A method for synthesizing a highly selective k opioid receptor full agonist

Abstract

The application discloses a synthesis method of a high-selectivity k opioid receptor complete agonist, and belongs to the technical field of pharmacy. The application comprises the following steps: (1) synthesizing resin by Fmoc / tBu solid-phase method; (2) cutting to obtain a tetrapeptide segment after full protection; (3) carrying out condensation reaction of the tetrapeptide segment and Boc-4-amino-1-amino-4-(piperidine)-4-carboxylic acid tert-butyl ester to obtain a full-protection crude peptide; (4) cutting the full-protection crude peptide by using a TFA mixed solution to obtain a crude peptide; and (5) carrying out HPLC purification, salt conversion, concentration, freeze-drying and the like on the crude peptide to obtain Difelikefalin. The synthesis is completed by adopting a solid-liquid combination mode, the operation is simple, and the method is very suitable for industrialized production of polypeptides. The HPLC purity of the method reaches more than 99%, and a single impurity is lower than 0.1%, thereby meeting the basic requirements of related substances of a bulk drug. The method is simple in operation, low in comprehensive synthesis cost, and very suitable for industrialized production of polypeptide bulk drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular to a method for synthesizing a highly selective ketoopioid receptor complete agonist (Difelikefalin). Background Technology

[0002] Difelikefalin (trade name KORSUVA) is a first-in-class, highly selective, complete kinase receptor (KOR) agonist that inhibits the activity of peripheral neurons that produce pruritus and has no significant activity against other receptors (including muco- or delta-opioid receptors), ion channels, or transporters. Furthermore, unlike small-molecule KOR agonists, Korsuva is a small synthetic peptide that primarily activates KORs expressed by peripheral neurons (PNS) and immune cells. The US FDA has approved difelikefalin (Korsuva) for the treatment of moderate to severe pruritus associated with chronic kidney disease (CKD-aP) in adults undergoing hemodialysis (HD). The original patent CN101627049B first disclosed the synthetic method of this compound. No other patent applications have been published. The original patent uses an all-solid-phase peptide synthesis method to synthesize this compound. This method requires a high equivalent of N-Boc-amino-(4-N-Fmoc-piperidinyl)carboxylic acid to react with CTC resin for loading. N-Boc-amino-(4-N-Fmoc-piperidinyl)carboxylic acid is a special amino acid with high cost. This loading method leads to a significant waste of this expensive amino acid, indirectly increasing the production cost of the active pharmaceutical ingredient and hindering industrial production. Therefore, the industry urgently needs a low-cost method for synthesizing difelikefalin. Summary of the Invention

[0003] To address the issues of high raw material production costs and hindering industrial production in the aforementioned background technologies, this invention proposes a method for synthesizing a highly selective κ-opioid receptor complete agonist.

[0004] Therefore, the present invention adopts the following technical solution: a method for synthesizing a highly selective κ-opioid receptor complete agonist, comprising the following steps:

[0005] Step 1: Synthesis via Fmoc / tBu solid-state method

[0006] Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-CTC tetrapeptide resin;

[0007] Step 2: The Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-OH tetrapeptide fragment was obtained by fully protected cleavage.

[0008] Step 3: The tetrapeptide fragment is condensed with Boc-4-amino-1-(piperidine)-4-carboxylic acid tert-butyl ester to obtain a fully protected crude peptide.

[0009] Step 4: The fully protected crude peptide is cleaved using a TFA mixed solution to obtain the crude peptide;

[0010] Step 5: The crude peptide was purified by HPLC, converted to salt, concentrated, and lyophilized to obtain Difelikefalin.

[0011] As a supplement and improvement to the above technical solution, the present invention also includes the following technical features.

[0012] The specific steps in Step One are as follows:

[0013] The synthesis of Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-CTC resin via the Fmoc / tBu solid-phase method refers to using CTC resin as the starting resin and sequentially connecting Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH, Fmoc-D-Phe-OH, and Boc-D-Phe-OH according to the Fmoc / tBu solid-phase method to obtain a tetrapeptide resin.

[0014] The specific steps in step two are as follows: using a fully protected cleavage method, the tetrapeptide resin is reacted with a 20% trifluoroethanol / dichloromethane solution or a 20% hexafluoroisopropanol / dichloromethane solution. After cleavage, the resin is filtered and concentrated by rotary evaporation to obtain the tetrapeptide fragment.

[0015] The specific steps in step three are as follows: A condensation reaction is carried out between a tetrapeptide fragment and Boc-4-amino-1-(piperidine)-4-carboxylic acid tert-butyl ester. The condensation reagent is one or a combination of several of DIC / HOBT, TBTU / DIEA, PyBOP / DIEA, EDCI / HOBT, and EDCI / HOSU. The condensation solvent is one or a combination of several of dichloromethane, chloroform, N,N-dimethylformamide, N-methylpyrrolidone, tetrahydrofuran, 2-methyltetrahydrofuran, dimethyl sulfoxide, methanol, and ethanol. The reaction temperature is 10–50 °C.

[0016] In step three, the reaction temperature is preferably 20–30°C.

[0017] The specific steps in step four are as follows: the TFA mixed solution is a combination of TFA and phenol, water, anisole, anisole sulfide, ethylene dithiol, triisopropylsilane and other scavenging agents, wherein the volume ratio of TFA is not less than 85%.

[0018] The present invention achieves the following beneficial effects: the Difelikefalin prepared by the method of the present invention has an HPLC purity of over 99%, with individual impurities below 0.1%, meeting the basic requirements for related substances in active pharmaceutical ingredients. This method is simple to operate, has low overall synthesis cost, and is very suitable for the industrial production of peptide active pharmaceutical ingredients. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the synthesis process of the present invention.

[0020] Figure 2 The HPLC chromatogram of the fully protected tetrapeptide fragment Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-OH.

[0021] Figure 3 MS spectrum of the fully protected tetrapeptide fragment Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-OH.

[0022] Figure 4 The image shows the HPLC chromatogram of the Difelikefalin product.

[0023] Figure 5 The image shows the MS spectrum of the Difelikefalin product. Detailed Implementation

[0024] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The described embodiments are only for illustration and explanation of the present invention and do not constitute the only limitation of the present invention.

[0025] like Figures 1-5 As shown:

[0026] Example 1

[0027] Preparation of Fmoc-D-Lys(Boc)-CTC resin

[0028] CTC resin (10 g, degree of substitution 1.00 mmol / g) was placed in a reactor and swollen with 100 ml of DCM for 30 min. The solvent was then removed under vacuum. Fmoc-D-Lys(Boc)-OH (7.26 g, 15 mmol, 1.5 eq) was added, and 30 ml of DCM was added to dissolve it under nitrogen. DIEA (5 ml, 30 mmol) was added dropwise, and the reaction was allowed to proceed for 1 h. After the reaction was complete, 10 ml of methanol was added to seal the reaction, and the reaction was allowed to continue for 10 min. The solvent was then removed under vacuum, and the mixture was washed alternately with DCM and methanol. Finally, the solvent was removed under vacuum to obtain Fmoc-D-Lys(Boc)-CTC wet resin. The resin was then dried under vacuum to obtain Fmoc-D-Lys(Boc)-CTC resin.

[0029] Example 2

[0030] Preparation of Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-CTC tetrapeptide resin

[0031] Fmoc-D-Lys(Boc)-CTC resin was added to the reactor, and DMF was added to swell the resin for 30 min. The solvent was then removed under vacuum. Fmoc-D-Leu-OH (5.30 g, 15 mmol), TBTU (4.82 g, 15 mmol), DIEA (5.2 ml, 30 mmol), and 30 ml DMF were added. The mixture was stirred at 25 ± 5 °C for 120 min. The resin was washed three times with DMF and then removed under vacuum. 50 ml of 20% Pip / DMF solution was added to remove Fmoc resin. After reacting for 30 min, the resin was washed and the solvent was removed under vacuum to obtain H2N-D-Leu-D-Lys(Boc)-CTC resin.

[0032] Repeat the above steps to sequentially couple Fmoc-D-Phe-OH and Boc-D-Phe-OH to obtain Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-CTC tetrapeptide resin. The dried resin weighs 16.50g.

[0033] Example 3

[0034] Preparation of the fully protected tetrapeptide fragment Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-OH

[0035] 16.50 g of Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-CTC tetrapeptide resin was added to the reactor, along with 165 ml of 20% hexafluoroisopropanol / DCM solution (10 ml per gram of resin). The reaction was carried out at 25 ± 5 °C for 4 hours. After the reaction, the resin was filtered, and the filtrate was directly concentrated by rotary evaporation and dried under vacuum using an oil pump to obtain 6.11 g of the tetrapeptide fragment with an HPLC purity of 98.13% and a yield of 81%.

[0036] A table of English abbreviations and their Chinese equivalents used in this article:

[0037]

[0038]

[0039] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for synthesizing a highly selective κ-opioid receptor complete agonist, characterized in that... Includes the following steps: Step 1: Synthesize via the Fmoc / tBu solid-state method; Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-CTC tetrapeptide resin; Step 2: The Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-OH tetrapeptide fragment was obtained by fully protected cleavage. Step 3: The tetrapeptide fragment is condensed with Boc-4-amino-1-(piperidine)-4-carboxylic acid tert-butyl ester to obtain a fully protected crude peptide. Step 4: The fully protected crude peptide is cleaved using a TFA mixed solution to obtain the crude peptide; Step 5: The crude peptide was purified by HPLC, converted to salt, concentrated, and lyophilized to obtain Difelikefalin; The specific steps in Step 1 are as follows: The synthesis of Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-CTC resin by the Fmoc / tBu solid-phase method refers to using CTC resin as the starting resin and sequentially connecting Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH, Fmoc-D-Phe-OH, and Boc-D-Phe-OH according to the Fmoc / tBu solid-phase method to obtain a tetrapeptide resin. The specific steps in step two are as follows: using a fully protected cleavage method, the tetrapeptide resin is reacted with a 20% trifluoroethanol / dichloromethane solution or a 20% hexafluoroisopropanol / dichloromethane solution. After cleavage, the resin is filtered and concentrated by rotary evaporation to obtain the tetrapeptide fragment. Step 3 specifically involves the following steps: A tetrapeptide fragment is condensed with tert-butyl Boc-4-amino-1-(piperidine)-4-carboxylic acid. The condensing agent is one or a combination of several of the following: DIC / HOBT, TBTU / DIEA, PyBOP / DIEA, EDCI / HOBT, and EDCI / HOSU. The condensing solvent is one or a combination of several of the following: dichloromethane, chloroform, N,N-dimethylformamide, N-methylpyrrolidone, tetrahydrofuran, 2-methyltetrahydrofuran, dimethyl sulfoxide, methanol, and ethanol. The reaction temperature is 10–50 °C. The specific steps in step four are as follows: the TFA mixed solution is a combination of TFA and phenol, water, anisole, anisole sulfide, ethylene dithiol, and triisopropylsilane scavenger, wherein the volume ratio of TFA is not less than 85%.

2. The method for synthesizing a highly selective κ-opioid receptor complete agonist according to claim 1, characterized in that, In step three, the reaction temperature is 20–30°C.

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