A process for the solid phase preparation of tirzepatide

Abstract

The application discloses a method for preparing Tirzepatide by solid phase, and belongs to the technical field of polypeptide medicine preparation. The method comprises the following steps: firstly, polypeptide fragment sequences of Tirzepatide are synthesized by solid phase; then, the polypeptide fragments are condensed by solid phase to obtain Tirzepatide peptide resin; the Tirzepatide peptide resin is subjected to acidolysis to obtain crude Tirzepatide; and finally, the crude Tirzepatide is purified to obtain pure Tirzepatide. In the application, the polypeptide is synthesized by three fragments at reasonable sites, so that the reaction period is shortened, the peptide loss caused by too long chain is reduced, and the problem that the conventional condensation is difficult to react is solved by using some special protected amino acid fragments.

Description

Technical Field

[0001] This invention belongs to the technical field of polypeptide drug preparation, and more specifically, relates to a method for solid-phase preparation of Tirzepatide. Background Technology

[0002] Tirzepatide, developed by Eli Lilly, is a dual agonist of both glucose-dependent insulinotropic peptide (GIP, also known as gastric inhibitory peptide) receptors and glucagon-like peptide-1 (GLP-1) receptors. Both GIP and GLP-1 are intestinal hormones that promote insulin secretion. Tirzepatide integrates the effects of these two insulin-stimulating mechanisms into a single molecule, representing a novel class of drugs for the treatment of type 2 diabetes. Tirzepatide, used once weekly, improves β-cell function and increases insulin sensitivity, showing improvements in both efficacy and tolerability in patients starting with lower doses and with smaller subsequent dose escalations. Clinical data on Tirzepatide indicate that it can significantly reduce A1C and weight levels in patients with type 2 diabetes, and it can also treat other metabolic diseases.

[0003] Tirzepatide has the following structure: Tyr 1 -Aib-Glu-Gly-Thr 5 -Phe-Thr-Ser-Asp-Tyr 10 -Ser-Ile-Aib-Leu-Asp 15 -Lys-Ile-Ala-Gln-Lys 20 (AEEA-AEEA-γ-Glu-Eicosanedioicacid)-Ala-Phe-Val-Gln-Trp 25 -Leu-Ile-Ala-Gly-Gly 30 -Pro-Ser-Ser-Gly-Ala 35 -Pro-Pro-Pro-Ser-NH2.

[0004] Solid-phase synthesis methods for Tirzepatide have been reported. Patents CN110903355A (application date: 2019.10.31, applicant: Chengdu Shengnuo Biopharmaceutical Co., Ltd.) and CN112592387A (application date: 2020.12.31, applicant: Jiangsu Nuotai Aosainuo Biopharmaceutical Co., Ltd.) disclose methods for preparing Tirzepatide. The method involves coupling the first amino acid to a resin, followed by sequential coupling of individual amino acids or fragments until a Tirzepatide peptide resin is obtained. The Tirzepatide resin is then acid-hydrolyzed to obtain crude Tirzepatide, and finally purified to obtain pure Tirzepatide.

[0005] The publicly available methods for preparing Tirzepatide all involve coupling amino acids or fragments one by one, which results in a long production cycle. Furthermore, as the synthesized peptide chain becomes longer, the reactivity of the coupling decreases, making it prone to peptide loss and increasing impurities. Moreover, the longer the peptide chain, the more impurities with properties similar to the product are present, making purification more difficult.

[0006] Therefore, designing and developing a Tirzepatide preparation method with high yield, short cycle, control of impurity generation, and reduced purification difficulty has significant practical and industrial value. Summary of the Invention:

[0007] In view of this, in order to solve the above-mentioned problems existing in the prior art, the purpose of this invention is to provide a method for solid-phase preparation of Tirzepatide. First, three polypeptide fragments are synthesized in the solid phase, and some special protective amino acid fragments are used in the synthesis. Then, Tirzepatide is obtained by solid-phase condensation, acid hydrolysis and purification. It has the advantages of high yield, short cycle, good stability and few impurities, and is suitable for large-scale industrial production.

[0008] To achieve the above-mentioned objectives, the specific technical solution of this invention is as follows:

[0009] A method for preparing Tirzepatide in a solid phase includes the following steps:

[0010] 1) First, solid-phase synthesis of polypeptide fragments 1Boc-AA(1~16)-OH, 2Fmoc-AA(17~26)-OH and 3H-(27~39)AA-AM Resin;

[0011] 2) Polypeptide fragment 2 is condensed starting with polypeptide fragment 3, and finally polypeptide fragment 1 is condensed on the product obtained above to obtain Tirzepatide peptide resin.

[0012] 3) Tirzepatide peptide resin was further acid-hydrolyzed to obtain crude Tirzepatide;

[0013] 4) Purify crude Tirzepatide to obtain pure Tirzepatide.

[0014] Preferably, the sequence of polypeptide fragment 1 is amino acids 1-16 of the Tirzepatide sequence, and the specific sequence of polypeptide fragment 1 is Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc).

[0015] The sequence of polypeptide fragment 2 is amino acids 17-26 of the Tirzepatide sequence. The specific sequence of polypeptide fragment 2 is Fmoc-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γ-Glu-Eicosanedioicacid(mon-tBu))-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu.

[0016] The sequence of polypeptide fragment 3 is amino acids 27-39 in the Tirzepatide sequence. The specific sequence of polypeptide fragment 3 is Fmoc-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu).

[0017] In this application, since the target polypeptide has two Lys (positions 16 and 20), to avoid the formation of isomer impurities due to the extensional side chain reaching position 16, amino acids 1 to 16 of fragment 1 were selected. When synthesizing polypeptides containing extensional side chains, the polypeptide fragment containing the side chain cannot be too long; otherwise, the steric hindrance contributed by the extensional side chain will increase the difficulty of synthesis, such as incomplete conversion of the starting materials. Therefore, amino acids 17 to 26 of fragment 2 were selected.

[0018] Preferably, the solid-phase preparation method for Tirzepatide described above includes the following specific steps:

[0019] (1)

[0020] H2N-Y 1 -Aib-EGT 5 FTSDY 10 SI-Aib-LD 15 KIAQ-K 20(AEEA-AEEA-γ-Glu-NH-CO-(CH2)18-COOH)-AFVQW 25 LIAGG 30 PSSGA 35 PPPS 39 -NH2 is synthesized in three fragments: polypeptide fragment 1, Boc-AA(1~16)-OH; polypeptide fragment 2, Fmoc-AA(17~26)-OH; and polypeptide fragment 3, H-(27~39)AA-AM Resin. During the synthesis of each fragment, amino acids are coupled sequentially from right to left, and fragment docking is performed from right to left.

[0021] Furthermore, in step (1), in the solid-phase synthesis of polypeptide fragment 1: Boc-AA(1~16)-OH, H-Lys(Boc)-CTC Resin is used as the starting material, and Fmoc-AA-OH is used for condensation; wherein the amino acids at positions 12 and 13 are condensed using the dipeptide Fmoc-Ile-Aib-OH; and the amino acids at positions 1 and 2 are condensed using the dipeptide Boc-Tyr(tBu)Aib-OH.

[0022] The synthesis of polypeptide fragment 2: Fmoc-AA(17-26)-OH was initiated using H-Leu-CTC Resin and condensed using Fmoc-AA-OH; the 20th amino acid was used...

[0023] Fmoc-Lys{(AEEA-AEEA-γ-Glu-NH-CO-(CH2)18-COOtBu}-OH, the 17th amino acid Fmoc is protected and retained, and is not removed;

[0024] The synthesis of polypeptide fragment 3: H-(27~39)AA-AM Resin was initiated using Fmoc-linker AMResin, condensed using Fmoc-AA-OH, and the last amino acid was deprotected by Fmoc.

[0025] That is, fragment 1 uses dichloro resin as the starting resin; fragment 2 uses dichloro resin as the starting resin; fragment 3 uses amino resin as the starting resin; the degree of substitution of dichloro resin and amino resin is 0.3-0.9 mmol / g, preferably 0.5-0.7 mmol / g.

[0026] Furthermore, the amino acid equivalents used when condensing polypeptide fragment 1 and polypeptide fragment 2 are 1.1 eq-3.5 eq, preferably 1.5-2.0 eq, and most preferably 1.5 eq; the condensing agent is any one or a mixture of HOBt, Cl-HOBt, HBTU, HATU, Oxymapure, DIC or DCC, preferably a combination of HOBt and HBTU; the reaction temperature is 10-60℃.

[0027] Furthermore, the condensing agent used in the synthesis of polypeptide fragment 3 is any one or a mixture of HOBt, Cl-HOBt, Oxymapure, DIC or DCC; the amino acid equivalents used in the condensation of the fragment are 1.1 eq-3.5 eq, preferably 1.5-2.0 eq, and most preferably 1.5 eq.

[0028] (2) During the synthesis process, the corresponding resins are coupled and protected amino acids or fragments in sequence; among them, polypeptide fragments 1 and 2 are cleaved from the resin, and polypeptide fragment 3 is attached to the resin; the resin-polypeptide fragment 3 condenses polypeptide fragment 2, and then condenses polypeptide fragment 1 to obtain Tirzepatide peptide resin.

[0029] Furthermore, the pyrolysis solution used in step (2) is a mixture of TFA and DCM, with a volume ratio of TFA to DCM of 0.1%-3%, and a pyrolysis time of 3-30 min.

[0030] (3) The crude Tirzepatide was cleaved from the Tirzepatide peptide resin. The cleavage time was 2-5 h and the cleavage temperature was 10-45℃.

[0031] (4) Purify crude Tirzepatide and dry it to obtain the Tirzepatide product.

[0032] In the solid-phase preparation method of Tirzepatide, in addition to using other conventional protected amino acids, the following special protected amino acids and fragments are also used in the synthesis of Tirzepatide polyresins:

[0033] (1)Boc-Tyr(tBu)-Aib-OH

[0034] (2)Fmoc-Ile-Aib-OH

[0035] (3)Fmoc-Gly-Gly-OH.

[0036] Another objective of this application is to protect Tirzepatide prepared using any of the above methods.

[0037] Compared with the prior art, the positive effects of the present invention are reflected in:

[0038] (i) This invention shortens the synthesis cycle by synthesizing in segments and solves the problems of generating too many missing peptides and difficulty in studying impurities when linking them one by one.

[0039] (II) This invention solves the problem of glycine being easily lost during the synthesis of polypeptide sequence fragment 3 by using the dipeptide Fmoc-Gly-Gly-OH.

[0040] (III) This invention solves the problem of low condensation efficiency of the next amino acid during the synthesis of polypeptide sequence fragment 1 by using dipeptides Fmoc-Ile-Aib-OH and Boc-Tyr(tBu)-Aib-OH.

[0041] (iv) This invention provides a new and efficient method for preparing Tirzepatide. This invention uses special protected amino acid fragments to solve the problems of low product purity and efficiency in large-scale preparation methods. Attached Figure Description

[0042] Figure 1 The mass spectrum of Tirzepatide prepared in Example 1;

[0043] Figure 2 The mass spectrum of Tirzepatide prepared in Example 1;

[0044] Figure 3 The image shows the HPLC chromatogram of the Tirzepatide product prepared in Example 1. Detailed implementation method:

[0045] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Operations not described in detail in this application are conventional operations in the art or do not have a specific impact on the effect of this application.

[0046] In a specific embodiment of the present invention, the English and Chinese names of the reagents are shown in the table below:

[0047]

[0048]

[0049] In this application, %, indicates the volume percentage content.

[0050] Unless otherwise specified, the techniques or conditions described in the following examples were performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0051] In this application, existing technology is used to sequentially link amino acids to synthesize the target molecule, linking ~3 amino acids per day; the whole fragment linking is completed in 12 days; the cutting and purification are completed in 2 days, for a total of 14 days, and the purity of the target product obtained is 97%.

[0052] The main reasons for synthesizing the target molecule in multiple fragments in this invention are as follows: First, considering the steric hindrance of the non-natural amino acid Aib at position 13, which may affect fragment linkage, position 13 should not be located at the edge during fragmentation. Second, the extended side chain is attached at position 20, and the extended side chain contains two AEEA, one Glu, and one 20-carbon diacid monotert-tert-ester structure. After being attached to the peptide chain, it will generate certain steric hindrance. Therefore, to prevent increased difficulty in linking multiple fragments, it is preferable that the amino acid at position 20 is located in the middle sequence of one of the fragments, and the sequence does not exceed 10 amino acids, i.e., 17–26. Therefore, the initial attempt was to segment the peptide into fragment 1 (positions 1–16), fragment 2 (positions 17–26), and fragment 3 (positions 27–39). In peptide synthesis, amino acids are usually linked one by one. However, when encountering sterically hindered amino acids located outside the beginning of the peptide chain (synthetic sequence), they may not be able to be linked smoothly, leading to incomplete reaction, ultimately resulting in reduced yield, deletion of peptides, and a series of other problems.

[0053] This invention involves synthesizing the target polypeptide in three separate fragments, which are then cross-linked to obtain the target polypeptide. The main reasons are as follows: We conducted a thorough analysis of the target polypeptide structure, which contains 39 amino acids, with its long-acting side chain located at the 20th position (Lys). When segmenting the polypeptide, we considered the following:

[0054] 1) The target product contains two Lys, at position 16 and position 20, and the extended side chain is connected to the Lys at position 20. In order to avoid the extended side chain being connected to position 16, the Lys at positions 16 and 20 need to exist on different fragments.

[0055] 2) The fragment containing the extended side chain cannot be too long, because the extended side chain itself contains two AEEA, one Glu, and one 18-diacid monotert-butyl ester structure, which will contribute certain steric hindrance, molecular weight, and other factors that are not conducive to peptide chain elongation during peptide synthesis (referring to the fragment after separation). Therefore, in order to reduce the difficulty of synthesizing this fragment and reducing the difficulty of linking fragments, we place the amino acid (Lys) with the extended side chain in the middle position of the peptide fragment. Therefore, we select amino acids 17-26 for fragment 2 and amino acids 27-39 for fragment 3.

[0056] 3) Fragment 1 was tested for amino acids 1 to 16. Experiments showed that the synthesis of fragment 1 was very smooth, and no amino acids that were difficult to connect appeared. Therefore, it was not further split into two or more fragments.

[0057] 4) In fragment 2, there is another amino group at Trp position 25. Although we used Boc for protection, considering that the protection may not be complete or that Boc may be accidentally removed during fragment synthesis, the extended side chain may be attached to the amino group of Trp position 25. This impurity in the structure is highly similar to the target peptide structure and has the same molecular weight, so it will be very difficult to remove in subsequent purification. Therefore, we chose to link the extended side chain to Lys before using it to synthesize fragment 2 to avoid these possible adverse reactions.

[0058] Example 1:

[0059] A method for preparing Tirzepatide in a solid phase includes the following steps:

[0060] H2N-Y 1 -Aib-EGT 5 FTSDY 10 SI-Aib-LD 15 KIAQ-K 20 (AEEA-AEEA-γ-Glu-NH-CO-(CH2)18-COOH)-AFVQW 25 LIAGG 30 PSSGA 35 PPPS 39 -NH2;

[0061] The synthesis process involves three fragments. During the synthesis of each fragment, amino acids are coupled sequentially from right to left, and fragment docking also proceeds from right to left.

[0062] 1. Synthesis of polypeptide fragment 1: Boc-AA(1~16)-OH

[0063] 1) The starting material used is H-Lys(Boc)-CTC Resin

[0064] 2) Condensation reagent and dosage:

[0065] name Dosage Fmoc-AA-OH 1.5eq HOBt 1.5eq HBTU 1.5eq DIPEA 3.0eq DMF /

[0066] 3) Reaction process.

[0067] Weigh 10g of H-Lys(Boc)-CTC Resin (5mmol), wash twice with DMF (100ml*2), weigh 3.09g of Fmoc-Asp(OtBu)-OH (7.5mmol), 1.01g of HOBt (7.5mmol), 2.84g of HBTU (7.5mmol), and 1.94g of DIPEA (15mmol). Dissolve and activate these four materials with DMF, then add them to the reactor containing the resin to allow them to react. After the reaction was completed, the mixture was washed sequentially with DMF (100 ml), MTBE (100 ml * 2), and DMF (100 ml * 2) to remove the condensing agent (HOBT, HBTU) and the base (DIPEA); then, it was washed twice with a DMF solution of 5% Pip / 1.25% DBU / 1% HOBT to remove Fmoc protection (100 ml / time, 10 min / time); finally, it was washed sequentially with DMF (100 ml * 2), MTBE (100 ml * 2), and DMF (100 ml * 2) to remove the reagents from the previous step.

[0068] 4) Repeat step 3) above, condensing the next protected amino acid or fragment sequentially from right to left; wherein the amino acids at positions 12 and 13 are condensed using the dipeptide Fmoc-Ile-Aib-OH; wherein the amino acids at positions 1 and 2 are condensed using the dipeptide Boc-Tyr(tBu)-Aib-OH.

[0069] The amino acids or fragments used are listed in the table below:

[0070] 15 Fmoc-Asp(OtBu)-OH 14 Fmoc-Leu-OH 12-13 Fmoc-Ile-Aib-OH 11 Fmoc-Ser(tBu)-OH 10 Fmoc-Tyr(tBu)-OH 9 Fmoc-Asp(OtBu)-OH 8 Fmoc-Ser(tBu)-OH 7 Fmoc-Thr(tBu)-OH 6 Fmoc-Phe-OH 5 Fmoc-Thr(tBu)-OH 4 Fmoc-Glu(OtBu)-OH 3 Fmoc-Gly-OH 1-2 Boc-Tyr(tBu)-Aib-OH

[0071] 5) After condensation, the resin was washed with DMF and MTBE and dried to obtain 22.3g of resin. The resin was lysed with 1% TFA / DCM solution (223ml*3, 5min each time), dried and set aside, which is fragment 1.

[0072] 2. Synthesis of polypeptide fragment 2: Fmoc-AA(17~26)-OH

[0073] 1) The starting material used is H-Leu-CTC Resin

[0074] 2) Condensation reagent and dosage:

[0075]

[0076]

[0077] 3) Using the same condensation and cleavage method as peptide fragment 1, peptide fragment 2 was obtained; after the reaction, it was washed with DMF, MTBE and DMF in sequence to remove the condensing agent and base; then the Fmoc protection was removed with a DMF solution of 5% Pip / 1.25% DBU / 1% HOBT, and the reagents from the previous step were washed with DMF, MTBE and DMF in sequence to remove the Fmoc protection.

[0078] 4) Repeat the above steps to condense the next amino acid; where 20 amino acids are used.

[0079] Fmoc-Lys{(AEEA-AEEA-γ-Glu-NH-CO-(CH2) 18 -COOtBu}-OH; the Fmoc protection at position 17 is retained and does not disappear;

[0080] The amino acids or fragments used are listed in the table below:

[0081] 25 Fmoc-Trp(Boc)-OH 24 Fmoc-Gln(Trt)-OH 23 Fmoc-Val-OH 22 Fmoc-Phe-OH 21 Fmoc-Ala-OH 20 Fmoc-Lys(AEEA-AEEA-γ-Glu-eicosanedioicacid(mon-tBu)-OH 19 Fmoc-Gln(Trt)-OH 18 Fmoc-Ala-OH 17 Fmoc-Ile-OH

[0082] 1) After condensation, cleave the resin (1% TFA / DCM), dry it for later use, and that is fragment 2.

[0083] 3. Synthesis of polypeptide fragment 3: H-(27~39)AA-AM Resin

[0084] 1) The starting material used is Fmoc-linker AM Resin

[0085] 2) Condensation reagent and dosage:

[0086]

[0087]

[0088] 3) Add 10g of Fmoc-linker AM Resin (3.5mmol) to the solid-phase reactor, wash twice with DMF (100ml*2), then remove Fmoc protection with a DMF solution of 5% Pip / 1.25% DBU / 1% HOBT (100ml*2, 10 minutes each time); then wash with DMF (100ml*2), MeOH (100ml*2), and DMF (100ml*2) in sequence to remove the reagents from the previous step.

[0089] 4) Add 2.01 g (5.25 mmol) of Fmoc-Ser(tBu)-OH and 1.42 g (10.5 mmol) of HOBt to the reactor cleaned in step 3). Dissolve them in an appropriate amount of DMF, then add 0.66 g (5.25 mmol) of DIC. React at approximately 25°C for 45 min, then add another 0.66 g (5.25 mmol) of DIC. Monitor the reaction hourly thereafter until complete. Repeat step 3) sequentially, condensing the next protected amino acid or fragment from right to left. The amino acids or fragments used are shown in the table below.

[0090]

[0091]

[0092] 5) The last amino acid (position 27) of this fragment is also deprotected by Fmoc to obtain "fragment 3-resin" for later use.

[0093] 4. Connect fragment 1, fragment 2, and fragment 3.

[0094] 1) Fragment 3 was condensed into fragment 2; the reagents and dosages used are as follows:

[0095] name Dosage Segment 2 1.5eq HOBT 3.0eq DIC 1.5eq DMF / DMSO / (45 minutes later) DIC 1.5eq

[0096] 2) Add 3 mmol of resin containing fragment 3 to the solid-phase reactor. After washing twice with DMF, dissolve fragment 2 (4.5 mmol) in a DMF / DMSO (3:1) solution and add it to the reactor. Add 0.57 g (4.5 mmol) of DIC and react at approximately 25°C for 45 min. Then add another 0.57 g (4.5 mmol) of DIC. Monitor the reaction hourly until complete. Then wash with DMF (100 ml), MeOH (100 ml * 2), and DMF (100 ml * 2) to remove the reagents from the previous step. Deprotect the Fmoc with a DMF solution of 5% Pip / 1.25% DBU / 1% HOBT. Then wash with DMF, MeOH, and DMF to remove the reagents from the previous step. 3) Repeat the above steps to condense fragment 1. The reagents and amounts used are as follows:

[0097] name Dosage Segment 1 1.5eq HOBT 3.0eq DIC 1.5eq DMF / (45 minutes later) DIC 1.5eq

[0098] 4) After the reaction is complete, wash the resin with DMF and MeOH and dry it to obtain "polypeptide compound-resin".

[0099] 5. Synthesize the target polypeptide

[0100] The obtained polypeptide resin was lysed at approximately 25°C for 3 hours in a TFA / Tis / H2O / DTT (92.5:2.5:2.5:2.5) solution with a volume 10 times its weight. The solution was filtered, and the filtrate was squeezed into a MTBE solution with a volume 10 times its weight (at approximately 0°C). A solid precipitated out and was filtered. The filter cake was washed three times with MTBE (5 ml / g) and dried under vacuum at approximately 30°C to obtain the crude product.

[0101] 6. Purification method: The crude peptide is purified by high-performance liquid chromatography (HPLC) column chromatography. The target product fraction is collected, concentrated, and lyophilized to obtain the TFA salt of the product. The desired salt can be substituted as needed.

[0102] Example 2: Resin Substitution Degree Experiment During the Synthesis of Fragment 1, Fragment 2, and Fragment 3

[0103] Based on Example 1, resin substitution degree experiments were conducted during the synthesis of fragments 1, 2, and 3. Specifically, using resin at a concentration of 1.22 mmol / g, amino acids located at the edge of the sequence during the synthesis of fragments 1, 2, and 3 were attached to the resin, and their substitution degree was tested. Fragments 1 and 2 were then linked to amino acids according to the ratios shown in Table 1; fragment 3 was linked to amino acids according to the ratios shown in Table 2. The final determined substitution degree was preferably 0.3–0.9 mmol / g, with an optimal value of 0.5–0.7 mmol / g.

[0104]

[0105]

[0106] The experimental results of the obtained degree of substitution are shown below:

[0107] 1. Resin substitution degree experiment during fragment 1 synthesis:

[0108]

[0109] 2. Resin substitution degree experiment during fragment 2 synthesis:

[0110]

[0111] 3. Resin substitution degree experiment during fragment 3 synthesis:

[0112]

[0113] Example 3: Experiment on the selection of dosage when connecting fragment 2 and fragment 1

[0114] Based on Example 1, we conducted experiments on the selection of dosages for the ligation of fragment 2 and fragment 1. For the ligation of fragment 2 and fragment 1, we selected HOBT as the condensing agent at a dosage of 3.0 eq, and DIC at a dosage of 1.5 eq + 1.5 eq. We screened different dosages for fragment 2 and fragment 1. According to publicly available technology, amino acid ligation in peptide sequences generally requires a dosage of not less than 1.1 equivalents; therefore, dosages below 1.1 equivalents were not considered. Higher dosages ensure complete reaction while effectively removing excess material during the process. Based on this, we examined the dosages of fragment 2 and fragment 1 and ultimately found that the dosages of fragment 2 and fragment 1 could be selected from 1.1 to 3.0 eq, preferably 1.5 to 2.0 eq.

[0115]

[0116] Example 4: Investigation of the amount of a single amino acid or polypeptide used in the synthesis of a single fragment

[0117] Referring to Example 3, the amounts of the dipeptides Boc-Tyr(tBu)-Aib-OH and Fmoc-Ile-Aib-OH in the synthesis of fragment 1, and the side-chain protected amino acid Lys{AEEA-AEEA-γ-Glu-Eicosanedioic acid(mon-tBu)} in the synthesis of fragment 2 were optimized. The final determined amounts were 1.1–3.5 eq, preferably 1.2–2.0 eq. Based on the selection of the dipeptide amounts, the amounts of other individual amino acids can be predicted, also preferably 1.1–3.5 eq, preferably 1.2–2.0 eq. The condensing agent or base selected during the experimental optimization process is shown in the table below:

[0118] name Dosage Fmoc-AA-OH Examine dosage HOBT 1.5eq HBTU 1.5eq DIPEA 3.0eq DMF /

[0119] Example 5: Investigation of the amount of a single amino acid or polypeptide used in the synthesis of a single fragment

[0120] Referring to Example 3, the amount of dipeptide Fmoc-Gly-Gly-OH used in the synthesis of fragment 3 was optimized, and its amount was ultimately determined to be 1.1–3.5 eq, preferably 1.2–2.0 eq. Based on the selection of the dipeptide amount, the amounts of other individual amino acids can be predicted, also preferably 1.1–3.5 eq, preferably 1.2–2.0 eq. The selection of condensing agents or bases during the optimization process is shown in the table below.

[0121] name Dosage Fmoc-AA-OH Examine dosage HOBT 3.0eq DIC 1.5eq DMF / (1 hour later) DIC 1.5eq

[0122] Example 6: Advantages of using dipeptide Fmoc-Gly-Gly-OH in fragment 3 synthesis

[0123] Referring to Example 5, the amount of amino acids or dipeptides used was 1.5 eq. The effects of linking Gly, Gly and using dipeptides to link Gly-Gly were compared. It was found that using dipeptides can significantly reduce the amount of Gly-deficient peptides.

[0124] sequence amino acids or polypeptides Gly-deficient peptide Purity of target fragment 3 1 Fmoc-Gly-Gly-OH 0 98.6％ 2 Gly 1～2％ 97％

[0125] Example 7: Selection of Condensing Agent

[0126] Based on the initial fragment synthesis experiments, Fragment 1 found that:

[0127] During the synthesis of fragment 2, the dipeptide Boc-Tyr(tBu)-Aib-OH and the dipeptide Fmoc-Ile-Aib-OH are laterally protected amino acids Lys{AEEA-AEEA-γ-Glu-Eicosanedioic acid(mon-tBu)}.

[0128] During the synthesis of fragment 3, the 32-bit and 33-bit Ser(tBu)

[0129] The above steps are the most difficult to connect, therefore we choose the amino acid or dipeptide steps above and examine the types of condensing agents.

[0130] When investigating fragments 1 and 2, the amount of condensing agent was uniformly selected as 1.5 eq to 2.0 eq, and the amount of base was 3.0 eq. The synthesis operation was carried out in the same manner, and it was finally found that the combination of HOBt and HBTU was optimal.

[0131]

[0132] For fragment 3, the amount of condensing agent was uniformly selected from 1.5 eq to 2.0 eq, and the synthesis operation was carried out in the same manner. Finally, it was found that the combination of HOBt and DIC was optimal.

[0133]

[0134] The foregoing basic examples and their further alternative examples of the present invention can be freely combined to form multiple embodiments, all of which are embodiments that can be adopted and are claimed by the present invention. In the present invention, each alternative example can be arbitrarily combined with any other basic example and alternative example. Those skilled in the art will recognize numerous combinations.

[0135] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A process for the solid phase preparation of Tirzepatide, characterized in that, Comprising the following steps: 1) solid phase synthesis of polypeptide fragment 1 Boc-AA(1~16)-OH, polypeptide fragment 2 Fmoc-AA(17~26)-OH and polypeptide fragment 3 H-(27~39)AA-AM Resin; 2) condensation of polypeptide fragment 2 with polypeptide fragment 3 as the starting fragment, and finally condensation of polypeptide fragment 1 on the aforementioned product to obtain Tirzepatide peptide resin; 3) acidolysis of Tirzepatide peptide resin to obtain crude Tirzepatide; 4) purification of crude Tirzepatide to obtain pure Tirzepatide; Wherein the sequence of polypeptide fragment 1 is: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp (OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc); The sequence of polypeptide fragment 2 is: Fmoc-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γ-Glu-Eicosanedioic acid(mon-tBu))-Ala-Phe-Val-Gln (Trt)-Trp (Boc)- Leu; The sequence of polypeptide fragment 3 is: Fmoc-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser (tBu).

2. The process for preparation of Tirzepatide as claimed in claim 1 wherein, In step 1), the solid phase synthesis of polypeptide fragment 1: Boc-AA(1~16)-OH, starting with H-Lys(Boc)-CTC Resin, condensing with Fmoc-AA-OH; wherein the 12th and 13th amino acids are condensed with dipeptide Fmoc-Ile-Aib-OH; the 1st and 2nd amino acids are condensed with dipeptide Boc-Tyr(tBu)Aib-OH; The synthesis of polypeptide fragment 2: Fmoc-AA(17~26)-OH, starting with H-Leu-CTC Resin, condensing with Fmoc-AA-OH; wherein the 20th amino acid is Fmoc-Lys{(AEEA-AEEA-γ-Glu-NH-CO-(CH2)18-COOtBu}-OH, and the 17th amino acid Fmoc is not removed; The synthesis of polypeptide fragment 3: H-(27~39)AA-AM Resin, starting with Fmoc-linker AM Resin, condensing with Fmoc-AA-OH, and the last amino acid is removed Fmoc protection; the substitution degree of dichloromethane resin and amino resin is 0.3-0.9mmol / g.

3. The process for the preparation of Tirzepatide as claimed in any one of claims 1-2, wherein Comprising the following specific steps: (1) H2N-Y 1 -Aib-EGT 5 FTSDY 10 SI-Aib-LD 15 KIAQ-K 20 (AEEA-AEEA-γ-Glu-NH-CO-(CH2)18-COOH)-AFVQW 25 LIAGG 30 PSSGA 35 PPPS 39 -NH2 three fragments, respectively, polypeptide fragment 1, Boc-AA (1~16)-OH; polypeptide fragment 2, Fmoc-AA (17~26)-OH; polypeptide fragment 3, H-(27~39) AA-AM Resin; each fragment synthesis, from right to left in turn coupling amino acids, fragment docking from right to left; (2) In the synthesis process, the corresponding resin is coupled with protected amino acids or fragments in turn; wherein polypeptide fragment 1 and 2 are cleaved from the resin, and polypeptide fragment 3 is connected to the resin; the resin-polypeptide fragment 3 is condensed with polypeptide fragment 2, and then condensed with polypeptide fragment 1 to obtain the Tirzepatide peptide resin; (3) Tirzepatide crude product is cleaved from the Tirzepatide peptide resin, the cleavage time is 2-5h, and the cleavage temperature is 10-45℃; (4) The Tirzepatide crude product is purified and dried to obtain the Tirzepatide product.

4. The process of preparing Tirzepatide as claimed in claim 3, The number of amino acid equivalents used when polypeptide fragment 1 and polypeptide fragment 2 are condensed is 1.1eq-3.5eq; the condensing agent is any one or a mixture of multiple of HOBt, Cl-HOBt, HBTU, HATU, Oxymapure, DIC or DCC, and the reaction temperature is 10-60℃.

5. The process of preparing Tirzepatide as claimed in claim 3, The condensing agent used when polypeptide fragment 3 is synthesized is any one or a mixture of multiple of HOBt, Cl-HOBt, Oxymapure, DIC or DCC; the number of amino acid equivalents used when the fragments are condensed is 1.1eq-3.5eq.

6. The process of preparing Tirzepatide as claimed in claim 3, wherein, The cleavage solution used for cleavage is a mixture of TFA and DCM, the volume ratio of TFA to DCM is 0.1%-3%, and the cleavage time is 3-30min.

7. The process of claim 4, wherein the process is carried out at a temperature of about 20 °C to about 30 °C. The condensing agent used when polypeptide fragment 1 and polypeptide fragment 2 are condensed is a combination of HOBt and HBTU.

8. The process of claim 3, wherein: In addition to using other conventional protected amino acids in the synthesis process of the Tirzepatide multi-resin, the following special protected amino acids and fragments are also used: (1) Boc-Tyr (tBu) -Aib-OH (2) Fmoc-Ile-Aib-OH (3) Fmoc-Gly-Gly-OH.

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