A method for the synthesis of icvastatin

Abstract

The application discloses a synthetic method of icaritin, and belongs to the technical field of polypeptide drug synthesis. The method uses Sieber resin as a solid phase carrier, and amino acid monomers and Fmoc-Nal-THP-Glu(OtBu)-OH fragments are coupled in a specific order to obtain a linear peptide resin through acetylation; a disulfide bond is formed on the solid phase through iodine oxidation to obtain a cyclic peptide resin, and then the target product is obtained through TFA mixed reagent cleavage and methyl tert-butyl ether sedimentation separation. The solid phase synthesis and cyclization process are optimized, the polymer impurities of difficult site coupling and liquid phase cyclization are avoided, the production cycle is shortened, the purity of the obtained crude peptide reaches 86.44%, the weight yield reaches 100%, and the method is suitable for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drug synthesis technology, specifically relating to a method for preparing Icotrokinra. Background Technology

[0002] Icotrokinra is an IL-23 receptor inhibitor developed by Johnson & Johnson and Protecton Therapeutics. A marketing application was submitted in the United States in July 2025 for the treatment of moderate to severe plaque psoriasis (PsO) in adults and pediatric patients aged 12 years and older. In China, it was launched on November 19, 2025, for the treatment of moderate to severe plaque psoriasis and was included in the priority review list.

[0003] Icotrokinra is a first-in-class targeted oral peptide drug that inhibits the IL-23 signaling pathway by selectively blocking the IL-23 receptor, thereby exerting its immunomodulatory effects. IL-23 is a key inflammatory factor in many autoimmune diseases, including psoriasis, and its pathway has long been primarily inhibited by injectable biologics (such as ustekinumab and guselkumab). Icotrokinra's structure is based on a cyclic peptide backbone, designed with dual optimization for gastrointestinal stability and receptor affinity. In vitro, it exhibits picomolar-level IL-23R binding capacity, demonstrating excellent selectivity and functional inhibitory efficiency. Traditionally, the IL-23 pathway has been a target for many biologics, but icotrokinra's advantage lies in its oral peptide formulation, offering a more convenient treatment option compared to existing subcutaneous or intravenous injections. Furthermore, Johnson & Johnson plans to expand icotrokinra to the treatment of other immune diseases, such as ulcerative colitis and Crohn's disease, further validating its broad application potential in the field of immunomodulation. Therefore, this drug has huge market demand in the future, and there is an urgent need for a more efficient synthesis method suitable for large-scale production to prepare the molecule.

[0004] The structural formula of Icosin is shown below:

[0005] The published process patents CN115298196A and CN120905324A both use Fmoc solid-phase synthesis to synthesize linear peptides. In both cases, Rink amide MBHA resin is selected as the resin. The process involves: removal of Fmoc protecting groups, resin washing, amino acid coupling, resin washing, and then sequential coupling with Fmoc-Sar-OH, Fmoc-3Pal-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-THP-OH, Fmoc-Nal-OH, Fmoc-AEF-OH, Fmoc-Pen(Trt)-OH, Fmoc-Lys(Ac)-OH, Fmoc-(7Me)Trp-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pen(Trt)-OH, and acetylation to synthesize linear peptide resins. Linear peptide resin is cleaved to obtain crude linear peptides, which are then oxidized with iodine in a liquid phase to obtain crude cyclic peptides.

[0006] This type of synthesis method has the following shortcomings: The process includes: solid-phase synthesis of linear peptides - linear peptide cleavage - liquid-phase oxidation of linear peptides - crude cyclic peptides. The process involves multiple steps and a long cycle time.

[0007] Rink amide MBHA resin is prone to producing M+163 impurities, which can affect product yield and finished product quality.

[0008] THP has a tetrahydropyran structure, which has large steric hindrance and is difficult to couple. Similarly, due to the large steric hindrance, the subsequent Fmoc-Nal-OH is also difficult to couple. Moreover, after THP removes the Fmoc protecting group, conventional colorimetric methods, such as the Kaiser test and tetrachlorobenzoquinone, are difficult to perform colorimetric analysis, making it impossible to accurately determine the reaction endpoint of Fmoc-Nal-OH.

[0009] The oxidation and cyclization of disulfide bonds occur under liquid phase conditions. When the concentration of linear peptides is too high, it will lead to the formation of a large number of intermolecular polymers; when the concentration of linear peptides is too low, the volume of the liquid phase reaction will be very large, which is not suitable for large-scale production. Summary of the Invention

[0010] To address the above problems, this invention creatively proposes: using Sieber resin as a solid-phase carrier, and Fmoc-Nal-THP-Glu(OtBu)-OH as a coupling unit, to perform oxidation and cyclization under solid-phase conditions.

[0011] This invention provides a method for synthesizing icosperidin, comprising the following steps: S1) Using Sieber resin as a solid-phase support, a solid-phase synthesis strategy is employed to sequentially couple amino acid monomers or structural units, followed by acetylation, to obtain a fully protected linear peptide resin; the amino acid monomers or structural units mentioned in step S1) are, in sequence: Fmoc-Sar-OH, Fmoc-3Pal-OH, Fmoc-Asn(Trt)-OH, Fmoc-Nal-THP- Glu(OtBu)-OH, Fmoc-AEF-OH, Fmoc-Pen(Trt)-OH, Fmoc-Lys(Ac)-OH, Fmoc-(7Me)Trp-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pen(Trt)-OH; S2) The linear peptide resin is oxidized to form disulfide bonds on the solid support to obtain a cyclic peptide resin. S3) The cyclic peptide resin is cleaved to separate the icospermia syringin.

[0012] Further, the degree of substitution of the Sieber resin in step S1) is 0.3~1.8 mmol / g. Preferably, it is 0.7-0.8 mmol / g.

[0013] Further, in step S1), the solid-phase synthesis strategy is a synthesis strategy that uses the Fmoc protecting group as the amino protecting group of the α-amino group of the amino acid monomer or the amino protecting group of the N-terminal amino group of the structural unit, and constructs the polypeptide chain directionally on the solid-phase support by cyclic operations of removing the Fmoc protecting group on the amino acid to be coupled, activating the amino group, and coupling.

[0014] Further, in step S1), in the solid-phase synthesis strategy, the removal of the Fmoc protecting group is a basic selective removal, the deprotecting agent is a 20% piperidine / N,N-dimethylformamide mixed solution, and the deprotecting conditions are to react twice at room temperature, each time for 10~15 min.

[0015] Further, in step S1), the cyclic operation process of the solid-phase synthesis strategy is as follows: after the solid-phase support is swollen and the protecting group is completely removed by the deprotecting reagent, the amino acid or structural unit containing Fmoc protection is activated by the activation system and then coupled with the solid-phase support. The deprotection and activation coupling operations are repeated, and the amino acid or structural unit of Icosin is coupled in the order from the C-terminus to the N-terminus to obtain a linear peptide resin.

[0016] Further, in step S1), the acetylation method is to add acetic anhydride and TEA, and react until complete.

[0017] Furthermore, in step S1), the molar ratio of the acetylated peptide resin to acetic anhydride and TEA is 1:(8-12):(8-12), preferably 1:10:10.

[0018] Furthermore, in step S1), methyl tert-butyl ether is added after acetylation to induce shrinkage.

[0019] Furthermore, the oxidation treatment in step S2) uses iodine (I2) as the oxidant.

[0020] Furthermore, in step S2), the molar ratio between the oxidant and the linear peptide resin obtained in step S1) is 4-8:1, preferably 5:1.

[0021] Further, the pyrolysis reagent used in step S3) is a mixture of trifluoroacetic acid (TFA), anisole, phenol and water.

[0022] Furthermore, in step S3), the volume ratio of each component in the pyrolysis reagent is 85-95:4-6:2-4:1.5-2.5 for trifluoroacetic acid: benzyl sulfide: phenol: water, preferably 90:5:3:2.

[0023] Further, in step S3), after the cyclic peptide resin obtained in step S2) is cleaved, the resin is removed by filtration, methyl tert-butyl ether is added, and after precipitation, it is filtered and separated to obtain Icosin.

[0024] Furthermore, the coupling of the amino acid monomers in step S1) employs an activation system. More specifically, in step S1), the activation system includes an activator and a condensing agent. The activator is selected from one of HOBt, HOAt, Oxyma Pure, and DIPEA, and the condensing agent is selected from one of DIC, PyBOP, PyAOP, HBTU, and HATU.

[0025] Further, in step S1), the activation system is one of the following: a combination of HOBt and DIC, a combination of HOAt and DIC, a combination of Oxyma Pure and DIC, a combination of PyBOP, HOBt, and DIPEA, a combination of PyAOP, HOBt, and DIPEA, a combination of HBTU, HOBt, and DIPEA, or a combination of HATU, HOBt, and DIPEA.

[0026] Furthermore, the molar ratio of each component in the activation system is amino acid: activator: condensing agent = 1.0:(0.8~1.4):(0.8~1.4), preferably 1.0:1.2:1.2.

[0027] Beneficial effects The process involves: solid-phase synthesis of cyclic peptides - cyclic peptide cleavage - crude cyclic peptide. This process has fewer steps, requires less production equipment, and shortens the production cycle.

[0028] The applicant discovered that the M+163 impurity generated by using Rink amide MBHA resin is due to the production of 2-(4-methylphenoxy)acetamide cations during the cleavage of the peptide resin. These cations bind to the tryptophan position 2 or other sites in the peptide chain. The applicant found that the linker structure of Sieber resin has good stability during cleavage and does not produce impurities from crude peptides and linker adsorption, thus improving the yield and product quality.

[0029] The applicant unexpectedly discovered that by using the fragment Fmoc-Nal-THP-Glu(OtBu)-OH, the yield was significantly improved.

[0030] Using iodine catalysis to remove the protecting group of the Trt side chain in Pen under solid-phase conditions simultaneously forms a disulfide bond, achieving two goals at once. Detailed Implementation

[0031] The following detailed description of the present invention through specific embodiments illustrates the preferred aspects of the invention. It should be noted that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. The following detailed description of the preferred features and performance of the present invention, in conjunction with the embodiments, provides further details.

[0032] Example 1 Synthesis of Cyclic Peptide Resins Sieber Resin resin (13.3 g, 10 mmol) with a degree of substitution of 0.75 mmol / g was added to the reaction column, and DMF was allowed to swell for 5 minutes. After 5 minutes, the DMF was removed, and the column was deprotected twice with 20% piperidine (10 minutes + 10 minutes). The solvent was then removed, and the column was washed five times with DMF before the solvent was removed again.

[0033] Fmoc-Sar-OH (9.3 g, 30 mmol) and HOBt (4.86 g, 36 mmol) were added to the reaction column, dissolved in DMF, and then DIC (5.63 mL, 36 mmol) was added. The reaction was carried out at room temperature for 1 hour. The reaction was confirmed to be complete by tetrachlorobenzoquinone. The mixture was filtered and washed 5 times with an appropriate amount of DMF. The above operation was repeated to couple other amino acids.

[0034] The specific coupling conditions for each amino acid are as follows:

[0035] The structural formula of Fmoc-Nal-THP-Glu(OtBu)-OH is shown below. .

[0036] After coupling was completed, methyl tert-butyl ether was added for shrinkage. The final yield was 36.3 g of peptide resin.

[0037] Cyclic peptide resin cleavage The lysis reagent used was TFA : anisole sulfide : phenol : H2O = 90 : 5 : 3 : 2 (volume ratio), with the volume of the lysis reagent being 8 times the weight of the resin. Lysis was performed at room temperature for 2 hours. The resin was removed by filtration. Methyl tert-butyl ether was added to the lysis buffer. The amount of methyl tert-butyl ether added was 8 times the volume of the lysis buffer (v / v). After sedimentation, filtration, and drying of the filter cake, 19.1 g of crude peptide product was finally obtained, with a yield of 100% by weight and a purity of 86.44%.

[0038] The purity and content were tested by HPLC. The results showed that the method of the present invention can achieve high purity and high yield without further purification.

[0039] Example 2 The difference from Example 1 is as follows: During coupling, Fmoc-Glu(OtBu)-OH, Fmoc-THP-OH, and Fmoc-Nal-OH are used instead of Fmoc-Nal-THP-Glu(OtBu)-OH. The coupling conditions for Fmoc-Glu(OtBu)-OH, Fmoc-THP-OH, and Fmoc-Nal-OH are consistent with the coupling conditions for the Fmoc-Nal-THP-Glu(OtBu)-OH fragment. The final yield of crude peptide product was 17.1g, with a weight yield of 90.3% and a purity of 63.07%.

[0040] Example 3 The difference from Example 1 is as follows: Rink amdie MBHA resin was selected as the carrier, and the selection and coupling method of other amino acids were the same as in Example 1.

[0041] The final yield of crude peptide product was 17.4g, with a weight yield of 91.8% and a purity of 63.12%.

[0042] The contents of crude peptides and impurities obtained in Examples 1-3 are shown in the table below.

[0043] This invention clearly verifies the independent effects and synergistic gain effects of the two core improvements through three sets of embodiments and single-variable comparisons: In Example 2, the pre-prepared Fmoc-Nal-THP-Glu(OtBu)-OH fragments were replaced with Glu(OtBu), THP, and Nal for stepwise coupling. The rest were processed using Sieber resin and solid-phase oxidation cyclization. The final crude peptide yield was reduced to 90.3% and the purity was only 63.07%. 3.41% of Des-Nal deletion impurities were detected, confirming that the THP group has large steric hindrance, the stepwise coupling reaction is incomplete, and the coupling endpoint cannot be determined by colorimetric analysis after deprotection. The pre-prepared integrated fragments can eliminate amino acid deletion impurities at the source and improve the integrity of the coupling.

[0044] Example 3 retained the three advantageous fragments, only replacing them with traditional Rinkamide MBHA resin. The crude peptide yield was 91.8% and the purity was 63.12%. However, 6.69% of difficult-to-remove M+163 resin-derived impurities were generated, indicating that the Rink resin linker arms easily generate active cations and add to the peptide chain during TFA cleavage. In contrast, the Sieber resin linker arms have higher cleavage stability and can completely avoid this unique process impurity. Comparing the two, optimizing only the fragments or simply changing the resin can only eliminate a single type of impurity, and there is a significant difference in crude product purity and yield. The only drawback is that Example 1, which simultaneously uses a Sieber solid-phase carrier and a pre-prepared three-fragment coupling unit, can achieve the detection of both key impurities, Des-Nal and M+163, with a crude peptide weight yield of 100% and a crude product purity of 86.44%. This fully demonstrates that the optimized resin selection and the pre-prepared functional fragment design have a synergistic effect. Both improvements are indispensable and together solve the industrial pain points of existing processes, such as high impurities, low yield, and high purification pressure. This also indirectly proves that the combined improvement scheme of this invention has outstanding technical advantages and large-scale production value.

[0045]

Claims

1. A method for synthesizing icosperidin, characterized in that, It includes the following steps: S1) Using Sieber resin as a solid-phase support, a solid-phase synthesis strategy was employed to sequentially couple Fmoc-protected amino acid monomers or amino acid fragment structural units from the N to C ends of the peptide sequence of Icosin, followed by acetylation to obtain a fully protected linear peptide resin; the amino acid fragment mentioned in step S1) is Fmoc-Nal-THP-Glu(OtBu)-OH: S2) The linear peptide resin is oxidized to form disulfide bonds on the solid support to obtain a cyclic peptide resin. S3) The cyclic peptide resin is cleaved to separate the icospermia syringin.

2. The synthesis method according to claim 1, characterized in that, The degree of substitution of the Sieber resin in step S1) is 0.3~1.8 mmol / g, preferably 0.7-0.8 mmol / g.

3. The synthesis method according to claim 1, characterized in that, In step S1), the solid-phase synthesis strategy is a synthesis strategy that uses the Fmoc protecting group as the amino protecting group of the α-amino group of the amino acid monomer or the amino protecting group of the N-terminal amino group of the amino acid fragment, and constructs the polypeptide chain directionally on the solid-phase support by cyclic operations of removing the Fmoc protecting group on the amino acid to be coupled, activating the amino group, and coupling.

4. The synthesis method according to claim 1, characterized in that, In step S1), the solid-phase synthesis strategy involves the removal of the Fmoc protecting group via alkaline selective removal. Preferably, the deprotecting agent is a 20% piperidine / N,N-dimethylformamide mixed solution, and the deprotecting condition is two reactions at room temperature.

5. The synthesis method according to claim 1, characterized in that, In step S1), the cyclic operation process of the solid-phase synthesis strategy is as follows: after the solid-phase support is swollen and the protecting group is completely removed by the deprotecting reagent, the amino acid monomer or structural unit containing Fmoc protection is activated by the activation system and then coupled with the solid-phase support. The deprotection and activation coupling operations are repeated, and the amino acids or structural units of the icosperidin are coupled in the order from the C-terminus to the N-terminus to obtain the linear peptide resin.

6. The synthesis method according to claim 1, characterized in that, The coupling of the amino acid monomers in step S1) is performed using an activation system; Preferably, in step S1), the activation system includes an activator and a condensing agent, wherein the activator is selected from HOBt, HOAt, and Oxyma Pure, and the condensing agent is selected from DIC, DIPEA, PyBOP, PyAOP, HBTU, and HATU. Preferably, in step S1), the activation system is one of the following: a combination of HOBt and DIC, a combination of HOAt and DIC, a combination of OxymaPure and DIC, a combination of PyBOP, HOBt, and DIPEA, a combination of PyAOP, HOBt, and DIPEA, a combination of HBTU, HOBt, and DIPEA, or a combination of HATU, HOBt, and DIPEA. Preferably, the molar ratio of each component in the activation system is amino acid: activator: condensing agent = 1.0:(0.8~1.4):(0.8~1.4).

7. The synthesis method according to claim 1, characterized in that, In step S1), the acetylation method is to add acetic anhydride and TEA, and react until complete; Preferably, in step S1), the molar ratio of peptide resin to acetic anhydride and TEA in the acetylation process is 1:(8-12):(8-12); Preferably, in step S1), methyl tert-butyl ether is added after acetylation to induce shrinkage.

8. The synthesis method according to claim 1, characterized in that, The oxidation treatment described in step S2) uses iodine (I2) as the oxidant; Preferably, the molar ratio between the oxidant and the linear peptide resin obtained in step S1) during the oxidation treatment in step S2) is 4-8:

1.

9. The synthesis method according to claim 1, characterized in that, The pyrolysis reagent used in step S3) is a mixture of trifluoroacetic acid (TFA), anisole, phenol and water; Preferably, in step S3), the volume ratio of each component in the pyrolysis reagent is 85-95:4-6:2-4:1.5-2.5 for trifluoroacetic acid: benzyl sulfide: phenol: water.

10. The synthesis method according to claim 1, characterized in that, In step S3), the cyclic peptide resin obtained in step S2) is cleaved, the resin is removed by filtration, methyl tert-butyl ether is added, and after precipitation, it is separated by filtration to obtain icosperidin.

Images