CN114524860A - Synthesis method and application of Etelcalcetide - Google Patents
Synthesis method and application of Etelcalcetide Download PDFInfo
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- ANIAZGVDEUQPRI-ZJQCGQFWSA-N etelcalcetide Chemical compound NC(N)=NCCC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)[C@@H](CCCN=C(N)N)NC(=O)[C@@H](CCCN=C(N)N)NC(=O)[C@@H](CCCN=C(N)N)NC(=O)[C@@H](C)NC(=O)[C@@H](CSSC[C@H](N)C(O)=O)NC(C)=O ANIAZGVDEUQPRI-ZJQCGQFWSA-N 0.000 title claims abstract description 42
- 229950006502 etelcalcetide Drugs 0.000 title claims abstract description 42
- 108091022127 etelcalcetide hydrochloride Proteins 0.000 title claims abstract description 42
- 238000001308 synthesis method Methods 0.000 title claims abstract description 12
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 27
- 238000010168 coupling process Methods 0.000 claims abstract description 22
- 238000005859 coupling reaction Methods 0.000 claims abstract description 22
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 20
- 230000008878 coupling Effects 0.000 claims abstract description 20
- 239000012634 fragment Substances 0.000 claims abstract description 20
- 150000001413 amino acids Chemical class 0.000 claims abstract description 18
- 239000007791 liquid phase Substances 0.000 claims abstract description 15
- 108010016626 Dipeptides Proteins 0.000 claims abstract description 12
- HNICLNKVURBTKV-MUUNZHRXSA-N (2r)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-MUUNZHRXSA-N 0.000 claims abstract description 11
- 125000006239 protecting group Chemical group 0.000 claims abstract description 7
- 239000003814 drug Substances 0.000 claims abstract description 5
- OVTLOLNDKQUMRH-QMMMGPOBSA-N (2r)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-3-[(3-nitropyridin-2-yl)disulfanyl]propanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CSSC1=NC=CC=C1[N+]([O-])=O OVTLOLNDKQUMRH-QMMMGPOBSA-N 0.000 claims abstract description 4
- 229940079593 drug Drugs 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 69
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 30
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 22
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 16
- 150000007530 organic bases Chemical class 0.000 claims description 16
- 239000002904 solvent Substances 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- 230000002194 synthesizing effect Effects 0.000 claims description 12
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 11
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 10
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 10
- 150000001875 compounds Chemical class 0.000 claims description 10
- 238000003776 cleavage reaction Methods 0.000 claims description 9
- 238000001953 recrystallisation Methods 0.000 claims description 9
- 150000007529 inorganic bases Chemical class 0.000 claims description 8
- 230000007017 scission Effects 0.000 claims description 8
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 6
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 6
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 claims description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 claims description 4
- 239000007822 coupling agent Substances 0.000 claims description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 4
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 4
- 238000010189 synthetic method Methods 0.000 claims description 3
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 claims description 2
- 239000007821 HATU Substances 0.000 claims description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 2
- 239000007810 chemical reaction solvent Substances 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 18
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 11
- 229920001184 polypeptide Polymers 0.000 abstract description 10
- 230000002209 hydrophobic effect Effects 0.000 abstract description 2
- 230000004048 modification Effects 0.000 abstract description 2
- 238000012986 modification Methods 0.000 abstract description 2
- 239000003960 organic solvent Substances 0.000 abstract description 2
- 238000005336 cracking Methods 0.000 abstract 1
- 239000012043 crude product Substances 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 43
- 238000003756 stirring Methods 0.000 description 28
- 239000000243 solution Substances 0.000 description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 22
- 239000000047 product Substances 0.000 description 21
- 239000012065 filter cake Substances 0.000 description 18
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 16
- 238000001914 filtration Methods 0.000 description 15
- 238000005406 washing Methods 0.000 description 14
- 239000011541 reaction mixture Substances 0.000 description 12
- 239000008367 deionised water Substances 0.000 description 10
- 229910021641 deionized water Inorganic materials 0.000 description 10
- 239000000203 mixture Substances 0.000 description 9
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 7
- 208000005770 Secondary Hyperparathyroidism Diseases 0.000 description 7
- 238000001035 drying Methods 0.000 description 7
- 239000000706 filtrate Substances 0.000 description 7
- 238000001291 vacuum drying Methods 0.000 description 7
- 239000007787 solid Substances 0.000 description 6
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 5
- 239000011575 calcium Substances 0.000 description 5
- 229910052791 calcium Inorganic materials 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 239000007858 starting material Substances 0.000 description 5
- 229910001868 water Inorganic materials 0.000 description 5
- GVIXTVCDNCXXSH-CQSZACIVSA-N (2r)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-azaniumylpentanoate Chemical compound OC(=O)[C@H](N)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C GVIXTVCDNCXXSH-CQSZACIVSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- 229910004373 HOAc Inorganic materials 0.000 description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 4
- 239000005457 ice water Substances 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 238000011031 large-scale manufacturing process Methods 0.000 description 4
- 108090000445 Parathyroid hormone Proteins 0.000 description 3
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 3
- 238000003760 magnetic stirring Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000011574 phosphorus Substances 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- QWXZOFZKSQXPDC-LLVKDONJSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-LLVKDONJSA-N 0.000 description 2
- 201000002980 Hyperparathyroidism Diseases 0.000 description 2
- 102000003982 Parathyroid hormone Human genes 0.000 description 2
- 102000007327 Protamines Human genes 0.000 description 2
- 108010007568 Protamines Proteins 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000001631 haemodialysis Methods 0.000 description 2
- 230000000322 hemodialysis Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 239000000199 parathyroid hormone Substances 0.000 description 2
- 229960001319 parathyroid hormone Drugs 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 229940048914 protamine Drugs 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 239000011345 viscous material Substances 0.000 description 2
- -1 4D-type Arg Chemical class 0.000 description 1
- 108010056764 Eptifibatide Proteins 0.000 description 1
- 235000013878 L-cysteine Nutrition 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 1
- 102100036893 Parathyroid hormone Human genes 0.000 description 1
- 229930003316 Vitamin D Natural products 0.000 description 1
- 206010047626 Vitamin D Deficiency Diseases 0.000 description 1
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000003281 allosteric effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000002092 calcimimetic effect Effects 0.000 description 1
- 239000007806 chemical reaction intermediate Substances 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 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 description 1
- 229960004468 eptifibatide Drugs 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000002990 parathyroid gland Anatomy 0.000 description 1
- 239000003495 polar organic solvent Substances 0.000 description 1
- 238000007867 post-reaction treatment Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 208000037920 primary disease Diseases 0.000 description 1
- 238000004537 pulping Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- YDJXDYKQMRNUSA-UHFFFAOYSA-N tri(propan-2-yl)silane Chemical compound CC(C)[SiH](C(C)C)C(C)C YDJXDYKQMRNUSA-UHFFFAOYSA-N 0.000 description 1
- 235000019166 vitamin D Nutrition 0.000 description 1
- 239000011710 vitamin D Substances 0.000 description 1
- 150000003710 vitamin D derivatives Chemical class 0.000 description 1
- 229940046008 vitamin d Drugs 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- 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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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
A synthesis method and application of Etelcalcetide, belonging to the technical field of drug synthesis. The invention comprises the following steps: (1) liquid phase synthesis of Fmoc-D-Ala-D-Arg (Pbf) -OH dipeptide fragment 1; (2) liquid phase synthesis of Fmoc-D-Arg (Pbf) -OH dipeptide fragment 2; (3) sequentially coupling amino acids: obtaining a dipeptide fragment A by dipeptide fragment 1, dipeptide fragment 2, dipeptide fragment 1 and N-Ac-D-Cys (Mmt) -OH; (4) removing the protecting group of Cys (Mmt) in the fragment A to obtain a fragment B, coupling with Boc-Cys (Npys) -OH, and cracking to obtain a crude product of Etelcalcetide. The invention can be applied to the direct modification of hydrophilic polypeptide, hydrophobic polypeptide and long-chain polypeptide and can be carried out in an organic solvent.
Description
Technical Field
The invention belongs to the technical field of drug synthesis, and particularly relates to a synthesis method and application of Etelcalcetide.
Background
Secondary Hyperparathyroidism (SHPT) is a disease in which hyperparathyroidism, elevation of blood parathyroid hormone, and thus aggravation of skeletal damage, are caused by abnormal calcium and phosphorus metabolism, vitamin D deficiency, or impairment of renal function. Current measures for the treatment of SHPT include active control of the primary disease that induces SHPT, restriction of dietary phosphorus intake, and, if necessary, phosphorus binders, active vitamin D analogs. Meanwhile, the calcium mimetic agent can effectively treat SHPT.
Eptifibatide (etelcalcide) is a calcimimetic used to treat SHPT in patients receiving hemodialysis. The administration was intravenous at the end of each dialysis session. It acts as an allosteric activator by binding and activating the calcium sensitive receptor (CaSR) in the parathyroid gland, inhibiting and reducing the secretion of parathyroid hormone, thereby achieving the goal of treating SHPT. The medicine is also the first intravenous calcium-simulating agent to be administrated by intravenous injection three times a week after the dialysis of a patient. After the patients with moderate and severe hyperparathyroidism hemodialysis receive the vilacatin treatment, the PTH level is obviously reduced. Etelcalcitide is a calcium mimetic agent that allosterically modulates the calcium sensitive receptor (CaSR). The main chain of the Etelcalcetide consists of 7D-type amino acids including 4D-type Arg, 2D-type Ala and acetylated D-type Cys, and the side chain is connected with L-cysteine through a disulfide bond to obtain the Etelcalcetide. The structure of Etelcalcetide is shown in FIG. 1, and the peptide sequence is shown in FIG. 2.
The synthesis of Etelcalcetide at home and abroad is reported, and for example, the patents with publication numbers of CN 105504012A, CN106928320, CN109280078B, CN110668984A, CN110498835A and CN110054662A all adopt solid-phase synthesis of Etelcalcetide. Although the solid-phase coupling process has the advantages of simple operation process, convenience for automatic production, high product yield and easiness in product separation, resin is required in the coupling process, the feeding multiple of amino acid usually needs to be excessive (3-5 equivalents) in order to ensure complete coupling, and the main chain of the Etelcalcetide is D-type amino acid, so that the price is relatively expensive compared with that of the conventional amino acid. Therefore, the solid phase synthesis method is too costly and is limited in production scale.
The patents with publication numbers CN106928321, WO2016154580A1 and CN111925418A all adopt liquid-phase synthesis of Etelcalcetide. The liquid phase synthesis method is suitable for large-scale production, has less amino acid consumption and achieves the aim of controlling the cost. However, the post-reaction treatment operation is complicated, the synthesis of insoluble peptides is difficult, the total yield is relatively low, polypeptide interchain disulfide bonds are formed in a liquid phase, the problem of disulfide bond mismatching occurs, the variety of side reactions and byproducts is large, the yield and the purity are not ideal, and the product yield is further reduced.
Disclosure of Invention
In view of the problems in the prior art, the invention aims to design and provide a synthetic method of Etelcalcetide and application thereof. The invention utilizes a liquid phase carrier method to synthesize the Etelcalcetide and uses two dipeptide fragments to improve the synthesis efficiency and reduce the defect impurities. The invention combines the advantages of solid phase synthesis and liquid phase synthesis, and achieves the purposes of simple operation, cost reduction, reduction of generation of defect impurities and suitability for large-scale production. And the operability is good, the effect of reducing impurities is good, the yield is improved, the cost is reduced, and the method is suitable for large-scale production.
In order to achieve the purpose, the invention adopts the following technical scheme:
a synthetic method of Etelcalcetide is characterized by comprising the following steps:
(1) amino acid is taken and put into a solvent for coupling and recrystallization, and Fmoc-D-Ala-D-Arg (Pbf) -OH dipeptide fragment is synthesized by liquid phase;
(2) putting amino acid into a solvent for coupling and recrystallization, and synthesizing Fmoc-D-Arg (Pbf) -OH dipeptide fragments by liquid phase;
(3) in terms of Dpm-NH2Is in liquid phaseSynthesizing a carrier, taking chloroform as a reaction solvent, and sequentially coupling the following amino acids: Fmoc-D-Ala-D-Arg (Pbf) -OH synthesized in the above step (1), Fmoc-D-Arg (Pbf) -OH synthesized in the above step (2), Fmoc-D-Ala-D-Arg (Pbf) -OH synthesized in the above step (1), N-Ac-D-Cys (Mmt) -OH to obtain N-Ac-D-Cys (Mmt) -D-Ala-D-Arg (Pbf) -D-Arg (Arg Pbf) (Pbf) -D-Ala-D-Arg (Pbf)2I.e., fragment A, of the formula 1;
(4) with 1% TFA/CHCl3Removing the protecting group of Cys (Mmt) in the fragment A by using the solution to obtain N-Ac-D-Cys (SH) -D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH2I.e. fragment B, is coupled with Boc-Cys (Npys) -OH and then cracked to obtain crude Etelcalcetide.
The method for synthesizing the Etelcalcetide comprises the steps (1) and (2) that the solvent comprises one or more of NMP, THF, DCM, ACN and DMF, the coupled system comprises one or more of DIC/HONb/organic base, DCC/HONb/organic base, EDCI/HOSu/organic base, DCC/HOSu/organic base, DIC/HONb/inorganic base and DCC/HONb/inorganic base, and the recrystallized solvent system comprises EtOH/H2O、DCM/Et2O、THF/Et2O、EA/Et2O、MeOH/Et2O、CH3CN/Et2O、EA/PE、THF/PE、CH3CN/H2One or more of O.
In the method for synthesizing Etelcalcetide, the organic base comprises at least one of DIPEA, triethylamine and N-methylmorpholine, preferably the organic base is DIPEA, and the inorganic base comprises Na2CO3Or NaHCO3Preferably the inorganic base is NaHCO3。
According to the synthesis method of Etelcalcetide, DCM is used as a solvent in the step (1), DIC/HONb/organic base is used as a coupling system, and EtOH/H is used as a recrystallization solvent system2O。
According to the synthesis method of the Etelcalcetide, the solvent in the step (2) is DCM, the coupling system is DIC/HONb/organic base, and the recrystallization solvent system is EA/PE.
The method for synthesizing Etelcalcetide comprises the step (3) of mixing amino acid with Dpm-NH2In a molar ratio of 1:1 to 3:1, the amino acid and Dpm-NH are preferably used2The molar ratio of (A) to (B) is 1.1: 1-1.3: 1, the coupling agent adopted for coupling comprises one or more of EDCI// HOBt, DIPCDI/HOBt, PyBop/HOBt/DIPEA, HBTU/HOBt/DIPEA, DIPCDI/HOAt, HATU/HOAt/DIPEA and PyAop/HOAt/DIPEA, and the coupling agent adopted for coupling is preferably EDCI// HOBt.
In the method for synthesizing Etelcalcetide, the volume of Mmt in the protecting group of Cys (Mmt) in the step (4) is TFA/CHCl 31% -5% of the volume of the solution, preferably the volume of Mmt in the protecting group of Cys (Mmt) is TFA/CHCl31-3% of the volume of the solution.
In the method for synthesizing Etelcalcetide, the cleavage reagent adopted in the step (4) comprises TFA, PhSMe, TIS, PhOH and H2O, PhOMe, preferably the cleavage reagent used for the cleavage is TFA: h2O: PhSMe: PhOMe: the volume ratio of TIS was 88:5:3:2: 2.
An Etelcalcetide synthesized by the method according to any one of claims 1 to 8.
Any one of the synthesis methods of Etelcalcetide is applied to synthesis of Etelcalcetide drugs.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention uses a liquid phase carrier Dpm-NH2The method is a homogeneous reaction, can be applied to the direct modification of hydrophilic polypeptide, hydrophobic polypeptide and long-chain polypeptide and can be carried out in an organic solvent, overcomes the defect that the synthesis scale of solid-phase polypeptide is limited, is suitable for large-scale production, and improves the production efficiency.
2. The amino acid equivalent used in each step of the reaction is 1.1eqv, compared with the solid phase polypeptide synthesis method, the method improves the utilization rate of the amino acid and saves the cost. The reaction intermediate in each step can be precipitated by using a polar organic solvent, compared with the traditional liquid phase polypeptide synthesis method, the post-treatment operation is simple, and the purity of the intermediate in each step is relatively high.
3. The invention uses two dipeptide fragments of Fmoc-D-Ala-D-Arg-OH and Fmoc-D-Arg-D-Arg-OH as starting materials to further improve the coupling efficiency. Greatly reduces the synthesis steps, reduces the generation of defective Arg impurities and reduces the synthesis difficulty of the peptide.
Drawings
FIG. 1 is a block diagram of Etelcalcetide;
FIG. 2 is a peptide sequence diagram of Etelcalcetide;
FIG. 3 shows Dpm-NH2The structural formula (1);
FIG. 4 is a scheme showing the synthesis scheme for Etelcalcetide;
FIG. 5 is an HPLC chart of the protamine;
FIG. 6 is a mass spectrum of the protamine.
Detailed Description
The invention will be further illustrated by the following figures and examples.
Example 1: synthesis of Fmoc-D-Ala-D-Arg (Pbf) -OH
Fmoc-D-Ala-OH (15.6g, 50mmol) and HOSu (6.33g, 55mmol) were dissolved in 200mL of THF, DCC (11.3g, 55mmol) was added in an ice-water bath, the ice bath was removed, and the mixture was stirred at room temperature for 5 hours. TLC showed the starting material was essentially completely reacted (PE: EA: HOAc ═ 1:1:0.05) the reaction mixture was filtered and the filtrate was ready for use.
200mL of deionized water dissolved Na2CO3(15.9g, 150mmol) and H-D-Arg (Pbf) -OH (21.32g, 50mmol) were slowly added to the newly prepared mixture, and the mixture was stirred at room temperature for 18 hours. Concentrating the reaction solution, adding 200mL deionized water to dilute the residual solution, EA extracting (250 × 3mL), acidifying 1mol/L hydrochloric acid solution of water layer to pH about 3, EA extracting, combining EA layers, washing the EA layer with 200mL 1mol/L hydrochloric acid solution, 200mL × 2mL deionized water for 2 times, washing the EA layer with 200mL saturated saline for 1 time, rapidly drying with 40g anhydrous sodium sulfate, filtering, concentrating, and using EtOH/H to concentrate the solution2And (3) recrystallizing O (2: 1). The product is filtered and dried in vacuum to obtain Fmoc-D-Ala-D-Arg (Pbf) -OH: 24.9g, HPLC: 98.2%, yield: 69.4 percent.
Example 2: synthesis of Fmoc-D-Ala-D-Arg (Pbf) -OH
Fmoc-D-Ala-OH (15.6g, 50mmol) and HONb (9.84g, 55mmol) were dissolved in 200mL DCM, DIC (8.5mL, 55mmol) was added in an ice-water bath, the ice bath was removed, and the mixture was stirred at room temperature for 5 h. TLC showed the starting material was essentially completely reacted (PE: EA: HOAc 1:1:0.05) the reaction mixture was filtered and the filtrate was ready for use.
H-D-Arg (Pbf) -OH (21.32g, 50mmol) was added to the above filtrate, magnetic stirring was started, and DIPEA (10.74mL, 65mmol) was added dropwise from the dropping funnel, and stirring was continued at room temperature for 10 hours. Concentrating the reaction solution, adding 200mL deionized water to dilute the residual solution, EA extracting (250 × 3mL), acidifying 1mol/L hydrochloric acid solution of water layer to pH about 3, EA extracting, combining EA layers, washing the EA layer with 200mL 1mol/L hydrochloric acid solution, 200mL × 2mL deionized water for 2 times, washing the EA layer with 200mL saturated saline for 1 time, rapidly drying with 40g anhydrous sodium sulfate, filtering, concentrating, and using EtOH/H to concentrate the solution2And (4) recrystallizing O (1: 4). The product was filtered and dried in vacuo to yield Fmoc-D-Ala-D-Arg (Pbf) -OH: 26.7g, HPLC: 98.9%, yield: 73.8 percent.
Example 3: synthesis of Fmoc-D-Arg (Pbf) -OH
Fmoc-D-Arg (Pbf) -OH (32.4g, 50mmol) and HOSu (6.33g, 55mmol) were dissolved in 200mL of THF, DCC (11.3g, 55mmol) was added in an ice-water bath, the ice bath was removed, and the mixture was stirred at room temperature for 5 hours. TLC showed the starting material was essentially completely reacted (PE: EA: HOAc 1:1:0.05) the reaction mixture was filtered and the filtrate was ready for use.
200mL of deionized water dissolved Na2CO3(15.9g, 150mmol) and H-D-Arg (Pbf) -OH (21.32g, 50mmol) were slowly added to the newly prepared mixture, and the mixture was stirred at room temperature for 18 hours. The reaction was concentrated, 200mL of deionized water was added to dilute the residue, EA extraction (250 × 3mL) was performed, the aqueous layer was acidified to pH about 3 with 1mol/L hydrochloric acid solution, EA extraction was performed, the EA layers were combined, washed with 200mL of 1mol/L hydrochloric acid solution, 200mL × 2mL of deionized water for 2 times, 200mL was washed with saturated brine for 1 time in this order, flash dried over 40g of anhydrous sodium sulfate, filtered and concentrated, and the concentrate was recrystallized from EA: PE (3: 1). Filtering the product, and drying in vacuum to obtain Fmoc-D-Arg (Pbf) -D-arg (pbf) -OH: 37.1g, HPLC: 98.9%, yield: 70.2 percent.
Example 4: synthesis of Fmoc-D-Arg (Pbf) -OH
Fmoc-D-Arg (Pbf) -OH (32.4g, 50mmol) and HONb (9.84g, 55mmol) were dissolved in 200mL of DCM, and DIC (8.5mL, 55mmol) was gradually added dropwise with ice-water bath, followed by removal of the ice bath and stirring at room temperature for 5 h. TLC showed the starting material was essentially completely reacted (PE: EA: HOAc 1:1:0.05) the reaction mixture was filtered and the filtrate was ready for use.
H-D-Arg (Pbf) -OH (21.32g, 50mmol) was added to the above filtrate, magnetic stirring was started, and DIPEA (10.74mL, 65mmol) was added dropwise from the dropping funnel, and stirring was continued at room temperature for 10 hours. Concentrating the reaction solution, adding 200mL deionized water to dilute the residual solution, EA extracting (250 × 3mL), acidifying 1mol/L hydrochloric acid solution of water layer to pH about 3, EA extracting, combining EA layers, washing the EA layer with 200mL 1mol/L hydrochloric acid solution, 200mL × 2mL deionized water for 2 times, washing the EA layer with 200mL saturated saline for 1 time, rapidly drying with 40g anhydrous sodium sulfate, filtering, concentrating, and using EtOH/H to concentrate the solution2And (4) recrystallizing O (1: 4). Filtering the product, and drying in vacuum to obtain Fmoc-D-Arg (Pbf) -OH: 39.8g, HPL C: 99.1%, yield: 75.3 percent.
Example 5: Fmoc-D-Ala-D-Arg (Pbf) -Dpm-NH2Synthesis of (2)
Weighing liquid phase carrier Dpm-NH2(8.3g,10.0mmol) was charged into a 250mL three-necked flask, and CHCl was added to the reaction flask3(80mL), HOBt (1.3g, 10.0mmol) and Fmoc-D-Ala-D-Arg (Pbf) -OH (8.64g, 12.0mmol) in example 2 were added in that order. Stirring and dissolving to clear. EDCI (2.6g, 13mmol) was added and stirring continued at room temperature for 3 h. The reaction was monitored by TLC (DCM: MeOH: HAc: 100:1: 0.5). After completion of the reaction, the reaction mixture was concentrated under reduced pressure at 30 ℃ to give a viscous product, and methanol (80mL) was added to the viscous product, followed by stirring for 2 hours. Filtration and washing of the filter cake three times with methanol (30 mL. times.3). Vacuum drying the filter cake at 40 ℃ for 5 hours to obtain Fmoc-D-Ala-D-Arg (Pbf) -Dpm-NH2(14.64g, yield 95.5%).
Example 6: Fmoc-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH2Synthesis of (2)
The compound of example 5 was weighed out as Fmoc-D-Ala-D-Arg (Pbf) -Dpm-NH2(12.27g, 8.01mmol) was charged in a 1.0L three-necked flask, chloroform (400mL) was added to the reaction flask, and after stirring to dissolve the solution, DBU (1.22g, 8.01mmol) was added thereto. The reaction was cooled to below 5 ℃ in an ice bath and diethylamine (6.56g, 90mmol) was slowly added dropwise with the temperature controlled not to exceed 5 ℃. After the addition was complete, the reaction was allowed to warm to room temperature and stirring was continued for 2 hours. The reaction was monitored by TLC (DCM: MeOH: HAc: 100:1: 0.5). After completion of the reaction, the reaction mixture was concentrated under reduced pressure at 30 ℃ to give a viscous substance, and acetonitrile (80mL) was added to the viscous substance, followed by stirring for 30 minutes. Filtration and washing of the filter cake twice with methanol (40 mL. times.2). Vacuum drying the filter cake at 40 deg.C for 2 hr to obtain solid H-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH2＝14.67g。
The solid was charged into a 500mL three-necked flask, chloroform (150mL) was added to the flask, and HO Bt (1.08g, 8.01mmol) and Fmoc-D-Arg (Pbf) -OH (10.15g, 9.61mm ol) in example 3 were sequentially added thereto. Stirring and dissolving to clear. The reaction was cooled to 0 ℃. EDCI (1.69g, 8.95mmol) was added and stirring was continued at 0-10 ℃ for 3 h. The reaction was monitored by TLC (DCM: MeOH: HAc: 100:1: 0.5). After completion of the reaction, the reaction mixture was concentrated under reduced pressure at 30 ℃ to give a viscous product, and methanol (40mL) was added to the viscous product, followed by stirring for 2 hours. Filtration and washing of the filter cake three times with methanol (40 mL. times.3). Vacuum drying the filter cake at 40 deg.C for 3 hr to obtain Fmoc-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH2(17.25g, yield 91.6%).
Example 7: Fmoc-D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH2Synthesis of (2)
The compound of example 6, Fmoc-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH, was weighed2(16.45g, 7.0mmol) was charged in a 1.0L three-necked flask, chloroform (200mL) was added to the reaction flask, and then DBU (1.06g, 7.0mmol) was added thereto with stirring. The reaction was cooled to below 5 ℃ in an ice bath and diethylamine (5.47g, 75mmol) was slowly added dropwise, the temperature was controlled not to exceed 5 ℃. After the addition was complete, the reaction was allowed to warm to room temperature and stirring was continued for 2 hours. The reaction was monitored by TLC (DCM: MeO H: HAc ═ 100:1: 0.5). After the reaction is completedThe reaction mixture was concentrated under reduced pressure at 30 ℃ to give a viscous product, and acetonitrile (70mL) was added to the viscous product, followed by stirring for 30 minutes. Filtration and washing of the filter cake twice with methanol (40 mL. times.2). Vacuum drying the filter cake at 40 deg.C for 2 hr to obtain solid H-D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH2＝15.95g。
The solid was charged into a 500mL three-necked flask, chloroform (160mL) was added to the reaction flask, and HO Bt (0.94g, 7.0mmol) and Fmoc-D-Ala-D-Arg (Pbf) -OH (16.04g, 8.4mmol) in example 3 were sequentially added thereto. Stirring and dissolving to clear. The reaction was cooled to 0 ℃. EDCI (1.45g, 7.7mmol) was added and stirring was continued at 0-10 ℃ for 3 h. The reaction was monitored by TLC (DCM: MeOH: HAc: 100:1: 0.5). After completion of the reaction, the reaction mixture was concentrated under reduced pressure at 30 ℃ to give a viscous product, and methanol (40mL) was added to the viscous product, followed by stirring for 2 hours. Filtration and washing of the filter cake three times with methanol (40 mL. times.3). Vacuum drying the filter cake at 40 deg.C for 3 hr to obtain Fmoc-D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH2(18.52g, yield 93.5%).
Example 8: N-Ac-D-Cys (Mmt) -D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH2Synthesis of (2)
The compound of example 7 was weighed Fmoc-D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH2(18.4g, 6.5mmol) was charged in a 1.0L three-necked flask, chloroform (200mL) was added to the reaction flask, and after stirring to dissolve the solution, DBU (0.98g, 6.5mmol) was added thereto. The reaction was cooled in an ice bath to below 5 ℃ and diethylamine (5.10g,70mmol) was slowly added dropwise with the temperature controlled not to exceed 5 ℃. After the addition was complete, the reaction was allowed to warm to room temperature and stirring was continued for 2 hours. The reaction was monitored by TLC (DCM: MeOH: HAc: 100:1: 0.5). After completion of the reaction, the reaction mixture was concentrated under reduced pressure at 30 ℃ to give a viscous product, and acetonitrile (80mL) was added to the viscous product, followed by stirring for 30 minutes. Filtration and the filter cake was washed twice with methanol (50 mL. times.2). The filter cake was dried under vacuum at 40 ℃ for 2 hours to give solid H-D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH2＝18.16g。
Adding the above solid to 500mIn a three-necked flask, chloroform (200mL) was added to the reaction flask, and HO Bt (0.87g, 6.5mmol) and N-Ac-D-Cys (Mmt) -OH (3.39g, 7.8mmol) were sequentially added thereto. Stirring and dissolving to clear. The reaction was cooled to 0 ℃. EDCI (1.35g, 7.2mmol) was added and stirring was continued at 0-10 ℃ for 3 h. The reaction was monitored by TLC (DC M: MeOH: HAc ═ 100:1: 0.5). After completion of the reaction, the reaction mixture was concentrated under reduced pressure at 30 ℃ to give a viscous product, and methanol (50mL) was added to the viscous product, followed by stirring for 2 hours. Filtration and washing of the filter cake three times with methanol (50 mL. times.3). Vacuum drying the filter cake at 40 deg.C for 3 hr to obtain N-Ac-D-Cys (Mmt) -D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH2(18.15g, yield 92.3%).
Example 9: N-Ac-D-Cys (SH) -D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dp m-NH2Synthesis of (2)
The compound N-Ac-D-Cys (Mmt) -D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH in example 8 was weighed2(18.0g,5.95mmol) was charged to a 1.0L three-necked flask, and 300mL of 1% TFA/CHCl was added3After magnetically stirring at room temperature for 5 minutes, the reaction mixture was concentrated under reduced pressure at 30 ℃ to give a viscous product, and acetonitrile (80mL) was added to the viscous product, followed by stirring for 30 minutes. Filtration and washing of the filter cake twice with methanol (50 mL. times.2); this process was repeated 2 times for a total of 3 times. Obtaining the compound N-Ac-D-Cys (SH) -D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH2(17.65g, yield 98.05%)
Example 10: c152H243N21O26S6Synthesis of (2)
The compound N-Ac-D-Cys (SH) -D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH in example 9 was weighed2(17.65g, 6.41mmol) was charged to a 1.0L three-necked flask, and chloroform (250mL) and Boc-Cys (Npys) -OH (2.64g, 7.05mmol) were added to the reaction flask. Stirring and dissolving to clear. The reaction was cooled to 0 ℃. DIPEA (2.0mL, 10mmol) was added and stirring continued at 20-30 deg.C for 24 h. The reaction was monitored by TLC (DCM: MeOH: HA c ═ 100:1: 0.5). After the reaction is completed, the reaction solution is decompressed and concentrated to be viscous at the temperature of 30 ℃ until the reaction solution is viscousMethanol (50mL) was added to the mixture, and the mixture was stirred for 2 hours. Filtration and washing of the filter cake three times with methanol (50 mL. times.3). Vacuum drying the filter cake at 40 deg.C for 3 hr to obtain compound C152H243N21O26S616.19 g, yield 85%, the structural formula is as follows.
Example 11: c38H73N21O10S2Synthesis of (2)
16.0g of the compound of example 10 was placed in a 1.0L reaction flask and 160mL of the cleavage reagent TFA H was added at a ratio of 10mL/g of the compound2O PhSMe, Anisole, TIS 88:5:3:2:2, and the cleavage reaction was performed with magnetic stirring at room temperature for 2.5 hours. The reaction was then poured slowly into frozen MTBE (1600mL), stirred for 30 minutes and allowed to stand in the refrigerator for 1 hour. Centrifuged and washed three times with ether (100 mL. times.3). Drying the obtained precipitate at 30 deg.C for 2 hr, pulping with methanol (100mL) for 2 hr, filtering, discarding the filter cake, and spin-drying the filtrate at 40 deg.C to obtain crude peptide (10.03g, 136.8%) which is Etelcalcetide (formula C)38H73N21O10S2). The crude peptide had an HPLC purity of about 89.34%.
After purification, 6.08g of Etelcalcetide refined peptide is obtained, the total yield is 82.95%, and the purity of the refined peptide is 99.37%. The chemical structural formula is as follows:
the abbreviations and English meanings referred to in the above examples 1 to 11 are shown in Table 1 below.
TABLE 1 abbreviations and English meanings
Claims (10)
1. A synthetic method of Etelcalcetide is characterized by comprising the following steps:
(1) amino acid and a compound are taken and placed in a solvent to form a coupling system for coupling reaction, then recrystallization is carried out, and Fmoc-D-Ala-D-Arg (Pbf) -OH dipeptide fragments are synthesized in a liquid phase;
(2) amino acid and compound are put into a solvent to form a coupling system for coupling reaction, and then recrystallization is carried out, and Fmoc-D-Arg (Pbf) -OH dipeptide fragments are synthesized in a liquid phase;
(3) in terms of Dpm-NH2The method is characterized in that a liquid-phase synthesis carrier is prepared by sequentially coupling the following amino acids by using chloroform as a reaction solvent: Fmoc-D-Ala-D-Arg (Pbf) -OH synthesized in the above step (1), Fmoc-D-Arg (Pbf) -OH synthesized in the above step (2), Fmoc-D-Ala-D-Arg (Pbf) -OH synthesized in the above step (1), N-Ac-D-Cys (Mmt) -OH to obtain N-Ac-D-Cys (Mmt) -D-Ala-D-Arg (Pbf) -D-Arg (Arg Pbf) (Pbf) -D-Ala-D-Arg (Pbf)2I.e., fragment A, of the formula 1;
(4) with 1% TFA/CHCl3Removing the protecting group of Cys (Mmt) in the fragment A by using the solution to obtain N-Ac-D-Cys (SH) -D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH2I.e. fragment B, is coupled with Boc-Cys (Npys) -OH and then cracked to obtain crude Etelcalcetide.
2. The method for synthesizing Etelcalcetide according to claim 1, wherein the solvent in step (1) and step (2) comprises one of NMP, THF, DCM, ACN and DMFOr a plurality of coupling systems, wherein the coupling system comprises one or more of DIC/HONb/organic base, DCC/HONb/organic base, EDCI/HOSu/organic base, DCC/HOSu/organic base, DIC/HONb/inorganic base, DCC/HONb/inorganic base, and the recrystallization solvent system comprises EtOH/H2O、DCM/Et2O、THF/Et2O、EA/Et2O、MeOH/Et2O、CH3CN/Et2O、EA/PE、THF/PE、CH3CN/H2One or more of O.
3. The synthesis method of Etelcalcetide as claimed in claim 2, wherein said organic base comprises at least one of DIPEA, triethylamine and N-methylmorpholine, preferably said organic base is DIPEA and said inorganic base comprises Na2CO3Or NaHCO3Preferably the inorganic base is NaHCO3。
4. The method for synthesizing Etelcalcetide according to claim 2, wherein the solvent in step (1) is DCM, the coupling system is DIC/HONb/organic base, and the solvent system for recrystallization is EtOH/H2O。
5. The method for synthesizing Etelcalcetide according to claim 2, wherein the solvent in step (2) is DCM, the coupling system is DIC/HONb/organic base, and the solvent system for recrystallization is EA/PE.
6. The method for synthesizing Etelcalcetide according to claim 1, wherein the amino acid in step (3) is reacted with Dpm-NH2Is 1:1 to 3:1, and preferably amino acid and Dpm-NH2The molar ratio of (A) to (B) is 1.1: 1-1.3: 1, the coupling agent adopted for coupling comprises one or more of EDCI// HOBt, DIPCDI/HOBt, PyBop/HOBt/DIPEA, HBTU/HOBt/DIPEA, DIPCDI/HOAt, HATU/HOAt/DIPEA and PyAop/HOAt/DIPEA, and the coupling agent adopted for coupling is preferably EDCI// HOBt.
7. The method of claim 1The method for synthesizing Etelcalcetide is characterized in that the volume of Mmt in the protecting group of Cys (Mmt) in the step (4) is TFA/CHCl31% -5% of the volume of the solution, preferably the volume of Mmt in the protecting group of Cys (Mmt) is TFA/CHCl31-3% of the volume of the solution.
8. The method for synthesizing Etelcalcetide as claimed in claim 1, wherein the cleavage reagent used in the cleavage in step (4) comprises TFA, PhSMe, TIS, PhOH, H2O, PhOMe, preferably the cleavage reagent used for the cleavage is TFA: h2O: PhSMe: PhOMe: the volume ratio of TIS was 88:5:3:2: 2.
9. Etelcalcetide characterized in that it is synthesized using the Etelcalcetide synthesis method according to any one of claims 1 to 8.
10. Use of the method of any one of claims 1 to 8 for the synthesis of an etelcalcide drug.
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