WO2017114238A1 - 一种合成Etelcalcetide的方法 - Google Patents

一种合成Etelcalcetide的方法 Download PDF

Info

Publication number
WO2017114238A1
WO2017114238A1 PCT/CN2016/111093 CN2016111093W WO2017114238A1 WO 2017114238 A1 WO2017114238 A1 WO 2017114238A1 CN 2016111093 W CN2016111093 W CN 2016111093W WO 2017114238 A1 WO2017114238 A1 WO 2017114238A1
Authority
WO
WIPO (PCT)
Prior art keywords
arg
pbf
ala
cys
fmoc
Prior art date
Application number
PCT/CN2016/111093
Other languages
English (en)
French (fr)
Inventor
刘飞孟
伍柯瑾
宓鹏程
陶安进
袁建成
Original Assignee
深圳翰宇药业股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳翰宇药业股份有限公司 filed Critical 深圳翰宇药业股份有限公司
Publication of WO2017114238A1 publication Critical patent/WO2017114238A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents

Definitions

  • the invention relates to the field of pharmaceutical synthesis, and in particular to a method for synthesizing Etelcalcetide.
  • Secondary hyperparathyroidism refers to chronic renal insufficiency, intestinal malabsorption syndrome, Fanconi syndrome and renal tubular acidosis, vitamin D deficiency or resistance, and pregnancy.
  • the parathyroid gland is stimulated by hypocalcemia, hypomagnesemia or hyperphosphatemia for a long time to secrete excess parathyroid hormone (PTH) to increase blood calcium, blood magnesium and reduce blood phosphorus.
  • PTH parathyroid hormone
  • Etelcalcetide is a novel calciimetic agent developed by Kai Pharmaceuticals, Inc. that inhibits the secretion of parathyroid hormone (PTH).
  • Primary hyperparathyroidism SHPT
  • SHPT Secondary hyperparathyroidism
  • CKD chronic kidney disease
  • PTH parathyroid hormone
  • Etelcalcetide binds to and activates calcium-sensitive receptors on the parathyroid glands to reduce levels of parathyroid hormone (PTH).
  • Etelcalcetide has three D-configuration arginine, two D-form alanine, one D-form arginine, one L-configuration cysteine and one D-configuration cysteine
  • the acid (block N is blocked by an acetyl group) in which the D-configuration cysteine is linked to the L-configuration cysteine by a disulfide bond (N-acetyl-D-cysteinyl-D-alanyl-D-arginyl -D-arginyl-D-arginyl-D-alanyl-D-Argininamide, disulfide with L-cysteine), whose structure is as follows:
  • the compound CN201080045024.9 first reported the compound, but no synthetic process report of the compound was observed.
  • the key to the synthesis of this compound lies in the construction of disulfide bonds.
  • the methods for constructing disulfide bonds used in conventional peptide synthesis include air oxidation, iodine/acetic acid system oxidation, hydrogen peroxide oxidation, etc. These methods are only suitable for cyclic peptides.
  • the construction of the disulfide bond, the disulfide bond of the cyclic peptide is often the two thiol groups in the molecule are connected by oxidation, and the reaction needs to be carried out by using a very dilute solution to reduce the occurrence of the intermolecular reaction.
  • Etelcalcetide requires an intermolecular thiol oxidation reaction. If the reaction is carried out according to the conventional disulfide bond construction method, the final product of desired yield and purity cannot be obtained. In addition, if the synthesis of the compound is synthesized by a common solid phase synthesis method, the sulfhydryl group is relatively easily removed to form a degradation impurity, and the purification is extremely difficult.
  • the present invention provides the following technical solutions:
  • a method of synthesizing Etelcalcetide comprising the following steps:
  • Step 1 Liquid phase synthesis of a linear heptapeptide which is N-terminally acetylated and C-terminally amidated in the amino acid sequence shown in SEQ ID NO: 1.
  • Step 2 L-Cys chlorination of NCS, the hydrogen on the sulfhydryl group is replaced by chlorine to form L-Cys (SCl);
  • Step 3 coupling the linear heptapeptide synthesized in step 1 and L-Cys (SCl) to form two Sulfide, obtained Etelcalcetide;
  • Steps 1 and 2 are in no particular order.
  • the invention synthesizes the Etelcalcetide main chain 7 peptide by liquid phase synthesis method, and prepares the active intermediate L-Cys (SCl) by NCS chlorination method, and uses the active intermediate to construct a disulfide bond to synthesize Etelcalcetide to ensure the specific selection of the reaction. Sex, high yield, less impurities.
  • step 1 is:
  • the protected amino acid is used as a raw material to synthesize a fully protected linear heptapeptide which is N-terminally acetylated and C-terminally amidated in the amino acid sequence shown in SEQ ID NO: 1 under the action of a coupling system, and then cleavage and removal of each amino acid.
  • the protecting group is obtained by obtaining a linear heptapeptide.
  • step 1 is:
  • N-Ac-D-Cys(Trt)-OH is N-terminally acetylated Protected cysteine.
  • step 1 is:
  • Step 1.1 the Fmoc-D-Arg (Pbf) -OH C-terminal amidation, to obtain Fmoc-D-Arg (Pbf) -NH 2 , then Fmoc-D-Arg (Pbf) -NH 2 as a C-terminal,
  • the amino acid sequence of the amino acid sequence shown in SEQ ID NO: 1 was synthesized by Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Ala-OH protecting amino acid under the action of isopropyl chloroformate/DIPEA coupling system.
  • the fully protected polypeptide A at positions 4-7 ie, Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH 2 ;
  • N-Ac-D-Cys(Trt)-OH is N-terminal, and Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Ala-OH is used to protect amino acids in DIC/HOSu/DIPEA three-agent coupling system or The full-protection polypeptide B from the N-terminus to the C-terminal position 1-3 of the amino acid sequence shown in SEQ ID NO: 1 by the HATU/DIPEA double-reagent coupling system, ie N-Ac-D-Cys(Trt)- D-Ala-D-Arg(Pbf)-OH;
  • Step 1.2 Coupling the fully protected polypeptide A and the full protective polypeptide B with a HATU/DIPEA double reagent coupling system to obtain a fully protected linear heptapeptide N-Ac-D-Cys(Trt)-D-Ala-D-Arg (Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH 2 ;
  • Step 1.3 cleavage of the fully protected linear heptapeptide to remove the protective amino acid protecting group to obtain the linear heptapeptide N-Ac-D-Cys-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala- D-Arg-NH 2 .
  • Etelcalcetide backbone 7 peptide protocol of the present invention 1-3 and 4-7 fragments are first synthesized according to the Etelcalcetide main chain peptide sequence (amino acid sequence of SEQ ID NO: 1 of the present invention), respectively, corresponding to full protection. Peptides B and A, and then the two polypeptide fragments are coupled to obtain a fully protected direct heptapeptide, numbered in the amino acid sequence from the N-terminus to the C-terminus of the Etelcalcetide backbone, as follows:
  • the protecting group of the present invention is a protecting group for protecting the amino acid main chain and the amino group, the carboxyl group, the sulfhydryl group and the like which interfere with the synthesis in the field of polypeptide synthesis, and prevents the amino group, the carboxyl group and the like from occurring in the process of preparing the target product.
  • the reaction produces impurities, and the amino acid protected by the protecting group is called a protected amino acid.
  • groups that require protection of the amino acid side chains, side chain structures, and how to couple the protecting groups are well known to those skilled in the art.
  • Amino acid representations conjugated to a protecting group in the present invention are also commonly used in the art and are well known to those skilled in the art, such as Fmoc-D-Arg(Pbf)-OH, Fmoc is an N-terminal protecting group for amino acids, in parentheses Pbf is an Arg side chain protecting group, and other protective amino acid synthetic starting materials of the present invention can be referred to unless otherwise specified.
  • Boc-L-Cys-OtBu protected amino acid Boc is its N-terminal protecting group and OtBu is its carboxylic acid protecting group.
  • the polypeptide fragments of interest are preferably synthesized by coupling one by one, and the coupling one by one means starting from the first amino acid, and the remaining amino acids are in the order of the amino acid sequence shown in SEQ ID NO: 1 and the previous one.
  • the coupled amino acid is subjected to a condensation reaction (condensation reaction of a main chain amino group and a carboxyl group) for coupling.
  • a condensation reaction condensation reaction of a main chain amino group and a carboxyl group
  • the Fmoc protecting group was terminated, and the Boc-L-Cys-OtBu carboxylic acid protecting group OtBu was removed with trifluoroacetic acid. Due to constant amino acid coupling, Synthetic polypeptide fragments are constantly changing, preferably, the molar ratio of each amino acid starting material to be coupled to a previously synthesized polypeptide fragment, the molar ratio between each two amino acid materials to be coupled, and each two polypeptides The molar ratio between the fragments is close to 1:1, and other applicable molar ratios can be adjusted according to the actual reaction.
  • amidation of the C-terminus of the linear heptapeptide can be carried out by amidation of the C-terminus of arginine (for example, reaction with aqueous ammonia) to carry out liquid phase synthesis as a C-terminus;
  • the N-terminus of cysteine is acetylated to carry out liquid phase synthesis as an N-terminus, and can also be synthesized using commercially available acetylated cysteine.
  • step 2 of the present invention is:
  • Boc-L-Cys-OtBu performs chlorination of NCS to replace the hydrogen on the sulfhydryl group with chlorine to form Boc-L-Cys(SCl)-OtBu.
  • the sulfhydryl group is relatively easy to remove to form a technical problem of degrading impurities, and the present invention solves the problem of yield reduction and purity reduction by reducing the reaction temperature to the greatest extent, so the step 1 of the present invention is as described in the first step of the present invention.
  • the liquid phase synthesis, the NCS chlorination in step 2, and the coupling reaction in step 3 are preferably carried out at 0-10 ° C, more preferably between 0 ° C ⁇ reaction temperature ⁇ 10 ° C.
  • the liquid phase synthesis in step 1, the NCS chlorination in step 2, and the coupling reaction in step 3 are both DMF, DCM or a mixture of the two (the volume ratio of the mixture is preferably 1:1). .
  • the cleavage is carried out by adding a mixed solution of dichloromethane and trifluoroacetic acid, and further preferably, the volume ratio of dichloromethane to trifluoroacetic acid is 15:85.
  • the present invention adopts the all-liquid phase method to synthesize Etelcalcetide, and in the liquid phase synthesis process, simple and easy purification means such as crystallization and beating can be used, and the quality of the intermediate is greatly improved.
  • the solid phase method uses less reagents and solvents, is environmentally friendly, and does not use expensive resins, which reduces costs.
  • the low-cost and readily available NCS forms a disulfide bond with cysteine to form a disulfide bond, which avoids the disadvantages of low conversion rate and low purity of the traditional method, and is beneficial to the large-scale production of Etelcalcetide.
  • the invention discloses a method for synthesizing Etelcalcetide, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method of the present invention has been described in terms of preferred embodiments, and it is obvious that those skilled in the art can modify and modify and combine the compounds and preparation methods described herein without departing from the scope of the present invention.
  • the technology of the present invention is described in terms of preferred embodiments, and it is obvious that those skilled in the art can modify and modify and combine the compounds and preparation methods described herein without departing from the scope of the present invention. The technology of the present invention.
  • each intermediate product can be purified by means of crystallization, column chromatography or the like, such as ethyl acetate heated under reflux, dissolved in n-hexane, cooled and crystallized, added with ethyl acetate, heated to reflux, and then cooled and crystallized (acetic acid).
  • Ethyl acetate beating), column chromatography (dichloromethane/methanol gradient elution); and fully protected linear heptapeptide can be purified by ethanol recrystallization, and the linear heptapeptide and the final product can be precipitated by diethyl ether.
  • Fmoc-D-Arg(Pbf)-OH 130 g, 200 mmol was added to 1000 ml of dichloromethane, cooled to less than 10 ° C in an ice water bath, and isopropyl chloroformate (27 g, 220 mmol, 1.1 equ.) was added and stirred. After 5 minutes, DIPEA (31 g, 242 mmol, 1.21 equ.) was slowly added dropwise, and the mixture was kept at a temperature below 10 ° C, stirred, and the reaction was monitored by TLC until the starting material disappeared.
  • Fmoc-D-Arg(Pbf)-OH 130 g, 200 mmol was added to 1000 ml of dichloromethane, the temperature of the reaction system was controlled at 30-40 ° C, and isopropyl chloroformate (27 g, 220 mmol, 1.1 equ.) was added. After stirring for 5 minutes, DIPEA (31 g, 242 mmol, 1.21 eq.) was slowly added dropwise, the mixture was maintained at a temperature of 30-40 ° C, stirred, and the reaction was monitored by TLC until the starting material disappeared.
  • Fmoc-D-Ala-OH (4.67 g, 15 mmol) was added to 100 ml of dichloromethane, stirred for 10 minutes, cooled to less than 10 ° C in an ice water bath, and isopropyl chloroformate (2.0 g, 16.5 mmol, 1.1) was added. After equ.), after stirring for 5 minutes, DIPEA (2.33 g, 18.1 mmol, 1.21 equ.) was slowly added dropwise, and the mixture was kept at a temperature below 10 ° C, stirred, and the reaction was monitored by TLC until the starting point disappeared.
  • Fmoc-D-Arg(Pbf)-NH 2 was removed from the Fmoc protecting group, and HD-Arg(Pbf)-NH 2 (7.0 g, 16.5 mmol) was dissolved in dichloromethane (50 ml), and then added to the above reaction solution. In the middle, the temperature of the system was kept below 10 ° C and stirring was continued for 10 hours until the starting point disappeared (TLC).
  • reaction mixture was washed once with a saturated sodium hydrogen carbonate solution, washed twice with water and dried over anhydrous sodium sulfate.
  • the obtained white solid was added to ethyl acetate (100 ml), and evaporated to reflux, and the solid was dissolved, and 50 ml of n-hexane was slowly added to the ethyl acetate solution, and the solution was refluxed for 0.5 hour, then slowly cooled to room temperature. The solid was precipitated and filtered to give 8.9 g of a white solid. The purity is 95.7%.
  • Fmoc-D-Ala-OH (4.67 g, 15 mmol) was added to 100 ml of dichloromethane, stirred for 10 minutes, cooled to less than 10 ° C in an ice water bath, and isopropyl chloroformate (2.0 g, 16.5 mmol, 1.1) was added. After stirring for 5 minutes, DIPEA (2.33 g, 18.1 mmol, 1.21 equ.) was slowly added dropwise, and the resulting mixture was kept at a system temperature of 30-40 ° C, stirred, and the reaction was monitored by TLC until the starting point disappeared.
  • Fmoc-D-Arg(Pbf)-NH 2 was removed from the Fmoc protecting group, and HD-Arg(Pbf)-NH 2 (7.0 g, 16.5 mmol) was dissolved in dichloromethane (50 ml), and then added to the above reaction solution. In the middle, keep the temperature of the system at 30-40 ° C and continue to stir until the material point disappears (TLC).
  • reaction mixture was washed once with a saturated sodium hydrogen carbonate solution, washed twice with water, dried over anhydrous sodium sulfate and evaporated
  • the obtained white solid was added to ethyl acetate (100 ml), and evaporated to reflux, and the solid was dissolved, and 50 ml of n-hexane was slowly added to the ethyl acetate solution, and the solution was refluxed for 0.5 hour, then slowly cooled to room temperature. The solid was precipitated and filtered to give 7.2 g of a white solid. The purity is 93.8%.
  • Fmoc-D-Ala-R-Arg(Pbf)-NH 2 (8.9 g 12 mmol) was added to dichloromethane (200 ml). After stirring for 5 min, piperidine (5.1 g, 60 mmol) was added dropwise and the reaction mixture was continued. Stir until Fmoc-D-Ala-R-Arg(Pbf)-NH 2 is completely gone (TLC).
  • Fmoc-D-Arg(Pbf)-OH (6.5 g, 10 mmol) was added to 100 ml of N,N-dimethylformamide, cooled to less than 10 ° C in an ice water bath, and isopropyl chloroformate (1.34 g, 11 mmol, 1.1 equ.), after stirring for 5 minutes, DIPEA (1.56 g, 12.1 mmol, 1.21 equ.) was slowly added dropwise, and the mixture was kept at a temperature below 10 ° C, stirred, and the reaction was monitored by TLC until the material disappeared.
  • Fmoc-D-Ala-D-Arg(Pbf)-NH 2 (8.9 g 12 mmol) was added to dichloromethane (200 ml). After stirring for 5 min, piperidine (5.1 g, 60 mmol) was added dropwise and the reaction mixture was continued. Stir until Fmoc-D-Ala-D-Arg(Pbf)-NH 2 disappears completely (TLC). After washing with dilute hydrochloric acid to a dichloromethane solution, it was neutral, and the mixture was separated, and the methylene chloride liquid was dried. The solvent was evaporated to give an oily substance, HD-Al aD-Arg(Pbf)-NH 2 (5.4 g, yield: 89.0%). The oil was treated with ethyl acetate / n-hexane to afford pure.
  • Fmoc-D-Arg(Pbf)-OH (6.5 g, 10 mmol) was added to 100 ml of N,N-dimethylformamide, cooled to less than 10 ° C in an ice water bath, and isopropyl chloroformate (1.34 g, 11 mmol, 1.1 equ.), after stirring for 5 minutes, DIPEA (1.56 g, 12.1 mmol, 1.21 equ.) was slowly added dropwise, and the mixture was cooled to below 10 ° C in an ice water bath, stirred, and the reaction was monitored by TLC until the material disappeared.
  • HD-Ala-D-Arg(Pbf)-NH 2 5.3 g, 10.3 mmol was dissolved in N,N-dimethylformamide (50 ml), and then added to the above reaction solution to maintain the system temperature 30-40 Stirring was continued for more than 10 hours at °C until the starting material disappeared (TLC). The resulting reaction solution was added to 5 volumes of water and allowed to stand overnight.
  • Fmoc-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH 2 (10.2 g 8.77 mmol) was added to dichloromethane (250 ml). After stirring for 5 min, piperidine (4.25) was added dropwise. g, 50 mmol), the reaction mixture was stirred until Fmoc-D-Ala-D-Arg(Pbf)-NH2 completely disappeared (TLC). Column chromatography (dichloromethane / methanol gradient), the solvent was removed to give a white solid HD-Arg (Pbf) -D- Ala-D-Arg (Pbf) -NH 2 (5.31g, yield 64.3%) .
  • Fmoc-D-Arg(Pbf)-OH (3.6 g, 5.5 mmol) was added to 80 ml of N,N-dimethylformamide, cooled to less than 10 ° C in an ice water bath, and isopropyl chloroformate (0.81 g) was added. After 6.5 mmol, 1.2 equ.), after stirring for 5 minutes, DIPEA (1.1 g, 8.3 mmol, 1.5 equ.) was slowly added dropwise, and the mixture was kept at a temperature below 10 ° C, stirred, and the reaction was monitored by TLC until the material disappeared.
  • HD-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH 2 (5.31 g, 5.64 mmol) was dissolved in N,N-dimethylformamide (50 ml), and then added to the above reaction solution In the middle, the temperature of the system was kept below 10 ° C and stirring was continued for 10 hours until the starting material disappeared (TLC).
  • Fmoc-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH 2 (10.2 g, 8.77 mmol) was added to dichloromethane (250 ml). After stirring for 5 min, piperidine was added dropwise. 4.25 g, 50 mmol), the reaction mixture was stirred until Fmoc-D-Ala-D-Arg(Pbf)-NH2 completely disappeared (TLC). Column chromatography (dichloromethane / methanol gradient), the solvent was removed to give HD-Arg (Pbf) -D- Ala-D-Arg (Pbf) -NH 2 (5.28g, 64.0% yield).
  • Fmoc-D-Arg(Pbf)-OH (3.6 g, 5.5 mmol) was added to 80 ml of N,N-dimethylformamide, cooled to less than 10 ° C in an ice water bath, and isopropyl chloroformate (0.81 g) was added. After 6.5 mmol, 1.2 equ.), after stirring for 5 minutes, DIPEA (1.1 g, 8.3 mmol, 1.5 equ.) was slowly added dropwise, and the mixture was kept at a temperature below 10 ° C, stirred, and the reaction was monitored by TLC until the material disappeared.
  • HD-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH 2 (5.31 g, 5.64 mmol) was dissolved in N,N-dimethylformamide (50 ml), and then added to the above reaction solution In the middle, the system temperature was maintained at 30-40 ° C and stirring was continued for more than 10 hours until the starting material disappeared (TLC).
  • N-Ac-D-Cys(Trt)-OH (4.06 g, 10 mmol) was added to dichloromethane (100 ml), cooled to less than 10 ° C in an ice water bath, and DIC (1.52 g, 12 mmol, 1.2 equ.), HOSU (1.80g, 12mmol, 1.2equ.), after stirring for 5 minutes, DIPEA (1.94g, 15mmol, 1.5equ.) was slowly added dropwise, the mixture was kept at a temperature below 10 ° C, stirred, and the reaction was monitored by TLC until the starting material disappear. Filtration and removal of the solvent under reduced pressure gave an oil.
  • the control sample was taken, and acetonitrile (150 mL) and DIPEA (1.94 g, 15 mmol, 1.5 equ.) were added to the oil. After stirring and stirring, H-D-Ala-OH (0.98 g, 11 mmol) was added to the above solution, and 80 mL was further added. After the water was mixed well, the temperature of the system was kept below 10 ° C and stirring was continued, and the control sample was compared until the disappearance of the raw material (TLC).
  • N-Ac-D-Cys(Trt)-OH (4.06 g, 10 mmol) was added to dichloromethane (100 ml), cooled to less than 10 ° C in an ice water bath, and DIC (1.52 g, 12 mmol, 1.2 equ.), HOSU (1.80g, 12mmol, 1.2equ.), after stirring for 5 minutes, DIPEA (1.94g, 15mmol, 1.5equ.) was slowly added dropwise, the mixture was kept at a temperature of 30-40 ° C, stirred, and the reaction was monitored by TLC until the starting material disappear. Filtration and removal of the solvent under reduced pressure gave an oil.
  • control sample was taken, and acetonitrile (150 mL) and DIPEA (1.94 g, 15 mmol, 1.5 equ.) were added to the oil. After stirring and dissolved, HD-Ala-OH (0.98 g, 11 mmol) was added to the above solution, and 80 mL of water was added thereto. After mixing well, the system temperature was maintained at 30-40 ° C and stirring was continued, and the control sample was compared until the starting material disappeared (TLC).
  • reaction solution was washed twice with water, dried and evaporated, and then evaporated, evaporated. After cooling to room temperature, it was filtered to give 3.82 g of a white solid.
  • N-Ac-D-Cys(Trt)-D-Ala-OH (4.06 g 8.5 mmol)
  • HD-Arg(Pbf)-OH (4.26 g, 10 mmol) was added to N,N-dimethylformamide (80 In ml)
  • the ice water bath was cooled to less than 10 ° C
  • HATU (3.81 g, 10 mmol, 1.2 equ.) was added, and after stirring for 5 minutes, DIPEA (1.64 g, 12.8 mmol, 1.5 equ.) was slowly added dropwise, and the resulting mixture was kept.
  • the temperature of the system was below 10 ° C, stirred, and the reaction was monitored by TLC until the starting material disappeared.
  • N-Ac-D-Cys(Trt)-D-Ala-OH (4.06 g 8.5 mmol)
  • HD-Arg(Pbf)-OH (4.26 g, 10 mmol) was added to N,N-dimethylformamide (80 In ml)
  • the reaction temperature was kept at 30-40 ° C
  • HATU (3.81 g, 10 mmol, 1.2 equ.) was added, and after stirring for 5 minutes, DIPEA (1.64 g, 12.8 mmol, 1.5 equ.) was slowly added dropwise, and the resulting mixture was kept in the reaction.
  • the temperature was 30-40 ° C, stirred, and the reaction was monitored by TLC until the starting material disappeared.
  • the reaction solution was added to 5 times by volume of water, and allowed to stand overnight, and filtered to give a white solid.
  • White solid was added to ethyl acetate (200 mL), and the mixture was heated to reflux, and the residual water was removed using a water separator, cooled, allowed to stand for crystallization, and filtered. 5.41 g of a white solid was obtained, the yield was 71.9%, and the purity was 98.8%.
  • DIPEA 0.6 g, 4.65 mmol, 1.5 equ.
  • DIPEA 0.6 g, 4.65 mmol, 1.5 equ.
  • Boc-L-Cys-OtBu (0.56g, 2.0mmol) was added to dichloromethane (20 ml), stirred and dissolved, cooled in an ice water bath, and added to a solution of triethylamine (3.1 g, 3.0 mmol of organic base) Then, NCS (3.0 g, 2.2 mmol) was added to the reaction system in portions, and the resulting mixture was further kept for 4 hours. No residue of the raw material was detected by TLC. Filtration was carried out, the solvent was removed from the filtrate, and N,N-dimethyl was added. The carbamide (20 ml) dissolved the residue.
  • Example 9 The crude heptapeptide obtained in the above Example 9 (1.86 g, 2.0 mmol) was added to N,N-dimethylformamide (50 ml), stirred and dissolved, cooled in an ice water bath, and the sample solution prepared in Example 8 was slowly added. The mixed reaction solution was kept warm and stirred for 6 hours. Trifluoroacetic acid (5 ml of OtBu protecting group) was slowly added to the reaction mixture, and the mixture was further stirred for 4 hours, and then slowly added to 500 ml of pre-cooled diethyl ether. The mixture was allowed to stand for 4 hours, and centrifuged to obtain 2.03 g of a crude peptide solid. The HPLC purity was 77.2%. The crude peptide was purified by HPLC to obtain a pure product of 1.32 g, yield 62.9%, purity 98.2%, MS: [M+H] + : 1049.

Landscapes

  • 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)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

一种合成Etelcalcetide的方法，所述方法液相合成在SEQ ID NO:1所示氨基酸序列上N末端乙酰化、C末端酰胺化的直链七肽；L-Cys进行NCS氯化，使巯基上的氢被氯取代，生成L-Cys(SCl)；直链七肽和L-Cys(SCl)偶联反应生成二硫化物，获得Etelcalcetide；所述方法采用全液相方法合成Etelcalcetide，与固相方法比较使用更少的试剂及溶剂，绿色环保，并且不使用价格高昂的树脂，降低了成本。同时以价廉易得的NCS与半胱氨酸形成活性中间体构建二硫键，避免了传统方法转化率低，纯度低的缺点，利于Etelcalcetide的大规模生产。

Description

一种合成Etelcalcetide的方法
本申请要求于2015年12月31日提交中国专利局、申请号为201511030047.3、发明名称为“一种合成Etelcalcetide的方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及医药合成领域，具体涉及一种合成Etelcalcetide的方法。
背景技术
继发性甲状旁腺功能亢进(SHPT，简称继发性甲旁亢)，是指在慢性肾功能不全、肠吸收不良综合征、Fanconi综合征和肾小管酸中毒、维生素D缺乏或抵抗以及妊娠、哺乳等情况下，甲状旁腺长期受到低血钙、低血镁或高血磷的刺激而分泌过量的甲状旁腺激素(PTH)，以提高血钙、血镁和降低血磷的一种慢性代偿性临床表现，长期的甲状旁腺增生最终导致形成功能自主的腺瘤。
Etelcalcetide是由Kai Pharmaceuticals,Inc.开发的一种新颖的拟钙剂(calcimimetic agent)，能够抑制甲状旁腺激素(PTH)的分泌。继发性甲旁亢(SHPT)是接受透析治疗的慢性肾脏病(CKD)患者中一种常见且严重的代偿失调疾病。目前已知，持续升高的甲状旁腺激素(PTH)与CKD患者的关键临床结局相关。Etelcalcetide可结合并激活甲状旁腺上的钙敏感受体，实现甲状旁腺激素(PTH)水平的降低。
Etelcalcetide有3个D构型的精氨酸、2个D构型的丙氨酸、1个D构型的精氨酰胺、1个L构型半胱氨酸与1个D构型半胱氨酸(N段被乙酰基封闭)构成，其中D构型半胱氨酸与L构型半胱氨酸以二硫键连接在一起(N-acetyl-D-cysteinyl-D-alanyl-D-arginyl-D-arginyl-D-arginyl-D-alanyl-D-Argininamide,disulfide with L-cysteine)，其结构如下所示：
Figure PCTCN2016111093-appb-000001
专利CN201080045024.9首度报道了该化合物，但是未见该化合物的合成工艺报道。该化合物合成的关键在于二硫键的构建，常规的肽合成中使用的构建二硫键的方法包括空气氧化法，碘/醋酸体系氧化法，双氧水氧化法等，这类方法仅适合于环肽的二硫键的构建，环肽的二硫键往往是分子内的两个巯基通过氧化的方法进行连接，需要通过使用极稀溶液来进行反应，减少分子间反应的发生。但是Etelcalcetide需要进行分子间巯基的氧化反应，若按照常规的二硫键构建方法进行反应，将无法得到理想收率和纯度的最终产物。此外，如果以普通的固相合成方法合成该化合物合成，其中的胍基较为容易脱除而形成降解杂质，纯化难度极大。
发明内容
有鉴于此，本发明的目的在于提供一种合成Etelcalcetide的方法，使得本发明所述方法能够提高Etelcalcetide及中间产物的收率，提高Etelcalcetide纯度和降低杂质。
为实现上述目的，本发明提供如下技术方案：
一种合成Etelcalcetide的方法，包括以下步骤：
步骤1、液相合成在SEQ ID NO:1所示氨基酸序列上N末端乙酰化、C末端酰胺化的直链七肽；
步骤2、L-Cys进行NCS氯化，使巯基上的氢被氯取代，生成L-Cys(SCl)；
步骤3、将步骤1合成的直链七肽和L-Cys(SCl)偶联反应生成二 硫化物，获得Etelcalcetide；
步骤1和步骤2不分先后。
本发明以液相合成方法合成Etelcalcetide主链7肽，并以NCS氯化的方法制备活性中间体L-Cys(SCl)，使用该活性中间体构建二硫键合成Etelcalcetide，保证反应的专一选择性，收率高，杂质少。
其中，作为优选，所述步骤1为：
以保护氨基酸为原料，在偶联体系作用下，液相合成在SEQ ID NO:1所示氨基酸序列上N末端乙酰化、C末端酰胺化的全保护直链七肽，然后裂解脱除各氨基酸的保护基，获得直链七肽。
进一步优选地，步骤1为：
以Fmoc-D-Arg(Pbf)-OH、Fmoc-D-Ala-OH、N-Ac-D-Cys(Trt)-OH保护氨基酸为原料，在氯甲酸异丙酯/DIPEA偶联体系作用下液相合成在SEQ ID NO:1所示氨基酸序列上N末端乙酰化、C末端酰胺化的全保护直链七肽N-Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2，然后裂解脱除各氨基酸的保护基，获得直链七肽N-Ac-D-Cys-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2；所述N-Ac-D-Cys(Trt)-OH为N端乙酰化的保护的半胱氨酸。
更优选地，步骤1为：
步骤1.1、将Fmoc-D-Arg(Pbf)-OH的C端酰胺化，获得Fmoc-D-Arg(Pbf)-NH2，然后以Fmoc-D-Arg(Pbf)-NH2为C端，用Fmoc-D-Arg(Pbf)-OH、Fmoc-D-Ala-OH保护氨基酸在氯甲酸异丙酯/DIPEA偶联体系作用下液相合成SEQ ID NO:1所示氨基酸序列N端到C端第4-7位的全保护多肽A，即Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2；
以N-Ac-D-Cys(Trt)-OH为N端，用Fmoc-D-Arg(Pbf)-OH、Fmoc-D-Ala-OH保护氨基酸在DIC/HOSu/DIPEA三试剂偶联体系或HATU/DIPEA双试剂偶联体系作用下液相合成SEQ ID NO:1所示氨基酸序列N端到C端第1-3位的全保护多肽B，即N-Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-OH；
步骤1.2、将全保护多肽A和全保护多肽B用HATU/DIPEA双试剂偶联体系偶联，获得全保护直链七肽N-Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)- D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2；
步骤1.3、全保护直链七肽裂解脱除各保护氨基酸保护基获得直链七肽N-Ac-D-Cys-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2。
在本发明优选的液相合成Etelcalcetide主链7肽方案中，首先按照Etelcalcetide主链肽序(本发明SEQ ID NO:1所示氨基酸序列)合成1-3和4-7片段，分别对应全保护多肽B和A，然后再将这2个多肽片段偶联得到全保护直连七肽，以Etelcalcetide主链N端到C端的氨基酸顺序编号，如下式：
H-D-Cys1-D-Ala2-D-Arg3-D-Arg4-D-Arg5-D-Ala6-D-Arg7-NH2
在SEQ ID NO:1所示氨基酸序列中，Xaa表示D型氨基酸，其中Xaa(1)＝D-Cys；Xaa(2，6)＝D-Ala；Xaa(3，4，5)＝D-Arg，括号中的数字表示SEQ ID NO:1所示氨基酸序列中氨基酸编号。
本发明所述保护基是在多肽合成领域中用以保护氨基酸主链以及侧链上氨基、羧基、巯基等干扰合成的基团的保护基团，防止氨基、羧基等在制备目标产物过程中发生反应，生成杂质，而被保护基保护的氨基酸称为保护氨基酸。在本技术领域，对于氨基酸侧链需要保护的基团、侧链结构以及如何偶联保护基为本领域技术人员公知。本发明中对偶联有保护基的氨基酸表示形式也是本领域常用表示形式，为本领域技术人员所熟知，如Fmoc-D-Arg(Pbf)-OH，Fmoc为氨基酸N端保护基，括号里的Pbf为Arg侧链保护基，本发明其他保护氨基酸合成原料除非特殊说明均可参照此解释。特别说明的是对于Boc-L-Cys-OtBu保护氨基酸，Boc为其N端保护基，OtBu为其羧酸保护基。
本发明在液相合成中优选通过逐一偶联方式合成目的多肽片段，逐一偶联是指以第一个氨基酸为起始，剩余氨基酸按照SEQ ID NO:1所示氨基酸序列的顺序逐个和前一个偶联的氨基酸发生缩合反应(主链氨基和羧基的缩合反应)进行偶联。在偶联中，由于每个氨基酸N端都有保护基，因此需要先脱除N端Fmoc保护基再偶联，这对本领域技术人员来说是公知常识，本发明优选加入哌啶脱除N端Fmoc保护基，用三氟醋酸脱除Boc-L-Cys-OtBu羧酸保护基OtBu。由于不断有氨基酸偶联，所 合成的多肽片段是不断变化的，作为优选，每个待偶联的氨基酸原料与之前已经合成的多肽片段的摩尔比、每两个待偶联的氨基酸原料之间的摩尔比以及每两个多肽片段之间的摩尔比接近于1:1，其他适用摩尔比可根据实际反应进行调整。
此外，所述直链七肽C末端的酰胺化可通过对精氨酸的C端进行酰胺化(如和氨水反应)，使其作为C端进行液相合成；而N末端的乙酰化可通过对半胱氨酸的N端进行乙酰化，使其作为N端进行液相合成，也可采用市售的乙酰化半胱氨酸进行合成。
作为优选，本发明步骤2为：
Boc-L-Cys-OtBu进行NCS氯化，使巯基上的氢被氯取代，生成Boc-L-Cys(SCl)-OtBu。
针对Etelcalcetide合成中，其胍基较为容易脱除而形成降解杂质的技术问题，本发明通过降低反应温度来最大程度解决胍基脱除导致收率和纯度降低的问题，故本发明步骤1所述液相合成、步骤2所述NCS氯化、步骤3所述偶联反应均优选在0-10℃中进行，更优先为0℃≤反应温度＜10℃之间。
作为优选，步骤1所述液相合成、步骤2所述NCS氯化、步骤3所述偶联反应均以DMF、DCM或两者的混合物(混合物中两者体积比优选1:1)为溶剂。
作为优选，所述裂解通过加入二氯甲烷和三氟醋酸的混合溶液进行裂解，进一步优选地，二氯甲烷和三氟醋酸的体积比为15:85。
以本发明所述方法合成的Etelcalcetide经HPLC纯化后纯度达到98％以上，收率在62％以上。
由以上技术方案可知，本发明采用全液相方法合成Etelcalcetide，在液相合成过程中，可使用结晶、打浆等简单易行的纯化手段，大大提高了中间体质量。与固相方法比较使用更少的试剂及溶剂，绿色环保，并且不使用价格高昂的树脂，降低了成本。同时以价廉易得的NCS与半胱氨酸形成活性中间体构建二硫键，避免了传统方法转化率低，纯度低的缺点，利于Etelcalcetide的大规模生产。
具体实施方式
本发明公开了一种合成Etelcalcetide的方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的化合物和制备方法进行改动或适当变更与组合，来实现和应用本发明技术。
在本发明具体实施方式中，所有保护氨基酸均可通过市售获得，本发明中的保护氨基酸购自于吉尔生化有限公司，申请文件中所用英文缩写对应的中文含义见表1。
表1英文缩写释义
缩写及英文 含义
NCS N-氯代丁二酰亚胺
Boc 叔丁氧羰基
Etelcalcetide 一种凯伊药品公司开发的新型多肽药物
Cys 半胱氨酸
-OtBu 叔丁酯
MS 质谱
Argininamide 精氨酰胺
Fmoc 芴甲氧羰基
Ac 乙酰基
DIPEA 二异丙基乙基胺
mmol 毫摩尔
Pbf 2,2,4,6,7-五甲基二氢苯并呋喃-5-磺酰氯
HPLC 高效液相
Equ. 当量
Kai Pharmaceuticals,Inc. 凯伊药品公司
在本发明液相合成中，各中间产物可通过结晶、柱层析等手段纯化，如乙酸乙酯加热回流溶解后加正己烷冷却析晶、加乙酸乙酯加热回流溶解后冷却析晶(乙酸乙酯打浆)、柱层析(二氯甲烷/甲醇梯度洗脱)；而全保护直链七肽可通过乙醇重结晶纯化，直链七肽和终产物可采用乙醚沉淀法。
下面结合实施例，进一步阐述本发明。
实施例1：Fmoc-D-Arg(Pbf)-NH2的合成
1、本发明方法
Fmoc-D-Arg(Pbf)-OH(130g,200mmol)加入到1000毫升二氯甲烷中，冰水浴冷却至低于10℃，加入氯甲酸异丙酯(27g,220mmol,1.1equ.),搅拌5分钟后，缓慢滴加DIPEA(31g,242mmol,1.21equ.)，所得混合物保持体系温度低于10℃，搅拌，TLC监控反应，直到原料点消失。
200毫升氨水缓慢加入到上述反应液中，保持反应温度低于20℃，继续搅拌2小时，分液，DCM层水洗两遍，无水硫酸钠干燥，移除溶剂后得到白色固体115g(收率88.5％)，HPLC纯度为95.2％。
2、对照方法
Fmoc-D-Arg(Pbf)-OH(130g,200mmol)加入到1000毫升二氯甲烷中，反应体系温度控制在30-40℃，加入氯甲酸异丙酯(27g,220mmol,1.1equ.),搅拌5分钟后，缓慢滴加DIPEA(31g,242mmol,1.21equ.)，所得混合物保持体系温度30-40℃，搅拌，TLC监控反应，直到原料点消失。
200毫升氨水缓慢加入到上述反应液中，保持反应温度低于20℃，继续搅拌2小时，分液，DCM层水洗两遍，无水硫酸钠干燥，移除溶剂后得到白色固体112.3g(收率86.4％)，HPLC纯度89.6％。
所得白色固体加入到乙酸乙酯(200mL),室温搅拌1小时，过滤，得白色固体104.8g，HPLC纯度94.8％。
实施例2：Fmoc-D-Ala-D-Arg(Pbf)-NH2的合成
Fmoc-D-Ala-OH(4.67g,15mmol)加入到100毫升二氯甲烷中，搅拌10分钟后，冰水浴冷却至低于10℃，加入氯甲酸异丙酯(2.0g,16.5mmol,1.1equ.),搅拌5分钟后，缓慢滴加DIPEA(2.33g,18.1mmol,1.21equ.)，所得混合物保持体系温度低于10℃，搅拌，TLC监控反应，直到原料点消失。
Fmoc-D-Arg(Pbf)-NH2脱除Fmoc保护基,H-D-Arg(Pbf)-NH2(7.0g,16.5mmol)加入二氯甲烷(50毫升)溶解，然后将其加入上述反应液中，保持体系温度低于10℃继续搅拌10小时，直到原料点消失(TLC)。
反应混合溶液用饱和碳酸氢钠溶液洗涤一次，水洗两遍，无水硫酸钠干燥，移除溶剂后得到白色固体11.1g。
将所得白色固体加入到乙酸乙酯(100毫升)中，加热至回流，全部固体溶解，50毫升正己烷缓慢加入到乙酸乙酯溶液中，所得溶液继续回流0.5小时，然后缓慢冷却至室温，大量固体析出，过滤得白色固体8.9g，收率81.0％。纯度95.7％。
2、对照方法
Fmoc-D-Ala-OH(4.67g,15mmol)加入到100毫升二氯甲烷中，搅拌10分钟后，冰水浴冷却至低于10℃，加入氯甲酸异丙酯(2.0g,16.5mmol,1.1equ.),搅拌5分钟后，缓慢滴加DIPEA(2.33g,18.1mmol,1.21equ.)，所得混合物保持体系温度30-40℃，搅拌，TLC监控反应，直到原料点消失。
Fmoc-D-Arg(Pbf)-NH2脱除Fmoc保护基,H-D-Arg(Pbf)-NH2(7.0g,16.5mmol)加入二氯甲烷(50毫升)溶解，然后将其加入上述反应液中，保持体系温度30-40℃继续搅拌，直到原料点消失(TLC)。
反应混合溶液用饱和碳酸氢钠溶液洗涤一次，水洗两遍，无水硫酸钠干燥，移除溶剂后得到白色固体10.4g。
将所得白色固体加入到乙酸乙酯(100毫升)中，加热至回流，全部固体溶解，50毫升正己烷缓慢加入到乙酸乙酯溶液中，所得溶液继续回流0.5小时，然后缓慢冷却至室温，大量固体析出，过滤得白色固体7.2g，收率65.5％。纯度93.8％。
实施例3：Fmoc-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2的合成
Fmoc-D-Ala-R-Arg(Pbf)-NH2(8.9g 12mmol)加入到二氯甲烷(200毫升)中，搅拌5分钟后，滴加哌啶(5.1g，60mmol)，反应液继续搅拌直到Fmoc-D-Ala-R-Arg(Pbf)-NH2完全消失(TLC)。使用稀盐酸洗涤至二氯甲烷溶液呈中性，分液，二氯甲烷液干燥，移除溶剂得油状物H-D-Ala-D-Arg(Pbf)-NH2(5.3g,收率85.8％)，将油状物用乙酸乙酯/正己烷处理得到纯品。
Fmoc-D-Arg(Pbf)-OH(6.5g,10mmol)加入到100毫升N,N-二甲基甲酰胺中，冰水浴冷却至低于10℃，加入氯甲酸异丙酯(1.34g,11mmol,1.1equ.),搅拌5分钟后，缓慢滴加DIPEA(1.56g,12.1mmol,1.21equ.)，所得混合物保持体系温度低于10℃，搅拌，TLC监控反应，直到原料消失。
油状物(H-D-Ala-D-Arg(Pbf)-NH2,5.3g,10.3mmol)加入N,N-二甲基甲酰胺(50毫升)溶解，然后将其加入上述反应液中，保持体系温度低于10℃继续搅拌10小时，直到原料消失(TLC)。将所得反应液加入到5倍体积的水中，静置过夜。
过滤，将所得白色固体加入到乙酸乙酯(150毫升)中，加热至回流0.5小时，然后缓慢冷却至室温，过滤得白色固体10.2g，收率87.7％。纯度93.7％。
2、对照方法
Fmoc-D-Ala-D-Arg(Pbf)-NH2(8.9g 12mmol)加入到二氯甲烷(200毫升)中，搅拌5分钟后，滴加哌啶(5.1g，60mmol)，反应液继续搅拌直到Fmoc-D-Ala-D-Arg(Pbf)-NH2完全消失(TLC)。使用稀盐酸洗涤至二氯甲烷溶液呈中性，分液，二氯甲烷液干燥，移除溶剂得油状物H-D-Al a-D-Arg(Pbf)-NH2(5.4g,收率89.0％)。将油状物用乙酸乙酯/正己烷处理得到纯品。
Fmoc-D-Arg(Pbf)-OH(6.5g,10mmol)加入到100毫升N,N-二甲基甲酰胺中，冰水浴冷却至低于10℃，加入氯甲酸异丙酯(1.34g,11mmol,1.1equ.),搅拌5分钟后，缓慢滴加DIPEA(1.56g,12.1mmol,1.21equ.)，所得混合物冰水浴冷却至低于10℃，搅拌，TLC监控反应，直到原料消失。
H-D-Ala-D-Arg(Pbf)-NH2,5.3g,10.3mmol加入N,N-二甲基甲酰胺(50毫升)溶解，然后将其加入上述反应液中，保持体系温度30-40℃继续搅拌10小时以上，直到原料消失(TLC)。将所得反应液加入到5倍体积的水中，静置过夜。
过滤，将所得白色固体加入到乙酸乙酯(150毫升)中，加热至回流0.5小时，然后缓慢冷却至室温，过滤得白色固体9.1g，收率78.2％。纯度92.6％。
实施例4：Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2(全保护多肽A)的合成
Fmoc-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2(10.2g 8.77mmol)加入到二氯甲烷(250毫升)中，搅拌5分钟后，滴加哌啶(4.25g，50mmol)，反应液继续搅拌直到Fmoc-D-Ala-D-Arg(Pbf)-NH2完全消失(TLC)。柱层析纯化(二氯甲烷/甲醇梯度洗脱)，移除溶剂得白色固体H-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2(5.31g,收率64.3％)。
Fmoc-D-Arg(Pbf)-OH(3.6g,5.5mmol)加入到80毫升N,N-二甲基甲酰胺中，冰水浴冷却至低于10℃，加入氯甲酸异丙酯(0.81g,6.6mmol,1.2equ.),搅拌5分钟后，缓慢滴加DIPEA(1.1g,8.3mmol,1.5equ.)，所得混合物保持体系温度低于10℃，搅拌，TLC监控反应，直到原料消失。
H-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2(5.31g,5.64mmol)加入N,N-二甲基甲酰胺(50毫升)溶解，然后将其加入上述反应液中，保持体系温度低于10℃继续搅拌10小时，直到原料消失(TLC)。
500毫升水缓慢加入到上述反应体系中，继续搅拌0.5小时，过滤，将所得白色固体加入到乙酸乙酯(150毫升)中，加热至回流2.0小时，然后冷却至室温，过滤得白色固体6.14g，收率70.3％。纯度96.7％。
2、对照方法
Fmoc-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2(10.2g,8.77mmol)加入到二氯甲烷(250毫升)中，搅拌5分钟后，滴加哌啶(4.25g，50mmol)，反应液继续搅拌直到Fmoc-D-Ala-D-Arg(Pbf)-NH2完全消失(TLC)。柱层析纯化(二氯甲烷/甲醇梯度洗脱)，移除溶剂得H-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2(5.28g,收率64.0％)。
Fmoc-D-Arg(Pbf)-OH(3.6g,5.5mmol)加入到80毫升N,N-二甲基甲酰胺中，冰水浴冷却至低于10℃，加入氯甲酸异丙酯(0.81g,6.6mmol,1.2equ.),搅拌5分钟后，缓慢滴加DIPEA(1.1g,8.3mmol,1.5equ.)，所得混合物保持体系温度低于10℃，搅拌，TLC监控反应，直到原料消失。
H-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2(5.31g,5.64mmol)加入N,N-二甲基甲酰胺(50毫升)溶解，然后将其加入上述反应液中，保持体系温度30-40℃继续搅拌10小时以上，直到原料消失(TLC)。
500毫升水缓慢加入到上述反应体系中，继续搅拌0.5小时，过滤，将所得白色固体加入到乙酸乙酯(150毫升)中，加热至回流2.0小时，然后冷却至室温，过滤得白色固体5.80g，收率66.4％。纯度95.1％。
实施例5：N-Ac-D-Cys(Trt)-D-Ala-OH的合成
N-Ac-D-Cys(Trt)-OH(4.06g 10mmol)加入到二氯甲烷(100毫升)中，冰水浴冷却至低于10℃，加入DIC(1.52g,12mmol,1.2equ.)、HOSu(1.80g,12mmol,1.2equ.),搅拌5分钟后，缓慢滴加DIPEA(1.94g,15mmol,1.5equ.)，所得混合物保持体系温度低于10℃，搅拌，TLC监控反应，直到原料消失。过滤，减压移除溶剂，得油状物。取对照样品，油状物中加入乙腈(150mL)、DIPEA(1.94g,15mmol,1.5equ.)，搅拌溶解后，H-D-Ala-OH(0.98g,11mmol)加入上述溶液中，另加入80mL 水，混合均匀后，保持体系温度低于10℃继续搅拌，与对照样品对照直到原料消失(TLC)。
反应液水洗两次，干燥，移除溶剂后，所得油状物加入到100毫升乙酸乙酯中，加热溶解，正己烷缓慢加入，直到体系有固体析出。冷却至室温后，过滤得到白色固体4.05g,收率84.9％，纯度97.2％。
2、对照方法
N-Ac-D-Cys(Trt)-OH(4.06g 10mmol)加入到二氯甲烷(100毫升)中，冰水浴冷却至低于10℃，加入DIC(1.52g,12mmol,1.2equ.)、HOSu(1.80g,12mmol,1.2equ.),搅拌5分钟后，缓慢滴加DIPEA(1.94g,15mmol,1.5equ.)，所得混合物保持体系温度30-40℃，搅拌，TLC监控反应，直到原料消失。过滤，减压移除溶剂，得油状物。取对照样品，油状物中加入乙腈(150mL)、DIPEA(1.94g,15mmol,1.5equ.)，搅拌溶解后，H-D-Ala-OH(0.98g,11mmol)加入上述溶液中，另加入80mL水，混合均匀后，保持体系温度30-40℃继续搅拌，与对照样品对照直到原料消失(TLC)。
反应液水洗两次，干燥，移除溶剂后，所得油状物加入到100毫升乙酸乙酯中，加热溶解，正己烷缓慢加入，直到体系有固体析出。冷却至室温后，过滤得到白色固体3.82g,收率80.1％，纯度95.4％。
实施例6：N-Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-OH(全保护多肽B)的合成
1、本发明方法
N-Ac-D-Cys(Trt)-D-Ala-OH(4.06g 8.5mmol)、H-D-Arg(Pbf)-OH(4.26g,10mmol)加入到N,N-二甲基甲酰胺(80毫升)中，冰水浴冷却至低于10℃，加入HATU(3.81g,10mmol,1.2equ.),搅拌5分钟后，缓慢滴加DIPEA(1.64g,12.8mmol,1.5equ.)，所得混合物保持体系温度低于10℃，搅拌，TLC监控反应，直到原料消失。
反应液加到5倍体积水中，静置过夜，过滤得到白色固体，白色固体加入到乙酸乙酯(200mL)中，加热回流，使用分水器移除残余水分， 冷却，静置析晶，过滤得到白色固体5.58g,收率74.2％，纯度98.9％。
2、对照方法
N-Ac-D-Cys(Trt)-D-Ala-OH(4.06g 8.5mmol)、H-D-Arg(Pbf)-OH(4.26g,10mmol)加入到N,N-二甲基甲酰胺(80毫升)中，保持反应温度30-40℃，加入HATU(3.81g,10mmol,1.2equ.),搅拌5分钟后，缓慢滴加DIPEA(1.64g,12.8mmol,1.5equ.)，所得混合物保持反应温度30-40℃，搅拌，TLC监控反应，直到原料消失。
反应液加到5倍体积水中，静置过夜，过滤得到白色固体，白色固体加入到乙酸乙酯(200mL)中，加热回流，使用分水器移除残余水分，冷却，静置析晶，过滤得到白色固体5.41g,收率71.9％，纯度98.8％。
实施例7：N-Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2(全保护直链七肽)的合成
1、本发明方法
Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2(6.14g 3.86mmol)加入到DMF(80毫升)中，搅拌5分钟后，滴加哌啶(1.62g，19mmol)，反应液继续搅拌直到Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2完全消失(TLC)。150毫升水缓慢加入到混合液中，过滤，所得固体用100毫升乙酸乙酯打浆，过滤的白色固体H-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2(4.23g,收率80.3％)。
N-Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-OH(2.74g,3.1mmol)、H-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2(4.23g,3.1mmol)加入到N,N-二甲基甲酰胺(100毫升)，冰水浴冷却至低于10℃，加入HATU(1.42g,3.72mmol,1.2equ.),搅拌5分钟后，缓慢滴加DIPEA(0.6g,4.65mmol,1.5equ.)，所得混合物保持体系温度低于10℃，搅拌，TLC监控反应，直到原料消失。
200毫升水缓慢加入到反应体系中，过滤，所得固体加入到乙醇中，加热回流，搅拌2小时，过滤得白色固体4.63g，收率66.4％，纯度95.1％。
2、对照方法
Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2(6.14g 3.86mmol)加入到DMF(80毫升)中，搅拌5分钟后，滴加哌啶(1.62g，19mmol)，反应液继续搅拌直到Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2完全消失(TLC)。150毫升水缓慢加入到混合液中，过滤，所得固体用100毫升乙酸乙酯打浆，过滤的白色固体H-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2(4.23g,收率80.3％)。
N-Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-OH(2.74g,3.1mmol)、H-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2(4.23g,3.1mmol)加入到N,N-二甲基甲酰胺(100毫升)，保持反应温度30-40℃，加入HATU(1.42g,3.72mmol,1.2equ.),搅拌5分钟后，缓慢滴加DIPEA(0.6g,4.65mmol,1.5equ.)，搅拌，TLC监控反应，直到原料消失。
200毫升水缓慢加入到反应体系中，过滤，所得固体加入到乙醇中，加热回流，搅拌2小时，过滤得白色固体4.41g，收率63.2％，纯度94.9％。
实施例8：Boc-L-Cys(SCl)-OtBu的合成
Boc-L-Cys-OtBu(0.56g，2.0mmol)加入到二氯甲烷(20毫升)中，搅拌溶解后，冰水浴冷却，三乙胺(3.1g，3.0mmol有机碱催化反应进行)加入溶液中，然后将NCS(3.0g,2.2mmol)分批加入反应体系中，所得混合液继续保温反应4小时，TLC检测无原料残留，过滤，将滤液移除溶剂，另加N,N-二甲基甲酰胺(20毫升)溶解残余物。
实施例9：N-Ac-D-Cys-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2(直链七肽)的合成
实施例7所得全保护直链七肽N-Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2(4.63g,2.56mmol)加入到二氯甲烷/三氟醋酸(50毫升，15:85)混合溶液中，反应液控温不高于10℃，继续搅拌4小时，将所得反应混合液缓慢加入预冷至0℃的乙醚(5 00毫升)中，过滤得直链七肽粗品1.86g，HPLC纯度：79.5％收率78.3％。MS:[M+H]+:929。
实施例10：Etelcalcetide的合成
上述实施例9所得直七肽粗品(1.86g，2.0mmol)加入到N,N-二甲基甲酰胺(50毫升)中，搅拌溶解，冰水浴冷却，缓慢加入实施例8制得的样品溶液，混合好的反应液继续保温搅拌6小时，反应液中缓慢加入三氟醋酸(5毫升脱除OtBu保护基)，混合液继续搅拌4小时，然后缓慢加入到500毫升预冷好的乙醚中，混合，静置4小时，离心过滤，得粗肽固体2.03g,HPLC纯度77.2％。粗肽利用HPLC制备纯化，得纯品1.32g，收率62.9％，纯度98.2％，MS:[M+H]+:1049。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Figure PCTCN2016111093-appb-000002

Claims (8)

  1. 一种合成Etelcalcetide的方法，其特征在于，包括以下步骤：
    步骤1、液相合成在SEQ ID NO:1所示氨基酸序列上N末端乙酰化、C末端酰胺化的直链七肽；
    步骤2、L-Cys进行NCS氯化，使巯基上的氢被氯取代，生成L-Cys(SCl)；
    步骤3、将步骤1合成的直链七肽和L-Cys(SCl)偶联反应生成二硫化物，获得Etelcalcetide；
    步骤1和步骤2不分先后。
  2. 根据权利要求1所述方法，其特征在于，所述步骤1为：
    以保护氨基酸为原料，在偶联体系作用下，液相合成在SEQ ID NO:1所示氨基酸序列上N末端乙酰化、C末端酰胺化的全保护直链七肽，然后裂解脱除各氨基酸的保护基，获得直链七肽。
  3. 根据权利要求2所述方法，其特征在于，所述步骤1为：
    以Fmoc-D-Arg(Pbf)-OH、Fmoc-D-Ala-OH、N-Ac-D-Cys(Trt)-OH保护氨基酸为原料，在氯甲酸异丙酯/DIPEA偶联体系作用下液相合成在SEQ ID NO:1所示氨基酸序列上N末端乙酰化、C末端酰胺化的全保护直链七肽N-Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2，然后裂解脱除各氨基酸的保护基，获得直链七肽N-Ac-D-Cys-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2；
    其中，所述N-Ac-D-Cys(Trt)-OH为N端乙酰化的保护的D型半胱氨酸。
  4. 根据权利要求3所述方法，其特征在于，所述步骤1为：
    步骤1.1、将Fmoc-D-Arg(Pbf)-OH的C端酰胺化，获得Fmoc-D-Arg(Pbf)-NH2，然后以Fmoc-D-Arg(Pbf)-NH2为C端，用Fmoc-D-Arg(Pbf)-OH、Fmoc-D-Ala-OH保护氨基酸在氯甲酸异丙酯/DIPEA偶联体系作用下液相合成SEQ ID NO:1所示氨基酸序列N端到C端第4-7位的全保护多肽A，即Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2；
    以N-Ac-D-Cys(Trt)-OH为N端，用Fmoc-D-Arg(Pbf)-OH、Fmoc-D-Ala-OH保护氨基酸在DIC/HOSu/DIPEA三试剂偶联体系或HATU/DIPEA双试剂偶联体系作用下液相合成SEQ ID NO:1所示氨基酸序列N端到C端第1-3位的全保护多肽B，即N-Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-OH；
    步骤1.2、将全保护多肽A和全保护多肽B用HATU/DIPEA双试剂偶联体系偶联，获得全保护直链七肽N-Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH2；
    步骤1.3、全保护直链七肽裂解脱除各保护氨基酸保护基获得直链七肽N-Ac-D-Cys-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2。
  5. 根据权利要求1所述方法，其特征在于，步骤2为：
    Boc-L-Cys-OtBu进行NCS氯化，使巯基上的氢被氯取代，生成Boc-L-Cys(SCl)-OtBu。
  6. 根据权利要求1所述方法，其特征在于，步骤1所述液相合成、步骤2所述NCS氯化、步骤3所述偶联反应均在0-10℃中进行。
  7. 根据权利要求1所述方法，其特征在于，步骤1所述液相合成、步骤2所述NCS氯化、步骤3所述偶联反应均以DMF、DCM或两者的混合物为溶剂。
  8. 据权利要求2-5任意一项所述方法，其特征在于，所述裂解通过加入二氯甲烷和三氟醋酸的混合溶液进行裂解。
PCT/CN2016/111093 2015-12-31 2016-12-20 一种合成Etelcalcetide的方法 WO2017114238A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201511030047.3 2015-12-31
CN201511030047.3A CN106928321B (zh) 2015-12-31 2015-12-31 一种合成Etelcalcetide的方法

Publications (1)

Publication Number Publication Date
WO2017114238A1 true WO2017114238A1 (zh) 2017-07-06

Family

ID=59224627

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/111093 WO2017114238A1 (zh) 2015-12-31 2016-12-20 一种合成Etelcalcetide的方法

Country Status (2)

Country Link
CN (1) CN106928321B (zh)
WO (1) WO2017114238A1 (zh)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021038431A1 (en) * 2019-08-26 2021-03-04 Auro Peptides Ltd An improved process for the preparation of etelcalcetide hydrochloride
CN112521450A (zh) * 2019-09-19 2021-03-19 深圳市健翔生物制药有限公司 一种固液相结合制备维拉卡肽的方法
WO2021109297A1 (zh) * 2019-12-03 2021-06-10 凯莱英医药集团（天津）股份有限公司 一种依特卡肽中间体及依特卡肽的合成方法
WO2022044030A1 (en) 2020-08-31 2022-03-03 Usv Private Limited An improved process for fmoc synthesis of etelcalcetide
CN114524860A (zh) * 2021-12-29 2022-05-24 深圳翰宇药业股份有限公司 一种Etelcalcetide的合成方法及其应用

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109306366B (zh) * 2017-07-26 2021-12-21 深圳翰宇药业股份有限公司 一种合成pt141的方法
CN110498835B (zh) * 2018-05-17 2021-06-08 深圳翰宇药业股份有限公司 一种合成etelcalcetide的方法
CN109280078B (zh) * 2018-10-30 2019-06-25 成都诺和晟泰生物科技有限公司 一种制备维拉卡肽的方法
CN112175042B (zh) * 2019-07-03 2022-05-31 深圳翰宇药业股份有限公司 Etelcalcetide的合成方法
EP3875466A1 (en) 2020-03-05 2021-09-08 Fresenius Kabi iPSUM S.r.l. Process for the synthesis of etelcalcetide
CN113480634A (zh) * 2021-08-09 2021-10-08 甘肃瑞德林生物有限公司 一种兰瑞肽的制备方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102711789A (zh) * 2009-07-29 2012-10-03 凯伊药品公司 用于降低甲状旁腺激素水平的治疗剂
WO2014210489A1 (en) * 2013-06-28 2014-12-31 Amgen Inc. Stable liquid formulation of amg 416 (velcalcetide)
WO2015154031A1 (en) * 2014-04-03 2015-10-08 Amgen Inc. Method for preparing amg 416
WO2016154580A1 (en) * 2015-03-26 2016-09-29 Amgen Inc. Solution phase method for preparing etelcalcetide

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102711789A (zh) * 2009-07-29 2012-10-03 凯伊药品公司 用于降低甲状旁腺激素水平的治疗剂
WO2014210489A1 (en) * 2013-06-28 2014-12-31 Amgen Inc. Stable liquid formulation of amg 416 (velcalcetide)
WO2015154031A1 (en) * 2014-04-03 2015-10-08 Amgen Inc. Method for preparing amg 416
WO2016154580A1 (en) * 2015-03-26 2016-09-29 Amgen Inc. Solution phase method for preparing etelcalcetide

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HE, JUNLING ET AL.: "The Research on Hyperparathyroidism Increase the Risk of Cardiovascular Disease in Chronic Kidney Disease", CHINESE JOURNAL OF CLINICIANS (ELECTRONIC EDITION, vol. 7, no. 24, 15 December 2013 (2013-12-15), pages 11585 - 11588 *
LOI, V.V. ET AL.: "Redox regulation by reversible protein S-thiolation in bacteria", FRONTIERS IN MICROBIOLOGY, vol. 6, 16 March 2015 (2015-03-16) *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021038431A1 (en) * 2019-08-26 2021-03-04 Auro Peptides Ltd An improved process for the preparation of etelcalcetide hydrochloride
CN112521450A (zh) * 2019-09-19 2021-03-19 深圳市健翔生物制药有限公司 一种固液相结合制备维拉卡肽的方法
WO2021109297A1 (zh) * 2019-12-03 2021-06-10 凯莱英医药集团（天津）股份有限公司 一种依特卡肽中间体及依特卡肽的合成方法
EP4071135A4 (en) * 2019-12-03 2023-02-01 Asymchem Laboratories (Tianjin) Co., Ltd. ETELCALCETIDE INTERMEDIATE AND METHOD OF SYNTHETIC ETELCALCETIDE
JP2023504848A (ja) * 2019-12-03 2023-02-07 アシムケム ラボラトリーズ （ティエンジン） カンパニー リミテッド エテルカルセチド中間体及びエテルカルセチドの合成方法
WO2022044030A1 (en) 2020-08-31 2022-03-03 Usv Private Limited An improved process for fmoc synthesis of etelcalcetide
CN114524860A (zh) * 2021-12-29 2022-05-24 深圳翰宇药业股份有限公司 一种Etelcalcetide的合成方法及其应用

Also Published As

Publication number Publication date
CN106928321A (zh) 2017-07-07
CN106928321B (zh) 2019-07-26

Similar Documents

Publication Publication Date Title
WO2017114238A1 (zh) 一种合成Etelcalcetide的方法
EP3398957B1 (en) Method for synthesizing etelcalcetide
WO2018033127A1 (zh) 一种低消旋杂质利拉鲁肽的合成方法
JP6350632B2 (ja) ペプチドの製造方法
PT1511761E (pt) Processo para produção peptídeos cíclicos
RU2525468C2 (ru) Пептидные лиганды соматостатиновых рецепторов
WO2017097194A1 (zh) 一种全固相制备卡贝缩宫素的方法
KR910001724B1 (ko) 향정신성 펩티드류의 제조방법
US9850285B2 (en) Process for preparing eptifibatide
CN109206481B (zh) 一种肽类化合物、其应用及含其的组合物
JP2024507884A (ja) ポリペプチド化合物及びその応用
US9150615B2 (en) Process for the preparation of leuprolide and its pharmaceutically acceptable salts
US20230021514A1 (en) Etelcalcetide intermediate and method for synthesizing etelcalcetide
JP4615221B2 (ja) ヘプタペプチドオキシトシンアナログを製造するための中間体及び方法
US20020165132A1 (en) Lanthionine bridged peptides
KR20150114111A (ko) 데스모프레신의 제조방법
JPH051798B2 (zh)
WO2019001459A1 (zh) 一种肽类化合物、其应用及含其的组合物
WO2020019128A1 (zh) 丙氨酸—赖氨酸—谷氨酸三肽衍生物及其应用
WO2013057736A2 (en) Preparation of eptifibatide peptide
US20070111930A1 (en) Process for preparing vapreotide
CN107698663B (zh) 一种维洛丝肽的液相合成方法
CN109438363B (zh) 一种环(亮氨酰-精氨酰)二肽盐的液相高纯度规模化合成方法
CN109810169B (zh) 一种Reltecimod的液相制备方法
JPH01197465A (ja) 新規アルギニン誘導体、その製法及びペプチド合成における使用法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16881027

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16881027

Country of ref document: EP

Kind code of ref document: A1