A method for synthesizing eptifibatide cleavage peptide impurities
Synthesizing the fragment of etibatide by solid phase synthesis and reacting with the pyridyl disulfide compound in the liquid phase solves the problem that the directional disulfide bonding reaction in the prior art is difficult to achieve, and the yield and purity of the impurities of etibatide fracture peptide are significantly improved.
Patent Information
- Application Number
- CN202411920930.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-25
- Publication Date
- 2025-04-04
AI Technical Summary
The prior art is difficult to achieve directional disulfide bonding reaction, resulting in low yield and purity of etepatide-breaking peptide impurities.
Fragments I and fragment II were synthesized by solid phase synthesis, and then reacted with pyridyl disulfide compound in the liquid phase to form a disulfide intermediate, and then disulfide bond exchange was performed to obtain etepatide cleavage peptide impurity.
Directional disulfide bond bonding reaction is achieved, the process is simple, convenient operation, gentle reaction conditions and short time, which significantly improves the yield and purity of the target impurities.
Smart Images
Figure CN119751554A_ABST
Abstract
Description
Technical field:
[0001] The invention belongs to the field of polypeptide drug synthesis, and in particular relates to a method for synthesizing eptifibatide cleavage peptide impurities. Background technology:
[0002] Eptifibatide is a small molecule cyclic heptapeptide derived from the venom dissociation factor of the Southeast Asian dwarf rattlesnake. It can reversibly bind to the platelet glycoprotein IIb / IIIa receptor and exert antiplatelet aggregation activity. The clinical indication is acute coronary syndrome, and it is an antithrombotic drug. Its adverse reactions mainly include thrombocytopenia and various bleeding risks. The original developer of Eptifibatide Injection is Schering-Plough Corporation of the United States.
[0003] Because eptifibatide contains a special sequence similar to RGD, there is a certain probability that it will break during the production and storage process, especially when it encounters high temperature or pH changes, which will cause the peptide bond to break and produce broken peptides.
[0004] Among the eptifibatide cleavage peptide impurities disclosed in the prior art, eptifibatide impurity 6, CAS: 358620-99-2, has a peptide sequence of:
[0005]
[0006] The compound can be used as a reference substance in quality control of eptifibatide.
[0007] During the peptide synthesis process, two thiol groups can be oxidized into corresponding disulfide bonds with the help of appropriate oxidants. The study of disulfide bond formation methods is one of the hot spots and difficulties in the field of peptide synthesis. In the application of peptide synthesis, the methods of intramolecular disulfide bond ring formation mainly include air oxidation, potassium ferrocyanide oxidation, iodine oxidation, hydrogen peroxide oxidation, dimethyl sulfoxide (DMSO) oxidation, and thallium trifluoroacetate oxidation, which are commonly used methods. However, for the disulfide bond formation reaction of bimolecules, the above methods are difficult to achieve directional disulfide bond synthesis. Therefore, the use of a better route to synthesize eptifibatide impurity 6 is of great significance for controlling the quality of eptifibatide. Summary of the invention:
[0008] The purpose of the present invention is to provide a method for synthesizing eptifibatide cleavage peptide impurities in view of the deficiencies in the prior art. The method is a directional disulfide bond-forming reaction, has a simple process, is easy to operate, has mild reaction conditions, and has a short reaction time, and can significantly improve the yield and purity of the target impurities.
[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0010] The invention discloses a method for synthesizing an eptifibatide cleavage peptide impurity, and the method is characterized in that the eptifibatide cleavage peptide impurity is an eptifibatide impurity 6. The method for synthesizing the eptifibatide cleavage peptide impurity comprises the following steps: a solid phase synthesis method is used to synthesize a fragment I, namely, Mpr-Har-Gly-Asp-OH, and a fragment II, namely, H-Trp-Pro-Cys-NH2; a liquid phase synthesis method is used to firstly generate a disulfide intermediate from the fragment I and a pyridyl disulfide compound; and then the fragment II is added to perform disulfide bond exchange to obtain the eptifibatide cleavage peptide impurity.
[0011] The method for synthesizing eptifibatide cleavage peptide impurities of the present invention further comprises the following preferred technical solutions:
[0012] 1. The resin used for fragment I is Wang Resin or 2-CTC Resin, and the resin used for fragment II is RinkAmideAM Resin;
[0013] 2. Solid phase synthesis of fragments I and II, the reaction solvent is one or a combination of DMF, NMP, DCM, ACN;
[0014] 3. Solid phase synthesis of fragments I and II, the condensing agent is any one of DIC / HOBt, DIC / Cl-HOBt, HBTU / DIEA, HATU / DIEA, PyBOP / DIEA, (HBTU+HOBt) / DIEA, (HATU+HOAt) / DIEA.
[0015] 4. Solid phase synthesis of fragments I and II, the reagent for deprotection of Fmoc was 20% PIP / DMF (v / v) solution;
[0016] 5. Solid phase synthesis of fragments I and II, the cleavage reagent is TFA:EDT:TIS:water (v / v / v / v) = 90:5:3:2;
[0017] 6. Pyridyl disulfide compounds are 2,2'-dithiobis(5-nitropyridine), dithiopyridine, 2,2'-dithiodipyridine and 4,4'-dithiodipyridine;
[0018] 7. The solvent used in liquid phase synthesis is any one of DMF, THF, DMSO, and ACN. The reaction temperature is 20-25°C and the reaction progress is monitored by TLC.
[0019] Compared with the prior art, the advantages of the present invention are:
[0020] The method of the present invention avoids the conventional disulfide bond non-directional bonding method, and first solid-phase synthesizes fragments I and II using cheap materials, and then performs liquid-phase directional synthesis with pyridyl disulfide compounds to obtain the target impurity. The method is a directional disulfide bond formation reaction, the process is simple, easy to operate, the reaction conditions are mild, the reaction time is short, and the yield and purity of the target impurity can be significantly improved. Description of the drawings:
[0021] Figure 1 This is the HPLC chromatogram of the refined peptide of Example 1.
[0022] Specific implementation cases
[0023] The meanings of the English abbreviations used in this invention
[0024]
[0025]
[0026] The present invention discloses a method for synthesizing eptifibatide cleavage peptide impurities. The embodiments are only a part of the present invention, not all of them. The purpose of the embodiments is to illustrate and explain the technical properties and characteristics of the present invention, but they cannot limit the protection scope of the present invention. All equivalent changes or modifications made according to the essence of the present invention are included in the protection scope of the present invention.
[0027] Example 1: Synthesis of Eptifibatide Fragmented Peptide Impurities
[0028] 1. Synthesis of fragment I Mpr-Har-Gly-Asp-OH
[0029] Weigh 9.43g of Wang Resin (sub = 0.53mmol / g, 5mmol), add DCM to swell for 30min, drain, add Fmoc-Asp(OtBu)-OH (10mmol, 2eq.) 4.11g, HOBt (11mmol, 2.2eq.) 1.49g, DIC (11mmol, 2.2eq.) 1.7mL, DMAP (1mmol, 0.2eq.) 0.12g, DMF 50mL, react at 25-30℃ for 5h, drain, wash 4 times with DMF, add acetylating agent, react for 2h to cap. After the reaction is completed, wash the resin 5 times with DMF (50mL), then add 20% PIP / DMF (v / v) solution, react at 20-25℃ for 30min, drain, and wash 6 times with DMF. Then, Fmoc-Gly-OH, Fmoc-Har(Pbf)-OH, and Mpr(Trt)-OH were coupled in sequence to obtain 14.97 g of peptide resin of fragment I. 149.7 mL of cleavage solution TFA:EDT:TIS:water (v / v / v / v)=90:5:3:2 was added, and the mixture was reacted at 20-25°C for 2 h, filtered, and the resin was washed with TFA. The filtrate was added to ice ether for precipitation, centrifuged, washed, and dried to obtain 2.51 g of solid fragment I.
[0030] 2. Synthesis of fragment II H-Trp-Pro-Cys-NH2
[0031] Weigh 5.56g of RinkAmideAM Resin (sub=0.9mmol / g, 5mmol), add DMF to swell for 30min, drain, add 20% PIP / DMF (v / v) solution, react at 20-25℃ for 30min, drain, wash 6 times with DMF, add Fmoc-Cys(Trt)-OH (10mmol, 2eq.), 1.49g of HOBt (11mmol, 2.2eq.), 1.7mL of DIC (11mmol, 2.2eq.), 50mL of DMF, react at 25-30℃ for 1.5-2h, drain, wash 4 times with DMF. Sequentially couple Fmoc-Pro-OH and Boc-Trp(Boc)-OH to obtain 9.68g of peptide resin of fragment II. Add 96.8 mL of the cleavage solution TFA:EDT:TIS:water (v / v / v / v) = 90:5:3:2, react at 20-25°C for 2 h, filter, wash the resin with TFA, add the filtrate to ice ether for precipitation, centrifuge, wash and dry to obtain 2.18 g of fragment II solid.
[0032] 3. Synthesis
[0033] Weigh 1.00 g of fragment Ⅰ and 0.6 g of DTNP, add them into 20 mL of DMSO and stir until dissolved. The reaction temperature is 20-25°C. Monitor the reaction by TLC until fragment Ⅰ disappears to obtain the intermediate.
[0034] Weigh 1.00 g of fragment II, add it into 20 mL of DMSO and stir until dissolved. After dissolution, pour it into the intermediate solution for disulfide bond exchange at a reaction temperature of 20-25°C and monitor the disappearance of the intermediate by TLC.
[0035] 4. Refined
[0036] The solution obtained in step 3 was added to 360 mL of purified water, filtered and purified by RP-HPLC, wherein mobile phase A was 0.1% TFA aqueous solution and mobile phase B was acetonitrile, and the components with a purity greater than 95% were collected, combined and concentrated until no fraction flowed out, and lyophilized to obtain 0.91 g of refined peptide dry powder, with a yield of 86.3%.
[0037] It was confirmed that the obtained refined peptide was eptifibatide impurity 6, CAS: 358620-99-2, and its peptide sequence was:
[0038]
[0039] Comparative Example 1:
[0040] Weigh 1.00 g of fragment I and 1.00 g of fragment II, add them to 10% acetic acid aqueous solution, stir to dissolve, slowly add 30 g / L iodine ethanol solution at room temperature until the solution turns yellow, let it stand for 30 minutes, add vitamin C aqueous solution to reduce excess iodine, filter the crude peptide and purify it using the same preparation system as in the example, collect the components with a purity greater than 95%, combine and concentrate until no fraction flows out, and lyophilize to obtain 0.25 g of refined peptide powder with a yield of 23.7%.
Claims
1. A method for synthesizing eptifibatide cleavage peptide impurities, characterized in that: The eptifibatide cleavage peptide impurity is eptifibatide impurity 6; its synthesis method is: using a solid phase synthesis method to synthesize fragment I, namely Mpr-Har-Gly-Asp-OH and fragment II, namely H-Trp-Pro-Cys-NH2, using a liquid phase synthesis method to react fragment I with a pyridyl disulfide compound to generate a disulfide intermediate, and then adding fragment II to perform disulfide bond exchange to obtain the eptifibatide cleavage peptide impurity.
2. The method for synthesizing eptifibatide cleavage peptide impurities according to claim 1, characterized in that: The resin used in fragment I is Wang Resin or 2-CTC Resin, and the resin used in fragment II is RinkAmideAM Resin.
3. The method for synthesizing eptifibatide cleavage peptide impurities according to claim 1, characterized in that: The fragments I and II are synthesized in solid phase, and the reaction solvent is one or a combination of DMF, NMP, DCM and THF.
4. The method for synthesizing eptifibatide cleavage peptide impurities according to claim 1, characterized in that: The fragments I and II are synthesized in solid phase, and the condensing agent is any one of DIC / HOBt, DIC / Cl-HOBt, HBTU / DIEA, HATU / DIEA, PyBOP / DIEA, (HBTU+HOBt) / DIEA, and (HATU+HOAt) / DIEA.
5. The method for synthesizing eptifibatide cleavage peptide impurities according to claim 1, characterized in that: Fragment I and fragment II were synthesized on solid phase and deprotected to form a 20% PIP / DMF (v / v) solution.
6. The method for synthesizing eptifibatide cleavage peptide impurities according to claim 1, characterized in that: Fragment I and fragment II were synthesized on solid phase, and the cleavage reagent was TFA:EDT:TIS:water (v / v / v / v)=90:5:3:
2.
7. The method for synthesizing eptifibatide cleavage peptide impurities according to claim 1, characterized in that: The pyridyl disulfide compound is selected from 2,2'-dithiobis(5-nitropyridine), dithiopyridine oxide, 2,2'-dithiodipyridine and 4,4'-dithiodipyridine.
8. The method for synthesizing eptifibatide cleavage peptide impurities according to claim 1, characterized in that: The solvent used in the liquid phase synthesis is any one of DMF, THF, DMSO and ACN, the reaction temperature is 20-25° C., and the reaction progress is monitored by TLC.