Sulfur-containing phthalimide alkenyl chloride compounds, synthesis method and application thereof

Abstract

The application discloses a kind of sulphur-containing phthalimide alkenyl chloride compounds as shown in formula (3) and synthesis method thereof, with diacetylene as shown in formula (1) and phthalimide sulphur chloride as shown in formula (2) as reaction raw material, without any catalyst additive, reaction obtains sulphur-containing phthalimide alkenyl chloride compound.The application has mild reaction condition, raw material is cheap and easy to obtain, reaction operation is simple, yield is higher, without any catalyst additive in reaction, it is economical and practical, and friendly to environment;Reaction substrate is easy to prepare;Reaction efficiency is high after reaction amplification.The sulphur-containing phthalimide alkenyl chloride compound described in the application can be used to prepare polypeptide binding reagent.The application also discloses polysulfide binding peptide compound obtained by using the sulphur-containing phthalimide alkenyl chloride compound as shown in formula (3) and synthesis method thereof.The application has wide application prospect and practical value.

Description

Technical Field

[0001] This invention belongs to the field of organic compound process application technology, specifically relating to a class of sulfur-containing phthalimide alkenyl chloride compounds and their synthesis methods and applications. Background Technology

[0002] Protein-protein interactions play a crucial role in modern drug development, but the inability of small molecules to bind specifically and tightly, and the impermeability of protein drugs to permeate membranes, present many limitations. Peptides containing α-helical structures can overcome these problems, primarily due to their stable secondary structure. Linear peptides, however, cannot maintain their original secondary conformation after detaching from their parent compound, resulting in impermeability and susceptibility to protease hydrolysis. Binding technology is currently a relatively reliable method for binding linear peptides into cyclic peptides, maintaining the α-helical structure while increasing peptide stability and improving membrane permeability, thus effectively addressing the druggability issues of peptide drugs.

[0003] Although many binding reagents based on cysteine ​​thiol groups have been reported, these existing binding reagents are mainly based on carbon-sulfur, carbon-nitrogen bonds, etc. to construct binding peptides. However, the main limitation of such methods is that the staples are irreversible, some active peptides require unbinding steps, and some reactions take a long time and are not compatible with living systems. Summary of the Invention

[0004] This invention overcomes the shortage of peptide binding reagents and innovatively develops a highly efficient method for synthesizing sulfur-containing phthalimide-based enyl chloride compounds. The sulfur-containing phthalimide-based enyl chloride compounds described in this invention are suitable peptide binding reagents, providing a solution for constructing α-helical polysulfide binding peptides. Furthermore, the binding peptides constructed based on disulfide bonds in this invention effectively address the reversibility issue, exhibit rapid reaction, compatibility with aqueous biological systems, and can be used to construct cyclic peptides, bicyclic peptides, and modify large protein molecules. Research has revealed that sulfur-containing phthalimide-based enyl chloride compounds are a unique class of peptide binding reagents, possessing characteristics such as being odorless, stable, and easy to prepare. Therefore, this invention designs a reaction method for preparing sulfur-containing phthalimide-based enyl chloride compounds through the addition reaction of diyne and phthalimide thiochloride.

[0005] This invention proposes a sulfur-containing phthalimide alkenyl chloride compound, the structure of which is shown in formula (3):

[0006]

[0007] Formula (3) can be used to construct polysulfide staple peptide compounds, such as polysulfide staple peptide compounds, whose structures are shown in Formula (5):

[0008]

[0009] wherein R 1 selected from linear alkyl, alkoxy, benzyloxy, ester, phenyl, p-toluenesulfinic acid, benzyl and its derivatives, substituted benzyl, fluorescein; R 2 selected from linear alkyl, ester, phenyl.

[0010] Preferably, R 1 selected from n-propyl, nitrogen substituted propyl, oxygen substituted propyl, sulfur substituted propyl, ester substituted propyl, n-hexyl, dioxygen substituted hexyl, ester substituted heptyl, difester substituted dodecane, ortho substituted aryl, meta substituted aryl, para substituted aryl, t-butyl ester substituted aryl, fluorescein, trisubstituted aryl.

[0011] Further preferably, R 1 selected from n-propyl, nitrogen p-toluenesulfinyl substituted propyl, oxygen substituted propyl, sulfur substituted propyl, ester substituted propyl, n-hexyl, dioxygen substituted hexyl, ester substituted heptyl, difester substituted dodecane, ortho disubstituted ether aryl, meta trisubstituted diether aryl, para oxygen sulfur disubstituted aryl, para disubstituted thioether aryl, para disubstituted t-butyl ester aryl, fluorescein, trisubstituted aryl diether ester, trisubstituted aryl triether; R 2 selected from n-propyl, trisubstituted aryl diether ester.

[0012] Further, the sulfur-containing phthalimide alkenyl chloride compound shown in formula (3) of the present application includes:

[0013]

[0014]

[0015]

[0016]

[0017] The polysulfide stapled peptide compound shown in formula (5) of the present application includes:

[0018] The compound shown in formula (5a) is synthesized by combining the polypeptide VCDPCA-CONH2 and the compound shown in formula (3a):

[0019]

[0020] The compound shown in formula (5b) is synthesized by combining the polypeptide ACSR YEVDCRGRGSACG-NH2 and the compound shown in formula (3q):

[0021]

[0022] The application also provides a synthesis method of the sulfur-containing phthalimide alkenyl chloride compound shown in the formula (3), as shown in the following reaction formula (a):

[0023]

[0024] wherein, R 1 selected from linear alkyl, alkoxy, benzyloxy, ester, phenyl, p-toluene phenyl sulfonic acid, benzyl and its derivatives, substituted benzyl, fluorescein.

[0025] Preferably, R 1 selected from n-propyl, nitrogen-substituted propyl, oxygen-substituted propyl, sulfur-substituted propyl, ester-substituted propyl, n-hexyl, dioxygen-substituted hexyl, ester-substituted heptyl, difester-substituted dodecane, ortho-substituted aryl, meta-substituted aryl, para-substituted aryl, t-butyl ester-substituted aryl, fluorescein, trisubstituted aryl.

[0026] According to the above reaction formula (a), the application uses diacetylene shown in the formula (1) and phthalimide sulfur chloride shown in the formula (2) as reaction raw materials, without any catalyst and additive, to react in an organic solvent to obtain the sulfur-containing phthalimide alkenyl chloride compound shown in the formula (3).

[0027] In the application, the molar ratio of the diacetylene shown in the formula (1) to the phthalimide sulfur chloride shown in the formula (2) is 1:(2.2-3.3); preferably, 1:2.2 or 1:3.3.

[0028] In the application, the organic solvent is one or more of 1,2-dichloromethane, dichloromethane, trichloromethane, etc.; preferably, dichloromethane.

[0029] In the application, the reaction is carried out at 0°C and room temperature.

[0030] In the application, the reaction time is 2-12 hours; preferably, 4 hours.

[0031] In the application, the sulfur-containing phthalimide alkenyl chloride compound can be used as a component of a polypeptide binding reagent.

[0032] The application also provides applications of the above-mentioned sulfur-containing phthalimide alkenyl chloride compound in polypeptide binding, construction of binding peptides, construction of cyclic peptides and bicyclic peptides, construction of biological probes, labeling experiments, etc.

[0033] In one embodiment, the reaction is carried out in dichloromethane as the reaction solvent without any catalyst and additive, using appropriate amount of diacetylene compound of formula (1) and phthalimide sulfuryl chloride of formula (2) as the substrates under room temperature air condition, the reaction condition is simple and mild, the starting material is easy to obtain, and the reaction is more efficient, green and practical for synthesizing the phthalimide alkynyl chloride compound containing sulfur.

[0034] In one embodiment, as shown in reaction formula (a), the synthesis reaction of the present application is carried out in reaction bottle A, diacetylene compound (X mmol), phthalimide sulfuryl chloride (Y mmol), dichloromethane (Z mL), the reaction system is reacted at 0°C for 15 min, and then stirred at room temperature for 2-12 h under air atmosphere; after the reaction is completed, the reaction solution is directly concentrated, and the target product is separated by column chromatography.

[0035] In the reaction, the activity of phthalimide sulfuryl chloride of formula (2) is very high, so it is necessary to slowly drop at low temperature to control the selectivity of the reaction and avoid side reactions, so the reaction needs to be carried out at 0°C in advance, and then at room temperature.

[0036] The present application also provides the phthalimide alkynyl chloride compound containing sulfur of formula (3) prepared by the above-mentioned synthesis method.

[0037] The optimal conditions of the phthalimide alkynyl chloride compound containing sulfur of formula (3) prepared by the synthesis method of the present application (the molar ratio of diacetylene compound of formula (1) and phthalimide sulfuryl chloride of formula (2) is 1:2.2 or 1:3.3, dichloromethane (0.1M) is used as the solvent, and the reaction temperature is first 0°C for 15 min, and then transferred to room temperature for 2-12 h) are as follows: the yield of reaction formula (b) is more than 60%.

[0038]

[0039] R is selected from linear alkyl, alkoxy, benzyloxy, ester, phenyl, p-toluenesulfinic acid, benzyl and its derivatives, substituted benzyl, fluorescein. 1 selected from linear alkyl, alkoxy, benzyloxy, ester, phenyl, p-toluenesulfinic acid, benzyl and its derivatives, substituted benzyl, fluorescein.

[0040] R is selected from linear alkyl, alkoxy, benzyloxy, ester, phenyl, p-toluenesulfinic acid, benzyl and its derivatives, substituted benzyl, fluorescein. 1 selected from linear alkyl, alkoxy, benzyloxy, ester, phenyl, p-toluenesulfinic acid, benzyl and its derivatives, substituted benzyl, fluorescein.

[0041] The application further provides a polypeptide binding reagent, which comprises a sulfur-containing phthalimide alkenyl chloride compound shown in formula (3):

[0042]

[0043] R is selected from linear alkyl, alkoxy, benzyloxy, ester, phenyl, p-toluenesulfonic acid, benzyl and its derivatives, substituted benzyl, fluorescein. 1 R is selected from linear alkyl, alkoxy, benzyloxy, ester, phenyl, p-toluenesulfonic acid, benzyl and its derivatives, substituted benzyl, fluorescein.

[0044] R is selected from linear alkyl, alkoxy, benzyloxy, ester, phenyl, p-toluenesulfonic acid, benzyl and its derivatives, substituted benzyl, fluorescein. 1 R is selected from linear alkyl, alkoxy, benzyloxy, ester, phenyl, p-toluenesulfonic acid, benzyl and its derivatives, substituted benzyl, fluorescein.

[0045] The application further provides that the sulfur-containing phthalimide alkenyl chloride compound shown in formula (3) can be used as a polypeptide binding reagent to further synthesize a polysulfur binding peptide compound with the thiol of a polypeptide side chain cysteine.

[0046] The application further provides a synthesis method of a polysulfur binding peptide compound, which comprises the following steps: taking a sulfur-containing phthalimide alkenyl chloride compound shown in formula (3) and a thiol-containing polypeptide shown in formula (4) as raw materials, and reacting in an aqueous solvent to obtain a polysulfur binding peptide compound shown in formula (5).

[0047]

[0048] The molar ratio of the sulfur-containing phthalimide alkenyl chloride compound to the thiol-containing polypeptide is 1.5:1; and / or,

[0049] The aqueous solvent comprises water and acetonitrile; and / or,

[0050] The reaction temperature is room temperature; and / or,

[0051] The reaction time is 1 min.

[0052] The present application has the following advantages: high reaction efficiency and high yield, wherein the yield of all cases is above 60%; the polypeptide stapling reagent is simple, stable, green and has no irritating smell; the reaction conditions are relatively mild; no catalyst additive is required in the reaction, which is economical and practical, and friendly to the environment; the reaction solvent is a low-boiling organic solvent, which is easy to remove; the reaction is in a gram-scale, which has practical value. The present application uses simple-to-prepare diacetylene and phthalimide thionyl chloride as raw materials for the reaction, and obtains sulfur-containing phthalimide alkenyl chloride compounds; the reaction operation is simple, the reaction conditions are relatively mild, and the reaction is suitable for large-scale industrial production. As a new type of stapling reagent, the sulfur-containing phthalimide alkenyl chloride compounds can construct cysteine-based mercapto multi-sulfur stapled peptides, including cyclic peptides, bicyclic peptides, and multi-sulfur stapled protein macromolecules. The stapled peptides constructed based on disulfide bonds can well solve the problems of polypeptide delivery and reversible stapling, and the reaction is rapid, compatible with aqueous life systems, and can be used for biological orthogonal reactions (the sulfur-containing phthalimide alkenyl chloride compounds shown in formula (3) are expected to be used in biological organisms for such rapid click chemistry reactions based on disulfide bond construction, and can be used in the fields of biological probes and labeling experiments. DETAILED DESCRIPTION

[0053] The present application will be further described in combination with the following specific examples, and the protection scope of the present application is not limited to the following examples. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and the protection scope is defined by the appended claims. The process, conditions, reagents, experimental methods, etc. for implementing the present application are the general knowledge and common sense in the art, and the present application has no special limitations. The data given in the following examples include specific operations, reaction conditions and products. The product purity is identified by nuclear magnetic resonance.

[0054] The synthesis reaction of the sulfur-containing phthalimide alkenyl chloride compounds of the present application includes the following steps:

[0055] As shown in reaction formula (a), the synthesis reaction of the present application is to add diacetylene, phthalimide thionyl chloride and organic solvent in a reaction bottle, the reaction system is stirred at 0℃ for 15 minutes, and then stirred at room temperature under air atmosphere for 2-12 hours; after the reaction is completed, the organic phase is concentrated, and the target product is separated by column chromatography.

[0056] As shown in Table 1, the sulfur-containing phthalimide alkenyl chloride compounds are all products synthesized by the method of the present application, and no public literature has disclosed these compounds.

[0057] Table 1 Sulfur-containing phthalimide alkenyl chloride compounds (3a-3q) and multi-sulfur stapled peptides (5a, 5b) of the present application

[0058]

[0059]

[0060]

[0061] Example 1

[0062] Synthesis of compound 3a:

[0063]

[0064] Dialkyne 1a (2 mmol, 184 mg) was dissolved in anhydrous DCM (16 mL). The mixture was cooled at 0 °C. Then phthalimidothiochloride 2 (4.4 mmol, 937 mg) was dissolved in DCM (6 mL) and added dropwise to the cooled mixture. The mixture was stirred at 0 °C for 15 min, then at room temperature for 2 h. After the reaction was completed by TLC, the solvent was removed by rotary evaporation, and column chromatography gave compound 3a (0.79 g, 76%) as a white solid. 1 H NMR (400 MHz, CDC13) δ 7.93-7.90 (m, 4H), 7.79-7.77 (m, 4H), 6.79 (s, 2H), 2.39 (t, J = 8.0 Hz, 4H), 2.08-2.00 (m, 2H); 13 C NMR (100 MHz, CDC13) δ 167.5, 137.9, 134.8, 131.9, 124.2, 124.1, 30.0, 24.7; IR (film) 2361, 1730, 1713, 1340, 1277, 1217, 1047, 712; HRMS (ESI) Calcd for C 23 H 16 C l2 N2O4S2Na[M+Na] + 540.9821, found 540.9814.

[0065] Example 2

[0066] Synthesis of compound 3b:

[0067]

[0068] Dissolve diacetylene 1b (2 mmol, 294 mg) in anhydrous DCM (16 mL). Cool the mixture at 0 °C. Then dissolve phthalimidothioyl chloride 2 (4.4 mmol, 937 mg) in DCM (6 mL) and add dropwise to the cooled mixture. Stir the mixture at 0 °C for 15 min, then at room temperature for 6 h. After the reaction is complete as monitored by TLC, remove the solvent by rotary evaporation and purify by column chromatography to give compound 3b as a white solid (1.18 g, 84%). 1 H NMR (400 MHz, CDC13) δ 7.83 - 7.75 (m, 10H), 7.30 (d, J = 8.0 Hz, 2H), 7.08 (s, 2H), 4.69 (s, 4H), 2.41 (s, 3H); 13 C NMR (100 MHz, CDC13) δ 167.6, 143.8, 136.1, 134.7, 134.5, 132.0, 131.6, 129.7, 127.7, 124.0, 50.0, 21.6; IR (film) 2362, 1734, 1705, 1354, 1277, 1163, 1045, 763, 750, 710, 571, 543, 526, 513; HRMS (ESI) Calcd for C 29 H 22 Cl2N3O6S3[M+H] + 674.0042, found 674.0032.

[0069] Example 3

[0070] Synthesis of compound 3c:

[0071]

[0072] Dissolve diacetylene 1c (2 mmol, 188 mg) in anhydrous DCM (16 mL). Cool the mixture at 0 °C. Then dissolve phthalimidothioyl chloride 2 (4.4 mmol, 937 mg) in DCM (6 mL) and add dropwise to the cooled mixture. Stir the mixture at 0 °C for 15 min, then at room temperature for 2 h. After the reaction is complete as monitored by TLC, remove the solvent by rotary evaporation and purify by column chromatography to give compound 3c as a white solid (0.87 g, 84%). 1 H NMR (400 MHz, CDC13) δ 7.88 (dd, J = 5.4, 3.0 Hz, 4H), 7.77 (dd, J = 5.4, 3.0 Hz, 4H), 6.73 (s, 2H), 4.31 (s, 4H); 13C NMR (100 MHz, CDC13) δ 167.3, 134.7, 133.9, 131.9, 126.7, 124.1, 68.6; IR (film) 3066, 1786, 1738, 1711, 1468, 1458, 1358, 1273, 1263, 1043, 866, 793, 710, 525; HRMS (ESI) Calcd for C 22 H 15 Cl2N2O5S2[M+H] + 520.9794, found 520.9792.

[0073] Example 4

[0074] Synthesis of compound 3d:

[0075]

[0076] Dialkyne 1d (2 mmol, 220 mg) was dissolved in anhydrous DCM (16 mL). The mixture was cooled at 0 °C. Then phthalimidothiochloride 2 (4.4 mmol, 937 mg) was dissolved in DCM (6 mL) and added dropwise to the cooled mixture. The mixture was stirred at 0 °C for 15 min, then at room temperature for 2 h. After the reaction was completed by TLC, the solvent was removed by rotary evaporation, and column chromatography gave compound 3d (0.89 g, 79%) as a white solid. 1 H NMR (400 MHz, CDC13) δ 7.90 (dd, J = 5.4, 3.0 Hz, 4H), 7.78 (dd, J = 5.4, 3.0 Hz, 4H), 6.85 (s, 2H), 3.66 (s, 4H); 13 C NMR (100 MHz, CDC13) δ 167.5, 134.8, 134.6, 132.0, 126.8, 124.1, 33.5; IR (film) 3067, 1869, 1740, 1711, 1275, 1263, 1043, 866, 712, 525; HRMS (ESI) Calcd for C 22 H 15 C l2 N2O4S3[M+H] + 536.9566, found 536.9557.

[0077] Example 5

[0078] Synthesis of compound 3e:

[0079]

[0080] Dissolve diacetylene 1e (2 mmol, 300 mg) in anhydrous DCM (16 mL). Cool the mixture at 0 °C. Then dissolve phthalimidothioyl chloride 2 (4.4 mmol, 937 mg) in DCM (6 mL) and add dropwise to the cooled mixture. Stir the mixture at 0 °C for 15 min, then at room temperature for 4 h. After the reaction is complete as monitored by TLC, remove the solvent by rotary evaporation and purify by column chromatography to give compound 3e as a white solid (0.89 g, 73%). 1 H NMR (400 MHz, CDC13) δ 7.88 (dd, J = 5.4, 3.0 Hz, 4H), 7.77 (dd, J = 5.4, 3.0 Hz, 4H), 6.98 (s, 2H), 3.71 - 3.65 (m, 1H), 3.62 (s, 3H), 2.78 (dd, J = 14.2, 8.2 Hz, 2H), 2.59 (dd, J = 14.2, 6.4 Hz, 2H); 13 C NMR (100 MHz, CDC13) δ 173.8, 167.5, 135.6, 134.8, 131.9, 128.1, 124.1, 52.1, 41.1, 32.9; IR (film) 2360, 1732, 1711, 1340, 1275, 1045, 866, 794, 735, 712, 696, 527; HRMS (ESI) Calcd for C 25 H 19 Cl2N2O6S2[M+H] + 577.0056, found 577.0049.

[0081] Example 6

[0082] Synthesis of compound 3f:

[0083]

[0084] Dissolve diacetylene 1f (2 mmol, 268 mg) in anhydrous DCM (16 mL). Cool the mixture at 0 °C. Then dissolve phthalimidothioyl chloride 2 (4.4 mmol, 937 mg) in DCM (6 mL) and add dropwise to the cooled mixture. Stir the mixture at 0 °C for 15 min, then at room temperature for 6 h. After the reaction is complete as monitored by TLC, remove the solvent by rotary evaporation and purify by column chromatography to give compound 3f as a white solid (0.93 g, 81%). 1H NMR (400 MHz, CDC13) δ 7.92 (dd, J = 5.4, 3.0 Hz, 4H), 7.79 (dd, J = 5.4, 3.0 Hz, 4H), 6.73 (s, 2H), 2.31 (t, J = 8.0 Hz 4H), 1.65-1.60 (m, 4H), 1.35-1.30 (m, 4H); 13 C NMR (100 MHz, CDC13) δ 167.6, 138.6, 134.8, 131.9, 124.1, 123.1, 30.4, 28.7, 26.9; IR (film) 2928, 1738, 1710, 1464, 1342, 1277, 1043, 790, 764, 714, 526; HRMS (ESI) Calcd for C 26 H 23 C l2 N2O4S2[M+H] + 561.0471, found 561.0469.

[0085] Example 7

[0086] Synthesis of compound 3g:

[0087]

[0088] Dialkyne lg (2 mmol, 276 mg) was dissolved in anhydrous DCM (16 mL). The mixture was cooled at 0 °C. Then phthalimidothioyl chloride 2 (4.4 mmol, 937 mg) was dissolved in DCM (6 mL) and added dropwise to the cooled mixture. The mixture was stirred at 0 °C for 15 min, then at room temperature for 6 h. After TLC detection of the reaction was completed, the solvent was removed by rotary evaporation, and column chromatography gave compound 3g (0.95 g, 82%) as a white solid. 1 H NMR (400 MHz, CDC13) δ 7.92 (dd, J = 5.4, 3.0 Hz, 4H), 7.79 (dd, J = 5.4, 3.0 Hz, 4H), 6.73 (s, 2H), 2.31 (t, J = 8.0 Hz 4H), 1.65-1.60 (m, 4H), 1.35-1.30 (m, 4H); 13 C NMR (100 MHz, CDC13) δ 167.6, 138.6, 134.8, 131.9, 124.1, 123.1, 30.4, 28.7, 26.9; IR (film) 2928, 1738, 1710, 1464, 1342, 1277, 1043, 790, 764, 714, 526; HRMS (ESI) Calcd for C 24 H19 Cl2N2O6S2[M+H] + 565.0056, found 565.0054.

[0089] Example 8

[0090] Synthesis of compound 3h:

[0091]

[0092] Dialkyne 1h (2 mmol, 328 mg) was dissolved in anhydrous DCM (16 mL). The mixture was cooled at 0 °C. Then phthalimidothiochloride 2 (4.4 mmol, 937 mg) was dissolved in DCM (6 mL) and added dropwise to the cooled mixture. The mixture was stirred at 0 °C for 15 min, then at room temperature for 6 h. After the reaction was completed by TLC, the solvent was removed by rotary evaporation, and column chromatography gave compound 3h (1.00 g, 85%) as a white solid. 1 H NMR (400 MHz, CDC13) δ 7.89 (dd, J = 5.4, 3.0 Hz, 4H), 7.79 - 7.76 (m, 4H), 6.93 (s, 1H), 6.74 (s, 1H), 4.38 (t, J = 6.4 Hz, 2H), 2.67 (t, J = 6.2 Hz, 2H), 2.35 - 2.30 (m, 2H), 2.24 (t, J = 7.2 Hz, 2H), 1.97 - 1.91 (m, 2H); 13 C NMR (100 MHz, CDC13) δ 172.7, 167.5, 167.5, 137.8, 135.0, 134.9, 134.9, 131.8, 127.3, 124.3, 124.1, 124.1, 124.0, 60.9, 33.0, 30.4, 29.6, 22.1; IR (film) 2360, 2341, 1558, 1275, 763, 750, 669; HRMS (ESI) Calcd for C 26 H 21 Cl2N2O6S2[M+H] + 591.0213, found 591.0204.

[0093] Example 9

[0094] Synthesis of compound 3i:

[0095]

[0096] Dissolve diacetylene 1i (2 mmol, 500 mg) in anhydrous DCM (16 mL). Cool the mixture at 0 °C. Then dissolve phthalimidothioyl chloride 2 (4.4 mmol, 937 mg) in DCM (6 mL) and add dropwise to the cooled mixture. Stir the mixture at 0 °C for 15 min, then at room temperature for 6 h. After the reaction is complete as monitored by TLC, remove the solvent by rotary evaporation and purify by column chromatography to give compound 3i as a white solid (1.24 g, 90%). 1 H NMR (400 MHz, CDC13) δ 7.91 (dd, J = 5.2, 3.0 Hz, 4H), 7.79 (dd, J = 5.4, 3.0 Hz, 4H), 6.79 (s, 2H), 4.26 (s, 4H), 2.39 (t, J = 7.2 Hz, 8H), 2.06 - 1.99 (m, 4H); 13 C NMR (100 MHz, CDC13) δ 172.7, 167.5, 137.7, 134.8, 131.8, 124.2, 124.1, 62.2, 33.0, 29.5, 22.2; IR (film) 2360, 2330, 1743, 1705, 1558, 1541, 1506, 1275, 1261, 763, 750; HRMS (ESI) Calcd for C 30 H 27 Cl2N2O8S2[M+H] + 677.0580, found 677.0571.

[0097] Example 10

[0098] Synthesis of compound 3j:

[0099]

[0100] Dissolve diacetylene 1j (2 mmol, 372 mg) in anhydrous DCM (16 mL). Cool the mixture at 0 °C. Then dissolve phthalimidothioyl chloride 2 (4.4 mmol, 937 mg) in DCM (6 mL) and add dropwise to the cooled mixture. Stir the mixture at 0 °C for 15 min, then at room temperature for 4 h. After the reaction is complete as monitored by TLC, remove the solvent by rotary evaporation and purify by column chromatography to give compound 3j as a white solid (0.86 g, 67%). 1 H NMR (400 MHz, CDC13) δ 7.86 (dd, J = 5.4, 3.0 Hz, 4H), 7.74 (dd, J = 5.4, 3.0 Hz, 4H), 6.90 (s, 2H), 6.75 (s, 4H), 4.81 (s, 4H); 13C NMR (100 MHz, CDC13) δ 167.3, 166.5, 148.5, 135.0, 134.6, 134.0, 132.2, 132.0, 127.5, 124.3, 124.0, 122.8, 116.9, 67.8; IR (film) 3066, 1786, 1740, 1713, 1496, 1467, 1340, 1275, 1043, 866, 792, 763, 748, 736, 711, 526; HRMS (ESI) Calcd for C 28 H 19 Cl2N2O6S2[M+H] + 613.0056, found 613.0052.

[0101] Example 11

[0102] Synthesis of compound 3k:

[0103]

[0104] Dialkyne 1k (2 mmol, 544 mg) was dissolved in anhydrous DCM (16 mL). The mixture was cooled at 0 °C. Then phthalimidothiochloride 2 (4.4 mmol, 937 mg) was dissolved in DCM (6 mL) and added dropwise to the cooled mixture. The mixture was stirred at 0 °C for 15 min, then at room temperature for 12 h. After the reaction was completed by TLC, the solvent was removed by rotary evaporation, and column chromatography gave compound 3k (0.89 g, 79%) as a white solid. 1 H NMR (400 MHz, CDC13) δ 7.86 (dd, J = 5.4, 3.0 Hz, 4H), 7.76 (dd, J = 5.4, 3.0 Hz, 4H), 7.07 (s, 2H), 6.98 (d, J = 2.0 Hz, 2H), 6.28 (t, J = 2.0 Hz, 1H), 4.24 (t, J = 6.2 Hz, 4H), 3.88 (s, 3H), 2.88 (t, J = 6.2 Hz, 4H); 13 C NMR (100 MHz, CDC13) δ 167.6, 166.5, 159.3, 134.9, 134.8, 131.8, 128.0, 124.3, 124.1, 108.1, 106.6, 64.9, 52.2, 31.1; IR (film) 3736, 2360, 2341, 1739, 1714, 1276, 714, 699; HRMS (ESI) Calcd for C 32 H 25 C l2 N2O8S2[M+H]+ 699.0424, found 699.0411.

[0105] Example 12

[0106] Synthesis of compound 3l:

[0107]

[0108] Dialkyne 1l (2 mmol, 404 mg) was dissolved in anhydrous DCM (16 mL). The mixture was cooled at 0 °C. Then phthalimidothiochloride 2 (4.4 mmol, 937 mg) was dissolved in DCM (6 mL) and added dropwise to the cooled mixture. The mixture was stirred at 0 °C for 15 min, then at room temperature for 6 h. After the reaction was completed by TLC, the solvent was removed by rotary evaporation, and column chromatography gave compound 3l (0.93 g, 72%) as a white solid. 1 H NMR (300 MHz, CDC13) δ 8.09 - 7.85 (m, 10H), 7.29 (s, 1H), 6.93 (s, 1H), 6.61 (d, J = 8.6 Hz, 2H), 5.00 (s, 2H), 3.89 (s, 2H); 13 C NMR (100 MHz, CDC13) δ 167.6, 167.2, 166.5, 158.0, 135.3, 134.9, 134.8, 134.7, 134.5, 133.0, 132.2, 132.0, 131.9, 129.3, 126.7, 125.1, 124.3, 124.1, 124.0, 115.1, 66.2, 37.8; IR (film) 2361, 1740, 1736, 1716, 1340, 1275, 764, 750, 712, 526; HRMS (ESI) Calcd for C 28 H 18 Cl2N2O5S3Na [M + Na] + 650.9647, found 650.9639.

[0109] Example 13

[0110] Synthesis of compound 3m:

[0111]

[0112] Dissolve diacetylene 1m (2 mmol, 436 mg) in anhydrous DCM (16 mL). Cool the mixture at 0 °C. Then dissolve phthalimidothioyl chloride 2 (4.4 mmol, 937 mg) in DCM (6 mL) and add dropwise to the cooled mixture. Stir the mixture at 0 °C for 15 min, then at room temperature for 6 h. After TLC monitoring of the reaction completion, remove the solvent by rotary evaporation and column chromatography to give compound 3m as a yellow solid (0.9 g, 70%). 1 H NMR (400 MHz, CDC13) δ 7.91 (dd, J = 5.4, 3.0 Hz, 4H), 7.79 (dd, J = 5.4, 3.0 Hz, 4H), 7.20 (s, 4H), 6.87 (s, 2H), 3.92 (s, 4H); 13 C NMR (100 MHz, CDC13) δ 167.5, 134.8, 134.3, 133.6, 132.3, 132.0, 130.2, 129.2, 127.4, 124.1, 36.4; IR (film) 3854, 3736, 3649, 2343, 2330, 1749, 1739, 1684, 1506, 764, 750; HRMS (ESI) Calcd for C 28 H 18 C l2 N2O4S4Na[M+Na] + 666.9419, found 666.9410.

[0113] Example 14

[0114] Synthesis of compound 3n:

[0115]

[0116] Dissolve diacetylene 1n (2 mmol, 708 mg) in anhydrous DCM (16 mL). Cool the mixture at 0 °C. Then dissolve phthalimidothioyl chloride 2 (4.4 mmol, 937 mg) in DCM (6 mL) and add dropwise to the cooled mixture. Stir the mixture at 0 °C for 15 min, then at room temperature for 12 h. After TLC monitoring of the reaction completion, remove the solvent by rotary evaporation and column chromatography to give compound 3n as a white solid (0.97 g, 62%). 1 H NMR (400 MHz, CDC13) δ 7.62-7.54 (m, 8H), 7.20 (d, J = 9.2 Hz, 2H), 6.78 (d, J = 5.0 Hz, 4H), 6.25 (d, J = 3.4 Hz, 2H), 1.40 (d, J = 4.0 Hz, 18H); 13C NMR (100MHz, CDCl3) δ177.0,177.0,166.5,135.3,135.1,134.9,134.8,134.3,134.3,131.6,123.0,129.2,124.4,124.3 ,123.3,69.0,69.0,39.2,27.2,27.2; IR(film)3050,2360,1734,1713,1275,1142,1051,866,823,714; HRMS(ESI)Calcd forC 38 H 38 C l2 N3O8S2[M+NH4] + 798.1472, found 798.1465.

[0117] Example 15

[0118] Synthesis of compound 3o:

[0119]

[0120] Diyne 1O (2 mmol, 984 mg) was dissolved in anhydrous DCM (16 mL). The mixture was cooled at 0 °C. Then, phthalimide thiochloro 2 (4.4 mmol, 937 mg) was dissolved in DCM (6 mL) and added dropwise to the cooled mixture. The mixture was stirred at 0 °C for 15 minutes, then stirred at room temperature for 6 hours. After the reaction was complete as detected by TLC, the solvent was removed by rotary evaporation, and column chromatography yielded a white solid compound 3O (1.65 g, 87%). 1 H NMR (300MHz, CDCl3) δ8.05-8.01(m,1H),7.90(dd,J=5.4,3.0Hz,4H),7.77(dd,J=5.4,3.0Hz,4H),7.70-7.61(m,2H ),7.16(d,J=7.0Hz,1H),7.08(s,2H),6.98(s,2H),6.83-6.77(m,4H),3.07(t,J=7.4Hz,4H),2.77(t,J=7.4Hz,4H); 13C NMR (100 MHz, CDC13) δ 170.3, 169.1, 167.6, 152.9, 152.0, 151.5, 136.6, 135.3, 134.9, 131.8, 130.1, 128.9, 126.6, 126.1, 125.2, 124.2, 124.1, 117.7, 116.4, 110.4, 81.7, 31.5, 25.8; IR (film) 2360, 1763, 1741, 1712, 1608, 1419, 1275, 1242, 1130, 1045, 866, 763, 713, 526; HRMS (ESI) Calcd for C 46 H 29 Cl2N2O 11 S2[M+H] + 919.0584, found 919.0577.

[0121] Example 16

[0122] Synthesis of compound 3p:

[0123]

[0124] Triyne 1p (1 mmol, 310 mg) was dissolved in anhydrous DCM (8 mL). The mixture was cooled at 0 °C. Then phthalimidothiochloride 2 (3.3 mmol, 703 mg) was dissolved in DCM (3 mL) and added dropwise to the cooled mixture. The mixture was stirred at 0 °C for 15 min, then at room temperature for 12 h. After the reaction was completed by TLC detection, the solvent was removed by rotary evaporation, and column chromatography gave compound 3p (0.6 g, 60%) as a white solid. 1 H NMR (400 MHz, CDC13) δ 7.91-7.85 (m, 6H), 7.79-7.75 (m, 6H), 7.06 (d, J = 3.8 Hz, 3H), 6.95 (d, J = 2.2 Hz, 2H), 6.23 (t, J = 2.2 Hz, 1H), 4.62 (t, J = 6.0 Hz, 2H), 4.22 (t, J = 6.2 Hz, 4H), 2.90-2.83 (m, 6H); 13C NMR (100 MHz, CDC13) δ 167.6, 165.8, 159.2, 134.9, 134.9, 134.8, 134.8, 131.8, 131.8, 131.5, 127.9, 127.9, 124.1, 108.1, 106.9, 64.9, 61.8, 31.2, 30.4; IR (film) 3736, 3649, 2360, 2343, 2330, 1734, 1716, 1653, 1541, 1506, 1458, 1275, 1261, 763, 750, 669; HRMS (ESI) Calcd for C 43 H 31 Cl3N3O 10 S3[M+H] + 950.0232, found 950.0228.

[0125] Example 17

[0126] Synthesis of compound 3q:

[0127]

[0128] Triyne 1q (1 mmol, 366 mg) was dissolved in anhydrous DCM (8 mL). The mixture was cooled at 0 °C. Then phthalimidothiochloride 2 (3.3 mmol, 703 mg) was dissolved in DCM (3 mL) and added dropwise to the cooled mixture. The mixture was stirred at 0 °C for 15 min, then at room temperature for 12 h. After the reaction was completed by TLC, the solvent was removed by rotary evaporation, and column chromatography gave compound 3q (0.79 g, 75%) as a white solid. 1 H NMR (400 MHz, CDC13) δ 7.92 (dd, J = 5.4, 3.0 Hz, 6H), 7.79 (dd, J = 5.4, 3.0 Hz, 6H), 6.96 (s, 3H), 6.81 (s, 3H), 3.03 (t, J = 7.6 Hz, 6H), 2.75 (t, J = 7.6 Hz, 6H); 13 C NMR (100 MHz, CDC13) δ 169.9, 167.6, 150.9, 136.6, 134.9, 131.8, 126.3, 124.2, 112.8, 31.4, 25.6; IR (film) 3736, 2360, 2343, 1734, 1716, 1653, 1541, 1506, 1458, 1275, 1261, 763, 750, 669; HRMS (ESI) Calcd for C 45 H 31 Cl3N3O12 S3[M+H] + 1006.0130, found 1006.0124.

[0129] Example 18

[0130] Synthesis of compound 5a:

[0131]

[0132] Peptide VCDPCA-CONH2(5 μmol, 3.0 mg) was dissolved in 0.5 mL H2O, CH3CN 2.5 mL was added. Reagent 3a (15 μmol, 7.7 mg) was dissolved in 2 mL CH3CN, then added dropwise to the peptide solution, and reacted for 1 min at room temperature under air (analytical HPLC monitoring). CH3CN was removed in vacuo, and the resulting mixture was dissolved in 3 mL H2O and 1 mL CH3CN. After filtration, the mixture was directly injected into preparative (RP-HPLC) and lyophilized to give stapled product 5a (3.7 mg, 89%). HRMS (ESI) Calcd for C 30 H 46 Cl2N7O8S4[M+H] + 830.1662, found 830.1662.

[0133] Example 19

[0134] Synthesis of compound 5b:

[0135]

[0136] Peptide ACSR YEVDCRGRGSACG-NH2(3 μmol, 5.6 mg) was dissolved in 0.5 mL H2O, CH3CN 2.5 mL was added. Reagent 3q (4.5 μmol, 4.5 mg) was dissolved in 2 mL CH3CN, then added dropwise to the peptide solution, and reacted for 1 min at room temperature under air (analytical HPLC monitoring). CH3CN was removed in vacuo, and the resulting mixture was dissolved in 3 mL H2O and 1 mL CH3CN. After filtration, the mixture was directly injected into preparative (RP-HPLC) and lyophilized to give stapled product 5b (2.3 mg, 33%).

[0137] Example 20

[0138] Reversibility experiments (unstapling)

[0139]

[0140] The disulfide bond can be regenerated into the original linear peptide VCDPCA-CONH2 by the action of reducing agent TCEP [tris (2-carboxyethyl) phosphine], which increases the flexibility of the reaction. The disassembly is mainly to adapt to some specific environment, such as the active peptide is unstable in vitro and difficult to penetrate the membrane. After disassembly, the active peptide can stably penetrate the cell membrane, and then disassemble in the cell to release the active peptide, so as to realize the effective drug delivery of the active peptide.

[0141] Step: The polypeptide 5a (33 μmol, 2.5 mg) was dissolved in a mixture of water / acetonitrile (2.7 mL / 0.3 mL) and loaded into a 5 mL vial, and TCEP [tris (2-carboxyethyl) phosphine] (7.5 μmol, 2.2 mg) was added. The resulting mixture was stirred at room temperature for 0.5 h until the reaction was complete, monitored by LC-MS. The mixture was directly injected into preparative (RP-HPLC) and lyophilized to obtain the polypeptide VA-6: VCDPCA-CONH2 (1.7 mg, 94%).

[0142] The protection scope of the present application is not limited to the above examples. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and are protected by the appended claims.

Claims

1. A sulfur-containing phthalimidine alkenyl chloride compound characterized in that, The structure is shown as formula (3): The sulfur-containing phthalimide alkenyl chloride compound shown as formula (3) is a compound shown as formula 3a-3q:

2. The use of the sulfur-containing phthalimide alkenyl chloride compound of claim 1 in the preparation of a polysulfide stapled peptide, a biological probe, or in a labeling experiment.

3. Use according to claim 2, wherein the compound is ###0002### The polysulfide stapled peptide is selected from a cyclic peptide, a bicyclic peptide.

4. A method for synthesizing a sulfur-containing phthalimide alkenyl chloride compound according to claim 1, characterized by, The diacetylene and phthalimide thiochloride shown as formula (1) and formula (2) are used as reaction raw materials in an organic solvent, and a sulfur-containing phthalimide alkenyl chloride compound shown as formula (3) is obtained by reaction; the reaction process is shown as reaction formula (a):

5. The method of synthesis of claim 4, wherein, The molar ratio of the diacetylene shown as formula (1) to the phthalimide thiochloride shown as formula (2) is 1:(2.2-3.3); and / or, The organic solvent is selected from one or more of 1,2-dichloromethane, dichloromethane, and trichloromethane; and / or, The reaction temperature is 0°C and room temperature; and / or, The reaction time is 2-12h.

6. A polypeptide binding reagent, characterized in that, The polypeptide stapling reagent comprises the sulfur-containing phthalimide alkenyl chloride compound of claim 1.

7. A polysulfide stapling peptide compound, characterized in that, The structure of the polysulfide stapled peptide compound is shown as formula (5): The polysulfide stapled peptide compound shown as formula (5) is a compound shown as formula 5a, 5b:

8. A method of synthesizing the polysulfide stapled peptide compound of claim 7, wherein, The sulfur-containing phthalimide alkenyl chloride compound shown as formula (3) and the thiol-containing polypeptide shown as formula (4) are used as reaction raw materials in an aqueous solvent, and the polysulfide stapled peptide compound shown as formula (5) in claim 7 is obtained by reaction, and the reaction process is shown as reaction formula (b):

9. The method of synthesis of claim 8, wherein, The molar ratio of the sulfur-containing phthalimide alkenyl chloride compound to the thiol-containing polypeptide is 1.5:1; and / or, The aqueous solvent comprises water and acetonitrile; and / or, The reaction temperature is room temperature; and / or, The reaction time is 1min.

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