A kind of aromatic diamine immobilized linker, its precursor, preparation method and application

Abstract

The present invention discloses an aryl diamine solid-supported linker, a precursor thereof, a preparation method thereof, and an application thereof in the preparation of a cyclic peptide. The aryl diamine solid-supported linker comprises a solid-phase carrier material, the solid-phase carrier material is directly connected to the aryl diamine through an active group thereon or indirectly connected to the aryl diamine through an intermediate connecting unit, and the aryl diamine has a structure shown in any one of formulas (1) to (6): #imgabs0# In formulas (1) to (6): X is C or N; R1 and R2 are independently H or a carbonyl compound structural unit in which an amino acid undergoes a condensation reaction with an amino group to remove a molecule of water, and at least one of R1 and R2 is H.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide synthesis, and in particular to an aromatic diamine immobilized linker, a precursor thereof, and a preparation method and application thereof. Background Art

[0002] Solid phase flow synthesis is an effective method for preparing cyclic peptide molecules. It has the advantages of simple operation, no need to separate and purify intermediates, and easy automation. Related prior arts include: Patent specification with publication number CN108218964A discloses a method for synthesizing cyclic peptides by enzymatic method on a solid phase carrier, including a method for synthesizing linear peptides on a solid phase carrier and cyclizing the linear peptides on the carrier by enzyme catalysis to form cyclic peptides; Patent specification with publication number CN116178503A discloses a method for preparing polypeptide cyclic peptides, through azide-alkyne cycloaddition reaction under specific conditions, a series of histamine head-to-tail cyclic peptides with triazole rings are prepared.

[0003] At present, there are four main methods for preparing cyclic peptides, namely "head-to-tail", "side chain-to-side chain", "head-to-side chain" and "side chain-to-tail" connection. Among them, the latter three methods all require the incorporation of special functional group amino acids or non-natural amino acids, thereby limiting the structural diversity and changing the structure of natural amino acids. The "head-to-tail" connection method can retain the natural amino acid structure of the cyclic peptide to the greatest extent.

[0004] In order to promote intramolecular acylation and inhibit intermolecular polymerization, low-concentration and medium-loaded resins are often used, but this still cannot solve the problems of long reaction time, low efficiency and purification difficulties in the preparation process. Summary of the invention

[0005] In view of the above technical problems and the deficiencies in the art, the present invention provides an aromatic diamine immobilized linker, a precursor thereof, and a preparation method and application thereof.

[0006] The present invention combines the method of synthesizing peptides by solid phase flow, selects the method of "head-to-tail" connection, develops "aryl diamine" as the main body of the linker, fixes it on a solid phase carrier material such as a resin, and forms an aryl triazole structure with a nitrite compound such as an "activator" nitrite, and then undergoes intramolecular cyclization to obtain a cyclized product. The present invention is successfully applied to the preparation of cyclopeptides by designing the structure and preparation process of the new "linker" and its precursor, and the process flow is also applicable to the high-temperature flow automated synthesis of cyclopeptides.

[0007] The present invention adopts an aromatic diamine immobilized linker to prepare a cyclic peptide. The entire process has mild conditions, simple operation, and does not require multiple separation and purification, so that technical problems such as low synthesis efficiency and poor selectivity in the reaction process can be effectively solved, and high-temperature flow automated synthesis of cyclic peptides is achieved, which effectively shortens the reaction time and can meet the high-throughput screening of cyclic peptide drugs.

[0008] In the present invention, the solid phase supporting material is not particularly limited and can be conventionally used in the art, and can specifically include any one or more combinations of the following: Wang resin, MBHA resin, Trityl resin, 2-CTC resin, Rink Amide resin, Sieber resin, DHP resin, Merrifield resin, NO2-Merr. resin, REM resin, ONb resin, HMNb resin, HMBA resin, HMPA resin, HMPB resin, PAM resin, Sasrin resin, HAL resin, CBHO resin, PAL resin, AMNb resin, ANP resin, BHA resin, Knorr resin, XAL resin, MoBHB resin, glass with controlled pore size, magnetic beads, etc.

[0009] In a first aspect, the present invention provides an aryl diamine solid-supported linker, comprising a solid-phase support material, wherein the solid-phase support material is directly connected to an aryl diamine through an active group thereon or indirectly connected via an intermediate connecting unit, wherein the aryl diamine has a structure shown in any one of formulas (1) to (6):

[0010]

[0011] In formulas (1) to (6):

[0012] X is C or N, preferably N. Compared with the benzene ring structure, the N-containing heterocyclic ring is beneficial to improving the selectivity in the preparation process of the cyclic peptide, and its selectivity is greater than 95:5;

[0013] R1 and R2 are independently H or a carbonyl compound structural unit in which an amino acid undergoes a condensation reaction with an amino group to remove a molecule of water, and at least one of R1 and R2 is H. The amino acids from which the carbonyl compound structural unit is derived may include any one or more of the following combinations: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (methionine) (Met), phenylalanine (Phe), tryptophan (Trp), proline (Pro), serine (Ser), threonine (Thr), cysteine ​​(Cys), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), histidine (His), selenocysteine ​​(Sec), and pyrrolysine (Pyl).

[0014] The active group may include at least one of an amino group, a halogen atom (such as Cl, etc.), and a hydroxyl group.

[0015] The intermediate connection unit may include any one or more of the following combined structures:

[0016] one or more linked amino acid segments;

[0017] n1 is any integer between 1 and 15;

[0018] R 2 is O, -N(R3)-, S, -C(=O)- or -S(=O)2-, R3 is H or C1-C4 alkyl;

[0019] n2 is any integer between 1 and 15;

[0020] R 3 is H, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted thienyl, C1-C4 alkyl substituted or unsubstituted naphthyl, C1-C4 alkyl substituted or unsubstituted furyl, C1-C4 alkyl substituted or unsubstituted indolyl, thiol, C2-C4 amide, C2-C4 carboxyl or C1-C4 alkylamino, and the substituent on the C1-C4 alkyl is selected from one or more of thiol, C1-C4 alkylthio, hydroxyl and halogen;

[0021] R 4 It is one or more of H, halogen, alkynyl, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1-C4 alkoxy, ester, hydroxyl, C2-C4 acyl, and cyano, and the substituents on the C1-C4 alkyl and the C1-C4 alkoxy are independently selected from one or more of thiol, C1-C4 alkylthio, hydroxyl, and halogen;

[0022] R 5 It is one or more of H, halogen, alkynyl, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1-C4 alkoxy, ester, hydroxyl, C2-C4 acyl, and cyano, and the substituents on the C1-C4 alkyl and the C1-C4 alkoxy are independently selected from one or more of thiol, C1-C4 alkylthio, hydroxyl, and halogen;

[0023] R 6 It is one or more of H, halogen, alkynyl, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1-C4 alkoxy, ester, hydroxyl, C2-C4 acyl, and cyano, and the substituents on the C1-C4 alkyl and the C1-C4 alkoxy are independently selected from one or more of thiol, C1-C4 alkylthio, hydroxyl, and halogen;

[0024] R 7 It is one or more of H, halogen, alkynyl, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1-C4 alkoxy, ester, hydroxyl, C2-C4 acyl, and cyano, and the substituents on the C1-C4 alkyl and the C1-C4 alkoxy are independently selected from one or more of thiol, C1-C4 alkylthio, hydroxyl, and halogen;

[0025] R 8 -(CH2) n -, -CH2(CH3)-, -CH2(CH2CH3)-, -CH2(CH2Ph)-, -CH2OCH2CH2- or -CH2(CH2CH2SCH3)-, n=1 to 20, or R 8 Any structure in formula (a) to (h):

[0026]

[0027] n3, n4, n5 are independently 1 or 2, R 10 , R 11 Each is independently C1~C4 alkyl or C1~C4 alkoxy;

[0028] R 9 -(CH2) m -, -CH2(CH3)-, -CH2(CH2CH3)-, -CH2(CH2Ph)-, -CH2OCH2CH2- or -CH2(CH2CH2SCH3)-, m=1-20, or R 9 It is any structure of formula (a) to (h).

[0029] In a second aspect, the present invention provides a method for preparing the aryl diamine immobilized linker according to the first aspect, comprising the steps of:

[0030] 1) The solid-phase support material is directly connected to any structure shown in formula (7) to (12) through the active groups on the solid-phase support material to obtain a solid-support linker precursor; or,

[0031] The solid-phase support material is first connected to the intermediate connection unit through the active groups on it, and then the structure shown in any one of formulas (7) to (12) is connected to the intermediate connection unit to obtain a solid-supported linker precursor;

[0032]

[0033] In formulas (7) to (12):

[0034] X is C or N, preferably N. Compared with the benzene ring structure, the N-containing heterocyclic ring is beneficial to improving the selectivity in the preparation process of the cyclic peptide, and its selectivity is greater than 95:5;

[0035] R4 and R5 are independently nitro, -NH2, -NHFmoc or -NHR6, R6 is a carbonyl compound structural unit in which an amino acid without protection or protected by Fmoc undergoes a condensation reaction with an amino group to remove a molecule of water, and R4 and R5 are not simultaneously selected from -NH2 and a carbonyl compound structural unit in which an amino acid without protection undergoes a condensation reaction with an amino group to remove a molecule of water;

[0036] 2) reductively aminate the nitro group on any one of the structures of formulae (7) to (12) of the solid-supported linker precursor and / or remove at least one Fmoc protection to obtain the aryl diamine solid-supported linker.

[0037] The reaction system used in step 2) nitro reductive amination may include sodium borohydride and weak acid system, sodium cyanoborohydride and weak acid system or sodium triacetoxyborohydride and weak acid system. Further, the weak acid may include dilute hydrochloric acid or glacial acetic acid. Furthermore, the concentration of the dilute hydrochloric acid may be 1 to 2 mmol / L.

[0038] The reducing agent used in step 2) nitro reductive amination may include: tetrahydroxydiboron and 4,4'-bipyridine system, Pd / C hydrogenation system, Raney Ni hydrogenation system, iron powder and acidic system, zinc powder and acidic system, SnCl2 and acidic system, TiCl3 and acidic system, lithium aluminum hydride system, sodium borohydride-Lewis acid system, sodium dithionite and alkaline system, hydrazine hydrate system.

[0039] In a third aspect, the present invention provides a solid-phase linker precursor, comprising a solid-phase support material, wherein the solid-phase support material is directly connected to a precursor structure through an active group thereon or indirectly connected via an intermediate connecting unit, wherein the precursor structure has a structure shown in any one of formulas (7) to (12):

[0040]

[0041] In formulas (7) to (12):

[0042] X is C or N, preferably N. Compared with the benzene ring structure, the N-containing heterocyclic ring is beneficial to improving the selectivity in the preparation process of the cyclic peptide, and its selectivity is greater than 95:5;

[0043] R4 and R5 are independently nitro, -NH2, -NHFmoc or -NHR6, R6 is a carbonyl compound structural unit in which an amino acid without protection or protected by Fmoc undergoes a condensation reaction with an amino group to remove a molecule of water, and R4 and R5 are not simultaneously selected from -NH2 and a carbonyl compound structural unit in which an amino acid without protection undergoes a condensation reaction with an amino group to remove a molecule of water. The optional range of the source amino acid of the carbonyl compound structural unit can refer to the description of the source amino acid of the carbonyl compound structural unit in the first aspect.

[0044] In the third aspect, the optional range of the active group and the intermediate connecting unit can refer to the description of the active group and the intermediate connecting unit in the first aspect.

[0045] In a fourth aspect, the present invention provides use of the aromatic diamine immobilized linker described in the first aspect or the immobilized linker precursor described in the third aspect in the preparation of a cyclic peptide.

[0046] In a fifth aspect, the present invention provides a method for preparing a cyclic peptide, using the aromatic diamine immobilized linker described in the first aspect, and specifically comprising the steps of:

[0047] S1, synthesizing a polypeptide chain on the aromatic diamine immobilized linker through an amino acid condensation reaction;

[0048] S2, treating the product of step S1 with a nitrous acid compound to form an aryl triazole structure;

[0049] S3, the product of step S2 is cyclized under alkaline conditions to generate a cyclopeptide.

[0050] In step S1, the condensing agent used in the amino acid condensation reaction may include at least one of a carbodiimide condensing agent, a phosphorus cation condensing agent, and a urea cation condensing agent. Further:

[0051] The carbodiimide type condensing agent may include at least one of N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl);

[0052] The phosphonium cation type condensing agent may include at least one of 1H-benzotriazole-1-yloxytripyrrolidino hexafluorophosphate (PyBOP), (7-azabenzotriazole-1-oxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), bis(2-oxo-3-oxazolidinyl)phosphinoyl chloride (BOP-Cl), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), and tripyrrolidinophosphonium bromide hexafluorophosphate (PyBrop);

[0053] The urea cationic condensing agent may include at least one of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), N,N,N',N'-tetramethyl-O-(7-azabenzotriazole-1-yl)uronium hexafluorophosphate (HATU), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TATU), O-[(ethoxycarbonyl)cyanomethylamine]-N,N,N',N'-tetramethylthiourea tetrafluoroborate (TOTU), and O-(benzotriazole-1-yl)-N,N,N',N'-dipyrrolyluronium hexafluorophosphate (HBPyU).

[0054] In step S1, the condensation activator used in the amino acid condensation reaction may include at least one of 1-hydroxy-7-azobenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-1H-1,2,3-triazole-4-carboxylic acid ethyl ester (HOCT), 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt), 2-hydroxypyridine-N-oxide (HOPO), 4-dimethylaminopyridine (DMAP), N-hydroxysuccinimide (HOSu), p-nitrophenol, N,N-diisopropylethylamine (DIEA), Oxyma and its derivatives.

[0055] In step S1 to step S3, the solvent of each reaction process can independently include at least one of N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), ethyl acetate (EA), tetrahydrofuran (THF), toluene, acetone, ether, acetonitrile, methanol, and water.

[0056] In step S1 to step S3, the reaction temperature of each reaction process can be independently 0 to 100°C.

[0057] In step S2, the nitrite compound may include at least one of sodium nitrite, nitrosyl sulfuric acid, methyl nitrite, ethyl nitrite, isopropyl nitrite, butyl nitrite, tert-butyl nitrite, amyl nitrite, isoamyl nitrite, and hexyl nitrite.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] 1. The "linker" synthesis method of the present invention can adopt solid phase synthesis technology, and in the process, there is no need to use explosive and dangerous palladium carbon, metal reagent zinc powder, iron powder, tin dichloride and other strong reducing agents. The reaction conditions are mild, the operation is simple, the safety is high, and the biological activity is high.

[0060] 2. The N, O, S heterocyclic linkers of the present invention effectively solve the chemical selectivity problem existing in the synthesis process, avoid the generation of a large number of bimolecular acylation products, and improve the reaction efficiency. The selectivity of monoacylation: diacylation reaction is not less than 90:10, among which the N heterocyclic linker is the best, and the selectivity of monoacylation: diacylation reaction is greater than 95:5. This property can effectively improve the reaction efficiency.

[0061] 3. The linker of the present invention can be used for manual synthesis of cyclic peptides, and also realizes high-temperature flow automated synthesis of cyclic peptides.

[0062] 4. The present invention does not require a special functional group amino acid to be reserved as a connection point in the chain peptide skeleton for the synthesis of cyclic peptides, thus breaking the limitation of the structural design of cyclic peptide drugs and realizing the "traceless" cyclization preparation of cyclic peptides.

[0063] 5. The present invention can be used for the synthesis of cyclopeptides of different magnitudes, including but not limited to the preparation of cyclopeptides of milligram, gram, kilogram, etc., and can be applied industrially. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Chromatographic profile to verify selectivity for heteroaryl diamine resins.

[0065] Figure 2 Chromatographic profile to verify selectivity for phenyldiamine resin. DETAILED DESCRIPTION

[0066] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The operating methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0067] Different linker synthesis methods and examples of cyclopeptide synthesis routes are described below.

[0068] a) “Nitrogen heterocycle” linker

[0069] The method utilizes peptide solid phase synthesis technology, firstly obtains the corresponding 5-nitro-6-aminonicotinamide compound through a condensation reaction between Rink Amide resin and 5-nitro-6-aminonicotinic acid, and then the 5-nitro-6-aminonicotinamide compound is subjected to nitro reduction under the action of tetrahydroxydiboron and 4,4'-bipyridine.

[0070] The technical solution route is shown as follows:

[0071]

[0072] in, Indicates Rink Amide resin.

[0073] b) "Benzene ring" linker

[0074] This method utilizes peptide solid phase synthesis technology, firstly obtains the corresponding 4-aminomethyl-2-nitroaniline compound through a reductive amination reaction of Rink Amide resin and 4-nitro-3-aminobenzaldehyde, and then the 4-aminomethyl-2-nitroaniline compound is subjected to nitro reduction under the action of tetrahydroxydiboron and 4,4'-bipyridine.

[0075] The technical solution route is shown as follows:

[0076]

[0077] in, Indicates Rink Amide resin.

[0078] c) Synthesis of natural cyclic peptides

[0079] This method uses peptide solid-phase synthesis technology to synthesize long-chain peptides. Under the action of isopentyl nitrite, 2,3-diaminopyridine generates a pyridine-triazole analogue with a high leaving ability. Subsequently, an intramolecular ring-closing reaction occurs under alkaline conditions to obtain a side-chain protected cyclic peptide. Finally, the protecting group is removed under acidic conditions to obtain a natural cyclic peptide.

[0080] The technical solution route is shown as follows:

[0081]

[0082] Here, Trt represents a triphenylmethyl protecting group.

[0083] Example 1

[0084]

[0085] (1) Prepared using a peptide synthesizer, 0.34mmol / g Rink Amide resin (300mg, 0.102mmol) was placed in a 10mL syringe with a sieve plate, and then 5mL DMF was added for swelling for 30min, the solvent was removed, 5-nitro-6-aminonicotinic acid or 3-nitro-4-aminobenzoic acid (0.41mmol), HATU (156mg, 0.41mmol), DIEA (110μL, 0.62mmol) were dissolved in 2mL DMF, added to the resin, and allowed to react for 1h. After the reaction, it was washed three times with DCM and DMF respectively.

[0086] (2) Tetrahydroxydiboron (28 mg, 0.31 mmol) and 4,4'-bipyridine (1 mg, 0.005 mmol) were dissolved in 2.5 mL DMF, then added to the resin obtained in (1) and reacted on a shaker for 30 min. After the reaction, the mixture was washed three times with DCM and DMF respectively.

[0087] 1a: The resin was cut with a cleavage solution (TFA:TIPS:EDT:H2O=94:1:2.5:2.5), the cleavage filtrate was collected, and the solution was evaporated by air flow to obtain a solid. Ether was added to the solid, and the solid was centrifuged and analyzed by LCMS: MS (ESI) exact mass calcd. for C6H9N4O[M+H] + :153.08;Found:153.16.

[0088] 2a: The resin was cut with a cleavage solution (TFA:TIPS:EDT:H2O=94:1:2.5:2.5), the cleavage filtrate was collected, and the solution was evaporated by air flow to obtain a solid. Ether was added to the solid, and the solid was centrifuged and analyzed by LCMS: MS (ESI) exact mass calcd. for C6H9N4O[M+H] + :152.08;Found:152.22.

[0089] Example 2

[0090]

[0091] (1) Prepared by using a peptide synthesizer, 0.34mmol / g Rink Amide resin (300mg, 0.102mmol) was placed in a 10mL syringe with a sieve plate, and then 5mL DMF was added for swelling for 30min. The solvent was removed, Fmoc-Gly-OH (92mg, 0.31mmol), HATU (118mg, 0.31mmol), and DIEA (110μL, 0.62mmol) were dissolved in 2mL DMF, and then added to the resin. The reaction was allowed to stand for 30min. After the reaction was completed, it was washed three times with DCM and DMF respectively. Finally, 20% piperidine was added to remove Fmoc.

[0092] (2) 5-Nitro-6-aminonicotinic acid or 3-nitro-4-aminobenzoic acid (0.41 mmol), HATU (156 mg, 0.41 mmol), and DIEA (110 μL, 0.62 mmol) were dissolved in 2 mL of DMF and then added to the resin obtained in (1). The resin was allowed to react for 1 h. After the reaction was completed, the resin was washed three times with DCM and DMF respectively.

[0093] (3) Tetrahydroxydiboron (28 mg, 0.31 mmol) and 4,4'-bipyridine (1 mg, 0.005 mmol) were dissolved in 2.5 mL DMF, then added to the resin obtained in (2) and reacted on a shaker for 30 min. After the reaction, the mixture was washed three times with DCM and DMF respectively.

[0094] 1b: The resin was cut with a cleavage solution (TFA:TIPS:EDT:H2O=94:1:2.5:2.5), the cleavage filtrate was collected, and the solution was evaporated by air flow to obtain a solid. Ether was added to the solid, and the solid was centrifuged and analyzed by LCMS: MS (ESI) exact mass calcd. for C8H 12 N5O2[M+H] + :210.10;Found:210.21.

[0095] 2b: The resin was cut with a cleavage solution (TFA:TIPS:EDT:H2O=94:1:2.5:2.5), the cleavage filtrate was collected, and the solution was evaporated by air flow to obtain a solid. Ether was added to the solid, and the solid was centrifuged and analyzed by LCMS: MS (ESI) exact mass calcd. for C9H 13 N4O2[M+H] + :209.10;Found:209.22.

[0096] Example 3

[0097]

[0098] (1) Prepared by using a peptide synthesizer, 0.34 mmol / g Rink Amide resin (300 mg, 0.102 mmol) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min. The solvent was removed, 4-amino-3-nitrophenylacetic acid (81 mg, 0.41 mmol), HATU (156 mg, 0.41 mmol), and DIEA (110 μL, 0.62 mmol) were dissolved in 2 mL of DMF and added to the resin. The reaction was allowed to stand for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively.

[0099] (2) Tetrahydroxydiboron (28 mg, 0.31 mmol) and 4,4'-bipyridine (1 mg, 0.005 mmol) were dissolved in 2.5 mL DMF, then added to the resin obtained in (1) and reacted on a shaker for 30 min. After the reaction, the mixture was washed three times with DCM and DMF respectively.

[0100] 2c: The resin was cut with a cleavage solution (TFA:TIPS:EDT:H2O=94:1:2.5:2.5), the cleavage filtrate was collected, and the solution was evaporated by air flow to obtain a solid. Ether was added to the solid, and the solid was centrifuged and analyzed by LCMS: MS (ESI) exact mass calcd. for C8H 12 N3O[M+H] + :166.10;Found:166.24.

[0101] Example 4

[0102]

[0103] (1) Prepared by using a peptide synthesizer, 0.34 mmol / g Rink Amide resin (300 mg, 0.102 mmol) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min. The solvent was removed, 6-amino-5-nitropyridine-3-carboxaldehyde or 4-amino-3-nitrobenzaldehyde (0.51 mmol) was dissolved in 2 mL of a mixed solution of DMF:MeOH:AcOH, and then added to the resin. Then, sodium cyanoborohydride (64 mg, 0.51 mmol) was added to the reaction system, and the reaction was carried out on a shaker for 48 h. After the reaction was completed, the mixture was washed three times with DCM, H2O and DMF respectively.

[0104] (2) Tetrahydroxydiboron (28 mg, 0.31 mmol) and 4,4'-bipyridine (1 mg, 0.005 mmol) were dissolved in 2.5 mL DMF, then added to the resin obtained in (1) and reacted on a shaker for 30 min. After the reaction, the mixture was washed three times with DCM and DMF respectively.

[0105] (3) Verification of 1d aromatic diamine resin: Preparation of chain peptide, first condensation: HATU (159.2 mg, 0.41 mmol), Fmoc-Gly-OH (124.7 mg, 0.41 mmol) and DIEA (110 μL, 0.62 mmol) were dissolved in DMF, then added to the resin obtained in (2), and allowed to react for 1 hour. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then 20% piperidine solution was added to remove Fmoc, the reaction time was 5 minutes, and it was washed three times with DCM and DMF respectively. Second condensation: HATU (159.2 mg, 0.41 mmol), Fmoc-Gln-OH (192.4 mg, 0.31 mmol) and DIEA (110 μL, 0.62 mmol) were dissolved in DMF, then added to the resin, and allowed to react for 1 hour. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then, 20% piperidine solution was added to remove Fmoc. The reaction time was 5 min, and the product was washed three times with DCM and DMF respectively.

[0106] Cyclization of peptide chain: 1.2 mL of isoamyl nitrite was dissolved in 2 mL of DMF, added to the resin obtained in (3), and reacted on a shaker for 1.5 h. After the reaction, it was washed three times with DCM and DMF respectively. Then 5% DIEA in DMF solution was added, and the reaction was continued on a shaker for 1.5 h. After the reaction was completed, the LCMS analysis results of the cyclopeptide product were as follows: MS (ESI) exact mass calculation for C 26 H 25 N3O3Na[M+Na] + :450.18;Found:450.22.

[0107] (4) Verification of 2d aromatic diamine resin: Preparation of chain peptide, first condensation: HBTU (159.2 mg, 0.41 mmol), Fmoc-Gly-OH (124.7 mg, 0.41 mmol) and DIEA (110 μL, 0.62 mmol) were dissolved in DMF, then added to the resin obtained in (2), and allowed to react for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then 20% piperidine solution was added to remove Fmoc, the reaction time was 5 min, and it was washed three times with DCM and DMF respectively. Second condensation: HBTU (159.2 mg, 0.41 mmol), Fmoc-Gln-OH (192.4 mg, 0.31 mmol) and DIEA (110 μL, 0.62 mmol) were dissolved in DMF, then added to the resin, and allowed to react for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then, 20% piperidine solution was added to remove Fmoc. The reaction time was 5 min, and the product was washed three times with DCM and DMF respectively.

[0108] Cyclization of peptide chain: 1.2 mL of isoamyl nitrite was dissolved in 2 mL of DMF, added to the resin obtained in (4), and reacted on a shaker for 1.5 h. After the reaction, it was washed three times with DCM and DMF respectively. Then, 5% DIEA in DMF solution was added, and the reaction was continued on a shaker for 1.5 h. After the reaction was completed, the LCMS analysis results of the cyclopeptide product were as follows: MS (ESI) exact mass calculation for C 26 H 25 N3O3Na[M+Na] + :450.18;Found:450.22.

[0109] Example 5

[0110]

[0111] (1) Prepared by using a peptide synthesizer, 0.34 mmol / g Rink Amide resin (300 mg, 0.102 mmol) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min. The solvent was removed, 4-hydroxymethylbenzoic acid (92 mg, 0.31 mmol), HATU (118 mg, 0.31 mmol), and DIEA (110 μL, 0.62 mmol) were dissolved in 2 mL of DMF and then added to the resin. The reaction was allowed to stand for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively.

[0112] (2) 5-Nitro-6-aminonicotinic acid (0.41 mmol), HATU (156 mg, 0.41 mmol), and DIEA (110 μL, 0.62 mmol) were dissolved in 2 mL of DMF and then added to the resin obtained in (1). The resin was allowed to react for 1 h. After the reaction was completed, the resin was washed three times with DCM and DMF respectively.

[0113] (3) Tetrahydroxydiboron (28 mg, 0.31 mmol) and 4,4'-bipyridine (1 mg, 0.005 mmol) were dissolved in 2.5 mL DMF, then added to the resin obtained in (2) and reacted on a shaker for 30 min. After the reaction, the mixture was washed three times with DCM and DMF respectively.

[0114] 1e: The resin was cut with a cleavage solution (TFA:TIPS:EDT:H2O=94:1:2.5:2.5), the cleavage filtrate was collected, and the solution was evaporated by air flow to obtain a solid. Ether was added to the solid, and the solid was centrifuged and analyzed by LCMS: MS (ESI) exact mass calcd.for C 14 H 15 N4O3[M+H] + :287.11;Found:287.25.

[0115] Example 6

[0116]

[0117] (1) Prepared by using a peptide synthesizer, 0.34 mmol / g Rink Amide resin (300 mg, 0.102 mmol) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min. The solvent was removed, 4-hydroxymethylbenzoic acid (92 mg, 0.31 mmol), HATU (118 mg, 0.31 mmol), and DIEA (110 μL, 0.62 mmol) were dissolved in 2 mL of DMF and then added to the resin. The reaction was allowed to stand for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively.

[0118] (2) 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-aminobenzoic acid (154 mg, 0.41 mmol), HATU (156 mg, 0.41 mmol), and DIEA (110 μL, 0.62 mmol) were dissolved in 2 mL of DMF and then added to the resin obtained in (1). The mixture was allowed to react for 1 h. After the reaction was completed, the mixture was washed three times with DCM and DMF respectively. Finally, 20% piperidine solution was added to remove Fmoc.

[0119] Verification of 2e aromatic diamine resin: HBTU (159.2 mg, 0.41 mmol), Fmoc-Gly-OH (124.7 mg, 0.41 mmol) and DIEA (110 μL, 0.62 mmol) were dissolved in DMF, then added to the resin obtained in (2) and allowed to react for 1 h. After the reaction, the mixture was washed three times with DCM and DMF respectively. A 20% piperidine solution was then added to remove Fmoc. The reaction time was 5 min, and the mixture was washed three times with DCM and DMF respectively.

[0120] The resin was cut with a cleavage solution (TFA:TIPS:EDT:H2O=94:1:2.5:2.5), the cleavage filtrate was collected, and the solution was evaporated by air flow to obtain a solid. Ether was added to the solid, and the solid was centrifuged and analyzed by LCMS: MS (ESI) exact mass calcd.for C 17 H 19 N4O4[M+H] + :343.14;Found:343.18.

[0121] Example 7

[0122]

[0123] (1) Prepared by using a peptide synthesizer, 0.34 mmol / g Rink Amide resin (300 mg, 0.102 mmol) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min. The solvent was removed, 4-chloromethylbenzoic acid (91 mg, 0.31 mmol), HATU (118 mg, 0.31 mmol), and DIEA (110 μL, 0.62 mmol) were dissolved in 2 mL of DMF and then added to the resin. The reaction was allowed to stand for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively.

[0124] (2) 4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-nitrophenol (313 mg, 0.9 mmol) and NaH (60%, 40 mg, 1 mmol) were placed in 5 mL THF and reacted at 65° C. for 3 hours. Then, the mixture was added to the resin obtained in (1) and 5 mL DMF was added. The reaction was continued at 65° C. for 2 hours. After the reaction was completed, the mixture was washed with water, DCM, and DMF three times each.

[0125] (3) Tetrahydroxydiboron (28 mg, 0.31 mmol) and 4,4'-bipyridine (1 mg, 0.005 mmol) were dissolved in 2.5 mL DMF, then added to the resin obtained in (2) and reacted on a shaker for 30 min. After the reaction, the mixture was washed three times with DCM and DMF respectively.

[0126] 2f' Resin verification: The resin was cut with a cleavage solution (TFA:TIPS:EDT:H2O=94:1:2.5:2.5), the cleavage filtrate was collected, and the solution was evaporated by air flow to obtain a solid. Ether was added to the solid, and the solid was centrifuged and analyzed by LCMS: MS (ESI) exact mass calcd.for C 14 H 14 N3O4[M+H] + :288.10;Found:288.09.

[0127] Example 8

[0128]

[0129] (1) Prepared by using a peptide synthesizer, 1.58 mmol / g 2-CTC resin (460 mg, 0.72 mmol) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min. The solvent was removed, 3-nitro-4-aminophenol (554.4 mg, 3.6 mmol), DMAP (294 mg, 2.4 mmol), and TEA (488 μL, 3.6 mmol) were dissolved in 6 mL of DCM, and then added to the resin. The reaction was allowed to proceed overnight on a shaker. After the reaction was completed, the resin was washed three times with DCM, water, and DMF, respectively.

[0130] (2) Ethyl viologen diiodide (337 mg, 0.72 mmol), Na2S2O4 (335 mg, 1.8 mmol) and K2CO3 (199 mg, 1.4 mmol) were dissolved in 4 mL of a mixed solvent of water and DMF (ratio 1:1), then added to the resin obtained in (1) and reacted on a shaker for 48 h. After the reaction, the mixture was washed three times with DCM, water and DMF respectively.

[0131] (3) Verification of 2g aromatic diamine resin: Preparation of chain peptide, first condensation: HATU (159.2mg, 0.41mmol), Fmoc-Gly-OH (124.7mg, 0.41mmol) and DIEA (110μL, 0.62mmol) were dissolved in DMF, then added to the resin obtained in (2), and allowed to react for 1h. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then 20% piperidine solution was added to remove Fmoc, the reaction time was 5min, and it was washed three times with DCM and DMF respectively. Second condensation: HATU (159.2mg, 0.41mmol), Fmoc-Gln-OH (192.4mg, 0.31mmol) and DIEA (110μL, 0.62mmol) were dissolved in DMF, then added to the resin, and allowed to react for 1h. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then, 20% piperidine solution was added to remove Fmoc. The reaction time was 5 min, and the product was washed three times with DCM and DMF respectively.

[0132] Cyclization of peptide chain: 1.2 mL of isoamyl nitrite was dissolved in 2 mL of DMF, added to the resin obtained in (3), and reacted on a shaker for 1.5 h. After the reaction, it was washed three times with DCM and DMF respectively. Then, a 5% DIEA DMF solution was added, and the reaction was carried out on a shaker for 1.5 h. After the reaction was completed, the corresponding cyclic peptide product was detected by LCMS to verify the successful preparation of the linker.

[0133] Example 9

[0134]

[0135] (1) Prepared by using a peptide synthesizer, 1.10 mmol / g Wang resin (300 mg, 0.3 mmol) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DCM was added for swelling for 30 min. The solvent was removed, 4-amino-3-nitrobenzenesulfonyl chloride (313 mg, 0.9 mmol) and TEA (125.1 μL, 0.9 mmol) were dissolved in 3 mL of DCM and then added to the resin. The temperature was raised to 60 ° C. After the reaction was completed for 2 h, it was washed three times with DCM and DMF respectively.

[0136] (2) Tetrahydroxydiboron (28 mg, 0.31 mmol) and 4,4'-bipyridine (1 mg, 0.005 mmol) were dissolved in 2.5 mL DMF, then added to the resin obtained in (1) and reacted on a shaker for 30 min. After the reaction, the mixture was washed three times with DCM and DMF respectively.

[0137] (3) Verification of 2h aromatic diamine resin: Preparation of chain peptide, first condensation: HBTU (159.2mg, 0.41mmol), Fmoc-Gly-OH (124.7mg, 0.41mmol) and DIEA (110μL, 0.62mmol) were dissolved in DMF, then added to the resin obtained in (2), and allowed to react for 1h. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then 20% piperidine solution was added to remove Fmoc, the reaction time was 5min, and it was washed three times with DCM and DMF respectively. Second condensation: HBTU (159.2mg, 0.41mmol), Fmoc-Gln-OH (192.4mg, 0.31mmol) and DIEA (110μL, 0.62mmol) were dissolved in DMF, then added to the resin, and allowed to react for 1h. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then, 20% piperidine solution was added to remove Fmoc. The reaction time was 5 min, and the product was washed three times with DCM and DMF respectively.

[0138] Cyclization of peptide chain: 1.2 mL of isoamyl nitrite was dissolved in 2 mL of DMF, added to the resin obtained in (3), and reacted on a shaker for 1.5 h. After the reaction, it was washed three times with DCM and DMF respectively. Then, a 5% DIEA DMF solution was added, and the reaction was carried out on a shaker for 1.5 h. After the reaction was completed, the corresponding cyclic peptide product was detected by LCMS to verify the successful preparation of the linker.

[0139] Example 10

[0140]

[0141] (1) Prepared by using a peptide synthesizer, 0.34 mmol / g Rink Amide resin (300 mg, 0.102 mmol) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min. The solvent was removed, 4-hydroxymethylbenzoic acid (92 mg, 0.31 mmol), HATU (118 mg, 0.31 mmol), and DIEA (110 μL, 0.62 mmol) were dissolved in 2 mL of DMF and then added to the resin. The reaction was allowed to stand for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively.

[0142] (2) 4-Amino-3-nitrobenzenesulfonyl chloride (313 mg, 0.9 mmol) and TEA (125.1 μL, 0.9 mmol) were dissolved in 3 mL DCM, then added to the resin obtained in (1), heated to 60°C, and after 2 h of reaction, washed three times with DCM and DMF respectively. Finally, nitro group reduction was performed, tetrahydroxydiboron (28 mg, 0.31 mmol) and 4,4'-bipyridine (1 mg, 0.005 mmol) were dissolved in 2.5 mL DMF, then added to the obtained resin, and reacted on a shaker for 30 min. After the reaction was completed, washed three times with DCM and DMF respectively.

[0143] 2i resin verification: The resin was cut with a cleavage solution (TFA:TIPS:EDT:H2O=94:1:2.5:2.5), the cleavage filtrate was collected, and the solution was evaporated by air flow to obtain a solid. Ether was added to the solid, and the solid was centrifuged and analyzed by LCMS: MS (ESI) exact mass calcd.for C 14 H 14 N3O4S[M+H] + :322.08;Found:322.22.

[0144] Example 11 Comparison of the selectivity of heteroaryl diamine resin and phenyl diamine resin

[0145] (1) Using a peptide synthesizer, 1a and 2a aromatic diamine resins (300 mg) were placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min.

[0146] (2) HATU (159.2 mg, 0.41 mmol), Fmoc-Gly-OH (124.7 mg, 0.41 mmol) and DIEA (110 μL, 0.62 mmol) were dissolved in DMF and then added to the syringe of the aromatic diamine resins 1a and 2a obtained in (1) and allowed to react for 1 h. After the reaction, the mixture was washed three times with DCM and DMF respectively. A 20% piperidine solution was then added to remove Fmoc. The reaction time was 5 min and the mixture was washed three times with DCM and DMF respectively.

[0147] (3) The aromatic diamine resins 1a and 2a were cleaved with a cleavage solution (TFA:TIPS:EDT:H2O=94:1:2.5:2.5), the cleavage filtrate was collected, and the solution was evaporated by air flow to obtain a solid. Ether was added to the solid, and the solid was precipitated and centrifuged and analyzed by liquid chromatography. The results are shown in Figure 1 and Figure 2 .

[0148] Conclusion: The ratio of monoacylation: diacylation products of 1a aromatic diamine resin is 96:4, and the ratio of monoacylation: diacylation products of 2a aromatic diamine resin is 85:15. Comparison of the liquid phase results of the two resins shows that the selectivity of heteroaryl diamine resin is much higher than that of phenyl diamine resin.

[0149] Example 12

[0150]

[0151] (1) Prepared by using a peptide synthesizer, 0.34 mmol / g Rink Amide resin (300 mg, 0.102 mmol) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min. The solvent was removed, 7-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-amino-2-naphthoic acid (131 mg, 0.31 mmol), HATU (118 mg, 0.31 mmol), and DIEA (110 μL, 0.62 mmol) were dissolved in 2 mL of DMF and then added to the resin. The reaction was allowed to stand for 1 h. After the reaction was completed, the resin was washed three times with DCM and DMF respectively.

[0152] (2) Add 20% piperidine solution to the aromatic resin in (1) to remove Fmoc. The reaction time is 5 min. Wash with DCM and DMF three times respectively. Repeat this reaction step once.

[0153] (3)1j Verification of aromatic diamine resin: Preparation of chain peptide, first condensation: HBTU (159.2 mg, 0.41 mmol), Fmoc-Gly-OH (124.7 mg, 0.41 mmol) and DIEA (110 μL, 0.62 mmol) were dissolved in DMF, then added to the resin obtained in (2), and allowed to react for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then 20% piperidine solution was added to remove Fmoc, the reaction time was 5 min, and it was washed three times with DCM and DMF respectively. Second condensation: HBTU (159.2 mg, 0.41 mmol), Fmoc-Gln-OH (192.4 mg, 0.31 mmol) and DIEA (110 μL, 0.62 mmol) were dissolved in DMF, then added to the resin, and allowed to react for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then, 20% piperidine solution was added to remove Fmoc. The reaction time was 5 min, and the product was washed three times with DCM and DMF respectively.

[0154] Cyclization of peptide chain: 1.2 mL of isoamyl nitrite was dissolved in 2 mL of DMF, added to the resin obtained in (3), and reacted on a shaker for 1.5 h. After the reaction, it was washed three times with DCM and DMF respectively. Then, a 5% DIEA DMF solution was added, and the reaction was carried out on a shaker for 1.5 h. After the reaction was completed, the corresponding cyclic peptide product was detected by LCMS to verify the successful preparation of the linker.

[0155] The selectivity test was carried out in the manner of Example 11. The selectivity of the monoacylated: diacylated products of 1j aromatic diamine resin was greater than 80:20, and the selectivity was close to that of phenyl diamine resin.

[0156] Example 13

[0157]

[0158] (1) Prepared by using a peptide synthesizer, 0.34 mmol / g Rink Amide resin (300 mg, 0.102 mmol) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min. The solvent was removed, 4-bromo-5-nitrothiophene-3-carboxylic acid (100 mg, 0.40 mmol), HATU (152 mg, 0.4 mmol), and DIEA (110 μL, 0.62 mmol) were dissolved in 2 mL of DMF and then added to the resin. The reaction was allowed to stand for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively.

[0159] (2) The above reaction resin was transferred to a sealable reaction tube, and then 2 mL of ethanol and 0.5 mL of NH 3· H2O (30%), stirred at 70°C for 24h, at this time, attention should be paid to the stirring speed to prevent the resin from breaking, and further, the speed is 100-200 rpm. After the reaction is completed, filter with a syringe with a sieve plate, wash with water, DMF and DCM three times each.

[0160] (3) Tetrahydroxydiboron (28 mg, 0.31 mmol) and 4,4'-bipyridine (1 mg, 0.005 mmol) were dissolved in 2.5 mL DMF, then added to the obtained resin and reacted on a shaker for 30 min. After the reaction, the resin was washed three times with DCM and DMF respectively.

[0161] (4) Verification of 4a aromatic diamine resin: Preparation of chain peptide, first condensation: HBTU (159.2 mg, 0.41 mmol), Fmoc-Gly-OH (124.7 mg, 0.41 mmol) and DIEA (110 μL, 0.62 mmol) were dissolved in DMF, then added to the resin obtained in (3), and allowed to react for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then 20% piperidine solution was added to remove Fmoc, the reaction time was 5 min, and it was washed three times with DCM and DMF respectively. Second condensation: HBTU (159.2 mg, 0.41 mmol), Fmoc-Gln-OH (192.4 mg, 0.31 mmol) and DIEA (110 μL, 0.62 mmol) were dissolved in DMF, then added to the resin, and allowed to react for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then, 20% piperidine solution was added to remove Fmoc. The reaction time was 5 min, and the product was washed three times with DCM and DMF respectively.

[0162] Cyclization of peptide chain: 1.2 mL of isoamyl nitrite was dissolved in 2 mL of DMF, added to the resin obtained in (4), and reacted on a shaker for 1.5 h. After the reaction, it was washed three times with DCM and DMF respectively. Then, a 5% DIEA DMF solution was added, and the reaction was carried out on a shaker for 1.5 h. After the reaction was completed, the corresponding cyclic peptide product was detected by LCMS to verify the successful preparation of the linker.

[0163] The selectivity test was carried out in the same manner as in Example 11. The selectivity of the monoacylated: diacylated products of 4a aromatic diamine resin was not less than 90:10, which was better than that of phenyl diamine resin.

[0164] Embodiment 14

[0165]

[0166] (1) Prepared by using a peptide synthesizer, 0.34 mmol / g Rink Amide resin (300 mg, 0.102 mmol) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min. The solvent was removed, 3-bromo-4-nitrothiophene-2-carboxylic acid (100 mg, 0.40 mmol), HATU (152 mg, 0.4 mmol), and DIEA (110 μL, 0.62 mmol) were dissolved in 2 mL of DMF and then added to the resin. The reaction was allowed to stand for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively.

[0167] (2) The above reaction resin was transferred to a sealable reaction tube, and then 2 mL of ethanol and 0.5 mL of NH 3· H2O (30%), stirred at 70°C for 24h, at this time, attention should be paid to the stirring speed to prevent the resin from breaking, and further, the speed is 100-200 rpm. After the reaction is completed, filter with a syringe with a sieve plate, wash with water, DMF and DCM three times each.

[0168] (3) Tetrahydroxydiboron (28 mg, 0.31 mmol) and 4,4'-bipyridine (1 mg, 0.005 mmol) were dissolved in 2.5 mL DMF, then added to the obtained resin and reacted on a shaker for 30 min. After the reaction, the resin was washed three times with DCM and DMF respectively.

[0169] (4) Verification of 4a aromatic diamine resin: Preparation of chain peptide, first condensation: HBTU (159.2 mg, 0.41 mmol), Fmoc-Gly-OH (124.7 mg, 0.41 mmol) and DIEA (110 μL, 0.62 mmol) were dissolved in DMF, then added to the resin obtained in (3), and allowed to react for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then 20% piperidine solution was added to remove Fmoc, the reaction time was 5 min, and it was washed three times with DCM and DMF respectively. Second condensation: HBTU (159.2 mg, 0.41 mmol), Fmoc-Gln-OH (192.4 mg, 0.31 mmol) and DIEA (110 μL, 0.62 mmol) were dissolved in DMF, then added to the resin, and allowed to react for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then, 20% piperidine solution was added to remove Fmoc. The reaction time was 5 min, and the product was washed three times with DCM and DMF respectively.

[0170] Cyclization of peptide chain: 1.2 mL of isoamyl nitrite was dissolved in 2 mL of DMF, added to the resin obtained in (4), and reacted on a shaker for 1.5 h. After the reaction, it was washed three times with DCM and DMF respectively. Then, a 5% DIEA DMF solution was added, and the reaction was carried out on a shaker for 1.5 h. After the reaction was completed, the corresponding cyclic peptide product was detected by LCMS to verify the successful preparation of the linker.

[0171] The selectivity test was carried out in the same manner as in Example 11. The selectivity of the monoacylated: diacylated products of 4a aromatic diamine resin was not less than 90:10, which was better than that of phenyl diamine resin.

[0172] Embodiment 15

[0173]

[0174] (1) Prepared by using a peptide synthesizer, 0.34 mmol / g Rink Amide resin (300 mg, 0.102 mmol) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min. The solvent was removed, 4-amino-5-nitrofuran-3-carboxylic acid (69 mg, 0.40 mmol), HATU (152 mg, 0.4 mmol), and DIEA (110 μL, 0.62 mmol) were dissolved in 2 mL of DMF and then added to the resin. The reaction was allowed to stand for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively.

[0175] (2) Tetrahydroxydiboron (28 mg, 0.31 mmol) and 4,4'-bipyridine (1 mg, 0.005 mmol) were dissolved in 2.5 mL DMF, then added to the obtained resin and reacted on a shaker for 30 min. After the reaction, the resin was washed three times with DCM and DMF respectively.

[0176] (3) Verification of 5a aromatic diamine resin: Preparation of chain peptide, first condensation: HBTU (159.2 mg, 0.41 mmol), Fmoc-Gly-OH (124.7 mg, 0.41 mmol) and DIEA (110 μL, 0.62 mmol) were dissolved in DMF, then added to the resin obtained in (3), and allowed to react for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then 20% piperidine solution was added to remove Fmoc, the reaction time was 5 min, and it was washed three times with DCM and DMF respectively. Second condensation: HBTU (159.2 mg, 0.41 mmol), Fmoc-Gln-OH (192.4 mg, 0.31 mmol) and DIEA (110 μL, 0.62 mmol) were dissolved in DMF, then added to the resin, and allowed to react for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then, 20% piperidine solution was added to remove Fmoc. The reaction time was 5 min, and the product was washed three times with DCM and DMF respectively.

[0177] Cyclization of peptide chain: 1.2 mL of isoamyl nitrite was dissolved in 2 mL of DMF, added to the resin obtained in (3), and reacted on a shaker for 1.5 h. After the reaction, it was washed three times with DCM and DMF respectively. Then, a 5% DIEA DMF solution was added, and the reaction was carried out on a shaker for 1.5 h. After the reaction was completed, the corresponding cyclic peptide product was detected by LCMS to verify the successful preparation of the linker.

[0178] The selectivity test was carried out in the same manner as in Example 11. The selectivity of the monoacylated: diacylated products of 5a aromatic diamine resin was not less than 90:10, which was better than that of phenyl diamine resin.

[0179] Example 16

[0180]

[0181] (1) Prepared by using a peptide synthesizer, 0.34 mmol / g Rink Amide resin (300 mg, 0.102 mmol) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min. The solvent was removed, 3-amino-4-nitrofuran-2-carboxylic acid (69 mg, 0.40 mmol), HATU (152 mg, 0.4 mmol), and DIEA (110 μL, 0.62 mmol) were dissolved in 2 mL of DMF and then added to the resin. The reaction was allowed to stand for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively.

[0182] (2) Tetrahydroxydiboron (28 mg, 0.31 mmol) and 4,4'-bipyridine (1 mg, 0.005 mmol) were dissolved in 2.5 mL DMF, then added to the obtained resin and reacted on a shaker for 30 min. After the reaction, the resin was washed three times with DCM and DMF respectively.

[0183] (3) Verification of 6a aromatic diamine resin: Preparation of chain peptide, first condensation: HBTU (159.2 mg, 0.41 mmol), Fmoc-Gly-OH (124.7 mg, 0.41 mmol) and DIEA (110 μL, 0.62 mmol) were dissolved in DMF, then added to the resin obtained in (3), and allowed to react for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then 20% piperidine solution was added to remove Fmoc, the reaction time was 5 min, and it was washed three times with DCM and DMF respectively. Second condensation: HBTU (159.2 mg, 0.41 mmol), Fmoc-Gln-OH (192.4 mg, 0.31 mmol) and DIEA (110 μL, 0.62 mmol) were dissolved in DMF, then added to the resin, and allowed to react for 1 h. After the reaction was completed, it was washed three times with DCM and DMF respectively. Then, 20% piperidine solution was added to remove Fmoc. The reaction time was 5 min, and the product was washed three times with DCM and DMF respectively.

[0184] Cyclization of peptide chain: 1.2 mL of isoamyl nitrite was dissolved in 2 mL of DMF, added to the resin obtained in (3), and reacted on a shaker for 1.5 h. After the reaction, it was washed three times with DCM and DMF respectively. Then, a 5% DIEA DMF solution was added, and the reaction was carried out on a shaker for 1.5 h. After the reaction was completed, the corresponding cyclic peptide product was detected by LCMS to verify the successful preparation of the linker.

[0185] The selectivity test was carried out in the manner of Example 11. The selectivity of the monoacylated: diacylated products of 6a aromatic diamine resin was not less than 90:10, which was better than that of phenyl diamine resin.

[0186] Example 17 Synthesis of Cyclo-GQ

[0187]

[0188] (1) Using a peptide synthesizer, 1a aromatic diamine resin (300 mg) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min.

[0189] (2) The linear peptide was prepared according to the following conditions:

[0190]

[0191] (3) Cyclization: Dissolve 1.2 mL of isoamyl nitrite in 2 mL of DMF, add to the swollen peptide resin, and react on a shaker for 1.5 h. After the reaction is complete, remove the solvent and rinse with DMF. Then add DIEA (100 μL) dissolved in 2 mL of DMF, react for 1.5 h, concentrate under reduced pressure, evaporate the solvent, and obtain a crude product, which is purified by HPLC. The LCMS analysis results of the cyclopeptide product are as follows: MS (ESI) exact mass calcd.for C 26 H 25 N3O3Na[M+Na] + :450.18;Found:450.22.

[0192] The cyclic peptide can also be synthesized by high temperature flow automation, and the reaction steps are as follows:

[0193] (1) Using a self-developed automated synthesis device, 1a aromatic diamine resin (20 mg) was placed in a reaction module.

[0194] (2) The reaction conditions are shown in the following table.

[0195]

[0196] Description in the table:

[0197] a) Preparation steps of long polypeptide chains:

[0198] Pump 1: delivers 0.4 M DMF solution of amino acid monomers and DMF;

[0199] Pump 2: delivers 0.38 M HATU in DMF and DMF;

[0200] Pump 3: delivers DIEA, 99.9% pure, and DMF;

[0201] b) Deprotection step:

[0202] Pump 2: delivering 20% ​​(volume ratio) piperidine and 0.5% (volume ratio) HCOOH in DMF;

[0203] c) Activation step:

[0204] Pump 3: delivering 50% (volume ratio) isoamyl nitrite in DMF solution;

[0205] d) Cyclization step:

[0206] Pump 3: delivering 1% (volume ratio) DIEA in DMF solution;

[0207] The volumes shown in the table refer to the volume of a single pump. If three pumps are used at the same time, the total volume will be three times that in the table.

[0208] In addition, the aromatic diamine resins in Examples 1 to 10 and Examples 12 to 16 can all successfully prepare Cyclo-GQ, and the steps are the same as those in Example 17, which will not be described in detail here. The aromatic diamine immobilized linker of the present invention has universal applicability in the preparation of cyclic peptides, and can be applied to the preparation of cyclic dipeptides and even cyclic heptapeptides, and the synthesis scheme is even applicable to cyclic peptide compounds with larger rings. In addition, the aromatic diamine immobilized linkers of the present invention can all be used for the high-temperature flow automation preparation of cyclic peptides.

[0209] Example 18 Synthesis of Cyclo-KT

[0210]

[0211] (1) Using a peptide synthesizer, 1a aromatic diamine resin (300 mg) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min.

[0212] (2) The linear peptide was prepared according to the following conditions:

[0213]

[0214] (3) Cyclization: Dissolve 1.2 mL of isoamyl nitrite in 2 mL of DMF, add to the swollen peptide resin, and react on a shaker for 1.5 h. After the reaction is complete, remove the solvent and rinse with DMF. Then add DIEA (100 μL) dissolved in 2 mL of DMF, react for 1.5 h, concentrate under reduced pressure, evaporate the solvent, and obtain a crude product, which is purified by HPLC. The LCMS analysis results of the cyclopeptide product are as follows: MS (ESI) exact mass calcd.for C 19 H 36 N3O5[M+H] + :386.26;Found:386.24.

[0215] The cyclic peptide can also be successfully prepared by high temperature flow automated synthesis. For the specific implementation scheme, please refer to Example 17.

[0216] Example 19 Synthesis of Cyclo-GDT

[0217]

[0218] (1) Using a peptide synthesizer, 1a aromatic diamine resin (300 mg) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min.

[0219] (2) The linear peptide was prepared according to the following conditions:

[0220]

[0221] (3) Cyclization: Dissolve 1.2 mL of isoamyl nitrite in 2 mL of DMF, add to the swollen peptide resin, and react on a shaker for 1.5 h. After the reaction is complete, remove the solvent and rinse with DMF. Then add DIEA (100 μL) dissolved in 2 mL of DMF, react for 1.5 h, concentrate under reduced pressure, evaporate the solvent, and obtain a crude product, which is purified by HPLC. The LCMS analysis results of the cyclopeptide product are as follows: MS (ESI) exact mass calcd.for C 36 H 62 N6O 12 Na[2M+Na] + :793.43;Found:793.32.

[0222] The cyclic peptide can also be successfully prepared by high temperature flow automated synthesis. For the specific implementation scheme, please refer to Example 17.

[0223] Example 20 Synthesis of Cyclo-GKYA

[0224]

[0225] (1) Using a peptide synthesizer, 1a aromatic diamine resin (300 mg) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min.

[0226] (2) The linear peptide was prepared according to the following conditions:

[0227]

[0228] (3) Cyclization: Dissolve 1.2 mL of isoamyl nitrite in 2 mL of DMF, add to the swollen peptide resin, and react on a shaker for 1.5 h. After the reaction is complete, remove the solvent and rinse with DMF. Then add DIEA (100 μL) dissolved in 2 mL of DMF, react for 1.5 h, concentrate under reduced pressure, evaporate the solvent, and obtain a crude product, which is purified by HPLC. The LCMS analysis results of the cyclopeptide product are as follows: MS (ESI) exact mass calcd.for C 58 H 90 N 10 O 14 Na[2M+Na] + :1173.65;Found:1173.39.

[0229] The cyclic peptide can also be successfully prepared by high temperature flow automated synthesis. For the specific implementation scheme, please refer to Example 17.

[0230] Example 21 Synthesis of Cyclo-FSNMG

[0231]

[0232] (1) Using a peptide synthesizer, 1a aromatic diamine resin (300 mg) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min.

[0233] (2) The linear peptide was prepared according to the following conditions:

[0234]

[0235] (3) Cyclization: Dissolve 1.2 mL of isoamyl nitrite in 2 mL of DMF, add to the swollen peptide resin, and react on a shaker for 1.5 h. After the reaction is complete, remove the solvent and rinse with DMF. Then add DIEA (100 μL) dissolved in 2 mL of DMF, react for 1.5 h, concentrate under reduced pressure, evaporate the solvent, and obtain a crude product, which is purified by HPLC. The LCMS analysis results of the cyclopeptide product are as follows: MS (ESI) exact mass calcd.for C 46 H 54 N6O7Na[M+Na] + :857.37;Found:857.23.

[0236] The cyclic peptide can also be successfully prepared by high temperature flow automated synthesis. For the specific implementation scheme, please refer to Example 17.

[0237] Example 22 Synthesis of Yunnanin C

[0238]

[0239] (1) Using a peptide synthesizer, 1a aromatic diamine resin (300 mg) was placed in a 10 mL syringe with a sieve plate, and then 5 mL of DMF was added for swelling for 30 min.

[0240] (2) The linear peptide was prepared according to the following conditions:

[0241]

[0242] (3) Cyclization: 1.2 mL of isoamyl nitrite was dissolved in 2 mL of DMF, and then added to the swollen peptide resin. The reaction was carried out on a shaker for 1.5 h. After the reaction was completed, the solvent was removed and DMF was used for washing. Then DIEA (100 μL) dissolved in 2 mL of DMF was added and the reaction was carried out for 1.5 h. The crude product was obtained and purified by HPLC.

[0243] (4) Deprotection: The crude product was dissolved in 1 mL of TFA:TIPS:EDT:H2O (ratio 94:1:2.5:2.5) solution and reacted on a shaker for 2 h to obtain the deprotected natural cyclic peptide. The reaction solution was evaporated by air flow to obtain a solid, and ether was added to the solid for precipitation and centrifugation. The solid was purified by HPLC. The LCMS analysis results of the cyclic peptide product are as follows: MS (ESI) exact mass calcd.for C 36 H 47 N7O9Na[M+Na] + :744.23;Found:744.34.

[0244] The cyclic peptide can also be successfully prepared by high temperature flow automated synthesis. For the specific implementation scheme, please refer to Example 17.

[0245] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An aromatic diamine solid-supported linker, comprising a solid-phase supporting material, wherein the solid-phase supporting material is directly connected to the aromatic diamine through an active group thereon or indirectly connected via an intermediate connecting unit, characterized in that: The aromatic diamine has a structure shown in formula (1): In formula (1): X is N; R1 and R2 are independently H or a carbonyl compound structural unit formed by a condensation reaction between an amino acid and an amino group to remove a molecule of water, and at least one of R1 and R2 is H.

2. The aryl diamine immobilized linker according to claim 1, characterized in that: The active group includes at least one of an amino group, a halogen atom, and a hydroxyl group; The intermediate connection unit includes any one or more of the following combined structures: one or more linked amino acid segments; n1 is any integer between 1 and 15; R 2 is O, -N(R3)-, S, -C(=O)- or -S(=O)2-, R3 is H or C1-C4 alkyl; n2 is any integer between 1 and 15; R 3 is H, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted thienyl, C1-C4 alkyl substituted or unsubstituted naphthyl, C1-C4 alkyl substituted or unsubstituted furyl, C1-C4 alkyl substituted or unsubstituted indolyl, thiol, C2-C4 amide, C2-C4 carboxyl or C1-C4 alkylamino, and the substituent on the C1-C4 alkyl is selected from one or more of thiol, C1-C4 alkylthio, hydroxyl and halogen; R 4 It is one or more of H, halogen, alkynyl, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1-C4 alkoxy, ester, hydroxyl, C2-C4 acyl, and cyano, and the substituents on the C1-C4 alkyl and the C1-C4 alkoxy are independently selected from one or more of thiol, C1-C4 alkylthio, hydroxyl, and halogen; R 5 It is one or more of H, halogen, alkynyl, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1-C4 alkoxy, ester, hydroxyl, C2-C4 acyl, and cyano, and the substituents on the C1-C4 alkyl and the C1-C4 alkoxy are independently selected from one or more of thiol, C1-C4 alkylthio, hydroxyl, and halogen; R 6 It is one or more of H, halogen, alkynyl, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1-C4 alkoxy, ester, hydroxyl, C2-C4 acyl, and cyano, and the substituents on the C1-C4 alkyl and the C1-C4 alkoxy are independently selected from one or more of thiol, C1-C4 alkylthio, hydroxyl, and halogen; R 7 It is one or more of H, halogen, alkynyl, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1-C4 alkoxy, ester, hydroxyl, C2-C4 acyl, and cyano, and the substituents on the C1-C4 alkyl and the C1-C4 alkoxy are independently selected from one or more of thiol, C1-C4 alkylthio, hydroxyl, and halogen; R 8 -(CH2) n -, -CH2(CH3)-, -CH2(CH2CH3)-, -CH2(CH2Ph)-, -CH2OCH2CH2- or -CH2(CH2CH2SCH3)-, n=1 to 20, or R 8 Any structure in formula (a) to (h): n3, n4, n5 are independently 1 or 2, R 10 , R 11 Each is independently a C1-C4 alkyl group or a C1-C4 alkoxy group; R 9 -(CH2) m -, -CH2(CH3)-, -CH2(CH2CH3)-, -CH2(CH2Ph)-, -CH2OCH2CH2- or -CH2(CH2CH2SCH3)-, m=1-20, or R 9 It is any structure of formula (a) to (h).

3. The method for preparing an aryl diamine immobilized linker according to claim 1 or 2, characterized in that: Includes steps: 1) The solid-phase support material is directly connected to the structure shown in formula (7) through the active groups on it to obtain a solid-phase support linker precursor; or, The solid-phase support material is first connected to the intermediate connection unit through the active groups on the solid-phase support material, and then the structure represented by formula (7) is connected to the intermediate connection unit to obtain a solid-phase support linker precursor; In formula (7): X is N; R4 and R5 are independently nitro, -NH2, -NHFmoc or -NHR6, R6 is a carbonyl compound structural unit in which an amino acid without protection or protected by Fmoc undergoes a condensation reaction with an amino group to remove a molecule of water, and R4 and R5 are not simultaneously selected from -NH2 and a carbonyl compound structural unit in which an amino acid without protection undergoes a condensation reaction with an amino group to remove a molecule of water; 2) reductively aminate the nitro group on the structure of formula (7) of the solid-supported linker precursor and / or remove at least one Fmoc protection to obtain the aryl diamine solid-supported linker.

4. A solid-supported linker precursor, comprising a solid-phase supporting material, wherein the solid-phase supporting material is directly connected to the precursor structure through the active groups thereon or indirectly connected via an intermediate connecting unit, characterized in that: The precursor structure has the structure shown in formula (7): In formula (7): X is N; R4 and R5 are independently nitro, -NH2, -NHFmoc or -NHR6, R6 is a carbonyl compound structural unit in which an amino acid without protected amino group or protected by Fmoc undergoes condensation reaction with an amino group to remove a molecule of water, and R4 and R5 are not simultaneously selected from -NH2 and a carbonyl compound structural unit in which an amino acid without protected amino group undergoes condensation reaction with an amino group to remove a molecule of water.

5. The immobilized linker precursor according to claim 4, characterized in that: The active group includes at least one of an amino group, a halogen atom, and a hydroxyl group; The intermediate connection unit includes any one or more of the following combined structures: one or more linked amino acid segments; n1 is any integer between 1 and 15; R2 is O, -N(R3)-, S, -C(=O)- or -S(=O)2-, R3 is H or C1-C4 alkyl; n2 is any integer between 1 and 15; R 3 is a substituted or unsubstituted C1-C4 alkyl group, a C1-C4 alkyl substituted or unsubstituted phenyl group, a C1-C4 alkyl substituted or unsubstituted thienyl group, a C1-C4 alkyl substituted or unsubstituted naphthyl group, a C1-C4 alkyl substituted or unsubstituted furyl group, a C1-C4 alkyl substituted or unsubstituted indolyl group, a thiol group, a C2-C4 amide group, a C2-C4 carboxyl group or a C1-C4 alkylamino group, wherein the substituent on the C1-C4 alkyl group is selected from one or more of a thiol group, a C1-C4 alkylthio group, a hydroxyl group and a halogen group; R 4 It is one or more of H, halogen, alkynyl, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1-C4 alkoxy, ester, hydroxyl, C2-C4 acyl, and cyano, and the substituents on the C1-C4 alkyl and the C1-C4 alkoxy are independently selected from one or more of thiol, C1-C4 alkylthio, hydroxyl, and halogen; R 5 It is one or more of H, halogen, alkynyl, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1-C4 alkoxy, ester, hydroxyl, C2-C4 acyl, and cyano, and the substituents on the C1-C4 alkyl and the C1-C4 alkoxy are independently selected from one or more of thiol, C1-C4 alkylthio, hydroxyl, and halogen; R 6 It is one or more of H, halogen, alkynyl, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1-C4 alkoxy, ester, hydroxyl, C2-C4 acyl, and cyano, and the substituents on the C1-C4 alkyl and the C1-C4 alkoxy are independently selected from one or more of thiol, C1-C4 alkylthio, hydroxyl, and halogen; R 7 It is one or more of H, halogen, alkynyl, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1-C4 alkoxy, ester, hydroxyl, C2-C4 acyl, and cyano, and the substituents on the C1-C4 alkyl and the C1-C4 alkoxy are independently selected from one or more of thiol, C1-C4 alkylthio, hydroxyl, and halogen; R 8 -(CH2) n -, -CH2(CH3)-, -CH2(CH2CH3)-, -CH2(CH2Ph)-, -CH2OCH2CH2- or -CH2(CH2CH2SCH3)-, n=1 to 20, or R 8 Any structure in formula (a) to (h): n3, n4, n5 are independently 1 or 2, R 10 , R 11 Each is independently a C1-C4 alkyl group or a C1-C4 alkoxy group; R 9 -(CH2) m -, -CH2(CH3)-, -CH2(CH2CH3)-, -CH2(CH2Ph)-, -CH2OCH2CH2- or -CH2(CH2CH2SCH3)-, m=1-20, or R 9 It is any structure of formula (a) to (h).

6. Use of the aryl diamine immobilized linker according to claim 1 or 2 or the immobilized linker precursor according to claim 4 or 5 in the preparation of a cyclic peptide.

7. A method for preparing a cyclic peptide, characterized in that: The aryl diamine solid-supported linker is used, and the aryl diamine solid-supported linker includes a solid-phase supporting material, and the solid-phase supporting material is directly connected to the aryl diamine through the active group thereon or indirectly connected through an intermediate connecting unit, and the aryl diamine has a structure shown in formula (1): In formula (1): X is C or N; R1 and R2 are independently H or a carbonyl compound structural unit formed by a condensation reaction between an amino acid and an amino group to remove a molecule of water, and at least one of R1 and R2 is H; The cyclic peptide preparation method specifically comprises the steps of: S1, synthesizing a polypeptide chain on the aromatic diamine immobilized linker through an amino acid condensation reaction; S2, treating the product of step S1 with a nitrous acid compound to form an aryl triazole structure; S3, the product of step S2 is cyclized under alkaline conditions to generate a cyclopeptide.

8. The method for preparing a cyclic peptide according to claim 7, characterized in that: The active group includes at least one of an amino group, a halogen atom, and a hydroxyl group; The intermediate connection unit includes any one or more of the following combined structures: one or more linked amino acid segments; n1 is any integer between 1 and 15; R 2 is O, -N(R3)-, S, -C(=O)- or -S(=O)2-, R3 is H or C1-C4 alkyl; n2 is any integer between 1 and 15; R 3 is H, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted thienyl, C1-C4 alkyl substituted or unsubstituted naphthyl, C1-C4 alkyl substituted or unsubstituted furyl, C1-C4 alkyl substituted or unsubstituted indolyl, thiol, C2-C4 amide, C2-C4 carboxyl or C1-C4 alkylamino, and the substituent on the C1-C4 alkyl is selected from one or more of thiol, C1-C4 alkylthio, hydroxyl and halogen; R 4 It is one or more of H, halogen, alkynyl, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1-C4 alkoxy, ester, hydroxyl, C2-C4 acyl, and cyano, and the substituents on the C1-C4 alkyl and the C1-C4 alkoxy are independently selected from one or more of thiol, C1-C4 alkylthio, hydroxyl, and halogen; R 5 It is one or more of H, halogen, alkynyl, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1-C4 alkoxy, ester, hydroxyl, C2-C4 acyl, and cyano, and the substituents on the C1-C4 alkyl and the C1-C4 alkoxy are independently selected from one or more of thiol, C1-C4 alkylthio, hydroxyl, and halogen; R 6 It is one or more of H, halogen, alkynyl, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1-C4 alkoxy, ester, hydroxyl, C2-C4 acyl, and cyano, and the substituents on the C1-C4 alkyl and the C1-C4 alkoxy are independently selected from one or more of thiol, C1-C4 alkylthio, hydroxyl, and halogen; R 7 It is one or more of H, halogen, alkynyl, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted or unsubstituted phenyl, C1-C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1-C4 alkoxy, ester, hydroxyl, C2-C4 acyl, and cyano, and the substituents on the C1-C4 alkyl and the C1-C4 alkoxy are independently selected from one or more of thiol, C1-C4 alkylthio, hydroxyl, and halogen; R 8 -(CH2) n -, -CH2(CH3)-, -CH2(CH2CH3)-, -CH2(CH2Ph)-, -CH2OCH2CH2- or -CH2(CH2CH2SCH3)-, n=1 to 20, or R 8 Any structure in formula (a) to (h): n3, n4, n5 are independently 1 or 2, R 10 , R 11 Each is independently a C1-C4 alkyl group or a C1-C4 alkoxy group; R 9 -(CH2) m -, -CH2(CH3)-, -CH2(CH2CH3)-, -CH2(CH2Ph)-, -CH2OCH2CH2- or -CH2(CH2CH2SCH3)-, m=1-20, or R 9 It is any structure of formula (a) to (h).

9. The method for preparing a cyclic peptide according to claim 7 or 8, characterized in that: In step S1: The condensing agent used in the amino acid condensation reaction includes at least one of a carbodiimide condensing agent, a phosphorus cation condensing agent, and a urea cation condensing agent; The carbodiimide type condensing agent includes at least one of N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; The phosphorus cation type condensing agent includes at least one of 1H-benzotriazole-1-yloxytripyrrolidino hexafluorophosphate, (7-azabenzotriazole-1-oxy)tripyrrolidinophosphine hexafluorophosphate, bis(2-oxo-3-oxazolidinyl)phosphinyl chloride, chlorotripyrrolidinophosphine hexafluorophosphate, and tripyrrolidinophosphonium bromide hexafluorophosphate; The urea cationic condensing agent includes at least one of benzotriazole-N,N,N',N'-tetramethyl uronium hexafluorophosphate, N,N,N',N'-tetramethyl-O-(7-azabenzotriazole-1-yl) uronium hexafluorophosphate, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyl uronium tetrafluoroborate, O-[(ethoxycarbonyl) cyanomethylamine]-N,N,N',N'-tetramethyl thiourea tetrafluoroborate, and O-(benzotriazole-1-yl)-N,N,N',N'-dipyrrolyl uronium hexafluorophosphate; The condensation activator used in the amino acid condensation reaction includes at least one of 1-hydroxy-7-azobenzotriazole, 1-hydroxybenzotriazole, 1-hydroxy-1H-1,2,3-triazole-4-carboxylic acid ethyl ester, 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine, 2-hydroxypyridine-N-oxide, 4-dimethylaminopyridine, N-hydroxysuccinimide, p-nitrophenol, N,N-diisopropylethylamine, Oxyma and its derivatives.

10. The method for preparing a cyclic peptide according to claim 7 or 8, characterized in that: In step S1 to step S3: The solvent of each reaction process independently includes at least one of N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, ethyl acetate, tetrahydrofuran, toluene, acetone, ether, acetonitrile, methanol and water; The reaction temperature of each reaction process is independently 0 to 100°C.

11. The method for preparing a cyclic peptide according to claim 7 or 8, characterized in that: In step S2, the nitrite compound includes at least one of sodium nitrite, nitrosyl sulfuric acid, methyl nitrite, ethyl nitrite, isopropyl nitrite, butyl nitrite, tert-butyl nitrite, amyl nitrite, isoamyl nitrite, and hexyl nitrite.

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