8.0Hz, 2H), 6.69-6.68 (m, 1H), 4.20-4.16 (m, 2H), 4.15-4.11 (m, 1H), 3.60 (s,3H), 3.00-2.94 (m, 1H), 2.88-2.82 (m, 1H), 1.34 (s, 9H), 1.00 (t, J = 7.1 Hz,3H). 13 C NMR (101 MHz, DMSO-d6) δ 172.60, 159.46, 155.40, 145.71, 144.05,143.80, 134.85, 129.82, 127.85, 120.34, 110.00, 78.28, 62.28, 55.25, 51.76,35.90, 28.12, 13.51. HRMS (m / z) (ESI): calcd for C 22 H 29 N4O6 [M+H] + 445.2082, found 445.2091.

[0045] Example 18: Preparation method and product characterization of ((S)-2-((tert-butoxycarbonyl)amino)-3-(4-(((E)-2-ethoxy-2-oxo-1-(1H-pyrazol-1-yl)ethylene)amino)phenyl)propionyl)-L-valine methyl ester (3r)

[0046] 0.3 mmol of methyl((S)-2-((tert-butyloxycarbonyl)amino)-3-(4-(((2-ethoxy-2-oxoethyl)amino)phenyl)propionyl)-L-valine, 0.6 mmol of pyrazole, 0.2 mmol of 2,2,6,6-tetramethylpiperidine oxide, and 0.6 mmol of sodium iodide were added to a 10 mL three-necked flask, dissolved in 6 mL of acetonitrile. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 15 mA and 30 °C with stirring for 6 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:5 elution) to obtain the target product 3r. The product was characterized as follows: Yellow oil (68%, 110.9 mg). 1 H NMR (500 MHz, DMSO-d6) δ 8.53 (d, J =2.8 Hz, 1H), 8.12 (d, J = 8.3 Hz, 1H), 7.92 (d, J = 1.6 Hz, 1H), 7.30 (d, J =8.2 Hz, 2H), 6.95-6.93 (m, 1H), 6.92-6.90 (m, 2H), 6.70-6.68 (m, 1H), 4.27-4.23 (m, 1H), 4.22-4.19 (m, 1H), 4.19-4.15 (m, 2H), 3.64 (s, 3H), 2.95-2.90(m, 1H), 2.76-2.70 (m, 1H), 2.09-2.03 (m, 1H), 1.31 (s, 9H), 1.00 (t, J = 7.0Hz, 3H), 0.91-0.88 (m, 6H). 13 C NMR (126 MHz, DMSO-d6) δ 172.18, 171.93,159.52, 155.29, 145.60, 143.99, 143.54, 135.53, 129.94, 127.92, 120.15,110.00, 78.01, 62.27, 57.28, 55.43, 51.76, 36.57, 30.13, 28.13, 18.90, 18.14,13.54. HRMS (m / z) (ESI): calcd for C 27 H 38 N5O7 [M+H] +544.2766, found 544.2772.

[0047] Example 19: Preparation method and product characterization of ((S)-2-((tert-butoxycarbonyl)amino)-3-(4-(((E)-2-ethoxy-2-oxo-1-(1H-pyrazol-1-yl)ethylene)amino)phenyl)propionyl)-L-methionine methyl ester (3S):

[0048] 0.3 mmol of methyl((S)-2-((tert-butyloxycarbonyl)amino)-3-(4-(((2-ethoxy-2-oxoethyl)amino)phenyl)propionyl)-L-methionine ester, 0.6 mmol of pyrazole, 0.2 mmol of 2,2,6,6-tetramethylpiperidine oxide, and 0.6 mmol of potassium iodide were added to a 10 mL three-necked flask, dissolved in 6 mL of an acetonitrile-water mixture (v:v = 5:1). Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 9 mA and 25 °C with stirring for 5 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:3 elution) to obtain the target product 3S. The product was characterized as follows: Yellow oil (60%, 103.6 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.53 (d, J =2.7 Hz, 1H), 8.36 (d, J = 7.8 Hz, 1H), 7.92 (d, J = 1.6 Hz, 1H), 7.30 (d, J =8.1 Hz, 2H), 6.93 (t, J = 8.7 Hz, 3H), 6.70-6.68 (m, 1H), 4.47-4.41 (m, 1H), 4.18 (q, J = 7.0 Hz, 3H), 3.64-3.63 (m, 3H), 2.97-2.92 (m, 1H), 2.77-2.71 (m,1H), 2.58-2.50 (m, 2H), 2.05 (s, 3H), 2.01-1.88 (m, 2H), 1.31 (s, 9H), 1.00 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 172.18, 172.11, 159.51,155.28, 145.59, 144.03, 143.55, 135.53, 129.92, 127.82, 120.19, 109.99,78.00, 62.27, 55.56, 52.01, 50.84, 36.56, 30.69, 29.41, 28.14, 14.62, 13.53.HRMS (m / z) (ESI): calcd for C 27 H 38 N5O7S [M+H] + 576.2486, found 576.2481.

[0049] Example 20: Preparation method and product characterization of ((S)-2-((tert-butoxycarbonyl)amino)-3-(4-(((E)-2-ethoxy-2-oxo-1-(1H-pyrazol-1-yl)ethylene)amino)phenyl)propionyl)-L-phenylalanine methyl ester (3t):

[0050] 0.3 mmol of methyl((S)-2-((tert-butyloxycarbonyl)amino)-3-(4-(((2-ethoxy-2-oxoethyl)amino)phenyl)propionyl)-L-phenylalanine ester, 0.6 mmol of pyrazole, 0.2 mmol of triethylenediamine, and 0.6 mmol of cuprous chloride were added to a 10 mL three-necked flask, dissolved in 6 mL of a mixture of acetonitrile and methanol (v:v = 5:1). Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 7 mA and 35 °C with stirring for 5 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:2 elution) to obtain the target product 3t. The product was characterized as follows: Yellow oil (61%, 108.3 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.54-8.52 (m,1H), 8.36-8.35 (m, 1H), 7.92-7.90 (m, 1H), 7.31-7.21 (m, 7H), 6.91-6.85 (m,3H), 6.69-6.67 (m, 1H), 4.56-4.48 (m, 1H), 4.21-4.15 (m, 3H), 3.59 (s, 3H), 3.11-2.96 (m, 2H), 2.91-2.86 (m, 1H), 2.72-2.66 (m, 1H), 1.30 (s, 9H), 1.01(t, J = 7.2 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 171.87, 171.84, 159.51,155.13, 145.58, 144.02, 143.53, 137.00, 135.43, 129.88, 129.13, HRMS (m / z) (ESI): calcd for C 31 H 38 N5O7 [M+H] + 592.2766, found 592.2772.

[0051] Example 21: Preparation method and product characterization of (S,E)-6-(4-((2-ethoxy-2-oxo-1-(1H-pyrazol-1-yl)ethylene)amino)benzyl)-2,2-dimethyl-4,7,10-trioxo-3-oxa-5,8,11-triazatridecane-13-olate (3u):

[0052] 0.3 mmol of ethyl(S)-(4-(2-((tert-butyloxycarbonyl)amino)-3-((2-((2-ethoxy-2-oxoethyl)amino)-2-oxoethyl)amino)-3-oxopropyl)phenyl)glycine ester, 0.6 mmol of pyrazole, 0.2 mmol of ferrocene, and 0.6 mmol of sodium iodide were added to a 10 mL three-necked flask, dissolved in 6 mL of acetonitrile. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 9 mA and 35 °C with stirring for 5 h. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:1 elution) to obtain the target product 3u. The product was characterized as follows: Yellow oil (70%, 120.2 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.53 (d, J =2.7 Hz, 1H), 8.28 (t, J = 5.7 Hz, 1H), 8.21 (t, J = 6.0 Hz, 1H), 7.92 (d, J =1.6 Hz, 1H), 7.28 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 8.2 Hz, 1H), 6.91 (d, J =8.2 Hz, 2H), 6.69-6.68 (m, 1H), 4.21-4.18 (m, 1H), 4.17-4.15 (m, 2H), 4.12-4.06 m, 2H), 3.87-3.84 (m, 2H), 3.80-3.76 (m, 2H), 3.02-2.97 (m, 1H), 2.78-2.71 (m, 1H), 1.31 (s, 9H), 1.21-1.16 (m, 3H), 1.01 (t, J = 7.1 Hz, 3H). 13 CNMR (101 MHz, DMSO-d6) δ 172.03, 169.65, 169.31, 159.52, 155.42, 145.58,144.02, 143.52, 135.60, 129.92, 127.82, 120.19, 109.97, 78.13, 62.27, 60.47,55.75, 41.82, 40.65, 36.74, 28.15, 14.07, 13.54. HRMS (m / z) (ESI): calcd forC 27 H37 N6O8 [M+H] + 573.2667, found 573.2659.

[0053] Example 22: Preparation method and product characterization of (S,E)-6-(4-((2-ethoxy-2-oxo-1-(1H-pyrazol-1-yl)ethylene)amino)benzyl)-2,2-dimethyl-4,7,10,13-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-olate (3v):

[0054] 0.3 mmol of (S)-4-(2-((tert-Butoxycarbonyl)amino)-3-((2-((2-(((2-ethoxy-2-oxoethyl)amino)-2-oxoethyl)amino)-2-oxoethyl)amino)-3-oxopropyl)phenylethylenediamine acetate, 0.6 mmol of pyrazole, 0.2 mmol of 2,2,6,6-tetramethylpiperidine oxide, and 0.6 mmol of potassium iodide were added to 10 mL three-necked flasks, dissolved in 6 mL of acetonitrile. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 6 mA and 30 °C with stirring for 6 h. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with ethyl acetate (3 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1:1 elution) to obtain the target product 3V. The product was characterized as follows: Yellow oil (67%, 126.6 mg). 1H NMR (600 MHz, DMSO-d6) δ 8.54 (d, J =2.7 Hz, 1H), 8.27-8.22 (m, 2H), 8.13 (t, J = 5.8 Hz, 1H), 7.92 (d, J = 1.6Hz, 1H), 7.28 (d, J = 8.0 Hz, 2H), 7.00 (d, J = 8.3 Hz, 1H), 6.91-6.90 (m,2H), 6.69-6.68 (m, 1H), 4.20-4.18 (m, 1H), 4.18-4.15 (m, 2H), 4.09-4.07 (m,2H), 3.84-3.82 (m, 2H), 3.78-3.74 (m, 4H), 3.02-2.98 (m, 1H), 2.78-2.72 (m,1H), 1.31 (s, 9H), 1.18 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 7.1 Hz, 3H). 13 C NMR(151 MHz, DMSO-d6) δ 172.12, 169.67, 169.26, 169.07, 159.51, 155.37, 145.56,144.02, 143.51, 135.61, 129.90, 127.83, 120.18, 109.98, 78.11, 62.26, 60.43,55.69, 45.68, 42.13, 41.72, 40.64, 28.15, 14.07, 13.54. HRMS (m / z) (ESI):calcd for C 29 H 40 N7O9 [M+H] + 630.2882, found 630.2891.

[0055] To investigate the pharmacological activity of the synthesized compounds, the MTT assay was used to determine the in vitro antitumor activity of some compounds against SKOV3 (human ovarian adenocarcinoma cells), HepG2 (human hepatocellular carcinoma cells), MDA-MB-231 (human breast cancer cells), and A549 (human non-small cell lung cancer cells) cell lines. The results showed that most compounds exhibited good antitumor activity against tumor cell lines. Compound 31 showed good inhibitory activity against SKOV3, HepG2, MDA-MB-231, and A549 cell lines, with an IC50 concentration of [missing value]. 50The values ​​were 9.94 ± 1.46, 24.37 ± 3.60, 17.11 ± 3.67 and 29.60 ± 1.27 μM, respectively. The activity assay showed that some of the obtained products had a certain inhibitory effect on cancer cell lines.

Claims

1. A method for achieving late-stage azaarylation of peptides through electrochemically mediated α-CH functionalization of glycine residues, characterized in that, The general synthetic formula for achieving late-stage nitrogen-arylization of the peptide by α-CH functionalization of the glycine residue is as follows: ， in: R 1 = Aromatic group, amino acid, polypeptide, natural product, or drug molecule; R 2 = Aryl or alkyl, or natural product or drug molecule; R 3 = Aryl or alkyl or halogen; The additives are: 2,2,6,6-tetramethylpiperidine oxide or ferrocene or ferric trifluoromethanesulfonate or triethylenediamine; the electrolytes are: sodium iodide or cuprous chloride or sodium bromide or potassium iodide or tetrabutylammonium iodide; the solvents are: acetonitrile or a mixture of acetonitrile and methanol or a mixture of acetonitrile and water.

2. The method for achieving late-stage azaarylation of peptides via electrochemically mediated α-CH functionalization of glycine residues according to claim 1, characterized in that, The synthetic process for achieving late-stage azaarylation of the peptide through α-CH functionalization of glycine residues is as follows: 0.3 mmol of glycine derivative, 0.6 mmol of nitrogen heterocyclic compound, 0.2 mmol of additive, and 0.6 mmol of electrolyte were added to 10 mL three-necked flasks, dissolved in 6 mL of solvent. Platinum sheets were used as the anode and cathode, respectively. The reaction was carried out at 20-40 °C with stirring for 3-6 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was extracted with 3 × 5 mL of ethyl acetate. The organic layer was dried with anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography to obtain the target product. During purification, the ratio of silica gel, ethyl acetate / petroleum ether was 1:1-40.

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