A method of preparing a south fragment of MK-0616

Abstract

The application provides a preparation method of MK-0616 south fragment, and relates to the technical field of polypeptide synthesis. The preparation method comprises the following steps: condensation reaction of alkyl primary alcohol 17 and protected amino acid 2 to obtain intermediate 18; deprotection reaction of the intermediate 18 to obtain intermediate 19; condensation reaction of the intermediate 19 and compound 4 to obtain intermediate 20; deprotection reaction of the intermediate 20 to obtain intermediate 21; condensation reaction of the intermediate 21 and compound 10 to obtain intermediate 22; deprotection reaction of the intermediate 22 to obtain intermediate 23; condensation reaction of the intermediate 23 and compound 14 to obtain intermediate 24; hydrolysis reaction of the intermediate 24 to obtain a crude product of MK-0616 south fragment; salt crystallization of the crude product of MK-0616 south fragment to obtain intermediate 15; and free acidization of the intermediate 15 to obtain a pure product of MK-0616 south fragment.

Description

Technical Field

[0001] This invention relates to the field of polypeptide synthesis technology, specifically to a method for preparing the MK-0616 fragment, and more particularly to a method for preparing the MK-0616 fragment using alkyl primary alcohol compounds as a liquid-phase synthetic carrier for polypeptides. Background Technology

[0002] MK-0616 (also known as Enlicitide) is a promising oral PCSK9 inhibitor developed by Merck & Co. It is used to treat hypercholesterolemia and other conditions related to PCSK9 activity, such as atherosclerosis and atherosclerotic cardiovascular disease. According to the latest information, all three Phase III clinical trials for hypercholesterolemia have been successful.

[0003] .

[0004] The structure of compound A shown below is that of the southern fragment of MK-0616: .

[0005] Merck & Co. has published two generations of original synthetic routes for the southern fragment of MK-0616 (compound A) (J. Am. Chem. Soc. 2025, 147, 13, 11036–11048). The following reaction formula is its optimized second-generation route:

[0006] Compound 1 was protected with an Ns group on a primary amine under alkaline conditions to give compound 2. Subsequently, thionyl chloride and methanol were used to give methyl ester compound 3. This methyl ester was then amide-condensed with compound 4 (N-tert-butoxycarbonyl-L-alanine), followed by lithium hydroxide hydrolysis of the methyl ester, removal of the Boc group by hydrochloric acid, and salt formation to give compound 5. Fragment 10 was synthesized by protecting the Boc group on compound 6, reacting with DMF via lithium halide exchange to give compound 8, followed by reductive amination with 4-penten-1-amine, and then condensation with succinic anhydride. Compound 5 and fragment 10 underwent amide condensation and Boc removal with TMSBr to give intermediate 11. Intermediate 11 was further condensed with dipeptide 14 (obtained by the condensation of compounds 12 and 13) to give crude intermediate A (MK-0616 South fragment crude). Crude intermediate A was then crystallized with dehydrorosin amine to give intermediate 15. Intermediate 15 releases dehydrorosin amine under lithium hydroxide conditions, and finally acidification completes the synthesis of A (MK-0616 fragment).

[0007] The original research process uses traditional liquid-phase synthesis of the MK-0616 South Fragment. This process is lengthy, has complicated post-processing operations, requires a large amount of solvent (requiring multiple extractions and solvent switching), and some intermediates require drying, resulting in high time costs and thus high costs for the final synthesized product.

[0008] Meanwhile, the solid-phase Fmoc strategy is often used to synthesize peptides, but it also has obvious disadvantages: (1) It requires 2 to 5 times or even higher Fmoc-protected amino acid feed. (2) After coupling an amino acid, a large amount of DMF solvent is required for washing (generally 3 to 6 washes). (3) The reaction is a heterogeneous reaction. (4) Due to the large volume of resin and the large amount of solvent used, the yield per batch is low.

[0009] In addition, the liquid-phase synthesis of benzyl alcohol phenol ether tags is also a common method for synthesizing peptides, which can reduce the amount of Fmoc-protected amino acids required compared to solid-phase synthesis. However, it also has some disadvantages: (1) It requires multiple sedimentation operations, which inevitably results in mechanical loss. (2) It is difficult to avoid the generation of impurities such as ammonolysis and DKP. (3) Removing the tag requires a strong acid, and the protecting group on the peptide chain will also be removed accordingly during the tag removal process.

[0010] In view of this, the present invention is hereby proposed. Summary of the Invention

[0011] The purpose of this invention is to provide a method for preparing the MK-0616 fragment. This method uses alkyl primary alcohol compounds as a liquid-phase synthesis carrier for peptides, overcoming the shortcomings of existing MK-0616 fragment synthesis processes, such as lengthy steps, cumbersome post-processing, large solvent consumption, and high costs.

[0012] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a method for preparing the MK-0616 southern fragment, the method comprising: Alkyl primary alcohol 17 undergoes a condensation reaction with protected amino acid 2 to give intermediate 18; Intermediate 18 undergoes a deprotection reaction to obtain intermediate 19; Intermediate 19 undergoes a condensation reaction with compound 4 to obtain intermediate 20; Intermediate 20 undergoes a deprotection reaction to obtain intermediate 21; Intermediate 21 undergoes a condensation reaction with compound 10 to obtain intermediate 22; Intermediate 22 undergoes a deprotection reaction to obtain intermediate 23; Intermediate 23 undergoes a condensation reaction with compound 14 to obtain intermediate 24; Intermediate 24 was hydrolyzed to obtain crude MK-0616 southern fragment; The crude product of the southern segment of MK-0616 was subjected to salt crystallization to obtain intermediate 15; Intermediate 15 was subjected to free acidification to obtain the pure MK-0616 southern fragment; The reaction formula for the preparation method is shown below: ; In the structural formulas of alkyl primary alcohol 17, intermediate 18, intermediate 19, intermediate 20, intermediate 21, intermediate 22, intermediate 23, and intermediate 24, n is an integer between 6 and 30.

[0013] Furthermore, the condensation reaction of the alkyl primary alcohol 17 with the protected amino acid 2 is carried out in the presence of a condensing agent and a base; wherein the condensing agent includes any one or a combination of at least two of EDCI, DMAP, T4P, HATU, HBTU, PyBOP, TCFH, and NMI; and the base includes any one or a combination of at least two of Py, DIPEA, 2,6-Lut, and NMM.

[0014] Furthermore, the molar ratio of the alkyl primary alcohol 17, the protected amino acid 2, the condensing agent, and the base is 1:(1.5~3):(1.5~4):(2~6).

[0015] Furthermore, the condensation reaction of the alkyl primary alcohol 17 with the protected amino acid 2 is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of dichloromethane, 2-methyltetrahydrofuran, and N,N-dimethylformamide.

[0016] Furthermore, the condensation reaction of the alkyl primary alcohol 17 with the protected amino acid 2 is carried out at a temperature of 0~30℃ and for a time of 2~10 h.

[0017] Furthermore, the deprotection reaction of the intermediate 18 is carried out in the presence of an acid; wherein the acid includes any one or a combination of at least two of HCl, TFA, TfOH, and HTFSI.

[0018] Furthermore, the molar ratio of intermediate 18 to acid is 1:(10~50).

[0019] Furthermore, the deprotection reaction of the intermediate 18 is carried out at a temperature of 0~30℃ and for a time of 1~8h.

[0020] Furthermore, the condensation reaction between intermediate 19 and compound 4 is carried out in the presence of a condensing agent; wherein the condensing agent includes any one or a combination of at least two of EDCI, HOBT, HOAT, T4P, HATU, HBTU, PyBOP, TCFH, and NMI.

[0021] Furthermore, the molar ratio of intermediate 19, compound 4 and condensing agent is 1:(1.1~1.5):(1.2~2).

[0022] Furthermore, the condensation reaction of intermediate 19 with compound 4 is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of dichloromethane, 2-methyltetrahydrofuran, and N,N-dimethylformamide.

[0023] Furthermore, the condensation reaction of intermediate 19 with compound 4 is carried out at a temperature of 0~30℃ and for a time of 1~3 h.

[0024] Furthermore, the deprotection reaction of the intermediate 20 is carried out in the presence of an acid; wherein the acid includes any one or a combination of at least two of HCl, TFA, TfOH, and HTFSI.

[0025] Furthermore, the molar ratio of intermediate 20 to acid is 1:(10~30).

[0026] Furthermore, the deprotection reaction of the intermediate 20 is carried out at a temperature of 10~30℃ and for a time of 1~8 h.

[0027] Furthermore, the condensation reaction of intermediate 21 with compound 10 is carried out in the presence of a condensing agent and a base; wherein the condensing agent includes any one or a combination of at least two of EDCI, HOBT, HOAT, T4P, HATU, HBTU, PyBOP, TCFH, and NMI; and the base includes any one or a combination of at least two of DIPEA, Py, and 2,6-Lut.

[0028] Further, the molar ratio of intermediate 21, compound 10, condensing agent and base is 1:(1.2~1.5):(1.2~2):(2~3).

[0029] Furthermore, the condensation reaction of intermediate 21 with compound 10 is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of dichloromethane, 2-methyltetrahydrofuran, and N,N-dimethylformamide.

[0030] Furthermore, the condensation reaction of intermediate 21 with compound 10 is carried out at a temperature of 0~30℃ and for a time of 2~10 h.

[0031] Furthermore, after the condensation reaction of intermediate 21 and compound 10 is completed, the following post-processing step is also included: adding to the reaction system N,N -Dimethylethylenediamine, which reacts with an excess of the active ester of compound 10.

[0032] Furthermore, in the post-processing step, the... N,N The addition equivalent of dimethylethylenediamine is 0.8~1.5.

[0033] Furthermore, in the post-processing step, with N,N The reaction of dimethylethylenediamine takes place at a temperature of 20-30°C for 2-4 hours.

[0034] Furthermore, the deprotection reaction of the intermediate 22 is carried out in the presence of an acid; wherein the acid includes any one or a combination of at least two of HCl, TFA, TfOH, and HTFSI.

[0035] Furthermore, the molar ratio of intermediate 22 to acid is 1:(10~30).

[0036] Furthermore, the deprotection reaction of the intermediate 22 is carried out at a temperature of 20~30°C and for a time of 1~8 h.

[0037] Furthermore, the condensation reaction between intermediate 23 and compound 14 is carried out in the presence of a condensing agent; wherein the condensing agent includes any one or a combination of at least two of EDCI, HOBT, HOAT, T4P, HATU, HBTU, PyBOP, TCFH, and NMI.

[0038] Furthermore, the molar ratio of intermediate 23, compound 14 and condensing agent is 1:(1.2~1.5):(1.2~2).

[0039] Furthermore, the condensation reaction of intermediate 23 with compound 14 is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of dichloromethane, 2-methyltetrahydrofuran, and N,N-dimethylformamide.

[0040] Furthermore, the condensation reaction of intermediate 23 and compound 14 is carried out at a temperature of 0~30℃ and for a time of 2~10 h.

[0041] Furthermore, after the condensation reaction of intermediate 23 and compound 14 is completed, the following post-processing step is also included: adding to the reaction system N,N -Dimethylethylenediamine, which reacts with an excess of the active ester of compound 14. Furthermore, in the post-processing step, the... N,N The addition equivalent of dimethylethylenediamine is 0.8~1.5.

[0042] Furthermore, in the post-processing step, with N,N The reaction of dimethylethylenediamine takes place at a temperature of 20-30°C for 2-4 hours.

[0043] Furthermore, the hydrolysis reaction is carried out in the presence of an alkali; wherein the alkali includes any one or a combination of at least two of LiOH, NaOH, and KOH.

[0044] Furthermore, the molar ratio of intermediate 24 to base is 1:(6~12).

[0045] Furthermore, the hydrolysis reaction is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of tetrahydrofuran, 2-methyltetrahydrofuran, and 1,4-dioxane.

[0046] Furthermore, the hydrolysis reaction is carried out at a temperature of -10 to 10°C for 4 to 10 hours.

[0047] Furthermore, the salt-forming reagent used in the salt crystallization is dehydrorosinamine; wherein, the molar ratio of the crude MK-0616 fragment to dehydrorosinamine is 1:(1.7~2.3).

[0048] Furthermore, the specific steps of the salt crystallization include: The crude MK-0616 southern fraction, dehydrorosin amine, and solvent were mixed and heated to the crystallization temperature. The crystallization temperature was then maintained for crystallization, followed by cooling and stirring.

[0049] Furthermore, the crystallization temperature is 50~55℃, and the crystallization time is 60~80 h.

[0050] Furthermore, the temperature of the cooling and stirring is 10~15℃, and the cooling and stirring time is 4~10 h.

[0051] Furthermore, the free acidification is carried out in the presence of a base; wherein the base includes any one or a combination of at least two of LiOH, NaOH, and KOH.

[0052] Furthermore, the molar ratio of intermediate 15 to base is 1:(1.5~4).

[0053] Furthermore, the free acidification is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of methanol, tetrahydrofuran, 2-methyltetrahydrofuran, and 1,4-dioxane.

[0054] Furthermore, the free acidification temperature is 10~20℃, and the free acidification time is 10~30 min.

[0055] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a simple and high-purity method for synthesizing the MK-0616 amino acid fragment using a liquid-phase Boc strategy, with an overall yield slightly higher than the original process. The introduction of alkyl alcohols significantly enhances the lipophilicity of the intermediates, while excess amino acid fragments, condensing agents, and byproducts can be removed by washing with water after acid removal from the Boc phase. The organic phase after washing can be directly fed into the next condensation reaction. Therefore, this process uses less solvent, is simple to operate, has high yield and high purity, and is suitable for scale-up. Attached Figure Description

[0056] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0057] Figure 1 The NMR spectrum of compound 21a provided in Example 1 of this invention.

[0058] Figure 2 The NMR spectrum of the MK-0616 southern fragment provided in Embodiment 1 of the present invention.

[0059] Figure 3 The HPLC spectrum of the MK-0616 fragment provided in Example 1 of this invention.

[0060] Figure 4 The mass spectrum of the MK-0616 southern fragment provided in Embodiment 1 of the present invention. Detailed Implementation

[0061] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0062] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0063] In a first aspect, the present invention provides a method for preparing the MK-0616 southern fragment, the method comprising: (1) Alkyl primary alcohol 17 undergoes a condensation reaction with protected amino acid 2 to obtain intermediate 18; (2) Intermediate 18 undergoes a deprotection reaction to obtain intermediate 19; (3) Intermediate 19 undergoes a condensation reaction with compound 4 to obtain intermediate 20; (4) Intermediate 20 undergoes a deprotection reaction to obtain intermediate 21; (5) Intermediate 21 undergoes a condensation reaction with compound 10 to obtain intermediate 22; (6) Intermediate 22 undergoes a deprotection reaction to obtain intermediate 23; (7) Intermediate 23 undergoes a condensation reaction with compound 14 to obtain intermediate 24; (8) Intermediate 24 undergoes hydrolysis to obtain crude MK-0616 South fragment; (9) The crude product of the MK-0616 South Fragment was subjected to salt crystallization to obtain intermediate 15; (10) Intermediate 15 was subjected to free acidification to obtain pure MK-0616 South fragment; The reaction formula for the preparation method is shown below: ; In the structural formulas of alkyl primary alcohol 17, intermediate 18, intermediate 19, intermediate 20, intermediate 21, intermediate 22, intermediate 23, and intermediate 24, n is an integer between 6 and 30, such as 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, etc.

[0064] It should be noted that excess amino acid active esters in each condensation reaction can be hydrolyzed or ammonolyzed with alkaline water (such as 5% sodium carbonate aqueous solution) or N,N-dimethylethylenediamine to prevent the formation of intercalation peptides. The individual amino acid fragments after hydrolysis or ammonolysis can be removed by washing with water after deBoc formation to salt under acidic conditions in the next step. Intermediates 18-24, due to their high lipid solubility, will remain in the organic phase. The entire process is a continuous induction process; only the final product, the MK-0616 fragment, requires recrystallization purification. The intermediates only require simple washing with water and sodium carbonate solution before being directly added to the next reaction.

[0065] As an optional implementation, in step (1), the alkyl primary alcohol 17 includes 1-hexanol (n=6), 1-heptanol (n=7), 1-octanol (n=8), 1-nonanol (n=9), 1-decanol (n=10), 1-undecanol (n=11), 1-dodecanool (n=12), 1-tridecanool (n=13), 1-tetradecanool (n=14), 1-pentadecanol (n=15), 1-hexadecanool (n=16), 1-heptadecanool (n=17), and 1-octadecanool (n=18). 8) Any one of the primary alcohols of 1-nonadecanol (n=19), 1-eicosylol (n=20), 1-mono-ol (n=21), 1-mono-diol (n=22), 1-mono-triol (n=23), 1-mono-tetra-ol (n=24), 1-mono-penta-ol (n=25), 1-mono-hexa-ol (n=26), 1-mono-hepta-ol (n=27), 1-mono-octa-ol (n=28), 1-mono-nonadecanol (n=29), 1-tria-ol (n=30) or their isomers.

[0066] In a preferred embodiment, in step (1), the alkyl primary alcohol 17 is 1-eicosadiol (n=22).

[0067] As an optional implementation, in step (1), the molar ratio of the alkyl primary alcohol 17 to the protected amino acid 2 is 1:(1.5~3), for example, it can be 1:1.5, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.5, 1:2.6, 1:2.8, 1:3, etc.

[0068] As an optional implementation, in step (1), the condensation reaction of the alkyl primary alcohol 17 with the protected amino acid 2 is carried out in the presence of a condensing agent; wherein the condensing agent includes any one or a combination of at least two of the following: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP), 1-butylphosphine anhydride (T4P), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP), N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (TCFH), and N-methylimidazolium (NMI).

[0069] As an optional implementation, in step (1), the condensing agent is a combination of EDCI and DMAP.

[0070] As an optional implementation, in step (1), the condensing agent is a combination of TCFH and NMI.

[0071] In a preferred embodiment, the condensing agent in step (1) is T4P.

[0072] As an optional implementation, in step (1), the molar ratio of the alkyl primary alcohol 17 to the condensing agent is 1:(1.5~4), for example, it can be 1:1.5, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.5, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.5, 1:3.6, 1:3.8, 1:4, etc.

[0073] As an optional implementation, in step (1), the condensation reaction of the alkyl primary alcohol 17 with the protected amino acid 2 is carried out in the presence of a base; wherein the base includes any one or a combination of at least two of pyridine (Py), N,N-diisopropylethylamine (DIPEA), 2,6-dimethylpyridine (2,6-Lut), and N-methylmorpholine (NMM).

[0074] As an optional implementation, in step (1), the molar ratio of the alkyl primary alcohol 17 to the base is 1:(2~6), for example, it can be 1:2, 1:2.2, 1:2.4, 1:2.5, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.5, 1:3.6, 1:3.8, 1:4, 1:4.2, 1:4.4, 1:4.5, 1:4.6, 1:4.8, 1:5, 1:5.2, 1:5.4, 1:5.5, 1:5.6, 1:5.8, 1:6, etc.

[0075] As an optional implementation, in step (1), the molar ratio of the alkyl primary alcohol 17, the protected amino acid 2, the condensing agent and the base is 1:(1.5~3):(1.5~4):(2~6).

[0076] As an optional implementation, in step (1), the condensation reaction of the alkyl primary alcohol 17 with the protected amino acid 2 is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of dichloromethane, 2-methyltetrahydrofuran, and N,N-dimethylformamide.

[0077] As an optional implementation, in step (1), the temperature of the condensation reaction between the alkyl primary alcohol 17 and the protected amino acid 2 is 0~30℃, for example, 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, etc., and the time of the condensation reaction is 2~10 h, for example, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, etc.

[0078] As an optional implementation, step (1) includes a post-treatment step of washing with an aqueous sodium carbonate solution after the condensation reaction is completed.

[0079] As an optional implementation, step (1) specifically includes the following post-processing steps after the condensation reaction is completed: adding an aqueous sodium carbonate solution to the reaction system, stirring, then allowing it to stand and separating the liquids; adding an aqueous hydrochloric acid solution to the separated organic phase, stirring, then allowing it to stand and separating the liquids; adding an aqueous sodium chloride solution to the separated organic phase, stirring, then allowing it to stand and separating the liquids; and obtaining an organic phase containing intermediate 18.

[0080] As an optional implementation, in step (2), the deprotection reaction of the intermediate 18 is carried out in the presence of an acid; wherein the acid includes any one or a combination of at least two of hydrochloric acid (HCl), trifluoroacetic acid (TFA), trifluoromethanesulfonic acid (TfOH), and bis(trifluoromethanesulfonyl)imide (HTFSI).

[0081] As an optional implementation, in step (2), when the acid is HCl, the reagent used for the deprotection reaction is a solution containing HCl, such as a 1,4-dioxane solution of HCl, an ethyl acetate (EA) solution of HCl, etc.

[0082] In a preferred embodiment, the acid in step (2) is TFA.

[0083] As an optional implementation, in step (2), the molar ratio of intermediate 18 to acid is 1:(10~50), for example, it can be 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, etc.

[0084] As an optional implementation, in step (2), the temperature of the deprotection reaction of the intermediate 18 is 0~30℃, for example, it can be 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, etc., and the time of the deprotection reaction is 1~8 h, for example, it can be 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, etc.

[0085] As an optional implementation, step (2) specifically includes the following post-processing steps after the deprotection reaction is completed: adding sodium chloride aqueous solution to the reaction system, stirring, then letting it stand and separating the liquids; adding sodium carbonate aqueous solution to the separated organic phase, stirring, then letting it stand and separating the liquids; to obtain an organic phase containing intermediate 19.

[0086] As an optional implementation, in step (3), the molar ratio of intermediate 19 to compound 4 is 1:(1.1~1.5), for example, it can be 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, etc.

[0087] As an optional implementation, in step (3), the condensation reaction of intermediate 19 with compound 4 is carried out in the presence of a condensing agent; wherein the condensing agent includes any one or a combination of at least two of the following: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), N-hydroxy-7-azabenzotriazole (HOAT), 1-butylphosphine (T4P), O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), 1H-benzotriazole-1-yloxytripyrrolyl hexafluorophosphate (PyBOP), N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (TCFH), and N-methylimidazole (NMI).

[0088] As an optional implementation, in step (3), the condensing agent is a combination of EDCI and HOBT.

[0089] As an optional implementation, in step (3), the condensing agent is a combination of EDCI and HOAT.

[0090] As an optional implementation, in step (3), the molar ratio of the intermediate 19 to the condensing agent is 1:(1.2~2), for example, it can be 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, etc.

[0091] As an optional implementation, in step (3), the molar ratio of intermediate 19, compound 4 and condensing agent is 1:(1.1~1.5):(1.2~2).

[0092] As an optional implementation, in step (3), the condensation reaction of intermediate 19 with compound 4 is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of dichloromethane, 2-methyltetrahydrofuran, and N,N-dimethylformamide.

[0093] As an optional implementation, in step (3), the temperature of the condensation reaction between intermediate 19 and compound 4 is 0~30℃, for example, it can be 0℃, 2℃, 4℃, 6℃, 8℃, 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, etc., and the time of the condensation reaction is 1~3 h, for example, it can be 1 h, 1.5 h, 2 h, 2.5 h, 3 h, etc.

[0094] As an optional implementation, step (3) includes a post-treatment step of washing with an aqueous sodium carbonate solution after the condensation reaction is completed.

[0095] As an optional implementation, step (3) specifically includes the following post-processing steps after the condensation reaction is completed: adding sodium carbonate aqueous solution to the reaction system, stirring, then letting it stand and separating the liquids; adding hydrochloric acid aqueous solution to the separated organic phase, stirring, then letting it stand and separating the liquids; to obtain an organic phase containing intermediate 20.

[0096] As an optional implementation, in step (4), the deprotection reaction of the intermediate 20 is carried out in the presence of an acid; wherein the acid includes any one or a combination of at least two of hydrochloric acid (HCl), trifluoroacetic acid (TFA), trifluoromethanesulfonic acid (TfOH), and bis(trifluoromethanesulfonyl)imide (HTFSI).

[0097] As an optional implementation, in step (4), when the acid is HCl, the reagent used for the deprotection reaction is a solution containing HCl, such as a 1,4-dioxane solution of HCl, an ethyl acetate (EA) solution of HCl, etc.

[0098] In a preferred embodiment, the acid in step (4) is TFA.

[0099] As an optional implementation, in step (4), the molar ratio of intermediate 20 to acid is 1:(10~30), for example, it can be 1:10, 1:15, 1:20, 1:25, 1:30, etc.

[0100] As an optional implementation, in step (4), the temperature of the deprotection reaction of the intermediate 20 is 10~30℃, for example, it can be 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, etc., and the time of the deprotection reaction is 1~8 h, for example, it can be 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, etc.

[0101] As an optional implementation, step (4) specifically includes the following post-processing steps after the deprotection reaction is completed: adding water to the reaction system, stirring, then letting it stand and separating the liquids; adding sodium carbonate aqueous solution to the separated organic phase, stirring, then letting it stand and separating the liquids; adding hydrochloric acid aqueous solution to the separated organic phase, stirring, then letting it stand and separating the liquids; and obtaining an organic phase containing intermediate 21.

[0102] As an optional implementation, in step (5), the molar ratio of intermediate 21 to compound 10 is 1:(1.2~1.5), for example, it can be 1:1.2, 1:1.3, 1:1.4, 1:1.5, etc.

[0103] As an optional implementation, in step (5), the condensation reaction of intermediate 21 with compound 10 is carried out in the presence of a condensing agent; wherein the condensing agent includes any one or a combination of at least two of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), N-hydroxy-7-azabenzotriazole (HOAT), 1-butylphosphine (T4P), O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), 1H-benzotriazole-1-yloxytripyrrolyl hexafluorophosphate (PyBOP), N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (TCFH), and N-methylimidazolium (NMI).

[0104] As an optional implementation, in step (5), the condensing agent is a combination of EDCI and HOBT.

[0105] As an optional implementation, in step (5), the condensing agent is a combination of EDCI and HOAT.

[0106] As an optional implementation, in step (5), the molar ratio of the intermediate 21 to the condensing agent is 1:(1.2~2), for example, it can be 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, etc.

[0107] As an optional implementation, in step (5), the condensation reaction between intermediate 21 and compound 10 is carried out in the presence of a base; wherein the base includes N,N - Any one or a combination of at least two of diisopropylethylamine (DIPEA), pyridine (Py), and 2,6-dimethylpyridine (2,6-Lut).

[0108] As an optional implementation, in step (5), the molar ratio of intermediate 21 to alkali is 1:(2~3), for example, it can be 1:2, 1:2.2, 1:2.4, 1:2.5, 1:2.6, 1:2.8, 1:3, etc.

[0109] As an optional implementation, in step (5), the molar ratio of intermediate 21, compound 10, condensing agent and base is 1:(1.2~1.5):(1.2~2):(2~3).

[0110] As an optional implementation, in step (5), the condensation reaction of intermediate 21 with compound 10 is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of dichloromethane, 2-methyltetrahydrofuran, and N,N-dimethylformamide.

[0111] As an optional implementation, in step (5), the temperature of the condensation reaction between the intermediate 21 and compound 10 is 0~30℃, for example, it can be 0℃, 2℃, 4℃, 6℃, 8℃, 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, etc., and the time of the condensation reaction is 2~10 h, for example, it can be 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, etc.

[0112] As an optional implementation, in step (5), after the condensation reaction of intermediate 21 and compound 10 is completed, the following post-processing step is further included: adding to the reaction system N,N -Dimethylethylenediamine, which reacts with an excess of the active ester of compound 10.

[0113] As an optional implementation, in step (5), the post-processing step, the N,N The addition equivalent of dimethylethylenediamine is 0.8 to 1.5, for example, it can be 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, etc.

[0114] It should be noted that in the post-processing step, the... N,N The addition equivalent of dimethylethylenediamine refers to the amount added based on the molar equivalent of 1 eq of compound 17 used.

[0115] As an optional implementation, in step (5), the post-processing step, and N,N The reaction temperature for dimethylethylenediamine is 20~30℃, for example, 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, etc., and the reaction time is 2~4h, for example, 2h, 2.5h, 3h, 3.5h, 4h, etc.

[0116] As an optional implementation, in step (6), the deprotection reaction of the intermediate 22 is carried out in the presence of an acid; wherein the acid includes any one or a combination of at least two of hydrochloric acid (HCl), trifluoroacetic acid (TFA), trifluoromethanesulfonic acid (TfOH), and bis(trifluoromethanesulfonyl)imide (HTFSI).

[0117] As an optional implementation, in step (6), when the acid is HCl, the reagent used for the deprotection reaction is a solution containing HCl, such as a 1,4-dioxane solution of HCl, an ethyl acetate (EA) solution of HCl, etc.

[0118] In a preferred embodiment, the acid in step (6) is TFA.

[0119] As an optional implementation, in step (6), the molar ratio of intermediate 22 to acid is 1:(10~30), for example, it can be 1:10, 1:15, 1:20, 1:25, 1:30, etc.

[0120] As an optional implementation, in step (6), the temperature of the deprotection reaction of the intermediate 22 is 20~30℃, for example, it can be 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, etc., and the time of the deprotection reaction is 1~8 h, for example, it can be 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, etc.

[0121] As an optional implementation, step (6) specifically includes the following post-processing steps after the deprotection reaction is completed: adding sodium chloride aqueous solution to the reaction system, stirring, then letting it stand and separating the liquids; adding sodium carbonate aqueous solution to the separated organic phase, stirring, then letting it stand and separating the liquids; to obtain an organic phase containing intermediate 23.

[0122] As an optional implementation, in step (7), the molar ratio of intermediate 23 to compound 14 is 1:(1.2~1.5), for example, it can be 1:1.2, 1:1.3, 1:1.4, 1:1.5, etc.

[0123] As an optional implementation, in step (7), the condensation reaction of intermediate 23 with compound 14 is carried out in the presence of a condensing agent; wherein the condensing agent includes any one or a combination of at least two of the following: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), N-hydroxy-7-azabenzotriazole (HOAT), 1-butylphosphine (T4P), O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), 1H-benzotriazole-1-yloxytripyrrolyl hexafluorophosphate (PyBOP), N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (TCFH), and N-methylimidazolium (NMI).

[0124] As an optional implementation, in step (7), the condensing agent is a combination of EDCI and HOBT.

[0125] As an optional implementation, in step (7), the condensing agent is a combination of EDCI and HOAT.

[0126] As an optional implementation, in step (7), the molar ratio of the intermediate 23 to the condensing agent is 1:(1.2~2), for example, it can be 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, etc.

[0127] As an optional implementation, in step (7), the molar ratio of intermediate 23, compound 14 and condensing agent is 1:(1.2~1.5):(1.2~2).

[0128] As an optional implementation, in step (7), the condensation reaction of intermediate 23 with compound 14 is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of dichloromethane, 2-methyltetrahydrofuran, and N,N-dimethylformamide.

[0129] As an optional implementation, in step (7), the temperature of the condensation reaction between the intermediate 23 and the compound 14 is 0~30℃, for example, it can be 0℃, 2℃, 4℃, 6℃, 8℃, 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, etc., and the time of the condensation reaction is 2~10 h, for example, it can be 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, etc.

[0130] As an optional implementation, in step (7), after the condensation reaction of intermediate 23 and compound 14 is completed, the following post-processing step is further included: adding to the reaction system N,N -Dimethylethylenediamine, which reacts with an excess of the active ester of compound 14.

[0131] As an optional implementation, in step (7), the post-processing step, the N,N The amount of dimethylethylenediamine added is 0.5~1.2, for example, it can be 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, etc.

[0132] It should be noted that in the post-processing step, the... N,N The addition equivalent of dimethylethylenediamine refers to the amount added based on the molar equivalent of 1 eq of compound 17 used.

[0133] As an optional implementation, in step (7), the post-processing step, and N,N The reaction temperature for dimethylethylenediamine is 20~30℃, for example, 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, etc., and the reaction time is 2~4h, for example, 2h, 2.5h, 3h, 3.5h, 4h, etc.

[0134] As an optional implementation, in step (7), with N,N After the reaction of -dimethylethylenediamine is completed, the following post-processing steps are also included: adding hydrochloric acid aqueous solution to the reaction system, stirring, then allowing it to stand and separating the liquid; adding sodium carbonate aqueous solution to the separated organic phase, stirring, then allowing it to stand and separating the liquid to obtain an organic phase containing intermediate 24; then concentrating the organic phase containing intermediate 24 to obtain crude intermediate 24.

[0135] As an optional implementation, in step (8), the hydrolysis reaction is carried out in the presence of an alkali; wherein the alkali includes any one or a combination of at least two of lithium hydroxide (LiOH), sodium hydroxide (NaOH), and potassium hydroxide (KOH).

[0136] As an optional implementation, in step (8), the molar ratio of intermediate 24 to alkali is 1:(6~12), for example, it can be 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, etc.

[0137] As an optional implementation, in step (8), the hydrolysis reaction is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of tetrahydrofuran (THF), 2-methyltetrahydrofuran (2-MeTHF), and 1,4-dioxane.

[0138] In a preferred embodiment, in step (8), the hydrolysis reaction is carried out in a solvent; wherein the solvent is THF.

[0139] As an optional implementation, in step (8), the temperature of the hydrolysis reaction is -10~10℃, for example, it can be -10℃, -8℃, -6℃, -4℃, -2℃, 0℃, 2℃, 4℃, 6℃, 8℃, 10℃, etc., and the time of the hydrolysis reaction is 4~10h, for example, it can be 4h, 5h, 6h, 7h, 8h, 9h, 10h, etc.

[0140] As an optional implementation, step (8) includes the following post-processing steps after the hydrolysis reaction is completed: adding methyl tert-butyl ether (MTBE) to the reaction system, stirring, then allowing it to stand, separating the liquid, and removing the organic phase; adding 2-MeTHF and sodium chloride aqueous solution to the aqueous phase after separation, stirring, then allowing it to stand, separating the liquid, and removing the aqueous phase; adjusting the pH of the organic phase to about 4-5 with hydrochloric acid aqueous solution, then washing it once with water and once with sodium chloride aqueous solution to obtain the organic phase (crude product of MK-0616 South fragment).

[0141] As an optional implementation, in step (9), the salt-forming reagent used for salt crystallization is dehydrorosin amine; wherein, the molar ratio of the crude MK-0616 fragment to dehydrorosin amine is 1:(1.7~2.3), for example, it can be 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, etc. As an optional implementation, the specific steps of salt crystallization in step (9) include: The crude MK-0616 southern fraction, dehydrorosin amine, and solvent were mixed and heated to the crystallization temperature. The crystallization temperature was then maintained for crystallization, followed by cooling and stirring.

[0142] As an optional implementation, in step (9), the crystallization temperature is 50~55℃, for example, it can be 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, etc., and the crystallization time is 60~80 h, for example, it can be 60 h, 65 h, 66 h, 70 h, 72 h, 75 h, 78 h, 80 h, etc.

[0143] As an optional implementation, in step (9), the temperature of the cooling and stirring is 10~15℃, for example, it can be 10℃, 11℃, 12℃, 13℃, 14℃, 15℃, etc., and the time of the cooling and stirring is 4~10 h, for example, it can be 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, etc.

[0144] As an optional implementation, step (9) further includes the following post-processing steps after cooling and stirring: filtration, washing the filter cake with acetonitrile, and drying it; subsequently, vacuum drying is performed to obtain intermediate 15 (white solid).

[0145] As an optional implementation, in step (10), the free acidification is carried out in the presence of an alkali; wherein the alkali includes any one or a combination of at least two of lithium hydroxide (LiOH), sodium hydroxide (NaOH), and potassium hydroxide (KOH).

[0146] As an optional implementation, in step (10), the molar ratio of intermediate 15 to alkali is 1:(1.5~4), for example, it can be 1:1.5, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.5, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.5, 1:3.6, 1:3.8, 1:4, etc.

[0147] As an optional implementation, in step (10), the free acidification is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of methanol, tetrahydrofuran (THF), 2-methyltetrahydrofuran (2-MeTHF), and 1,4-dioxane.

[0148] In a preferred embodiment, in step (10), the free acidification is carried out in a solvent; wherein the solvent is methanol.

[0149] As an optional implementation, in step (10), the temperature of free acidification is 10~20℃, for example, it can be 10℃, 12℃, 14℃, 15℃, 16℃, 18℃, 20℃, etc., and the time of free acidification is 10~30 min, for example, it can be 10 min, 15 min, 20 min, 25 min, 30 min, etc.

[0150] As an optional implementation, step (10) further includes the following post-processing steps after the free acidification is completed: adding methyl tert-butyl ether (MTBE) and water, stirring, then allowing to stand, separating, and removing the organic phase; adding methyl tert-butyl ether (MTBE) to the separated aqueous phase, stirring, then allowing to stand, separating, and removing the organic phase; adding 2-methyltetrahydrofuran (2-MeTHF) to the separated aqueous phase, and adjusting the pH of the system to about 4-5 with hydrochloric acid aqueous solution, stirring; then washing once with water, and once with sodium chloride aqueous solution to obtain the organic phase; drying the organic phase with anhydrous sodium sulfate, filtering, rinsing, and concentrating the filtrate to dryness to obtain the pure product of MK-0616 South fragment (white foamy solid).

[0151] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0152] Example 1 This embodiment provides a method for synthesizing the MK-0616 south fragment using 1-eicosadiol as a starting material. The synthetic route of the preparation method is shown below:

[0153] The preparation method specifically includes the following steps: (1) Add 1-cocadiol (100 g) and dichloromethane (1000 mL) to a reaction flask, dissolve at 40 °C, then cool to 25 °C and add 2 (215 g, 1.8 eq), pyridine (121 g, 5.0 eq), and T4P (552 g, 1.8 eq, 50% EA solution) in sequence, and then keep the reaction at 25 °C for 6 h; after confirming that the raw materials have reacted completely, add 5 wt% sodium carbonate aqueous solution (1000 mL) and stir for 1 h, then let stand and separate the liquids; add 1 M hydrochloric acid aqueous solution (1000 mL) to the separated organic phase and stir for 10 min, then let stand and separate the liquids; add saturated sodium chloride aqueous solution (1000 mL) to the separated organic phase and stir for 10 min, then let stand and separate the liquids to obtain the organic phase (crude intermediate 18a).

[0154] (2) Add the organic phase from the previous step into the reaction flask, add TFA (300 mL) at 25°C, and keep the reaction at 25°C for 5 h after the addition is complete; after confirming that the raw materials have reacted completely, stop stirring, add 5 wt% sodium chloride aqueous solution (1000 mL) and stir for 15 min, then let stand and separate the liquids; add 5 wt% sodium carbonate aqueous solution (1000 mL) to the separated organic phase and stir for 0.5 h, then let stand and separate the liquids to obtain the organic phase (crude intermediate 19a).

[0155] (3) Add the organic phase from the previous step to the reaction flask, and add 4 (69.5 g, 1.2 eq), HOBt (53.8 g, 1.3 eq), and EDCI (88.0 g, 1.5 eq) sequentially at 5°C; maintain the reaction at 5°C for 2 h; after confirming that the reaction of the raw materials is complete, add 5 wt% sodium carbonate aqueous solution (1000 mL) and stir for 1 h, then let stand and separate the liquids; add 1 M hydrochloric acid aqueous solution (1000 mL) to the separated organic phase and stir for 10 min, then let stand and separate the liquids to obtain the organic phase (crude intermediate 20a).

[0156] (4) Add the organic phase from the previous step to the reaction flask, add TFA (300 mL) at 25°C, and maintain the reaction at 25°C for 5 h after the addition is complete; after confirming that the reactants have reacted completely, stop stirring, add water (1000 mL) and stir for 15 min, then let stand and separate the liquids; add 5 wt% sodium carbonate aqueous solution (1000 mL) to the separated organic phase and stir for 10 min, then let stand and separate the liquids; add 1 M hydrochloric acid aqueous solution (1000 mL) to the separated organic phase and stir for 10 min, then let stand and separate the liquids to obtain the organic phase (crude intermediate 21a); ​​the NMR spectrum of compound 21a is shown below. Figure 1 As shown.

[0157] (5) The organic phase obtained in the previous step (crude intermediate 21) was added to the reaction flask, and 10 (167 g, 1.3 eq), HOBt (57.9 g, 1.4 eq), EDCI (88.0 g, 1.5 eq), and DIPEA (119 g, 3.0 eq) were added sequentially at 5 °C. The reaction was maintained at 5 °C for 4 h. After confirming that the reaction of the starting material was completed, N,N-dimethylethylenediamine (21.6 g, 0.8 eq) was added at 5 °C, and then the mixture was stirred at 25 °C for 2 h to obtain the organic phase (crude intermediate 22a).

[0158] (6) Then, add TFA (300 mL) to the organic phase of the previous step at 25°C. After the addition is complete, keep the reaction at 25°C for 5 h. After confirming that the raw materials have reacted completely, stop stirring, add 5 wt% sodium chloride aqueous solution (1000 mL) and stir for 15 min. Then let stand and separate the liquids. Add 5 wt% sodium carbonate aqueous solution (1000 mL) to the separated organic phase and stir for 0.5 h. Then let stand and separate the liquids to obtain the organic phase (crude intermediate 23a).

[0159] (7) Add the organic phase from the previous step to the reaction flask, and add 14 (156 g, 1.25 eq), HOBt (57.9 g, 1.4 eq), and EDCI (88.0 g, 1.5 eq) sequentially at 5°C. Maintain the reaction at 5°C for 6 h. After confirming that the reaction of the raw materials is complete, add N,N-dimethylethylenediamine (13.5 g, 0.5 eq) at 5°C, and then stir at 25°C for 2 h. Add 1 M hydrochloric acid aqueous solution (1000 mL) to the reaction system and stir for 10 min. Then let it stand and separate the liquid to obtain the organic phase. Add 5 wt% sodium carbonate aqueous solution (1000 mL) to the separated organic phase and stir for 20 min. Then let it stand and separate the liquid to obtain the organic phase. Then concentrate the organic phase directly under reduced pressure to obtain crude intermediate 24a (HPLC purity 95.1%).

[0160] (8) Add crude intermediate 24 to the reaction flask, add THF (1000 mL) to dissolve it, add an aqueous solution of LiOH (73.3 g, 10.0 eq LiOH and 400 g water) at -5℃, and keep the reaction at -5℃ for 6 h after the addition is complete; after confirming that the raw material reaction is complete, stop stirring, add MTBE (1000 mL) and stir for 20 min, then let stand and separate to remove the organic phase; add 2-MeTHF (1000 mL) and saturated sodium chloride (300 mL) to the aqueous phase after separation and stir for 30 min, then let stand and separate to remove the aqueous phase; adjust the pH of the organic phase to about 4 with 1 N hydrochloric acid aqueous solution, then wash once with water (500 mL) and once with saturated sodium chloride aqueous solution (500 mL) to obtain the organic phase (crude product of MK-0616 South Fragment, referred to as crude intermediate A).

[0161] (9) The organic phase (crude intermediate A) obtained in the previous step was concentrated until no more liquid flowed out. Acetonitrile (2.2 L) was added to dissolve it, followed by the addition of an acetonitrile solution of dehydrorosinamine (175 g, 2.0 eq of dehydrorosinamine and 1.1 L of acetonitrile). The temperature was raised to 55 °C and maintained at 55 °C for crystallization for 70 h. Then the temperature was lowered to 15 °C and stirred for 6 h. The mixture was filtered, the filter cake was washed with 400 mL of acetonitrile, dried under vacuum, and then placed in a vacuum drying oven to dry, yielding intermediate 15 (white solid).

[0162] (10) Dissolve intermediate 15 obtained in the previous step in methanol (600 mL), then add LiOH aqueous solution (1M, 800 mL) at 10°C, and stir at 10°C for 30 min after the addition is complete; then add MTBE (800 mL) and water (900 mL), stir at 10°C for 20 min, let stand, and separate the liquid (remove the organic phase); add MTBE (800 mL) to the aqueous phase, stir at 10°C for 20 min, let stand, separate the liquid, and remove the organic phase; add 2-MeTHF (800 mL) to the aqueous phase, and adjust the pH of the system to about 4 with 1 N hydrochloric acid aqueous solution, and stir at 10°C for 20 min; then wash once with water (500 mL) and once with saturated sodium chloride aqueous solution (500 mL) to obtain the organic phase; add 500 g of anhydrous sodium sulfate to the organic phase and dry for 2 h, filter, rinse, and concentrate the filtrate to dryness to obtain a white foamy solid MK-0616 fragment (210 g). g, based on a total yield of 65.3% for 1-coedadiol and an HPLC purity of 98.1%); the NMR spectrum of the MK-0616 fragment is shown below. Figure 2 As shown, the HPLC spectrum is as follows: Figure 3 As shown, the mass spectrum is as follows Figure 4 As shown.

[0163] Example 2 This embodiment provides a method for synthesizing the MK-0616 south fragment using 1-dodecyl alcohol as a starting material. The synthetic route of the preparation method is shown below:

[0164] The preparation method described in this embodiment is the same as in Example 1, except that 1-dodecyl alcohol was used as the starting material to synthesize the MK-0616 fragment, with a total yield of 55.8% and an HPLC purity of 96.9%.

[0165] Example 3 This embodiment provides a method for synthesizing the MK-0616 fragment using 2-butyl-1-octanol as a starting material. The synthetic route of the preparation method is shown below:

[0166] The preparation method described in this embodiment is the same as in Example 1, except that 2-butyl-1-octanol (an isomer of 1-dodecyl alcohol) was used as the starting material to synthesize the MK-0616 fragment, with an overall yield of 57.5% and an HPLC purity of 97.3%.

[0167] Example 4 This embodiment provides a method for synthesizing the MK-0616 fragment using 1-tetradecyl alcohol as a starting material. The synthetic route of the preparation method is shown below:

[0168] The preparation method described in this embodiment is the same as in Example 1, except that MK-0616 fragment was synthesized using 1-tetradecyl alcohol as the starting material, with a total yield of 58.3% and an HPLC purity of 97.0%.

[0169] Example 5 This embodiment provides a method for synthesizing the MK-0616 fragment using 1-hexadecyl alcohol as a starting material. The synthetic route of the preparation method is shown below:

[0170] The preparation method described in this embodiment is the same as in Example 1, except that 1-hexadecyl alcohol was used as the starting material to synthesize the MK-0616 fragment, with a total yield of 57.6% and an HPLC purity of 96.6%.

[0171] Example 6 This embodiment provides a method for synthesizing the MK-0616 south fragment using 1-octadecyl alcohol as a starting material. The synthetic route of the preparation method is shown below:

[0172] The preparation method described in this embodiment is the same as in Example 1, except that 1-octadecyl alcohol was used as the starting material to synthesize the MK-0616 fragment, with a total yield of 60.2% and an HPLC purity of 97.3%.

[0173] Example 7 This embodiment provides a method for synthesizing the MK-0616 fragment using 1-eicosyl alcohol as a starting material. The synthetic route of the preparation method is shown below:

[0174] The preparation method described in this embodiment is the same as in Example 1, except that 1-eicosyl alcohol was used as the starting material to synthesize the MK-0616 fragment, with a total yield of 61.5% and an HPLC purity of 97.4%.

[0175] Example 8 This embodiment provides a method for synthesizing the MK-0616 fragment using 1-tetracodone as a starting material. The synthetic route of the preparation method is shown below:

[0176] The preparation method described in this embodiment is the same as in Example 1, except that 1-tetracosanol was used as the starting material to synthesize the MK-0616 fragment, with a total yield of 60.5% and an HPLC purity of 97.8%.

[0177] Example 9 This embodiment provides a method for synthesizing the MK-0616 south fragment using 1-trianediol as a starting material. The synthetic route of the preparation method is shown below:

[0178] The preparation method described in this embodiment is the same as in Example 1, except that 1-trianediol was used as the starting material to synthesize the MK-0616 fragment, with a total yield of 63.2% and an HPLC purity of 98.2%.

[0179] Example 10 This embodiment provides a method for preparing MK-0616 fragments using 1-coedadiol as the starting material. In step (1), the condensing agent T4P is replaced with EDCI (1.5 eq) and DMAP (0.2 eq), and the other steps are the same as in the embodiment. The MK-0616 fragment was synthesized with an overall yield of 62.9% and an HPLC purity of 97.3%.

[0180] Example 11 This embodiment provides a method for preparing MK-0616 fragments using 1-dienoyl alcohol as the starting material. In steps (2), (4), and (6), TFA is replaced with an equal volume of 1,4-dioxane hydrochloric acid solution. Other steps are the same as in the embodiment. The MK-0616 fragment was synthesized with an overall yield of 59.1% and an HPLC purity of 98.2%.

[0181] Example 12 This embodiment provides a method for synthesizing the MK-0616 fragment using 1-eicosyl alcohol as the starting material. In step (8), the solvent for the hydrolysis reaction is replaced with an equal volume of 2-MeTHF. The other steps are the same as in the embodiment. The MK-0616 fragment was synthesized with an overall yield of 64.8% and an HPLC purity of 98.2%.

[0182] Example 13 This embodiment provides a method for preparing MK-0616 fragments using 1-eicosyl alcohol as a starting material. In step (10), methanol, the solvent for free acidification, is replaced with an equal volume of THF. Other steps are the same as in the embodiment. The MK-0616 fragment was synthesized with an overall yield of 64.6% and an HPLC purity of 97.8%.

[0183] Example 14 This embodiment provides a method for preparing MK-0616 fragments using 1-eicosyl alcohol as the starting material. In step (5), N,N-dimethylethylenediamine is not added after the condensation reaction is completed. Other steps are the same as in the embodiment. The MK-0616 fragment was synthesized with an overall yield of 60.2% and an HPLC purity of 97.2%.

[0184] Example 15 This embodiment provides a method for preparing MK-0616 fragments using 1-eicosyl alcohol as the starting material. In step (7), N,N-dimethylethylenediamine is not added after the condensation reaction is completed. Other steps are the same as in the embodiment. The MK-0616 fragment was synthesized with an overall yield of 63.5% and an HPLC purity of 97.9%.

[0185] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing the MK-0616 southern fragment, characterized in that, The preparation method includes: Alkyl primary alcohol 17 undergoes a condensation reaction with protected amino acid 2 to give intermediate 18; Intermediate 18 undergoes a deprotection reaction to obtain intermediate 19; Intermediate 19 undergoes a condensation reaction with compound 4 to give intermediate 20; Intermediate 20 undergoes a deprotection reaction to obtain intermediate 21; Intermediate 21 undergoes a condensation reaction with compound 10 to obtain intermediate 22; Intermediate 22 undergoes a deprotection reaction to obtain intermediate 23; Intermediate 23 undergoes a condensation reaction with compound 14 to obtain intermediate 24; Intermediate 24 was hydrolyzed to obtain crude MK-0616 southern fragment; The crude product of the southern segment of MK-0616 was subjected to salt crystallization to obtain intermediate 15; Intermediate 15 was subjected to free acidification to obtain the pure MK-0616 southern fragment; The reaction formula for the preparation method is shown below: ； In the structural formulas of alkyl primary alcohol 17, intermediate 18, intermediate 19, intermediate 20, intermediate 21, intermediate 22, intermediate 23, and intermediate 24, n is an integer between 6 and 30.

2. The method for preparing the MK-0616 southern fragment according to claim 1, characterized in that, The condensation reaction of the alkyl primary alcohol 17 with the protected amino acid 2 is carried out in the presence of a condensing agent and a base; wherein the condensing agent includes any one or a combination of at least two of EDCI, DMAP, T4P, HATU, HBTU, PyBOP, TCFH, and NMI; and the base includes any one or a combination of at least two of Py, DIPEA, 2,6-Lut, and NMM. And / or, the molar ratio of the alkyl primary alcohol 17, the protected amino acid 2, the condensing agent and the base is 1:(1.5~3):(1.5~4):(2~6); And / or, the condensation reaction of the alkyl primary alcohol 17 with the protected amino acid 2 is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of dichloromethane, 2-methyltetrahydrofuran, and N,N-dimethylformamide; And / or, the condensation reaction of the alkyl primary alcohol 17 with the protected amino acid 2 is carried out at a temperature of 0~30℃ and for a time of 2~10 h.

3. The method for preparing the MK-0616 southern fragment according to claim 1, characterized in that, The deprotection reaction of intermediate 18 is carried out in the presence of an acid; wherein the acid includes any one or a combination of at least two of HCl, TFA, TfOH, and HTFSI; And / or, the molar ratio of the intermediate 18 to the acid is 1:(10~50); And / or, the deprotection reaction of the intermediate 18 is carried out at a temperature of 0~30°C for a time of 1~8 h.

4. The method for preparing the MK-0616 southern fragment according to claim 1, characterized in that, The condensation reaction of intermediate 19 with compound 4 is carried out in the presence of a condensing agent; wherein the condensing agent includes any one or a combination of at least two of EDCI, HOBT, HOAT, T4P, HATU, HBTU, PyBOP, TCFH, and NMI. And / or, the molar ratio of intermediate 19, compound 4 and condensing agent is 1:(1.1~1.5):(1.2~2); And / or, the condensation reaction of intermediate 19 with compound 4 is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of dichloromethane, 2-methyltetrahydrofuran, and N,N-dimethylformamide; And / or, the condensation reaction of intermediate 19 with compound 4 is carried out at a temperature of 0~30°C and for a time of 1~3h.

5. The method for preparing the MK-0616 southern fragment according to claim 1, characterized in that, The deprotection reaction of intermediate 20 is carried out in the presence of an acid; wherein the acid includes any one or a combination of at least two of HCl, TFA, TfOH, and HTFSI; And / or, the molar ratio of the intermediate 20 to the acid is 1:(10~30); And / or, the deprotection reaction of the intermediate 20 is carried out at a temperature of 10~30°C for a time of 1~8 h.

6. The method for preparing the MK-0616 southern fragment according to claim 1, characterized in that, The condensation reaction of intermediate 21 with compound 10 is carried out in the presence of a condensing agent and a base; wherein the condensing agent includes any one or a combination of at least two of EDCI, HOBT, HOAT, T4P, HATU, HBTU, PyBOP, TCFH, and NMI; and the base includes any one or a combination of at least two of DIPEA, Py, and 2,6-Lut. And / or, the molar ratio of the intermediate 21, compound 10, condensing agent and base is 1:(1.2~1.5):(1.2~2):(2~3); And / or, the condensation reaction of intermediate 21 with compound 10 is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of dichloromethane, 2-methyltetrahydrofuran, and N,N-dimethylformamide; And / or, the condensation reaction of the intermediate 21 with compound 10 is carried out at a temperature of 0~30°C for a time of 2~10h.

7. The method for preparing the MK-0616 southern fragment according to claim 1 or 6, characterized in that, After the condensation reaction of intermediate 21 and compound 10 is completed, the following post-processing step is further included: adding to the reaction system N,N -Dimethylethylenediamine, which reacts with an excess of the active ester of compound 10; And / or, in the post-processing step, the N,N The addition equivalent of dimethylethylenediamine is 0.8~1.5; And / or, in the post-processing step, with N,N The reaction of dimethylethylenediamine takes place at a temperature of 20-30°C for 2-4 hours.

8. The method for preparing the MK-0616 southern fragment according to claim 1, characterized in that, The deprotection reaction of intermediate 22 is carried out in the presence of an acid; wherein the acid includes any one or a combination of at least two of HCl, TFA, TfOH, and HTFSI; And / or, the molar ratio of the intermediate 22 to the acid is 1:(10~30); And / or, the deprotection reaction of the intermediate 22 is carried out at a temperature of 20~30°C for a time of 1~8 h.

9. The method for preparing the MK-0616 southern fragment according to claim 1, characterized in that, The condensation reaction between intermediate 23 and compound 14 is carried out in the presence of a condensing agent; wherein the condensing agent includes any one or a combination of at least two of EDCI, HOBT, HOAT, T4P, HATU, HBTU, PyBOP, TCFH, and NMI. And / or, the molar ratio of the intermediate 23, compound 14 and condensing agent is 1:(1.2~1.5):(1.2~2); And / or, the condensation reaction of intermediate 23 with compound 14 is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of dichloromethane, 2-methyltetrahydrofuran, and N,N-dimethylformamide; And / or, the condensation reaction of intermediate 23 with compound 14 is carried out at a temperature of 0~30°C and for a time of 2~10h.

10. The method for preparing the MK-0616 southern fragment according to claim 1, characterized in that, After the condensation reaction of intermediate 23 and compound 14 is completed, the following post-processing step is further included: adding to the reaction system N,N -Dimethylethylenediamine, which reacts with an excess of the active ester of compound 14; And / or, in the post-processing step, the N,N The addition equivalent of dimethylethylenediamine is 0.8~1.5; And / or, in the post-processing step, with N,N The reaction of dimethylethylenediamine takes place at a temperature of 20-30°C for 2-4 hours.

11. The method for preparing the MK-0616 southern fragment according to claim 1, characterized in that, The hydrolysis reaction is carried out in the presence of an alkali; wherein the alkali includes any one or a combination of at least two of LiOH, NaOH, and KOH. And / or, the molar ratio of the intermediate 24 to the base is 1:(6~12); And / or, the hydrolysis reaction is carried out in a solvent; wherein the solvent comprises any one or a combination of at least two of tetrahydrofuran, 2-methyltetrahydrofuran, and 1,4-dioxane; And / or, the hydrolysis reaction is carried out at a temperature of -10 to 10°C for 4 to 10 hours.

12. The method for preparing the MK-0616 southern fragment according to claim 1, characterized in that, The salt-forming reagent used for the salt crystallization is dehydrorosinamine; wherein, the molar ratio of the crude MK-0616 fragment to dehydrorosinamine is 1:(1.7~2.3).

13. The method for preparing the MK-0616 southern fragment according to claim 1, characterized in that, The specific steps for salt crystallization include: The crude MK-0616 southern fraction, dehydrorosin amine, and solvent were mixed and heated to the crystallization temperature. The crystallization temperature was then maintained for crystallization, followed by cooling and stirring.

14. The method for preparing the MK-0616 southern fragment according to claim 13, characterized in that, The crystallization temperature is 50~55℃, and the crystallization time is 60~80 h; And / or, the temperature of the cooling and stirring is 10~15℃, and the time of the cooling and stirring is 4~10 h.

15. The method for preparing the MK-0616 southern fragment according to claim 1, characterized in that, The free acidification is carried out in the presence of a base; wherein the base includes any one or a combination of at least two of LiOH, NaOH, and KOH; And / or, the molar ratio of the intermediate 15 to the base is 1:(1.5~4); And / or, the free acidification is carried out in a solvent; wherein the solvent includes any one or a combination of at least two of methanol, tetrahydrofuran, 2-methyltetrahydrofuran, and 1,4-dioxane; And / or, the free acidification temperature is 10~20℃, and the free acidification time is 10~30 min.

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