CN112521450A

CN112521450A - Method for preparing vilacatide by solid-liquid phase combination

Info

Publication number: CN112521450A
Application number: CN201910886294.5A
Authority: CN
Inventors: 胡鹏; 刘自成; 付玉清; 李文静; 龙娓芳
Original assignee: Shenzhen Jx Bio Pharmaceutical Co ltd
Current assignee: Shenzhen Jx Bio Pharmaceutical Co ltd
Priority date: 2019-09-19
Filing date: 2019-09-19
Publication date: 2021-03-19

Abstract

The invention discloses a method for preparing vilacard peptide by solid-liquid phase combination, which comprises the steps of adopting a solid-liquid combination mode, selecting cheap side chain protective amino acid and acid-sensitive solid-phase carrier resin to synthesize main chain peptide resin, selectively removing protective groups and resin of Cys side chains, activating main chain Cys sulfydryl, directly reacting with L-Cys-OH with low price, and finally cracking and removing Arg protective groups to obtain the vilacard peptide. Less synthesis impurities, high yield, easy purification, cheap and easily obtained raw materials, low synthesis cost and suitability for large-scale production.

Description

Method for preparing vilacatide by solid-liquid phase combination

Technical Field

The invention relates to the field of polypeptide synthesis, in particular to a method for preparing vilacatide by solid-liquid phase combination.

Technical Field

Secondary hyperparathyroidism (SHPT, secondary hyperthyroidism for short) refers to the condition of chronic renal insufficiency, intestinal malabsorption syndrome, Fanconi syndrome and renal tubular acidosis, vitamin D deficiency or resistance, pregnancy, lactation and the like, in which parathyroid gland is stimulated by hypocalcemia, hypomagnesemia or hyperphosphatemia for a long time to secrete excessive parathyroid hormone (PTH) so as to improve a chronic compensatory clinical manifestation of blood calcium, blood magnesium and blood phosphorus, and long-term parathyroid hyperplasia finally leads to the formation of adenoma with autonomous function.

Vilacatide, the english name etelcalcitide, is a calcium-sensitive receptor agonist, developed by Kai Pharmaceuticals INC, a proprietary company of AMGEN INC, for treating secondary hyperparathyroidism induced by hemodialysis treatment of adult chronic kidney disease patients, which binds and activates the calcium-sensitive receptor on the parathyroid gland to achieve a reduction in parathyroid hormone (PTH) levels. The drug is approved by the FDA in the United states and marketed in 2017, 2 and 7 months, is the first approved drug for treating the secondary hyperparathyroidism of dialysis patients in 12 years, and has better effectiveness and safety than the marketed cinacalcet which is a similar product.

The structure of vilacatide is shown below:

the key point of the synthesis of the compound lies in the construction of intermolecular disulfide bonds, and in the current preparation method of the compound, patent document CN105504012 discloses a solid-phase synthesis method of vilaca peptide, which comprises the steps of solid-phase synthesis of main chain peptide resin, selective removal of Cys side chain protecting groups, synthesis of full-protection peptide resin by reaction with Y-Cys (NPys) -OH, and finally cracking and purification to obtain the vilaca peptide. Wherein, the amino acid material Y-Cys (NPys) -OH is expensive in cost, and the dosage of disulfide bond formation in solid phase synthesis is large and is not easy to control. Patent document CN108218957 discloses a method for synthesizing vilaca peptide by solid-liquid phase combination, which comprises selecting acid-sensitive amino resin to synthesize main chain peptide resin, removing Mmt protecting group and resin of Cys side chain in dilute acid cracking solution, adding Boc-Cys (npys) -OH to form disulfide bond, finally cracking and removing Arg protecting group, and purifying to obtain the product. Wherein, the amino acid materials Fmoc-D-Cys (Mmt) -OH and Boc-Cys (NPys) -OH are expensive, the synthesis cost is high, and the practicability is lacked. Patent CN106795201 discloses a method for preparing vilaca peptide by solid-liquid phase combination, which comprises the steps of firstly synthesizing a main chain peptide resin in a solid phase, then cutting the peptide, activating a Cys side chain sulfhydryl group by using 2, 2-dithiodipyridine, then reacting with L-Cys-OH, and purifying to obtain the vilaca peptide. Wherein, when the side chain protecting groups of Cys and Arg of the main chain are cracked simultaneously, more impurities are generated, and the yield is low.

In combination with the above documents, the existing preparation method of vilacatide has many impurities, the price of amino acid materials is high, the synthesis cost is high, and the industrial large-scale production is not facilitated.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a novel method for preparing the vilacatide by combining a solid phase and a liquid phase, which can effectively reduce synthetic impurities, reduce the production cost and is beneficial to the mass production of the vilacatide.

In order to realize the purpose of the invention, the invention provides the following technical scheme:

a method for preparing vilacatide by solid-liquid phase combination comprises the following steps:

step 1: coupling the resin with Fmoc-D-Arg (X) -OH to obtain Fmoc-D-Arg (X) -resin 1;

step 2: sequentially coupling the Fmoc-D-Arg (X) -resin 1 obtained in the step 1 with D-Ala-OH, D-Arg (X) -OH, D-Ala-OH and D-Cys (Y) -OH, and acetylating amino at a nitrogen end to obtain fully-protected Vila-peptide main chain resin;

and step 3: taking the Vila peptide main chain resin obtained in the step 2, adding lysis solution 1 to remove a protecting group Y and cut the resin, and activating a main chain Cys sulfydryl to obtain activated Vila peptide main chain peptide;

and 4, step 4: and (3) dissolving the activated vila peptide main chain peptide obtained in the step (3) in a solution, coupling with L-Cys-OH to obtain fully protected vila peptide, and adding a lysis solution 2 for lysis to obtain the vila peptide.

Preferably, the solid phase carrier resin in step 1 is an acid-sensitive amino resin selected from Rink Amide resin and Sieber resin. More preferably, the solid-phase carrier resin in step 1 is Sieber resin, which is more sensitive to acid, and 1% TFA can be completely cleaved to ensure complete cleavage of the resin.

Preferably, the side chain protecting group of Arg is Pbf, and the side chain protecting group of Cys is selected from Trt and Mmt. Trt and Mmt are acid-sensitive protecting groups and can be easily removed under acidic conditions. More preferably, the side chain protecting group of Cys is Trt, which is cheap and can greatly reduce the production cost.

Preferably, the lysis solution 1 in the step 3 is a mixed solution of TFA, TIS and DCM, and more preferably the volume ratio of TFA, TIS and DCM is 0-10: 0-20: 70-100. The cracking liquid can selectively remove acid-sensitive resin and acid-sensitive amino acid protecting groups.

Preferably, in step 3, the removal of the protecting group Y, the cleavage of the resin and the activation of the main chain Cys thiol group are performed simultaneously. The cracking and the activation are not interfered with each other, and the reaction time can be shortened by carrying out the cracking and the activation simultaneously.

Preferably, the solution of step 4 is selected from water, acetonitrile/water, ethanol/water. More preferably, the solution in step 3 is acetonitrile/water, the activated vilacatide backbone peptide is completely dissolved in the acetonitrile/water, and the acetonitrile/water has a low boiling point, so that the concentration is convenient.

Preferably, in step 4, the lysis solution 2 is TFA, TIS, H₂And (3) a mixed solution of O. Further preferred are TFA, TIS, H₂The volume ratio of O is 80-100: 0-10.

The invention adopts a solid-liquid combined mode, selects cheap amino acid with side chain protection and acid-sensitive solid-phase carrier resin to synthesize main chain peptide resin, selectively removes protective groups and resin of Cys side chains, activates main chain Cys sulfydryl, then directly reacts with cheap L-Cys-OH, and finally cleaves and removes Arg protective groups to obtain the vilacard peptide. Less synthesis impurities, high yield, easy purification, cheap and easily obtained raw materials, low synthesis cost and suitability for large-scale production.

Drawings

FIG. 1 is an HPLC chromatogram of crude vilacatide prepared in example 10

FIG. 2 is an HPLC chromatogram of purified vilacatide protide from example 13

Detailed Description

The present invention will be described in further detail with reference to specific examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

The meanings of the abbreviations used in the present invention are listed in the following table:

Fmoc	fmoc group
		Fmoc-AA	Fmoc-protected amino acids
HOBT	1-hydroxybenzotriazoles
		DIC	N, N-diisopropylcarbodiimide
Boc	Tert-butyloxycarbonyl radical
		DMF	N, N-dimethylformamide
TFA	Trifluoroethanol
		DCM	Methylene dichloride
Pbf	2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl
		Mmt	4-Methoxytrityl group
Trt	Trityl radical
		Pys	2-pyridylthio
TIS	Tri-isopropyl silane
		Sieber resin	Fmoc-amino-ton-3-yloxy-methyl resin

Example 1 Synthesis of Fmoc-D-Arg (Pbf) -Rink Amide resin

Rink Amide resin (5.95g, 5mmol) with substitution degree of 0.84mmol/g was weighed into a polypeptide solid phase reactor, washed three times with DMF (30ml), drained, and swelled with DMF (60ml) for 30 min. And (3) after filtering, adding 20% piperidine/DMF solution to remove the Fmoc protecting group, detecting the reaction condition by ninhydrin, turning the resin to blue, completely deprotecting, draining the solvent, and adding DMF to wash the resin for 6 times.

Fmoc-D-Arg (Pbf) -OH (8.12g,12.5mmol) and HOBt (2.54g,18.75mmol) were weighed and dissolved in 40ml of DMF, DIC (2.86g,22.50mmol) was added for activation for 5min, and then the activated amino acid solution was added to the above solid phase reactor and stirred at room temperature for 2 h. And detecting the reaction condition by ninhydrin to obtain colorless resin, completely condensing, draining the solvent, and washing the resin 4 times by adding DMF (dimethyl formamide). And (3) adding 20% piperidine/DMF solution to remove the Fmoc protecting group, detecting ninhydrin, turning the resin to blue, completely deprotecting, and washing the resin with DMF for 6 times.

EXAMPLE 2 Synthesis of Fmoc-D-Arg (Pbf) -Sieber resin

Sieber resin (7.04g, 5mmol) with substitution 0.71mmol/g was weighed into a polypeptide solid phase reactor, washed three times with DMF (35ml), drained, and swollen with DMF (75ml) for 30 min. And (3) after filtering, adding 20% piperidine/DMF solution to remove the Fmoc protecting group, detecting the reaction condition by ninhydrin, turning the resin to blue, completely deprotecting, draining the solvent, and adding DMF to wash the resin for 6 times.

Fmoc-D-Arg (Pbf) -OH (8.12g,12.5mmol) and HOBt (2.54g,18.75mmol) were weighed and dissolved in 45ml of DMF, DIC (2.86g,22.50mmol) was added for activation for 5min, and then the activated amino acid solution was added to the above solid phase reactor and stirred at room temperature for 2 h. And detecting the reaction condition by ninhydrin to obtain colorless resin, completely condensing, draining the solvent, and washing the resin 4 times by adding DMF (dimethyl formamide). And (3) adding 20% piperidine/DMF solution to remove the Fmoc protecting group, detecting ninhydrin, turning the resin to blue, completely deprotecting, and washing the resin with DMF for 6 times.

Example 3 Synthesis of Ac-D-Cys (Trt) -D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Rink Amide resin

Fmoc-D-Arg (Pbf) -Rink Amide resin obtained in example 1 was coupled with Fmoc-D-Ala-OH, Fmoc-D-Arg (Pbf) -OH, Fmoc-D-Ala-OH and Fmoc-D-Cys (Trt) -OH in this order in the same manner as in example 1.

And (4) removing the Fmoc protecting group after the coupling is finished. Acetic anhydride (1.55g,10mmol) was weighed, 40ml DMF and DIEA (1.96g,10mmol) were added, the mixture was added to the resin to react, ninhydrin detection was performed, the resin was colorless, and DMF was used to wash the resin 6 times. After washing 3 times with DCM, 40 ml/time, the peptide resin was taken out and dried to yield 15.54g of peptide resin.

Example 4

Synthesis of Ac-D-Cys (Trt) -D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Sieber resin

Fmoc-D-Arg (Pbf) -Sieber resin obtained in example 2 was used to couple Fmoc-D-Ala-OH, Fmoc-D-Arg (Pbf) -OH, Fmoc-D-Ala-OH and Fmoc-D-Cys (Trt) -OH in this order in the same manner as in example 2.

And (4) removing the Fmoc protecting group after the coupling is finished. Acetic anhydride (1.55g,10mmol) was weighed, 45ml DMF and DIEA (1.96g,10mmol) were added, the mixture was added to the resin to react, ninhydrin detection was performed, the resin was colorless, and DMF was used to wash the resin 6 times. After washing 3 times with DCM, 45 ml/time, the peptide resin was taken out and dried to obtain 16.84g of peptide resin.

Example 5

Synthesis of Ac-D-Cys (Mmt) -D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Rink Amide resin

Fmoc-D-Arg (Pbf) -Rink Amide resin obtained in example 1 was coupled with Fmoc-D-Ala-OH, Fmoc-D-Arg (Pbf) -OH, Fmoc-D-Ala-OH and Fmoc-D-Cys (Mmt) -OH in this order in the same manner as in example 1.

And (4) removing the Fmoc protecting group after the coupling is finished. Acetic anhydride (1.55g,10mmol) was weighed, 40ml DMF and DIEA (1.96g,10mmol) were added, the mixture was added to the resin to react, ninhydrin detection was performed, the resin was colorless, and DMF was used to wash the resin 6 times. After washing 3 times with DCM, 40 ml/time, the peptide resin was taken out and dried to yield 15.78g of peptide resin.

Example 6

Synthesis of Ac-D-Cys (Mmt) -D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Sieber resin

Fmoc-D-Arg (Pbf) -Sieber resin obtained in example 2 was used to couple Fmoc-D-Ala-OH, Fmoc-D-Arg (Pbf) -OH, Fmoc-D-Ala-OH and Fmoc-D-Cys (Mmt) -OH in this order in the same manner as in example 2.

And (4) removing the Fmoc protecting group after the coupling is finished. Acetic anhydride (1.55g,10mmol) was weighed, 45ml DMF and DIEA (1.96g,10mmol) were added, the mixture was added to the resin to react, ninhydrin detection was performed, the resin was colorless, and DMF was used to wash the resin 6 times. The peptide resin was then washed 3 times with DCM, 45 ml/time, and after washing was complete, the peptide resin was removed and dried to give 16.65g of peptide resin.

Example 7 activated Vilata peptide backbone peptides

Ac-D-Cys(Pys)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH₂Synthesis of (2)

50ml of frozen TFA/TIS/DCM-10.0%/20.0%/70.0% lysate was prepared, added to a 100ml round-bottomed flask, and 5.0g of the peptide resin of example 3 was weighed out and added to the above lysate and reacted at room temperature for 2 h. After filtration, 2' 2-dithiodipyridine (0.39g, 1.78mmol) was added to the filtrate to react for 30 min. The reaction mixture was added to 500ml of cold isopropyl ether and precipitated, followed by centrifugal washing to obtain 3.09g of a white solid with an HPLC purity of 84.68%.

Example 8 activated Vilata peptide backbone peptides

50ml of frozen TFA/TIS/DCM 6.5%/10.0%/83.5% lysate was prepared, added to a 100ml round-bottomed flask, and 5.0g of the peptide resin of example 4 was weighed out and added to the above cleavage, and at the same time,' 2-dithiodipyridine (0.39g, 1.78mmol) was added and reacted at room temperature for 2 h. The reaction solution was filtered, and the filtrate was added to 500ml of cold isopropyl ether to precipitate, followed by centrifugal washing to obtain 3.28g of a white solid with an HPLC purity of 89.24%.

Example 9 activated Vilata peptide backbone peptides

50ml of frozen TFA/TIS/DCM 2.0%/2.0%/96.0% lysate was prepared, added to a 100ml round-bottomed flask, and 5.0g of the peptide resin of example 5 was weighed out and added to the above lysate and reacted at room temperature for 2 h. After filtration, 2' 2-dithiodipyridine (0.39g, 1.78mmol) was added to the filtrate to react for 30 min. The reaction solution was added to 500ml of cold isopropyl ether, and the mixture was precipitated and washed by centrifugation to give 3.06g of a white solid having an HPLC purity of 82.65%.

Example 10 Ac-D-Cys (H-L-Cys-OH) -D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH₂Synthesis of crude peptide

2.95g of the activated Vilata peptide backbone obtained in example 7 was dissolved in 30ml of water, and H-L-Cys-OH (0.50g,2.9mmol) was added thereto to react for 30 min. The reaction was controlled to completion by HPLC, concentrated under reduced pressure, and 30ml of a lysate with a TFA/TIS/H2O ratio of 95.0%/2.5%/2.5% was added to conduct cleavage reaction for 1 hour. The reaction solution was added to 300ml of cold isopropyl ether and settled, and centrifuged to obtain 1.56g of pale yellow solid with a yield of 91.2% and an HPLC purity of 77.48%, and the HPLC chromatogram is shown in FIG. 1.

Example 11 Ac-D-Cys (H-L-Cys-OH) -D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH₂Synthesis of crude peptide

2.95g of the activated vilacatide backbone peptide obtained in example 8 was dissolved in 30ml of acetonitrile/water (4/1), and H-L-Cys-OH (0.50g,2.9mmol) was added to the solution to react for 30 min. The reaction was quenched by HPLC, concentrated under reduced pressure to about 1.5ml solvent, and cleaved by adding 27ml TFA and 1.5ml TIS at a ratio of TFA/TIS/H2O to 90.0%/5.0%/5.0% lysate for 1H. The reaction solution was added to 300ml of cold isopropyl ether for sedimentation, and centrifuged to obtain 1.84g of a pale yellow solid with a yield of 99.8% and an HPLC purity of 82.71%, and the HPLC chromatogram was similar to that of FIG. 1.

Example 12 Ac-D-Cys (H-L-Cys-OH) -D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH₂Synthesis of crude peptide

2.95g of the activated Vilata peptide backbone obtained in example 9 was dissolved in 30ml of an ethanol/water (4/1) solution, and H-L-Cys-OH (0.50g,2.9mmol) was added thereto and reacted for 30 min. The reaction was controlled to completion by HPLC, and the mixture was concentrated under reduced pressure to about 3ml of a solvent, and 24ml of TFA and 3ml of TIS were added to the solvent in a ratio of 80.0%/10.0%/10.0% of TFA/H2O to conduct cleavage reaction for 1 hour. The reaction solution is added into 300ml of cold isopropyl ether for sedimentation, and the mixture is centrifugally washed to obtain 1.68g of light yellow solid, the yield is 92.3 percent, the HPLC purity is 80.25 percent, and the HPLC chromatogram is similar to that of the product shown in the figure 1.

Example 13 Ac-D-Cys (H-L-Cys-OH) -D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH₂Purification of (2)

And (2) carrying out reversed-phase HPLC purification on the crude Vilata peptide obtained in example 10, dissolving the crude Vilata peptide, filtering, balancing a chromatographic column with water, loading, carrying out gradient elution by using a 0.1% trifluoroacetic acid/acetonitrile system, merging fractions with the purity of more than 98%, carrying out reduced pressure concentration on the merged fractions to remove acetonitrile, and then carrying out salt conversion. The salt conversion step comprises: balancing the chromatographic column with water, loading, passing through the column with 50mM ammonium chloride solution, performing ion exchange for 15min, performing gradient elution with 0.05% hydrochloric acid/acetonitrile system, combining fractions with purity of more than 99%, concentrating, and lyophilizing to obtain vilacatide refined peptide 1.04g, with yield of 58.6%, HPLC purity of 99.94%, and HPLC chromatogram shown in FIG. 2.

Claims

1. A method for preparing vilacatide by solid-liquid phase combination is characterized by comprising the following steps:

step 1: coupling solid phase carrier resin and Fmoc-D-Arg (X) -OH to obtain Fmoc-D-Arg (X) -resin;

step 2: sequentially coupling D-Ala-OH, D-Arg (X) -OH, D-Ala-OH and D-Cys (Y) -OH with Fmoc-D-Arg (X) -resin obtained in the step 1, and acetylating amino at a nitrogen end to obtain fully-protected Vila card peptide main chain resin;

and step 3: taking the Vila peptide main chain resin obtained in the step 2, adding lysis solution 1 to remove a protecting group Y and cut the resin, adding an activating agent to activate a main chain Cys sulfydryl, and obtaining activated Vila peptide main chain;

2. The method for preparing vilacatide according to claim 1, wherein: the solid phase carrier resin in the step 1 is acid-sensitive amino resin selected from Rink Amide resin and Sieber resin.

3. The method for preparing vilacatide according to claim 1, wherein: x is Pbf, Y is selected from Trt and Mmt.

4. The method for preparing vilacatide according to claim 1, wherein: and 3, the lysis solution 1 is a mixed solution of TFA, TIS and DCM.

5. The method for preparing vilacatide according to claim 4, wherein: the volume ratio of the TFA, the TIS and the DCM is 0-10: 0-20: 70-100.

6. The method for preparing vilacatide according to claim 1, wherein: the solution in the step 4 is selected from water, acetonitrile/water and ethanol/water.

7. The method for preparing vilacatide according to claim 1, wherein: step 4, the lysis solution 2 is TFA, TIS and H₂And (3) a mixed solution of O.

8. The method for preparing vilacatide according to claim 7, wherein: the TFA, TIS, H₂The volume ratio of O is 80-100: 0-10.