A diphenylphosphonoxybenzhydrylamine label-assisted liquid-phase eptifibatide total synthesis method

Abstract

A diphenylphosphonoxybenzhydrylamine tag-assisted liquid-phase eptifibatide total synthesis method utilizes a diphenylphosphonoxybenzhydrylamine tag instead of a solid-phase peptide synthesis polymer resin as a peptide synthesis carrier and amino acid carboxyl-terminal protecting group for eptifibatide total synthesis. The method includes amide coupling of the Fmoc-protected diphenylphosphonoxybenzhydrylamine tag with an amino acid, preparation of an eptifibatide peptide chain assisted by the diphenylphosphonoxybenzhydrylamine tag, cleavage and removal of the diphenylphosphonoxybenzhydrylamine tag and side chain protecting groups, and oxidative cyclization of linear eptifibatide to prepare eptifibatide. The diphenylphosphonoxybenzhydrylamine tag in the present invention has good solubility in the reaction medium and assists in peptide chain precipitation and purification, thereby achieving a simple, efficient, green and economical synthesis of eptifibatide.

Description

Technical Field

[0001] The present invention belongs to the field of organic synthesis and polypeptide chemical synthesis, and relates to a synthesis method of eptifibatide, in particular to a liquid-phase total synthesis method of eptifibatide based on a diphenylphosphonoxybenzhydrylamine label. Background Art

[0002] Recent epidemiological surveys have shown that the incidence of myocardial infarction in China is as high as 0.06%, making it a major killer among middle-aged and elderly people. Eptifibatide, a cyclic peptide cardiovascular drug, boasts high efficacy and low toxicity and side effects. It has been widely used clinically, and demand is increasing annually. As a novel GPIIb / IIIa antagonist, eptifibatide improves peripheral blood flow and tissue perfusion. It is recognized as a "gold medal" antiplatelet drug and plays an irreplaceable role in the treatment of acute coronary syndrome (ACS) and ST-segment elevation myocardial infarction.

[0003] Eptifibatide was originally developed by COR Therapeutic, a US company, and was launched in the US in 1998. Its compound molecule is a cyclic heptapeptide found in the venom of the Southeastern pygmy rattlesnake (Sistrurus miliarius barbouri). Its chemical name is N 6 -(aminoiminomethyl)-N 2 -(3-Mercapto-1-oxypropyl)-L-lysyl-glycyl-L-α-aspartyl-L-tryptophanyl-L-prolyl-L-cysteinamide, a cyclic (1→6) disulfide, is a synthetic cyclic peptide. Its amino acid sequence from the N→C terminus is: mercaptopropionic acid (Mpa1), homoarginine (Har2), glycine (Gly3), aspartic acid (Asp4), tryptophan (Trp5), proline (Pro6), and cysteine ​​(Cys7). The specific chemical structure and amino acid sequence are shown below.

[0004]

[0005] Eptifibatide is a carbon-terminally amidated peptide, meaning the carboxyl terminus of the peptide chain is amino-amidated. Currently, the synthesis of eptifibatide primarily involves liquid-phase peptide synthesis (LPPS) and solid-phase peptide synthesis (SPPS). During the carbon-to-nitrogen (C→N) extension of the eptifibatide peptide chain, the carboxyl group of the first amino acid (Cys) at the carboxyl terminus must first form an amide bond with a carrier group. This allows the peptide chain to be directly amidated after final cleavage.

[0006] Solid-phase synthesis of eptifibatide molecules primarily utilizes carbon-terminally amidated peptide synthesis resins formed by linking common polymers with the linker arm Rink Amide (chemical name: 2-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)(2,4-dimethoxyphenyl)methyl)phenoxy)acetic acid), such as Rink Amide MBHA resin, Rink Amide AM resin, or MBHA resin, for the synthesis of eptifibatide linear peptides. These resins all contain a dibenzhydrylamine reactive group that can directly amidate with the carboxyl group of an Fmoc amino acid to form an amide bond. After cleavage, terminally amidated peptide chains are directly obtained. Examples include the solid-phase preparation method for eptifibatide based on a Rink Amide MBHA resin support disclosed in CN 110498834A, and the preparation methods for eptifibatide based on a Fmoc-based resin support disclosed in CN 105585613A and CN 105037496A. However, the high price of solid-phase resin carriers, the complicated preparation route and the low loading rate (0.3-2.0 mmol / g) limit the large-scale production of eptifibatide.

[0007] In traditional liquid-phase synthesis of eptifibatide peptide chains, chromatography or recrystallization purification is typically required after each amino acid coupling, as exemplified by the methods for liquid-phase preparation of eptifibatide disclosed in CN 102924569A and CN 103450346A. Consequently, these liquid-phase eptifibatide preparation methods consume significant amounts of chemical reagents and time, resulting in the emission of reagent pollutants and hindering large-scale production and environmental protection.

[0008] With the growing market demand for eptifibatide APIs and the serious environmental pollution caused by the discharge of waste materials such as polymers and reagents during solid-phase and traditional liquid-phase peptide synthesis, the current synthesis of eptifibatide lacks a green, economical, and scalable preparation method, both in terms of economic cost and social benefit. Therefore, it is necessary to explore and develop green synthesis methods that can combine the advantages of liquid-phase and solid-phase peptide synthesis, thereby achieving scalable liquid-phase preparation and sustainable green production of carbon-terminal amidated eptifibatide. Summary of the Invention

[0009] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a liquid-phase total synthesis method of eptifibatide based on a diphenylphosphonoxybenzhydrylamine label-assisted method.

[0010] Compared with traditional solid-phase and liquid-phase synthesis of eptifibatide, the method of the present invention utilizes diphenylamine-type small molecule tags instead of traditional polymer resins as carriers to extend the eptifibatide peptide chain. Such tags can assist in the precipitation of the eptifibatide chain in a specific solvent to achieve the purpose of purification, which can greatly reduce the waste of chemical reagents, amino acids and coupling reagent raw materials, and mainly solve the green environmental protection and economic deficiencies faced by the current solid-phase and liquid-phase synthesis preparation processes of eptifibatide.

[0011] The diphenylphosphonoxybenzhydrylamine tag-assisted liquid-phase total synthesis of eptifibatide described in the present invention specifically uses diphenylphosphonoxybenzhydrylamine (TAG-NH2) represented by the following general structural formula (I) as the tag molecule:

[0012]

[0013] Wherein, the substituent R is selected from H, OPOPh2, C1-C3 alkyl, C1-C3 alkoxy, halogen atom or NO2.

[0014] Eptifibatide was prepared according to the following steps:

[0015] 1) Treating diphenylphosphinoyloxy Fmoc-diphenylmethane (TAG-NH-Fmoc) in an Fmoc-protected state represented by the following general structural formula (II) with a de-Fmoc reagent to remove the Fmoc protecting group to obtain the tag molecule diphenylphosphinoyloxy diphenylmethane;

[0016]

[0017] 2) Under the action of a coupling reagent, the tag molecule is subjected to an amide coupling reaction with Fmoc-protected cysteine ​​Fmoc-Cys(Trt)-OH to generate a tag-loaded intermediate compound Fmoc-Cys(Trt)-CONH-TAG, and the Fmoc protecting group is removed again to obtain the Fmoc-protected intermediate H2N-Cys(Trt)-CONH-TAG;

[0018] 3) Under the action of a coupling reagent, the Fmoc-protected intermediate H2N-Cys(Trt)-CONH-TAG is subjected to an amide coupling reaction with Fmoc-protected proline Fmoc-Pro-OH to obtain a tag-loaded intermediate compound Fmoc-Pro-Cys(Trt)-CONH-TAG, and the Fmoc protecting group is removed again to obtain the Fmoc-protected intermediate H2N-Pro-Cys(Trt)-CONH-TAG;

[0019] 4) Using Fmoc-Trp(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Har-OH and Mpa(Trt)-OH as raw materials, respectively, under the action of coupling reagents and de-Fmoc reagents, repeatedly performing amide coupling reactions and removing Fmoc protecting groups to obtain the tag-loaded eptifibatide precursor compound Mpa(Trt)-Har-Gly-Asp(tBu)-Trp(Boc)-Pro-Cys(Trt)-CONH-TAG;

[0020] 5) Using the eptifibatide precursor compound represented by Mpa(Trt)-Har-Gly-Asp(tBu)-Trp(Boc)-Pro-Cys(Trt)-CONH-TAG as a raw material, an acidic cleavage reagent is used for cleavage treatment to remove the diphenylphosphonooxybenzhydrylamine tag group and the Trt, tBu, and Boc protecting groups on the side chain of the eptifibatide precursor compound to obtain an unprotected linear eptifibatide compound Mpa-Har-Gly-Asp-Trp-Pro-Cys-CONH2;

[0021] 6) The linear eptifibatide compound is subjected to oxidative cyclization of the intramolecular disulfide bond in an alkaline solution, or in the presence of an oxidizing agent, to prepare the target eptifibatide product.

[0022] In addition to using fluorenylmethoxycarbonyl (Fmoc) to protect the α-amino groups of various amino acids used for amide coupling tag loading, this invention also protects reactive side-chain groups on amino acids. Specifically, the side-chain thiol groups of cysteine ​​(Cys) and mercaptopropionic acid (Mpa) are protected with a trityl (Trt) group, the side-chain indole group of tryptophan (Trp) is protected with a tert-butyloxycarbonyl (Boc) group, and the side-chain carboxyl group of aspartic acid (Asp) is protected with a tert-butyl (tBu) group. Furthermore, the side-chain guanidinium group of homoarginine (Har) can be protected with an aromatic sulfonyl Pbf protecting group, or the exposed guanidinium group can be left unprotected.

[0023] The Fmoc protecting group removal reaction is carried out by using a Fmoc removal reagent and stirring the reaction at 15 to 40° C. for 0.5 to 3 hours to remove the Fmoc group.

[0024] Furthermore, the Fmoc removal reagent is one or more of a diethylamine / acetonitrile mixture, a piperidine / acetonitrile mixture, and a 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) / dichloromethane mixture.

[0025] Furthermore, the present invention utilizes a low-polarity ether or alkane solvent to precipitate and purify the diphenylphosphonooxybenzhydrylamine tag molecule, each tag-loaded intermediate compound, and the Fmoc-deprotected intermediate. The tag molecule, the tag-loaded intermediate compound, and the Fmoc-deprotected intermediate all rapidly precipitate in the low-polarity ether or alkane solvent, and are then separated from impurities such as the removed Fmoc residue by solid-liquid filtration. Repeating this process multiple times can achieve simple and rapid purification.

[0026] Specifically, the low-polarity ether or alkane solvent is one or more of petroleum ether, n-hexane, cyclohexane, diethyl ether, and tert-butyl ether.

[0027] More specifically, in the present invention, before using a low-polarity ether or alkane solvent to precipitate and purify the reaction product, the reaction product may preferably be washed multiple times with a NaHCO3 aqueous solution and deionized water.

[0028] The amide coupling reaction of the present invention can be carried out under the action of various conventional coupling reagents, and the present invention is not particularly limited thereto. Preferably, the coupling reagent used in the present invention is one or more of EDCl / HOBt / DIEA, DCC / HOSU, DIC / HOBt, and PyBOP / DIEA.

[0029] Furthermore, the amide coupling reaction of the present invention is carried out using one or more of dichloromethane, chloroform and tetrahydrofuran as the solvent system.

[0030] Furthermore, the amide coupling reaction is preferably carried out at 0-40° C. with stirring for 0.5-5 h.

[0031] In the present invention, the eptifibatide precursor compound is subjected to a cleavage treatment with an acidic cleavage reagent for 3 to 5 hours to completely cleave and remove the diphenylphosphonoxybenzhydrylamine tag group and the Trt, tBu, and Boc protecting groups on the side chain of the eptifibatide precursor compound. The acidic cleavage reagent can be any conventional acidic cleavage reagent used for removing amino acid protecting groups and is not particularly limited in the present invention. For example, it can be a mixture of trifluoroacetic acid / 1,2-ethanedithiol / water / anisole (TFA / EDT / H2O / PhOMe), a mixture of trifluoroacetic acid / triisopropylsilane / water (TFA / Tis / H2O), or a mixture of hydrofluoric acid / p-cresol / 4-thiocresol (HF / p-cresol / p-thiocresol).

[0032] Furthermore, the unprotected linear eptifibatide compound obtained by cleavage of the eptifibatide precursor compound can be purified by precipitation using cold ether, cold isopropyl ether, or other ice-cold lower ether solvents.

[0033] Furthermore, the oxidative cyclization of the linear eptifibatide compound in the alkaline solution of the present invention is specifically to dissolve the linear eptifibatide compound in a DMSO / ultrapure water mixture, adjust the pH to alkaline with NH4OH, and stir at room temperature to carry out the oxidative cyclization of the disulfide bond within the linear eptifibatide molecule.

[0034] Furthermore, the linear eptifibatide compound of the present invention can also be oxidatively cyclized using an oxidizing agent, wherein the oxidizing agent is one or more of DMSO oxidation, air oxidation, hydrogen peroxide, and I2 oxidation.

[0035] Preferably, the present invention uses a preparative chromatography column to purify the finally prepared eptifibatide target product.

[0036] The present invention utilizes a readily soluble small molecule tag, diphenylphosphonoxybenzhydrylamine, as a carboxyl-terminal carrier for eptifibatide synthesis, replacing traditional solid-phase peptide synthesis resins. This provides a tag-assisted liquid-phase eptifibatide total synthesis method. The readily soluble small molecule tag not only serves as an amino acid carboxyl-terminal protecting group to assist in liquid-phase extension of the eptifibatide chain, but also allows the tag molecule itself and the attached eptifibatide chain to precipitate and crystallize in a specific solvent system, allowing the eptifibatide precursor to be purified through simple solid-liquid filtration separation.

[0037] Therefore, the eptifibatide synthesis process assisted by the soluble small molecule tag of the present invention not only realizes the liquid-phase amide coupling reaction, but also completes the solid-liquid precipitation separation and purification, fully combining the advantages of solid-phase peptide synthesis SPPS and liquid-phase peptide synthesis LPPS, and solves the problems faced by eptifibatide in the preparation of SPPS and LPPS, such as large waste of raw materials, high economic cost, difficulty in large-scale production and preparation, and large discharge of solid-phase polymer resin waste causing serious environmental pollution. It realizes the green synthesis of eptifibatide in a simple and efficient manner, and is highly consistent with the "Guiding Opinions on Promoting the Green Development of the API Industry" jointly issued by four national departments. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a flow chart of the diphenylphosphonoxybenzhydrylamine label-assisted liquid-phase total synthesis of eptifibatide. Implementation Method

[0039] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention so that those skilled in the art can better understand and utilize the present invention, but are not intended to limit the scope of protection of the present invention.

[0040] Unless otherwise specified, the production processes, experimental methods or detection methods involved in the embodiments of the present invention are all conventional methods in the prior art, and their names and / or abbreviations are conventional names in the field and are very clear and unambiguous in the relevant fields of use. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment to implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0041] 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 prepared according to conventional methods well known to those skilled in the art.

[0042] like Figure 1 As shown, the present invention embodiment synthesizes eptifibatide according to the following route.

[0043] (1) Deprotection of diphenylphosphonooxy Fmoc-diphenylmethane to prepare small molecule diphenylphosphonooxy diphenylmethane tag;

[0044]

[0045] (2) The diphenylphosphonooxybenzhydrylamine tag molecule is coupled to the Fmoc amino acid amide;

[0046]

[0047] (3) Preparation of eptifibatide peptide chain assisted by diphenylphosphonooxybenzhydrylamine label;

[0048]

[0049] (4) Cleavage and removal of diphenylphosphonooxybenzhydrylamine tag and side chain protecting groups;

[0050]

[0051] (5) Preparation of eptifibatide by oxidative cyclization of linear eptifibatide;

[0052]

[0053] The specific meanings of some commonly used abbreviations appearing in the specification of the present invention are as follows.

[0054] Fmoc: fluorenylmethoxycarbonyl.

[0055] EDCl: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0056] HOBt: 1-hydroxybenzotriazole.

[0057] DCC: N,N-dicyclohexylcarbonyldiimide.

[0058] DIC: 1,3-diisopropylcarbodiimide.

[0059] PyBOP: 1H-Benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate.

[0060] DIEA: N,N-diisopropylethylamine.

[0061] HOSU: N-hydroxysuccinimide.

[0062] Trt: trityl.

[0063] Boc: tert-butyloxycarbonyl.

[0064] tBu: tert-butyl.

[0065] TAG: tag. Example

[0066] Example 1

[0067] Step 1: Deprotection of 4,4'-bis(diphenylphosphinoyloxy)-Fmoc-diphenylmethane to prepare TAG-NH2 tag.

[0068] Weigh Fmoc-protected 4,4'-bis(diphenylphosphinoyloxy)-Fmoc-dibenzhydrylamine (8.4 g, 10 mmol), add it to 15 mL of acetonitrile solution and stir at room temperature. Then add 5 mL of diethylamine (DEA) to the solution system and continue stirring for 1 hour.

[0069] After the removal of Fmoc, the solvent was removed by concentration under reduced pressure. The residue was precipitated with petroleum ether three times and filtered and purified to obtain 6.1 g of the target product, 4,4'-bis(diphenylphosphinoyloxy)benzhydrylamine, with a yield of approximately 99%.

[0070] 1 H NMR (400 MHz, CDCl3), d 7.89-7.84 (m, 8H), 7.53-7.50 (m, 4H), 7.46-7.42 (m, 8H), 7.16-7.08 (m, 8H), 5.02 (s, 1H), 1.80 (s, 2H)ppm; 31 P NMR (162MHz, CDCl3), d 30.44 ppm; 13 C NMR (100 MHz, CDCl3), d149.7, 141.6, 132.5, 131.8,130.3, 128.7, 128.2, 120.7, 58.4ppm.

[0071] HRMS (ESI) m / z calcd for C 37 H 31 NO4P2Na + (M+Na) + 638.16205, found 638.16229.

[0072] Step 2: Fmoc-Cys(Trt)-CONH-TAG was prepared by coupling 4,4'-bis(diphenylphosphinoyloxy)benzhydrylamine tag with Fmoc-Cys(Trt)-OH amide.

[0073] Fmoc-Cys(Trt)-OH (6.38 g, 10.9 mmol), EDCl (2.27 g, 11.9 mmol), and HOBt (1.61 g, 11.9 mmol) were weighed in sequence and dissolved in 30 mL of dichloromethane. The mixture was stirred in an ice bath for 30 min. 4,4'-bis(diphenylphosphinoyloxy)benzhydrylamine (6.1 g, 9.9 mmol) was then added. The mixture was heated to room temperature and stirred for 1 h. The reaction endpoint was detected by TLC.

[0074] The reaction product was washed with 20% NaCl solution and 20% Na2CO3 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 three times to obtain 11.1 g of purified Fmoc-Cys(Trt)-CONH-TAG with a yield of approximately 95%.

[0075] HRMS (ESI) m / z calcd for C 74 H 60 N2O7P2SNa + (M+Na) + 1205.34887, found1205.34985.

[0076] Step 3: Preparation of eptifibatide peptide chain MPa(Trt)-Har-Gly-Asp(tBu)-Trp(Boc)-Pro-Cys(Trt)-CONH-TAG by 4,4'-bis(diphenylphosphinoyl)benzhydrylamine tag-assisted extension.

[0077] Repeat the Fmoc deprotection method in step 1 and use 25% diethylamine / acetonitrile system to remove the Fmoc protecting group on the coupling product of 4,4'-bis(diphenylphosphinoyl)benzhydrylamine and amino acid Fmoc-Cys(Trt)-CONH-TAG obtained in step 2 to obtain the deprotected product.

[0078] The amide coupling reaction in step 2 was repeated, and the amide coupling of Fmoc-Pro-OH was performed using an EDCl / HOBt coupling reagent system to obtain the TAG-loaded dipeptide intermediate Fmoc-Pro-Cys(Trt)-CONH-TAG.

[0079] The above-mentioned de-Fmoc protection and amide coupling were used as a cycle, and the above steps were continuously repeated. Fmoc-Trp(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Har-OH and Mpa(Trt)-OH were sequentially coupled to the de-Fmoc protected intermediate by amide coupling, and finally 11.8 g of eptifibatide precursor compound MPa(Trt)-Har-Gly-Asp(tBu)-Trp(Boc)-Pro-Cys(Trt)-CONH-TAG loaded with 4,4'-bis(diphenylphosphinoyloxy)benzhydrylamine tag was prepared, with a total yield of about 57%.

[0080] HRMS (ESI) m / z calcd for C 119 H 124 N 11 O 15 P2S2 + (M+H) + 2073.82231, found2073.82446.

[0081] Step 4: Cleavage and removal of the label and side chain protecting groups on the precursor compound to prepare linear eptifibatide MPa-Har-Gly-Asp-Trp-Pro-Cys-CONH2.

[0082] The linear eptifibatide precursor compound Mpa(Trt)-Har-Gly-Asp(tBu)-Trp(Boc)-Pro-Cys(Trt)-CONH-TAG (11.8 g, 5.7 mmol) prepared in step 3 above and containing a tag and a side chain protecting group was added to a 100 mL round-bottom flask, and then 20 mL of a cleavage reagent prepared by mixing trifluoroacetic acid / 1,2-ethanedithiol / water / anisole (TFA / EDT / H2O / PhOMe=90 / 2.5 / 2.5 / 5, v / v / v / v) was added, and the reaction was stirred at room temperature for 3 h.

[0083] After the cleavage reaction, the mixture was concentrated under reduced pressure at room temperature to remove most of the trifluoroacetic acid. 30 mL of cold ether was added to the concentrate to precipitate a white precipitate. Centrifugation afforded a white solid, which was then washed with cold ether and assisted by ultrasonic precipitation three times. After drying, 24.56 g of crude linear eptifibatide peptide MPa-Har-Gly-Asp-Trp-Pro-Cys-CONH was obtained with a yield of 96%.

[0084] HRMS (ESI) m / z calcd for C 35 H 51 N 11 O9S2Na + (M+Na) + 856.32048, found856.32135.

[0085] Step 5: Prepare eptifibatide by oxidative cyclization of linear eptifibatide.

[0086] Linear eptifibatide Mpa-Har-Gly-Asp-Trp-Pro-Cys-CONH2 (830 mg, 1.0 mmol) was dissolved in 1000 mL of 5% DMSO aqueous solution, and NH4OH solution was added dropwise to adjust the pH to 8. The reaction was stirred at room temperature for 12 h. The cyclization process was monitored by HPLC. After the cyclization was completed, the reaction was stopped to obtain a crude eptifibatide mixture.

[0087] The crude mixture was concentrated and purified by preparative chromatography (chromatographic conditions: mobile phase: acetonitrile / water 0.1% TFA; C18 column; wavelength 220 nm, flow rate 5 mL / min) to obtain 515 mg of the target product eptifibatide with a yield of 62%.

[0088] Example 2

[0089] Step 1: Deprotection of 4-diphenylphosphonooxy-Fmoc-diphenylmethane to prepare TAG-NH2 tag.

[0090] Weigh Fmoc-protected 4-diphenylphosphonooxy-Fmoc-dibenzhydrylamine (6.2 g, 10 mmol), add it to 15 mL of acetonitrile solution and stir at room temperature. Then add 5 mL of diethylamine (DEA) to the solution system and continue stirring for 1 hour.

[0091] After the Fmoc removal was completed, the solvent was removed by concentration under reduced pressure. The residue was precipitated with petroleum ether three times and filtered and purified to obtain 3.9 g of the target tagged molecule, 4-diphenylphosphinoyldibenzhydrylamine, with a yield of 98%.

[0092] Step 2: 4-diphenylphosphinoylbenzhydrylamine tag molecule is coupled with Fmoc-Cys(Trt)-OH amide to prepare Fmoc-Cys(Trt)-CONH-TAG.

[0093] Fmoc-Cys(Trt)-OH (6.31 g, 10.8 mmol), EDCl (2.25 g, 11.8 mmol), and HOBt (1.60 g, 11.8 mmol) were weighed in sequence and dissolved in 30 mL of dichloromethane. The mixture was stirred in an ice bath for 30 min. 4-Diphenylphosphinoyloxybenzhydrylamine (3.9 g, 9.8 mmol) was then added. The mixture was heated to room temperature and stirred for 1 h. The reaction endpoint was detected by TLC.

[0094] The reaction product was washed with 20% NaCl solution and 20% Na2CO3 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 three times to obtain 9.1 g of purified Fmoc-Cys(Trt)-CONH-TAG with a yield of 96%.

[0095] Step 3: Preparation of eptifibatide peptide chain Mpa(Trt)-Har-Gly-Asp(tBu)-Trp(Boc)-Pro-Cys(Trt)-CONH-TAG by 4-diphenylphosphonoxybenzhydrylamine tag molecule-assisted extension.

[0096] Repeat the Fmoc deprotection method in step 1, and use 25% diethylamine / acetonitrile system to remove the Fmoc protecting group on the coupling product of 4-diphenylphosphonoxybenzhydrylamine and amino acid Fmoc-Cys(Trt)-CONH-TAG obtained in step 2 to obtain the deprotected product.

[0097] The amide coupling reaction in step 2 was repeated, and the amide coupling of Fmoc-Pro-OH was performed using an EDCl / HOBt coupling reagent system to obtain the TAG-loaded dipeptide intermediate Fmoc-Pro-Cys(Trt)-CONH-TAG.

[0098] The above-mentioned de-Fmoc protection and amide coupling were used as a cycle, and the above steps were continuously repeated. Fmoc-Trp(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Har-OH and Mpa(Trt)-OH were sequentially amide-coupled on the de-Fmoc protected intermediate to finally prepare 10.2 g of eptifibatide precursor compound MPa(Trt)-Har-Gly-Asp(tBu)-Trp(Boc)-Pro-Cys(Trt)-CONH-TAG loaded with 4-diphenylphosphinoylbenzhydrylamine tag molecule with a total yield of 55%.

[0099] Step 4: Cleavage and removal of the label molecule and side chain protecting groups on the precursor compound to prepare linear eptifibatide Mpa-Har-Gly-Asp-Trp-Pro-Cys-CONH2.

[0100] The linear eptifibatide precursor compound Mpa(Trt)-Har-Gly-Asp(tBu)-Trp(Boc)-Pro-Cys(Trt)-CONH-TAG (10.2 g, 5.5 mmol) prepared in step 3 above and containing a tag molecule and a side chain protecting group was added to a 100 mL round-bottom flask, and then 20 mL of a cleavage reagent prepared by mixing trifluoroacetic acid / 1,2-ethanedithiol / water / anisole (TFA / EDT / H2O / PhOMe=90 / 2.5 / 2.5 / 5, v / v / v / v) was added, and the reaction was stirred at room temperature for 3 h.

[0101] After the cleavage reaction, the mixture was concentrated under reduced pressure at room temperature to remove most of the trifluoroacetic acid. 30 mL of cold ether was added to the concentrate to precipitate a white precipitate. Centrifugation yielded a white solid, which was then washed with cold ether and assisted by ultrasonic precipitation three times. After drying, 24.35 g of crude linear eptifibatide peptide MPa-Har-Gly-Asp-Trp-Pro-Cys-CONH was obtained with a yield of 96%.

[0102] Step 5: Prepare eptifibatide by oxidative cyclization of linear eptifibatide.

[0103] Linear eptifibatide Mpa-Har-Gly-Asp-Trp-Pro-Cys-CONH2 (830 mg, 1.0 mmol) was dissolved in 1000 mL of 5% DMSO aqueous solution, and NH4OH solution was added dropwise to adjust the pH to 8. The reaction was stirred at room temperature for 12 h. The cyclization process was monitored by HPLC. After the cyclization was completed, the reaction was stopped to obtain a crude eptifibatide mixture.

[0104] The crude mixture was concentrated and purified by preparative chromatography (chromatographic conditions: mobile phase: acetonitrile / water 0.1% TFA; C18 column; wavelength 220 nm, flow rate 5 mL / min) to obtain 523 mg of the target product eptifibatide with a yield of 63%.

[0105] Example 3

[0106] Step 1: Deprotection of 4,4'-bis(diphenylphosphinoyloxy)-Fmoc-diphenylmethane to prepare TAG-NH2 tag molecule.

[0107] Weigh Fmoc-protected 4,4'-bis(diphenylphosphinoyloxy)-Fmoc-dibenzhydrylamine (8.4 g, 10 mmol), add it to 15 mL of acetonitrile solution and stir at room temperature. Then add 5 mL of diethylamine (DEA) to the solution system and continue stirring for 1 hour.

[0108] After Fmoc removal, the solvent was removed by concentration under reduced pressure. The residue was precipitated with petroleum ether three times and filtered and purified to obtain 6.1 g of the target tagged molecule, 4,4'-bis(diphenylphosphinoyloxy)benzhydrylamine, with a yield of approximately 99%.

[0109] Step 2: 4,4'-bis(diphenylphosphinoyloxy)benzhydrylamine tag molecule is coupled with Fmoc-Cys(Trt)-OH amide to prepare Fmoc-Cys(Trt)-CONH-TAG.

[0110] Fmoc-Cys(Trt)-OH (6.38 g, 10.9 mmol), EDCl (2.27 g, 11.9 mmol), and HOBt (1.61 g, 11.9 mmol) were weighed in sequence and dissolved in 30 mL of dichloromethane. The mixture was stirred in an ice bath for 30 min. 4,4'-bis(diphenylphosphinoyloxy)benzhydrylamine (6.1 g, 9.9 mmol) was then added. The mixture was heated to room temperature and stirred for 1 h. The reaction endpoint was detected by TLC.

[0111] The reaction product was washed with 20% NaCl solution and 20% Na2CO3 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 three times to obtain 11.1 g of purified Fmoc-Cys(Trt)-CONH-TAG with a yield of approximately 95%.

[0112] Step 3: Preparation of eptifibatide peptide chain Mpa(Trt)-Har-Gly-Asp(tBu)-Trp(Boc)-Pro-Cys(Trt)-CONH-TAG by 4,4'-bis(diphenylphosphinoyl)benzhydrylamine tag molecule-assisted extension.

[0113] Repeat the Fmoc deprotection method in step 1 and use 25% diethylamine / acetonitrile system to remove the Fmoc protecting group on the coupling product of 4,4'-bis(diphenylphosphinoyl)benzhydrylamine and amino acid Fmoc-Cys(Trt)-CONH-TAG obtained in step 2 to obtain the deprotected product.

[0114] The amide coupling reaction in step 2 was repeated, and the amide coupling of Fmoc-Pro-OH was performed using an EDCl / HOBt coupling reagent system to obtain the TAG-loaded dipeptide intermediate Fmoc-Pro-Cys(Trt)-CONH-TAG.

[0115] The above-mentioned de-Fmoc protection and amide coupling were used as a cycle, and the above steps were continuously repeated. Fmoc-Trp(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Har-OH and Mpa(Trt)-OH were sequentially amide-coupled on the de-Fmoc protected intermediate to finally prepare 11.8 g of eptifibatide precursor compound MPa(Trt)-Har-Gly-Asp(tBu)-Trp(Boc)-Pro-Cys(Trt)-CONH-TAG loaded with 4,4'-bis(diphenylphosphinoyloxy)benzhydrylamine tag molecule, with a total yield of about 57%.

[0116] Step 4: Cleavage and removal of the label molecule and side chain protecting groups on the precursor compound to prepare linear eptifibatide Mpa-Har-Gly-Asp-Trp-Pro-Cys-CONH2.

[0117] The linear eptifibatide precursor compound Mpa(Trt)-Har-Gly-Asp(tBu)-Trp(Boc)-Pro-Cys(Trt)-CONH-TAG (11.8 g, 5.7 mmol) prepared in step 3 above and containing a tag molecule and a side chain protecting group was added to a 100 mL round-bottom flask, and then 20 mL of a cleavage reagent prepared by mixing trifluoroacetic acid / 1,2-ethanedithiol / water / anisole (TFA / EDT / H2O / PhOMe=90 / 2.5 / 2.5 / 5, v / v / v / v) was added, and the reaction was stirred at room temperature for 3 h.

[0118] After the cleavage reaction, the mixture was concentrated under reduced pressure at room temperature to remove most of the trifluoroacetic acid. 30 mL of cold ether was added to the concentrate to precipitate a white precipitate. Centrifugation afforded a white solid, which was then washed with cold ether and assisted by ultrasonic precipitation three times. After drying, 24.56 g of crude linear eptifibatide peptide MPa-Har-Gly-Asp-Trp-Pro-Cys-CONH was obtained with a yield of 96%.

[0119] Step 5: Prepare eptifibatide by oxidative cyclization of linear eptifibatide.

[0120] Linear eptifibatide Mpa-Har-Gly-Asp-Trp-Pro-Cys-CONH2 (830 mg, 1.0 mmol) was dissolved in 1000 mL of water, and glacial acetic acid solution was added dropwise to adjust the pH to 6.0. The reaction was carried out in an open air oxidation reaction at room temperature. The cyclization process was monitored by HPLC. After the cyclization was completed, the reaction was stopped to obtain a crude eptifibatide mixture.

[0121] The crude mixture was concentrated and purified by preparative chromatography (chromatographic conditions: mobile phase: acetonitrile / water 0.1% TFA; C18 column; wavelength 220 nm, flow rate 5 mL / min) to obtain 515 mg of the target product eptifibatide with a yield of 62%.

[0122] In the above three embodiments, Example 2 replaces the 4,4'-bis(diphenylphosphinoyloxy)benzhydrylamine tag molecule with 4-diphenylphosphinoyloxybenzhydrylamine compared to Example 1 and Example 3. The overall synthesis yield of linear eptifibatide is comparable, but during the precipitation and purification of the product, the 4,4'-bis(diphenylphosphinoyloxy)benzhydrylamine tag molecule significantly exhibits a better precipitation effect, which can greatly improve the synthesis efficiency of the eptifibatide peptide chain. However, the 4,4'-bis(diphenylphosphinoyloxy)benzhydrylamine tag molecule has a higher synthesis cost than 4-diphenylphosphinoyloxybenzhydrylamine, so the economic efficiency of Examples 1 and 3 is not as good as that of Example 2.

[0123] In Example 3, compared with Example 1 and Example 2, the oxidative cyclization method of linear eptifibatide was changed. Example 1 and Example 2 used DMSO oxidative cyclization, while Example 3 used air oxidative cyclization. The yields of the two oxidative cyclizations were comparable.

[0124] The above embodiments of the present invention do not describe all details in detail, nor do they limit the present invention to the above embodiments. Various changes, modifications, substitutions, and variations made by those skilled in the art without departing from the principles and purpose of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. A diphenylphosphonoxybenzhydrylamine tag-assisted liquid-phase total synthesis of eptifibatide, using diphenylphosphonoxybenzhydrylamine represented by the following general structural formula (I) as the tag molecule: ， in, The substituent R is selected from H or OPOPh2; Eptifibatide was prepared according to the following steps: 1) Treating diphenylphosphinoyloxy Fmoc-benzhydrylamine in an Fmoc-protected state represented by the following general structural formula (II) with a de-Fmoc reagent to remove the Fmoc protecting group, followed by precipitation and purification with petroleum ether to obtain the tag molecule diphenylphosphinoyloxybenzhydrylamine; ， 2) In the presence of a coupling reagent, the tag molecule is subjected to an amide coupling reaction with Fmoc-protected cysteine ​​Fmoc-Cys(Trt)-OH, and petroleum ether precipitation is used to obtain a tag-loaded intermediate compound Fmoc-Cys(Trt)-CONH-TAG. The Fmoc protecting group is removed again, and petroleum ether precipitation is used to obtain a de-Fmoc-protected intermediate H2N-Cys(Trt)-CONH-TAG. 3) Under the action of a coupling reagent, the Fmoc-protected intermediate H2N-Cys(Trt)-CONH-TAG is subjected to an amide coupling reaction with Fmoc-protected proline Fmoc-Pro-OH, and the tag-loaded intermediate compound Fmoc-Pro-Cys(Trt)-CONH-TAG is obtained by precipitation with petroleum ether. The Fmoc protecting group is removed again, and the Fmoc-protected intermediate H2N-Pro-Cys(Trt)-CONH-TAG is obtained by precipitation with petroleum ether. 4) Using Fmoc-Trp(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Har-OH and Mpa(Trt)-OH as raw materials, respectively, under the action of coupling reagents and de-Fmoc reagents, repeatedly performing amide coupling reactions and removing Fmoc protecting groups to obtain the tag-loaded eptifibatide precursor compound Mpa(Trt)-Har-Gly-Asp(tBu)-Trp(Boc)-Pro-Cys(Trt)-CONH-TAG; 5) Using the eptifibatide precursor compound represented by Mpa(Trt)-Har-Gly-Asp(tBu)-Trp(Boc)-Pro-Cys(Trt)-CONH-TAG as a raw material, an acidic cleavage reagent is used for cleavage treatment to remove the diphenylphosphonooxybenzhydrylamine tag group and the Trt, tBu, and Boc protecting groups on the side chain of the eptifibatide precursor compound to obtain an unprotected linear eptifibatide compound Mpa-Har-Gly-Asp-Trp-Pro-Cys-CONH2; 6) The linear eptifibatide compound is subjected to oxidative cyclization of the intramolecular disulfide bond in an alkaline solution, or in the presence of an oxidizing agent, to prepare the target eptifibatide product.

2. The diphenylphosphonoxybenzhydrylamine label-assisted liquid phase eptifibatide total synthesis method according to claim 1, characterized in that One or more of diethylamine / acetonitrile mixture, piperidine / acetonitrile mixture, and 1,8-diazabicyclo[5.4.0]undec-7-ene / dichloromethane mixture are used as Fmoc removal reagents, and the reaction is stirred at 15-40° C. for 0.5-3 h to remove the Fmoc protecting group.

3. The diphenylphosphonoxybenzhydrylamine label-assisted liquid phase eptifibatide total synthesis method according to claim 1, characterized in that One or more of dichloromethane, chloroform, and tetrahydrofuran are used as solvents, and one or more of EDCl / HOBt / DIEA, DCC / HOSU, DIC / HOBt, and PyBOP / DIEA are used as coupling reagents. The amide coupling reaction is carried out at 0-40° C. with stirring for 0.5-5 h.

4. The diphenylphosphonoxybenzhydrylamine label-assisted liquid-phase total synthesis of eptifibatide according to claim 1, characterized in that a trifluoroacetic acid / 1,2-ethanedithiol / water / anisole mixture, a trifluoroacetic acid / triisopropylsilane / water mixture, or a hydrofluoric acid / p-methylphenol / 4-toluenethiophenol mixture is used as an acidic cleavage reagent to cleave the eptifibatide precursor compound for 3 to 5 hours to remove the label group and side chain protecting groups on the eptifibatide precursor compound.

5. The diphenylphosphonoxybenzhydrylamine label-assisted liquid phase eptifibatide total synthesis method according to claim 1, characterized in that The unprotected linear eptifibatide compound was purified by precipitation using ice-cold lower ether solvents.

6. The diphenylphosphonoxybenzhydrylamine label-assisted liquid phase eptifibatide total synthesis method according to claim 5, characterized in that The ice-cold lower ether solvent is cold ether or cold isopropyl ether.

7. The diphenylphosphonoxybenzhydrylamine label-assisted liquid phase eptifibatide total synthesis method according to claim 1, characterized in that The oxidizing agent is one or more of DMSO, air, hydrogen peroxide, and I2.

8. The diphenylphosphonoxybenzhydrylamine label-assisted liquid-phase eptifibatide total synthesis method according to claim 1, characterized in that Eptifibatide was purified using preparative chromatography.

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