CN118344369A - Method for preparing eptifibatide impurity I based on diphenylphosphonooxy label in auxiliary mode - Google Patents

Abstract

The invention relates to a method for preparing eptifibatide impurity I based on diphenylphosphonooxy label assistance, which comprises the steps of carrying out liquid-phase esterification coupling reaction on TAG label molecules and Boc-Pro-OH, carrying out Boc protection removal, carrying out Fmoc-Trp (Boc) -OH amidation coupling reaction to obtain linear dipeptide, removing Fmoc protecting group, carrying out self-cleavage-cyclization to remove TAG, and finally removing Boc protection to prepare eptifibatide impurity I. The preparation method is simple, the industrial preparation is easy, the total yield of the eptifibatide impurity I is 74%, and the purity of the product is more than 97%.

Description

Method for preparing impurity I of eptifibatide based on diphenylphosphonyloxy tag

Technical Field

The invention belongs to the fields of pharmaceutical technology and polypeptide chemical synthesis technology, and relates to a mild and efficient preparation method of eptifibatide impurity I.

Background technique

Eptifibatide is a naturally derived cyclic heptapeptide. As a highly selective platelet glycoprotein IIb/IIIa receptor inhibitor, it has the characteristics of short plasma half-life, rapid onset of antiplatelet effect, and rapid reversibility of platelet inhibition after cessation of treatment. It is clinically used to treat acute coronary syndrome (unstable angina/non-ST-segment elevation myocardial infarction), including patients who will receive drug treatment or are scheduled to undergo percutaneous coronary intervention (PCI). Its specific structure is as follows:

During the production and storage of eptifibatide, the eptifibatide impurity I with the following structure may appear, which may affect the efficacy and safety of the eptifibatide drug. The synthesis and preparation of eptifibatide impurity I helps to understand the nature and content range of possible impurities during the research process of eptifibatide preparation technology, so that corresponding quality control strategies can be formulated to ensure that the drug quality meets the standards.

CN 114685607A discloses a method for preparing eptifibatide impurity I, but it is necessary to first use a solid phase synthetic resin to synthesize the dipeptide chain of the impurity, and then go through a series of complex procedures such as acid cleavage, group protection, deprotection, peptide cyclization, etc. to complete the preparation. The synthetic route is relatively complex, which greatly reduces the preparation efficiency of eptifibatide impurity I.

In the field of organic synthesis, it has always been an important and arduous challenge to seek and develop environmentally friendly, simple to operate and economically feasible synthesis methods. The preparation method of eptifibatide impurity I disclosed in the above patent document is not only not easy to industrialize, but also does not achieve a good atomic utilization rate. Therefore, it is urgent to develop a green, economical and efficient synthesis process route of eptifibatide impurity I, further reduce the use of raw materials, and realize the simple and efficient production and preparation of eptifibatide impurity I.

Summary of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for preparing eptifibatide impurity I based on a diphenylphosphonyloxy tag to prepare eptifibatide impurity I simply and efficiently.

To achieve the above-mentioned purpose of the invention, the method for preparing eptifibatide impurity I based on diphenylphosphonyloxy tag-assisted preparation of the present invention specifically uses a tag (TAG)-assisted peptide synthesis method to prepare a linear dipeptide, and self-cleaves and cyclizes under alkaline conditions to prepare eptifibatide impurity I, specifically comprising:

S1: Esterification coupling of Boc-Pro-OH and TAG

Under the action of the esterification coupling reagent, the TAG tag molecule undergoes a liquid phase esterification coupling reaction with the Boc-protected proline Boc-Pro-OH to generate the TAG tag-loaded intermediate Boc-Pro-O-TAG;

S2: Removal of Boc protecting group

Under the action of an acidic deprotection reagent, Boc-Pro-O-TAG is deprotected to obtain a deprotected intermediate H ₂ N-Pro-O-TAG;

S3: Amidation coupling of Fmoc-Trp(Boc)-OH and H ₂ N-Pro-O-TAG

Under the action of amide coupling reagent, H ₂ N-Pro-O-TAG undergoes amidation coupling reaction with Fmoc-protected tryptophan Fmoc-Trp(Boc)-OH to generate TAG tag-loaded linear dipeptide Fmoc-Trp(Boc)-Pro-O-TAG;

S4: Linear dipeptide self-cleavage-cyclization

Under the action of alkaline reagent, the Fmoc protecting group of Fmoc-Trp(Boc)-Pro-O-TAG is removed, and TAG is removed by self-cleavage-cyclization to form a Boc-protected cyclodipeptide derivative cyclo [Trp(Boc)-Pro];

S5: Removal of Boc protecting group

Under the action of an acidic deprotection reagent, cyclo [Trp(Boc)-Pro] is deprotected from Boc, and cyclo [Trp-Pro], i.e., eptifibatide impurity I, is prepared after purification.

The TAG tag molecule involved in the preparation method of the present invention is a diphenylphosphinoyloxy group-based benzophenone oxime compound TAG=N-OH represented by the following general structural formula (I):

Or it is a benzyl alcohol compound TAG-OH based on a diphenylphosphinoyloxy group as shown in the following general structural formula (II):

The substituent R is selected from H, OPOPh ₂ , C1-C3 alkyl, C1-C3 alkoxy, halogen atom or NO ₂ .

Furthermore, the substituent R is preferably selected from H, OPOPh ₂ , CH ₃ , OCH ₃ , Cl, F or NO ₂ .

The specific synthetic route of the above-mentioned eptifibatide impurity I is as follows:

In the above method of the present invention, the esterification coupling reagent used in the esterification coupling reaction is various conventional esterification coupling reagents, and the present invention has no special limitation thereto. Specifically, the esterification coupling reagent used in the present invention can be one or more of the conventional coupling combination reagents EDCl/DMAP, DIC/DMAP, and DCC/DMAP. In some specific embodiments, the esterification coupling reagent is preferably EDCl/DMAP.

In the above method of the present invention, the amide coupling reagent used in the amidation coupling reaction is also various conventional amidation coupling reagents, and the present invention does not specifically limit it. Specifically, the amide coupling reagent used in the present invention can be one or more of the conventional coupling combination reagents EDCl/HOBt, EDCl/HOBt/DIPEA, DIC/HOBt, DCC/HOSU, HATU/HOAt/DIPEA, and PyBOP/DIPEA for amidation. In some specific embodiments, the amide coupling reagent is preferably EDCl/HOBt.

More specifically, the esterification coupling reaction is preferably carried out under stirring at 0 to 40° C. for 0.5 to 10 h; and the amidation coupling reaction is preferably carried out under stirring at 0 to 40° C. for 1 to 2 h.

In the above method of the present invention, the acidic deprotection reagent for removing Boc protection can be any one of dioxane hydrochloride/dichloromethane, trifluoroacetic acid/dichloromethane, hydrochloric acid/methanol, etc. In some specific embodiments, the acidic deprotection reagent is preferably trifluoroacetic acid/dichloromethane.

Furthermore, the specific reaction conditions for removing the Boc protection are stirring the reaction at 0-10° C. for 3-6 hours.

In the above method of the present invention, the alkaline reagent used for self-cleavage and cyclization of linear dipeptides can be any one of a diethylamine/acetonitrile mixture, a piperidine/acetonitrile mixture, and a 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) solution. In some specific embodiments, the alkaline reagent is preferably a diethylamine/acetonitrile mixture.

Furthermore, the specific reaction conditions of the self-cleavage-cyclization are stirring the reaction at 30-50° C. for 1-3 hours.

In the preparation method of the present invention, the reaction of each step is carried out in a suitable solvent system. The solvent system suitable for each reaction in the present invention is an organic solvent that can form a good phase separation with water, such as one or more of chloroform, dichloromethane, etc.

Furthermore, the eptifibatide impurity I in the present invention is obtained after HPLC purification.

Specifically, the chromatographic conditions for the HPLC purification are to use a reverse phase C18 BP, 4.6×250 mm, 5 μm chromatographic column, with 0.1% trifluoroacetic acid/water as mobile phase A and 0.1% trifluoroacetic acid/acetonitrile as mobile phase B, and gradient elution at a detection wavelength of 280 nm.

In the preparation method of the present invention, the tag (TAG) plays the following three roles in assisting the synthesis of eptifibatide impurity I:

1) As a temporary protecting group for the carbon-terminal carboxyl group (COOH) of the amino acid in the peptide chain, it can undergo self-cleavage and cyclization in an alkaline environment to form a cyclodipeptide derivative, and the removed label molecule can be directly recovered and recycled after purification;

2) Compared with other carboxyl protecting groups, it has the effect of improving the solubility of the polypeptide chain in the medium, and can assist in the precipitation and purification of the eptifibatide impurity I intermediate in a specific precipitation solvent, avoiding the use of chromatography for purification of the intermediate;

3) Based on the polybenzene ring system of TAG, it is easy to monitor the intermediate reaction process of homogeneous reaction by TLC.

The present invention prepares eptifibatide impurity I by using a tag-assisted polypeptide synthesis technology, realizes a homogeneous reaction and a self-cleavage-cyclization reaction by utilizing the properties of the tag molecule, and the tag molecule can be recycled, thereby improving the atomic utilization rate, reducing the use of raw materials and simplifying the synthesis steps. The preparation route is simple, the reaction conditions are mild, and the industrial preparation is easy. The total yield of preparing eptifibatide impurity I reaches 74%, and the product purity is more than 97%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a synthetic route diagram of impurity I of eptifibatide.

FIG. 2 is a mass spectrum of the TAG-tag loaded linear dipeptide Fmoc-Trp(Boc)-Pro-O-TAG prepared in Example 1.

FIG3 is a mass spectrum of eptifibatide impurity I cyclo [Trp-Pro].

FIG4 is a mass spectrum of the TAG-tag loaded intermediate Boc-Pro-O-N=TAG prepared in Example 2.

FIG5 is a mass spectrum of the linear dipeptide Fmoc-Trp(Boc)-Pro-O-N=TAG loaded with a TAG tag prepared in Example 2.

FIG6 is a mass spectrum of the cyclodipeptide derivative cyclo [Trp(Boc)-Pro].

Implementation

The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to more clearly illustrate the technical solution of the present invention so that those skilled in the art can well understand and utilize the present invention, rather than limiting the scope of protection of the present invention.

The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and/or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

The present invention provides a method for preparing eptifibatide impurity I based on a diphenylphosphonyloxy tag, the synthesis route of which is shown in FIG1 , wherein the tag molecule involved is a diphenylphosphonyloxybenzophenone oxime compound or a diphenylphosphonyloxybenzyl alcohol compound.

First, Boc-protected proline Boc-Pro-OH is used as raw material, and a liquid phase esterification coupling reaction is carried out with a TAG tag molecule under the action of an esterification coupling reagent to generate a TAG tag-loaded intermediate Boc-Pro-O-TAG, and then the Boc protection is removed under the action of an acidic deprotection reagent to obtain a de-Boc protected intermediate _H2N -Pro-O-TAG.

Next, the prepared _H2N -Pro-O-TAG and Fmoc-protected tryptophan Fmoc-Trp(Boc)-OH were used as raw materials to further undergo an amidation coupling reaction under the action of an amide coupling reagent to generate a TAG-tag loaded linear dipeptide Fmoc-Trp(Boc)-Pro-O-TAG.

The alkaline reagent acts on Fmoc-Trp(Boc)-Pro-O-TAG to remove the Fmoc protecting group and undergo a self-cleavage-cyclization reaction to remove TAG to form a Boc-protected cyclodipeptide derivative cyclo [Trp(Boc)-Pro], and the tag molecule is recovered for reuse.

Finally, the Boc protection of cyclo [Trp (Boc) -Pro] was removed by an acidic deprotection reagent, and the product was eluted and purified by HPLC to prepare the target product eptifibatide impurity I.

The meanings of the English abbreviations involved in the present invention are as follows:

Boc: tert-butyloxycarbonyl

DBU: 1,8-diazabicyclo-bicyclo(5,4,0)-7-undecene

DCC: N,N-dicyclohexylcarbodiimide

DIC: 1,3-Diisopropylcarbodiimide

DIPEA: N,N-diisopropylethylamine

DMAP: 4-dimethylaminopyridine

EDCl: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

Fmoc: fluorenylmethoxycarbonyl

HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

HOAt: 1-Hydroxy-7-azobenzotriazole

HOBt: 1-Hydroxybenzotriazole

HOSU: N-hydroxysuccinimide

Pro: Proline

PyBOP: 1H-Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate

Trp: Tryptophan

Example

Example 1

Step 1: Synthesis of Boc-Pro-O-TAG

Boc-Pro-OH (2.37 g, 11 mmol), EDCl (2.3 g, 12 mmol), and DMAP (146 mg, 1.2 mmol) were weighed in sequence and dissolved in 30 mL of dichloromethane. The mixture was stirred in an ice bath for 30 min. 4-diphenylphosphonyloxybenzyl alcohol label molecule (3.24 g, 10 mmol) was added, and the mixture was heated to room temperature and stirred for 2 h. The reaction endpoint was detected by TLC.

The reaction product was washed with saturated NH ₄ Cl solution and 10% Na ₂ CO ₃ solution, respectively, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to 3 mL. 30 mL of petroleum ether was added dropwise to produce a white precipitate and the solution was filtered off. The above precipitation operation was repeated 3 times to obtain a purified Boc-Pro-O-TAG intermediate for use with a yield of 96%.

Step 2: Synthesis of _H2N -Pro-O-TAG

The Boc-Pro-O-TAG prepared in step 1 was dissolved in 6 mL of dichloromethane solution, stirred at 0°C for 10 min, and then 6 mL of trifluoroacetic acid was added. The reaction was continued to be stirred at 0°C for 3 h and then the reaction was terminated. The reaction endpoint was detected by TLC.

The reaction solution was concentrated under reduced pressure, and the product was redissolved in 30 mL of dichloromethane, and then washed once with 10% NaHCO ₃ and 20% NaCl solutions in sequence. The obtained organic phase was concentrated under reduced pressure to obtain the purified H ₂ N-Pro-O-TAG intermediate for standby use, with a yield of 98%.

Step 3: Synthesis of Fmoc-Trp(Boc)-Pro-O-TAG

Fmoc-Trp(Boc)-OH (5.53 g, 10.5 mmol), EDCl (2.3 g, 12 mmol), and HOBt (1.62 g, 12 mmol) were weighed in sequence and dissolved in 30 mL of dichloromethane. The mixture was stirred in an ice bath for 30 min. H ₂ N-Pro-O-TAG prepared in step 2 was added. The mixture was heated to room temperature and stirred for 1 h. The end point of the reaction was detected by TLC.

The reaction product was washed with 10% Na ₂ CO ₃ solution and 20% NaCl solution, respectively, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to 3 mL. 30 mL of petroleum ether was added dropwise to produce a white precipitate and the solution was filtered off. The above precipitation operation was repeated 3 times to obtain the purified linear dipeptide Fmoc-Trp(Boc)-Pro-O-TAG for use with a yield of 97%.

The structure of the product was confirmed by the mass spectrum in Figure 2, HRMS (ESI) m/z calcd for C ₅₅ H ₅₃ N ₃ O ₉ P ⁺ (M+H) ⁺ 930.35139, found 930.35156.

Step 4: Synthesis of cyclo [Trp(Boc)-Pro]

The Fmoc-Trp(Boc)-Pro-O-TAG prepared in step 3 was dissolved in 6 mL of acetonitrile, and 6 mL of diethylamine solution was added at 45°C. The Fmoc group was removed by stirring for 1 h. After concentration under reduced pressure, the mixture was dissolved in 3 mL of dichloromethane. 30 mL of petroleum ether was added dropwise to produce a white precipitate and the solution was filtered off. The above precipitation operation was repeated 3 times to obtain a purified cyclodipeptide derivative cyclo [Trp(Boc)-Pro] for use with a yield of 94%.

HRMS (ESI) m/z calcd for C ₂₁ H ₂₆ N ₃ O ₄ ⁺ (M+H) ⁺ 384.19178, found 384.19171.

Step 5: Synthesis of Eptifibatide Impurity I

The cyclo [Trp(Boc)-Pro] prepared in step 4 was dissolved in 6 mL of dichloromethane solution, stirred at 0°C for 10 min, and then 6 mL of trifluoroacetic acid was added. The reaction was continued to be stirred at 0°C for 3 h and then the reaction was terminated. The reaction endpoint was detected by TLC.

After the reaction solution was concentrated under reduced pressure, 30 mL of dichloromethane was used to redissolve the product, and then 10% NaHCO ₃ and 20% NaCl solutions were used to wash the product once respectively. The obtained organic phase was concentrated under reduced pressure and purified by chromatography to obtain pure eptifibatide impurity I with a yield of 84%.

The mass spectrum of the product is shown in Figure 3. HRMS (ESI) m/z calcd for C ₁₆ H ₁₈ N ₃ O ₂ ⁺ (M+H) ⁺ 284.13935, found 284.13950.

Example 2

Step 1: Synthesis of Boc-Pro-O-N=TAG

Boc-Pro-OH (2.37 g, 11 mmol), EDCl (2.3 g, 12 mmol), and DMAP (146 mg, 1.2 mmol) were weighed in turn and dissolved in 30 mL of dichloromethane. The mixture was stirred in an ice bath for 30 min. 4,4'-diphenylphosphonyloxybenzophenone oxime label molecule (6.30 g, 10 mmol) was added. The mixture was heated to room temperature and stirred for 3 h. The reaction endpoint was detected by TLC.

The reaction product was washed with saturated NH ₄ Cl solution and 10% Na ₂ CO ₃ solution, respectively, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to 3 mL. 30 mL of petroleum ether was added dropwise to produce a white precipitate and the solution was filtered off. The above precipitation operation was repeated 3 times to obtain a purified Boc-Pro-ON=TAG intermediate for use with a yield of 95%.

The mass spectrum in Figure 4 verifies the Boc-Pro-ON=TAG structure of the intermediate: HRMS (ESI) m/z calculated for C ₄₇ H ₄₅ N ₂ O ₈ P ₂ ⁺ (M+H) ⁺ 827.26457, found 827.26489.

Step 2: Synthesis of H ₂ N-Pro-ON=TAG

The Boc-Pro-O-N=TAG prepared in step 1 was dissolved in 8 mL of dichloromethane solution, stirred at 0°C for 10 min, and then 8 mL of trifluoroacetic acid was added. The reaction was continued to be stirred at 0°C for 3.5 h and then the reaction was terminated. The reaction endpoint was detected by TLC.

The reaction solution was concentrated under reduced pressure, and the product was redissolved in 30 mL of dichloromethane, and then washed once with 10% NaHCO ₃ and 20% NaCl solutions in sequence. The obtained organic phase was concentrated under reduced pressure to obtain the purified H ₂ N-Pro-ON=TAG intermediate for standby use, with a yield of 96%.

Step 3: Synthesis of Fmoc-Trp(Boc)-Pro-O-N=TAG

Fmoc-Trp(Boc)-OH (5.53 g, 10.5 mmol), EDCl (2.3 g, 12 mmol), and HOBt (1.62 g, 12 mmol) were weighed in sequence and dissolved in 30 mL of dichloromethane. The mixture was stirred in an ice bath for 30 min. H ₂ N-Pro-ON=TAG obtained in step 2 was added. The mixture was heated to room temperature and stirred for 1 h. The end point of the reaction was detected by TLC.

The reaction product was washed with 10% Na ₂ CO ₃ solution and 20% NaCl solution, respectively, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to 3 mL. 30 mL of petroleum ether was added dropwise to produce a white precipitate and the solution was filtered off. The above precipitation operation was repeated 3 times to obtain a purified linear dipeptide Fmoc-Trp(Boc)-Pro-ON=TAG for use with a yield of 98%.

The structure of the linear dipeptide was confirmed by the mass spectrum in Figure 5, HRMS (ESI) m/z calcd for C ₇₃ H ₆₅ N ₄ O ₁₁ P ₂ ⁺ (M+H) ⁺ 1235.41196, found 1235.41296.

Step 4: Synthesis of cyclo [Trp(Boc)-Pro]

The Fmoc-Trp(Boc)-Pro-ON=TAG prepared in step 3 was dissolved in 6 mL of acetonitrile, and 6 mL of diethylamine solution was added at 45°C. The Fmoc group was removed by stirring for 1 h. After concentration under reduced pressure, the mixture was dissolved in 3 mL of dichloromethane. 30 mL of petroleum ether was added dropwise to produce a white precipitate and the solution was filtered off. The above precipitation operation was repeated 3 times to obtain a purified cyclodipeptide derivative cyclo [Trp(Boc)-Pro] for use. The yield was 96%.

HRMS (ESI) m/z calcd for C ₂₁ H ₂₆ N ₃ O ₄ ⁺ (M+H) ⁺ 384.19178, found 384.19128. The mass spectrum in Figure 6 verified the structure of the cyclic dipeptide derivative.

Step 5: Synthesis of Eptifibatide Impurity I

The cyclo [Trp(Boc)-Pro] prepared in step 4 was dissolved in 6 mL of dichloromethane solution, stirred at 0°C for 10 min, and then 6 mL of trifluoroacetic acid was added. The reaction was continued to be stirred at 0°C for 3 h and then the reaction was terminated. The reaction endpoint was detected by TLC.

After the reaction solution was concentrated under reduced pressure, 30 mL of dichloromethane was used to redissolve the product, and then 10% NaHCO ₃ and 20% NaCl solutions were used to wash the product once respectively. The obtained organic phase was concentrated under reduced pressure and purified by chromatography to obtain pure eptifibatide impurity I with a yield of 85%.

cyclo [Trp-Pro]: HRMS (ESI) m/z calcd for C ₁₆ H ₁₇ N ₃ O ₂ Na ⁺ (M+Na) ⁺ 306.12130, found 306.12103.

Example 3

Step 1: Synthesis of Boc-Pro-O-TAG

Boc-Pro-OH (4.74 g, 22 mmol), EDCl (4.6 g, 24 mmol), and DMAP (292 mg, 2.4 mmol) were weighed in sequence and dissolved in 50 mL of dichloromethane. The mixture was stirred in an ice bath for 30 min. 4-diphenylphosphonyloxybenzyl alcohol label molecule (6.5 g, 20 mmol) was added, and the mixture was heated to room temperature and stirred for 2 h. The reaction endpoint was detected by TLC.

The reaction product was washed with saturated NH ₄ Cl solution and 10% Na ₂ CO ₃ solution, respectively, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to 5 mL. 50 mL of petroleum ether was added dropwise to produce a white precipitate and the solution was filtered off. The above precipitation operation was repeated 3 times to obtain a purified Boc-Pro-O-TAG intermediate for use with a yield of 97%.

Step 2: Synthesis of _H2N -Pro-O-TAG

The Boc-Pro-O-TAG prepared in step 1 was dissolved in 10 mL of dichloromethane solution, stirred at 0°C for 10 min, and then 10 mL of trifluoroacetic acid was added. The reaction was continued to be stirred at 0°C for 3 h and then the reaction was terminated. The reaction endpoint was detected by TLC.

The reaction solution was concentrated under reduced pressure, and the product was redissolved in 50 mL of dichloromethane, and then washed once with 10% NaHCO ₃ and 20% NaCl solutions in sequence. The obtained organic phase was concentrated under reduced pressure to obtain a purified H ₂ N-Pro-O-TAG intermediate for standby use, with a yield of 98%.

Step 3: Synthesis of Fmoc-Trp(Boc)-Pro-O-TAG

Fmoc-Trp(Boc)-OH (11.1 g, 21 mmol), EDCl (4.6 g, 24 mmol), and HOBt (3.24 g, 24 mmol) were weighed in sequence and dissolved in 30 mL of dichloromethane. The mixture was stirred in an ice bath for 30 min. H ₂ N-Pro-O-TAG prepared in step 2 was added. The mixture was heated to room temperature and stirred for 1 h. The reaction endpoint was detected by TLC.

The reaction product was washed with 10% Na ₂ CO ₃ solution and 20% NaCl solution, respectively, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to 5 mL. 50 mL of petroleum ether was added dropwise to produce a white precipitate and the solution was filtered off. The above precipitation operation was repeated 3 times to obtain the purified linear dipeptide Fmoc-Trp(Boc)-Pro-O-TAG for use with a yield of 98%.

Step 4: Synthesis of cyclo [Trp(Boc)-Pro]

The Fmoc-Trp(Boc)-Pro-O-TAG prepared in step 3 was dissolved in 10 mL of acetonitrile, 10 mL of diethylamine solution was added at 45 °C, and the Fmoc group was removed by stirring for 1 h. After concentration under reduced pressure, it was dissolved in 5 mL of dichloromethane, and 50 mL of petroleum ether was added dropwise to produce a white precipitate and the solution was filtered off. The above precipitation operation was repeated 3 times to obtain a purified cyclodipeptide derivative cyclo [Trp(Boc)-Pro] for use, with a yield of 95%.

HRMS (ESI) m/z calcd for C ₂₁ H ₂₆ N ₃ O ₄ Na ⁺ (M+Na) ⁺ 407.18155, found 407.18234.

Step 5: Synthesis of Eptifibatide Impurity I

The cyclo [Trp(Boc)-Pro] prepared in step 4 was dissolved in 10 mL of dichloromethane solution, stirred at 0°C for 10 min, and then 10 mL of trifluoroacetic acid was added. The reaction was continued to be stirred at 0°C for 3 h and then the reaction was terminated. The reaction endpoint was detected by TLC.

After the reaction solution was concentrated under reduced pressure, 50 mL of dichloromethane was used to redissolve the product, and then 10% NaHCO ₃ and 20% NaCl solutions were used to wash the product once each. The obtained organic phase was concentrated under reduced pressure and purified by chromatography to obtain pure eptifibatide impurity I with a yield of 84%.

In the above three embodiments, in Example 2, the 4-diphenylphosphinoyloxybenzyl alcohol tag molecule in Examples 1 and 3 is replaced by 4,4'-diphenylphosphinoyloxybenzophenone oxime. The overall yield of the three methods for preparing eptifibatide impurity I is comparable, but during the precipitation and purification of the product, the 4,4'-diphenylphosphinoyloxybenzophenone oxime tag molecule obviously exhibits a better precipitation effect, which can greatly improve the synthesis efficiency of the eptifibatide peptide chain. However, the 4,4'-diphenylphosphinoyloxybenzophenone oxime tag molecule has a higher synthesis cost than 4-diphenylphosphinoyloxybenzyl alcohol, so the economic efficiency of Example 2 is not as good as that of Examples 1 and 3.

In addition, the oxime ester bond formed by the ketoxime tag and the peptide chain is not as stable as the ester bond formed by benzyl alcohol. Therefore, the self-cleavage-cyclization efficiency is higher when the ketoxime tag is used to synthesize the impurity I of eptifibatide. However, when preparing the peptide chain of eptifibatide, the requirements for the environment, such as air humidity, are also higher.

Compared with Examples 1 and 2, Example 3 enlarged the preparation batch of eptifibatide impurity I, and the overall yield of eptifibatide impurity I after the enlarged batch remained stable, indicating that the tag molecule has great potential in the large-scale preparation of eptifibatide impurity I.

The above embodiments of the present invention do not describe all the details in detail, nor limit the present invention to the above embodiments. Various changes, modifications, substitutions and variations made by ordinary technicians in this field without departing from the principles and purpose of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A method for preparing eptifibatide impurity I based on diphenylphosphonooxy label in an assisted manner is to prepare linear dipeptide by using a label-assisted polypeptide synthesis method, and prepare the eptifibatide impurity I by self-cleavage-cyclization under alkaline conditions, and comprises the following steps:

S1: esterification coupling of Boc-Pro-OH with TAG

Under the action of an esterification coupling reagent, carrying out liquid-phase esterification coupling reaction on the TAG TAG molecules and the proline Boc-Pro-OH protected by Boc to generate an intermediate Boc-Pro-O-TAG loaded by the TAG TAG;

S2: removal of Boc protecting groups

Removing Boc protection from Boc-Pro-O-TAG under the action of an acidic deprotection reagent to obtain a Boc-removed intermediate H ₂ N-Pro-O-TAG;

S3: fmoc-Trp (Boc) -OH amidated coupling with H ₂ N-Pro-O-TAG

Under the action of an amide coupling reagent, carrying out amidation coupling reaction on H ₂ N-Pro-O-TAG and Fmoc-protected tryptophan Fmoc-Trp (Boc) -OH to generate a linear dipeptide Fmoc-Trp (Boc) -Pro-O-TAG loaded with a TAG label;

s4: linear dipeptide self-cleavage-cyclization

Under the action of an alkaline reagent, fmoc protecting groups of Fmoc-Trp (Boc) -Pro-O-TAG are removed, and TAG is removed from the cleavage-cyclization to form a cyclic dipeptide derivative cyclo [ Trp (Boc) -Pro ] with Boc protection;

S5: removal of Boc protecting groups

Under the action of an acidic deprotection reagent, removing Boc protection from the cyclo [ Trp (Boc) -Pro ], and purifying to obtain the cyclo [ Trp-Pro ], namely eptifibatide impurity I;

wherein the TAG TAG molecule is a benzophenone oxime compound based on diphenyl phosphonooxy group shown in the following structural general formula (I):

，

Or a diphenyl phosphonooxy group-based benzyl alcohol compound represented by the following structural formula (II):

，

Wherein the substituent R is selected from H, OPOPh ₂, C1-C3 alkyl, C1-C3 alkoxy, halogen atom or NO ₂.

2. The method according to claim 1, characterized in that the substituent R is selected from H, OPOPh ₂、CH₃、OCH₃, cl, F or NO ₂.

3. The method according to claim 1, wherein the esterification coupling reagent is one or more of EDCl/DMAP, DIC/DMAP and DCC/DMAP.

4. The method according to claim 1, wherein the amide coupling reagent is one or more of EDCl/HOBt, EDCl/HOBt/DIPEA, DIC/HOBt, DCC/HOSU, HATU/HOAt/DIPEA, pyBOP/DIPEA.

5. The method according to claim 1, characterized in that the acidic deprotection reagent is any one of dioxane/dichloromethane, trifluoroacetic acid/dichloromethane, hydrochloric acid/methanol.

6. The method according to claim 1, wherein the basic reagent is any one of a diethylamine/acetonitrile mixture, a piperidine/acetonitrile mixture, and a1, 8-diazabicyclo [5.4.0] undec-7-ene solution.

7. The method according to claim 1, wherein the esterification coupling reaction is performed at 0 to 40 ℃ with stirring for 0.5 to 10 hours.

8. The method according to claim 1, wherein the amidation coupling reaction is performed by stirring at 0 to 40 ℃ for 1 to 2 hours.

9. The process according to claim 1, wherein the reaction is stirred at 0-10 ℃ for 3-6 h to remove Boc protection.

10. The method according to claim 1, wherein the self-cleavage-cyclization reaction is carried out by stirring at 30 to 50 ℃ for 1 to 3 hours.