Efficient preparation method of thioester and epithioester compounds

Abstract

The invention discloses a preparation method of thioester and epithioester compounds, and belongs to the technical field, the preparation method of the thioester compounds comprises the following steps: in an organic solvent, under the participation of alkali, reacting an alpha-carbonyl alkenyl ester compound with a thiol or thiophenol compound at 0-50 DEG C to obtain the thioester compounds; the preparation method of the episulfide compound comprises the following steps: reacting a sulfydryl-containing carboxylic acid compound with an allene ketone compound or an alkyne amide compound in dichloroethane at 0-70 DEG C to obtain a sulfydryl-containing alkenyl ester compound; then, in an organic solvent, the alkenyl ester compound containing sulfydryl reacts at the temperature of 0-50 DEG C under the participation of alkali and / or an additive, and the episulfide compound is obtained; the method for preparing thioester and epithioester provided by the invention has the advantages of low cost, simple operation, mild reaction conditions, wide substrate adaptability, no racemization of products and the like.

Description

Technical Field

[0001] This invention relates to the fields of organic chemistry and chemical biology, and in particular to an efficient method for preparing thioesters and cyclic thioesters. Background Technology

[0002] In recent years, the development of new organic small molecule drugs has become increasingly difficult. Meanwhile, peptide and protein drugs and diagnostic reagents, characterized by high targeting selectivity, easy absorption, and low toxicity, have received widespread attention from the academic and pharmaceutical communities. Peptides have become a research hotspot in the post-genomic era and an important source of new drug development. Peptide drugs developed in recent years, such as telpolide and smegglutide, are typical examples. The natural chemical linking method invented by Kent et al. in 1994 provides a powerful pathway for the synthesis of long-chain peptides and proteins, and thioesters are used as key intermediates in acyl transfer reactions in this method. Thioester compounds not only play a vital role in chemical biology but also hold a crucial position in synthetic chemistry, serving as precursors for the synthesis of acids, esters, aldehydes, ketones, amides, and heterocyclic compounds.

[0003] Macrolides are widely found in natural products, antibiotics, and anticancer drugs with important biological activities. For example, erythromycin and azithromycin are typical examples. Macrocyclic thioesters, as bioisosteres of macrolides, have great potential in drug development. Many compounds with important medicinal value, such as the antibiotics thiolactomycin and nosiheptide, and the anticancer drug thiocoraline, belong to the cyclic thioester class. The latter two are typical examples of cyclic thioester peptides. Furthermore, thioesters, due to their ease of hydrolysis, can also be used as prodrugs in drug development. Cyclic thioester peptides in organisms, also known as autoinducible peptides (AIPs), are key signaling molecules regulating quorum behavior and are an important component of quorum sensing mechanisms. Non-Staphylococcal AIPs, due to their ability to inhibit quorum sensing and virulence gene expression in Staphylococcus aureus, have attracted considerable attention from researchers as potential antitoxins. Due to the very small secretion of aminotransferases (AIPs) and the complex growth and culture processes of some microorganisms, the structural identification and functional study of AIPs in different organisms remain challenging. With the increasing demand for peptide and protein products in the pharmaceutical, scientific research, and other fields, the efficient preparation of peptide thioesters and cyclic peptide thioesters is of great significance.

[0004] In recent years, synthetic chemists have focused on the crucial area of ​​efficient preparation of peptide thioesters and cyclic thioesters. For example, peptide thioesters can also be prepared by activating the relatively inert main-chain amide bonds followed by thiolysis, using activated N-acylurea linkers, main-chain pyroglutamylimide linkers, and immobilized N-mercaptoethoxyglycine (MEGA) linkers. Although these methods have been applied to the synthesis of various proteins, they are limited by the use of non-natural amino acids, cumbersome synthetic steps, the generation of byproducts, or the poor activity and stability of the linkers, hindering large-scale promotion and application. Additionally, peptide thioesters can also be prepared by activating protective peptides in solution, using metal reagents, Lawson's reagent, and light-controlled linkers, but these methods are all limited by racemization during the preparation process.

[0005] Professor Liu Lei's team at Tsinghua University has made a breakthrough in the preparation of peptide thioesters. They first converted peptide hydrazides into acyl azides, then efficiently prepared peptide thioesters through in-situ thiolysis, and applied them to the natural chemical linking method, ultimately achieving the precise synthesis of long-chain peptides and proteins. However, in this method, the preparation of acyl azides needs to be carried out at low temperatures.

[0006] On the other hand, traditional methods for preparing macrocyclic thioesters, such as those using sulfur transfer reagents, external sulfur sources, and ring expansion strategies, typically require complex procedures and stringent reaction conditions. This makes these methods difficult to apply to the preparation of structurally complex cyclic thioester peptide natural products or modified cyclic thioester peptide drugs. However, peptide condensing agents have played a crucial role in the total synthesis of the anticancer natural product Thiocoraline, the peptide antibiotic Nosiheptide, and the first synthesis of an AIP molecule. Furthermore, cyclic thioester peptides can be directly prepared on a solid-phase support via chemoselective thiol-thioester exchange, but the use of hydrofluoric acid limits this method. In contrast, the strategy for synthesizing cyclic thioester peptides on a solid-phase support based on the Dawson peptide thioester preparation method has some applicability. The Olsen team used this method to synthesize a series of AIPs from the canine pathogen Streptococcus schleigh. However, this method still has some limitations; its synthesis process is relatively cumbersome and inefficient.

[0007] In addition to the solid-phase support synthesis strategies developed based on specific methods, cyclic thioester peptides can also be constructed through photocatalysis or enzyme catalysis. The former has certain advantages in constructing non-natural cyclic thioester peptides, while the latter is limited by substrate specificity and enzyme activity conditions, resulting in poor method universality.

[0008] In addition, the applicant's patent CN112430199A, filed in 2019, discloses a method for preparing thioester compounds mediated by acetylacetamide. However, the intermediate formed by the reaction of acetylacetamide in this method has low activity, and the subsequent thiolysis reaction is slow.

[0009] Peptide thioesters, as key intermediates in natural chemical linkages (NCLs), play an important role in related fields. Cyclic peptide thioesters, on the other hand, not only require further investigation into their existence and mechanisms of action in different organisms, but also hold immense potential in drug development. However, the preparation of both peptide thioesters and cyclic thioesters faces limitations to varying degrees, including but not limited to cumbersome preparation processes, demanding conditions, slow rates, racemization during the reaction, low yields, and dimerization during the synthesis of cyclic thioesters.

[0010] Therefore, the synthesis of thioesters and cyclic thioesters of peptides is a scientific problem that urgently needs to be solved in the field of peptide synthesis. Summary of the Invention

[0011] One of the objectives of this invention is to provide a method for preparing thioester compounds to solve the above-mentioned problems.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a method for preparing thioester compounds, comprising the following steps:

[0013] In an organic solvent, with the presence of a base, α-carbonyl alkenyl ester compound 3 reacts with thiols or thiophenols 4 at 0–50 °C to yield thioester compound 5, as shown in the following reaction formula:

[0014] ,in:

[0015] R 1 Selected from C1~C 22 Alkyl, C4~C 10The alkyl group, substituted alkyl group, heterocyclic alkyl group, alkenyl group, alkynyl group, protected α-aminoalkyl group, protected β-aminoalkyl group, protected γ-aminoalkyl group, and protected polypeptide chain alkyl group are preferred; preferably methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, adamantyl, vinyl, 1-propynyl, 2-propynyl, 1-butenyl, 2-butenyl, 3-butenyl, styryl, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, phenylethynyl, benzene One of the following: alkyl, naphthyl, anthraceneyl, phenanthryl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-fluorophenyl, 3-chlorophenyl, 3-bromophenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, pyridyl, pyrroleyl, indolyl, indolylmethyl, indazole, furanyl, benzofuranyl, thienyl, benzothienyl, quinolinyl, styrylyl, phenylethynyl, benzyl, 11-hydroxyundecyl, pentadecyl, protected α-aminoalkyl, protected β-aminoalkyl, protected γ-aminoalkyl, protected polypeptide chain alkyl;

[0016] R 2 Selected from C1~C 10 One of alkyl, aryl, substituted aryl, heterocyclic aryl, and substituted heterocyclic aryl; preferably one of phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-methylphenyl, 4-methoxyphenyl, 2-methylphenyl, 2-methoxyphenyl, 3,5-dimethoxyphenyl, 3-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3,5-dinitrophenyl, pentafluorophenyl, 4-trifluoromethylphenyl, 3,5-dichlorophenyl, 1-naphthyl, 2-naphthyl, furanyl, and thiopheneyl;

[0017] R 3 It is selected from one of hydrogen, alkyl, halogen, acyl, cyano, and alkoxycarbonyl; preferably one of H, methyl, formyl, acetyl, propionyl, cyano, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl.

[0018] R 4 It is selected from one of hydrogen, alkyl, halogen, acyl, cyano, and alkoxycarbonyl; or one of H, methyl, formyl, acetyl, propionyl, cyano, methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, and benzyloxycarbonyl.

[0019] R 5 It is selected from one of hydrogen, alkyl, halogen, acyl, cyano, and alkoxycarbonyl; preferably one of H, methyl, formyl, acetyl, propionyl, cyano, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl.

[0020] R 6 Selected from C1-C8 alkyl groups, C3-C 10Cycloalkyl, aryl, substituted aryl, cysteine ​​residue, protected cysteine ​​residue, or polypeptide containing cysteine ​​residue; preferably one of ethyl, n-hexyl, cyclohexyl, phenyl, naphthyl, benzyl, p-tolyl, p-methoxyphenyl, p-chlorophenyl, p-bromophenyl, 2-pyridyl, 2-thiophenyl, 2-furanyl, cysteine ​​residue, protected cysteine ​​residue, or polypeptide containing cysteine ​​residue.

[0021] As a preferred technical solution, compound 3 is prepared by reacting carboxylic acid compound 1 and allenone compound 2 in one or a mixture of two solvents selected from dichloroethane, dichloroethane, acetonitrile, tetrahydrofuran, and 2-methyltetrahydrofuran.

[0022] As a preferred technical solution, compound 2 is selected from one of the following compounds:

[0023]

[0024] In a preferred embodiment of the present invention, allenone compound 2 is an aryl allenone, more preferably an aryl allenone without substitution on the aryl group, and the allenone compound 2 recommended to be is 1-phenylbutane-2,3-diene-1-one.

[0025] It should be noted that in the preparation of the following cyclic thioester compounds, the allenone compound 2 used is also selected from the above compounds and is preferably 1-phenylbutane-2,3-diene-1-one.

[0026] As a preferred technical solution, the alkali is selected from one or more of N,N-diisopropylethylamine (DIEA), triethylamine (NEt3), sodium carbonate, potassium carbonate, 4-dimethylaminopyridine (DMAP), cesium carbonate, sodium hydroxide, potassium hydroxide, imidazole, N-methylimidazolium, and pyridine; preferably N,N-diisopropylethylamine (DIEA); the amount of alkali used is 0 to 1 equivalent.

[0027] As a preferred technical solution, the organic solvent is selected from one or more of dichloromethane (DCM), dichloroethane (DCE), trichloroethane, dimethyl sulfoxide (DMSO), methanol, acetonitrile, N,N-dimethylformamide (DMF), tetrahydrofuran, n-pentane, cyclohexane, diethyl ether, petroleum ether, and ethyl acetate; preferably dimethyl sulfoxide.

[0028] Alternatively, the two-step reaction can be carried out in a "one-pot" manner. That is, after the reaction of carboxylic acid compound 1 and allenone compound 2 in dichloroethane (DCE), the active intermediate (α-carbonyl alkenyl ester compound 3) generated does not need to be separated. After removing the reaction solvent dichloroethane from the first step, thiol or thiophenol compound 4 is directly added to carry out the next step of the reaction, and the synthesis of allenone-mediated thioester compound 5 can be achieved at room temperature.

[0029] The second objective of this invention is to provide a method for preparing cyclic thioester compounds, the technical solution of which includes the following steps:

[0030] A thiol-containing carboxylic acid compound 7 and an allenone compound 2 or an acetylide compound 9 are reacted in one or a mixture of two solvents selected from dichloroethane, dichloroethane, acetonitrile, tetrahydrofuran, and 2-methyltetrahydrofuran at 0–70 °C to prepare a thiol-containing α-carbonyl alkenyl ester compound 8 or 10; subsequently, in an organic solvent, the thiol-containing α-carbonyl alkenyl ester compound 8 or 10 is reacted at 0–50 °C with the participation of a base and / or additives to obtain a cyclic thioester compound 11, as shown in the following reaction formula:

[0031] ,in,

[0032] PG 1 Selected from H, S t One of Bu, Trt, and Acm;

[0033] PG 2 Selected from one of H, Fmoc, Boc, and Cbz;

[0034] R 2 Selected from C1~C 10 One of alkyl, aryl, substituted aryl, heterocyclic aryl, and substituted heterocyclic aryl; preferably one of phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-methylphenyl, 4-methoxyphenyl, 2-methylphenyl, 2-methoxyphenyl, 3,5-dimethoxyphenyl, 3-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3,5-dinitrophenyl, pentafluorophenyl, 4-trifluoromethylphenyl, 3,5-dichlorophenyl, 1-naphthyl, 2-naphthyl, furanyl, and thiopheneyl;

[0035] R 3 It is selected from one of hydrogen, alkyl, halogen, acyl, cyano, and alkoxycarbonyl; preferably one of H, methyl, formyl, acetyl, propionyl, cyano, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl.

[0036] R 4 It is selected from one of hydrogen, alkyl, halogen, acyl, cyano, and alkoxycarbonyl; preferably one of H, methyl, formyl, acetyl, propionyl, cyano, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl.

[0037] R 5It is selected from one of hydrogen, alkyl, halogen, acyl, cyano, and alkoxycarbonyl; preferably one of H, methyl, formyl, acetyl, propionyl, cyano, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl.

[0038] R 6 Selected from C1~C 10 Alkyl, aryl, or substituted aromatic ring groups; preferably methyl, ethyl, phenyl, heterocyclic aryl, or halocyclic aryl groups;

[0039] R 7 Selected from hydrogen, aryl, C1-C8 alkyl, C1-C8 alkynyl, and C1-C8 alkenyl; preferably hydrogen, phenyl, methyl, propyl, isobutyl, ethynyl, and vinyl;

[0040] As an electron-withdrawing group, EWG is selected from C1-C5 alkylsulfonyl, C1-C5 alkylyl, and C6-C5 alkylsulfonyl groups. 10 arylsulfonyl group, C6~C 10 The aromatic acyl, nitrile, or nitro group; one of methanesulfonyl, ethanesulfonyl, benzenesulfonyl, substituted benzenesulfonyl, nitrile, or nitro group; preferably one or two of the following compounds: Further preferred options are N-ethynyl-N-methyl-p-toluenesulfonamide or N-ethynyl-N-methylmethanesulfonamide;

[0041] The heteroatom of the heterocyclic group is O, N or S, and the number of heteroatoms is 1 or 2; the substituent of the substituted aryl or substituted aromatic ring group is selected from C1 to C8 alkyl, C1 to C8 alkoxy, halogen, phenyl, benzyl, benzyloxy, cyano, and the number of substituents is an integer from 1 to 3.

[0042] As a preferred technical solution, the additive is selected from triphenylphosphine (PPh3), tributylphosphine (PBu3), tricarboxyethylphosphine (TCEP), tri(2-carbonylethyl)phosphine hydrochloride, tri(2-cyanoethyl)phosphine, dithiothreitol (DTT), 2-mercaptoethanol, and 3-(diphenylphosphine)propionic acid.

[0043] As a further preferred technical solution, when using allenone compounds, the additive is tricarboxyethylphosphine (TCEP); when using acetylamides, additives are basically not required. If some reactions are slightly slow due to steric hindrance and solubility issues, a small amount of tributylphosphine (PBu3) can be added to prevent the formation of disulfide bonds.

[0044] As a preferred technical solution, the alkali is selected from N,N-diisopropylethylamine (DIEA), triethylamine (NEt3), sodium carbonate, potassium carbonate, 4-dimethylaminopyridine (DMAP), cesium carbonate, sodium hydroxide, potassium hydroxide, imidazole, and pyridine, preferably N,N-diisopropylethylamine (DIEA).

[0045] As a preferred technical solution, the organic solvent is selected from one or more of dichloromethane (DCM), trichloroethane, dimethyl sulfoxide (DMSO), methanol, acetonitrile, N,N-dimethylformamide (DMF), tetrahydrofuran, n-pentane, diethyl ether, and petroleum ether; when allenone compounds are used, acetonitrile is preferred as the organic solvent, and when acetylenide compounds are used, dimethyl sulfoxide (DMSO) is preferred as the organic solvent.

[0046] Compared with existing technologies, the method of the present invention does not involve the use of non-natural amino acids, the reaction conditions are simple and mild, the synthesis steps are simple, the intermediates formed by the reaction have high activity and good stability, and no racemization occurs during the reaction process, which can be promoted and used on a large scale.

[0047] Compared to CN112430199A, the intermediate formed by the reaction of this invention, namely the alkenyl ester formed by carboxylic acid and allenone, is not only easier to purify, but also has higher activity and faster subsequent thiolysis reaction.

[0048] Compared with existing technologies, the advantages of this invention are as follows: This invention provides a method for preparing thioester compounds from carboxylic acids and thiols or thiophenols as raw materials under the catalysis of a base and mediated by allenone condensing agents; it also allows for a "one-pot" reaction, where the intermediate generated after the reaction of carboxylic acids and allenones does not need to be separated. After removing the solvent from the first step under vacuum, thiols or thiophenols and the solvent required for the second step are directly added to carry out the next reaction, thus achieving the synthesis of allenone-mediated thioester compounds. Furthermore, this invention also provides a method for preparing cyclic thioesters after reacting a thiol-containing carboxylic acid with allenones or acetylenides to prepare an alkenyl ester compound, under the action of a base and additives. Similarly, the preparation of cyclic thioesters can also be achieved using a two-step one-pot method, where after the reaction of the thiol-containing carboxylic acid with allenones or acetylenides is completed, separation is not required. After removing the solvent from the first step under vacuum, solvent, base, and additives are directly added to achieve the preparation of cyclic thioesters. The synthesis method provided by this invention has mild reaction conditions, simple operation, and wide substrate adaptability. For carboxylic acid compounds with a chiral carbon atom at the α-position of the carboxyl group, no racemization occurs during the formation of active intermediates or thioesters or cyclic thioesters, and it has broad application prospects in the development of peptide and cyclic peptide drugs. Attached Figure Description

[0049] Figure 1 The HPLC spectrum of a racemic mixture (L:D=1:1) of compound 9a is shown.

[0050] Figure 2 The HPLC spectrum of compound 9a is shown below.

[0051] Figure 3The HPLC spectrum of a racemic mixture (L:D=1:1) of compound 11a is shown.

[0052] Figure 4 The HPLC spectrum of compound 11a is shown below.

[0053] Figure 5 From top to bottom, the HPLC spectra are as follows: raw material polypeptide acid, raw material polypeptide acid reacting with MYMsA to prepare activated ester, preparation of cyclic thioester 19a in DMSO, purified cyclic thioester 19a, and a pair of non-corresponding isomers of cyclic thioester 19a, 19a'. Detailed Implementation

[0054] To explain the technical content, objectives, and effects of the present invention in detail, the following specific embodiments are provided to further illustrate the content of the present invention. However, the content of the present invention is far more than the following examples.

[0055] Examples 1-8 below describe the preparation of α-carbonyl alkenyl ester compounds.

[0056] Example 1: Synthesis of compound 1a

[0057]

[0058] Boc-Ala-OH (0.2 mmol), 1-phenylbutane-2,3-dien-1-one (0.24 mmol), and dichloroethane (DCE, 1 mL) were added to a clean 4 mL reaction flask. The mixture was stirred at room temperature and monitored by TLC. After 8 hours, the reaction was completed. The solvent was concentrated and purified by column chromatography to obtain the target product 1a, a light red oil with a yield of 95%.

[0059] 1 HNMR(400MHz, CDCl3)δ7.90(d,J=7.1Hz,2H),7.54(t,J=7.4Hz,1H),7.44(t,J=7.6Hz,2H), 6.80(s,1H),5.09(d,J=7.6Hz,1H),4.42(p,J=7.5Hz,1H),2.39(s,3H),1.51-1.42(m,12H); 13 CNMR (100MHz, CDCl3) δ190.1,171.0,163.3,155.1,138.4,132.9,128.5,128.1,113.7,80.2,49.5,28.2,18.6,18.0.

[0060] Example 2: Synthesis of compound 2a

[0061]

[0062] Add Fmoc-Ser to a clean 4mL reaction flask. t Bu)-OH (0.2 mmol), 1-phenylbutane-2,3-dien-1-one (0.24 mmol), and dichloroethane (DCE, 1 mL) were stirred at room temperature and monitored by TLC. The reaction was completed in 6 hours. After the reaction, the solvent was concentrated and purified by column chromatography to obtain the target product 2a, a light red solid with a yield of 95%.

[0063] 1 HNMR(400MHz, CDCl3)δ7.92(d,J=7.6Hz,2H),7.78(d,J=7.5Hz,2H),7.64(t,J=6.4Hz ,2H),7.55(t,J=7.3Hz,1H),7.49-7.38(m,4H),7.33(t,J=7.4Hz,2H),6.82(s,1H),5 .75(d,J=8.2Hz,1H),4.69(d,J=8.8Hz,1H),4.54-4.37(m,2H),4.28(t,J=7.1Hz,1H) ,3.98(dd,J=8.9,2.6Hz,1H),3.69(dd,J=8.9,2.6Hz,1H),2.44(s,3H),1.23(s,9H); 13 CNMR (100MHz, CDCl3) δ190.0,168.3,163.4,156.0,143.6,141.2,138.4,132.8,128.5 ,128.1,127.7,127.0,125.0,119.9,113.8,73.6,67.2,62.2,54.8,47.1,27.3,18.7.

[0064] Example 3: Synthesis of compound 3a

[0065]

[0066] Add Fmoc-Leu-OH (0.2 mmol), 1-phenylbutane-2,3-dien-1-one (0.24 mmol), and dichloroethane (DCE, 1 mL) to a clean 4 mL reaction flask. Stir at room temperature and monitor the reaction using TLC. After 5 hours, the reaction is complete. After the reaction is complete, concentrate the solvent and purify the product 3a by column chromatography to obtain a pale red solid with a yield of 94%.

[0067] 1HNMR(400MHz, CDCl3)MHz, CDCl3) δ7.92(d,J=7.7Hz,2H),7.77(d,J=7.5Hz,2H),7.65-7.57(m,2H),7.60-7.50(m,1H),7.49-7.36(m,4H),7.31(t, J=7.4Hz,2H),6.81(s,1H),5.29(d,J=8.6Hz,1H),4.59-4.43(m,3H),4.2 5(t,J=6.9Hz,1H),2.42(s,3H),1.82-1.62(m,3H),1.02(d,J=5.7Hz,6H); 13 CNMR (100MHz, CDCl3) δ190.1,170.7,163.2,156.0,143.7,143.6,141.3,138.4,132.9,128. 5,128.1,127.7,127.0,124.9,120.0,113.8,67.0,52.7,47.1,41.2,24.8,22.9,21.6,18.7.

[0068] Example 4: Synthesis of compound 4a

[0069]

[0070] In a clean 4 mL reaction flask, add Boc-Ala-Leu-OH (0.2 mmol), 1-phenylbutane-2,3-dien-1-one (0.24 mmol), and dichloroethane (DCE, 1 mL). Stir at room temperature and monitor the reaction using TLC. After 18 hours, the reaction is complete. After the reaction is complete, concentrate the solvent and purify the product 4a by column chromatography to obtain a white solid with a yield of 65%.

[0071] 1 HNMR(400MHz, CDCl3)δ7.90(d,J=6.9Hz,2H),7.73(d,J=8.3Hz,1H),7.58-7.49(m,1H),7.49-7.40(m,2H),7.29(d,J=8.0Hz,1H),6.81-6.76(m,1H ),5.13-5.05(m,1H),4.72-4.61(m,1H),4.28-4.17(m,1H),2.38(s,3H), 1.77-1.69(m,2H),1.43(s,9H),1.37(d,J=7.0Hz,3H),1.00-0.92(m,6H); 13CNMR(100MHz,CDCl3)δ=190.2,170.2,163.3,138.4,132.9,129.6,128.5,128 .1,127.2,113.8,80.2,51.0,49.8,40.8,28.2,24.8,22.9,21.6,21.4,18.7.

[0072] Example 5: Synthesis of compound 5a

[0073]

[0074] 1-Methyl-3-indazolecarboxylic acid (0.2 mmol), 1-phenylbutane-2,3-dien-1-one (0.24 mmol), and dichloroethane (DCE, 1 mL) were added to a clean 4 mL reaction flask. The mixture was stirred at room temperature and monitored by TLC. After 24 hours, the reaction was completed. The solvent was concentrated and purified by column chromatography to obtain the target product 5a, a pale yellow oil with a yield of 84%.

[0075] 1 HNMR(400MHz, CDCl3)δ8.23(d,J=8.2Hz,1H),7.96(d,J=7.2Hz,2H),7.55(t,J=7.2Hz,1H),7.52 -7.49(m,2H),7.46(t,J=7.6Hz,2H),7.40-7.35(m,1H),7.03(s,1H),4.22(s,3H),2.61(s,3H); 13 CNMR (100MHz, CDCl3) δ190.3,163.8,159.8,141.1,138.7,133.4,132.8,128.6,128.2,127.2,124.0,123.7,121.9,114.2,109.7,36.6,19.2.

[0076] Example 6: Synthesis of compound 6a

[0077]

[0078] In a clean 4 mL reaction flask, benzothiophene-2-carboxylic acid (0.2 mmol), 1-phenylbutane-2,3-dien-1-one (0.24 mmol), and dichloroethane (DCE, 1 mL) were added. The mixture was stirred at room temperature and monitored by TLC. After 13 hours, the reaction was completed. The solvent was concentrated and purified by column chromatography to obtain the target product 6a, a pale red oil with a yield of 81%.

[0079] 1HNMR(400MHz, CDCl3)δ8.19(s,1H),8.00-7.87(m,4H),7.61-7.40(m,5H),7.01(s,1H),2.55(s,3H); 13 CNMR (100MHz, CDCl3) δ190.2,163.4,160.1,142.7,138.6,138.5,132.9,132.2,132.2,128.6,128.2,127.5,125.8,125.2,122.8,113.9,19.0.

[0080] Example 7: Synthesis of compound 7a

[0081]

[0082] A magnetic stir bar was placed in a 5 mL reaction flask, and the long-chain polypeptide acid Fmoc-Phe-Thr(OtBu)-Ser(O) was added. t Bu)-Asp(OtBu)-Val-Ser(O t 0.05 mmol of Bu-OH, 0.25 mmol of 1-phenylbutane-2,3-dien-1-one, and 2 mL of reaction solvent DCE were stirred at 50 °C and monitored by HPLC. After the reaction was completed, a large amount of petroleum ether was added to the reaction system, and then centrifuged to achieve solid-liquid separation. Petroleum ether was added again for washing and centrifugation was repeated 3 times. The solid obtained after centrifugation was completely removed from the solvent under vacuum pump, which is compound 7a, a light red solid with a yield of 99%.

[0083] 1HNMR(400MHz,DMSO-d6)δ8.47(d,J=7.0Hz,1H),8.15(d,J=8.1Hz,1H),7.88 (dd,J=15.2,7.7Hz,5H),7.80-7.57(m,6H),7.53(t,J=7.9Hz,2H),7.39(t,J =7.6Hz,2H),7.35-7.22(m,5H),7.17(t,J=7.4Hz,1H),6.88(s,1H),4.68(t, J=6.4Hz,1H),4.55(t,J=5.5Hz,1H),4.43-4.27(m,4H),4.15(dd,J=12.7,8. 4Hz,3H),4.00-3.94(m,1H),3.75(dd,J=9.4,5.4Hz,1H),3.59(dd,J=9.4,4. 7Hz,1H),3.56-3.45(m,2H),3.08(d,J=13.7Hz,1H),2.82(t,J=11.7Hz,1H), 2.71-2.61(m,1H),2.47-2.39(m,1H),2.30(s,3H),2.05-1.93(m,1H),1.33( s,9H),1.15(s,18H),1.09(s,9H),1.01(d,J=6.6Hz,3H),0.92-0.80(m,6H). 13 CNMR(100MHz,DMSO-d6)δ189.4,171.7,171.1,169.8,169.3,169.2,169.1,168.2,163.6,155.9, 143.7,140.7,138.2,137.9,133.3,129.3,128.9,128.0,127.9,127.6,127.1,126.3,125.3,120. 1,113.5,80.2,74.1,73.2,73.1,66.7,65.8,61.9,61.2,57.5,57.1,56.1,53.2,53.2,49.5,46.5 ,37.5,37.2,30.9,28.0,27.6,27.1,27.1,19.1,18.5,18.5,17.8;HRMS(ESI-TOF)m / z:calcdforC 69 H 92 N6NaO 15 + [M+Na] + :1267.6513,found1267.6505.

[0084] Example 8: Synthesis of compound 8a

[0085]

[0086] Place a magnetic stir bar in a 5 mL reaction flask, and add the long-chain polypeptide acid Fmoc-Cys(SS) required for the reaction. t 0.04 mmol of Bu-Leu-Ala-Phe-Leu-OH, 0.2 mmol of 1-phenylbutane-2,3-dien-1-one, and 2 mL of LCE were used as reaction solvents. The reaction was stirred at 50 °C, and the reaction progress was monitored by HPLC. After the reaction was completed, a large amount of petroleum ether was added to the reaction system, and then the mixture was centrifuged to achieve solid-liquid separation. The mixture was washed with petroleum ether again and centrifuged again. This process was repeated 3 times. After centrifugation, the solid obtained was completely removed from the solvent under vacuum pump to obtain compound 8a, a light red solid with a yield of 98%.

[0087] 1 HNMR(400MHz,DMSO-d6)δ8.52(d,J=7.1Hz,1H),8.05(d,J=8.0Hz,1H),7.98-7.92(m,4H),7.89(d,J=7.6Hz,2H),7.78(d,J=8.4Hz,1H),7.7 2(d,J=7.5Hz,2H),7.65(t,J=7.4Hz,1H),7.54(t,J=7.6Hz,2H),7.41(t,J=7.5Hz,2H),7.32(t,J=7.5Hz,2H),7.27-7.19(m,4H),7.18-7.11 (m,1H),6.92(s,1H),4.61-4.48(m,1H),4.42-4.15(m,7H),3.12-3.01(m,2H),2.98-2.89(m,1H),2.87-2.78(m,1H),2.27(s,3H),1.75-1. 51(m,4H),1.50-1.34(m,2H),1.27(s,9H),1.13(d,J=7.1Hz,3H),0.93(d,J=5.0Hz,3H),0.87(d,J=4.9Hz,3H),0.81(dd,J=9.5,6.5Hz,6H); 13CNMR(100MHz,DMSO-d6)δ189.5,171.9,171.5,171.3,170.2,170.0,163.7,156 .0,143.8,140.7,137.9,137.5,133.3,129.2,128.9,128.1,128.0,127.7,127 .1,126.3,125.3,120.2,113.6,65.9,54.3,53.4,51.1,50.8,48.2,47.7,46.6,42.5,40.5,38.9,37.4,29.6,24.2,24.1,23.1,22.9,21.5,21.3,18.5,18.0.

[0088] Examples 9-18 below describe the preparation of thioesters or cyclic thioesters from α-carbonyl alkenyl esters and thiols or thiophenols under the catalysis of an alkaline base.

[0089] Example 9: Synthesis of compound 9a

[0090]

[0091] In a clean 4 mL reaction flask, add α-carbonyl alkenyl ester compound (0.2 mmol), benzyl mercaptan (0.24 mmol), dimethyl sulfoxide (DMSO, 1 mL), and DIEA (0.02 mmol). Stir at room temperature and monitor using TLC. The reaction is complete in 0.5 hours. After the reaction is complete, the target product 9a is obtained by column chromatography as a white solid with a yield of 98% and >99%ee.

[0092] 1 HNMR (400MHz, CDCl3) δ7.33-7.19(m,5H),4.99(d,1H),4.48-4.35(m,1H),4.17-3.99(m,2H),1.44(s,9H),1.37(d,J=7.2Hz,3H); 13 CNMR(100MHz, CDCl3)δ201.3,154.9,137.1,128.8,128.6,127.3,80.3,56.2,33.2,28.3,18.7.

[0093] The racemic mixture of compound 9a (L:D = 1:1) was analyzed by HPLC under the following conditions: a normal-phase chiral IC column (Chiralpak® IC250 × 4.6 mm); hexane as mobile phase A and isopropanol as mobile phase B, with isocratic elution maintained at 5% for 30 minutes; flow rate = 1.0 mL / min; detection wavelength = 220 nm. Results are as follows: Figure 1 As shown. Figure 1 In the HPLC chromatogram, peaks 1 and 2 accounted for 49.74% and 50.26% of the total area, respectively, and their peak heights were 2064.234 mAv and 1748.615 mAv, respectively. The HPLC chromatogram of compound 9a is shown below. Figure 2 As shown, Figure 2 In the figure, the peak area ratios of peak 1 and peak 2 are 99.99% and 0.01%, respectively, and the peak heights are 2213.970 mAv and 0.093 mAv, respectively, indicating that no racemization occurs.

[0094] Example 10: Synthesis of compound 10a

[0095]

[0096] In a clean 4 mL reaction flask, add α-carbonyl alkenyl ester compound (0.2 mmol), benzyl mercaptan (0.24 mmol), dimethyl sulfoxide (DMSO, 1 mL), and DIEA (0.02 mmol). Stir at room temperature and monitor using TLC. The reaction is complete in 5 min. After the reaction, the target product 10a is obtained by column chromatography as a white solid with a yield of 99% and 99% ee.

[0097] 1 HNMR (400MHz, CDCl3) δ7.28(d,J=8.0Hz,2H),7.21(d,J=8.0Hz,2H),5.05(d,J=8. 4Hz,1H),4.51(p,J=7.5Hz,1H),2.37(s,3H),1.48(s,9H),1.43(d,J=7.2Hz,3H); 13 CNMR(100MHz, CDCl3)δ200.4,154.9,139.7,134.6,130.0,123.6,80.3,56.2,28.3,21.3,18.7.

[0098] Example 11: Synthesis of compound 11a

[0099]

[0100] In a clean 4 mL reaction flask, add α-carbonyl alkenyl ester compound (0.2 mmol), benzyl mercaptan (0.24 mmol), dimethyl sulfoxide (DMSO, 1 mL), and DIEA (0.02 mmol). Stir at room temperature and monitor using TLC. The reaction is complete in 10 min. After the reaction is complete, the target product 11a is obtained by column chromatography as a white solid with a yield of 99% and >99%ee.

[0101] 1 HNMR(400MHz,CD2Cl2)δ7.41-7.16(m,10H),5.73(d,J=9.0Hz,1H),5.15(s,2H),4.55 -4.43(m,1H),4.20-4.02(m,2H),3.95-3.84(m,1H),3.59-3.47(m,1H),1.08(s,9H); 13 CNMR (100MHz, CDCl3) δ199.8,156.0,137.2,136.2,128.8,128.5,128.5,128.2,128.1,127.2,73.5,67.3,61.9,61.0,33.3,27.2.

[0102] The racemic mixture of compound 11a (L:D = 1:1) was analyzed by HPLC using a normal-phase IC chiral column, a Chiralpak® IC250 × 4.6 mm column; n-hexane was used as mobile phase A, and isopropanol as mobile phase B, with isocratic elution. The proportion of mobile phase B remained constant at 10% for 27 minutes; the flow rate was 1.0 mL / min; and the detection wavelength was 220 nm. Results are as follows: Figure 3 As shown. Figure 3 In the HPLC chromatogram, peaks 1 and 2 accounted for 49.74% and 50.26% of the total area, respectively, with peak heights of 2064.234 mAv and 1748.615 mAv, respectively. The HPLC chromatogram of compound 11a is shown below. Figure 4 As shown, Figure 4 In the middle, only peak 1 is shown, with a peak area of ​​100%, indicating that no racemization occurred.

[0103] Example 12: Synthesis of compound 12a

[0104]

[0105] In a clean 4 mL reaction flask, add α-carbonyl alkenyl ester compound (0.2 mmol), benzyl mercaptan (0.24 mmol), and dimethyl sulfoxide (DMSO, 1 mL). Stir at room temperature and monitor the reaction using TLC. The reaction is complete in 10 min. After the reaction is complete, the target product 12a is obtained by column chromatography as a white solid with a yield of 99% and >99%ee.

[0106] 1HNMR(400MHz, CDCl3)δ7.32-7.17(m,8H),7.10-7.04(m,2H),4.91(d,J=8.9Hz, 1H),4.65(q,J=7.2Hz,1H),4.17-3.98(m,2H),3.16-2.97(m,2H),1.39(s,9H); 13 CNMR(100MHz, CDCl3)δ200.4,154.9,137.1,135.5,129.3,128.9,128.5,128.5,127.2,127.0,80.3,60.8,38.3,33.3,28.2.

[0107] Example 13: Synthesis of compound 13a

[0108]

[0109] In a clean 4 mL reaction flask, add α-carbonyl alkenyl ester compound (0.2 mmol), benzyl mercaptan (0.24 mmol), dimethyl sulfoxide (DMSO, 1 mL), and DIEA (0.02 mmol). Stir at room temperature and monitor using TLC. After 20 min, the reaction is complete. After the reaction is complete, the target product 13a is obtained by column chromatography as a white solid with a yield of 64% and a ratio >99:1dr.

[0110] 1 HNMR(400MHz, CDCl3)δ7.32-7.17(m,5H),6.95(d,J=8.3Hz,1H),5.21(d,J=7.7Hz,1H),4.76-4.65(m,1H),4.28-4.20(m, 1H),4.08(d,J=2.8Hz,2H),1.77-1.57(m,2H),1.59-1.47(m,1H),1.42(s,9H),1.36(d,J=7.0Hz,3H),0.93-0.85(m,6H); 13 CNMR(100MHz, CDCl3)δ200.3,172.7,155.6,136.9,128.8,128.5,127.2,80.1,57.5,49.8,41.3,33.2,28.2,24.6,23.0,21.3,17.6.

[0111] Example 14: Synthesis of compound 14a

[0112]

[0113] In a clean 4 mL reaction flask, add α-carbonyl alkenyl ester compound (0.2 mmol), benzyl mercaptan (0.24 mmol), dimethyl sulfoxide (DMSO, 1 mL), and DIEA (0.02 mmol). Stir at room temperature and monitor using TLC. The reaction is complete in 10 min. After the reaction is complete, the target product 14a is obtained by column chromatography as a white solid with a yield of 74%.

[0114] 1 HNMR(400MHz, CDCl3)δ8.28(dt,J=8.1,1.1Hz,1H),7.51-7.38(m,4H),7.39-7.27(m,3H),7.26-7.21(m,1H),4.36(s,2H),4.15(s,3H); 13 CNMR (100MHz, CDCl3) δ185.6,141.2,140.5,137.7,129.0,128.5,127.2,127.1,123.7,122.1,121.9,109.4,36.5,32.3.

[0115] Example 15: Synthesis of compound 15a

[0116]

[0117] In a clean 4 mL reaction flask, add α-carbonyl alkenyl ester compound (0.2 mmol), benzyl mercaptan (0.24 mmol), dimethyl sulfoxide (DMSO, 1 mL), and DIEA (0.02 mmol). Stir at room temperature and monitor using TLC. The reaction is complete in 15 min. After the reaction is complete, the target product 15a is obtained by column chromatography as a white solid with a yield of 87%.

[0118] 1 HNMR (400MHz, CDCl3) δ8.02(s,1H),7.87-7.82(m,2H),7.46-7.36(m,4H),7.34-7.29(m,2H),7.28-7.23(m,1H),4.35(s,2H); 13 CNMR (100MHz, CDCl3) δ184.6,141.8,141.1,138.6,137.1,129.0,128.7,128.2,127.4,127.3,125.8,125.1,122.8,33.6.

[0119] Example 16: Synthesis of compound 16a

[0120]

[0121] α-Carbonyl alkenyl ester compound 7a was added to a 5 mL reaction flask and a magnetic stir bar was placed inside. DMSO was added as the reaction solvent, along with 2 eq. of benzyl mercaptan and 0.025 eq. of DIEA. The reaction was monitored by HPLC. After the reaction was completed, polypeptide thioester 16a was obtained by recrystallization and semi-preparative separation and purification, with a yield of 99% and a ratio >99:1dr.

[0122] 1 HNMR(400MHz,DMSO-d6)δ8.41(d,J=8.0Hz,1H),8.15(d,J=8.1Hz,1H),7.87(d,J=7.5Hz,3H),7.78-7.59(m,4H),7.43-7.37(m,2H),7.34-7.14(m, 13H),4.66(q,J=7.3Hz,1H),4.57-4.49(m,1H),4.42-4.32(m,3H),4.31- 4.27(m,1H),4.19-4.10(m,3H),4.07(d,J=3.7Hz,2H),4.00-3.91(m,1H), 3.66-3.60(m,1H),3.54-3.47(m,3H),3.07(dd,J=13.9,3.8Hz,1H),2.80(dd,J=13.8,10.8Hz,1H),2.64(dd,J=15.9,6.3Hz,1H),2.42(dd,J=15.9 ,7.5Hz,1H),2.13-1.93(m,1H),1.32(s,9H),1.14(s,9H),1.09-1.06(m, 18H),1.00(d,J=6.3Hz,3H),0.84(d,J=6.7Hz,3H),0.79(d,J=6.8Hz,3H); 13 CNMR(100MHz,DMSO-d6)δ198.8,171.7,171.3,169.9,169.4,169.2,169.1,155.9,143.7,14 0.7,138.2,137.7,129.3,128.7,128.5,128.1,127.7,127.1,126.3,125.4,125.3,120.2,8 0.3,74.1,73.1,66.7,65.8,61.9,61.5,59.5,57.5,57.1,56.1,53.2,49.5,46.6,37.5,37. 2,32.0,30.6,29.1,28.0,27.7,27.1,27.1,19.4,18.5,17.4;HRMS(ESI-TOF)m / z:calcdforC66 H 90 N6NaO 13 S + [M+Na] + :1229.6179,found1229.6174.

[0123] Example 17: Two-step one-pot method for preparing compound 17a

[0124]

[0125] First, a magnetic stir bar was placed in a 4 mL reaction flask, and 0.24 mmol of lithocholic acid, 0.2 mmol of 1-phenylbutane-2,3-dien-1-one, and 1 mL of reaction solvent DCE were added. The reaction was monitored by TLC. After the reaction was completed, the reaction solvent DCE was removed under reduced pressure using a rotary evaporator. Then, 1 mL of DMSO was added as the reaction solvent, followed by 0.24 mmol of benzyl mercaptan and 0.02 mmol of DIEA. The reaction was stirred at room temperature and monitored by TLC. After the reaction was completed, compound 17a was obtained by column chromatography.

[0126] 1 HNMR (400MHz, CDCl3) δ7.35–7.11(m,5H),4.14–4.04(m,2H),2.96–2.76(m,3H),2.69-2.58(m,1H),2.57-2.47(m,1H),2.41-1.7 4(m,15H),1.59(td,J=14.4,4.7Hz,1H),1.49-1.40(m,1H),1.38(s,3H),1.32-1.18(m,2H),1.03(s,3H),0.83(d,J=6.6Hz,3H). 13 CNMR(100MHz, CDCl3)δ211.9,209.1,208.7,199.1,137.8,128.8,128.6,127.2,56.9,51.8,49.0,46.8,45.6,45.5,45 .0,42.8,41.1,38.7,36.5,36.0,35.4,35.3,33.2,31.2,27.7,25.2,21.9,18.7,11.8;HRMS(ESI-TOF)m / z:calcdforC 31 H 40 NaO4S + [M+Na] + :531.2540,found531.2531.

[0127] Example 18: Synthesis of cyclic thioester compound 18a

[0128]

[0129] 0.04 mmol of α-carbonyl alkenyl ester compound 8a was added to a 25 mL reaction flask with a magnetic stirrer. 8 mL of acetonitrile was added as the reaction solvent, followed by DIEA and TCEP. The reaction progress was monitored by HPLC. After the reaction was completed, the polypeptide cyclic thioester compound 18a was obtained by semi-preparative separation and purification with a yield of 78% and a ratio of >99:1dr.

[0130] 1 HNMR(400MHz,DMSO-d6)δ8.75(d,J=8.5Hz,1H),8.10-7.98(m,4H),7.90(d,J=7.5Hz,2H) ,7.71(dd,J=7.6,3.2Hz,2H),7.45-7.38(m,2H),7.36-7.25(m,4H),7.23-7.14(m,3H),4. 34-4.14(m,5H),4.06-3.91(m,2H),3.19-2.99(m,3H),2.81(t,J=12.2Hz,1H),1.77-1.5 7(m,2H),1.55-1.31(m,4H),1.24-1.17(m,4H),0.85(d,J=6.4Hz,3H),0.81-0.72(m,9H); 13 CNMR(100MHz,DMSO-d6)δ200.0,171.9,171.7,170.7,169.6,155.6,143.8,140.8,137.4,129.2,128.3,127.7,127.1,126.5,125.3,120.2 ,66.0,57.2,56.6,54.0,52.0,49.9,46.6,40.5,36.5,29.1,28.8,24.2,23.8,23.4,22.7,21.4,20.9,17.5;HRMS(ESI-TOF)m / z:calcdforC 42 H 51 N5NaO7S + [M+Na] + :792.3401,found792.3399.

[0131] Examples 19-20 below describe the preparation of cyclic thioester compounds using a two-step, one-pot method with acetylacetamide compounds.

[0132] Example 19: Preparation of cyclic thioester compound 19a

[0133]

[0134] A magnetic stir bar was placed in a 25 mL reaction flask, and 0.04 mmol of the long-chain polypeptide acid Fmoc-Cys-Leu-Ala-Phe-Leu-OH, 0.2 mmol of N-ethynyl-N-methylmethanesulfonamide (MYMsA), and 2 mL of LDCE were added as reaction solvent. The reaction was stirred at 50 °C, and the reaction progress was monitored by HPLC. After the reaction was completed, the solvent was completely removed under vacuum pump to obtain the intermediate α-carbonyl alkenyl ester compound. Then, 8 mL of reaction solvent DMSO was added to the reaction flask, followed by 0.025 eq. DIEA and 2 eq. Bu3P. The reaction progress was monitored by HPLC. After the reaction was completed, the polypeptide cyclic thioester compound 19a was obtained by semi-preparative separation and purification with a yield of 50% and a ratio >99:1dr.

[0135] 1 HNMR(400MHz,DMSO-d6)δ8.78(d,J=8.4Hz,1H),8.24-7.79(m,6H),7.70(d,J=7.4Hz,2H),7.48-7.12(m,9H),4. 34-4.16(m,6H),4.06-3.75(m,2H),3.15-3.02(m,2H),2.90-2.77(m,1H),1.57-1.09(m,8H),0.91-0.65(m,8H). 13 CNMR(101MHz,DMSO-d6)δ199.9,171.8,171.8,170.6,169.6,155.6,143.8,140.8,137.5,129.3,128.3,127.8,127.2,126.6,125. 3,120.3,66.1,55.6,54.7,54.0,52.0,50.0,46.6,36.6,29.1,28.3,24.3,22.8,21.5,17.3,16.5;HRMS(ESI-TOF)m / z:calcdforC 39 H 45 N5NaO7S + [M+Na] + :750.2932,found750.2916.

[0136] HPLC analysis was performed on the following components: raw material polypeptide acid, the reaction of raw material polypeptide acid with MYMsA to prepare activated ester, the preparation of cyclic thioester 19a in DMSO, purified cyclic thioester 19a, and a pair of non-corresponding isomers of cyclic thioester 19a, under the following detection conditions: Jupiter ® A 5 μm C18300 Å, 250 × 4.6 mm column was used. The mobile phase consisted of 0.039% TFA (v / v) and 10% acetonitrile-water as mobile phase A, and 0.045% TFA (v / v) aqueous solution as mobile phase B. Gradient elution was employed: mobile phase A maintained at 50-100% (v / v) over 30 min; flow rate was 1.0 mL / min; detection wavelength was 214 nm. Results are as follows: Figure 5 As shown. Figure 5 In the first image, the retention time is 11.813 min and the peak area is 100%. In the second image, the retention times of peak 1 and peak 2 are 11.803 and 15.243 min, respectively, and the peak area ratios of peak 1 and peak 2 are 2.81% and 97.19%, respectively. In the third image, the retention time is 13.933 min and the peak area is 100%. In the fourth image, the retention time is 13.950 min and the peak area is 100%. In the fifth image, the retention times of peak 1 and peak 2 are 13.573 and 13.947 min, respectively, and the peak area ratios of peak 1 and peak 2 are 54.66% and 45.34%, respectively.

[0137] Example 20: Preparation of cyclic thioester compound 20a

[0138]

[0139] A magnetic stir bar was placed in a 25 mL reaction flask, and 0.04 mmol of the long-chain polypeptide acid Fmoc-Glu-Leu-Phe-Gly-Val-Ala-Cys-NH2, 0.2 mmol of N-ethynyl-N-methylmethanesulfonamide (MYMsA), and 2 mL of LDCE were added as the reaction solvent. The reaction was stirred at 50 °C, and the reaction progress was monitored by HPLC. After the reaction was completed, the solvent was completely removed under vacuum to obtain the intermediate α-carbonyl alkenyl ester compound. Then, 8 mL of DMSO was added to the reaction flask, followed by 0.15 eq. DIEA. The reaction progress was monitored by HPLC. After the reaction was completed, the polypeptide cyclic thioester compound 20a was obtained by semi-preparative separation and purification, with a yield of 57% and a ratio >99:1dr.

[0140] 1HNMR(400MHz,DMSO-d6)δ8.40(t,J=6.0Hz,1H),8.06(d,J=6.1Hz,1H),7.92(dd,J=34.4,7.0Hz,4H),7.74-7.67( m,2H),7.62(d,J=8.4Hz,1H),7.47-7.37(m,4H),7.35-7.16(m,9H),4.42-4.16(m,6H),4.08(d,J=8.1Hz,3H),3.9 1(dd,J=16.8,6.6Hz,1H),3.48-3.39(m,1H),3.14-3.00(m,2H),2.98-2.89(m,1H),2.69-2.54(m,2H),2.13-2.00 (m,1H),1.97-1.80(m,2H),1.63-1.51(m,1H),1.36(q,J=7.4,6.9Hz,2H),1.26-1.17(m,4H),0.88-0.75(m,12H); 13 CNMR(100MHz,DMSO-D6)δ198.2,172.9,172.1,172.0,171.7,171.4,171.0,169 .2,156.0,144.0,140.9,137.8,129.2,128.3,127.9,127.3,126.6,125.5,120. 3,65.9,58.3,55.2,53.4,51.9,51.8,48.4,46.7,42.7,40.4,40.4,36.4,30.9 ,30.0,27.9,24.1,23.1,21.5,19.3,18.9,16.7;HRMS(ESI-TOF)m / z:calcdforC 48 H 60 N8NaO 10 S + [M+Na] + :963.4045,found963.4044.

[0141] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a thioester compound, characterized in that, Includes the following steps: In an organic solvent, with the presence of a base, α-carbonyl alkenyl ester compound 3 reacts with thiols or thiophenols 4 at 0–50 °C to yield thioester compound 5, as shown in the following reaction formula: ,in: R 1 Selected from C1~C 22 Alkyl, C4~C 10 The alkyl group, substituted alkyl group, heterocyclic alkyl group, alkenyl group, alkynyl group, protected α-aminoalkyl group, protected β-aminoalkyl group, protected γ-aminoalkyl group, and protected polypeptide chain alkyl group are preferred; preferably methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, adamantyl, vinyl, 1-propynyl, 2-propynyl, 1-butenyl, 2-butenyl, 3-butenyl, styryl, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, phenylethynyl, benzene One of the following: alkyl, naphthyl, anthraceneyl, phenanthryl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-fluorophenyl, 3-chlorophenyl, 3-bromophenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, pyridyl, pyrroleyl, indolyl, indolylmethyl, indazole, furanyl, benzofuranyl, thienyl, benzothienyl, quinolinyl, styrylyl, phenylethynyl, benzyl, 11-hydroxyundecyl, pentadecyl, protected α-aminoalkyl, protected β-aminoalkyl, protected γ-aminoalkyl, protected polypeptide chain alkyl; R 2 Selected from C1~C 10 One of alkyl, aryl, substituted aryl, heterocyclic aryl, and substituted heterocyclic aryl; preferably one of phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-methylphenyl, 4-methoxyphenyl, 2-methylphenyl, 2-methoxyphenyl, 3,5-dimethoxyphenyl, 3-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3,5-dinitrophenyl, pentafluorophenyl, 4-trifluoromethylphenyl, 3,5-dichlorophenyl, 1-naphthyl, 2-naphthyl, furanyl, and thiopheneyl; R 3 It is selected from one of hydrogen, alkyl, halogen, acyl, cyano, and alkoxycarbonyl; preferably one of H, methyl, formyl, acetyl, propionyl, cyano, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl. R 4 It is selected from one of hydrogen, alkyl, halogen, acyl, cyano, and alkoxycarbonyl; or one of H, methyl, formyl, acetyl, propionyl, cyano, methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, and benzyloxycarbonyl. R 5 It is selected from one of hydrogen, alkyl, halogen, acyl, cyano, and alkoxycarbonyl; preferably one of H, methyl, formyl, acetyl, propionyl, cyano, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl. R 6 Selected from C1-C8 alkyl groups, C3-C 10 Cycloalkyl, aryl, substituted aryl, cysteine ​​residue, protected cysteine ​​residue, or polypeptide containing cysteine ​​residue; preferably one of ethyl, n-hexyl, cyclohexyl, phenyl, naphthyl, benzyl, p-tolyl, p-methoxyphenyl, p-chlorophenyl, p-bromophenyl, 2-pyridyl, 2-thiophenyl, 2-furanyl, cysteine ​​residue, protected cysteine ​​residue, or polypeptide containing cysteine ​​residue.

2. The method according to claim 1, characterized in that, Compound 3 is prepared by reacting carboxylic acid compound 1 and allenone compound 2 in one or a mixture of two of the following solvents: dichloroethane, dichloroethane, acetonitrile, tetrahydrofuran, and 2-methyltetrahydrofuran.

3. The method according to claim 2, characterized in that, Compound 2 is selected from one of the following compounds: Preferably, it is 1-phenylbutane-2,3-diene-1-one.

4. The method according to claim 1, characterized in that, The alkali is selected from one or more of N,N-diisopropylethylamine, triethylamine, sodium carbonate, potassium carbonate, 4-dimethylaminopyridine, cesium carbonate, sodium hydroxide, potassium hydroxide, imidazole, N-methylimidazolium, and pyridine; preferably N,N-diisopropylethylamine; the amount of alkali used is 0 to 1 equivalent.

5. The method according to claim 1, characterized in that, The organic solvent is selected from one or more of dichloromethane, dichloroethane, trichloroethane, dimethyl sulfoxide, methanol, acetonitrile, N,N-dimethylformamide, tetrahydrofuran, n-pentane, cyclohexane, diethyl ether, petroleum ether, and ethyl acetate; preferably dimethyl sulfoxide.

6. A method for preparing a cyclic thioester compound, characterized in that, Includes the following steps: A thiol-containing carboxylic acid compound 7 and an allenone compound 2 or an acetylide compound 9 are reacted in one or a mixture of two solvents selected from dichloroethane, dichloroethane, acetonitrile, tetrahydrofuran, and 2-methyltetrahydrofuran at 0–70 °C to prepare a thiol-containing α-carbonyl alkenyl ester compound 8 or 10; subsequently, in an organic solvent, the thiol-containing α-carbonyl alkenyl ester compound 8 or 10 is reacted at 0–50 °C with the participation of a base and / or additives to obtain a cyclic thioester compound 11, as shown in the following reaction formula: in, PG 1 Selected from H, S t One of Bu, Trt, and Acm; PG 2 Selected from one of H, Fmoc, Boc, and Cbz; R 2 Selected from C1~C 10 One of alkyl, aryl, substituted aryl, heterocyclic aryl, and substituted heterocyclic aryl; preferably one of phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-methylphenyl, 4-methoxyphenyl, 2-methylphenyl, 2-methoxyphenyl, 3,5-dimethoxyphenyl, 3-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3,5-dinitrophenyl, pentafluorophenyl, 4-trifluoromethylphenyl, 3,5-dichlorophenyl, 1-naphthyl, 2-naphthyl, furanyl, and thiopheneyl; R 3 It is selected from one of hydrogen, alkyl, halogen, acyl, cyano, and alkoxycarbonyl; preferably one of H, methyl, formyl, acetyl, propionyl, cyano, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl. R 4 It is selected from one of hydrogen, alkyl, halogen, acyl, cyano, and alkoxycarbonyl; preferably one of H, methyl, formyl, acetyl, propionyl, cyano, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl. R 5 It is selected from one of hydrogen, alkyl, halogen, acyl, cyano, and alkoxycarbonyl; preferably one of H, methyl, formyl, acetyl, propionyl, cyano, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl. R 6 Selected from C1~C 10 Alkyl, aryl, or substituted aromatic ring groups; preferably methyl, ethyl, phenyl, heterocyclic aryl, or halocyclic aryl groups; R 7 Selected from hydrogen, aryl, C1-C8 alkyl, C1-C8 alkynyl, and C1-C8 alkenyl; preferably hydrogen, phenyl, methyl, propyl, isobutyl, ethynyl, and vinyl; The electron-withdrawing group EWG is selected from C1~C5 alkylsulfonyl, C1~C5 alkylyl, C6~C 10 arylsulfonyl group, C6~C 10 The aromatic acyl, nitrile, or nitro group; one of methanesulfonyl, ethanesulfonyl, benzenesulfonyl, substituted benzenesulfonyl, nitrile, or nitro group; preferably one or two of the following compounds: Further preferred options are N-ethynyl-N-methyl-p-toluenesulfonamide or N-ethynyl-N-methylmethanesulfonamide; The heteroatom of the heterocyclic group is O, N or S, and the number of heteroatoms is 1 or 2; the substituent of the substituted aryl or substituted aromatic ring group is selected from C1 to C8 alkyl, C1 to C8 alkoxy, halogen, phenyl, benzyl, benzyloxy, cyano, and the number of substituents is an integer from 1 to 3.

7. The method according to claim 6, characterized in that, The additive is selected from one of the following: triphenylphosphine, tributylphosphine, tricarboxyethylphosphine, tri(2-carbonylethyl)phosphine hydrochloride, tri(2-cyanoethyl)phosphine, dithiothreitol, 2-mercaptoethanol, and 3-(diphenylphosphine)propionic acid.

8. The method according to claim 7, characterized in that, When allenone compounds are used, the additive is tricarboxyethylphosphine; when acetylide compounds are used, no additive is required.

9. The method according to claim 6, characterized in that: The base is selected from N,N-diisopropylethylamine, triethylamine, sodium carbonate, potassium carbonate, 4-dimethylaminopyridine, cesium carbonate, sodium hydroxide, potassium hydroxide, imidazole, and pyridine, preferably N,N-diisopropylethylamine.

10. The method according to claim 6, characterized in that, The organic solvent is selected from one or more of dichloromethane, trichloroethane, dimethyl sulfoxide, methanol, acetonitrile, N,N-dimethylformamide, tetrahydrofuran, n-pentane, diethyl ether, and petroleum ether; when allenone compounds are used, acetonitrile is preferred as the organic solvent, and when acetylenide compounds are used, dimethyl sulfoxide is preferred as the organic solvent.

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