Synthetic method for tirzepatide

Abstract

The present invention provides a synthetic method for tirzepatide. The method uses three special materials Fmoc-Lys(AEEA-AEEA-γ-Glu-eicosanedioic acid)-OH, Fmoc-Ile-Aib-OH and Boc-Tyr-Aib-OH.

Description

A synthetic method of tilportide Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular to a method for synthesizing Tirzepatide. Background Art

[0002] Tirzepatide is a dual-target agonist of GLP-1 and GIP developed by Eli Lilly and Company. It was approved by the FDA on May 13, 2022 and is available in the United States.

[0003] Its structure (peptide sequence): NH2-Tyr-Aib-Glu-Gly-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Asn-Lys(AEEA-AE EA-γ-Glu-eicosanedioic acid)-Ala-Phe-Val-Asn-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2

[0004] Among them, AEEA is a special amino acid: 2-(2-(2-aminoethoxy)ethoxy)acetic acid.

[0005] The details are shown in the figure below:

[0006] The existing synthesis processes of tilpotide can be roughly divided into two categories:

[0007] The first is the liquid-phase splicing method: Eli Lilly and Company, in patent application number CN113330024A, used a liquid-phase splicing method to synthesize tilpoxetine. The four fully protected fragments spliced ​​in the liquid phase are fragment (1-14), fragment (15-21), fragment (22-29), and fragment (30-39). Fragments (22-29) and (30-39) are liquid-phase spliced ​​and treated using a nanofiltration device to obtain fragment (22-39). Fragment (22-39) is then liquid-phase condensed with fragment (15-21) and nanofiltered to obtain fragment (15-39). Finally, fragment (15-39) is liquid-phase condensed with fragment (1-14) and subsequently deprotected to obtain the crude peptide. The quality control of the four fully protected fragments in this process is difficult, especially for the identification of racemic impurities. Furthermore, purification of the intermediates in the liquid-phase reaction is difficult.

[0008] The second type is Fmoc solid-phase synthesis. This type of synthesis method has been widely reported, including sequential Fmoc solid-phase synthesis, Lys deprotection, and subsequent side chain coupling. Another method involves fragment condensation.

[0009] 1) Patent CN113330024A: Telpotide was synthesized using a standard Fmoc solid-phase synthesis strategy, in which the Lys side chain at position 20 was protected with ivDde. Hydrazine hydrate was used to selectively remove Lys at position 20, followed by the ligation of (AEEA)2-γ-Glu-C 20 The method is limited by the long coupling time, which generally takes 4-8 hours for conventional amino acids and even up to 18 hours for special amino acids.

[0010] 2) Patents CN112592387B and CN114736271A use serine-containing pseudo-proline dipeptides or DMB-protected glycine as special materials for step-wise coupling, effectively suppressing shrinkage during peptide resin synthesis. However, condensation of DMB-containing glycine results in dipeptide defects at that site and the next.

[0011] 3) Patents CN115651075B and CN110903355A use fragments (1-4) as the overall unit, aiming to address the difficulty in coupling amino acids 1 and 2. However, fragments (1-4) require additional solid-phase or liquid-phase synthesis, which is cost-effective for large-scale production.

[0012] 4) Patents CN116120403A and CN115991742A use large fragments such as (1-16), (17-26), (27-39), or (1-14), (22-29) as splicing units. The disadvantages of this type of method are that large fragments require additional synthesis steps and require a large amount of fragment peptides. In addition to cost issues, fully protected large fragments also pose quality control issues.

[0013] 5) Patents CN115181173A, CN115181174A, and CN115160429A use 2-6 or 5-7 peptides containing specific sites, which can partially address the defect problem. However, these methods also have significant shortcomings. First, the large amount of peptide fragments required, followed by cost and quality control issues. Summary of the Invention

[0014] To address the problems of low tilpotide synthesis efficiency, low purity, difficulty in obtaining raw materials, and high purification difficulty in the prior art, the present invention provides a method for synthesizing tilpotide using a solid-phase stepwise coupling strategy. The method of the present invention avoids the use of complex starting materials. The special materials used include Fmoc-Lys(AEEA-AEEA-γ-Glu-eicosanedioic acid)-OH, Fmoc-Ile-Aib-OH, and Boc-Tyr-Aib-OH. The use of these simple materials can not only ensure condensation efficiency but also effectively control the quality of the fragments, ultimately obtaining the final product with a high content yield.

[0015] The synthetic route of the present invention is as follows:

[0016] Specifically, the present invention provides a solid phase synthesis method of tilpoxetine, which comprises the following steps:

[0017] S1) using an amide resin as a solid phase support and an Fmoc solid phase synthesis strategy to sequentially couple Fmoc-protected amino acids or special materials to obtain a fully protected tilpoxetine peptide resin;

[0018] S2) cracking, settling, filtering, and drying the synthesized fully protected tilpotide peptide resin to obtain tilpotide;

[0019] The amino acids or special materials protected by Fmoc are sequentially coupled as follows: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-eicosanedioic acid(mon-tBu))-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-Aib-OH, Fmoc-Ser(tBu)-OH, Fm oc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Boc-Tyr-Aib-OH.

[0020] Further, the method of the Fmoc solid phase synthesis strategy in step S1) includes:

[0021] S11) removing the Fmoc protecting group;

[0022] S12) coupling Fmoc-protected amino acids or special materials;

[0023] Wherein, in S11), 20% piperidine / DMF is used as the Fmoc removal reagent; 20% piperidine / DMF is a mixed solution with a volume ratio of piperidine to DMF of 1:4.

[0024] The method for coupling the Fmoc-protected amino acid or special material in S12) is to perform the coupling under the action of an amino acid or special material coupling agent.

[0025] Furthermore, the deprotection method in S11) is to add an Fmoc removal reagent for deprotection for 5 to 10 minutes, wash with DMF, then add the Fmoc removal reagent again for deprotection for 5 to 10 minutes and wash with DMF.

[0026] Furthermore, the coupling agent for coupling the protected amino acid is a combination of DIC and A or a combination of DIPEA, A and B, wherein A is Oxyma, HOBt or HOAt, and B is one of PyBOP, PyAOP, HATU, HBTU, and TBTU; preferably, it is a combination of DIC and HOBt.

[0027] Furthermore, when coupling the protected amino acid, the molar amount of the coupling agent is 1-1.5 times the molar amount of the amino acid.

[0028] Furthermore, the coupling agent for coupling the special materials Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-eicosanedioic acid(mon-tBu))-OH, Fmoc-Ile-Aib-OH, and Boc-Tyr(tBu)-Aib-OH is a combination of DIC, HOBt, and DIPEA.

[0029] Furthermore, when coupling the special material, the molar ratio of the special material, DIC, HOBt and DIPEA is 1:1-1.5:1-1.5:0.1-0.5, preferably 1:1.2:1.2:0.1.

[0030] Furthermore, the amide resin in S1) is Rink amide resin, Rink amide AM resin, Rink amide MBHA resin, or Sieber resin, among which Rink amide MBHA resin and Sieber resin are preferred.

[0031] Furthermore, the degree of substitution of the amide resin in S1) is 0.2 to 0.8 mmol / g, preferably 0.4 to 0.6 mmol / g.

[0032] Furthermore, the reaction temperature in S1) is room temperature or 20-35°C.

[0033] Furthermore, the cleavage in S2) is performed using a mixed solution of TFA, TIPS, EDT and H2O, preferably with a volume ratio of TFA, TIPS, EDT and H2O of 91:3:3:3.

[0034] Furthermore, in S2), the sedimentation method is to add an excess of methyl tert-butyl ether to the cleavage reaction system for sedimentation. Preferably, the amount of methyl tert-butyl ether added is 5-10 times the volume of the cleavage reaction system. Beneficial effects

[0035] 1) The Tirzepatide synthesis method of the present invention has simple synthesis steps, and the starting materials are readily available. The stepwise coupling avoids the use of large peptide fragments, and the quality of the starting materials is easy to control.

[0036] 2) For special materials that are difficult to couple, using small special fragments for synthesis and adding a small amount of DIPEA can significantly improve the coupling efficiency.

[0037] 3) The Tirzepatide synthesis method of the present invention can ultimately synthesize Tirzepatide with a high content yield while maintaining a high purity, has obvious cost advantages, and is conducive to large-scale synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a synthetic route of the present invention.

[0039] FIG2 is the HPLC result of the crude peptide of telpotide synthesized in Example 1.

[0040] FIG3 is the HPLC result of the crude peptide of tilpoxetine synthesized in Comparative Example 1. DETAILED DESCRIPTION

[0041] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0042] 1 , the present invention provides a solid phase synthesis method of Tirzepatide, which comprises the following steps:

[0043] S1) using an amide resin as a solid phase support and an Fmoc solid phase synthesis strategy to sequentially couple Fmoc-protected amino acids or special materials to obtain a fully protected tilpoxetine peptide resin;

[0044] The amino acids or special materials protected by Fmoc are sequentially coupled as follows: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-eicosanedioic acid(mon-tBu))-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-Aib-OH, Fmoc-Ser(tBu)-OH, Fm oc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Boc-Tyr-Aib-OH.

[0045] The method of the Fmoc solid phase synthesis strategy in step S1) comprises:

[0046] S11) Removal of Fmoc protecting group: using 20% ​​piperidine / DMF as an Fmoc removal reagent; 20% piperidine / DMF is a mixed solution with a piperidine:DMF volume ratio of 1:4; the deprotection method comprises adding an Fmoc removal reagent for deprotection for 5 to 10 minutes, washing with DMF, and then adding the Fmoc removal reagent again for deprotection for 5 to 10 minutes and washing with DMF.

[0047] S12) coupling an Fmoc-protected amino acid or a special material; the coupling agent for coupling the protected amino acid is a combination of DIC and HOBt, and the amount of DIC and HOBt is 1.2 times the molar amount of the protected amino acid.

[0048] The coupling agents for the special materials Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-eicosanedioic acid(mon-tBu))-OH, Fmoc-Ile-Aib-OH, and Boc-Tyr(tBu)-Aib-OH were a combination of DIC, HOBt, and DIPEA. The molar ratio of the special materials, DIC, HOBt, and DIPEA during the coupling was 1:1.2:1.2:0.1.

[0049] The fully protected tilpotide peptide resin synthesized in step S2) is cleaved, sedimented, filtered, and dried to obtain tilpotide; the cleavage is performed using a mixed solution of TFA, TIPS, EDT, and H2O, with the volume ratio of TFA, TIPS, EDT, and H2O being 91:3:3:3. In step S2), the sedimentation is performed by adding 8 times the volume of methyl tert-butyl ether to the cleavage reaction system for sedimentation; and the filtration is performed using filter cloth.

[0050] Furthermore, the method may further include a step of purifying the synthesized tilpotide.

[0051] Example 1

[0052] 23.3 g of Rink amide MBHA Resin amino acid resin with a substitution degree of 0.43 mmol / g was selected and added to the reaction column. DMF was added to swell for 30 minutes, dried, and 20% piperidine / DMF solution was added to remove Fmoc twice (10 minutes + 10 minutes). After removal, DMF was washed 5 times. Fmoc-Ser(tBu)-OH (11.5 g, 30 mmol) and HOBt (4.86 g, 36 mmol) were weighed and added to the reaction column. DMF was dissolved, and DIC (5.63 mL, 36 mmol) was added. The reaction was allowed to react at room temperature for 1 hour. The reaction was complete when detected by ninhydrin. The reaction was filtered and washed 5 times with an appropriate amount of DMF. The above operation was repeated for coupling of other amino acids and special materials. The reaction time for conventional amino acids was 1 hour. When coupling the special materials Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-eicosanedioic acid(mon-tBu))-OH, Fmoc-Ile-Aib-OH, and Boc-Tyr-Aib-OH, 0.2 eq. DIPEA was added to promote the reaction. The coupling times were 3 hours, 3 hours, and 2 hours, respectively. After the coupling was completed, methyl tert-butyl ether was added to shrink the resin, and finally 91.5 g of peptide resin was obtained:

[0053] Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tB u)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-eicosane dioic acid(mon-tBu))-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink amide MBHA resin.

[0054] The specific coupling conditions are as follows:

[0055] Cleavage Step: This technique uses a cleavage reagent ratio of TFA / TIDPS / EDT / H2O (91 / 3 / 3 / 3), with the volume of the cleavage reagent being 8 times the weight of the resin. Cleavage is carried out at room temperature for 2 hours. The resin is removed by filtration. Methyl tert-butyl ether is added to the cleavage solution at a volume of 8 times the volume of the cleavage solution (v / v). After sedimentation, filtration, and cake drying, 48.0 g of crude peptide product is obtained, with a yield of 99.8%, a purity of 81.73%, and a content of 77.4%. (Figure 2)

[0056] Comparative Example 1

[0057] 23.3g of Rink amide MBHA Resin amino acid resin with a substitution degree of 0.43mmol / g was selected and added to the reaction column. DMF was added to swell for 30 minutes, dried, and 20% piperidine / DMF solution was added to remove Fmoc twice (10 minutes + 10 minutes). After removal, DMF was washed 5 times. Fmoc-Ser(tBu)-OH (11.5g, 30mmol) and HOBt (4.86g, 36mmol) were weighed and added to the reaction column. DMF was dissolved and DIC (5.63mL, 36mmol) was added. The reaction was allowed to react at room temperature for 1 hour. The reaction was detected by ninhydrin. The reaction was filtered and washed 5 times with an appropriate amount of DMF. The above operation was repeated to couple other amino acids. After the coupling was completed, methyl tert-butyl ether was added to shrink the resin, and finally 72g of peptide resin was obtained:

[0058] Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tB u)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-eicosane dioic acid(mon-tBu))-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink amide MBHA resin.

[0059] The specific coupling conditions are as follows:

[0060] Cleavage Step: This technique uses a cleavage reagent ratio of TFA / TIDPS / EDT / H2O (91 / 3 / 3 / 3), with the volume of the cleavage reagent being 8 times the weight of the resin. Cleavage is carried out at room temperature for 2 hours. The resin is removed by filtration. Methyl tert-butyl ether is added to the cleavage solution at a volume of 8 times the volume of the cleavage solution (v / v). After sedimentation, filtration, and cake drying, 29.0 g of crude peptide product is obtained, with a yield of 60.3%, a purity of 47.68%, and a content of 32.0%. (Figure 3)

[0061] Comparison of Example 1 with Comparative Example 1 demonstrates that the present invention utilizes specialized material fragments to improve synthesis efficiency, shortening peptide resin synthesis from 43 steps to 37, increasing yield from 60% to 99.8%, and nearly doubling purity. The present invention achieves significant improvements in synthesis efficiency and purity simply by adjusting the synthesis sequence and utilizing specialized materials.

Claims

1. A solid phase synthesis method of tepote, characterized in that: The solid phase synthesis invention comprises the following steps: S1) using an amide resin as a solid phase carrier and using an Fmoc solid phase synthesis strategy to sequentially couple Fmoc-protected amino acids or special materials to obtain a fully protected telpoxetine peptide resin; S2) the synthesized fully protected telpotide peptide resin is cracked, precipitated, filtered and dried to obtain telpotide; The amino acids or special materials protected by Fmoc are sequentially coupled as follows: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-eicosanedioic acid(mon-tBu))-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-Aib-OH, Fmoc-Ser(tBu)-OH, Fm oc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Boc-Tyr-Aib-OH.

2. The solid phase synthesis method according to claim 1, characterized in that The method of the Fmoc solid phase synthesis strategy in step S1) comprises: S11) removing the Fmoc protecting group; S12) coupling Fmoc-protected amino acids or special materials; Wherein, in S11), 20% piperidine / DMF is used as the Fmoc removal reagent; 20% piperidine / DMF is a mixed solution in which the volume ratio of piperidine to DMF is 1:4; The method for coupling the Fmoc-protected amino acid or special material in S12) is to carry out the coupling under the action of an amino acid or special material coupling agent.

3. The solid phase synthesis method according to claim 2, characterized in that The deprotection method in S11) is to add an Fmoc removal reagent for deprotection for 5 to 10 minutes, wash with DMF, add the Fmoc removal reagent again for deprotection for 5 to 10 minutes and wash with DMF.

4. The solid phase synthesis method according to claim 2, characterized in that The coupling agent for coupling the protected amino acid is a combination of DIC and A or a combination of DIPEA, A and B, wherein A is Oxyma, HOBt or HOAt, and B is one of PyBOP, PyAOP, HATU, HBTU, and TBTU; preferably, it is a combination of DIC and HOBt.

5. The solid phase synthesis method according to claim 4, characterized in that When coupling the protected amino acid, the molar amount of the coupling agent is 1-1.5 times the molar amount of the amino acid.

6. The solid phase synthesis method according to claim 2, characterized in that The coupling agent for coupling the special materials Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-eicosanedioic acid(mon-tBu))-OH, Fmoc-Ile-Aib-OH and Boc-Tyr(tBu)-Aib-OH is a combination of DIC, HOBt and DIPEA.

7. The solid phase synthesis method according to claim 6, characterized in that The molar ratio of special materials, DIC, HOBt and DIPEA when coupling special materials is 1:1-1.5:1-1.5:0.1-0.

5. The preferred ratio is 1:1.2:1.2:0.

1.

8. The solid phase synthesis method according to claim 1, characterized in that The amide resin in S1) is Rink amide resin, Rink amide AM resin, Rink amide MBHA resin, or Sieber resin. Among them, Rink amide MBHA resin and Sieber resin are preferred; Preferably, the degree of substitution of the amide resin in S1) is 0.2 to 0.8 mmol / g.

9. The solid phase synthesis method according to claim 1, characterized in that S2) is cleaved by a mixed solution of TFA, TIPS, EDT and H2O, Preferably, the volume ratio of TFA, TIPS, EDT and H2O is 91:3:3:

3.

10. The solid phase synthesis method according to claim 1, characterized in that S2) The sedimentation method is to add excess methyl tert-butyl ether into the cracking reaction system for sedimentation; Preferably, the amount of methyl tert-butyl ether added is 5-10 times the volume of the cracking reaction system.

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