CN102827249A - Liquid-phase synthetic method of eptifibatide - Google Patents
Liquid-phase synthetic method of eptifibatide Download PDFInfo
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- CN102827249A CN102827249A CN2011101586539A CN201110158653A CN102827249A CN 102827249 A CN102827249 A CN 102827249A CN 2011101586539 A CN2011101586539 A CN 2011101586539A CN 201110158653 A CN201110158653 A CN 201110158653A CN 102827249 A CN102827249 A CN 102827249A
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- 108010056764 Eptifibatide Proteins 0.000 title claims abstract description 43
- 229960004468 eptifibatide Drugs 0.000 title claims abstract description 42
- 239000007791 liquid phase Substances 0.000 title claims abstract description 22
- GLGOPUHVAZCPRB-LROMGURASA-N eptifibatide Chemical compound N1C(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CCCCNC(=N)N)NC(=O)CCSSC[C@@H](C(N)=O)NC(=O)[C@@H]2CCCN2C(=O)[C@@H]1CC1=CN=C2[C]1C=CC=C2 GLGOPUHVAZCPRB-LROMGURASA-N 0.000 title claims abstract 13
- 238000010189 synthetic method Methods 0.000 title claims abstract 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 26
- 108010033276 Peptide Fragments Proteins 0.000 claims abstract description 13
- 102000007079 Peptide Fragments Human genes 0.000 claims abstract description 13
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 12
- 125000006239 protecting group Chemical group 0.000 claims abstract description 10
- 239000012634 fragment Substances 0.000 claims abstract description 4
- 230000001590 oxidative effect Effects 0.000 claims abstract description 3
- 230000001681 protective effect Effects 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- 230000015572 biosynthetic process Effects 0.000 claims description 23
- 238000003786 synthesis reaction Methods 0.000 claims description 23
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 21
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 108010016626 Dipeptides Proteins 0.000 claims description 10
- 230000003647 oxidation Effects 0.000 claims description 9
- 238000007254 oxidation reaction Methods 0.000 claims description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- 150000002148 esters Chemical class 0.000 claims description 5
- KQSSATDQUYCRGS-UHFFFAOYSA-N methyl glycinate Chemical compound COC(=O)CN KQSSATDQUYCRGS-UHFFFAOYSA-N 0.000 claims description 5
- 229910052740 iodine Inorganic materials 0.000 claims description 4
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 3
- 239000011630 iodine Substances 0.000 claims description 3
- YEDNBEGNKOANMB-REOHCLBHSA-N (2r)-2-amino-3-sulfanylpropanamide Chemical compound SC[C@H](N)C(N)=O YEDNBEGNKOANMB-REOHCLBHSA-N 0.000 claims description 2
- WTKQMHWYSBWUBE-UHFFFAOYSA-N (3-nitropyridin-2-yl) thiohypochlorite Chemical compound [O-][N+](=O)C1=CC=CN=C1SCl WTKQMHWYSBWUBE-UHFFFAOYSA-N 0.000 claims description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 2
- DKIDEFUBRARXTE-UHFFFAOYSA-N 3-mercaptopropanoic acid Chemical compound OC(=O)CCS DKIDEFUBRARXTE-UHFFFAOYSA-N 0.000 claims description 2
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims description 2
- -1 aspartyl Chemical group 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 claims description 2
- HQEIPVHJHZTMDP-JEDNCBNOSA-N methyl (2s)-pyrrolidine-2-carboxylate;hydrochloride Chemical compound Cl.COC(=O)[C@@H]1CCCN1 HQEIPVHJHZTMDP-JEDNCBNOSA-N 0.000 claims description 2
- 238000007243 oxidative cyclization reaction Methods 0.000 claims description 2
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 claims description 2
- 238000007127 saponification reaction Methods 0.000 claims description 2
- 125000005454 tryptophanyl group Chemical group 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 238000006482 condensation reaction Methods 0.000 abstract 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- CZKPOZZJODAYPZ-LROMGURASA-N eptifibatide Chemical compound N1C(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CCCCNC(=N)N)NC(=O)CCSSC[C@@H](C(N)=O)NC(=O)[C@@H]2CCCN2C(=O)[C@@H]1CC1=CNC2=CC=CC=C12 CZKPOZZJODAYPZ-LROMGURASA-N 0.000 description 31
- 238000006243 chemical reaction Methods 0.000 description 23
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 15
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 15
- 238000003756 stirring Methods 0.000 description 14
- 239000000243 solution Substances 0.000 description 12
- 238000001308 synthesis method Methods 0.000 description 11
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 10
- 230000002829 reductive effect Effects 0.000 description 10
- 238000004809 thin layer chromatography Methods 0.000 description 10
- 229920001184 polypeptide Polymers 0.000 description 8
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- 238000001035 drying Methods 0.000 description 6
- 239000011347 resin Substances 0.000 description 6
- 229920005989 resin Polymers 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 5
- 239000000543 intermediate Substances 0.000 description 5
- 239000007790 solid phase Substances 0.000 description 5
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- FIRHQRGFVOSDDY-UHFFFAOYSA-N ethyl 1-hydroxytriazole-4-carboxylate Chemical compound CCOC(=O)C1=CN(O)N=N1 FIRHQRGFVOSDDY-UHFFFAOYSA-N 0.000 description 4
- BLWYXBNNBYXPPL-YFKPBYRVSA-N methyl (2s)-pyrrolidine-2-carboxylate Chemical compound COC(=O)[C@@H]1CCCN1 BLWYXBNNBYXPPL-YFKPBYRVSA-N 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000010532 solid phase synthesis reaction Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N Diisopropyl ether Chemical compound CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 238000009903 catalytic hydrogenation reaction Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 3
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 3
- 208000004476 Acute Coronary Syndrome Diseases 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 235000017550 sodium carbonate Nutrition 0.000 description 2
- ZRBPBPZOUBWCMM-JEDNCBNOSA-N (2r)-3-(acetamidomethylsulfanyl)-2-aminopropanamide;hydrochloride Chemical compound Cl.CC(=O)NCSC[C@H](N)C(N)=O ZRBPBPZOUBWCMM-JEDNCBNOSA-N 0.000 description 1
- 206010002388 Angina unstable Diseases 0.000 description 1
- 101710149643 Integrin alpha-IIb Proteins 0.000 description 1
- 102100025306 Integrin alpha-IIb Human genes 0.000 description 1
- QUOGESRFPZDMMT-UHFFFAOYSA-N L-Homoarginine Natural products OC(=O)C(N)CCCCNC(N)=N QUOGESRFPZDMMT-UHFFFAOYSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QUOGESRFPZDMMT-YFKPBYRVSA-N L-homoarginine Chemical compound OC(=O)[C@@H](N)CCCCNC(N)=N QUOGESRFPZDMMT-YFKPBYRVSA-N 0.000 description 1
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 1
- 101800001442 Peptide pr Proteins 0.000 description 1
- 108010035030 Platelet Membrane Glycoprotein IIb Proteins 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 208000007814 Unstable Angina Diseases 0.000 description 1
- 150000003862 amino acid derivatives Chemical group 0.000 description 1
- 230000000702 anti-platelet effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229940056984 integrilin Drugs 0.000 description 1
- 201000004332 intermediate coronary syndrome Diseases 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Peptides Or Proteins (AREA)
Abstract
The invention relates to a liquid-phase synthetic method of eptifibatide. The method comprises the following steps: (1) synthesizing a fragment 1 in a liquid-phase way; (2) synthesizing a peptide fragment 2 in a liquid-phase way; (3) carrying out a condensation reaction for the peptide fragment 1 and the peptide fragment 2 to obtain a protective peptide fragment 3; (4) carrying out a condensation reaction for the peptide fragment 3 and H-Cys(R4)-NH2 to obtain a peptide fragment 4; (5) removing protective group of the peptide fragment 4 to obtain a linear peptide; and (6) oxidizing and cyclizing a disulfide bond of the linear peptide to obtain the eptifibatide. The method facilitates realization of large-scale production and is relatively low in production cost.
Description
Technical Field
The invention relates to the technical field of polypeptide synthesis, in particular to a liquid-phase synthesis method of eptifibatide.
Background
Eptifibatide consists of seven amino acids or amino acid derivative residues, contains a modified KGD sequence, forms a ring by intramolecular disulfide bonds, and has high affinity and specificity for GP IIb/IIIa receptors. Eptifibatide, marketed in the united states in 1998 under the trade name Integrilin, is a clinically useful drug developed by structural modification of Barbourin as an anti-platelet aggregation drug for the treatment of Acute Coronary Syndrome (ACS), which mechanism works by inhibiting platelet aggregation, specifically by blocking the platelet receptor GPIIb/IIIa. Platelet aggregation can impede blood supply to the heart, causing unstable angina and possibly myocardial infarction. Eptifibatide has material for platelets, avoids the interference of other common cardiovascular methods, and can eliminate the effect after credit, so the eptifibatide is more and more widely applied clinically.
The synthesis method of eptifibatide can be mainly divided into a solid-phase synthesis method and a liquid-phase synthesis method, and the liquid-phase synthesis method is mainly realized by polypeptide fragment synthesis.
The synthesis of eptifibatide using solid phase methods is described in US5318899, US5686570, US5747447, WO2005121164, WO2006045483A2 and WO2006119388A2, among others. The solid phase method for synthesizing eptifibatide described in WO2005121164 is to attach protected Cys derivative to solid phase carrier, prepare Mpr (Acm) -Lys (Boc) -Gly-Asp (OtBu) -Trp-Pro-Cys (Acm) -Resin by conventional solid phase polypeptide synthesis method, and then cleave it from Resin by using cleavage reagent. The eptifibatide precursor with two sulfydryl groups protected by Acm can be obtained by converting Lys into homoarginine, and finally, intramolecular disulfide bond can be formed while removing the Acm protecting group by adopting iodine oxidation, so that eptifibatide is obtained. The solid phase peptide synthesis method is usually adopted to crack the crude eptifibatide precursor peptide from resin and then carry out oxidative cyclization to obtain eptifibatide, and a patent also introduces that a certain method is adopted to lead the eptifibatide precursor to form disulfide bond on the resin, so that the eptifibatide can be cracked from the resin to obtain the eptifibatide. The excess multiple of amino acids used in the synthesis of eptifibatide by the solid-phase polypeptide method is large (usually 4-5 times), meanwhile, solvent lotion resin such as DMF is adopted in the synthesis process, intermediate peptide cannot be separated, the eptifibatide can only be purified by an RP-HPLC method after being cyclized in a liquid phase, and no separation and purification process is adopted in the middle, which means that the synthesis cost is very high, and the eptifibatide can be synthesized by a liquid-phase synthesis method as a polypeptide only containing 7 amino acids.
It is generally considered that the liquid phase synthesis method is more feasible than the solid phase synthesis method for mass production of eptifibatide. Firstly, an intermediate is continuously separated in the liquid phase synthesis process, the purity of the intermediate obtained in each step is higher, and the quality of a finished product can be ensured by controlling the purity of the intermediate and the like; secondly, compared with a solid phase polypeptide synthesis method, the liquid phase polypeptide synthesis method can save more solvent cost and has more advantages for enterprises; thirdly, because the eptifibatide chain is not long, the solubility problem is small when the liquid phase synthesis method is adopted, so that a large amount of intermediates can be prepared, and a good foundation is laid for the industrial production of eptifibatide. A number of methods for the liquid phase synthesis of eptifibatide are also described, for example in US7674768 et al.
Disclosure of Invention
The invention provides a method for preparing eptifibatide in a liquid phase, which is characterized by firstly synthesizing two tripeptide chains, condensing the tripeptide chains to form hexapeptide, then reacting the hexapeptide with a Cys derivative to obtain an eptifibatide precursor, and cyclizing to obtain eptifibatide;
the specific method of the present invention comprises the steps of:
1) liquid phase synthesis of fragment 1: mpa (X) -Har (Y) -Gly-OH;
2) liquid-phase synthesis of peptide fragment 2: R1-Asp (R2) -Trp (R3) -Pro-OH;
3) condensing the peptide segment 1 and the peptide segment 2 to obtain a protective peptide segment 3:
Mpa(X)-Har(Y)-Gly-Asp(R2)-Trp(R3)-Pro-OH；
4) condensing the peptide fragment 3 with H-Cys (R4) -NH2 to obtain peptide fragment 4:
Mpa(X)-Har(Y)-Gly-Asp(R2)-Trp(R3)-Pro-Cys(R4)-NH2；
5) deprotecting peptide stretch 4 to give a linear peptide:
mpa (X) -Har-Gly-Asp-Trp-Pro-Cys (R4) -NH2 or
Mpa (X) -Har-Gly-Asp-Trp-Pro-Cys-NH2 or
Mpa-Har-Gly-Asp-Trp-Pro-Cys (R4) -NH2 or
Mpa-Har-Gly-Asp-Trp-Pro-Cys-NH2；
6) Oxidizing and cyclizing the linear peptide into a disulfide bond to prepare eptifibatide:
wherein,
mpa is mercaptopropionic acid;
har is a homoaminoacyl;
gly is glycyl;
asp is aspartyl;
trp is tryptophanyl;
pro is prolyl;
Cys-NH2 is cysteine amide;
x, Y, R1, R2, R3 and R4 are protecting groups and are preferably defined as follows:
r1 is selected from Fmoc, CbZ or Boc;
r2 is selected from tBu or Bn;
r3 is selected from H or Boc;
r4 is selected from Acm, Trt or Npys;
x is selected from Acm or Trt;
y is selected from Pbf, Mtr, Mbs or (Boc) 2.
The synthesis of the peptide chain can be carried out by adopting a common method for liquid phase polypeptide synthesis, and the synthesis is very easy.
The peptide segment 1 is synthesized by firstly reacting H-Gly-OMe HCl with alpha-amino protected Har (Y) to obtain alpha-amino protected Har (Y) -Gly-OMe dipeptide derivative, removing the alpha-amino protecting group of the dipeptide derivative, then reacting with Mpa (X) -OH to obtain Mpa (X) -Har (Y) -Gly-OMe tripeptide derivative, and saponifying under the condition of NaOH/MeOH/H2O to obtain Mpa (X) -Har (Y) -Gly-OH.
The peptide segment 1 can also be synthesized by adopting Mpa (X) -OH activated ester to firstly react with H-Har (Y) -OH to obtain Mpa (X) -Har (Y) -OH dipeptide, and then the dipeptide is activated to react with unprotected Gly to obtain Mpa (X) -Har (Y) -Gly-OH.
The peptide segment 1 can also be synthesized by adopting Mpa (X) -OH activated ester to react with H-Har (Y) -Gly-OH to obtain Mpa (X) -Har (Y) -Gly-OH, and the Mpa (X) -Har (Y) -Gly-OH is obtained by reacting the activated ester with unprotected Gly after activation.
The peptide segment 2 can be synthesized by firstly adopting H-Pro-OMe HCl to react with Trp (R3) protected by alpha-amino to obtain Trp-Pro-OMe dipeptide derivative protected by alpha-amino, removing the alpha-amino protecting group of the dipeptide derivative, then reacting with Asp protected by alpha-amino and side chain carboxyl to obtain R1-Asp (R2) -Trp (R3) -Pro-OMe, and removing R1 to obtain H-Asp (R2) -Trp (R3) -Pro-OMe.
According to the protection of Mpa, Har, Asp, Trp and Cys in the peptide segment 4, different methods can be adopted to selectively remove or completely remove the Mpa, Har, Asp, Trp and Cys.
If the eptifibatide linear peptide precursor with the protecting group completely removed is obtained, air oxidation, DMSO oxidation, iodine oxidation, 1% NH4HCO3 solution oxidation, or the like can be used.
Drawings
FIG. 1 is an HPLC chromatogram of a crude eptifibatide product;
FIG. 2 is an HPLC chromatogram of a fine eptifibatide.
Detailed Description
Certain embodiments of the present invention will be further illustrated with reference to specific examples, which should not be construed as limiting the scope of the invention.
Example 1: synthesis of amino acid methyl ester hydrochloride
(1) Synthesis of H-Gly-OMe & HCl
0.1mmol of H-Gly-OH was dissolved in anhydrous methanol (50ml) and stirred in an ice-salt bath, and 2eq of SOCl was slowly added dropwise2The dropwise addition was completed in the above solution for 30 minutes. And removing the deicing salt bath after the dropwise addition is finished, stirring at room temperature overnight, and adopting TLC (thin layer chromatography) for controlling the reaction process. After the reaction is finished, decompressing, concentrating and recrystallizing to obtain 10.23g of H-Gly-OMe, wherein the product is white crystal;
(2) synthesis of H-Pro-OMe & HCl
0.1mmol of H-Pro-OH was dissolved in anhydrous methanol (50ml) in an ice-salt bath and stirred, 2eq of SOCl was slowly added dropwise2The dropwise addition was completed in the above solution for 30 minutes. And removing the deicing salt bath after the dropwise addition is finished, stirring at room temperature overnight, and adopting TLC (thin layer chromatography) for controlling the reaction process. After the reaction, the reaction solution is decompressed, concentrated and recrystallized to obtain 10.94g of H-Pro-OMe & HCl;
example 2: synthesis of H-Trp-Pro-OMe
1mmol H-Pro-OMe & HCl was suspended in DCM and 3eq Et was added with stirring3N; 1.2eq CBz-Trp-OH and 1.5eq HOCt were dissolved in anhydrous DCM, and 1.5eq DCC was slowly added dropwise to the above solution with stirring for 30 min. After filtration, the solution was added to H-Pro-OMe/Et3In the mixture of N/DCM, stir overnight at room temperature and check the reaction by TLC. The reaction solution was filtered and then treated with 0.3M NaHCO3Washing for 4 times, washing for 3 times with 0.2M HCl, washing with saturated salt water for 1 time, adding anhydrous sodium sulfate, drying, and concentrating under reduced pressure to obtain white solid Cbz-Trp-Pro-OMe 0.41g;
dissolving 0.2mmol of Cbz-Trp-Pro-OMe in 10ml of MeOH, adding Pd/C30 mg, introducing hydrogen for catalytic hydrogenation, detecting complete reaction by TLC after 3h, filtering Pd/C, and concentrating under reduced pressure to obtain 52.67mg of oily liquid.
Example 3: synthesis of H-Asp (OtBu) -Trp-Pro-OMe
Dissolving 1mmol H-Trp-Pro-OMe in 10ml anhydrous DCM, and stirringThen add 1.5eq Et3N; 1.2eq Cbz-Asp (OtBu) -OH and 1.2eq HOCt were dissolved in 20ml of anhydrous DCM, 1.5eq DIC was slowly added dropwise to the above solution over 30min with stirring, and after 30min of activation, the mixed solution was added dropwise to the mixed solution and stirred at room temperature overnight. The reaction solution was filtered and then treated with 0.5M NaHCO30.1M HCl and brine, anhydrous MgSO4Drying, concentrating under reduced pressure to obtain oil, and crystallizing to obtain white Cbz-Asp (OtBu) -Trp-Pro-OMe 0.53 g.
1mmol of Cbz-Asp (OtBu) -Trp-Pro-OMe was dissolved in 30ml of anhydrous methanol, 50mg of Pd/C was added thereto, and then hydrogen gas was introduced thereinto to conduct catalytic hydrogenation, the reaction was allowed to proceed overnight, and the reaction was monitored by TLC. Pd/C was filtered off, and the filtrate was concentrated under reduced pressure to give 0.45g of oily liquid H-Asp (OtBu) -Trp-Pro-OMe.
Example 4: synthesis of H-Har-Gly-OMe
Cbz-Har-Gly-OMe is synthesized by the method of example 2, the feeding amount is respectively 1.2mmol of Cbz-Har-OH and 1mmol of H-Gly-OMe & HCl, and 0.41g of white solid Cbz-Har-Gly-OMe is obtained;
dissolving 1mmol Cbz-Har-Gly-OMe in 30ml anhydrous methanol, adding 50mg Pd/C, introducing hydrogen for catalytic hydrogenation, reacting overnight, and monitoring the reaction by TLC. Pd/C was filtered off, and the filtrate was concentrated under reduced pressure to give 0.32g of H-Har-Gly-OMe.
Example 5: mpa (Acm) -Har-Gly-OH synthesis
Mpa (Acm) -Har-Gly-OMe was synthesized by the method of example 3, the feeding amounts were respectively 1mmol Mpa (Acm) -OH and 1mmol H-Har-Gly-OMe, and Mpa (Acm) -Har-Gly-OMe 0.475g was obtained;
dissolving 1mmol Mpa (Acm) -Har-Gly-OMe in 20ml anhydrous methanol, adding Ba (OH) under stirring2Solution (1M, 2ml), TLC monitor the reaction, 4h reaction completion. Adding a certain amount of dilute hydrochloric acid to neutralize excessive Ba (OH)2After the solvent was removed under reduced pressure, 10ml of dilute hydrochloric acid was added to obtain a white solid, which was then filtered and washed with saline and isopropyl ether to obtain Mpa (Acm) -Har-Gly-OH0.44g.
Example 6: mpa (Acm) -Har-Gly-Asp (OtBu) -Trp-Pro-OH synthesis
(1)1mmol of H-Asp (OtBu) -Trp-Pro-OMe was dissolved in 20ml of anhydrous DCM and 1.2mmol of Et3N was added with stirring;
(2) dissolving 1.5mmol Mpa (Acm) -Har-Gly-OH and 2mmol HOCt in 10ml anhydrous DCM, slowly adding 2mmol DIC (dropwise adding within 30 min) under stirring, reacting for 30min, adding the solution into (1), stirring for reacting overnight, and monitoring the reaction by TLC. After completion of the reaction, the mixture was filtered, extracted with Na2CO3, dilute hydrochloric acid and saturated brine, and then anhydrous MgSO4Drying, concentrating under reduced pressure, and crystallizing to obtain Mpa (Acm) -Har-Gly-Asp (OtBu) -Trp-Pro-OMe 0.78 g.
(3) Preparation of Mpa (Acm) -Har-Gly-Asp (OtBu) -Trp-Pro-OH
0.2g Mpa (Acm) -Har-Gly-Asp (OtBu) -Trp-Pro-OMe was dissolved in 30ml of anhydrous methanol, and 1M Ba (OH) was added thereto with stirring2TLC monitored the extent of reaction progress and 4h reaction was complete. After adding dilute hydrochloric acid, the solvent was removed under reduced pressure, and then dilute hydrochloric acid was added thereto to obtain a white solid (Mpa), (Acm) -Har-Gly-Asp (OtBu) -Trp-Pro-OH (0.17 g).
Example 7: mpa (Acm) -Har-Gly-Asp-Trp-Pro-Cys (Acm) -NH2Preparation of
(1) Dissolving 1mmol of H-Cys (Acm) -NH2 in anhydrous DCM, and adding 1.5eq DIEA under stirring;
(2) dissolving 2mmol and 1.5mmol of (Acm) -Har-Gly-Asp (OtBu) -Trp-Pro-OH and HOCt in 20ml of anhydrous DCM respectively (adding a small amount of anhydrous DMF if the anhydrous DMF is not dissolved), slowly adding DIC under stirring, reacting for 30min, adding the mixed solution into (1), reacting overnight, and monitoring the reaction degree by TLC. The reaction was completed, and the mixture was extracted with Na2CO3, dilute hydrochloric acid and saturated brine in this order, and then anhydrous MgSO4Drying, drying under reduced pressure, and recrystallizing to obtain solid Mpa (Acm) -Har-Gly-Asp (OtBu) -Trp-Pro-Cys (Acm) -NH20.5g；
(3)Mpa(Acm)-Har-Gly-Asp-Trp-Pro-Cys(Acm)-NH2Preparation of
0 is added.5g Mpa(Acm)-Har-Gly-Asp(OtBu)-Trp-Pro-Cys(Acm)-NH2Put into a 100ml round bottom flask, 15ml 95% TFA was added: 5% H2After the O reaction for 1 hour, directly pouring the lysate into glacial isopropyl ether, filtering the obtained solid, washing the solid for 3 times by using the glacial isopropyl ether, and drying the solid under reduced pressure to obtain 0.46g Mpa (Acm) -Har-Gly-Asp-Trp-Pro-Cys (Acm) -NH2。
Example 8: disulfide bond formation
10g Mpa (Acm) -Har-Gly-Asp-Trp-Pro-Cys (Acm) -NH2Dissolving in 100ml of purified water, and slowly adding 5% I dropwise under stirring2And (3) monitoring the reaction of the solution by HPLC, purifying a target product by adopting reverse phase liquid chromatography after the reaction is completed, and freeze-drying to obtain 1.2g of a target compound.
Claims (9)
1. A synthetic method of eptifibatide comprises the following steps:
1) liquid phase synthesis of fragment 1: mpa (X) -Har (Y) -Gly-OH;
2) liquid-phase synthesis of peptide fragment 2: R1-Asp (R2) -Trp (R3) -Pro-OH;
3) condensing the peptide segment 1 and the peptide segment 2 to obtain a protective peptide segment 3: mpa (X) -Har (Y) -Gly-Asp (R2) -Trp (R3) -Pro-OH;
4) condensing the peptide fragment 3 with H-Cys (R4) -NH2 to obtain peptide fragment 4: mpa (x) -har (y) -Gly-Asp (R2) -Trp (R3) -Pro-Cys (R4) -NH 2;
5) deprotecting peptide stretch 4 to give a linear peptide: mpa (x) -Har-Gly-Asp-Trp-Pro-Cys (R4) -NH2 or Mpa (x) -Har-Gly-Asp-Trp-Pro-Cys-NH2 or Mpa-Har-Gly-Asp-Trp-Pro-Cys (R4) -NH2 or Mpa-Har-Gly-Asp-Trp-Pro-Cys-NH 2;
6) oxidizing and cyclizing the linear peptide into a disulfide bond to prepare eptifibatide:
wherein,
mpa is mercaptopropionic acid;
har is a homoaminoacyl;
gly is glycyl;
asp is aspartyl;
trp is tryptophanyl;
pro is prolyl;
Cys-NH2 is cysteine amide;
x, Y, R1, R2, R3 and R4 are protecting groups.
2. The method of synthesizing eptifibatide of claim 1, wherein the protecting groups X, Y, R1, R2, R3 and R4 are each defined as follows:
r1 is selected from Fmoc, CbZ or Boc;
r2 is selected from tBu or Bn;
r3 is selected from H or Boc;
r4 is selected from Acm, Trt or Npys;
x is selected from Acm or Trt;
y is selected from Pbf, Mtr, Mbs or (Boc) 2.
3. The method for synthesizing eptifibatide as claimed in claim 1, wherein the step 1) is carried out by: H-Gly-OMe HCl and Har (Y) protected by alpha-amino group react to obtain Har (Y) -Gly-OMe dipeptide derivative protected by alpha-amino group, the alpha-amino protecting group of the dipeptide derivative is removed and then reacts with Mpa (X) -OH to obtain Mpa (X) -Har (Y) -Gly-OMe tripeptide derivative, and the tripeptide derivative is saponified under the condition of NaOH/MeOH/H2O to obtain Mpa (X) -Har (Y) -Gly-OH.
4. The method for synthesizing eptifibatide as claimed in claim 1, wherein the step 1) is carried out by: the activated ester of Mpa (X) -OH is reacted with H-Har (Y) -OH to obtain Mpa (X) -Har (Y) -OH, and the Mpa (X) -Har (Y) -OH is activated and then reacted with unprotected Gly to obtain Mpa (X) -Har (Y) -Gly-OH.
5. The method for synthesizing eptifibatide as claimed in claim 1, wherein the step 1) is carried out by: mpa (X) -OH activated ester reacts with H-Har (Y) -Gly-OH to obtain Mpa (X) -Har (Y) -Gly-OH.
6. The method for synthesizing eptifibatide as claimed in claim 1, wherein the step 2) is carried out by: reacting H-Pro-OMe HCl with alpha-amino protected Trp (R3) to obtain alpha-amino protected Trp-Pro-OMe dipeptide derivative, removing the alpha-amino protecting group of the dipeptide derivative, reacting with alpha-amino and side chain carboxyl protected Asp to obtain R1-Asp (R2) -Trp (R3) -Pro-OMe, and removing R1 to obtain H-Asp (R2) -Trp (R3) -Pro-OMe.
7. The method for synthesizing eptifibatide as claimed in claim 1, wherein the step 3) is carried out by: mpa (X) -Har (Y) -Gly-OH and H-Asp (R2) -Trp (R3) -Pro-OMe are reacted to obtain Mpa (X) -Har (Y) -Gly-Asp (R2) -Trp (R3) -Pro-OMe, and the Mpa (X) -Har (Y) -Gly-Asp (R2) -Trp (R3) -Pro-OH is obtained by saponification under the condition of NaOH/MeOH/H2O.
8. The method for synthesizing eptifibatide as claimed in claim 1, wherein the step 4) is carried out by: mpa (X) -Har (Y) -Gly-Asp (R2) -Trp (R3) -Pro-OH and H-Cys (R4) -NH2 to obtain Mpa (X) -Har (Y) -Gly-Asp (R2) -Trp (R3) -Pro-Cys (R4) -NH 2.
9. The method for synthesizing eptifibatide as claimed in claim 1, wherein the oxidative cyclization in step 6) comprises air oxidation, DMSO oxidation, air oxidation in 1% NH4HCO3 and iodine oxidation.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005100381A2 (en) * | 2004-04-08 | 2005-10-27 | Millennium Pharmaceuticals, Inc. | Processes for preparing eptifibatide and pertinent intermediate compounds |
| CN101747412A (en) * | 2009-12-30 | 2010-06-23 | 江苏诺泰制药技术有限公司 | Synthesis and preparation process of eptifibatide |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005100381A2 (en) * | 2004-04-08 | 2005-10-27 | Millennium Pharmaceuticals, Inc. | Processes for preparing eptifibatide and pertinent intermediate compounds |
| CN101747412A (en) * | 2009-12-30 | 2010-06-23 | 江苏诺泰制药技术有限公司 | Synthesis and preparation process of eptifibatide |
Non-Patent Citations (1)
| Title |
|---|
| 廖永祥: "血小板糖蛋白IIb/IIIa受体拮抗剂依替巴肽的合成研究", 《中国优秀硕士论文全文数据库（医药卫生科技辑）》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105037496A (en) * | 2015-09-17 | 2015-11-11 | 四川吉晟生物医药有限公司 | Preparation method for eptifibatide |
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