A (2S, 4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline and a method for synthesizing the same

Abstract

The application relates to the technical field of organic synthesis and pharmaceutical intermediates, in particular to a (2S, 4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline and a synthesis method thereof. The method comprises the following steps: performing silicon-based protection on (2S, 4R)-4-hydroxypyrrolidine-2-carboxylic acid; refluxing and dehydrating the alkyl aldehyde to construct a tetrahydropyrrolooxazolone bicyclic intermediate; after the silicon-based group is removed, etherification is performed with 2-haloacetic acid allyl ester under strong alkali to introduce a side chain; and finally, acid ring-opening hydrolysis and Fmoc protection are performed to obtain the target product. The amino group and the carboxyl group are protected in situ by constructing a bicyclic structure, the sensitive allyloxycarbonylmethyl side chain is introduced in a non-water system, and the hydrolysis of the side chain ester bond is effectively avoided. The product prepared by the application has high purity and good stereoselectivity, and an efficient and low-cost industrialization approach is provided for the synthesis of high-performance polypeptide blocks.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis and pharmaceutical intermediates technology, specifically to a (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline and its synthesis method. Background Technology

[0002] With the rapid development of peptide drugs, protein engineering, and bioconjugation technologies, the demand for peptides with complex structures and specific functions is increasing. The traditional 20 natural amino acids have limitations in meeting the needs for precise chemical modification, conformational fixation, or the introduction of functional linkers into peptides. Therefore, designing and synthesizing non-natural amino acids with orthogonally reactive side chains has become one of the core technologies driving the development of this field.

[0003] Hydroxyproline is a key component of biomacromolecules such as collagen, and its 4-hydroxyl group provides a natural site for chemical modification. By specifically derivatizing this hydroxyl group, a programmable "chemical handle" can be introduced into the polypeptide backbone, enabling post-modification such as cyclization, labeling, vector linkage, or functional molecule coupling without affecting the main polypeptide sequence. Allyl esters are particularly favored for their excellent orthogonal reactivity—they remain stable under both acidic (e.g., TFA) and basic (e.g., piperidine) conditions in polypeptide synthesis, but can be selectively removed under mild palladium catalysis or nucleophilic conditions.

[0004] Fmoc-4-(O-allyloxycarbonylmethyl)hydroxyproline is a high-level peptide synthesis building block designed based on this concept. Its molecular structure integrates the Fmoc protecting group, the rigid proline backbone, and the 4-position (O-allyloxycarbonylmethyl) ether side chain. This compound shows great potential for the synthesis of complex peptides (such as bound peptides, cell-penetrating peptides, and antibody-drug conjugate linkers) and 3CLpro inhibitors. However, due to the sensitivity of the allyl ester side chain to alkaline environments, conventional ester derivative synthesis strategies present challenges in controlling side reactions and maintaining side chain stability.

[0005] Patent CN110551178A discloses a method for synthesizing proline-containing cyclic peptides, which involves coupling Fmoc-3-carboxyl-Pro-OAll and preparing cyclic peptides according to the Fmoc solid-phase synthesis strategy. While this technique involves the application of the Fmoc protection strategy and allyl protecting groups, its modification site is located at the 3-position of proline, and it focuses on the cyclization process on a solid-phase support. For non-natural amino acid building blocks requiring the introduction of a specific ether side chain (O-allyloxycarbonylmethyl) at the 4-position, this method provides limited molecular structural reference and does not address the challenge of accurately constructing complex 4-position side chains while maintaining product stereoselectivity during liquid-phase synthesis.

[0006] Patent CN102336697A discloses a method for synthesizing (2S,4S)-4-hydroxyproline, which achieves configurational transformation through carboxyl esterification, nitrogen protection, and hydroxyl esterification of (2S,4R)-4-hydroxyproline. This method primarily focuses on the isomerization and basic protection of hydroxyproline, lacking specific reaction design for introducing a highly orthogonal (O-allyloxycarbonylmethyl) functional side chain. Particularly when dealing with the allyloxycarbonyl side chain, which is highly susceptible to alkaline hydrolysis, the conventional hydrolysis and purification conditions used in this process easily lead to compromised side chain stability, leaving room for optimization in product purity control and overall yield improvement.

[0007] Therefore, developing a synthetic method that is simple to operate, has mild conditions, high stereoselectivity, ideal overall yield, and can effectively protect sensitive side chains is of great value for promoting the widespread application of high-performance peptide building blocks. Summary of the Invention

[0008] This application provides a (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline and its synthetic method. The aim is to pretreat (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid with a silicon-based protecting group, construct a tetrahydropyrroloxazosone rigid intermediate using an alkyl aldehyde reagent, introduce an allyloxycarbonylmethyl group into the proline side chain under specific fluoride ion deprotection and etherification reaction conditions, and finally obtain the target product through acidic ring opening and the introduction of the Fmoc protecting group.

[0009] In a first aspect, this application provides (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline and a method for synthesizing the same, comprising the following steps:

[0010] S10: Dissolve (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid as shown in formula (Ⅰ) in a solvent, add a silicon-based protecting group reagent and a base, and react at -10~100℃ until the reaction is complete. After the reaction solution is concentrated and crystallized, (2S,4R)-4-(silyloxy)pyrrolidine-2-carboxylic acid as shown in formula (Ⅱ) is obtained.

[0011] S20: Dissolve (2S,4R)-4-(silyloxy)pyrrolidine-2-carboxylic acid as shown in formula (II) in a solvent, add alkyl aldehyde or substituted alkyl aldehyde, heat and reflux to remove water until the reaction is complete, and then wash, concentrate and crystallize the reaction solution to obtain (6R,7aS)-6-(silyloxy)-3-(substituted alkyl)tetrahydro-1H,3H-pyrrolo[1,2-c]oxazol-1-one as shown in formula (III);

[0012] S30: Dissolve (6R,7aS)-6-(silyloxy)-3-(substituted alkyl)tetrahydro-1H,3H-pyrrolo[1,2-c]oxazol-1-one as shown in formula (III) in a solvent, add a fluoride ion deprotecting agent to remove the silyl protecting group, then add a base and 2-haloacetic acid allyl ester, and react at -20~50℃ until the reaction is complete. The reaction solution is extracted, washed, concentrated and crystallized to obtain (6R,7aS)-6-(2-allyloxycarbonylmethoxy)-3-trichloromethyltetrahydropyrrolooxazolone as shown in formula (IV);

[0013] S40: Dissolve (6R,7aS)-6-(2-allyloxycarbonylmethoxy)-3-trichloromethyltetrahydropyrroloxazosone as shown in formula (IV) in a solvent, add acid, and react at 0~100℃ until the reaction is complete. After adjusting the pH of the reaction solution to a weak base, add Fmoc protecting group reagent and stir until the reaction is complete. The reaction solution is extracted, washed, concentrated and crystallized to obtain (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline as shown in formula (V).

[0014] The compound represented by formula (Ⅰ) is: ,

[0015] The compound represented by formula (II) is: ,

[0016] The compound represented by formula (Ⅲ) is: ,

[0017] The compound represented by formula (IV) is: ;

[0018] The compound represented by formula (V): In the formula, R1 is any silicon-based protecting group, and R2 is any alkyl or substituted alkyl group.

[0019] According to this application, a silicon-based protecting group is introduced in step S10 to shield the 4-position hydroxyl group from interference with subsequent cyclization reactions. Step S20 utilizes the simultaneous reaction of an alkyl aldehyde with the amino and carboxyl groups of proline to construct a rigid tetrahydropyrroloxazolone bicyclic structure. This structure serves as an N,C-double protecting group in subsequent steps, avoiding the damage to the allyl ester side chain caused by hydrolysis following carboxyl esterification in conventional synthesis. Step S30 employs fluoride ion-selective removal of the silicon group and a nucleophilic substitution reaction using 2-haloacetic acid allyl ester in an anhydrous strong base system to introduce an allyloxycarbonyl methyl group at the 4-position oxygen atom. Step S40 utilizes acid-catalyzed ring-opening to restore the amino and carboxyl groups, followed by the introduction of an Fmoc group under a controlled weak base environment, thus avoiding the saponification effect of strongly alkaline aqueous solutions on the allyl ester bond.

[0020] Preferably, in step S10, the silicon-based protecting group reagent includes one or more of trimethylchlorosilane, tert-butyldimethylchlorosilane, tert-butyldiphenylchlorosilane, tert-butyldimethylsilyltrifluoromethanesulfonate, and triisopropylchlorosilane; the base includes one or more of imidazole, N,N-diisopropylethylamine, triethylamine, and pyridine.

[0021] Preferably, in step S10, the molar ratio of (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid, the silicon-based protecting group reagent, and the base is 1:(1.1-1.5):(1.2-1.8).

[0022] Specifically, when tert-butyldimethylchlorosilane is used as a silicon-based reagent, the silicon atom forms a stable silyl ether bond with the hydroxyl group, masking the nucleophilicity of the 4-position hydroxyl group. At the same time, the carboxyl group remains free or forms a salt under alkaline conditions, providing a chemical basis for subsequent cyclization reactions.

[0023] Preferably, in step S20, the alkyl aldehyde or substituted alkyl aldehyde includes one or more of trichloroacetaldehyde and pentylaldehyde.

[0024] Preferably, in step S20, the molar ratio of (2S,4R)-4-(silyloxy)pyrrolidine-2-carboxylic acid represented by formula (II) to the alkyl aldehyde or substituted alkyl aldehyde is 1:(1.05-1.2).

[0025] Preferably, in step S20, the solvent used is chloroform, toluene, or dichloromethane.

[0026] Specifically, when trichloroacetaldehyde is used, the aldehyde group of trichloroacetaldehyde undergoes condensation cyclization with the amino and carboxyl groups of the compound of formula (II) to form an oxazolone skeleton with a bicyclic structure. The construction of this skeleton protects both the nitrogen atom and carboxyl group of proline and fixes the spatial conformation of the molecule, allowing the modification of the 4-position side chain to be carried out in a confined spatial environment.

[0027] Preferably, in step S30, the fluoride ion deprotecting reagent includes one or more of tetrabutylammonium fluoride, pyridine hydrogen fluoride complex, cesium fluoride, potassium fluoride, and tetramethylammonium fluoride; the base includes one or more of sodium hydride, 1,8-diazobisspirocyclic [5.4.0]undecyl-7-ene, and (tert-butylimino)tris(pyrrolidine)phosphine; and the 2-haloacetic acid allyl ester is one or more of 2-chloroacetic acid allyl ester, 2-bromoacetic acid allyl ester, and 2-iodoacetic acid allyl ester.

[0028] Preferably, in step S30, the molar ratio of (6R,7aS)-6-(silyloxy)-3-(substituted alkyl)tetrahydro-1H,3H-pyrrolo[1,2-c]oxazol-1-one, fluoride ion deprotecting agent, base and 2-haloacetic acid allyl ester of formula (III) is 1:(1.0-1.1):(1.3-1.6):(1.1-1.3).

[0029] Specifically, in the process, the strong affinity of fluoride ions for silicon atoms is first used to break the silane-ether bond, generating a hydroxyl intermediate in situ. Then, a strong base is added to remove the proton from the hydroxyl group, generating an oxon. This oxon attacks the α-carbon atom of 2-haloacetic acid allyl ester, constructing an ether bond via an SN2 mechanism. Due to the presence of the oxazolone ring, the reaction is carried out in a non-aqueous alkaline system, avoiding the hydrolysis of the allyl ester group in an aqueous alkaline environment.

[0030] Preferably, in step S40, the acid used is one or more of hydrochloric acid, sulfuric acid, trifluoroacetic acid, p-toluenesulfonic acid, ferric chloride, and trimethylsilyltrifluoromethanesulfonate; the base is any inorganic or organic base; the Fmoc protecting agent is one or more of 9-fluorenylmethyl chloroformate, 9-fluorenmethyl-N-succinimide carbonate, and N-(9-fluorenylmethoxycarbonyl)benzotriazole ester; the molar ratio of (6R,7aS)-6-(2-allyloxycarbonylmethoxy)-3-trichloromethyltetrahydropyrroloxazosone of formula (IV) to the Fmoc protecting agent is 1:(1.05-1.2).

[0031] In a second aspect, this application provides (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline, which is prepared according to the method described in any embodiment of the first aspect.

[0032] According to this application, since the (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline is prepared according to any embodiment of the first aspect, its structure contains an Fmoc-protected nitrogen atom, a free 2-position carboxyl group, and an allyloxycarbonylmethyl side chain connected by an ether bond at the 4-position, the product purity is not less than 99.0%, and it maintains the (2S,4R) stereoconfiguration.

[0033] The technical solution provided by this invention involves first constructing a tetrahydropyrroloxazolone bicyclic intermediate to simultaneously mask the amino and carboxyl groups of proline. This structural feature not only provides a rigid molecular skeleton but also allows for etherification reactions in an anhydrous, strongly basic organic system (such as NaH / THF or BTPP / DMF) during the subsequent introduction of allyloxycarbonyl methyl groups. This anhydrous environment prevents the allyl ester groups from contacting water molecules, thus blocking the alkaline-catalyzed ester hydrolysis side reaction pathway.

[0034] In step S10, tert-butyldimethylchlorosilane or tert-butyldiphenylchlorosilane is selected as a protecting group. Utilizing its greater steric hindrance, it guides trichloroacetaldehyde from the reverse side of the hydroxyl group during the subsequent cyclization process in step S20, maintaining the original conformation of the hydroxyl group at position 4. Simultaneously, the silyl ether bond can be selectively cleaved by fluoride ions in step S30 without affecting other potentially sensitive functional groups present in the molecule.

[0035] In step S20, trichloroacetaldehyde condenses with (2S,4R)-4-(silyloxy)pyrrolidine-2-carboxylic acid under reflux. The strong electron-inductive effect of the trichloromethyl group increases the stability of the oxazolone ring, enabling it to withstand strongly alkaline conditions in the subsequent etherification reaction. The water separation operation physically removes the water molecules generated in the reaction, shifting the chemical equilibrium towards the cyclization product.

[0036] In step S30, tetrabutylammonium fluoride is used to remove the silicon group, and the released 4-hydroxyl group reacts in situ with a strong base. By controlling the dropping rate of 2-haloacetic acid allyl ester and the reaction temperature (from -20 to 10 °C), the probability of nucleophilic attack on the carbonyl group of allyl ester is reduced, and the reaction is focused on the nucleophilic substitution of the α-carbon atom.

[0037] In step S40, the hydrolysis of the oxazolone ring is mediated by a strong protic acid (such as 6M hydrochloric acid or 2M sulfuric acid). The acidic environment leads to the protonation of the acetal center on the oxazolone ring, followed by the attack of water molecules, which causes the ring structure to disintegrate, releasing trichloroacetaldehyde as a byproduct. This process takes place in an acidic medium, where the allyl ester group exhibits good chemical stability. The subsequent Fmoc protection step is carried out at a weakly alkaline (pH = 8 to 9) aqueous / organic phase interface, where rapid stirring brings Fmoc-OSu into contact with the free amino group, completing the carbamate esterification of the nitrogen atom.

[0038] This invention solves the technical problem of easy hydrolysis of allyl ester groups in conventional synthetic routes through the specific technical means described above. The total synthetic route avoids the destruction of side chain ester bonds by the strong alkaline environment in aqueous solutions. By controlling the conformation of intermediates and precisely switching reaction conditions, high purity and high yield of (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline are achieved.

[0039] The (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline prepared in this invention serves as a high-performance building block for peptide synthesis, and its application value is reflected in the following aspects:

[0040] 1. In ADC drug development, this building block can serve as the core unit of a linker. The carboxyl group at the 4-position of its side chain can couple with cytotoxic drugs (such as MMAE, DM1, etc.) via an amide or ester bond, while the Fmoc-protected amino group participates in peptide chain assembly, ultimately coupling with an antibody via the N-terminus or C-terminus. The allyl ester protecting group of the side chain provides excellent orthogonality for the coupling process, allowing for the site-specific introduction of toxin molecules without affecting other parts of the peptide chain.

[0041] 2. In the synthesis of PROTACs, this building block is an important raw material for constructing VHL ligands. VHL protein ligands typically contain a hydroxyproline backbone. Through the (O-allyloxycarbonylmethyl) side chain introduced in this invention, the length and chemical properties of the linker can be easily adjusted, thereby optimizing the degradation efficiency of PROTACs molecules on target proteins (such as coronavirus 3CL protease).

[0042] In cyclic peptide drug research, the 4-carboxyl handle provided by this building block can undergo lactamation with the lysine side chain amino group or the N-terminal amino group in the peptide chain sequence to construct macrocyclic peptides with specific conformational constraints. This cyclization method helps enhance the protease stability of the peptide in vivo and improves its bioavailability.

[0043] 3. The synthesis method of this invention not only fills the gap in the synthesis technology of this compound, but also achieves breakthroughs in key technical indicators through process optimization throughout the entire process:

[0044] Product purity: Through efficient crystallization process, the purity of the finished product is consistently above 99.0%, meeting the requirements of high-specification pharmaceutical intermediates.

[0045] 4. Stereoselectivity: The entire reaction is carried out under mild or controlled conditions, avoiding racemization of the chiral center, and the enantiomeric excess (ee value) of the product is greater than 99%.

[0046] Production Costs: The raw materials used, such as 4-hydroxyproline, trichloroacetaldehyde, and Fmoc-OSu, are all bulk chemicals, widely available and inexpensive. The process route does not require ultra-low temperature (e.g., -78°C) or ultra-high pressure environments, has low equipment requirements, and offers good economic benefits.

[0047] Environmental friendliness: Solvents used in the reaction process, such as dichloromethane, toluene, and tetrahydrofuran, can all be recycled. Stepwise crystallization and extraction reduce the amount of waste liquid generated, meeting the requirements of green chemistry.

[0048] 5. Path orthogonality: By utilizing the specific reaction between silicon-based protecting groups and fluoride ions, combined with the simultaneous masking of amino and carboxyl groups by the oxazolone ring, chemical orthogonality of side chain modification is achieved.

[0049] 6. Stability control: The rigid structure of the oxazolone ring allows for etherification in an anhydrous organic base system, avoiding the hydrolysis risk of allyl esters in traditional aqueous synthesis.

[0050] 7. Conformation retention: The synthesis process did not involve configuration inversion reactions at the 2- and 4-position chiral centers. 1H-NMR and mass spectrometry analysis confirmed that the product retained the absolute configuration of (2S, 4R).

[0051] 8. Easy to scale up: The reaction conditions in each step are mild. Except for step S30, which involves column chromatography, the rest of the steps can be purified by recrystallization, making it suitable for industrial production on a kilogram-scale basis.

[0052] In summary, this application provides a (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline and its synthesis method. Through an innovative bicyclic protection strategy and orthogonal deprotection process, it successfully achieves the efficient and high-purity synthesis of the target molecule, providing a key chemical tool for the biomedical field. Attached Figure Description

[0053] Figure 1 This is a flowchart illustrating the preparation of (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline and its synthesis method according to the present invention.

[0054] Figure 2 The image shows the ¹H-NMR spectrum of the target product (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline, which was finally synthesized in Example 1.

[0055] Figure 3 The ¹H-NMR spectrum of the key intermediate obtained in the second step of Example 2, (6R,7aS)-6-((tert-butyldiphenylsilyl)oxy)-3-(trichloromethyl)tetrahydro-1H,3H-pyrrolo[1,2-c]oxazol-1-one.

[0056] Figure 4 The ¹H-NMR spectrum of the target product (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline, which was finally synthesized in Example 2, is shown. Detailed Implementation

[0057] The various embodiments or implementation schemes in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments.

[0058] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0059] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0060] (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline is an important building block for peptide synthesis, containing a nitrogen atom protected by Fmoc, a free 2-position carboxyl group, and an allyloxycarbonylmethyl side chain linked by an ether bond at position 4. This compound has applications in the synthesis of complex peptides and antibody-drug conjugates (ADCs). However, the allyl ester bond in the 4-position side chain is extremely sensitive to alkaline environments, and hydrolysis or loss of the side chain ester bond is highly likely during conventional amino acid protecting group conversion. Furthermore, the 2-position carboxyl group and nitrogen atom on the proline backbone require effective shielding during side chain modification to avoid side reactions.

[0061] Based on this, this application provides (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline and its synthesis method. By constructing a specific tetrahydropyrroloxazolone bicyclic intermediate, in-situ dual protection of the nitrogen atom and carboxyl group of the proline skeleton is achieved. Furthermore, by utilizing a silicon-based protection strategy and an orthogonal deprotection process, sensitive side-chain functional groups are introduced under mild conditions.

[0062] Firstly, see... Figure 1 This application provides (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline and a method for synthesizing the same, comprising the following steps:

[0063] Step S10: Dissolve (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid as shown in formula (I) in an organic solvent, and react with a silicon-based protecting group reagent under the action of an alkaline auxiliary agent to obtain (2S,4R)-4-(silyloxy)pyrrolidine-2-carboxylic acid as shown in formula (II).

[0064] In step S10, the molar ratio of (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid, the silicon-based protecting agent, and the basic auxiliary agent is 1:(1.1-1.5):(1.2-1.8). The organic solvent is selected from one or more of dichloromethane, acetonitrile, dimethylformamide, and tetrahydrofuran. The basic auxiliary agent includes one or more of imidazole, N,N-diisopropylethylamine, triethylamine, and pyridine. The silicon-based protecting agent includes one or more of trimethylchlorosilane, tert-butyldimethylchlorosilane, tert-butyldiphenylchlorosilane, tert-butyldimethylsilyltrifluoromethanesulfonate, and triisopropylchlorosilane. The reaction temperature is controlled between -10°C and 100°C, preferably between 20°C and 50°C. The reaction time is 2 to 24 hours. Specifically, in dichloromethane solvent, the concentration of the reaction system is maintained at 0.5 to 1.0 mol / L, and the mixture is stirred at 25°C for 12 to 18 hours. The generated hydrochloride is removed by filtration, and the filtrate is concentrated and then recrystallized in a mixed solvent of methyl tert-butyl ether and water.

[0065] Step S20: The compound shown in formula (II) is reacted with an alkyl aldehyde or a substituted alkyl aldehyde under dehydration conditions to obtain (6R,7aS)-6-(silyloxy)-3-(substituted alkyl)tetrahydro-1H,3H-pyrrolo[1,2-c]oxazol-1-one shown in formula (III).

[0066] In step S20, the molar ratio of compound (II) to alkyl aldehyde or substituted alkyl aldehyde is 1:1.05 to 1.2. The alkyl aldehyde or substituted alkyl aldehyde includes one or more of trichloroacetaldehyde, pentylaldehyde, and isobutyraldehyde. The reaction is carried out in chloroform, toluene, dichloromethane, or benzene. During the reaction, an azeotropic dehydration process is performed using a water separator, or dehydrating agents such as molecular sieves, anhydrous magnesium sulfate, or acetic anhydride are added. The reaction temperature is 30 to 110°C. In a specific embodiment, chloroform is used as the solvent, and a catalytic amount of p-toluenesulfonic acid (0.01 to 0.05 equivalents) is added. The mixture is refluxed at 61 to 65°C and dehydrated for 4 to 8 hours. After the reaction, the solvent and excess trichloroacetaldehyde are removed by vacuum concentration, and the residue is recrystallized from methyl tert-butyl ether or n-hexane.

[0067] Step S30: Dissolve the compound shown in formula (III) in an ether or amide solvent, add a fluoride ion deprotecting agent to remove the silicon protecting group, and then react it with 2-haloacetic acid allyl ester in the presence of a strong base to obtain (6R,7aS)-6-(2-allyloxycarbonylmethoxy)-3-trichloromethyltetrahydropyrroloxazosone shown in formula (IV).

[0068] In step S30, the molar ratio of compound (III), fluoride ion deprotecting reagent, strong base, and 2-haloacetic acid allyl ester is 1:(1.0-1.1):(1.3-1.6):(1.1-1.3). The solvent is selected from dry tetrahydrofuran, dimethylformamide, or dimethyl sulfoxide. The fluoride ion deprotecting reagent includes one or more of tetrabutylammonium fluoride, pyridine hydrogen fluoride complex, cesium fluoride, potassium fluoride, and tetramethylammonium fluoride. The strong base includes one or more of sodium hydride, 1,8-diazobispyrocyclo[5.4.0]undecyl-7-ene (DBU), and (tert-butylimino)tris(pyrrolidine)phosphine (BTPP). The 2-haloacetic acid allyl ester includes 2-chloroacetic acid allyl ester, 2-bromoacetic acid allyl ester, and 2-iodoacetic acid allyl ester. The reaction temperature is -20 to 50°C. In the specific operation, a fluoride ion deprotecting agent is first added at 0 to 5°C and reacted for 1 to 2 hours. Then, a strong base is added in batches, and the temperature is maintained below 10°C while stirring for 1 hour. Finally, 2-haloacetic acid allyl ester is added dropwise. After the reaction is complete, excess base is quenched by adding dilute hydrochloric acid, and the mixture is extracted using methyl tert-butyl ether. The organic layer is then washed with saturated brine and dried with anhydrous sodium sulfate.

[0069] Step S40: The compound shown in formula (IV) is hydrolyzed and ring-opened under acidic conditions, and then the pH of the system is adjusted to weakly alkaline. Fmoc protecting agent is added to protect the N-terminus to obtain (2S,4R)-1-Fmoc-4-(O-allyloxymethyl)-proline shown in formula (V).

[0070] In step S40, the molar ratio of compound (IV) to the Fmoc protecting agent is 1:(1.05-1.2). The acidic conditions utilize one or more of hydrochloric acid, sulfuric acid, trifluoroacetic acid, and p-toluenesulfonic acid. The hydrolysis step uses an acid solution with a concentration of 2 to 6 mol / L, refluxed at 80 to 100°C for 2 to 4 hours. The ring-opening reaction temperature is 20 to 100°C. After cooling, the pH of the hydrolysate is adjusted to 8 to 9 using sodium bicarbonate, sodium carbonate, or sodium hydroxide. The Fmoc protecting agent includes one or more of 9-fluorenyl chloroformate (Fmoc-Cl), 9-fluorenylmethyl-N-succinimide carbonate (Fmoc-OSu), and N-(9-fluorenylmethoxycarbonyl)benzotriazole ester. The protection reaction is carried out in a mixture of water and an organic solvent (such as tetrahydrofuran, acetone, or dioxane) at a temperature of 0 to 40°C. The reaction time was 4 to 12 hours. The product was purified by recrystallization in an ethyl acetate / n-heptane (volume ratio 1:3 to 1:5) system, and the drying temperature was controlled at 40 to 45 °C.

[0071] In the silicon-based protection stage (S10), the effects of TBSCl and TBDPSCl were compared. Experiments showed that intermediate formula II obtained using TBDPSCl exhibited better chemical stability in subsequent steps, especially under long reflux conditions in S20, where the silicon-based shedding rate was less than 1%, while the shedding rate of TBSCl under the same conditions was approximately 5% to 8%. Therefore, TBDPSCl is preferred as the protective agent.

[0072] In the bicyclic construction stage (S20), the effects of different solvents on dehydration efficiency were compared. When chloroform was used as the solvent, the water separation rate was slow due to the low boiling point of the azeotrope formed with water (approximately 56°C), requiring the reaction to last for more than 24 hours. However, by using toluene or adding sodium acetate as a catalyst in glacial acetic acid, the reaction time could be shortened to less than 12 hours, and the conversion rate of compound III was increased to over 98%.

[0073] In the side-chain etherification stage (S30), the choice of base is crucial to the yield. If conventional potassium carbonate or sodium hydroxide is used, the unavoidable presence of trace amounts of water in the system will lead to extensive hydrolysis of 2-haloacetic acid allyl ester. This invention uses sodium hydride or a strong organic base (tert-butylimino)tris(pyrrolidine)phosphine, operated in anhydrous THF or DMF, achieving a nucleophilic substitution yield of over 85% with minimal byproducts.

[0074] In the final product purification stage (S40), by screening the recrystallization solvent ratio, it was determined that when the volume ratio of ethyl acetate to n-heptane was 1:3, the crystal form of the precipitated product was the most regular, and the purity was increased from 92% of the crude product to over 99%, and residual Fmoc-OSu and trichloroacetaldehyde condensate could be effectively removed.

[0075] The (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline prepared in this invention offers significant advantages in peptide synthesis. Due to its allyl ester protection form with a carboxyl group at the 4-position, the allyl group can be selectively removed under mild conditions using catalytic amounts of tetra(triphenylphosphine)palladium and phenylsilane after solid-phase peptide synthesis, exposing the free carboxyl group. This carboxyl group can then be further coupled with fluorescent molecules, biotin, or drug loadings, or cyclized with other amino groups on the peptide chain to construct bound peptides. This orthogonal protection strategy provides great flexibility for the design of complex peptides.

[0076] Furthermore, the method of this invention strictly controls the stereochemistry during the preparation process. Characterization of the product by 1H NMR and MS confirmed that the molecular structure of the product is completely consistent with the theoretical design. The NMR spectrum showed a distinct aromatic hydrogen signal from the Fmoc group at 7.3 to 7.8 ppm, a methylene hydrogen signal from the allyl group at 5.9 ppm, a terminal olefin hydrogen signal at 5.3 ppm, and the region from 4.7 to 3.7 ppm encompassing the proline backbone hydrogen and the methylene hydrogen in the side chain. Mass spectrometry analysis showed that the molecular ion peak M+H was 452.2, consistent with the molecular weight requirement of (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline.

[0077] In summary, the method provided in this application cleverly resolves the contradiction between protecting and introducing sensitive functional groups by constructing a tetrahydropyrroloxazolone intermediate, thus opening up a new technical route for the synthesis of high-performance peptide building blocks.

[0078] Secondly, the present invention provides structural features of the intermediates and final products involved in the above-described method.

[0079] In the compound of formula (II), R1 is a silicon-based protecting group, specifically selected from one of trimethylsilyl (TMS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS).

[0080] In the compound of formula (Ⅲ), R2 is an alkyl or haloalkyl group, specifically selected from trichloromethyl, tert-butyl, and isopropyl.

[0081] The compound of formula (V) is (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline, with a molecular weight of 451.47 and a chemical formula of C. 25 H 25 NO.7

[0082] The technical solution of the present invention will be described in detail below through specific embodiments.

[0083] Example 1: In a 1L reaction flask, (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid (50g, 381.3mmol) was suspended in 500mL of dichloromethane. Tert-butyldimethylchlorosilane (74.7g, 495.7mmol) and imidazole (38.9g, 572.0mmol) were added. The reaction was stirred at 25°C for 15 hours. After the reaction was complete, the solid produced was removed by filtration, and the filtrate was concentrated under reduced pressure at 35°C. 500mL of methyl tert-butyl ether and 500mL of deionized water were added to the residue, and the mixture was mechanically stirred for 1 hour and then allowed to stand at 5°C for 16 hours. The precipitated solid was collected by filtration and dried under vacuum at 40°C for 12 hours to obtain 75.8g of white powder (2S,4R)-4-((tert-butyldimethylsilyl)oxy)pyrrolidine-2-carboxylic acid, with a purity of 98.0%. (Product mass spectrometry molecular weight: M++H=246.1)

[0084] The (2S,4R)-4-((tert-butyldimethylsilyl)oxy)pyrrolidine-2-carboxylic acid (75 g, 305.6 mmol) obtained above was added to a 2L three-necked flask equipped with a water separator, and 750 mL of chloroform was added. Trichloroacetaldehyde (49.6 g, 336.2 mmol) was added. The mixture was heated to 62 °C and refluxed, and water separation was continued until no water was discharged from the water separator, which took about 6 hours. After the reaction solution was cooled to 25 °C, the solvent was removed by vacuum distillation. 600 mL of methyl tert-butyl ether was added to the residue, and the mixture was stirred for 2 hours. The mixture was filtered, washed with cold ether, and dried at 40 °C to obtain 105.1 g of (6R,7aS)-6-((tert-butyldimethylsilyl)oxy)-3-(trichloromethyl)tetrahydro-1H,3H-pyrrolo[1,2-c]oxazol-1-one, with a product purity of 98.6%. (Product mass spectrometry molecular weight: M++H=374.0)

[0085] Weigh 77.4 g (281.15 mmol, 5% water) of tetrabutylammonium fluoride into a 2 L flask, add 600 mL of toluene, and remove water by azeotropic distillation. Add 1000 mL of anhydrous tetrahydrofuran to the flask to dissolve the residue. Add the prepared oxazolone intermediate (105.1 g, 281.15 mmol) and stir at 25 °C for 1 hour. Cool the reaction system to 0-5 °C in an ice-water bath, and add sodium hydride (16.9 g, 421.7 mmol, 60% dispersed in mineral oil) in 5 portions. After the addition is complete, stir at 25 °C for 1 hour. Cool again to 0-10 °C, and slowly add allyl 2-bromoacetate (60.4 g, 337.4 mmol). After the addition is complete, react at 25 °C for 8 hours. Pour the reaction solution into 1000 mL of 1 mol / L hydrochloric acid and extract with 500 mL of methyl tert-butyl ether. The organic phases were combined, washed with 300 mL of saturated brine, and dried over anhydrous sodium sulfate. After concentration, the product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, volume ratio 3:1) to give 79.6 g of an oily substance (6R,7aS)-6-(2-allyloxycarbonylmethoxy)-3-trichloromethyltetrahydropyrroloxazosone, with a purity of 97.5%. (Mass spectrometry molecular weight: M++H=358.0)

[0086] The above-mentioned oily substance (79.6 g, 222.1 mmol) was added to a 500 mL round-bottom flask, along with 200 mL of deionized water and 200 mL of concentrated hydrochloric acid (12 mol / L). The mixture was heated to 95-100 °C and refluxed for 3 hours. After the reaction was complete, the mixture was cooled to 25 °C. Under ice bath cooling, solid sodium bicarbonate was added to adjust the pH of the solution to 8.0-8.5. 100 mL of tetrahydrofuran was added, followed by Fmoc-OSu (82.4 g, 244.3 mmol). The mixture was stirred at 25 °C for 12 hours. After the reaction was complete, the pH was adjusted to 5.0 with 2 mol / L hydrochloric acid. The mixture was extracted with 500 mL of methyl tert-butyl ether. The organic layers were combined and concentrated under reduced pressure. The residue was dissolved in 200 mL of ethyl acetate, and 600 mL of n-heptane was added dropwise with stirring. The mixture was stirred at 25 °C for 16 hours. Filter and vacuum dry at 40℃ to obtain 92.2g of solid (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline with a purity of 99.0% and a mass spectrometry molecular weight of M++H=452.2.

[0087] See Figure 2¹H-NMR (500MHz, CDCl3) δ (ppm): 7.8 (m, 2H), 7.6 (m, 2H), 7.4–7.3 (m, 4H), 6.8 (s, 1H), 5.9 (m, 1H), 5.3 (m, 2H), 4.7–3.7 (m, 11H), 2.4–2.2 (m, 2H). This corresponds to the ¹H-NMR spectrum of the final synthesized target product in Example 1: (2S, 4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline. Example 1 used tert-butyldimethylchlorosilane as a silicon protecting group, and obtained the final product through a four-step reaction. Figure 2 The 1H NMR data (δ(ppm): 7.8(m,2H) to 2.4-2.2(m,2H)) are used to characterize the structure of the target compound and to confirm the chemical structure of the final product.

[0088] Example 2: In a 2L reaction flask, (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid (50 g, 381.3 mmol) was added to 500 mL of acetonitrile. Tert-butyldiphenylchlorosilane (115 g, 419.4 mmol) and N,N-diisopropylethylamine (59.1 g, 457.6 mmol) were added. The reaction mixture was stirred at 45°C for 20 hours. The reaction solution was filtered, and the filtrate was concentrated at 40°C. 500 mL of methyl tert-butyl ether and 500 mL of water were added, and the mixture was stirred for 1 hour and allowed to stand for 16 hours. The solid was collected by filtration and dried at 40°C to obtain 115.5 g of (2S,4R)-4-((tert-butyldiphenylsilyl)oxy)pyrrolidine-2-carboxylic acid, with a product purity of 98.5%. (Mass spectrometry molecular weight: M++H=370.2)

[0089] The above-mentioned silicon-based protected product (115.5 g, 312.6 mmol) was added to 300 mL of glacial acetic acid. Anhydrous sodium acetate (28.2 g, 343.9 mmol) and trichloroacetaldehyde (50.7 g, 343.9 mmol) were added. The mixture was heated to 110 °C and refluxed for 5 hours. After the reaction was completed, the reaction solution was poured into 1000 mL of ice water and stirred. The precipitated solid was collected by filtration. The filter cake was washed with anhydrous ethanol and dried at 45 °C to obtain 140.0 g of (6R,7aS)-6-((tert-butyldiphenylsilyl)oxy)-3-(trichloromethyl)tetrahydro-1H,3H-pyrrolo[1,2-c]oxazol-1-one, with a purity of 99.1% and a mass spectrometry molecular weight of M++H=498.1.

[0090] See Figure 3¹H-NMR (500MHz, CDCl3) δ (ppm): 7.7 (m, 4H), 7.5 (m, 2H), 7.4 (m, 4H), 5.0 (s, 1H), 4.3 (m, 1H), 4.4 (m, 1H), 3.4 (dd, 1H), 2.9 (dd, 1H), 2.3 (m, 1H), 2.0 (m, 1H), 1.1 (s, 9H)). This corresponds to the ¹H-NMR spectrum of the key intermediate obtained in the second step of Example 2: (6R,7aS)-6-((tert-butyldiphenylsilyl)oxy)-3-(trichloromethyl)tetrahydro-1H,3H-pyrrolo[1,2-c]oxazol-1-one. Example 2 uses tert-butyldiphenylchlorosilane as a silicon-based protecting group. In the second step, the cyclization reaction between the silicon-protected hydroxyproline and trichloroacetaldehyde was completed to generate the tetrahydropyrroloxazolone intermediate. Figure 4 The proton NMR spectrum data serves as the basis for structural verification of this intermediate. The characteristic peak at δ1.1 (s, 9H) also corresponds to the hydrogen signal of tert-butyl, which can corroborate the structure of the intermediate.

[0091] The above-mentioned oxazolone intermediate (70.0 g, 140.3 mmol) was dissolved in 210 mL of N,N-dimethylformamide. Cesium fluoride (23.4 g, 154.3 mmol) was added, and the mixture was heated to 50 °C and stirred for 2 hours. Allyl 2-chloroacetate (22.7 g, 168.4 mmol) was added. BTPP (57.0 g, 182.4 mmol) was added dropwise under ice bath conditions. After the addition was complete, the reaction mixture was stirred at 25 °C for 10 hours. The reaction mixture was poured into 600 mL of 1 mol / L hydrochloric acid and extracted twice with 500 mL of methyl tert-butyl ether. The organic phases were combined, washed with 300 mL of saturated brine, and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate, volume ratio 4:1) to give 37.7 g of oily (6R,7aS)-6-(2-allyloxycarbonylmethoxy)-3-trichloromethyltetrahydropyrroloxazosone, with a purity of 98.0%. (Mass spectrometry molecular weight: M++H=358.0)

[0092] The resulting oily substance (37.7 g, 105.1 mmol) was added to 100 mL of 2 mol / L sulfuric acid solution. The mixture was refluxed at 100 °C for 4 hours. After cooling to 25 °C, the pH was adjusted to 8.2 with sodium bicarbonate. 50 mL of tetrahydrofuran and Fmoc-Cl (30.0 g, 115.8 mmol) were added. The mixture was stirred at 20–25 °C for 6 hours. The pH of the reaction solution was adjusted to 5.0 with 2 mol / L hydrochloric acid. The mixture was extracted twice with 100 mL of methyl tert-butyl ether. The organic phases were combined and concentrated. The residue was dissolved in 100 mL of ethyl acetate, and 300 mL of n-heptane was added dropwise. The mixture was stirred at 25 °C for 16 hours, filtered, and dried at 40 °C to give 47 g of (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline with a purity of 99.1%. The molecular weight of the product according to mass spectrometry was M++H=452.2. See Figure 4 ¹H-NMR (500MHz, CDCl3) δ (ppm): 7.8 (m, 2H), 7.6 (m, 2H), 7.4–7.3 (m, 4H), 6.8 (s, 1H), 5.9 (m, 1H), 5.3 (m, 2H), 4.7–3.7 (m, 11H), 2.4–2.2 (m, 2H). This corresponds to the ¹H-NMR spectrum of the target product finally synthesized in Example 2: (2S, 4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline.

[0093] Although Example 2 used a different silicon-based protecting group (tert-butyldiphenylsilyl) than Example 1, the final synthesized target product was completely consistent with that of Example 1. Figure 4 hydrogen spectrum data and Figure 3 The results were identical, further verifying the consistency and purity of the final product structure obtained under different process routes of this synthesis method.

[0094] Example 3: In a 1 L reaction flask, (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid (50 g, 381.3 mmol) was dissolved in 500 mL of tetrahydrofuran, and triisopropylchlorosilane (88.1 g, 457.6 mmol) and pyridine (45.2 g, 572.0 mmol) were added. The reaction was carried out at 50 °C for 18 hours. After the reaction was completed, the mixture was concentrated, washed with ethyl acetate and water, and the organic phase was dried and concentrated to give (2S,4R)-4-((triisopropylsilyl)oxy)pyrrolidine-2-carboxylic acid.

[0095] The above product (80 g, 278.3 mmol) and tert-pentanal (28.8 g, 334.0 mmol) were dissolved in 500 mL of toluene, and 10 g of 4A molecular sieve was added. The mixture was refluxed at 110 °C for 10 hours. The molecular sieve was removed by filtration, the residue was concentrated, and recrystallized from n-hexane to give (6R,7aS)-6-((triisopropylsilyl)oxy)-3-(tert-butyl)tetrahydro-1H,3H-pyrrolo[1,2-c]oxazol-1-one.

[0096] The intermediate (70 g, 196.9 mmol) was dissolved in 300 mL of dimethyl sulfoxide, and potassium fluoride (13.7 g, 236.3 mmol) was added. The reaction was carried out at 40 °C for 3 hours. Allyl 2-iodoacetate (53.4 g, 236.3 mmol) was added, followed by DBU (36.0 g, 236.3 mmol), and the reaction was carried out at 25 °C for 12 hours. Subsequent processing was the same as in Example 1 to obtain (6R,7aS)-6-(2-allyloxycarbonylmethoxy)-3-tert-butyltetrahydropyrroloxazosone.

[0097] The above product (50 g, 168.1 mmol) was hydrolyzed in 3 mol / L hydrochloric acid at 80 °C for 5 h, and then reacted with Fmoc-OSu (62.3 g, 184.9 mmol) in a THF / H2O system. After adjusting the pH, extraction, and recrystallization, (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline was obtained.

[0098] Comparative Example 1: Using a conventional etherification method: (2S,4R)-1-Fmoc-4-hydroxyproline was directly dissolved in dimethylformamide, 2.5 equivalents of sodium hydride were added, and after stirring at 0°C for 1 hour, allyl 2-bromoacetate was added dropwise. The reaction was carried out at 25°C.

[0099] Comparative Example 2: A stepwise protection method was adopted: (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid was first protected with N-Fmoc and C-terminal methyl ester, then reacted with allyl 2-bromoacetate under alkaline conditions, and finally C-terminal deprotection was performed.

[0100] The products of each example and comparative example were analyzed, including HPLC purity, overall yield, and chiral retention (diastereomer ratio) determined by nuclear magnetic resonance (NMR). The results are shown in Table 1 below:

[0101] Table 1 Test Results

[0102]

[0103] As shown in Table 1, the method provided in this embodiment of the invention is significantly superior to the comparative example in both yield and purity. Examples 1 and 2 introduced the side chain by constructing an oxazolone bicyclic intermediate in an anhydrous organic base system. Comparative Example 1 directly etherified the substrate with the Fmoc protecting group and free carboxyl group under strong base conditions, resulting in severe racemization and extensive hydrolysis of the allyl ester group in the side chain. Comparative Example 2, due to its cumbersome steps and the difficulty in maintaining the stability of the allyl ester in the C-terminal deprotection process, resulted in a lower final yield.

[0104] This invention constructs orthogonally reactive non-natural amino acid building blocks through a specific silicon-based protection and oxazolone cyclization strategy. This synthetic method utilizes the conformational anchoring effect of the oxazolone ring in the etherification reaction to introduce an allyl ester side chain under strongly alkaline anhydrous conditions, thus circumventing the hydrolysis pathway of the side chain ester bond. By sequentially switching between acidic ring-opening and Fmoc protection, high-purity target products were obtained. This compound has application value in peptide drug development, ADC linker design, and the synthesis of 3CLpro inhibitors.

[0105] In the above embodiments, the reaction temperature in step S10 can be adjusted within the range of -10 to 100°C, preferably 20 to 50°C. The amount of alkaline auxiliaries is typically 1.2 to 1.8 times the molar amount of amino acids. In the dehydration cyclization of step S20, the use of a water separator or the addition of a dehydrating agent can improve the conversion rate. In step S30, the type of fluoride ion reagent and the moisture content affect the deprotection effect; azeotropic dehydration treatment of tetrabutylammonium fluoride with toluene can improve the reactivity. In the etherification reaction, BTPP, as a non-nucleophilic strong base, exhibits solubility and reaction homogeneity in DMF solvent. The hydrolysis conditions in step S40 are controlled under a strongly acidic environment to promote the cleavage of the acetal structure, while the subsequent pH adjustment process needs to be carried out slowly. In the selection of Fmoc protecting reagents, the byproduct N-hydroxysuccinimide generated by Fmoc-OSu is easily removed by washing with water.

[0106] The (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline prepared in this invention exhibits selective removal of the allyl ester group at the 4-position of its side chain under palladium catalysis, exposing a carboxyl group. This provides a chemical handle for the side-chain cyclization of peptides or the coupling of functional molecules. The synthesis of this building block provides raw materials for the construction of complex peptides and PROTAC molecules.

[0107] The synthetic method provided by this invention achieves the protection of functional groups through the structural design of intermediates and the control of the reaction environment. The process route is compact and has a high raw material utilization rate. The introduction of recrystallization purification reduces reliance on large-scale column chromatography.

[0108] 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 synthesizing (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline, characterized in that, Includes the following steps: S10: Dissolve (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid as shown in formula (Ⅰ) in a solvent, add a silicon-based protecting group reagent and a base, and react at -10~100℃ until the reaction is complete. After the reaction solution is concentrated and crystallized, (2S,4R)-4-(silyloxy)pyrrolidine-2-carboxylic acid as shown in formula (Ⅱ) is obtained. S20: Dissolve (2S,4R)-4-(silyloxy)pyrrolidine-2-carboxylic acid as shown in formula (II) in a solvent, add alkyl aldehyde or substituted alkyl aldehyde, heat and reflux to remove water until the reaction is complete, and then wash, concentrate and crystallize the reaction solution to obtain (6R,7aS)-6-(silyloxy)-3-(substituted alkyl)tetrahydro-1H,3H-pyrrolo[1,2-c]oxazol-1-one as shown in formula (III); S30: Dissolve (6R,7aS)-6-(silyloxy)-3-(substituted alkyl)tetrahydro-1H,3H-pyrrolo[1,2-c]oxazol-1-one as shown in formula (III) in a solvent, add a fluoride ion deprotecting agent to remove the silyl protecting group, then add a base and 2-haloacetic acid allyl ester, and react at -20~50℃ until the reaction is complete. The reaction solution is extracted, washed, concentrated and crystallized to obtain the compound shown in formula (IV); S40: Dissolve the compound shown in formula (IV) in a solvent, add acid, and react at 0~100℃ until the reaction is complete. After adjusting the reaction solution to a weak base, add Fmoc protecting group reagent and stir until the reaction is complete. The reaction solution is extracted, washed, concentrated and crystallized to obtain (2S,4R)-1-Fmoc-4-(O-allyloxycarbonylmethyl)-proline shown in formula (V). The compound represented by formula (Ⅰ) is: , The compound represented by formula (II) is: , The compound represented by formula (Ⅲ) is: , The compound represented by formula (IV): ; The compound represented by formula (V) is: .

2. The method according to claim 1, characterized in that, In step S10, the silicon-based protecting group reagent is selected from trimethylchlorosilane, tert-butyldimethylchlorosilane, tert-butyldiphenylchlorosilane, tert-butyldimethylsilyltrifluoromethanesulfonate, or triisopropylchlorosilane; the base is selected from imidazole, N,N-diisopropylethylamine, triethylamine, or pyridine.

3. The method according to claim 2, characterized in that, In step S10, the molar ratio of (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid, silicon-based protecting group reagent, and base is 1:1.1-1.5:1.2-1.

8.

4. The method according to claim 1, characterized in that, In step S20, the alkyl aldehyde or substituted alkyl aldehyde is selected from trichloroacetaldehyde or terpentaldehyde.

5. The method according to claim 4, characterized in that, In step S20, the molar ratio of (2S,4R)-4-(silyloxy)pyrrolidine-2-carboxylic acid as shown in formula (II) to the alkyl aldehyde or substituted alkyl aldehyde is 1:1.05-1.

2.

6. The method according to claim 1, characterized in that, In step S20, the solvent used is chloroform, toluene, or dichloromethane.

7. The method according to claim 1, characterized in that, In step S30, the fluoride ion deprotecting reagent is selected from tetrabutylammonium fluoride, pyridine hydrogen fluoride complex, cesium fluoride, potassium fluoride, or tetramethylammonium fluoride; the base is selected from sodium hydride, 1,8-diazobisspirocyclo[5.4.0]undecyl-7-ene, or (tert-butylimino)tris(pyrrolidine)phosphine; and the 2-haloacetic acid allyl ester is selected from 2-chloroacetic acid allyl ester, 2-bromoacetic acid allyl ester, or 2-iodoacetic acid allyl ester.

8. The method according to claim 7, characterized in that, In step S30, the molar ratio of (6R,7aS)-6-(silyloxy)-3-(substituted alkyl)tetrahydro-1H,3H-pyrrolo[1,2-c]oxazol-1-one, fluoride ion deprotecting agent, base and 2-haloacetic acid allyl ester of formula (III) is 1:1.0-1.1:1.3-1.6:1.1-1.

3.

9. The method according to claim 1, characterized in that, In step S40, the acid is selected from hydrochloric acid, sulfuric acid, trifluoroacetic acid, p-toluenesulfonic acid, ferric chloride, or trimethylsilyltrifluoromethanesulfonate; the base is selected from any inorganic or organic base; the Fmoc protecting reagent is selected from 9-fluorenylmethyl chloroformate, 9-fluorenmethyl-N-succinimide carbonate, or N-(9-fluorenylmethoxycarbonyl)benzotriazole ester; the molar ratio of the compound of formula (IV) to the Fmoc protecting reagent is 1:1.05-1.2.

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