A method for preparing teriparatide

Abstract

The application provides a preparation method of teriparatide. Specifically, the method comprises the following steps: (a) providing Fmoc-Asp (amino resin)-Phe-OtBu; (b) coupling Fmoc-Asp (amino resin)-Phe-OtBu with amino acid precursors of amino acids at positions 32-1 of a teriparatide peptide sequence in sequence in the presence of a coupling agent to obtain a side chain-protected teriparatide peptide resin; and (c) cleaving the teriparatide peptide resin in the presence of a cleavage reagent to obtain a crude teriparatide. The teriparatide prepared by the method has less by-products, high yield and is easy to purify.

Description

Technical Field

[0001] This invention relates to the field of polypeptide synthesis, and in particular to a method for preparing teriparatide. Background Technology

[0002] Teriparatide is a 1-34 amino acid fragment of the N-terminal region of endogenous parathyroid hormone, and its peptide sequence is as follows:

[0003] H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH. This product, developed by Eli Lilly and Company, is primarily used to treat primary osteoporosis, postmenopausal osteoporosis in women, and osteoporosis following hypogonadism in men. It is manufactured using a bio-fermentation method.

[0004] In October 2017, the FDA approved five bio-fermented polypeptide drugs—glucagon, liraglutide, nesiritide, teriparatide, and teduglutide—as chemically synthesized drugs. For these long peptides, the conventional synthesis method is currently solid-phase synthesis.

[0005] Teriparatide's first C-terminal amino acid is phenylalanine, which is prone to racemization during coupling. When coupled with WangResin, the catalyst DMAP is required, further exacerbating the risk of racemization, making this site difficult to separate. Furthermore, in actual production, it was found that after de-Fmoc removal from Asn at position 33, the two C-terminal amino acids are easily removed via a diazinonone mechanism, generating a dipeptide-deficient peptide at positions 33-34. The larger the synthesis scale, the higher the proportion of the dipeptide-deficient peptide, significantly reducing the production yield.

[0006] Patent 201210213044.3 divides PTH into five segments by fragment coupling and couples them segment by segment according to the peptide sequence. All five peptide fragments need to be synthesized and purified by themselves, making it difficult to obtain a stable supply channel through suppliers, which is not conducive to industrial production.

[0007] Patent 201510005427.5 adopts the traditional solid-phase synthesis method, which synthesizes teriparatide stepwise from the C-terminus to the N-terminus of the peptide chain. Although the operation is simple, it cannot avoid the 33-34 dipeptide deletion peptide generated by the diazinon mechanism, nor can it avoid the racemization of the 34-position Phe terminal, resulting in low purity of crude product. Furthermore, when conducting quality studies of the refined peptide, it is necessary to separate the 34-position Phe racemic impurity and the 33-position Asn racemic impurity.

[0008] Patent 201410262511.0 uses pseudoproline dipeptide at positions 16-17 of the peptide chain to avoid the generation of specific process impurities, but it cannot avoid the loss of dipeptide at positions 33-34, nor can it avoid the racemization of Phe at position 34.

[0009] Patent 201310403743.9 uses a stepwise coupling method to synthesize the main chain by a one-step ester-amine conversion between the 17-position Ser and the 16-position Asn. The coupling efficiency is improved by reducing steric hindrance. However, the one-step ester-amine conversion reaction has a conversion rate problem, resulting in low process yield and ultimately increased cost.

[0010] Therefore, there is a need in the art to provide a method for preparing teriparatide that avoids racemization, produces fewer byproducts, and yields a high amount of teriparatide. Summary of the Invention

[0011] The purpose of this invention is to provide a method for preparing teriparatide that avoids racemization, produces fewer byproducts, and has a high yield.

[0012] In a first aspect, the present invention provides a method for preparing teriparatide, comprising the following steps:

[0013] (a) Provide Fmoc-Asp (amino resin)-Phe-OtBu;

[0014] (b) In the presence of a coupling agent, Fmoc-Asp (amino resin)-Phe-OtBu is sequentially coupled with the amino acid precursor at positions 32-1 of the teriparatide peptide sequence to obtain a side-chain protected teriparatide peptide resin; and

[0015] (c) The teriparatide peptide resin is cleaved in the presence of a cleavage reagent to obtain crude teriparatide.

[0016] 2. The preparation method according to claim 1, characterized in that, in step (b), the amino acid precursors of the teriparatide peptide at positions 32-1 are, in sequence, Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(pbf)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(Boc)-OH, Fmoc-Val-OH, Fmoc-Arg(pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-M The side-chain protected teriparatide peptide resin structures obtained from Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, and Fmoc-Ser(tBu)-OH are as follows:

[0017] Fmoc-Ser(tBu)-Val-Ser(tBu)-Glu(OtBu)-Ile-Gln(Trt)-Leu-Met-His(Trt)-Asn(Trt)-Leu-Gly-Lys(Boc)-His(Trt)-Leu-Asn(Trt)-Ser(tBu) -Met-Glu(OtBu)-Arg(pbf)-Val-Glu(OtBu)-Trp(Boc)-Leu-Arg(pbf)-Lys(Boc)-Lys(Boc)-Leu-Gln(Trt)-Asp(OtBu)-Val-His(Trt)-Asp(Amide Resin)-Phe-OtBu.

[0018] In another preferred embodiment, before step (a), the step further includes: (a1) in the presence of a coupling agent, It reacts with an amino resin to obtain the Fmoc-Asp (amino resin)-Phe-OtBu.

[0019] In another preferred embodiment, in step (a), the degree of substitution of the amino resin in Fmoc-Asp (amino resin)-Phe-OtBu is 0.3 to 0.5 mmol / g, such as 0.35 mmol / g or 0.4 mmol / g.

[0020] In another preferred embodiment, in step (a), the amino resin in Fmoc-Asp (amino resin)-Phe-OtBu is selected from the group consisting of Rink Amide Resin, Rink Amide AM Resin, and Rink Amide MBHA Resin.

[0021] In another preferred embodiment, in step (b), the coupling agents for each coupling reaction are independently selected from the group consisting of: HOBt / DIC, HOAt / DIC, HOBt / HBTU / DIEA, PyBOP / DIEA, TBTU / DIEA, or combinations thereof.

[0022] In another preferred embodiment, in step (b), each coupling reaction independently includes one or more of the following features:

[0023] (1) The amino acid precursor is first de-Fmoc group removed before the reaction. Preferably, the de-Fmoc reagent is 20% piperidine / DMF (volume ratio).

[0024] (2) The molar ratio of resin to amino acid precursor is 1:2.0-3.5, preferably 1:2.5-3.0, and the amount of resin used is calculated based on its total degree of substitution;

[0025] (3) The molar ratio of the amino acid precursor to the coupling agent is 1:1-1.5, preferably 1:1.1-1.2;

[0026] (4) The reaction solvent is a mixed solution of DMF / DCM, preferably a mixed solution of DMF / DCM in a volume ratio of 1:1; and / or

[0027] (5) DIC or DCC activating coupling agent is also added to the reaction solution. Preferably, the molar ratio of DIC to coupling agent is 1-1.2:1.

[0028] In another preferred embodiment, in step (b), each coupling reaction independently includes one or more of the following features:

[0029] (1) The reaction is carried out in an inert gas atmosphere, preferably N2;

[0030] (2) The reaction temperature is 25±10℃, preferably 25±5℃;

[0031] (3) The reaction time is 1-6 hours, preferably 2-3 hours; and / or

[0032] (4) After a reaction is completed, the reaction solution is separated and the resin is washed in order to proceed to the next reaction or to end the reaction.

[0033] In another preferred embodiment, in step (c), the cleavage reagent is a TFA solution containing 5 to 10% by volume a scavenger, wherein the scavenger is selected from the group consisting of dithiothreitol, anisole, phenol, mercaptoethanol, water, and triisopropylsilane, or combinations thereof.

[0034] In another preferred embodiment, the method further includes a purification step to obtain a purified teriparatide; preferably, the purification step may include steps selected from the group consisting of reversed-phase chromatography, nanofiltration, lyophilization, or a combination thereof.

[0035] In another preferred embodiment, the purification method includes one or more of the following steps:

[0036] (1) Chromatographic purification: C18 preparative column, mobile phase A: 0.1% TFA / water, mobile phase B: 0.1% TFA / acetonitrile, gradient elution; and / or

[0037] (2) Chromatographic purification: C18 preparative column, with 0.1% acetic acid / water as phase A and 0.1% acetic acid / acetonitrile as phase B, gradient elution.

[0038] In a second aspect, the present invention provides an intermediate for the preparation of teriparatide, said intermediate being Fmoc-Asp (amino resin)-Phe-OtBu:

[0039]

[0040] In another preferred embodiment, the amino resin in Fmoc-Asp (amino resin)-Phe-OtBu is selected from the group consisting of Rink Amide Resin, Rink Amide AM Resin, and Rink Amide MBHA Resin.

[0041] In a third aspect, the present invention provides the use of Fmoc-Asp (amino resin)-Phe-OtBu for the preparation of teriparatide.

[0042] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0043] Figure 1 A schematic diagram of the process for preparing teriparatide using conventional solid-phase synthesis methods in this field;

[0044] Figure 2 This is a schematic diagram of the process for preparing teriparatide using the synthetic method of the present invention. Detailed Implementation

[0045] Through extensive and in-depth research, and through numerous screenings and tests, the inventors have provided a method for preparing teriparatide. This invention utilizes a specific Fmoc-Asp-Phe-OtBu dipeptide precursor, directly coupling the Asp carboxyl group to an amino resin to form Fmoc-Asp (amino resin)-Phe-OtBu. This Fmoc-Asp-Phe-OtBu is used as a raw material for the synthesis of teriparatide. Unexpectedly, using this raw material not only avoids racemization of Phe at position 34 and Asn at position 33, but also avoids the formation of dipeptide deletion peptides at positions 33-34, thereby reducing reaction byproducts and improving yield and product purity. This invention is based on this principle.

[0046] the term

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0048] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0049] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0050] As used herein, the terms “room temperature” or “normal temperature” refer to a temperature of 4-40°C, preferably 25±5°C, and unless otherwise specified, the operating temperature may be normal.

[0051] As is used herein, the terms “Fmoc-Asp (Amide Resin)-Phe-OtBu” and “Fmoc-Asp (Amide Resin)-Phe-OtBu”, “intermediate of the present invention”, are used interchangeably.

[0052] Teriparatide

[0053] Teriparatide is a 1-34 amino acid fragment of the N-terminal region of endogenous parathyroid hormone, and its peptide sequence is as follows:

[0054] H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH.

[0055] In this invention, the peptide sequence of teriparatide starts at the N-terminus, i.e., Ser is the first position and Phe is the 34th position.

[0056] Fmoc-Asp (Amino Resin)-Phe-OtBu

[0057] This invention provides a key intermediate for the preparation of teriparatide, Fmoc-Asp (amino resin)-Phe-OtBu:

[0058]

[0059] The present invention does not have any particular requirements for the preparation method of the intermediate, and the Fmoc-Asp-Phe-OtBu amino acid precursor of the present invention can be used. It is prepared with amino resins under commonly used solid-phase synthesis conditions. For example, in the presence of a coupling agent, the carboxyl group of Fmoc-Asp-Phe-OtBu is reacted with the amino group of the amino resin (in the deprotected state) to undergo an amide reaction, thereby obtaining the Fmoc-Asp (amino resin)-Phe-OtBu.

[0060] In this invention, there are no particular requirements for the type of amino resin; it can be any solid-phase synthesis support capable of amide modification of carboxyl groups. The pretreatment procedures (such as washing, swelling, and Fmoc removal) required for amino resins before solid-phase synthesis are readily known to those skilled in the art, or can be performed according to the product instructions. Preferably, the amino resin can be (including but not limited to) Rink Amide Resin, Rink Amide AM Resin, or Rink Amide MBHA Resin.

[0061] The intermediate of this invention directly links the carboxyl group of the Asp group of the Fmoc-Asp-Phe-OtBu amino acid precursor to the amino resin, eliminating the need for Phe to participate in the subsequent coupling reaction. This prevents racemization of Phe at position 34 during the coupling reaction. Furthermore, the tert-butyl group on Phe has sufficient steric foci to prevent the formation of diazinonone and inhibit the formation of dipeptide-deficient peptides. This effectively improves the solid-state synthesis yield of teriparatide, reduces byproduct formation, and simplifies the purification process.

[0062] Preparation method of teriparatide

[0063] This invention provides a method for preparing teriparatide, comprising: a coupling reaction of an amino resin with an Fmoc-Asp-Phe-OtBu amino acid precursor as a starting step, or directly using Fmoc-Asp (amino resin)-Phe-OtBu as a starting material.

[0064] Furthermore, based on Fmoc-Asp (amino resin)-Phe-OtBu, teriparatide is sequentially coupled with the corresponding amino acid precursors according to its peptide sequence to obtain crude teriparatide. The amino acid precursor used to synthesize the amino acid at positions 32-1 can be a commonly used amino acid precursor in the art. Typically, the N-segment of the amino acid precursor is protected by Fmoc, Boc, etc., and deprotection is required before the reaction; moreover, the reactive group in the amino acid precursor that is not desired to participate in the coupling reaction is protected by other protecting groups (such as Boc, Trt, OtBu, tBu, Pbf, etc.), which is easily understood and understood by those skilled in the art.

[0065] Preferably, the precursors used to synthesize the amino acid at position 32-1 are, in order: Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(pbf)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(Boc)-OH, Fmoc-Val-OH, Fmoc-Arg(pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH. Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-H is(Trt)-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Ser(tBu)-OH.

[0066] In this invention, the coupling reaction of each amino acid precursor can be carried out using conventional methods in the art, typically including: removal of Fomc, and coupling reaction in the presence of a coupling agent. Commonly used coupling agents include (but are not limited to): HOBt / DIC, HOAt / DIC, HOBt / HBTU / DIEA, PyBOP / DIEA, TBTU / DIEA, or combinations thereof.

[0067] In addition, after the coupling reaction is completed, the process includes a step of cleaving the obtained teriparatide peptide resin to remove the protecting group and the resin. The cleavage reaction can be carried out under cleavage conditions commonly used in the art. For example, cleavage can be carried out in the presence of a cleavage reagent. Preferably, the cleavage reagent can be a TFA solution with 5-10% (v / v) scavenger added. More preferably, the scavenger is selected from the group consisting of dithiothreitol, anisole, phenol, mercaptoethanol, water, and triisopropylsilane, or combinations thereof.

[0068] The main advantages of this invention include:

[0069] 1. The method of the present invention cleverly avoids the de-rotation of Phe at position 34 and Asn at position 33, thereby improving product quality and greatly reducing the pressure of subsequent removal of related substances.

[0070] 2: The method of the present invention also avoids the generation of dipeptide deletion peptides at positions 33-34, increasing the overall yield by more than 10%.

[0071] 3. The method of the present invention uses readily available raw materials, is simple to operate, easy to control in terms of quality, has a high yield, few by-products, and the products are easy to purify.

[0072] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0073] The abbreviations used in the specification and claims have the following meanings:

[0074] Fmoc 9-fluorenyloxycarbonyl

[0075] Rink Amide AM Resin AM Amino Resin

[0076] Rink Amide MBHA ResinMBHA Amino Resin

[0077] tBu tert-butyl

[0078] Trt triphenylmethyl

[0079] DCM dichloromethane

[0080] DMF N,N-dimethylformamide

[0081] DMAP 4-Dimethylaminopyridine

[0082] DIEA N,N-Diisopropylethylamine

[0083] DIC N,N-diisopropylcarbodiimide

[0084] HBTU Benzotriazole-N,N,N',N'-Tetramethylurea hexafluorophosphate

[0085] TBTU O-benzotriazole-N,N,N',N'-tetramethylureatetrafluoroboric acid

[0086] HOBT 1-Hydroxybenzotriazole

[0087] HOAT 1-hydroxy-7-azobenzotriazole

[0088] TFA (trifluoroacetic acid)

[0089] Example 1: Synthesis of Fmoc-Asp(Rink Amide AM Resin)-Phe-OtBu

[0090] 171.42 g (Sub = 0.35 mmol / g) of Rink Amide AM Resin was weighed and placed in a solid-phase reactor. The resin was washed twice with DMF, and swollen with DCM for 30 min, 1.7 L / time. After the resin swelled, it was deprotected twice with 20% piperidine / DMF, 1.7 L / time, the first reaction for 5 min and the second for 10 min. After deprotection, it was washed 6 times with DMF, 1.7 L / time. A small sample was tested with Kaiser reagent; the resin turned deep blue.

[0091] Weigh 100.44 g (180.0 mmol, 3.0 eq) of Fmoc-Asp-Phe-OtBu and 26.73 g (198.0 mmol, 3.3 eq) of HOBt, dissolve them in 800.0 ml of a 1:1 DMF / DCM mixture, pre-freeze to -5 to 5°C, add 31.68 ml (198.0 mmol, 3.3 eq) of DIC and activate for 5 min.

[0092] After the resin washing was completed, the reaction was carried out under mechanical stirring and nitrogen protection at a reaction temperature of 25°C for 2.0 h. The reaction solution was then removed, and the resin was washed 6 times with DMF (1.7 L each time). A small sample was taken and tested with Kaiser reagent, and the resin was colorless.

[0093] Example 2: Synthesis of Fmoc-Asp(Rink Amide MBHA Resin)-Phe-OtBu

[0094] 131.58 g (Sub = 0.38 mmol / g) of Rink Amide MBHAResin was weighed and placed in a solid-phase reactor. The resin was washed twice with DMF, and swollen with DCM for 30 min, 1.3 L / time. After the resin swelled, it was deprotected twice with 20% piperidine / DMF, 1.3 L / time, the first reaction for 5 min and the second for 10 min. After deprotection, it was washed 6 times with DMF, 1.3 L / time. A small sample was tested with Kaiser reagent; the resin turned deep blue.

[0095] Weigh 83.70 g (150.0 mmol, 3.0 eq) of Fmoc-Asp-Phe-OtBu and 22.28 g (165.0 mmol, 3.3 eq) of HOBt, dissolve them in 600.0 ml of a 1:1 DMF / DCM mixture, pre-freeze to -5 to 5 °C, add 26.40 ml (165.0 mmol, 3.3 eq) of DIC and activate for 5 min.

[0096] After the resin washing was completed, the reaction was carried out under mechanical stirring and nitrogen protection at a reaction temperature of 25°C for 2.0 h. The reaction solution was then removed, and the resin was washed 6 times with DMF (1.3 L / wash). A small sample was taken and tested with Kaiser reagent, and the resin was colorless.

[0097] Example 3: Preparation of Teriparatide Peptide Resin

[0098] The Fmoc-Asp(Rink Amide AM Resin)-Phe-OtBu synthesized in Example 1 was deprotected twice with 20% piperidine / DMF, 1.7 L / time, for 5 min the first time and 10 min the second time; after deprotection, it was washed 6 times with DMF, 1.7 L / time. A small sample was tested with Kaiser's reagent, and the resin turned dark blue.

[0099] Weigh 111.42 g (180 mmol, 3.0 eq) of Fmoc-His(Trt)-OH and 26.73 g (198.0 mmol, 3.3 eq) of HOBt, dissolve them in 800.0 ml of DMF / DCM solution with a volume ratio of 1:1, pre-freeze to -5 to 5°C, add 31.68 ml (198.0 mmol, 3.3 eq) of DIC and activate for 5 min.

[0100] After the resin washing was completed, the reaction was carried out under mechanical stirring and nitrogen protection at a reaction temperature of 25°C for 2.0 h. The reaction solution was then removed, and the resin was washed 6 times with DMF (1.7 L each time). A small sample was taken and tested with Kaiser reagent, and the resin was colorless.

[0101] Following a similar coupling method to the 32-position His, amino acids are sequentially linked according to the teriparatide peptide sequence: Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(pbf)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(Boc)-OH, Fmoc-Val-OH, Fmoc-Arg(pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmo c-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-His (Trt)-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Ser(tBu)-OH. After the peptide resin synthesis was completed, the 1-position Ser amino protecting group Fmoc was removed. The resin was washed 4 times with DMF, 2 times with DCM, and 3 times with methanol, 1.7 L / wash. The resin was then vacuum dried at 25°C for 8-12 h. 584.1 g of PTH peptide resin was obtained by weighing.

[0102] Example 4: Preparation of Teriparatide Peptide Resin

[0103] In Example 2, the synthesized Fmoc-Asp(Rink Amide MBHA Resin)-Phe-OtBu was deprotected twice with 20% piperidine / DMF, 1.3 L / time, for 5 min the first time and 10 min the second time. After deprotection, it was washed 6 times with DMF, 1.3 L / time. A small sample was tested with Kaiser's reagent, and the resin turned dark blue.

[0104] Weigh 92.85 g (150 mmol, 3.0 eq) of Fmoc-His(Trt)-OH and 22.28 g (165.0 mmol, 3.0 eq) of HOBt, dissolve them in 600.0 ml of DMF / DCM solution with a volume ratio of 1:1, pre-freeze to -5 to 5°C, add 26.40 ml (165.0 mmol, 3.3 eq) of DIC and activate for 5 min.

[0105] After the resin washing was completed, the reaction was carried out under mechanical stirring and nitrogen protection at a reaction temperature of 25°C for 2.0 h. The reaction solution was then removed, and the resin was washed 6 times with DMF (1.3 L / wash). A small sample was taken and tested with Kaiser reagent, and the resin was colorless.

[0106] Following a similar coupling method to the 32-position His, amino acids are sequentially linked according to the teriparatide peptide sequence: Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(pbf)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(Boc)-OH, Fmoc-Val-OH, Fmoc-Arg(pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmo c-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-His (Trt)-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Ser(tBu)-OH. After the peptide resin synthesis was completed, the 1-position Ser amino protecting group Fmoc was removed. The resin was washed 4 times with DMF, 2 times with DCM, and 3 times with methanol, 1.3 L / wash. The resin was then vacuum dried at 25°C for 8-12 h. 471.1 g of PTH peptide resin was obtained by weighing.

[0107] Example 5: Preparation of crude teriparatide peptide

[0108] 142.85 g of Fmoc-Phe-Wang Resin with an initial Substrate concentration of 0.35 mmol / g was weighed and placed in a solid-phase synthesis vessel. Fmoc was deprotected twice with 20% piperidine / DMF, 1.3 L each time, for 5 min the first time and 10 min the second time. After deprotection, the resin was washed 6 times with DMF, 1.3 L each time. A small sample was tested with Kaiser's reagent; the resin turned deep blue.

[0109] Weigh 89.55 g (150 mmol, 3.0 eq) of Fmoc-Asn(Trt)-OH and 22.28 g (165.0 mmol, 3.0 eq) of HOBt, dissolve them in 600.0 ml of DMF / DCM solution (1:1 volume ratio), pre-freeze to -5 to 5°C, add 26.40 ml (165.0 mmol, 3.3 eq) of DIC, and activate for 5 min.

[0110] After the resin washing was completed, the reaction was carried out under mechanical stirring and nitrogen protection at a reaction temperature of 25°C for 2.0 h. The reaction solution was then removed, and the resin was washed 6 times with DMF (1.7 L each time). A small sample was taken and tested with Kaiser reagent, and the resin was colorless.

[0111] Following a similar coupling method to the 33rd Asn, amino acids are sequentially linked according to the teriparatide peptide sequence: Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(pbf)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(Boc)-OH, Fmoc-Val-OH, Fmoc-Arg(pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Me t-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc -His(Trt)-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Ser(tBu)-OH. After the peptide resin synthesis was completed, the 1-position Ser amino protecting group Fmoc was removed. The resin was washed 4 times with DMF, 2 times with DCM, and 3 times with methanol, 1.7 L / wash. The resin was then vacuum dried at 25°C for 8-12 h, and 565.1 g of PTH peptide resin was obtained by weighing.

[0112] Example 6: Preparation of crude teriparatide peptide

[0113] Weigh 200g of the teriparatide peptide resin prepared in Example 3 for later use.

[0114] Prepare 2000 ml of lysis reagent with a volume ratio of TFA:TIS:water:DTT = 92:2:5:1, and pre-freeze to 0±5℃ for later use.

[0115] Under nitrogen protection and stirring, peptide resin was added to the lysis agent and stirred at 25°C for 2.0 h. The reaction was then stopped, and the mixture was filtered. The resin was washed twice with TFA (200 ml each time). The filtrates were combined and slowly poured into 12 L of pre-cooled diethyl ether. The mixture was stirred and allowed to stand at room temperature for 0.5–1.0 h. The ether precipitate was centrifuged at 3000 rpm for 3 min, and washed / centrifuged three times with diethyl ether. The resulting solid was dried to obtain 88.6 g, with a yield of 104.85%. Analysis of the crude peptide sample showed a purity of 76.3%, and no dipeptide deletion peptide due to the 34,33 diazinonone mechanism was detected.

[0116] Example 7: Preparation of crude teriparatide peptide

[0117] Weigh 200g of the teriparatide peptide resin prepared in Example 4 for later use.

[0118] Prepare 2000 ml of lysis reagent with a volume ratio of TFA:TIS:water:DTT = 92:2:5:1, and pre-freeze to 0±5℃ for later use.

[0119] Under nitrogen protection and stirring, peptide resin was added to the lysis agent and stirred at 25°C for 2.0 h. The reaction was then stopped, and the mixture was filtered. The resin was washed twice with TFA (200 ml each time). The filtrates were combined and slowly poured into 12 L of pre-cooled diethyl ether. The mixture was stirred and allowed to stand at room temperature for 0.5–1.0 h. The ether precipitate was centrifuged at 3000 rpm for 3 min each time, and washed / centrifuged three times with diethyl ether. The resulting solid was dried to obtain 91.3 g, with a yield of 104.94%. Analysis of the crude peptide sample showed a purity of 77.1%, and no dipeptide deletion peptide caused by the 34,33 diazinonone mechanism was detected.

[0120] Example 8: Preparation of crude teriparatide peptide

[0121] Weigh 200g of the teriparatide peptide resin prepared in Example 5 for later use.

[0122] Prepare 2000 ml of lysis reagent with a volume ratio of TFA:TIS:water:DTT = 92:2:5:1, and pre-freeze to 0±5℃ for later use.

[0123] Under nitrogen protection and stirring, peptide resin was added to the lysis agent and stirred at 25°C for 2.0 h. The reaction was then stopped, and the mixture was filtered. The resin was washed twice with TFA (200 ml each time). The filtrates were combined and slowly poured into 12 L of pre-cooled diethyl ether. The mixture was stirred and allowed to stand at room temperature for 0.5–1.0 h. The ether precipitate was centrifuged at 3000 rpm for 3 min, and washed / centrifuged three times with diethyl ether. The resulting solid was dried to obtain 77.01 g, with a yield of 88.52%. Analysis of the crude peptide sample showed a purity of 62.31%, with 12.55% of the peptides being dipeptide deletions due to the diazinoperone mechanism at positions 34 and 33.

[0124] Example 9: Purification of crude teriparatide peptide

[0125] Weigh 30.0g of the crude peptide obtained in Example 6, dissolve it in 3000ml of 5% acetic acid / water, and filter it through a 0.45um filter membrane.

[0126] C with an inner diameter of 150mm 18 The preparative column was used with mobile phase A being 0.1% TFA / water and mobile phase B being 0.1% TFA / acetonitrile at a flow rate of 600 mL / min and a detection wavelength of 210 nm. Gradient elution was performed, and fractions were collected based on the chromatographic peak times. Fractions with a purity of 97.0% were considered pure and qualified.

[0127] A pure, qualified fraction in a C with an inner diameter of 150 mm 18 On the preparative column, 0.1% acetic acid / water was used as phase A and 0.1% acetic acid / acetonitrile was used as phase B. The flow rate was 600 ml / min and the detection wavelength was 210 nm. Gradient elution was used for two-step purification. The fraction at the top of the preparative peak was collected. The purity of the intermediate control analysis was >99%. The fraction with less than 0.1% of single impurities was the qualified fraction of the two-purity product.

[0128] The two qualified fractions were subjected to rotary evaporation, nanofiltration, and freeze-drying to obtain 12.61 g of teriparatide peptide, with a total yield of 44.1%. Related substances in the peptide were analyzed by reversed-phase UPLC, and the purity was 99.6%. Neither the racemic Phe impurity at position 34 nor the racemic Asn impurity at position 33 was detected.

[0129] Example 10: Purification of crude teriparatide peptide

[0130] Weigh 30.0g of the crude peptide obtained in Example 7, dissolve it in 3000ml of 5% acetic acid / water, and filter it through a 0.45um filter membrane.

[0131] C with an inner diameter of 150mm 18 The preparative column was used with mobile phase A being 0.1% TFA / water and mobile phase B being 0.1% TFA / acetonitrile at a flow rate of 600 mL / min and a detection wavelength of 210 nm. Gradient elution was performed, and fractions were collected based on the chromatographic peak times. Fractions with a purity of 97.0% were considered pure and qualified.

[0132] A pure, qualified fraction in a C with an inner diameter of 150 mm 18 On the preparative column, 0.1% acetic acid / water was used as phase A and 0.1% acetic acid / acetonitrile was used as phase B. The flow rate was 600 ml / min and the detection wavelength was 210 nm. Gradient elution was used for two-step purification. The fraction at the top of the preparative peak was collected. The purity of the intermediate control analysis was >99%. The fraction with less than 0.1% of single impurities was the qualified fraction of the two-purity product.

[0133] The two qualified fractions were subjected to rotary evaporation, nanofiltration, and freeze-drying to obtain 12.96 g of teriparatide peptide, with a total yield of 43.9%. Related substances in the peptide were analyzed by reversed-phase UPLC, and the purity was 99.6%. Neither the racemic Phe impurity at position 34 nor the racemic Asn impurity at position 33 was detected.

[0134] Example 11: Purification of crude teriparatide peptide

[0135] Weigh 30.0g of the crude peptide obtained in Example 8, dissolve it in 3000ml of 5% acetic acid / water, and filter it through a 0.45um filter membrane.

[0136] C with an inner diameter of 150mm 18 The preparative column was used with mobile phase A being 0.1% TFA / water and mobile phase B being 0.1% TFA / acetonitrile at a flow rate of 600 mL / min and a detection wavelength of 210 nm. Gradient elution was performed, and fractions were collected based on the chromatographic peak times. Fractions with a purity of 97.0% were considered pure and qualified.

[0137] A pure, qualified fraction in a C with an inner diameter of 150 mm 18 On the preparative column, 0.1% acetic acid / water was used as phase A and 0.1% acetic acid / acetonitrile was used as phase B. The flow rate was 600 ml / min and the detection wavelength was 210 nm. Gradient elution was used for two-step purification. The fraction at the top of the preparative peak was collected. The fraction with a purity >99% was considered a qualified distillate.

[0138] The qualified fraction was subjected to rotary evaporation, nanofiltration, and freeze-drying to obtain 8.30 g of teriparatide peptide, with a total yield of 28.12%. Related substances in the peptide were determined by reversed-phase UPLC, showing a purity of 99.25%, with 0.11% of racemic Phe impurities at position 34 and 0.08% of racemic Asn impurities at position 33.

[0139] In summary, the method of the present invention successfully avoids racemization of Phe at position 34 and Asn at position 33, and also avoids the generation of dipeptide-deficient peptides. This results in the preparation of teriparatide products free from difficult-to-separate racemic impurities, significantly improving the yield and making the product easy to purify. It is very suitable for the industrial production of teriparatide.

[0140] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A process for the preparation of teriparatide, characterized in that, Includes the following steps: (a) providing Fmoc-Asp (aminolinked resin) -Phe-OtBu: , Furthermore, the amino resin in Fmoc-Asp (amino resin)-Phe-OtBu is Rink Amide AM Resin or Rink Amide MBHA Resin; (b) In the presence of a coupling agent, Fmoc-Asp (amino resin)-Phe-OtBu is sequentially coupled with the amino acid precursor at positions 32-1 of the teriparatide peptide sequence to obtain a side-chain protected teriparatide peptide resin; and (c) The teriparatide peptide resin is cleaved in the presence of a cleavage reagent to obtain crude teriparatide. In step (b), the amino acid precursors of the teriparatide peptide at positions 32-1 are, in sequence, Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(pbf)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, and Fmoc-Glu(Boc)-OH. )-OH, Fmoc-Val-OH, Fmoc-Arg(pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fm oc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-O The side-chain protected teriparatide peptide resin structures obtained from H, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, and Fmoc-Ser(tBu)-OH are as follows: Fmoc-Ser(tBu)-Val-Ser(tBu)-Glu(OtBu)-Ile-Gln(Trt)-Leu-Met-His (Trt)-Asn(Trt)-Leu-Gly-Lys(Boc)-His(Trt)-Leu-Asn(Trt)-Ser(tBu)-Met-Glu(OtBu)-Arg(pbf)-Val-Glu(OtBu)- Trp(Boc)-Leu-Arg(pbf)-Lys(Boc)-Lys(Boc)-Leu-Gln(Trt)-Asp(OtBu)-Val-His(Trt)-Asp(AmideResin)-Phe-OtBu; Before step (a), there is also a step: (al) in the presence of a coupling agent, reacting with an amino resin, thereby obtaining said Fmoc-Asp (amino resin) -Phe-OtBu.

2. The production method according to claim 1, characterized by, In step (a), the amino resin in Fmoc-Asp (amino resin)-Phe-OtBu is Rink Amide AM Resin.

3. The preparation method according to claim 1, characterized in that, In step (b), the coupling agents for each coupling reaction are independently selected from the group consisting of: HOBt / DIC, HOAt / DIC, HOBt / HBTU / DIEA, PyBOP / DIEA, TBTU / DIEA, or combinations thereof.

4. The production method according to claim 1, characterized by, In step (c), the cleavage reagent is a TFA solution containing 5-10% scavenging agent by volume, wherein the scavenging agent is selected from the group consisting of dithiothreitol, anisole, phenol, mercaptoethanol, water, and triisopropylsilane, or combinations thereof.

5. The method of claim 1, wherein, The method further includes a purification step to obtain a high-quality teriparatide; the purification step may include steps selected from the group consisting of reversed-phase chromatography, nanofiltration, lyophilization, or a combination thereof.

6. The use of Fmoc-Asp (amino resin)-Phe-OtBu in the preparation of teriparatide. ， Furthermore, the amino resin in Fmoc-Asp (amino resin)-Phe-OtBu is Rink Amide AM Resin or Rink Amide MBHA Resin.

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