A preparation method of eptifibatide impurity I

Abstract

The present invention relates to the field of polypeptide technology, and in particular to a method for preparing eptifibatide impurity I. The preparation process of the present invention has high purity and yield of eptifibatide impurity I, a simple synthesis route, good controllability, low cost, and is easy for industrial production. Experiments show that the total yield of eptifibatide impurity I obtained by the preparation method of the present invention reaches 56%, and the purity is above 98%.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and in particular to a method for preparing eptifibatide impurity I. Background Art

[0002] Eptifibatide is a cyclic heptapeptide found in the venom of the dwarf rattlesnake. It can selectively block the binding of glycoprotein IIb / IIIa to adhesion proteins, has a certain weak inhibitory effect on adhesion-related receptors, and can prevent platelet aggregation caused by adenosine diphosphate and adrenaline at low concentrations. It is mainly used clinically for acute coronary syndrome and percutaneous coronary intervention. Its peptide sequence is as follows:

[0003]

[0004] Impurity I is produced during the preparation and storage of eptifibatide, thus affecting the safety of the drug. In the process of developing the production process of eptifibatide, it is necessary to prepare a reference substance of impurity I for the study of its product quality. The structure of impurity I is as follows:

[0005]

[0006] There are a large number of literature reports on methods for preparing eptifibatide, but there is no relevant literature report on methods for preparing impurity I. Summary of the invention

[0007] In view of this, the present invention provides a method for preparing eptifibatide impurity I. The total yield of eptifibatide impurity I obtained by the preparation method of the present invention is 56%, the purity is above 98%, the synthesis route is simple, the cost is low, and it is easy to industrialize production.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] A method for preparing eptifibatide impurity I, characterized by comprising:

[0010] Step 1: coupling protected amino acids X-Pro-OH and Y-Trp(Y)-OH one by one from the C-terminus to the N-terminus on a solid phase resin to obtain a peptide resin, which is cleaved by a cleavage solution to obtain compound 1 of formula I;

[0011] Step 2: Under the catalytic action of an organic base, compound 1 reacts with 2-bromoacetophenone to obtain compound 2 of formula II;

[0012] Step 3: removing the Boc protecting group from the compound 2 to obtain a compound 3 having a structure of formula III;

[0013] Step 4: Compound 3 is reacted under the catalytic action of an organic base to obtain a crude product of eptifibatide impurity I, which is then purified by HPLC to obtain eptifibatide impurity I having a structure of formula IV;

[0014] The lysate is a combination of TFE and DCM, or a combination of TFA, TIS, and DCM;

[0015]

[0016] The resin of the present invention is 2-Cl-Trt resin. In some specific embodiments, the resin is 2-Cl-Trt resin, and the substitution degree of the resin is preferably 0.1 to 1.6 mmol / g.

[0017] The present invention further comprises the step of swelling the solid phase resin with a solvent before coupling the protected amino acids X-Pro-OH and Y-Trp(Y)-OH one by one from the C-terminus to the N-terminus on the solid phase resin in step 1. The solvent used for the swelling can be any type well known in the art, including but not limited to DMF.

[0018] The swollen resin is coupled with protected amino acid X-Pro-OH to obtain X-Pro-resin.

[0019] Wherein, in X-Pro-OH and Y-Trp(Y)-OH, X is Fmoc and Y is Boc. In some specific embodiments, X is Fmoc and Y is Boc.

[0020] Before coupling Y-Trp(Y)-OH on X-Pro-resin, the X protecting group at the N-terminus of X-Pro-resin is first removed with a removing agent, and then Y-Trp(Y)-OH is coupled to obtain the peptide resin Y-Trp(Y)-Pro-resin.

[0021] In some embodiments, X is Fmoc, and the removing agent used to remove the Fmoc protecting group is a 20% by volume piperidine solution; the solvent of the piperidine solution is one or more of NMP, THF, DCM and DMF. In some specific embodiments, the solvent of the piperidine solution is DMF.

[0022] In the present invention, the coupling agent used to couple the protected amino acids X-Pro-OH and Y-Trp(Y)-OH on the solid phase resin is selected from any one of the following combinations:

[0023] DIPCDI and HOBt; PyBop, HOBt and DIPEA; HBTU, HOBt and DIPEA; DIPCDI and HOAt; HATU, HOAt and DIPEA; PyAop, HOAt and DIPEA.

[0024] The obtained peptide resin is cleaved with a cleavage solution to obtain compound 1 of formula I.

[0025] In some specific embodiments, the lysate is composed of TFE and DCM in a volume ratio of 2:8. In other specific embodiments, the lysate is composed of TFA, TIS and DCM in a volume ratio of 1:1:98.

[0026] After obtaining compound 1, under the catalytic action of an organic base, compound 1 is reacted with 2-bromoacetophenone to obtain compound 2 of formula II structure. Wherein, the organic base is one or more of triethylamine, diisopropylethylamine, pyridine and N-methylmorpholine. In some specific embodiments, the organic base is triethylamine.

[0027] After obtaining compound 2, the Boc protecting group of compound 2 is removed in the presence of an acid to obtain compound 3. The acid is one or more of hydrogen chloride, sulfuric acid and trifluoroacetic acid. In some specific embodiments, the acid is hydrogen chloride.

[0028] In some embodiments, the solvent in the reaction system for generating compound 3 can be any common solvent in the art, and can be selected from a combination of one or more of ethyl acetate, methanol, ethanol, tetrahydrofuran, dichloromethane, ether, methyl tert-butyl ether, isopropyl acetate, commercially available ethyl acetate solution of hydrogen chloride, methanol solution of hydrogen chloride, ethanol solution of hydrogen chloride, aqueous solution of hydrogen chloride. A mixed solvent of aqueous hydrogen chloride solution + methanol or ethanol or tetrahydrofuran is acceptable. Including but not limited to this.

[0029] The obtained compound 3 is reacted under the catalytic action of an organic base to obtain a crude product of eptifibatide impurity I. In some embodiments, the organic base is one or more of triethylamine, diisopropylethylamine, pyridine and N-methylmorpholine. In some specific embodiments, the organic base is triethylamine.

[0030] The crude product of eptifibatide impurity I is purified by HPLC to obtain eptifibatide impurity I of formula IV.

[0031] The chromatographic conditions of the HPLC purification are:

[0032] Octadecylsilane bonded silica gel was used as the stationary phase, 0.2% acetic acid solution was used as mobile phase A, methanol was used as mobile phase B, the detection wavelength was 280 nm, and gradient elution was used.

[0033] The invention adopts a new synthesis route, and the purity and yield of the obtained eptifibatide impurity I are both high, the synthesis route is simple, the controllability is good, the cost is low, and it is easy to industrialize production. Experiments show that the total yield of the eptifibatide impurity I obtained by the preparation method of the invention is 56%, and the purity is above 98%. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 shows the mass spectrum of compound 1;

[0035] Figure 2 shows the mass spectrum of compound 2;

[0036] Figure 3 shows the mass spectrum of compound 3;

[0037] Figure 4 The typical chromatogram of the refined peptide of impurity I of eptifibatide is shown;

[0038] Figure 5 4 shows the mass spectrum of impurity I of eptifibatide;

[0039] Figure 6 The impurity I of eptifibatide 1 H NMR spectrum;

[0040] Figure 7 The impurity I of eptifibatide 13 C NMR spectrum. DETAILED DESCRIPTION

[0041] The present invention provides a method for preparing impurity I of eptifibatide. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0042] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0043] The present invention provides a method for preparing an impurity I of eptifibatide, comprising the following steps:

[0044] 1. Synthesis of Compound 1

[0045] (1) Place the resin (such as 2-Cl-Trt resin) into a solid phase reaction column, wash twice with a solvent, swell for 30 minutes, and remove the solution. Dissolve Fmoc-Pro-OH and DIPEA in a solvent, add them to the reaction column, react at room temperature for 2 hours, then add methanol, react at room temperature for 1 hour, remove the solution, and wash with the solvent three times. Add 20% piperidine solution to remove the Fmoc protecting group, stir at room temperature for 5 minutes, remove the solution, repeat the operation once, and wash with the solvent six times.

[0046] (2) Boc-Trp(Boc)-OH, PyBOP, HOBt and solvent were slowly activated by adding DIPEA under ice bath, and then the solution was added to the reaction column and reacted at room temperature for 2 hours. The solution was removed, washed with DMF three times, shrunk with methanol, and dried under reduced pressure to obtain the peptide resin.

[0047] (3) The lysate (a dichloromethane solution containing 20% ​​trifluoroethanol) and the peptide resin prepared in step (2) were mixed, reacted at room temperature for 3 hours, filtered, and washed with dichloromethane. The combined filtrate was concentrated to dryness under reduced pressure to obtain compound 1, whose mass spectrum was consistent with the theoretical result.

[0048] 2. Synthesis of Compound 2

[0049] Compound 1, 2-bromoacetophenone and ethyl acetate were added to a glass bottle, triethylamine was added under ice bath, the reaction was allowed to proceed overnight at room temperature, and the reaction was monitored by TLC. After the reaction of compound 1 was complete, it was washed with 10% citric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution respectively, the organic phase was concentrated to dryness under reduced pressure, and purified by silica gel column chromatography to obtain compound 2, the mass spectrum of which was consistent with the theory.

[0050] 3. Synthesis of Compound 3

[0051] Compound 2 was mixed with 2M hydrogen chloride ethyl acetate solution under ice bath, reacted at room temperature for 1 hour, and the reaction was monitored by TLC. After the reaction of compound 2 was complete, it was concentrated to dryness under reduced pressure to obtain compound 3, and the mass spectrum was consistent with the theory.

[0052] 4. Synthesis of Eptifibatide Impurity I

[0053] Compound 3, triethylamine and solvent were mixed and reacted at room temperature overnight. The reaction was monitored by HPLC until compound 3 was completely reacted. The mixture was concentrated to dryness under reduced pressure to obtain crude peptide of eptifibatide impurity I.

[0054] 5. Preparation of Eptifibatide Impurity I Peptide

[0055] The crude peptide of impurity I of eptifibatide obtained in step 4 was prepared and purified by high performance liquid chromatography, with octadecylsilane bonded silica gel as the stationary phase, 0.1% trifluoroacetic acid solution as mobile phase A, acetonitrile as mobile phase B, monitoring wavelength of 280 nm, gradient elution to collect the target peak fraction, concentrated and lyophilized to obtain the refined peptide of impurity I of eptifibatide, and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy, which was consistent with the theory.

[0056] The specific synthetic route is as follows:

[0057]

[0058] In the present invention, the meanings of the English abbreviations are shown in Table 1.

[0059] Table 1

[0060] Abbreviations and English meaning HOAt 1-Hydroxy-7-azobenzotriazole DIPCDI Diisopropylcarbodiimide HOB 1-Hydroxybenzotriazole HATU 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate HBTU Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate DIPEA N,N-Diisopropylethylamine PyBOP Benzotriazol-1-yl-oxytripyrrolidino hexafluorophosphate DMF N,N-Dimethylformamide NMP N-Methylpyrrolidone THF Tetrahydrofuran DCM Dichloromethane EA Ethyl acetate TFA Trifluoroacetic acid TEA Triethylamine TIS Triisopropylsilane

[0061] The present invention will be further described below in conjunction with embodiments:

[0062] Example 1 Synthesis of Compound 1

[0063] Weigh 2-Cl-Trt resin (85.7 g, Sub = 1.4 mmol / g) and load it into the solid phase reaction column, wash it twice with DMF, swell it with DMF for 30 minutes, and remove the solution. Weigh Fmoc-Pro-OH 81.0 g (240 mmol), DIPEA 125.7 mL (480 mmol) and 300 ml DMF, add them into the reaction column, react at room temperature for 2 hours, then add 50 mL methanol, react at room temperature for 1 hour, remove the solution, and wash it with DMF three times. Add 20% piperidine solution to remove the Fmoc protecting group, stir and react at room temperature for 5 minutes, remove the solution, repeat the operation once, and wash it with DMF six times.

[0064] Weigh Boc-Trp(Boc)-OH 97.1 g (240 mmol), PyBOP 124.9 g (240 mmol), HOBt 38.9 g (288 mmol) and 300 ml DMF, slowly add DIPEA 125.7 mL (480 mmol) under ice bath for activation, add the solution into the reaction column, react at room temperature for 2 hours. Draw off the solution, wash three times with DMF, shrink with methanol, and dry under reduced pressure to obtain 127 g of peptide resin.

[0065] Weigh 1300 mL of 20% trifluoroethanol / dichloromethane solution and the above peptide resin into a glass bottle, react at room temperature for 3 hours, filter, and wash the resin with 600 mL of dichloromethane. The combined filtrate is concentrated to dryness under reduced pressure to obtain compound 1 (59.6 g), the mass spectrum is shown in Figure 1 , which is consistent with the theory.

[0066] Example 2 Synthesis of Compound 2

[0067] Weigh compound 1 (59.6 g, 118 mmol), 2-bromoacetophenone 23.65 g (178 mmol) and 600 ml of ethyl acetate into a glass bottle, add 75 ml of triethylamine (535 mmol) under ice bath, react at room temperature overnight, and monitor the reaction by TLC. After the reaction of compound 1 is complete, wash it with 10% citric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution respectively, the organic phase is concentrated to dryness under reduced pressure, and purified by silica gel column chromatography to obtain compound 2 (59 g), the mass spectrum is shown in Figure 2 , which is consistent with the theory.

[0068] Example 3 Synthesis of Compound 3

[0069] Weigh compound 2 (59 g) into a glass bottle, add 600 ml of 2M hydrogen chloride ethyl acetate solution under ice bath, react at room temperature for 1 hour, and monitor the reaction by TLC. After the reaction of compound 2 is complete, concentrate to dryness under reduced pressure to obtain compound 3 (49 g). The mass spectrum is shown in Figure 3 , which is consistent with the theory.

[0070] Example 4 Synthesis of Eptifibatide Impurity I

[0071] Compound 3 (49 g), 100 ml of triethylamine and 500 ml of tetrahydrofuran were weighed into a glass bottle and reacted at room temperature overnight. The reaction was monitored by HPLC. After the reaction of compound 3 was complete, the mixture was concentrated to dryness under reduced pressure to obtain crude peptide of eptifibatide impurity I.

[0072] Example 5 Preparation of Eptifibatide Impurity I Peptide

[0073] The crude peptide of impurity I obtained in Example 4 was prepared and purified by high performance liquid chromatography, with octadecylsilane bonded silica gel as the stationary phase, 0.1% trifluoroacetic acid solution as the mobile phase A, acetonitrile as the mobile phase B, monitoring wavelength of 280nm, gradient elution to collect the target peak fraction, concentrated and freeze-dried to obtain 19g of impurity I refined peptide, with a total yield of 56% and a purity of 98.71%. The chromatogram is as follows Figure 4 The typical characteristic peak data are shown in Table 2, and the mass spectrum is shown in Figure 5 As shown, the hydrogen and carbon spectra are Figures 6-7 As shown, the mass spectra and NMR spectra are consistent with the theory.

[0074] Table 2

[0075] Name RT Area Height %Area 1 10.051 25372 1031 0.10 2 10.875 24467002 1933140 98.71 3 11.339 175008 14196 0.71 4 11.550 23797 4284 0.10 5 11.723 60328 4124 0.24 6 12.058 21909 1812 0.09 7 12.392 9012 748 0.04 8 13.443 5411 640 0.02

[0076] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing eptifibatide impurity I, characterized in that: include: Step 1: coupling protected amino acids X-Pro-OH and Y-Trp(Y)-OH one by one from the C-terminus to the N-terminus on a solid phase resin to obtain a peptide resin, which is cleaved by a cleavage solution to obtain compound 1 of formula I; Step 2: Under the catalytic action of an organic base, compound 1 reacts with 2-bromoacetophenone to obtain compound 2 of formula II; Step 3: removing the Boc protecting group from the compound 2 to obtain a compound 3 having a structure of formula III; Step 4: Compound 3 is reacted under the catalytic action of an organic base to obtain a crude product of eptifibatide impurity I, which is then purified by HPLC to obtain eptifibatide impurity I having a structure of formula IV; The lysate is a combination of TFE and DCM, or a combination of TFA, TIS, and DCM; Formula I; Formula II; Formula III; Formula IV; In step 2 and step 4, the organic base is triethylamine; Step 3: the Boc protecting group of compound 2 is removed in the presence of an acid, wherein the acid is hydrogen chloride.

2. The preparation method according to claim 1, characterized in that: The lysate consists of TFE and DCM in a volume ratio of 2:

8.

3. The preparation method according to claim 1, characterized in that: The lysate consists of TFA, TIS and DCM in a volume ratio of 1:1:

98.

4. The preparation method according to claim 1, characterized in that: The resin is 2-Cl-Trt resin.

5. The preparation method according to claim 1, characterized in that: The X is Fmoc; the Y is Boc.

6. The preparation method according to claim 5, characterized in that: The removing agent used for removing the Fmoc protecting group is a piperidine solution with a volume percentage of 20%; the solvent of the piperidine solution is one or more of NMP, THF, DCM and DMF.

7. The preparation method according to claim 1, characterized in that: The coupling agent is selected from any one of the following combinations: DIPCDI and HOBt; PyBop, HOBt and DIPEA; HBTU, HOBt and DIPEA; DIPCDI and HOAt; HATU, HOAt and DIPEA; PyAop, HOAt and DIPEA.

8. The preparation method according to claim 1, characterized in that: The chromatographic conditions of the HPLC purification are: Octadecylsilane bonded silica gel was used as the stationary phase, 0.2% acetic acid solution was used as mobile phase A, methanol was used as mobile phase B, the detection wavelength was 280 nm, and gradient elution was used.

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