阅读说明：本技术 乌拉立肽的制备方法 (Preparation method of ularitide ) 是由 董华建 郭德文 文永均 于 2019-10-31 设计创作，主要内容包括：本发明提供了一种制备高纯度乌拉立肽方法,方法中采用了特殊的保护氨基酸片段：Boc-Thr(tBu)-Ala-Pro-Arg(Pbf)-Ser(tBu)-Leu-Arg(Pbf)-Arg(Pbf)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-OH,缩短了制备工艺周期,提高了规模化制备中产品的纯度和收率。(The invention provides a method for preparing high-purity ularitide, which adopts special protective amino acid fragments: Boc-Thr (tBu) -Ala-Pro-Arg (Pbf) -Ser (tBu) -Leu-Arg (Pbf) -Ser (tBu) -Cys (Trt) -Phe-Gly-OH, shortens the preparation process period and improves the purity and yield of products in large-scale preparation.)

1. The preparation method of the ularitide comprises the following steps: adopting Fmoc-Tyr (tBu) resin as initial resin, preparing the ularitide peptide resin by a solid-phase polypeptide synthesis method, carrying out acidolysis on the ularitide peptide resin to obtain a linear ularitide peptide crude product, carrying out iodine oxidation process cyclization, and finally carrying out purification and freeze-drying to obtain a pure ularitide product:

Thr-Ala-Pro-Arg-Ser ⁵-Leu-Arg-Arg-Ser-Ser ¹⁰-Cys-Phe-Gly-Gly-

Arg ¹⁵-Met-Asp-Arg-Ile-Gly ²⁰-Ala-Gln-Ser-Gly-Leu ²⁵-Gly-Cys-

Asn-Ser-Phe ³⁰-Arg-Tyr-OH，Cyclic(11，17)-Disulfide。

2. the method for preparing ularitide as claimed in claim 1, wherein: when the Thr at the 1 st position to the Gly at the 13 th position are accessed together, the corresponding protected amino acids are Boc-Thr (tBu) -Ala-Pro-Arg (Pbf) -Ser (tBu) -Leu-Arg (Pbf) -Ser (tBu) -Cys (Trt) -Phe-Gly-OH.

3. The method for preparing ularitide as claimed in claim 1, wherein: the Fmoc-Tyr (tBu) resin is obtained by coupling carrier resin with Fmoc-Tyr (tBu); wherein the substitution value of the Fmoc-Tyr (tBu) -resin is 0.2-1.0 mmol/g resin; the preferred substitution value is 0.3 to 0.5mmol/g resin.

4. The method for preparing ularitide as claimed in claim 1, wherein: the dosage of the Fmoc-protected amino acid or the protected amino acid fragment is 1.2-6 times of the total mole number of the charged resin; preferably 2.5 to 3.5 times.

5. A method for producing ularitide according to any one of claims 1-4, wherein: and (3) carrying out acidolysis on the ularitide peptide resin, and simultaneously removing the resin and a side chain protecting group to obtain the ularitide linear peptide crude product.

6. The method for preparing ularitide as claimed in claim 1, wherein: dissolving the crude product of the ularitide linear peptide by using acetic acid, filtering, and oxidizing and cyclizing by using an oxidizing agent to obtain a ularitide crude product solution. The volume percentage concentration of the acetic acid is 20-40%, and the preferred concentration is 30%. The oxidant is iodine or H ₂O ₂Or DMSO, preferably iodine.

7. The method for preparing ularitide as claimed in claim 1, wherein: purifying the crude product of the ularitide by high performance liquid chromatography and freeze-drying to obtain a pure product of the ularitide.

Technical Field

The invention belongs to the technical field of preparation methods of polypeptide medicaments, and particularly relates to a preparation method of ularitide.

Background

Urapide is one of the endogenous natriuretic peptide family members present in plasma and urine, which family includes ANP, BNP, CNP, DNP, vasodilator and Urapide. ANP, BNP and ularitide can exert physiological and pharmacological effects through atrial Natriuretic Peptide Receptor (NPRA), activate guanylate cyclase, increase cyclic guanosine phosphate (cGMP) levels, trigger RAAS system inhibition, vasodilation, fibrosis and the loosening effect (lusitropy). ANP, BNP and CNP can be combined with atrial natriuretic peptide receptor 3(NPR-C) to realize the control of the level of the ularitide in the blood plasma. The heart, kidney, vascular smooth muscle cells and other organs all express NPR-A receptors, and the kidney distal tubule can synthesize ularitide so as to respond to the increase of serum sodium concentration. The ularitide can be combined with NPR-A receptor in the inner marrow collecting duct and play A physiological role. Ularitide is associated with inhibition of sodium reabsorption, increased excretion of urine and sodium, pre-glomerular vasodilation, post-glomerular vasodilation, and maintenance of glomerular filtration rate. Clinical research evidence shows that the ularitide can act on kidney, heart and blood vessels. Because the receptor pathways of ulipriside and nesiritide are the same, ulipriside may not lead to a better clinical prognosis, however, earlier treatment against deleterious pathological mechanisms may translate into a good long-term prognosis.

The ularitide has the following structure:

Thr-Ala-Pro-Arg-Ser ⁵-Leu-Arg-Arg-Ser-Ser ¹⁰-Cys-Phe-Gly-Gly-Arg ¹⁵-Met-Asp-Arg-Ile-Gly ²⁰-Ala-Gln-Ser-Gly-Leu ²⁵-Gly-Cys-Asn-Ser-Phe ³⁰-Arg-Tyr-OH，Cyelic(11，17)-Disulfide

the preparation method of the ularitide has been reported, and the invention provides an efficient preparation method of the ularitide to prepare a high-purity product so as to meet the medical application.

Disclosure of Invention

The invention provides a novel high-efficiency preparation method, which adopts Fmoc-Tyr (tBu) resin as initial resin and uses special protected amino acid segments, thereby shortening the preparation process period and improving the product purity and yield in a large-scale preparation method.

The preparation method of the ularitide provided by the invention comprises the following steps: Fmoc-Tyr (tBu) resin is used as starting resin and is prepared by a solid-phase polypeptide synthesis method, the ularitide peptide resin is obtained by the polypeptide solid-phase synthesis method, the ularitide peptide resin is subjected to acidolysis to obtain a ularitide linear peptide crude product, the ularitide linear peptide crude product is subjected to cyclization by an iodine oxidation process, and finally, the ularitide pure product is obtained by purification and freeze-drying.

In addition to other conventional protected amino acids, the following special protected amino acid fragments are used in the synthetic process of the ularitide multi-resin:

Boc-Thr(tBu)-Ala-Pro-Arg(Pbf)-Ser(tBu)-Leu-Arg(Pbf)-Arg(Pbf)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-OH。

the ularitide resin is as follows:

Boe-Thr(tBu)-Ala-Pro-Arg(Pbf)-Ser(tBu) ⁵-Leu-Arg(Pbf)-Arg(Pbf)-Ser(tBu)-Ser(tBu) ¹⁰-Cys(Trt)-Phe-Gly-Gly-Arg(Pbf) ¹⁵-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly ²⁰-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu ²⁵-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe ³⁰-Arg (Pbf) -Tyr (tBu) -resin

In the preparation method of the ularitide, the dosage of the Fmoc-protected amino acid or the protected amino acid fragment is 1.2-6 times of the total mole number of the charged resin; preferably 2.5 to 3.5 times.

In the preparation method of the ularitide, the Fmoc-Tyr (tBu) resin is obtained by coupling carrier resin with Fmoc-Tyr (tBu); wherein the substitution value of the Fmoc-Gly-resin is 0.2-1.0 mmol/g resin, and the preferable substitution value is 0.3-0.5 mmol/g resin.

Further preferably, the carrier resin is a Trityl-Cl type resin or a hydroxyl type resin, wherein the Trityl-Cl type resin is preferably a Trityl-Cl resin, a 4-methylitrityl-Cl resin, a 4-Methoxytrityl-Cl (4-Methoxytrityl chloride) resin or a 2-Cl Trity-Cl (2-chlorotrityl chloride) resin; the hydroxyl resin is preferably Wang resin or p-hydroxymethylphenoxymethyl polystyrene (HMP) resin.

As a preferred embodiment of the present invention, when the support resin is trityl chloride resin, the coupling method of Fmoc-Tyr (tBu) and the support resin is as follows: the carboxyl of Fmoc-Tyr (tBu) and Cl-substituted alkyl in the resin are subjected to esterification reaction under the action of alkali so as to be connected with the protected amino acid.

As a preferable embodiment of the present invention, when the support resin is a hydroxyl type resin, the coupling method of Fmoc-Tyr (tBu) to the support resin is: the carboxyl of Fmoc-Tyr (tBu) and the hydroxyl in the resin are esterified under the action of a coupling agent, an activating agent and a base catalyst to join in the protected amino acid.

In a preferred embodiment of the present invention, the solid-phase coupling synthesis method comprises: and (3) after the Fmoc protecting group of the protected amino acid-resin obtained in the previous step is removed, carrying out coupling reaction with the next protected amino acid. The coupling reaction time is 60-300 minutes, and preferably 100-140 minutes.

Preferably, the resin and the side chain protecting group are removed simultaneously by the resin of the urotropine resin through acidolysis to obtain a crude product of the urotropine:

more preferably, the acidolysis agent used in the acidolysis of the ularitide resin is a mixed solvent of trifluoroacetic acid (TFA), 1, 2-Ethanedithiol (EDT) and water; wherein the volume ratio of the mixed solvent is as follows: 80-95% of TFA, 1-10% of EDT and the balance of water.

More preferably, the volume ratio of the mixed solvent is: 89-91% of TFA, 4-6% of EDT and the balance of water. Optimally, the volume ratio of the mixed solvent is as follows: TFA 90%, EDT 5%, balance water.

The dosage of the acidolysis agent is 4-15 mL per gram of the urapidil resin; preferably, 9-11 mL of acidolysis agent is needed for each gram of liulipristin resin. The time for cracking by using the acidolysis agent is 1-6 hours, preferably 3-4 hours at room temperature.

Further, dissolving the crude product of the ularitide linear peptide by using acetic acid, filtering, and oxidizing and cyclizing by using an oxidizing agent to obtain a solution of the ularitide crude product. The volume percentage concentration of the acetic acid is 20-40%, and the preferred concentration is 30%. The oxidant is iodine or H ₂O ₂Or DMSO, preferably iodine.

Further, the crude product of the ularitide is purified by high performance liquid chromatography and freeze-dried to obtain a pure product of the ularitide, and the specific method comprises the following steps:

purifying by high performance liquid chromatography, wherein a chromatographic filler for purification is 10 mu m reverse phase C18, alternately purifying by two mobile phase systems, the first mobile phase system is 0.1% TFA/aqueous solution-0.1% TFA/acetonitrile solution, the second mobile phase system is 50mmol ammonium acetate/aqueous solution-acetonitrile, the flow rate of a 77mm 250mm chromatographic column is 90mL/min, eluting by a gradient system, circularly injecting and purifying, sampling a crude product solution in the chromatographic column, starting mobile phase elution, collecting a main peak to evaporate acetonitrile, and filtering by a 0.45 mu m filter membrane to obtain a urotropine purified intermediate concentrated solution;

performing salt exchange by high performance liquid chromatography, wherein the mobile phase system is 1% acetic acid/water solution-acetonitrile, the chromatographic packing for purification is reversed phase C18 with 10 μm, and the flow rate of 77mm × 250mm chromatographic column is 90mL/min (corresponding flow rate can be adjusted according to chromatographic columns with different specifications); adopting gradient elution and circulation sample loading method, loading sample in chromatographic column, starting mobile phase elution, collecting atlas, observing change of absorbance, collecting main peak of salt exchange and analyzing liquid phase to detect purity, combining main peak solutions of salt exchange, concentrating under reduced pressure to obtain acetic acid water solution of ularitide, and freeze-drying to obtain pure product of ularitide.

The method of the invention directly uses the following special protected amino acids:

Boc-Thr(tBu)-Ala-Pro-Arg(Pbf)-Ser(tBu)-Leu-Arg(Pbf)-Arg(Pbf)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-OH。

the production period is shortened, the purity of the crude product is greatly improved, the product yield is improved, the purity of the obtained product is more than 99.0 percent, and compared with the prior art, the process has wide practical value and application prospect.

Detailed Description

The invention discloses a method for synthesizing ularitide, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as appropriate variations and combinations of the methods described herein, may be made and the techniques of the present invention employed without departing from the spirit and scope of the invention.

In the specific embodiment of the present invention, the Chinese meanings corresponding to the English abbreviations used in the application documents are shown in Table 1.

TABLE 1

The invention is further illustrated by the following examples.