Preparation method of polypeptide side chain

Abstract

The invention discloses a preparation method of a polypeptide side chain, which comprises the following steps: reacting a compound as shown in a formula I to obtain C00 or D00, carrying out AEA-OH multi-step reaction to obtain H00, and respectively obtaining polypeptide side chains X00 or Y00 under the action of a condensing agent and an acid-binding agent under the action of D00 or C00 and H00, compared with a traditional process with AEA-AEA as a starting material, the production of Des-AEA key impurities is better controlled, and compared with the traditional process, the production of the Des-AEA key impurities is further reduced, so that the pressure of subsequent GLP-1 polypeptide production and quality research is greatly reduced; compared with a normal polypeptide side chain, the Des-AEA has the advantages that only one AEA fragment is deleted in the structure, and the Des-AEA has high similarity in the structure, so that the Des-AEA shows relatively strong synchronism with a main component in the subsequent polypeptide production and purification process, and a lot of inconvenience is brought to quality control and quality research of raw material medicines or preparations.

Description

Technical Field

[0001] The present invention belongs to the technical field of GLP-1 polypeptide synthesis, and specifically relates to a preparation method applicable to semaglutide side chains, tirzepatide side chains and retatrutide side chains. Background Art

[0002] GLP-1 receptor agonists, GIP / GLP-1 dual receptor agonists and GIP / GLP-1 / GCGR triple receptor agonists are currently hot spots in drug development. The main indications include type II diabetes and weight loss indications, and other indications are also under development. This series includes blockbuster products such as semaglutide, telpotide and retalutide. This type of peptide is generally around 40 amino acids, and long-chain fatty acids (octadecanoic acid / eicosandioic acid) are connected to the 20-position lysine residue through a hydrophilic linker (gamma-glutamic acid and AEEA-AEEA / AEEA). Lipid modification is one of the chemical modification strategies to enhance the drug properties of peptide drugs, mainly by increasing the binding to albumin, thereby greatly increasing the half-life of the peptide. The potential advantages of lipid modification also include enhanced delivery, route of administration, improved pharmacological efficacy and reduced immunogenicity.

[0003] The side chain structure of semaglutide is shown below:

[0004]

[0005] The side chain structure of tepoxetine is shown below:

[0006]

[0007] In the prior art, patent CN114805543A discloses a method for preparing semaglutide side chains, and the starting material used is AEEA-AEEA. There is H-AEEA-OH impurity in the AEEA-AEEA synthesis route, which is difficult to control. If high-purity AEEA-AEEA is obtained, the cost is very high, so it is expensive. If H-AEEA-OH impurity exists, the impurities derived after the reaction are very difficult to remove, and the process involves multiple oily substances, the process operation is complicated, and it is not suitable for large-scale industrial production; patent CN113330024 A discloses a method for preparing telpotide side chains, and the intermediates are all oily substances, the process operation is complicated, and the post-treatment adopts column chromatography separation, which has high cost and three waste pressures, and is not suitable for large-scale industrial production.

[0008] Therefore, providing a method for preparing semaglutide side chains, telpotide side chains and retaluide side chains with controllable process, high economy, green environmental protection, high product quality, and suitability for industrial production has become a key issue to be solved by those skilled in the art. Summary of the Invention

[0009] The object of the present invention is to overcome the defects in the prior art, such as high cost, low synthesis efficiency, great environmental protection pressure, and being unfavorable for industrial-scale production, and to provide a preparation method applicable to the side chains of semaglutide, tirzepatide, and retatrutide. This synthesis method uses inexpensive and readily available raw materials, has mild reaction conditions, simple purification operations, high synthesis efficiency, is environmentally friendly, and is more suitable for industrial production.

[0010] According to the present invention, the specific preparation method includes the following steps:

[0011]

[0012] D00 or C00 reacts with H00 under the action of a condensing agent and an acid-binding agent to form compound X00 or Y00 respectively;

[0013] In the formula X00, n is selected from 4 to 20, such as 4 to 10, such as 8, 9 or 10;

[0014] In the formula Y00, m is selected from 4 to 20, such as 4 to 10, such as 8, 9 or 10.

[0015] Preferably, the condensing agent is selected from any one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate, and is further preferably 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0016] Preferably, the acid-binding agent is selected from any one of sodium carbonate, sodium acetate, sodium bicarbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine, pyridine, 2,6-dimethylpyridine, N-methylmorpholine, N-ethylmorpholine, and is further preferably N-methylmorpholine.

[0017] Preferably, the molar ratio of compound D00: compound H00: condensing agent: acid-binding agent is 1:0.8 to 1.2:1 to 3:1.5 to 20; and is further preferably 1:1:1.2:1.5.

[0018] Preferably, the reaction temperature is 0 to 50 °C, and is further preferably 20 to 30 °C; the reaction time is 1 to 8 h, and is further preferably 2 to 4 h.

[0019] Furthermore, as a specific implementation manner, the preparation method of the D00 includes the following steps: compound C00 reacts with H-AEEA-OH under the condition of an acid-binding agent, and then forms carboxylate D00 under the action of an inorganic base;

[0020]

[0021] Preferably, the acid-binding agent is selected from one or any combination of sodium carbonate, sodium acetate, sodium bicarbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine, pyridine, 2,6-dimethylpyridine, N-methylmorpholine, and N-ethylmorpholine.

[0022] Preferably, the molar ratio of compound C00:H-AEEA-OH:acid-binding agent is 1:0.9-1.4:1.5-50; more preferably 1:1:10.

[0023] Preferably, the reaction solvent is one or any combination of ethyl acetate, tetrahydrofuran, acetonitrile, DMF, 2-methyltetrahydrofuran, acetone, chloroform, methyl isobutyl ketone, and water in any proportion; more preferably 2-methyltetrahydrofuran.

[0024] Preferably, the inorganic base is selected from lithium salts, sodium salts, and potassium salts; further, the lithium salts are selected from one or any combination of lithium hydroxide, lithium carbonate, lithium bicarbonate, lithium acetate, and lithium phosphate; the sodium salts are selected from one or any combination of sodium hydroxide, sodium carbonate, sodium bicarbonate, sodium acetate, sodium phosphate, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium oxalate, and sodium formate; the potassium salts are selected from one or any combination of potassium hydroxide, potassium carbonate, potassium bicarbonate, potassium acetate, potassium phosphate, potassium dihydrogen phosphate, potassium hydrogen phosphate, potassium oxalate, potassium formate, and potassium fluoride; more preferably a mixture of one or any combination of lithium hydroxide, lithium carbonate, lithium acetate, sodium hydroxide, sodium carbonate, sodium acetate, potassium bicarbonate, potassium formate, and potassium fluoride.

[0025] Preferably, the reaction temperature is 0-50 °C, more preferably 20-30 °C; the reaction time is 1-8 h, more preferably 2-4 h.

[0026] As a specific embodiment, the preparation method of C00 is carried out according to the following steps:

[0027] (1) The long-chain alkane diacid monoter-butyl ester shown in formula I reacts with compound R 1 to form compound A00;

[0028] (2) Compound A00 reacts with L-glutamic acid 1-tert-butyl ester to form compound B00;

[0029] (3) Compound B00 reacts with compound R 2 to form compound C00;

[0030]

[0031] In step (1), preferably, in the long-chain alkane diacid monoter-butyl ester shown in formula I, n is 4-20, preferably 4-10, for example, n is 4, 5, 6, 7, 8, 9, 10.

[0032] In step (1), preferably, the compound R 1 is selected from 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate, N-hydroxysuccinimide or pentafluorophenol.

[0033] In step (1), preferably, in A00, R 1 ' is selected from

[0034] In step (1), preferably, the acid-binding agent is selected from one or any combination of sodium carbonate, sodium acetate, sodium bicarbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine, pyridine, 2,6-dimethylpyridine, N-methylmorpholine, and N-ethylmorpholine.

[0035] In step (1), preferably, the condensation reaction includes using a condensing agent, and the condensing agent is selected from one or any combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, and O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate;

[0036] In step (1), the molar ratio of the long-chain alkane diacid monoter-butyl ester shown in formula I to the compound R 1 , the acid-binding agent, and the condensing agent is 1:1-1.5:0-1.5:0-1.5; when the starting material II is 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate, the condensing agent may not be added.

[0037] In step (1), preferably, the reaction temperature is 0-50 °C, more preferably 20-30 °C; the reaction time is 1-8 h, more preferably 2-4 h.

[0038] In step (2), preferably, the reaction further includes adding an acid-binding agent, and the acid-binding agent is selected from any one of sodium acetate, sodium bicarbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine, pyridine, 2,6-dimethylpyridine, N-methylmorpholine, or N-ethylmorpholine.

[0039] In step (2), preferably, the molar ratio of the compound A00 to L-glutamic acid 1-tert-butyl ester and the acid-binding agent is 1:0.9-1.4:1.5-5.

[0040] In step (2), preferably, the reaction temperature is 0 to 50 °C, more preferably 20 to 30 °C; the reaction time is 1 to 8 h, more preferably 2 to 4 h.

[0041] In step (3), preferably, the compound R 2 is selected from 2-succinimidyloxy-1,1,3,3-tetramethyluronium tetrafluoroborate, N-hydroxysuccinimide or pentafluorophenol; more preferably 2-succinimidyloxy-1,1,3,3-tetramethyluronium tetrafluoroborate.

[0042] In step (3), preferably, the functional group R of C00 2 is selected from

[0043] In step (3), preferably, the acid-binding agent is selected from one or any combination of sodium carbonate, sodium acetate, sodium bicarbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine, pyridine, 2,6-dimethylpyridine, N-methylmorpholine, N-ethylmorpholine;

[0044] In step (3), preferably, the condensation reaction includes using a condensing agent, and the condensing agent is selected from one or any combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate;

[0045] In step (3), the molar ratio of the compound B00 to the compound R 2 , the acid-binding agent, and the condensing agent is 1:1 to 1.5:0 to 1.5:0 to 1.5.

[0046] In step (3), preferably, the reaction temperature is 0 to 50 °C, more preferably 20 to 30 °C; the reaction time is 1 to 8 h, more preferably 2 to 4 h.

[0047] As a specific embodiment, the preparation method of the H00 includes the following steps:

[0048] (1) H-AEEA-OH reacts with Boc anhydride to form E00;

[0049] (2) E00 reacts with the compound R 3 to form the compound F00;

[0050] (3) F00 reacts with Fmoc-lysine hydrochloride to form G00;

[0051] (4) G00 is obtained to form an amine salt compound H00 under the catalytic condition of an acid.

[0052]

[0053] In step (1), preferably, the acid-binding agent is selected from any one of sodium carbonate, sodium acetate, sodium bicarbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine, pyridine, 2,6-dimethylpyridine, N-methylmorpholine, and N-ethylmorpholine, and more preferably sodium bicarbonate;

[0054] In step (1), preferably, the reaction solvent is one or a mixed solvent of any several of ethyl acetate, tetrahydrofuran, acetonitrile, DMF, 2-methyltetrahydrofuran, acetone, chloroform, methyl isobutyl ketone, and water; more preferably, it is tetrahydrofuran and water (V / V = 1:1);

[0055] In step (1), preferably, the molar ratio of H-AEEA-OH:Boc anhydride:acid-binding agent is 1:1 to 3:1.5 to 20; more preferably, it is 1:1.5:2.

[0056] In step (1), preferably, the reaction temperature is 0 to 50 °C, more preferably 20 to 30 °C; the reaction time is 2 to 24 h, more preferably 8 to 12 h.

[0057] In step (2), preferably, compound R 3 is selected from 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate, N-hydroxysuccinimide, or pentafluorophenol; more preferably, it is pentafluorophenol.

[0058] In step (2), preferably, R 3 ' in F00 is selected from

[0059] In step (2), preferably, the acid-binding agent is selected from any one of sodium carbonate, sodium acetate, sodium bicarbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine, pyridine, 2,6-dimethylpyridine, N-methylmorpholine, and N-ethylmorpholine.

[0060] And / or, in step (2), preferably, the condensation reaction includes using a condensing agent, and the condensing agent is selected from any one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, and O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate;

[0061] In step (2), preferably, the reaction further includes adding a catalyst, and the catalyst is DMAP.

[0062] In step (2), preferably, the reaction solvent is ethyl acetate, tetrahydrofuran, acetonitrile, DMF, 2-methyltetrahydrofuran, acetone, methyl isobutyl ketone, water, or a mixed solvent of any one or several of them in any proportion.

[0063] In step (2), preferably, the molar ratio of compound E00: compound R 3 : acid-binding agent: condensing agent: catalyst is 1: 1 to 1.5: 0 to 1.5: 0 to 1.5: 0 to 0.5.

[0064] In step (2), preferably, the reaction temperature is 0 to 50 °C, more preferably 20 to 30 °C; the reaction time is 1 to 8 h, more preferably 2 to 4 h.

[0065] In step (3), preferably, the acid-binding agent is selected from any one of sodium carbonate, sodium acetate, sodium bicarbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine, pyridine, 2,6-dimethylpyridine, N-methylmorpholine, N-ethylmorpholine, and more preferably N,N-diisopropylethylamine.

[0066] In step (3), preferably, the molar ratio of compound F00: Fmoc-lysine hydrochloride: acid-binding agent is 1: 0.9 to 1.4: 1.5 to 10; more preferably 1: 1: 2.

[0067] In step (3), preferably, the reaction solvent is ethyl acetate, tetrahydrofuran, acetonitrile, DMF, 2-methyltetrahydrofuran, acetone, methyl isobutyl ketone, water, or a mixed solvent of any one or several of them in any proportion.

[0068] In step (3), preferably, the reaction temperature is 0 to 50 °C, more preferably 20 to 30 °C; the reaction time is 1 to 8 h, more preferably 2 to 4 h.

[0069] In step (4), preferably, the acid is sulfuric acid, hydrochloric acid, acetic acid, trifluoroacetic acid, methanol solution of HCl, ethanol solution of HCl, isopropanol solution of HCl, ethyl acetate solution of HCl, dioxane solution of HCl, oxalic acid, citric acid, tartaric acid, succinic acid, p-toluenesulfonic acid, or a mixture of any one or several of them in any proportion and its mixture with water; more preferably methanol solution of HCl, ethanol solution of HCl, isopropanol solution of HCl, ethyl acetate solution of HCl, dioxane solution of HCl; even more preferably dioxane solution of HCl.

[0070] In step (4), preferably, the reaction temperature is 0 to 50 °C, more preferably 20 to 30 °C; the reaction time is 1 to 8 h, more preferably 2 to 4 h.

[0071] In step (4), preferably, the ratio of the reactants is 1:1 to 5 by volume of compound G00 to acid; more preferably 1:2.

[0072] Compared with the prior art, the present invention has various beneficial effects:

[0073] The present invention breaks through the conventional technical design barriers and synthesizes various GLP-1 single-target and multi-target key raw materials including the side chain of semaglutide, the side chain of tirzepatide, the side chain of retatrutide, and novel polypeptide side chains in a clever way. The synthesis process is simple and controllable, with low material cost, strong atom economy, and is suitable for large-scale industrial production.

[0074] The synthesis process conditions of the present invention are mild, without involving special reaction conditions such as high temperature and high pressure, ultra-low temperature or hydrogenation. The use of acid in the process is also controlled, with low equipment corrosion, higher process and material safety, and better meeting the requirements of ICH Q3D;

[0075] The post-treatment of the present invention is simple. The purification process basically adopts the crystallization process, abandoning the traditional column chromatography separation technology, with more efficient production and low three-waste pressure;

[0076] Compared with the traditional process starting from AEEA-AEEA, the present invention better controls the generation of Des-AEEA key impurities, further reduces them compared with the traditional process, and greatly reduces the pressure on the subsequent production and quality research of GLP-1 polypeptides; Des-AEEA only lacks one AEEA fragment in structure compared with the normal polypeptide side chain, with high structural similarity. Therefore, in the subsequent production and purification processes of polypeptides, it shows strong synchronization with the main component, bringing many inconveniences to the quality control and quality research of APIs or preparations.

[0077] The present invention further improves the quality of the relevant polypeptide side chains. The product purity reaches more than 99%, without Des-AEEA impurities, and the single impurity is controlled below 0.2%.

[0078] Compared with the prior art, the present invention has low raw material cost, high reaction yield, strong operability, and is green and environmentally friendly, suitable for industrial production. Detailed Embodiments

[0079] The following specific embodiments further illustrate the present invention in detail. The following embodiments are used to understand the method and core idea of the present invention. For those skilled in the art, any possible changes or substitutions made without departing from the concept of the present invention fall within the protection scope of the present invention. The experimental methods without specific conditions noted in the embodiments of the present invention are usually conventional conditions, or the conditions recommended by the raw material or commodity manufacturers; the reagents without indicated sources are usually conventional reagents that can be purchased through commercial channels.

[0080] The chemical names corresponding to some of the abbreviations used in the present invention are as follows:

[0081] Semaglutide side chain Des-AEEA impurity: (25S,41S)-52-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-23-(tert-butoxycarbonyl)-2,2-dimethyl-4,21,26,35-tetraoxo-3,30,33-trioxa-22,27,36-triazatetratetracontane-42-carboxylic acid

[0082] Tirzepatide side chain Des-AEEA impurity: (25S,43S)-52-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-25-(tert-butoxycarbonyl)-2,2-dimethyl-4,23,28,37-tetraoxo-3,32,35-trioxa-24,29,38-triazatetratetracontane-44-carboxylic acid

[0083] Fmoc-L-LYS-AEEA-AEEA.TFA ： (S)-23-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-amino-8,17-dioxo-3,6,12,15-tetraoxa-9,18-diazatetracosane-24-carboxylic acid trifluoroacetate

[0084] H-AEEA-OH: 2-(2-(2-aminoethoxy)ethoxy)acetic acid

[0085] TSTU: O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate

[0086] DMAP: 4-dimethylaminopyridine

[0087] DIPEA: N,N-diisopropylethylamine

[0088] Fmoc-lys: N-(9-fluorenylmethoxycarbonyl)-L-lysine

[0089] THF: Tetrahydrofuran

[0090] PFP: Pentafluorophenol

[0091] EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0092] ACN: Acetonitrile

[0093] NMM: N-methylmorpholine

[0094] (Boc) 2 O: Di-tert-butyl dicarbonate

[0095] DCC: N,N'-Dicyclohexylcarbodiimide

[0096] CDI: N,N'-Carbonyldiimidazole

[0097] Py: Pyridine

[0098] K 3 PO 4 : Potassium phosphate

[0099] 2,6-Lutidine: 2,6-Dimethylpyridine

[0100] NEM: N-Ethylmorpholine

[0101] DBU: 1,8-Diazabicyclo[5.4.0]undec-7-ene

[0102] NMI: N-Methylimidazole

[0103] Imidazole: Imidazole

[0104] EEDQ: 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline

[0105] T3P: 1-n-Propylphosphonic anhydride

[0106] TCFH: Tetramethylchlorouronium hexafluorophosphate

[0107] TCT: Trichlorotriazine

[0108] Example 1

[0109] This example discloses the synthesis method of compound A00, specifically: 3.99 g of monoterbutyl sebacate (10 mmol) and 3.61 g of TSTU (12 mmol) as raw materials were put into a 100 ml three-necked flask, 32 ml of THF was added, stirred and dissolved, cooled to 10 - 20 °C, 1.55 g of DIPEA (12 mmol) was added. After adding, stirred and reacted at 20 - 30 °C for 4 h. TLC plate was used to confirm the completion of the reaction. The reaction product was quenched with water, adjusted to pH = 2 - 3 with dilute hydrochloric acid aqueous solution, filtered, the wet product was dried, the dried product was dissolved by heating with ethanol, cooled to 0 °C and crystallized for 6 h, suction filtered to obtain a solid, dried, to obtain 4.50 g of A00 (C-20), with a yield of 90.79% and a purity of 99.58%.

[0110] 1 1H NMR (DMSO-d6): 2.81 (s, 4H); 2.59 (s, 2H); 2.16 (m, 2H); 1.24 - 1.60 (m, 39H); LC-MS target [M+Na]+ = 490.3145, measured [M+H]+ = 490.3124.

[0111] Using monoterbutyl octadecanedioate as raw material and preparing according to the method of Example 1, A00(C-18) can be obtained with a yield of 89.77% and a purity of 98.98%.

[0112] 1 H NMR (DMSO-d6): 2.80 (s, 4H); 2.50 (s, 2H); 2.15 (m, 2H); 1.24 - 1.60 (m, 35H); LC-MS target [M+Na]+ = 518.3458, measured [M+H]+ = 518.3420.

[0113] Using monoterbutyl docosanedioate as raw material and preparing according to the method of Example 1, A00(C-22) can be obtained with a yield of 91.07% and a purity of 98.36%.

[0114] 1 H NMR (CDCl 3 ): 2.82 - 2.84 (m, 4H); 2.60 (t, 2H, J = 7.5Hz); 2.20 (t, 2H, J = 7.5Hz); 1.71 - 1.77 (m, 2H); 1.56 - 1.58 (m, 2H); 1.44 (s, 9H);; 1.25 (s, 32H); LC-MS target [M+Na]+ = 546.3771, measured [M+Na]+ = 546.3764.

[0115] Example 2

[0116] This example discloses a synthesis method of compound B00. Specifically: 4.96 g of A00(C-20) (10 mmol) and 2.03 g of L-glutamic acid-1-tert-butyl ester (10 mmol) as raw materials are put into a 100 ml three-necked flask, 50 ml of 2-methyltetrahydrofuran is added, stirred and dissolved, cooled to 10 - 20 °C, 1.94 g of DIPEA (15 mmol) is slowly added dropwise with the internal temperature not exceeding 20 °C. After addition, stir and react at 20 - 30 °C for 3 h. TLC is used to check the completion of the reaction. The reaction solution is quenched with dilute hydrochloric acid aqueous solution, separated, washed with water, dried with Na 2 SO 4 dried, filtered, concentrated to dryness, dissolved by heating with acetonitrile, cooled to 0 °C for crystallization for 6 h, filtered by suction to obtain a solid, dried, and 5.15 g of B00(C-20) is obtained with a yield of 92.55% and a purity of 99.20%.

[0117] 11H NMR (DMSO-d6): 12.17 (s, 1H); 8.05 (d, 1H, J = 7.5 Hz); 4.11 - 4.16 (m, 1H); 2.20 - 2.30 (m, 2H); 2.16 (t, 2H, J = 7.5 Hz); 2.09 (t, 2H, J = 7.5 Hz); 1.86 - 1.93 (m, 1H); 1.69 - 1.77 (m, 1H); 1.41 - 1.51 (m, 4H); 1.39 (s, 18H); 1.23 (s, 28H); LC-MS target [M + H]+ = 584.4448, found [M + H]+ = 584.4532.

[0118] Using A00 (C-18) as the raw material and prepared according to the method of Example 2, B00 (C-18) can be obtained with a yield of 91.11%; purity 98.55%.

[0119] 1 1H NMR (DMSO-d6): 12.14 (s, 1H); 8.03 (d, 1H, J = 8.0 Hz); 4.11 - 4.16 (m, 1H); 2.21 - 2.30 (m, 2H); 2.13 - 2.17 (m, 2H); 2.08 - 2.12 (m, 2H); 1.86 - 1.93 (m, 1H); 1.69 - 1.77 (m, 1H); 1.41 - 1.52 (m, 4H); 1.39 - 1.40 (m, 18H); 1.22 (s, 24H); LC-MS target [M + H]+ = 556.4135, found [M + H]+ = 556.4196.

[0120] Using A00 (C-22) as the raw material and prepared according to the method of Example 2, B00 (C-22) can be obtained with a yield of 90.35%; purity 99.02%.

[0121] 1 1H NMR (DMSO-d6): 12.14 (br s, 1H); 8.06 (d, 1H, J = 7.5 Hz); 4.11 - 4.16 (m, 1H); 2.19 - 2.31 (m, 2H); 2.16 (t, 2H, J = 7.2 Hz); 2.09 (t, 2H, J = 7.2 Hz); 1.86 - 1.93 (m, 1H); 1.69 - 1.77 (m, 1H); 1.43 - 1.51 (m, 4H); 1.39 (s, 18H); 1.23 (s, 32H); LC-MS target [M + H]+ = 612.4761, found [M + H]+ = 612.4838.

[0122] Example 3

[0123] This example discloses a synthesis method of compound C00, specifically as follows: 5.84 g of raw material B00 (C-20) (10 mmol) and 3.61 g of TSTU (12 mmol) were put into a 100 ml three-necked flask, 30 ml of tetrahydrofuran was added, stirred and dissolved, cooled to 10 - 20 °C, 1.55 g of DIPEA (12 mmol) was added. After addition, it was stirred and reacted at 20 - 30 °C for 3 h. TLC plate was used to confirm the completion of the reaction. The reaction solution was quenched with dilute hydrochloric acid aqueous solution, adjusted to pH = 2 - 3 with dilute hydrochloric acid aqueous solution, filtered, the wet product was dried, the dried product was dissolved by heating with ethanol, cooled to 0 °C and crystallized for 6 h, filtered by suction to obtain a solid, dried, to obtain 5.93 g of C00 (C-20), with a yield of 90.84%; purity 98.90%.

[0124] 1 H NMR (DMSO-d6): 8.13 (d, 1H, J = 7.5 Hz); 4.16 - 4.19 (m, 1H); 2.74 - 2.81 (m, 5H); 2.65 - 2.71 (m, 1H); 2.15 (t, 2H, J = 7.2 Hz); 2.11 (t, 2H, J = 7.5 Hz); 2.00 - 2.08 (m, 1H); 1.86 - 1.93 (m, 1H); 1.47 - 1.48 (m, 4H); 1.39 (d, 18H, J = 5.0 Hz); 1.22 (s, 28H); LC-MS target [M + H]+ = 681.4612, measured [M + H]+ = 681.4683.

[0125] Using B00 (C-18) as the raw material, prepared according to the method of Example 3, C00 (C-18) can be obtained, with a yield of 89.19%; purity 98.47%.

[0126] 1 H NMR (DMSO-d6): 8.11 (d, 1H, J = 8.0 Hz); 4.17 - 4.22 (m, 1H); 2.74 - 2.82 (m, 5H); 2.65 - 2.72 (m, 1H); 2.10 - 2.16 (m, 4H); 2.02 - 2.09 (m, 1H); 1.85 - 1.93 (m, 1H); 1.48 - 1.53 (m, 4H); 1.39 - 1.40 (d, 18H, J = 8.5 Hz); 1.24 (s, 24H); LC-MS target [M + Na]+ = 675.4197, measured [M + Na]+ = 675.4204.

[0127] Using B00 (C-22) as the raw material, prepared according to the method of Example 3, C00 (C-22) can be obtained, with a yield of 87.41%; purity 98.05%.

[0128] 1 1H NMR (DMSO-d6): δ 8.13 (d, 1H, J = 7.5 Hz); 4.14 - 4.18 (m, 1H); 2.75 - 2.81 (m, 5H); 2.64 - 2.71 (m, 1H); 2.16 (t, 2H, J = 7.2 Hz); 2.10 (t, 2H, J = 7.2 Hz); 1.99 - 2.06 (m, 1H); 1.84 - 1.92 (m, 1H); 1.41 - 1.51 (m, 4H); 1.39 (d, 18H, J = 3.5 Hz); 1.22 (s, 32H); LC-MS target [M + H]+ = 709.4925, found [M + H]+ = 709.4998.

[0129] Example 4

[0130] This example discloses a synthesis method of compound D00, specifically: 6.81 g of C00 (C-20) (10 mmol) and 1.63 g of AEEA (10 mmol) as raw materials were put into a 100 ml three-necked flask, 68 ml of 2-methyltetrahydrofuran was added, stirred and dissolved, cooled to 10 - 20 °C, and 10.1 g of N-methylmorpholine (100 mmol) was slowly added dropwise with the internal temperature not exceeding 20 °C. After addition, the reaction was stirred at 20 - 30 °C for 4 h. TLC was used to confirm the completion of the reaction. The reaction solution was quenched with dilute hydrochloric acid aqueous solution, separated, washed with brine, concentrated to dryness, dissolved in 65 ml of acetonitrile, and then 1 M lithium hydroxide aqueous solution was added to adjust the pH to 9 - 10. The mixture was concentrated to dryness and entrained with acetonitrile multiple times until KF < 0.1%. After filtration, acetonitrile was added for slurrying, cooled to -20 °C and stirred for 6 h, and the solid was obtained by suction filtration, dried to obtain 7.19 g of D00 (C-20), with a yield of 97.82% and a purity of 98.44%.

[0131] Using C00 (C-18) as the raw material, prepared according to the method of Example 4, D00 (C-18) was obtained with a yield of 95.21% and a purity of 98.87%.

[0132] Using C00 (C-22) as the raw material, prepared according to the method of Example 4, D00 (C-22) was obtained with a yield of 95.99% and a purity of 98.75%.

[0133] Example 5

[0134] This example discloses a synthesis method of compound E00, specifically: 9.78 g of H-AEEA-OH (60 mmol) and 19.62 g of (Boc) 290 mmol of O was added to a 250 ml three-necked flask, 75 ml of THF and 75 ml of purified water were added, and the mixture was stirred until dissolved. The temperature was lowered to 0 - 10 °C, and 12.74 g of sodium carbonate (120 mmol) was added in 5 batches at intervals of 10 min, about 2.5 g each time. The reaction solution temperature did not exceed 20 °C. After addition, the mixture was stirred at 20 - 30 °C for 8 h. TLC plate was used to confirm the completion of the reaction. The reaction solution was adjusted to pH = 3 - 4 with dilute hydrochloric acid aqueous solution, and extracted twice with 100 ml of 2-methyltetrahydrofuran each time. The 2-methyltetrahydrofuran extracts were combined, washed with saturated brine, and dried over Na 2 SO 4 filtered, concentrated to dryness to obtain 14.23 g of E00, with a yield of 95.06% and a purity of 99.55%.

[0135] Example 6

[0136] This example discloses a method for synthesizing compound F00. Specifically: 13.16 g of E00 (50 mmol), 9.20 g of PFP (50 mmol), and 0.31 g of DMAP (2.5 mmol) were added to a 250 ml three-necked flask, 130 ml of 2-methyltetrahydrofuran was added, and the mixture was stirred until dissolved. The temperature was lowered to 0 - 10 °C, and 11.52 g of EDCI (60 mmol) was added in 5 batches at intervals of about 5 min, about 2.3 g each time. The reaction solution temperature did not exceed 20 °C. After addition, the mixture was stirred at 20 - 30 °C for 2 h. TLC plate was used to confirm the completion of the reaction. The reaction solution was quenched with dilute hydrochloric acid aqueous solution, and the layers were separated. The organic layer was washed with 5% NaHCO 3 aqueous solution, washed with water, the layers were separated, and dried over Na 2 SO 4 filtered, concentrated to dryness to obtain 19.21 g of F00, with a yield of 89.47% and a purity of 97.78%.

[0137] Example 7

[0138] This example discloses a method for synthesizing compound G00. Specifically: 17.17 g of F00 (40 mmol) and 16.20 g of Fmoc-lys hydrochloride (40 mmol) were added to a 250 ml three-necked flask, 170 ml of 2-methyltetrahydrofuran was added, and the mixture was stirred until dissolved. The temperature was lowered to 0 - 10 °C, and 10.32 g of DIPEA (80 mmol) was slowly added dropwise. The reaction solution temperature did not exceed 20 °C. After addition, the mixture was stirred at 20 - 30 °C for 2 h. TLC plate was used to confirm the completion of the reaction. The reaction solution was quenched with dilute hydrochloric acid aqueous solution, and the layers were separated. The organic layer was washed with 10% Na 2 CO 3 aqueous solution, washed with hydrochloric acid, washed with water, the layers were separated, and dried over Na 2 SO 4Dry, filter, concentrate to dryness, add acetonitrile, warm up to dissolve, cool down to 0 °C, crystallize for 6 h, filter by suction to obtain solid, dry to obtain 19.92 g of G00, yield 88.15%; purity 98.53%.

[0139] Example 8

[0140] This example discloses a method for synthesizing compound H00. Specifically: Put 18.41 g of G00 (30 mmol) of raw materials into a 250 ml three-necked flask, add 37 ml of THF, stir to dissolve, cool down to -10 - 0 °C, slowly dropwise add 37 ml of a dioxane solution of 4M HCl, and the reaction solution does not exceed 10 °C. After adding, stir and react at 20 - 30 °C for 3 h. Confirm the completion of the reaction by TLC plate spotting. Concentrate the reaction solution to dryness. Add ether to the concentrate and slurry for 2 h. Filter by suction to obtain solid, dry to obtain 15.04 g of H00, yield 91.15%; purity 97.98%.

[0141] Example 9

[0142] Repeat the patent to prepare the intermediate Fmoc-L-LYS-AEEA-AEEA.TFA and the side chain of semaglutide. The specific operations are as follows:

[0143] Add 5 g of G00 (6.59 mmol) to a 100 ml reaction flask, add 10 g of DCM, control the temperature at 25 °C and slowly add 15 g of TFA. After adding, keep the temperature and react for 5 h. Observe by TLC plate spotting, and the reaction is complete; Distill off dichloromethane and a large amount of TFA under reduced pressure to obtain 5.11 g of Fmoc-L-LSY-AEAE-AEEA.TFA, yield 101%

[0144] Take 5.02 g of Fmoc-L-LYS-AEEA-AEEA.TFA (6.48 mmol), 3.81 g of C00 (C-18) (5.83 mmol), and 25 ml of dichloromethane and put them into a 100 ml three-necked flask. Control the temperature at 20 °C and slowly add 1.31 g of triethylamine (12.97 mmol). After adding, keep the temperature and react for 5 h. Observe by TLC plate spotting, and the reaction is complete. The reaction product is pickled, washed with water, dried, filtered, concentrated, and cooled to 0 °C with ethyl acetate:n-heptane mass ratio of 4:1, and crystallized for 4 h. A large amount of mucus precipitates, making it difficult to filter and unable to obtain the target product.

[0145] Further, the inventor cooled the crystallization solution to -20°C, stirred it for a long time to crystallize, filtered it at low temperature to obtain a powdery solid, dried it to obtain the side chain of semaglutide X00-1 (n is 8), with a yield of 74.05% (this yield is greatly affected by environmental factors and can be obtained well when the environmental temperature is close to 0°C or even lower. When the environmental temperature rises, the solid will turn into mucus at a visible speed and remain in the funnel); the purity is 99.01%. Among them, the key impurity desAEEA is 0.09%.

[0146] Example 10

[0147] Referring to steps (3) and (4) of Example 2 in Patent CN118546077A,

[0148] First step: Dissolve C00(C-20) (100 g, 0.15 mol) in dichloromethane (1000 ml), add H-AEEA-OH (26.40 g, 0.16 mol), dropwise add DIPEA (38.0 g, 0.29 mol), react at 20 - 30°C for 4 h, add 0.5N hydrochloric acid (600 ml), separate the layers, wash the organic layer with water, and directly feed it into the next step; concentrate and dry the organic phase, with a purity of 98.24% and a yield of 99.95%.

[0149] Second step: Take the organic layer from the previous step, add pentafluorophenol (40.5 g, 0.22 mol), cool it to 0 - 10°C, stir and add EDCI (42.1 g, 0.22 mol), raise the temperature to 20 - 30°C and react for 4 h, add 0.5N hydrochloric acid (500 ml) for washing, wash the organic layer with water, and directly feed it into the next step, with an HPLC purity of 97.32%;

[0150] Third step: Take the organic layer from the previous step, add H-AEEA-OH (26.40 g, 0.16 mol), dropwise add DIPEA (38.0 g, 0.29 mol), react at 20 - 30°C for 4 hours, add 0.5N hydrochloric acid (600 ml), separate the layers, wash the organic layer with water, and can directly feed it into the next step; concentrate and dry the organic phase, with a yield of 98.64%.

[0151] Fourth step: Take the organic layer from the previous step, add pentafluorophenol (40.5 g, 0.22 mol), cool it to 0 - 10°C, stir and add EDCI (42.1 g, 0.22 mol), raise the temperature to 20 - 30°C and react for 4 hours, add 0.5N hydrochloric acid (500 ml) for washing, wash the organic layer with water, and purify it by column chromatography with ethyl acetate and n-heptane to obtain the target compound, with a yield of 76.64% and an HPLC purity of 98.85%;

[0152] Step 5: Dissolve the product from the previous step (100.00 g, 0.11 mol) in dichloromethane (1000 ml), add Fmoc-lysine hydrochloride (48.5 g, 0.12 mol), dropwise add N,N-diisopropylethylamine (28.4 g, 0.22 mol), react at 20 - 30 °C for 4 hours, add 0.5 N hydrochloric acid (600 ml), separate the layers, wash the organic layer with 1% sodium bicarbonate solution (600 ml), wash the organic layer with saturated brine (600 ml), concentrate to dryness after drying to obtain the side chain of tirzepatide, HPLC purity 98.32%. The key impurity desAEEA is at 0.52%.

[0153] Example 11

[0154] This example discloses a method for synthesizing compound X00-2 (the side chain of tirzepatide). Specifically: Put 7.35 g of D00 (C-20) (10 mmol) and 1.51 g of N-methylmorpholine (15 mmol) into a 100 ml three-necked flask, add 50 ml of methyltetrahydrofuran, stir to dissolve, cool down to -5 - 0 °C, then add 2.30 g of EDCI (12 mmol), react at -5 - 0 °C for 1 h, then add 5.50 g of H00 (10 mmol). After adding, stir and react at 20 - 30 °C for 2 h. Confirm the completion of the reaction by TLC plate spotting. Quench the reaction solution with dilute hydrochloric acid aqueous solution, separate the layers, wash with saturated brine, 2 SO 4 dry, filter, concentrate to dryness, dissolve in acetonitrile at room temperature, cool down to -20 °C and stir for crystallization for 3 h, filter to obtain a solid, and vacuum dry the solid to obtain 10.44 g of X00-2 (n is 9, that is, the side chain of tirzepatide), with a yield of 85.29%; purity 99.34%, all single impurities are <0.2%, and there is no des AEEA impurity.

[0155] 11H NMR (DMSO-d6): 12.86 (br s, 1H); 8.05 (d, 1H, J = 7.5 Hz); 7.88 - 7.91 (m, 3H); 7.62 - 7.74 (m, 5H); 7.42 (t, 2H, J = 7.5 Hz); 7.33 (t, 2H, J = 7.2 Hz); 4.21 - 4.29 (m, 3H); 4.04 - 4.08 (m, 1H); 3.90 - 3.94 (m, 1H); 3.86 (d, 4H, J = 6.0 Hz); 3.51 - 3.57 (m, 8H); 3.45 (t, 2H, J = 5.8 Hz); 3.41 (t, 2H, J = 5.8 Hz); 3.29 (q, 2H, J = 6.0 Hz); 3.21 (q, 2H, J = 5.5 Hz); 3.07 - 3.15 (m, 2H); 2.09 - 2.17 (m, 6H); 1.86 - 1.93 (m, 1H); 1.68 - 1.78 (m, 2H); 1.58 - 1.66 (m, 1H); 1.41 - 1.51 (m, 6H); 1.39 (s, 18H); 1.22 (s, 30H); LC-MS target [M + H]+ = 1224.7556, found [M + H]+ = 1224.7637.

[0156] The effects of different types of condensing agents and acid-binding agents on purity and yield were investigated respectively, as shown in Table 1 specifically:

[0157] Table 1 Effects of different types of condensing agents and acid-binding agents on purity and yield

[0158]

[0159]

[0160] Example 12

[0161] This example discloses a synthesis method of compound X00-1 (the side chain of semaglutide), specifically: 7.07 g of D00 (C-18) (10 mmol) and 1.51 g of N-methylmorpholine (15 mmol) as raw materials were put into a 100 ml three-necked flask, 50 ml of methyltetrahydrofuran was added, stirred and dissolved, cooled to -5 - 0 °C, then 2.30 g of EDCI (12 mmol) was added, reacted at -5 - 0 °C for 1 h, then 5.50 g of H00 (10 mmol) was added. After adding, stirred and reacted at 20 - 30 °C for 2 h. TLC was used to confirm the completion of the reaction. The reaction solution was quenched with dilute hydrochloric acid aqueous solution, separated, washed with saturated brine, Na 2 SO 4Dry, filter, concentrate to dryness, add acetonitrile and dissolve at room temperature, cool to -20 °C, stir and crystallize for 3 h, filter by suction to obtain a solid, and vacuum dry the solid to obtain 10.37 g of X00-1 (n is 8, i.e., the side chain of semaglutide), with a yield of 86.71%; the purity is 99.26%, all single impurities are <0.2%, and there is no desAEEA impurity.

[0162] 1 H NMR (DMSO-d6): 12.50 (br s, 1H); 8.06 (d, 1H, J = 7.5 Hz); 7.89 - 7.93 (m, 3H); 7.62 - 7.74 (m, 4H); 7.57 (d, 1H, J = 7.0 Hz); 7.42 (t, 2H, J = 7.3 Hz); 7.33 (t, 2H, J = 7.3 Hz); 4.21 - 4.29 (m, 3H); 4.04 - 4.08 (m, 1H); 3.86 - 3.93 (m, 5H); 3.53 - 3.57 (m, 8H); 3.44 - 3.47 (m, 2H); 3.41 (t, 2H, J = 5.8 Hz); 3.29 (q, 2H, J = 6.0 Hz); 3.21 (q, 2H, J = 5.5 Hz); 3.09 - 3.13 (m, 2H); 2.07 - 2.17 (m, 6H); 1.86 - 1.93 (m, 1H); 1.68 - 1.78 (m, 2H); 1.58 - 1.63 (m, 1H); 1.42 - 1.51 (m, 6H); 1.39 (s, 18H); 1.22 (s, 26H), LC-MS target [M+Na]+ = 1218.7141, measured [M+Na] =: 1218.7153.

[0163] Example 13

[0164] This example discloses a synthesis method of compound X00-3, specifically: Put 7.63 g of D00 (C-20) (10 mmol) and 1.51 g of N-methylmorpholine (15 mmol) into a 100 ml three-necked flask, add 50 ml of methyltetrahydrofuran, stir to dissolve, cool to -5 - 0 °C, then add 2.30 g of EDCI (12 mmol), react at -5 - 0 °C for 1 h, then add 5.50 g of H00 (10 mmol). After adding, stir and react at 20 - 30 °C for 2 h. Confirm the completion of the reaction by TLC plate spotting. Quench the reaction solution with dilute hydrochloric acid aqueous solution, separate the layers, wash with saturated brine, Na 2 SO 4Dry, filter, concentrate to dryness, add acetonitrile and dissolve at room temperature, cool to -20 °C, stir for crystallization for 3 h, filter by suction to obtain a solid, and vacuum dry the solid to obtain 9.87 g of X00-3 (n is 10), with a yield of 88.87%; purity 99.15%, all single impurities are <0.2%, and there is no desAEEA impurity.

[0165] 1 1H NMR (DMSO-d6): 12.58 (br s, 1H); 8.06 (d, 1H, J = 7.5 Hz); 7.89 - 7.92 (m, 3H); 7.63 - 7.74 (m, 5H); 7.42 (t, 2H, J = 7.5 Hz); 7.33 (t, 2H, J = 7.2 Hz); 4.21 - 4.28 (m, 3H); 4.04 - 4.07 (m, 1H); 3.85 - 3.94 (m, 5H); 3.51 - 3.57 (m, 8H); 3.45 (t, 2H, J = 6.0 Hz); 3.41 (t, 2H, J = 5.8 Hz); 3.28 (q, 2H, J = 6.0 Hz); 3.20 (q, 2H, J = 5.8 Hz); 3.08 - 3.12 (m, 2H); 2.07 - 2.17 (m, 6H); 1.87 - 1.91 (m, 1H); 1.67 - 1.78 (m, 2H); 1.60 - 1.65 (m, 1H); 1.42 - 1.51 (m, 6H); 1.39 (s, 18H); 1.22 (s, 34H); LC-MS target [M + Na]+ = 1274.7767, measured [M + Na]+: 1224.7764.

[0166] Example 14

[0167] This example discloses a synthesis method of compound Y00-1 (the side chain of retatrutide), specifically: Put 6.81 g of C00 (C-20)

[0168] (10 mmol) and 1.51 g of N-methylmorpholine (15 mmol) into a 100 ml three-necked flask, add 50 ml of 2-methyltetrahydrofuran, stir to dissolve, cool to -5 - 0 °C, and then add 5.50 g of H00 (10 mmol). After adding, stir and react at 20 - 30 °C for 2 h. Confirm the completion of the reaction by TLC plate spotting. Quench the reaction solution with dilute hydrochloric acid aqueous solution, separate the layers, wash with saturated brine, and dry with Na 2 SO 4 Dry, filter, concentrate to dryness, add acetonitrile and dissolve at room temperature, cool to -20 °C, stir for crystallization for 3 h, filter by suction to obtain a solid, and vacuum dry the solid to obtain 9.19 g of Y00-1 (n is 9, that is, the side chain of retatrutide), with a yield of 85.17%; purity 99.04%. All single impurities are <0.2%.

[0169] 1 1H NMR (DMSO-d6): 12.57 (br s, 1H); 8.05 (d, 1H, J = 7.5 Hz); 7.89 - 7.92 (m, 3H); 7.63 - 7.74 (m, 4H); 7.42 (t, 2H, J = 7.5 Hz); 7.33 (t, 2H, J = 7.3 Hz); 4.21 - 4.28 (m, 3H); 4.04 - 4.07 (m, 1H); 3.89 - 3.93 (m, 1H); 3.85 (s, 2H); 3.53 - 3.56 (m, 4H); 3.40 (t, 2H, J = 6.0 Hz); 3.20 (q, 2H, J = 5.8 Hz); 3.08 - 3.12 (m, 2H); 2.07 - 2.17 (m, 6H); 1.87 - 1.91 (m, 1H); 1.68 - 1.76 (m, 2H); 1.60 - 1.63 (m, 1H); 1.40 - 1.51 (m, 6H); 1.38 (s, 18H); 1.22 (s, 30H). LC-MS target [M+Na]+ = 1101.6705, found [M+Na]+: 1101.6737.

[0170] Example 15

[0171] This example discloses a synthesis method of compound Y00-2, specifically: 6.53 g of C00(C-18) (10 mmol) and 1.51 g of N-methylmorpholine (15 mmol) as raw materials were added into a 100 ml three-necked flask, 50 ml of 2-methyltetrahydrofuran was added, stirred and dissolved, cooled to -5 - 0 °C, then 5.50 g of H00 (10 mmol) was added. After adding, the mixture was stirred and reacted at 20 - 30 °C for 2 h. TLC was used to confirm the completion of the reaction. The reaction solution was quenched with dilute hydrochloric acid aqueous solution, separated, washed with saturated brine, and dried with Na 2 SO 4 filtered, concentrated to dryness, dissolved in acetonitrile at room temperature, cooled to -20 °C and stirred for crystallization for 3 h, filtered by suction to obtain a solid, and the solid was dried in vacuum to obtain 8.66 g of Y00-2 (n is 8), with a yield of 86.28%; the purity was 99.38%, and all single impurities were <0.2%.

[0172] 11H NMR (DMSO-d6): 12.58 (br s, 1H); 8.06 (d, 1H, J = 7.5 Hz); 7.89 - 7.93 (m, 3H); 7.64 - 7.74 (m, 4H); 7.42 (t, 2H, J = 7.5 Hz); 7.33 (t, 2H, J = 7.5 Hz); 4.21 - 4.29 (m, 3H); 4.02 - 4.08 (m, 1H); 3.90 - 3.94 (m, 1H); 3.86 (s, 2H); 3.51 - 3.54 (m, 4H); 3.41 - 3.42 (m, 2H); 3.18 - 3.22 (m, 2H); 3.08 - 3.12 (m, 2H); 2.08 - 2.17 (m, 6H); 1.88 - 1.92 (m, 1H); 1.69 - 1.77 (m, 2H); 1.60 - 1.63 (m, 1H); 1.41 - 1.48 (m, 6H); 1.39 (s, 18H); 1.22 (s, 26H). LC-MS target [M + H]+ = 1051.6504, measured [M + H]+ = 1051.6576.

[0173] Example 16

[0174] This example discloses a synthesis method of compound Y00-3. Specifically: 7.09 g of C00 (C-22) (10 mmol) and 1.51 g of N-methylmorpholine (15 mmol) as raw materials were put into a 100 ml three-necked flask, 50 ml of 2-methyltetrahydrofuran was added, and the mixture was stirred to dissolve. The temperature was lowered to -5 - 0 °C, and then 5.50 g of H00 (10 mmol) was added. After addition, the mixture was stirred and reacted at 20 - 30 °C for 2 h. TLC was used to monitor the reaction until completion. The reaction solution was quenched with dilute hydrochloric acid aqueous solution, separated by liquid-liquid extraction, washed with saturated brine, and dried over Na 2 SO 4 dried, filtered, concentrated to dryness, dissolved in acetonitrile at room temperature, cooled to -20 °C, stirred for crystallization for 3 h, filtered by suction to obtain a solid, and the solid was dried under vacuum to obtain 9.43 g of Y00-3 (n = 10), with a yield of 85.25%; the purity was 99.38%, and all single impurities were < 0.2%.

[0175] 11H NMR (DMSO-d6): 12.57 (br s, 1H); 8.06 (d, 1H, J = 7.5 Hz); 7.87 - 7.93 (m, 3H); 7.64 - 7.75 (m, 4H); 7.42 (t, 2H, J = 7.5 Hz); 7.34 (t, 2H, J = 7.3 Hz); 4.21 - 4.29 (m, 3H); 4.03 - 4.08 (m, 1H); 3.90 - 3.95 (m, 1H); 3.86 (s, 2H); 3.55 (d, 4H, J = 5 Hz); 3.41 - 3.42 (m, 2H); 3.20 (q, 2H, J = 5.5 Hz); 3.09 - 3.13 (m, 2H); 2.08 - 2.16 (m, 6H); 1.88 - 1.92 (m, 1H); 1.69 - 1.77 (m, 2H); 1.61 - 1.64 (m, 1H); 1.41 - 1.51 (m, 6H); 1.39 (s, 18H); 1.22 (s, 34H); LC-MS target [M+Na]+ = 1129.7028, found [M+Na]+ = 1129.7014.

[0176] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a polypeptide side chain, characterized in that: The method comprises the following steps: D00 or C00 and H00 generate compound X00 or Y00 respectively under the action of condensation agent and acid binding agent; In the formula X00, n is selected from 4 to 20, such as 4 to 10, such as 8, 9 or 10; In the formula Y00, m is selected from 4 to 20, such as 4 to 10, such as 8, 9 or 10.

2. The preparation method according to claim 1, characterized in that: The condensing agent is selected from one or any combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and O-benzotriazole-tetramethyluronium hexafluorophosphate.

3. The preparation method according to claim 1, characterized in that: The acid binding agent is selected from one or any combination of sodium carbonate, sodium acetate, sodium bicarbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine, pyridine, 2,6-lutidine, N-methylmorpholine and N-ethylmorpholine.

4. The preparation method according to claim 1, characterized in that: The molar ratio of the compound D00:compound H00:condensing agent:acid binding agent is 1:0.8-1.2:1-3:1.5-20.

5. The preparation method according to claim 1, characterized in that: The reaction temperature is 0 to 50° C., more preferably 20 to 30° C.; the reaction time is 1 to 8 hours, more preferably 2 to 4 hours.

6. The preparation method according to claim 1, characterized in that: The preparation method of D00 comprises the following steps: compound C00 reacts with H-AEEA-OH under the condition of an acid binding agent, and then forms a carboxylate D00 under the action of an inorganic base; 7. The preparation method according to claim 6, characterized in that: The acid binding agent is selected from one or any combination of sodium carbonate, sodium acetate, sodium bicarbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine, pyridine, 2,6-lutidine, N-methylmorpholine and N-ethylmorpholine.

8. The preparation method according to claim 6, characterized in that: The molar ratio of the compound C00:H-AEEA-OH:acid binding agent is 1:0.9-1.4:1.5-50.

9. The preparation method according to claim 6, characterized in that: The reaction solvent is one or a mixed solvent of any of the following in any proportion: ethyl acetate, tetrahydrofuran, acetonitrile, DMF, 2-methyltetrahydrofuran, acetone, methyl isobutyl ketone, and water.

10. The preparation method according to claim 6, characterized in that: The inorganic base is selected from lithium salts, sodium salts, and potassium salts; further, the lithium salt is selected from one of lithium hydroxide, lithium carbonate, lithium bicarbonate, lithium acetate, and lithium phosphate, or a mixture of any of them in any proportion; the sodium salt is selected from one of sodium hydroxide, sodium carbonate, sodium bicarbonate, sodium acetate, sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium oxalate, and sodium formate, or a mixture of any of them in any proportion; the potassium salt is selected from one of potassium hydroxide, potassium carbonate, potassium bicarbonate, potassium acetate, potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium oxalate, potassium formate, and potassium fluoride, or a mixture of any of them in any proportion; further preferably, it is one of lithium hydroxide, lithium carbonate, lithium acetate, sodium hydroxide, sodium carbonate, sodium acetate, potassium bicarbonate, potassium formate, and potassium fluoride, or a mixture of any of them in any proportion.

11. The method according to claim 6, characterized in that: The preparation method of the compound C00 comprises the following steps: (1) The long-chain alkane diacid mono-tert-butyl ester represented by formula I is reacted with compound R1 to generate compound A00; the compound R1 is selected from 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate, N-hydroxysuccinimide or pentafluorophenol; in A00, R1' is selected from (2) Compound A00 reacts with L-glutamic acid-1-tert-butyl ester to generate compound B00; (3) Compound B00 reacts with compound R2 to generate compound C00; compound R2 is selected from 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate, N-hydroxysuccinimide or pentafluorophenol; in said C00, R2' is selected from 12. The method according to claim 11, characterized in that: In step (1), in the long-chain alkane diacid mono-tert-butyl ester represented by formula I, n is 4-20, preferably 4-10; And / or, in step (1), preferably, the acid binding agent is selected from one or any combination of sodium carbonate, sodium acetate, sodium bicarbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine, pyridine, 2,6-lutidine, N-methylmorpholine, and N-ethylmorpholine; And / or, in step (1), preferably, the condensation reaction comprises using a condensation agent, wherein the condensation agent is selected from any one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate; And / or, in step (1), the molar ratio of the long-chain alkane diacid mono-tert-butyl ester represented by formula I to compound R1, acid binding agent and condensing agent is 1:1-1.5:0-1.5:0-1.5; And / or, in step (1), the reaction temperature is 0 to 50°C, more preferably 20 to 30°C; the reaction time is 1 to 8 hours, more preferably 2 to 4 hours. And / or, in step (2), the reaction further comprises adding an acid binding agent, wherein the acid binding agent is selected from any one of sodium acetate, sodium bicarbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine, pyridine, 2,6-lutidine, N-methylmorpholine or N-ethylmorpholine; And / or, in step (2), the molar ratio of the compound A00 to L-glutamic acid-1-tert-butyl ester and the acid-binding agent is 1:0.9-1.4:1.5-5; And / or, in step (2), the reaction temperature is 0 to 50°C, more preferably 20 to 30°C; the reaction time is 1 to 8 hours, more preferably 2 to 3 hours. And / or, in step (3), preferably, the acid binding agent is selected from one or any combination of sodium carbonate, sodium acetate, sodium bicarbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine, pyridine, 2,6-lutidine, N-methylmorpholine, and N-ethylmorpholine; And / or, in step (3), preferably, condensing the reaction comprises using a condensing agent, wherein the condensing agent is selected from any one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate; And / or, in step (3), the molar ratio of the compound B00 to the compound R2, the acid binding agent, and the condensing agent is 1:1-1.5:0-1.5:0-1.5; And / or, in step (3), the reaction temperature is 0 to 50°C, more preferably 20 to 30°C; the reaction time is 1 to 8 hours, more preferably 3 to 4 hours.

13. The method of claim 1, wherein: The preparation method of H00 comprises the following steps: (1) H-AEEA-OH reacts with Boc anhydride to produce E00; (2) E00 reacts with compound R3 to generate compound F00; the compound R3 is selected from 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate, N-hydroxysuccinimide or pentafluorophenol; R3′ in F00 is selected from (3) F00 reacts with Fmoc-lysine hydrochloride to generate G00; (4) G00 obtains an amine salt compound H00 under acid catalysis; 14. The method according to claim 13, characterized in that: In step (1), the acid binding agent is selected from one of sodium carbonate, sodium acetate, sodium bicarbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine, pyridine, 2,6-lutidine, N-methylmorpholine, N-ethylmorpholine or a mixture of any of them; And / or, in step (1), the reaction solvent is one or a mixed solvent of any of the following in any proportion: ethyl acetate, tetrahydrofuran, acetonitrile, DMF, 2-methyltetrahydrofuran, acetone, chloroform, methyl isobutyl ketone, or water; And / or, in step (1), the molar ratio of H-AEEA-OH:Boc anhydride:acid binding agent is 1:1-3:1.5-20; And / or, in step (1), the reaction temperature is 0 to 50°C, more preferably 20 to 30°C; the reaction time is 2 to 24h, more preferably 8 to 12h; And / or, in step (2), the acid binding agent is selected from one or any combination of sodium carbonate, sodium acetate, sodium bicarbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine, pyridine, 2,6-lutidine, N-methylmorpholine, and N-ethylmorpholine; And / or, in step (2), preferably, condensing the reaction comprises using a condensing agent, wherein the condensing agent is selected from one or any combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate; And / or, in step (2), the reaction solvent is one or a mixed solvent of any of the following in any proportion: ethyl acetate, tetrahydrofuran, acetonitrile, DMF, 2-methyltetrahydrofuran, acetone, methyl isobutyl ketone, or water; And / or, in step (2), the reaction further comprises adding a catalyst, wherein the catalyst is DMAP; And / or, in step (2), the molar ratio of compound E00:compound R3:acid binding agent:condensing agent:catalyst is 1:1-1.5:0-1.5:0-1.5:0-0.5; And / or, in step (2), the reaction temperature is 0 to 50°C, more preferably 20 to 30°C; the reaction time is 1 to 8 hours, more preferably 2 to 4 hours; And / or, in step (3), the acid binding agent is selected from any one of sodium carbonate, sodium acetate, sodium bicarbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine, pyridine, 2,6-lutidine, N-methylmorpholine, and N-ethylmorpholine; And / or, in step (3), the molar ratio of the compound F00:Fmoc lysine hydrochloride:acid binding agent is 1:0.9-1.4:1.5-10; And / or, in step (3), the reaction solvent is one or a mixed solvent of any of the following in any proportion: ethyl acetate, tetrahydrofuran, acetonitrile, DMF, 2-methyltetrahydrofuran, acetone, methyl isobutyl ketone, or water; And / or, in step (3), the reaction temperature is 0 to 50°C, more preferably 20 to 30°C; the reaction time is 1 to 8 hours, more preferably 2 to 4 hours; And / or, in step (4), the acid is one of sulfuric acid, hydrochloric acid, acetic acid, trifluoroacetic acid, methanol solution of HCl, ethanol solution of HCl, isopropanol solution of HCl, ethyl acetate solution of HCl, dioxane solution of HCl, oxalic acid, citric acid, tartaric acid, succinic acid, p-toluenesulfonic acid, or a mixture of any of the above in any proportion, and a mixture of the above with water; more preferably, methanol solution of HCl, ethanol solution of HCl, isopropanol solution of HCl, ethyl acetate solution of HCl, or dioxane solution of HCl; And / or, in step (4), the reaction temperature is 0 to 50°C, more preferably 20 to 30°C; the reaction time is 1 to 8 hours, more preferably 2 to 4 hours; And / or, in step (4), the volume ratio of compound G00:acid is 1:1-5.

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