Synthesis of teduglutide

Abstract

The present invention provides a novel synthetic approach for the solid phase synthesis of peptides with a C-terminal aspartic acid, which avoids the formation of various impurities by anchoring the side chain carboxylic acid group of the aspartic acid to the solid support, thereby resulting in high yields and facilitating the purification process. The present invention further provides the use of free amino acids as antioxidants and reducing agents in the cleavage cocktail to counteract the formation of oxidative impurities formed during the overall cleavage and isolation of the peptide from the solid support.

Description

Technical Field

[0001] Related applications: This application claims the benefit of priority of Indian Patent Application IN202141000174, filed on January 4, 2021, which is incorporated herein by reference.

[0002] Technical field The present invention relates to an efficient solid-phase synthesis of teduglutide represented by Formula-I.

Chemical Formula

Background Art

[0003] background GATTEX® (teduglutide) is a glucagon-like peptide 2 (GLP-2) analog indicated for the treatment of adult patients and pediatric patients 1 year of age and older with short bowel syndrome (SBS) who are dependent on parenteral support. The active ingredient of injectable GATTEX® (teduglutide) is teduglutide, a 33-amino acid glucagon-like peptide 2 (GLP-2) analog produced using recombinant DNA technology-modified Escherichia coli strains.

[0004] The chemical composition of teduglutide is L-histidyl-L-glycyl-L-aspartyl-L-glycyl-L-seryl-L-phenylalanyl-L-seryl-L-aspartyl-L-glutamyl-L-methionyl-L-asparaginyl-. L-threonyl-L-isoleucyl-L-leucyl-L-aspartyl-L-asparaginyl-L-leucyl-L-alanyl-L-alanyl-L-arginyl-L-aspartyl-L-phenylalanyl-L-isoleucyl-L-asparaginyl-L-tryptophanyl-L-leucyl-L-isoleucyl-L-glutaminyl-L-threonyl-L-lysyl-L-isoleucyl-L-threonyl-L-aspartic acid. The molecular weight of teduglutide is 3752 daltons. The teduglutide active pharmaceutical ingredient is a colorless to pale straw-colored liquid.

[0005] U.S. Patent No. 5,789,379B1 discloses teduglutide and a method for producing the same. Many prior art publications teach the fragment phase synthesis of teduglutides. WO2012028602A1 discloses a fragment phase synthesis of teduglutide, in which amino acid fragments are first synthesized at positions 1-4 and 5-33, and then coupled to obtain teduglutide.

[0006] CN104072605A discloses three fragment-phase synthesis methods for teduglutide. The first method involves first synthesizing amino acid fragments at positions 1-9, 10-18, and 19-33, and then combining these fragments to obtain teduglutide. The second method involves first synthesizing amino acid fragments at positions 1-4, 5-12, 13-20, and 21-33, and then coupling these fragments to obtain teduglutide. The third method involves first synthesizing amino acid fragments at positions 1-4, 5-9, 10-18, 19-26, and 27-33, and then coupling these fragments to obtain teduglutide. CN104418949A discloses a method for preparing teduglutide, which involves first synthesizing amino acid fragments at positions 1-3 and 4-33, and then coupling them to obtain teduglutide.

[0007] CN104817638 discloses a method for synthesizing fragments 1-2, 3-4, and 5-33 and conjugating them to obtain teduglutide. CN104072603 discloses the synthesis of teduglutide by coupling the His residue with fragments 2-33. [Overview of the project] [Problems that the invention aims to solve]

[0008] The synthesis method described in the above patent involves first synthesizing fragments and then linking them to synthesize teduglutide. These methods require the synthesis of two or more polypeptide fragments to be linked. This approach requires linking the fragments one by one on a solid support, after which the complete protection is cleaved, and then the fragments are linked one by one. The biggest problem is that when the fragments undergo complete protected cleavage, some side chain protecting groups are lost, and impurities from the fragments may be introduced during subsequent liquid-phase linking. At the same time, if the purity of the completely protected fragments is low, these fragments may need to be purified, further complicating the process. Secondly, in the fragment condensation process, multiple amounts of fragments are usually required to ensure the reaction completes, resulting in a large amount of material waste. Furthermore, racemization of amino acids is often unavoidable in the fragment ligation process, which also presents a significant challenge for subsequent purification. Advantages of the present invention / specification:

[0009] Peptide synthesis can be achieved by a standard solid-phase approach using the Fmoc- / tBu strategy. However, several side reactions occur during synthesis, affecting the yield and quality of the final product.

[0010] This invention provides a novel synthetic approach for solid-phase synthesis of peptides having C-terminal aspartic acid, which avoids the formation of various impurities by immobilizing the side-chain carboxylic acid group of aspartic acid on a solid support, thereby increasing yield and facilitating the purification process. One of the major impurities is the formation of diketopiperazine (DKP) impurities. DKP is generated by one of the side reactions in solid-phase peptide synthesis that results in deletion sequences. Intermolecular cyclization occurs when the Fmoc group is deprotected from the N-terminus of the dipeptide bound to the Wang resin.

[0011] The present invention is useful for the solid-phase synthesis of a wide range of peptides having a C-terminal aspartic acid. A notable example of this class of peptides is arginylglycylaspartate (RGD) peptides, which are found in many matrix proteins, including fibronectin, fibrinogen, vitronectin, and osteopontin. Eptifibatide is another example. [Means for solving the problem]

[0012] This invention / specification solves the above-mentioned problems in the production of teduglutide using a standard procedure, by immobilizing the first amino acid, aspartic acid, on the α-carboxyl group of Fmoc-Asp(OtBu)-OH via the side-chain carboxyl group (Fmoc-Asp-OtBu) to the Wang resin, thereby minimizing the formation of DKP impurities. When using Fmoc-Asp-OtBu, it is necessary to form a 7-membered ring, which is difficult, but when using Fmoc-Asp(OtBu)-OH, the formation of a kinetically stable 6-membered ring diketopiperazine is observed, which causes peptide leaching from the Wang resin and generates Thr-Asp clip impurities. Therefore, this aspect of the invention / specification provides a synthesis of teduglutide in which the formation of diketopiperazine impurities is avoided.

[0013] a) Role in fixing the side-chain carboxyl group of aspartic acid to the solid support: Diketopiperazine (DKP) impurities: One common hurdle is the formation of diketopiperazine (DKP) impurities. DKP is produced by one of the side reactions in solid-phase peptide synthesis that results in the deletion sequence. Intermolecular cyclization occurs when the Fmoc group is deprotected from the N-terminus of the dipeptide bound to the Wang resin. In the case of teduglutide, since the first two amino acids are aspartic acid and threonine, intermolecular cyclization causes the peptide to leach from the resin, and the third amino acid in the sequence binds to the Wang resin, forming a DesThr-Asp impurity.

[0014] [ka]

[0015] In the present invention / specification, for the α-carboxyl group of Fmoc-Asp(OtBu)-OH, the first amino acid, aspartic acid, is fixed to Wang resin via the side-chain carboxyl group (Fmoc-Asp-OtBu). By doing so, standard procedures are used to solve the above problems in the production method of teduglutide and minimize the formation of DKP impurities. When using Fmoc-Asp-OtBu, it is necessary to form a 7-membered ring, which is difficult. However, when using Fmoc-Asp(OtBu)-OH, the formation of a kinetically stable 6-membered ring diketopiperazine is observed, which causes the leaching of the peptide from Wang resin and the generation of Thr-Asp clipped impurities. Therefore, this aspect of the present invention / specification provides the synthesis of teduglutide in which the formation of diketopiperazine impurities is avoided.

[0016] [Table 1]

[0017] b) Role of free amino acids and antioxidants in the TFA cocktail for cleaving the peptide from the resin:

[0018] The presence of tryptophan and methionine in the sequence leads to oxidation during cleavage and isolation of the peptide from the resin, which results in the generation of impurities, a decrease in yield, and the addition of purification steps to remove these impurities, making the process uneconomical. One aspect of the present invention / specification solves the above problems by incorporating the corresponding free amino acids into the cleavage cocktail. The addition of free tryptophan and methionine amino acids has been found to be advantageous in minimizing oxidative impurities. The present invention / specification further includes the use of antioxidants selected from, for example, BHT in the cleavage cocktail. These additional scavengers share the oxidative burden with the peptide of interest and thus minimize oxidative impurities. The present invention is particularly useful in peptides containing tryptophan and methionine in sequences that are susceptible to oxidation during peptide cleavage and isolation. Non-limiting examples of the present invention are teduglutide, glucagon, and the like.

[0019]

Chemical formula

[0020]

Chemical formula

[0021] c) Advantages of using Boc-His-OH: Conventional methods utilize double protection for histidine, such as Fmoc-His(Trt)-OH, di-Boc group, Boc-His(Trt)-OH, and Boc-His(Bom)-OH. Coupling of diprotected His is slow due to steric hindrance and can lead to racemization. Furthermore, diprotected amino acid derivatives require additional purification by chromatography or crystallization to separate unwanted τ isomers, making large-scale production cost-inefficient. On the other hand, the unprotected imidazole group of His can cause peptide elongation via the π nitrogen of the imidazole ring, and significant racemization is possible if His is in the middle of the sequence. Since His is the last amino acid in the teduglutide sequence, diprotection of His is not essential. This avoids the use of expensive diprotected amino acids. Moreover, the coupling conditions currently in use largely avoid racemization. Therefore, the use of histidine amino acids with free side chains does not cause problems during the teduglutide production process.

[0022] d) Advantages of using γ-valerolactone as the solvent and COMU / HCTU / T3P as the coupling agent: In peptide synthesis, the choice of coupling reagent varies based on the sequence and the incoming amino acids. COMU / HCTU / T3P are more advanced coupling reagents used during coupling. COMU, in particular, is a more efficient reagent, reducing racemization compared to HATU and HBTU, and possessing a lower safety profile. On the other hand, HCTU and T3P are efficient and cost-effective. In this invention / specification, when COMU / HCTU / T3P is used as a coupling agent in combination with γ-valerolactone as a solvent, the reagent is found to be stable, and these conditions suppressed racemization. The above combination is also environmentally friendly.

[0023] e) Advantages of using ACN and MIBK during peptide isolation after total cleavage: After overall cleavage, the TFA cocktail containing the peptide was concentrated, and the crude peptide was precipitated using ethers such as MTBE, diethyl ether, and diisopropyl ether as poor solvents. When ethers are used as poor solvents for peptide precipitation, the assay rate of the crude peptide decreases to 20-25%. This is because side-chain protecting groups and scavenger adducts precipitate with the target peptide. The assay rate of the crude peptide is increased by precipitation of the crude peptide with ACN or MIBK. The adducts remain in the mother liquor and are removed during filtration. Furthermore, teduglutide is susceptible to oxidation due to the presence of amino acids such as methionine, tryptophan, and histidine in its sequence. Ethers contain trace amounts of peroxides, which are known to lead to an increase in oxidative impurities. The use of acetonitrile significantly contributed to the reduction of oxidative impurities. A comparison of crude peptide purity and assays is shown in the table below.

[0024] [Table 2]

[0025] f) Role of formic acid and DBU in Fmoc deprotection using piperidine: Aspartimide impurities: One of the main side reactions involved in teduglutide synthesis is the formation of multiple aspartimide impurities due to the presence of five aspartic acid units in the sequence. Aspartimide impurities are generated at each stage of deprotection, as piperidine and other strong bases are used.

[0026] [ka]

[0027] One aspect of the present invention / specification solves the above problem in a method for producing teduglutide, by introducing a formic acid solution together with a piperidine solution during Fmoc deprotection, thereby minimizing the formation of aspartamide impurities. Since piperidine abstracts a proton from formic acid to form a piperidinium ion, the formation of aspartamide is minimized without affecting Fmoc deprotection. [ka]

[0028] Advantages of using DBU: At certain stages of the process, shrinkage of the resin was observed during Fmoc deprotection due to the presence of hydrophobic amino acids. This resulted in incomplete deprotection of the Fmoc group, which in turn contributed to the formation of missing impurities. To overcome the problem of incomplete deprotection of the Fmoc group, 1-3% DBU was incorporated into the reagent containing formic acid and piperidine. With the incorporation of DBU, efficient swelling of the resin was observed, and the Fmoc group was completely deprotected.

[0029] the purpose The object of the present invention / specification is to develop a simple, robust, and commercially viable process for preparing teduglutide represented by formula I, with the help of inorganic salts as needed. [Brief explanation of the drawing]

[0030] [Figure 1] Figure 1 shows the HPLC chromatogram of teduglutide.

[0031] Summary of the present invention One aspect of the present invention is, a) The first amino acid, aspartic acid, is immobilized on the Wang resin via the carboxyl group of its side chain. b) A step of preparing teduglutide by sequentially coupling side-chain protected amino acids in the presence of a coupling agent and optionally in the presence of one or more chaotropic salts, c) Crude teduglutide is obtained by removing the protecting group and cleaving the peptide from the resin. d) Optional step of purifying crude teduglutide. A process for preparing teduglutide containing is disclosed.

[0032] In one aspect, the present invention e) The first amino acid, aspartic acid, is immobilized on the Wang resin via the carboxyl group of its side chain. g) A step of preparing teduglutide by sequentially coupling side-chain protected amino acids in the presence of a coupling agent and optionally in the presence of one or more chaotropic salts, g) Crude teduglutide is obtained by removing the protecting group and cleaving the peptide from the resin. h) Optional step of purifying crude teduglutide. A process for preparing teduglutide containing is disclosed.

[0033] In another aspect, the present invention a) The first amino acid, aspartic acid, is immobilized on Wang resin via the carboxyl group of its side chain (Fmoc-Asp-OtBu). b) Prepare teduglutide by sequentially coupling side-chain protected amino acids in the presence of a coupling agent and optionally a chaotropic salt. c) Use of piperidine:formic acid:DBU mixture for deprotection of the Fmoc group, d) Crude teduglutide is obtained by removing the protecting group and cleaving the peptide from the resin. e) Optionally, purify the crude teduglutide. A process for the preparation of teduglutide, including the steps involved, is disclosed.

[0034] In another aspect, the present invention a) The first amino acid, aspartic acid, is immobilized on the Wang resin via the carboxyl group of its side chain (Fmoc-Asp-OtBu). b) A step of preparing teduglutide by sequentially coupling side-chain protected amino acids in the presence of a coupling agent and one or more chaotropic salts, c) Crude teduglutide is obtained by cleaving the peptide from the resin using a protecting group removal and cleavage cocktail. d) Use free amino acids in the cleavage cocktail to reduce oxidative impurities. e) A step of purifying crude teduglutide, Includes, Here, we disclose a process for the preparation of teduglutide, in which the free amino acid is selected from methonine, tryptophan, and histidine.

[0035] In another aspect, the present invention is a) The first amino acid, aspartic acid, is immobilized on the Wang resin via the carboxyl group of its side chain (Fmoc-Asp-OtBu). b) Steps to prepare teduglutide by sequentially coupling side-chain protected amino acids in the presence of a coupling agent and one or more chaotropic salts, c) Use of piperidine:formic acid:DBU mixture for deprotection of the Fmoc group, d) Crude teduglutide is obtained by removing the protecting group and cleaving the peptide from the resin. e) Use of free amino acids in cleavage cocktails to reduce oxidative impurities, f) Purification of crude teduglutide This includes, where the free amino acid is selected from methonine, tryptophan, and histidine. A process for preparing teduglutide is disclosed.

[0036] In yet another aspect, the present invention discloses a method for cleaving teduglutide from a solid support using a cleavage solution comprising an antioxidant, an amino acid, and a TFA cocktail. The amino acid in the above embodiment of the present invention is selected from methonine, tryptophan, and histidine. The antioxidant according to the above embodiment of the present invention is selected from butylated hydroxytoluene (BHT). In yet another aspect, the present invention discloses a method for preparing teduglutide using monoprotected histidine.

[0037] The protecting group in the above embodiment of the present invention is tert-butyloxycarbonyl (Boc). The approach used is a continuous solid-phase peptide synthesis of teduglutide, in which an inorganic salt is involved along with a selective coupling agent and additives during the coupling process. This method completes the coupling and deprotection reactions, reduces racemization, controls isomer impurities very close to the target molecule, and facilitates the peptide purification process.

[0038] Detailed description of the present invention Detailed description of the invention The present invention relates to an efficient method for producing teduglutide by sequential coupling using a solid-phase approach. This method involves sequentially coupling protected amino acids to prepare teduglutide, subsequently removing the protecting group, cleaving the peptide from a solid support, and purifying the resulting crude teduglutide.

[0039] The present invention is illustrated by the following examples. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0040] [Table 3]

[0041] The process outline is as follows: [ka]

[0042] example: Example 1: Synthesis of teduglutide by immobilizing aspartic acid (the first amino acid) on Wang resin via the α-carboxyl group of Asp. The synthesis was carried out by loading the C-terminal amino acid Fmoc-Asp(OtBu)-OH into Wang resin using DIPC and catalytic amounts of DMAP in the presence of MDC as a solvent. Unreacted functional sites were capped using acetic anhydride and DIPEA. Fmoc deprotection was performed twice for 5+10 minutes using a 10-20% piperidine solution in DMF. Peptide elongation was performed by sequentially adding amino acids according to the teduglutide sequence. The resulting peptide-protected resin is as follows. Boc-His(Trt)-Gly-Asp(OtBu)-Gly-Ser(tBu)-Phe-Ser(tBu)-Asp(OtBu)-Glu(OtBu)-Met-Asn(Trt)-Thr(tBu)-Ile-Leu-Asp(OtBu)-Asn(Trt) -Leu-Ala-Ala-Arg(Pbf)-Asp(OtBu)-Phe-Ile-Asn(Trt)-Trp(Boc)-Leu-Ile-Gln(Trt)-Thr(tBu)-Lys(Boc)-Ile-Thr(tBu)-Asp(OtBu)-Wang resin Complete cleavage was performed using a TFA:TIS:phenol cocktail, and the peptide was isolated using ether. A crude peptide with a purity of 28.77% was obtained.

[0043] Example 2: Synthesis of teduglutide by immobilizing aspartic acid (the first amino acid) on Wang resin via the side-chain carboxyl group of Asp. Step 1: Fmoc-Asp-O t Bu 33 - Synthesis of Wang resin Wang resin (0.3-0.6 mmol / g, packed volume) was packed into a peptide synthesis vessel using a 10V MDC, drained, and 7V MDC was added, followed by swelling for 1 hour. The solvent was completely drained. Fmoc-Asp-OtBu (2.0-4.0 equivalents) was dissolved in MDC and transferred to the reaction vessel. DMAP (0.01-0.1 equivalents) was dissolved in MDC and added to the peptide synthesis vessel, followed by the addition of DIPC (4.0-8.0 equivalents). Esterification was carried out at room temperature for 1.0-3.0 hours. The reaction mass was drained, and the resin packed with amino acids was washed with MDC and then with DMF. Unreacted functional sites were capped using acetic anhydride and DIPEA.

[0044] Step 2: Fmoc deprotection of the packing amino acids was carried out by washing the resin with 5-15% piperidine in DMF for 5 and / or 10 minutes. The resin was washed with a DMF solution (2 × 7v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7v).

[0045] Fmoc-Thr(tBu)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, or TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0046] Step 3: Fmoc deprotection of the packing amino acids was performed by washing the resin with 5–15% piperidine in DMF for 5 and / or 10 minutes. The resin was washed with a DMF solution (2 × 7v) of 0.01–0.1 M HOBt.H2O, followed by DMF (5 × 7v). Fmoc-Ile-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0047] Step 4: Fmoc deprotection of the packing amino acids was carried out by washing the resin with 5-15% piperidine in DMF for 5 and / or 10 minutes. The resin was then washed with 0.01-0.1 M HOBt.H2O (2 × 7v) in DMF, followed by DMF (5 × 7v). Fmoc-Lys(Boc)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0048] Step 5: Fmoc deprotection of the packing amino acids was carried out by washing the resin with 5-15% piperidine in DMF for 5 and / or 10 minutes. The resin was washed with a DMF solution (2 × 7v) of 0.01-0.1 M HOBt.H2O, followed by DMF (5 × 7v).

[0049] Fmoc-Thr(tBu)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, or TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0050] Step 6: Fmoc deprotection of the packing amino acids was carried out by washing the resin with 5-20% piperidine in DMF for 5 and / or 10 minutes. The resin was washed with a DMF solution (2 × 7v) of 0.01-0.1 M HOBt.H2O, followed by DMF (5 × 7v).

[0051] Fmoc-Gln(Trt)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0052] Step 7: Fmoc deprotection of the packing amino acids was carried out by washing the resin with 5-20% piperidine in DMF for 5 and / or 10 minutes. The resin was washed with a DMF solution (2 × 7v) of 0.01-0.1 M HOBt.H2O, followed by DMF (5 × 7v).

[0053] Fmoc-Ile-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0054] Step 8: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or with a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or with 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0055] Fmoc-Leu-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0056] Step 9: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or with a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or with 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0057] Fmoc-Trp(Boc)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0058] Step 10: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0059] Fmoc-Asn(Trt)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0060] Step 11: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0061] Fmoc-Ile-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0062] Step 12: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or with a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or with 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0063] Fmoc-Phe-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clump was discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0064] Step 13: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or with a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or with 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7v).

[0065] Fmoc-Asp(OtBu)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, or TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0066] Step 14: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt, H2O, followed by DMF (5 × 7 v).

[0067] Fmoc-Arg(Pbf)-OH (2.0 to 4.0 equivalents) was coupled using a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2 to 4.0 equivalents), and additives such as Oxyma Pure and HOBt·H2O, preferably HOBt·H2O (2.0 to 4.0 equivalents), using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, preferably MgCl2 (0.05 to 0.5 equivalents), in the presence of DMF and / or NMP and / or MDC as a solvent. The coupling reaction was carried out at 25 to 40 °C for 1 to 2 hours. The reaction mass was discharged and washed three times with DMF and / or NMP and / or MDC. Completion of the coupling was monitored by HPLC and / or Kaiser test.

[0068] Step 15: Fmoc deprotection of the loaded amino acid was carried out by washing with 5 to 20% piperidine in DMF, or a DMF solution of 0.1 M to 1 M formic acid and 5 to 20% piperidine, or 0.1 M to 1 M formic acid, 1 to 2% DBU, and 5 to 20% piperidine in DMF for 5 minutes and / or for 10 minutes. The resin was washed with a DMF solution of 0.01 to 0.1 M HOBt·H2O (2 × 7v), followed by DMF (5 × 7v).

[0069] Fmoc-Ala-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, or TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0070] Step 16: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or with a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or with 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7v).

[0071] Fmoc-Ala-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, or TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0072] Step 17: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or with a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or with 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0073] Fmoc-Leu-OH (2.0-4.0 equivalents) was coupled with a base such as DIPEA, NMM, or TMP, preferably DIPEA and a chaotropic salt, using a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and additives such as Oximapure and HOBt·H2O, preferably HOBt·H2O (2.0-4.0 equivalents). The coupling reaction was carried out in the presence of DMF and / or NMP and / or MDC as the solvent, with MgCl2, ZnCl2, CuCl2, preferably MgCl2 (0.05-0.5 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clump was discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0074] Step 18: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0075] Fmoc-Asn(Trt)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0076] Step 19: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0077] Fmoc-Asp(OtBu)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, or TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0078] Step 20: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0079] Fmoc-Leu-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0080] Step 21: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0081] Fmoc-Ile-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0082] Step 22: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or with a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or with 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0083] Fmoc-Thr( tBu)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0084] Step 23: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or with a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or with 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0085] Fmoc-Asn(Trt)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0086] Step 24: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0087] Fmoc-Met-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, or TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0088] Step 25: Fmoc deprotection of the packing amino acids was carried out by washing the resin with 5-20% piperidine in DMF or 0.1-1M formic acid, 1-3% DBU, and 5-20% piperidine in DMF for 5 and / or 10 minutes. The resin was washed with a DMF solution (2 × 7v) of 0.01-0.1M HOBt, H2O, followed by DMF (5 × 7v).

[0089] Fmoc-Glu(OtBu)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0090] Step 26: Fmoc deprotection of the packing amino acids was carried out by washing the resin with 5-20% piperidine in DMF or 0.1M-1M formic acid, 1-3% DBU, and 5-20% piperidine in DMF for 5 and / or 10 minutes. The resin was washed with 0.01-0.1M HOBt, DMF solution of H2O (2×7v), followed by DMF (5×7v).

[0091] Fmoc-Asp(OtBu)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, or TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0092] Step 27: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or with a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or with 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0093] Fmoc-Ser(tBu)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0094] Step 28: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0095] Fmoc-Phe-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clump was discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0096] Step 29: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0097] Fmoc-Ser(tBu)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0098] Step 30: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0099] Fmoc-Gly-OH (2.0-4.0 equivalents) was coupled with a base such as DIPEA, NMM, or TMP, preferably DIPEA and a chaotropic salt, using a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and additives such as Oximapure and HOBt·H2O, preferably HOBt·H2O (2.0-4.0 equivalents). The coupling reaction was carried out in the presence of DMF and / or NMP and / or MDC as the solvent, with MgCl2, ZnCl2, CuCl2, preferably MgCl2 (0.05-0.5 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clump was discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0100] Step 31: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or with a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or with 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0101] Fmoc-Asp(OtBu)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, or TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0102] Step 32: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0103] Fmoc-Gly-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, or TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0104] Step 33: Fmoc deprotection of the packing amino acids was carried out by washing for 5 and / or 10 minutes with 5-20% piperidine in DMF, or with a DMF solution of 0.1 M-1 M formic acid and 5-20% piperidine, or with 0.1 M-1 M formic acid, 1-2% DBU, and 5-20% piperidine in DMF. The resin was washed with a DMF solution (2 × 7 v) of 0.01 to 0.1 M HOBt.H2O, followed by DMF (5 × 7 v).

[0105] Boc-His(Trt)-OH (2.0-4.0 equivalents) was coupled in the presence of DMF and / or NMP and / or MDC as a solvent, using a base such as DIPEA, NMM, TMP, preferably DIPEA, and a chaotropic salt such as MgCl2, ZnCl2, CuCl2, or CuCl2, preferably MgCl2 (0.05-0.5 equivalents), with a coupling agent such as HBTU, COMU, DEPBT, and DIC, preferably HBTU (2-4.0 equivalents) and an additive such as Oximapure and HOBt.H2O, preferably HOBt.H2O (2.0-4.0 equivalents). The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction clumps were discharged and washed three times with DMF and / or NMP and / or MDC. Completion of coupling was monitored by HPLC and / or Kaiser test.

[0106] The peptidyl resin obtained after sequentially adding amino acids according to the sequence was washed twice with DMF, MDC, methanol, and MTBE. The resin was vacuum-dried in a VTD and completely cleaved using TFA:TIS:phenol in a ratio of 80:10:10. Cleavage was carried out under a nitrogen atmosphere at 20-30°C for 3-4 hours. The reaction mass was filtered, and the filtrate containing the peptide and TFA cocktail was concentrated. The peptide was isolated using a solvent selected from MTBE, diisopropyl ether, MIBK, ACN, and mixtures thereof. The precipitated solid was centrifuged and / or filtered, washed with a solvent selected from MTBE, diisopropyl ether, MIBK, ACN, and any mixture thereof, vacuum-dried in a VTD, and purified by RP-HPLC. Crude purity: 39.99%.

[0107] Example 3: Synthesis of crude peptides The process remains the same as described in Example 2 up to step 32. For the final amino acid, i.e., His, coupling was carried out using Boc-His-OH (2.0-4.0 equivalents) and a coupling agent such as HCTU, COMU, DEPBT, T3P, and DIC, preferably COMU (2-4.0 equivalents) and DMAP (0.1-0.5 equivalents), with a base such as DIPEA, NMM, TMP, preferably DIPEA. Chaotropic salts such as MgCl2, ZnCl2, CuCl2, preferably MgCl2 (0.05-0.5 equivalents) were used in the presence of a solvent selected from γ-valerolactone (GVL), DMF, NMP, MDC, and any mixture thereof. The coupling reaction was carried out at 25-40°C for 1-2 hours. The reaction mass was discharged and washed three times with DMF and / or NMP and / or MDC.

[0108] The peptidyl resin obtained after sequentially adding amino acids according to the sequence was washed twice with DMF, MDC, methanol, and MTBE. The resin was dried under vacuum in a VTD and completely cleaved using TFA:TIS:phenol:NH4I:DMS in a ratio of 80:7.5:7.5:5:5. Cleavage was carried out under a nitrogen atmosphere at 20-30°C for 3-4 hours. The reaction mass was filtered, and the filtrate containing the peptide and TFA cocktail was concentrated. The peptide was isolated using a solvent selected from MTBE, diisopropyl ether, MIBK, ACN, and any mixture thereof. The precipitated solid was centrifuged and / or filtered, washed with a solvent selected from MTBE, diisopropyl ether, MIBK, ACN, and any mixture thereof, vacuum-dried in a VTD, and collected for purification by RP-HPLC. Crude purity: 46.69%

[0109] Example 4: Cutting using TFA: TIS:Phenol:NH4I:DMS (80:7.5:7.5:5:5) The process is the same as described in Example 2 up to step 32. Coupling of the 33rd amino acid, i.e., His, was carried out as in Example 3. Dried peptidyl resin (5 g) was taken and treated with a cocktail containing TFA:TIS:phenol:NH4I:DMS in a ratio of 80:7.5:7.5:5:5 at 10-15 v. Cutting was carried out under a nitrogen atmosphere at 20-30°C for 3-4 hours. The reaction mass was filtered, and the filtrate containing the peptide and TFA cocktail was concentrated. The peptide was isolated using a solvent selected from MTBE, diisopropyl ether, MIBK, ACN, and any mixture thereof. The precipitated solid was centrifuged and / or filtered, washed with a solvent selected from MTBE, diisopropyl ether, MIBK, ACN, and any mixture thereof, vacuum dried on VTD, and purified by RP-HPLC. Crude purity: 47.7%, API content: 0.64 g

[0110] Example 5: Cutting using TFA: TIS: Phenol: NH4I: DMS: Met (75:5:5:5:5:5) The process was the same as described in Example 2. Coupling of the 33rd amino acid, i.e., His, was carried out as in Example 3. 5 g of dried peptidyl resin was taken and treated with a cocktail containing TFA:TIS:phenol:NH4I:DMS:Met in a ratio of 75:5:5:5:5:5 at 10-15V. Cutting was carried out under a nitrogen atmosphere at 20-30°C for 3-4 hours. The reaction mass was filtered, and the filtrate containing the peptide and TFA cocktail was concentrated. The peptide was isolated using a solvent selected from MTBE, diisopropyl ether, MIBK, ACN, and any mixture thereof. The precipitated solid was centrifuged and / or filtered, washed with a solvent selected from MTBE, diisopropyl ether, MIBK, ACN, and any mixture thereof, vacuum-dried on a VTD, and purified by RP-HPLC. Crude purity: 48.94%, API content: 0.74 g

[0111] Example 6: Cutting using TFA: TIS:Phenol:NH4I:DMS:Trp(75:5:5:5:5:5) The process was the same as described in Example 2. The bonding of the 33rd amino acid, i.e., His, was carried out as in Example 3. A dried peptidyl resin (5 g) was taken and treated with a cocktail containing TFA:TIS:phenol:NH4I:DMS:Trp in a ratio of 75:5:5:5:5:5 at 10-15 v. Cutting was carried out under a nitrogen atmosphere at 20-30°C for 3-4 hours. The reaction mass was filtered, and the filtrate containing the peptide and TFA cocktail was concentrated. The peptide was isolated using a solvent selected from MTBE, diisopropyl ether, MIBK, ACN, and any mixture thereof. The precipitated solid was centrifuged and / or filtered, washed with a solvent selected from MTBE, diisopropyl ether, MIBK, ACN, and any mixture thereof, vacuum dried on VTD, and purified by RP-HPLC. Crude purity: 43.85%, API content: 0.76 g

[0112] Example 7: Cutting using TFA: TIS: Phenol: NH4I: DMS: Trp: Met (75:5:5:5:5:2.5:2.5) The process is the same as described in Example 2. Coupling of the 33rd amino acid, i.e., His, was performed as in Example 3. Dried peptidyl resin (5 g) was taken and treated with a cocktail containing TFA:TIS:phenol:NH4I:DMS:Trp:Met in a ratio of 75:5:5:5:5:2.5:2.5 at 10-15 v. Cutting was carried out under a nitrogen atmosphere at 20-30°C for 3-4 hours. The reaction mass was filtered, and the filtrate containing the peptide and TFA cocktail was concentrated. The peptide was isolated using a solvent selected from MTBE, diisopropyl ether, MIBK, ACN, and any mixture thereof. The precipitated solid was centrifuged and / or filtered, washed with a solvent selected from MTBE, diisopropyl ether, MIBK, ACN, and any mixture thereof, vacuum dried on VTD, and purified by RP-HPLC. Crude purity: 46.06%, API content: 0.75 g

[0113] Example 8: Cutting using TFA: TIS: Phenol: NH4I: DMS: Trp: Met: Water (75:4:5:5:5:2.5:2.5:1) The process was the same as described in Example 2. Coupling of the 33rd amino acid, i.e., His, was carried out as in Example 3. Dried peptidyl resin (5 g) was taken and treated with 10-15 volumes of a cocktail containing TFA:TIS:phenol:NH4I:DMS:Trp:Met:water in a ratio of 75:4:5:5:5:2.5:2.5:1. Cutting was carried out under a nitrogen atmosphere at 20-30°C for 3-4 hours. The reaction mass was filtered, and the filtrate containing the peptide and TFA cocktail was concentrated. The peptide was isolated using a solvent selected from MTBE, diisopropyl ether, MIBK, ACN, and any mixture thereof. The precipitated solid was centrifuged and / or filtered, washed with a solvent selected from MTBE, diisopropyl ether, MIBK, ACN, and any mixture thereof, vacuum dried on VTD, and purified by RP-HPLC. Crude purity: 48.09%, API content: 0.72 g.

[0114] Example 9: Cutting using TFA: TIS: Phenol: NH4I: DMS: Trp: Met: BHT (74: 7.5: 7.5: 2.5: 2.5: 2.5: 2.5: 1) The process was the same as described in Example 2. Coupling of the 33rd amino acid, i.e., His, was carried out as in Example 3. Dried peptidyl resin (200 g) was taken and treated with a cocktail containing TFA:TIS:phenol:NH4I:DMS:Trp:Met:BHT in a ratio of 74:7.5:7.5:2.5:2.5:2.5:2.5:1 at 10-15 v. Cutting was carried out under a nitrogen atmosphere at 20-30°C for 3-4 hours. The reaction mass was filtered, and the filtrate containing the peptide and TFA cocktail was concentrated. The peptide was isolated using a solvent selected from MTBE, diisopropyl ether, MIBK, ACN, and any mixture thereof. The precipitated solid was centrifuged and / or filtered, washed with a solvent selected from MTBE, diisopropyl ether, MIBK, ACN, and any mixture thereof, vacuum dried on VTD, and purified by RP-HPLC. Crude purity: 52.54%, API content: 36.40g.

Claims

1. a) The first amino acid, aspartic acid, is immobilized on the Wang resin via the carboxyl group of its side chain. b) A step of preparing teduglutide by sequentially coupling side-chain protected amino acids in the presence of a coupling agent and optionally in the presence of one or more chaotropic salts, and c) Step of obtaining crude teduglutide by cleaving the protecting group and peptide from Wang resin. A process for preparing teduglutide containing [the specified substance].

2. The process according to claim 1, wherein the aspartic acid in step a) is Fmoc-Asp-OtBu.

3. d) The process according to claim 1 or 2, further comprising the step of purifying crude teduglutide.

4. The process according to claim 1, wherein step b) is to sequentially deprotect the Fmoc group from the side-chain protected amino acid using a mixture of piperidine, formic acid and 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU).

5. The process according to claim 1, wherein the side-chain protecting amino acid in step b) is Boc-His-OH or Boc-His(Trt)-OH.

6. The process according to claim 1, wherein step b) comprises the use of one or more chaotropic salts selected from MgCl₂, CuCl₂, and ZnCl₂.

7. The process according to claim 1, wherein the cleavage of the protecting group and the peptide from the Wang resin in step c) is carried out using a cleavage cocktail comprising a free amino acid and a TFA cocktail.

8. The process according to claim 1, wherein the cleavage of the protecting group and the peptide from the Wang resin in step c) is carried out using a cleavage cocktail comprising an antioxidant, free amino acids and a TFA cocktail.

9. The process according to claim 7 or 8, wherein the free amino acid is selected from methonine, tryptophan, and histidine.

10. The process according to claim 8, wherein the antioxidant is butylated hydroxytoluene (BHT).

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