Preparation method of Retatrutide

Abstract

The invention discloses a preparation method of Retatrutide, and belongs to the technical field of polypeptide synthesis. The invention relates to a preparation method of Retatrutide, which comprises the following steps: by taking Sieber amide resin as a coupling resin carrier, sequentially coupling and inoculating Fmoc protected amino acid and dipeptide and tripeptide protected amino acid fragments, and then cutting the resin to prepare a peptide fragment I, namely an intermediate M1; cTC resin is used as a coupling resin carrier, Fmoc protected amino acid and oligopeptide protected amino acid fragments are sequentially coupled and connected, then the resin is cut, and a peptide fragment II, namely an intermediate M2, is prepared; coupling the intermediate M1 and the intermediate M2 to prepare full-protection Retatrutide, namely an intermediate M3, and cracking the intermediate M3 by using a cracking agent to remove a protective functional group, so as to obtain a crude Retatrutide product; and dissolving, filtering, purifying, and freeze-drying to obtain a Retatrutide refined product. Two long peptide fragments and a plurality of protected amino acid fragments are used for preparing Retatrutide, the generation of impurities is reduced, the single impurity is small, the purification difficulty is reduced, the product purity is ensured, and the purity of the obtained product is greater than 98.0%.

Description

Technical Field

[0001] The invention relates to the technical field of polypeptide synthesis, and in particular to a method for preparing Retatrutide. Background Art

[0002] Retatrutide, also known as retaglutide, is a multi-receptor agonist of glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide 1 (GLP-1) and glucagon (GCG).

[0003] It can be seen that Retatrutide is a hypoglycemic drug, mainly used to improve blood sugar control in patients with type 2 diabetes. It is a glucagon-like peptide-1 (GLP-1) analog, which has similar effects to natural GLP-1 and achieves the effect of lowering blood sugar by simulating the effects of natural GLP-1.

[0004] It has significant effects on promoting insulin secretion, inhibiting glucagon secretion, delaying gastric emptying, and suppressing appetite.

[0005] Therefore, Retatrutide, taking advantage of its properties, has a wide range of usage scenarios as a raw material for pharmaceutical products. Retatrutide is generally prepared chemically during the production process. However, the existing preparation methods are prone to impurities during the preparation process, which increases the difficulty of purification and makes it impossible to guarantee the purity of the product, thereby keeping the purity of the prepared Retatrutide at a low level for a long time. Summary of the invention

[0006] The purpose of the present invention is to solve the problem in the prior art that impurities are easily generated during the preparation process, which increases the difficulty of purification and makes it impossible to ensure the purity of the product, thereby keeping the purity of the prepared Retatrutide at a low level for a long time, and a preparation method of Retatrutide is proposed.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for preparing Retatrutide comprises the following steps:

[0009] Step 1: Using Sieber amide resin as a coupling resin carrier, sequentially coupling and connecting Fmoc-protected amino acids and tripeptide-protected amino acid fragments, and then cutting the resin to prepare peptide fragment 1, which is intermediate M1;

[0010] Step 2: Using CTC resin as a coupling resin carrier, sequentially coupling and connecting Fmoc-protected amino acids and short peptide-protected amino acid fragments, and then cutting the resin to prepare peptide fragment 2, i.e., intermediate M2;

[0011] Step 3: Couple the intermediate M1 and the intermediate M2 to prepare the fully protected Retatrutide, namely the intermediate M3.

[0012] The intermediate M3 is cleaved with a cleavage agent to remove the protective functional group to obtain crude Retatrutide;

[0013] Step 4: Dissolve, filter, purify and freeze-dry the crude Retatrutide to obtain refined Retatrutide.

[0014] Preferably, the dipeptide and tripeptide protected amino acid fragments are: Fmoc-Pro-Pro-Pro-OH, Fmco-Ser(tbu)-Gly-OH, Fmoc-Gly-Gly-OH;

[0015] The intermediate M1 is:

[0016] NH2-Phe-Ile-Glu(otbu)-Tyr(tbu)-Leu-Leu-Glu(otbu)-Gly-Gly-

[0017] Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH2.

[0018] Preferably, the short peptide protected amino acid fragment is Fmoc-Gln(trt)-Aib-OH, Fmoc-Ile-αMeL-OH, Fmoc-Gln(trt)-Gly-OH, Boc-Tyr(tbu)-Aib-OH, Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-OH;

[0019] The intermediate M2 is:

[0020] Boc-Tyr(tbu)-Aib-Gln(trt)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Se r(tbu)-Ile-αMeL-Leu-Asp(otbu)-Lys(boc)-{Boc-Eicosanedioicacid-γ-Glu(otbu)-AEEA-Lys}

[0021] -Ala-Gln(trt)-Aib-Ala-COOH.

[0022] Preferably, the intermediate M3 is:

[0023] Boc-Tyr(tbu)-Aib-Gln(trt)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tbu)-Ile-αMeL-Leu-Asp(otbu)-Ly s(boc)-{Boc-Eicosanedioicacid-γ-Glu(otbu)-AEEA-Lys}-Ala-Gln(trt)-Aib-Ala-Phe-Ile-Glu(otbu)-Tyr(tbu)-Leu-Leu-Glu(ot bu)-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH2.

[0024] Preferably, the protected amino acid fragment is Fmoc-Pro-Pro-Pro-OH, Fmco-Ser(tbu)-Gly-OH, Fmoc-Gly-Gly-OH, Fmoc-Gln(trt)-Aib-OH, Fmoc-Ile-αMeL-OH, Fmoc-Gln(trt)-Gly-OH, Boc-Tyr(tbu)-Aib-OH, Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-OH.

[0025] Preferably, in step (1), the amount of the protected amino acid and the protected amino acid fragment is 1.5 to 4 times the total molar number of the charged resin; in step (2), the amount of the protected amino acid and the protected amino acid fragment is 3 to 5 times the total molar number of the charged resin.

[0026] Preferably, in step 1, the deprotection reagent for removing Fmoc protection is a PIP / DMF mixed solution, the mixed solution contains 20% to 30% (V / V) piperidine, the amount of the deprotection reagent is 1.2 to 1.5 times the volume of the peptide resin, the deprotection number is 1, and the deprotection time is 20 to 30 minutes; in step 2, the deprotection reagent for removing Fmoc protection is a PIP / DMF mixed solution, the mixed solution contains 20% to 30% (V / V) piperidine, the amount of the deprotection reagent is 1.2 to 1.5 times the volume of the peptide resin, the deprotection number is 2, the first deprotection time is 5 to 10 minutes, and the second deprotection time is 10 to 20 minutes.

[0027] Preferably, in step 1, the coupled Sieber amide resin has a resin substitution degree in the range of 0.3 to 0.8 mmol / g; in step 2, the coupled CTC resin has a resin substitution degree in the range of 0.5 to 0.8 mmol / g.

[0028] Preferably, in step three, the cracking agent is a mixed solvent of trifluoroacetic acid, 1,2-ethanedithiol, triisopropylsilane and water; the mixed solvent contains 90% to 95% TFA, 1% to 4% EDT and 1% to 4% TIS, the amount of the cracking agent consumed per gram of intermediate M3 is 5 to 10 ml, and the cracking time is 2 to 3 hours at room temperature.

[0029] Preferably, the crude solution of Retatrutide is purified by high performance liquid chromatography, the purification chromatographic column is a reverse octadecyl bonded silica gel; the mobile phases are ammonium acetate solution and acetonitrile solution, respectively; the salt conversion step is carried out by high performance liquid chromatography, and the mobile phase system is ammonium acetate solution-acetonitrile solution.

[0030] Compared with the prior art, the present invention provides a method for preparing Retatrutide, which has the following beneficial effects:

[0031] 1. The preparation method of Retatrutide uses two long peptide fragments and multiple protected amino acid fragments to prepare Retatrutide, which reduces the generation of impurities. The single impurity is relatively small, which reduces the difficulty of purification and ensures the purity of the product. The purity of the obtained product is greater than 98.0%. Compared with the prior art, the process of the present invention has the characteristics of simple reaction operation and mild reaction conditions, and has broad practical value and application prospects;

[0032] 2. The preparation method of Retatrutide uses industrially produced protected amino acids and independently synthesized protected amino fragments as raw materials for coupling reactions, thus avoiding the problems of peptide resin folding and low coupling yield in the prior art during synthesis, shortening the production process steps for Retatrutide and reducing production costs;

[0033] 3. The preparation method of Retatrutide, the overall access of the side chain functional groups, avoids the coupling difficulties caused by the folding of the main chain peptide chain, and improves the synthesis yield of the peptide resin. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a flow chart for preparing intermediate M1 proposed by the present invention;

[0035] Figure 2 This is a flow chart for preparing intermediate M2 proposed by the present invention;

[0036] Figure 3This is a flow chart for preparing intermediate M3 proposed by the present invention. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0038] In addition, reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0039] Example:

[0040] Reference Figure 1-Figure 3 , a method for preparing Retatrutide, comprising the following steps:

[0041] Step 1: Using Sieber amide resin as a coupling resin carrier, sequentially coupling and connecting Fmoc-protected amino acids and dipeptide and tripeptide-protected amino acid fragments, and then cutting the resin to prepare peptide fragment 1, which is intermediate M1;

[0042] The dipeptide and tripeptide protected amino acid fragments are: Fmoc-Pro-Pro-Pro-OH, Fmco-Ser(tbu)-Gly-OH, and Fmoc-Gly-Gly-OH;

[0043] Intermediate M1 is:

[0044] NH2-Phe-Ile-Glu(otbu)-Tyr(tbu)-Leu-Leu-Glu(otbu)-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH2.

[0045] The process for preparing intermediate M1 is:

[0046] Preparation process 1: Sieber amide carrier resin (0.6mmol / g, 20mmol) was added to the reactor and washed with DMF three times, 10min / time; Fmoc protection was removed once with 20% PIP / DMF solution, and after detection, DMF was washed 6 times.

[0047] Dissolve Fmoc-Ser(tbu)-OH (40 mmol), HOBt (80 mmol), and DIC (80 mmol) in DMF, add into a solid phase reactor, and react at room temperature for 1-4 h (the reaction end point is based on the result of ninhydrin method). After the reaction is completed, wash with DMF for 6 times, 2-3 min / time; and prepare Fmoc-Ser(tbu)-resin.

[0048] Preparation process 2: The Fmoc-Ser(tbu)-resin prepared in preparation process 1 was deprotected once with 20% PIP / DMF solution. After detection, the resin was washed with DMF 6 times.

[0049] Fmoc-Pro-Pro-Pro-OH (40 mmol), HOBt (40 mmol), Pybop (60 mmol), and DIEA (100 mmol) were dissolved in DMF, added into a solid phase reactor, and reacted at room temperature for 1-4 h (the reaction end point was based on the result of ninhydrin method). After the reaction was completed, the product was washed with DMF for 6 times to obtain Fmoc-Pro-Pro-Pro-Ser(tbu)-resin.

[0050] Preparation process 3: The same reaction method as in preparation process 1 is used to couple a single protected amino acid, and the same reaction method as in preparation process 2 is used to couple a protected amino acid fragment.

[0051] Solid phase coupling one by one: Fmoc-Ala-OH, Fmoc-Ser(tbu)-Gly-OH, Fmoc-Ser(tbu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Glu(otbu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Tyr(tbu)-OH, Fmoc-Glu(otbu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH

[0052] Fmoc-Phe-Ile-Glu(otbu)-Tyr(tbu)-Leu-Leu

[0053] -Glu(otbu)-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-resin.

[0054] After removing the Fmoc protection by the same method, the resin was washed 4 times with DMF and DCM respectively, and then dried in vacuum to obtain NH2-Phe-Ile-Glu(otbu)-Tyr(tbu)

[0055] -Leu-Leu-Glu(otbu)-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-resin.

[0056] Preparation process 4: The dried resin obtained in preparation process 3 was cleaved with 5% TFA / DCM at room temperature for 1-2 hours, filtered, the filtrate was concentrated, precipitated with methyl tert-butyl ether, filtered, and dried to obtain intermediate M1:

[0057] (NH2-Phe-Ile-Glu(otbu)-Tyr(tbu)-Leu-Leu-Glu(otbu)-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH2), 32.26g, HPLC purity 70.1%.

[0058] The amount of the protected amino acid and the protected amino acid fragment is 1.5 to 4 times the total molar number of the resin fed.

[0059] The step-by-step coupling reaction process uses the ninhydrin colorimetric reaction method for in-process control, strictly controlling the reaction time and reducing the generation of impurities during the synthesis process.

[0060] The reagent for the coupling reaction is one of DIC / HOBT, Pybop / HOBT / DIEA, HATU / HOBT / DIEA and OP / DIC; the preferred coupling reagent for connecting a single protected amino acid is DIC / HOBT; the preferred coupling reagent for connecting a fragment is Pybop / HOBT / DIEA; the amount of the coupling reagent is 4 to 6 times the molar number of the carrier resin; the solvent for the coupling reaction is one of DMF, DMSO, DCM or a mixture thereof.

[0061] Step 2: Using CTC resin as a coupling resin carrier, sequentially coupling and connecting Fmoc-protected amino acids and short peptide-protected amino acid fragments, and then cutting the resin to prepare peptide fragment 2, namely intermediate M2;

[0062] The short peptide protected amino acid fragments are Fmoc-Gln(trt)-Aib-OH, Fmoc-Ile-αMeL-OH, Fmoc-Gln(trt)-Gly-OH, Boc-Tyr(tbu)-Aib-OH, and Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-OH.

[0063] Intermediate M2 is:

[0064] Boc-Tyr(tbu)-Aib-Gln(trt)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tbu)-Ile-αMe L-Leu-Asp(otbu)-Lys(boc)-{Boc-Eicosanedioicacid-γ-Glu(otbu)-AEEA-Lys}-Ala-Gln(trt)-Aib-Ala-COOH.

[0065] The process for preparing intermediate M2 is:

[0066] Preparation process 5: Add CTC carrier resin (0.7mmol / g, 40mmol) into the reactor and wash it with DMF three times, 10min / time; dissolve Fmoc-Ala-OH (80mmol) and DIEA (200mmol) in DMF, add them into the solid phase reactor, react at room temperature for 2-4h, after the reaction is completed, wash with DMF 6 times, 2-3min / time, wash with DCM 3 times, take out and dry to obtain Fmoc-Glu(otbu)-Gly-resin; send it for detection of its degree of substitution.

[0067] Preparation process 6: Fmoc-Ala-resin (20mmol) was added back into the reactor, washed with DMF three times, 10min / time, deprotected with 20% PIP / DMF solution twice, and washed with DMF 6 times after the deprotection was completed after detection; Fmoc-Gln(trt)-Aib-OH (60mmol), HOBt (60mmol), Pybop (80mmol), and DIEA (120mmol) were dissolved in DMF, added to the solid phase reactor, and reacted at room temperature for 1-6h (the reaction end point was based on the result of the ninhydrin method), and the reaction was completed, and washed with DMF 6 times, 2-3min / time; Fmoc-Gln(trt)-Aib-Ala-resin was prepared.

[0068] Preparation process 7: The Fmoc-Gln(trt)-Aib-Ala-resin prepared in preparation process 6 was deprotected once with 20% PIP / DMF solution, and after detection, the resin was washed with DMF 6 times;

[0069] Fmoc-Ala-OH (60 mmol), HOBt (100 mmol), and DIC (100 mmol) were dissolved in DMF, added into a solid phase reactor, and reacted at room temperature for 1-4 h (the reaction end point was based on the result of the ninhydrin method). After the reaction was completed, the mixture was washed with DMF for 6 times, 2-3 min / time, to obtain Fmoc-Ala-Gln(trt)-Aib-Ala-resin.

[0070] Preparation process 8: A single protected amino acid is coupled by the same reaction method as in preparation process 7, and a protected amino acid fragment is coupled by the reaction method of preparation process 6; Fmoc-Lys(Dde)-OH, Fmoc-Lys(boc)-OH, Fmoc-Asp(otbu)-OH, Fmoc-Leu-OH, Fmoc-Ile-αMeL-OH, Fmoc-Ser(tbu)-OH, Fmoc-Tyr(tbu)-OH, Fmoc-Asp(otbu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gln(trt)-Gly-OH, and Boc-Tyr(tbu)-Aib-OH are coupled one by one on the solid phase to prepare Boc-Tyr(tbu)

[0071] -Aib-Gln(trt)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tbu)-Ile-αMeL-Leu-Asp(otbu)-Lys(boc)-Lys(Dde)-Ala-Gln(trt)-Aib-Ala-resin.

[0072] Preparation 9: Boc-Tyr(tbu)-Aib-Gln(trt) in Preparation 8

[0073] -Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tbu)-Ile-αMeL-Leu

[0074] -Asp(otbu)-Lys(boc)-Lys(Dde)-Ala-Gln(trt)-Aib-Ala-resin, deprotect Dde twice with 5% hydrazine hydrate / DMF solution, and after detection, wash with DMF 8 times; Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-OH (60mmol), HOBt (60mmol), Pybop (80mmol), DIEA (120mmol) are added to a solid phase reactor and reacted at room temperature for 1-4h (the reaction end point is based on the result of the ninhydrin method). After the reaction is completed, wash with DMF 6 times and DCM 3 times, take out, and dry in vacuum to prepare Boc-Tyr(tbu)-Aib-Gln(trt)-Gly-Thr(tbu)-Phe-Thr(tbu)

[0075] -Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tbu)-Ile-αMeL-Leu-Asp(otbu)-Lys(boc)-{Boc-Eicosanedioic acid-γ-Glu

[0076] (otbu)-AEEA-Lys}-Ala-Gln(trt)-Aib-Ala-resin.

[0077] Preparation process 10: The dried resin obtained in preparation process 9 was cleaved and cut with 1% TFA / DCM at room temperature for 1-2 hours, filtered, and the filtrate was concentrated. It was precipitated with methyl tert-butyl ether, filtered, and dried to obtain intermediate M2: (Boc-Tyr(tbu)-Aib-Gln(trt)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tbu)-Ile-αMeL-Leu-Asp(otbu)-Lys(boc)-{Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-Lys}-Ala-Gln(trt)-Aib-Ala-COOH), 53.5 g, HPLC purity 69.4%.

[0078] The amount of the protected amino acid and the protected amino acid fragment is 2 to 5 times the total molar number of the resin fed.

[0079] In steps 1 and 2, the deprotection reagent for removing Fmoc protection is a PIP / DMF mixed solution, the mixed solution contains 20% to 30% (V / V) piperidine, the amount of the deprotection reagent is 1.2 to 1.5 times the volume of the peptide resin, the deprotection number is 1, and the deprotection time is 20 to 30 minutes;

[0080] In step 1, the coupled Sieber amide resin has a resin substitution degree ranging from 0.3 to 0.8 mmol / g;

[0081] In step 2, the coupled CTC resin has a resin substitution degree ranging from 0.5 to 0.8 mmol / g.

[0082] In step 1 and step 2, the cleavage agent is a TFA / DCM mixed solvent; the mixed solution contains 1% to 5% (V / V) TFA, the amount used is 5 to 10 times the volume of the peptide resin, and the cleavage time is 30 to 90 minutes;

[0083] Step 3: Couple the intermediate M1 and the intermediate M2 to prepare the fully protected Retatrutide, namely the intermediate M3.

[0084] The intermediate M3 is cleaved with a cleavage agent to remove the protective functional group to obtain crude Retatrutide;

[0085] Intermediate M3 is:

[0086] Boc-Tyr(tbu)-Aib-Gln(trt)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tbu)-Ile-αMeL-Leu-Asp(otbu)-Lys(boc)-{Boc-Eicosanedioicacid-γ-G lu(otbu)-AEEA-Lys}-Ala-Gln(trt)-Aib-Ala-Phe-Ile-Glu(otbu)-Tyr(tbu)-Leu-Leu-Glu(otbu)-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH2.

[0087] The process for preparing intermediate M3 is:

[0088] Preparation process 11: M2 (5 mmol), Pybop (6 mmol), and HOBT (5 mmol) were dissolved in DMF, and DIEA (30 mmol) was added to prepare M2 activation solution; M1 (5 mmol) was dissolved in DMF solution, and the M2 activation solution was added dropwise to the M1 solution to couple the two peptide fragments. The reaction process was monitored by HPLC. After the coupling was completed, the solid was precipitated and filtered to obtain the solid, which was dried to obtain the intermediate M3: (Boc-Tyr(tbu)-Aib-Gln(trt)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tbu)-Ile-αMeL-Leu-Asp(otbu)-Lys(boc)-{Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-Lys}-Ala-Gln(trt)-Aib-Ala-Phe-Ile-Glu(otbu)-Tyr(tbu)-Leu-Leu-Glu( otbu)-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH2), 25.5g, HPLC purity 55.2%.

[0089] Preparation process 12: Prepare 200 ml of lysis solution (188 ml of TFA, 4 ml of EDT, 4 ml of TIS, and 4 ml of H2O), add 25.5 g of M3 into the lysis solution, and react at room temperature for 2 hours.

[0090] After the reaction was completed, the mixture was concentrated under reduced pressure, precipitated with methyl tert-butyl ether, filtered, and dried in vacuo to obtain 10.8 g of crude Retatrutide peptide (HPLC purity: 51.1%).

[0091] In step three, the cracking agent is a mixed solvent of trifluoroacetic acid, 1,2-ethanedithiol, triisopropylsilane and water; the mixed solvent contains TFA 90% to 95%, EDT 1% to 4% and TIS 1% to 4%, the amount of the cracking agent consumed per gram of intermediate M3 is 5 to 10 ml, and the cracking time is 2 to 3 hours at room temperature.

[0092] Step 4: Dissolve, filter, purify and freeze-dry the crude Retatrutide to obtain the refined Retatrutide.

[0093] Preparation process 13: purification, salt conversion, concentration and lyophilization of Retatrutide.

[0094] The crude product of Retatrutide was dissolved with ammonium acetate solution, filtered with a 0.45 μm mixed microporous filter membrane after dissolution, and purified for later use;

[0095] Purification was carried out by high performance liquid chromatography. The purification chromatographic column was a C18 column with a diameter of 10 cm, a filler of 10 um eicosyl bonded silica gel, a pore size of , and a mobile phase of ammonium acetate aqueous solution and acetonitrile, respectively. The flow rate was 120 ml / min, the sample load was 5 to 10 g, and the detection wavelength of the chromatograph was 220 nm.

[0096] Take the concentrated solution of Retatrutide purified intermediate and filter it through a 0.45 μm filter membrane for later use;

[0097] The salt was exchanged by high performance liquid chromatography, the mobile phase system was 0.1% ammonia / water solution-acetonitrile solution, and the flow rate was 20 mL / min; gradient elution and cyclic loading method were adopted, the sample was loaded on the chromatographic column, the mobile phase elution was started, the spectrum was collected, the change of absorbance was observed, the main peak of the salt exchange was collected and the purity was detected by analytical liquid phase, the main peak solutions of the salt exchange were combined, concentrated under reduced pressure, and a Retatrutide solution was obtained, which was freeze-dried to obtain 1.95 g of a refined Retatrutide with a purity of 99.0%.

[0098] It should be noted that the protected amino acid fragments are existing, purchased on the market or independently synthesized.

[0099] The crude solution of Retatrutide was purified by high performance liquid chromatography, the chromatographic column for purification was reverse octadecyl bonded silica gel, and the mobile phase was ammonium acetate solution and acetonitrile solution.

[0100] After analysis of experimental data, the present invention adopts two long peptide fragments and liquid phase coupling, which can reduce the difficulty of coupling one by one in total synthesis and improve the product yield; Gly is coupled with a protective fragment to avoid similar diglyceride impurities that are difficult to purify, thereby reducing the difficulty of purification; αMeL and Ile are connected in the form of a protective fragment to avoid the problem of huge steric hindrance between Ile solid phase coupling and improve the synthesis yield.

[0101] The protected amino acids and protected amino acid fragments used in the preparation of peptide fragments of the present invention are: Fmoc-Ser(tbu)-OH, Fmoc-Pro-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Ser(tbu)-Gly-OH, Fmoc-Ser(tbu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Glu(otbu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Tyr(tbu)-OH, Fmoc-Glu(otbu)-OH, Fmoc-Ile-OH, and Fmoc-Phe-OH;

[0102] The protected amino acids and protected amino acid fragments used in the preparation of the peptide fragment II of the present invention are: Fmoc-Ala-OH, Fmoc-Gln(trt)-Aib-OH, Fmoc-Ala-OH, Dde-Lys(Fmoc)-OH or Fmoc-Lys(Dde)-OH, Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-OH, Fmoc-Lys(boc)-OH, Fmoc-Asp(otbu)-OH, Fmoc-Leu-OH, Fmoc-Ile-αMeL-OH, Fmoc-Ser(tbu)-OH, Fmoc-Tyr(tbu)-OH , Fmoc-Asp(otbu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gln(trt)-Gly-OH, Boc-Tyr(tbu)-Aib-OH.

[0103] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the disclosure of the embodiments of the present invention (including the claims) is limited to these examples. In the spirit of the embodiments of the present invention, the technical features in the above embodiments or different embodiments may also be combined, and there are many other changes in different aspects of the embodiments of the present invention as above, which are not provided in detail for the sake of simplicity. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present invention should be included in the protection scope of the embodiments of the present invention.

Claims

1. A method for preparing Retatrutide, characterized in that: The steps include: Step 1: Using Sieber amide resin as a coupling resin carrier, sequentially coupling and connecting Fmoc-protected amino acids, dipeptides and tripeptides protected amino acid fragments, and then cutting the resin to prepare peptide fragment 1, which is intermediate M1; Step 2: Using CTC resin as a coupling resin carrier, sequentially coupling and connecting Fmoc-protected amino acids and short peptide-protected amino acid fragments, and then cutting the resin to prepare peptide fragment 2, namely intermediate M2; Step 3: Couple the intermediate M1 and the intermediate M2 to prepare the fully protected Retatrutide, namely the intermediate M3. The intermediate M3 is cleaved with a cleavage agent to remove the protective functional group to obtain crude Retatrutide; Step 4: Dissolve, filter, purify and freeze-dry the crude Retatrutide to obtain refined Retatrutide.

2. The method for preparing Retatrutide according to claim 1, characterized in that: The dipeptide and tripeptide protected amino acid fragments are: Fmoc-Pro-Pro-Pro-OH, Fmco-Ser(tbu)-Gly-OH, Fmoc-Gly-Gly-OH; The intermediate M1 is: NH2-Phe-Ile-Glu(otbu)-Tyr(tbu)-Leu-Leu-Glu(otbu)-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH2.

3. The method for preparing Retatrutide according to claim 1, characterized in that: The short peptide protected amino acid fragments are Fmoc-Gln(trt)-Aib-OH, Fmoc-Ile-αMeL-OH, Fmoc-Gln(trt)-Gly-OH, Boc-Tyr(tbu)-Aib-OH, Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-OH; The intermediate M2 is: Boc-Tyr(tbu)-Aib-Gln(trt)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tbu)-Ile-αMeL-Leu-Asp(otbu)-Lys(boc)-{Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-Lys}-Ala -Gln(trt)-Aib-Ala-COOH.

4. The method for preparing Retatrutide according to claim 1, characterized in that: The intermediate M3 is: Boc-Tyr(tbu)-Aib-Gln(trt)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Tyr(tbu)-Ser(tbu)-Ile-αMeL-Leu-Asp(otbu)-Lys(boc)-{Boc-Eicosanedioic acid-γ -Glu(otbu)-AEEA-Lys}-Ala-Gln(trt)-Aib-Ala-Phe-Ile-Glu(otbu)-Tyr(tbu)-Leu-Leu-Glu(otbu)-Gly-Gly-Pro-Ser(tbu)-Ser(tbu)-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-NH2.

5. The method for preparing Retatrutide according to claim 3, characterized in that: The protected amino acid fragments are Fmoc-Pro-Pro-Pro-OH, Fmco-Ser(tbu)-Gly-OH, Fmoc-Gly-Gly-OH, Fmoc-Gln(trt)-Aib-OH, Fmoc-Ile-αMeL-OH, Fmoc-Gln(trt)-Gly-OH, Boc-Tyr(tbu)-Aib-OH, and Boc-Eicosanedioic acid-γ-Glu(otbu)-AEEA-OH.

6. The method for preparing Retatrutide according to claim 1, characterized in that: In step 1, the amount of the protected amino acid and the protected amino acid fragment is 1.5 to 4 times the total molar number of the resin fed; In step 2, the amount of the protected amino acid and the protected amino acid fragment is 2 to 5 times the total molar number of the resin fed.

7. The method for preparing Retatrutide according to claim 1, characterized in that: In steps 1 and 2, the deprotection reagent for removing Fmoc protection is a PIP / DMF mixed solution, the mixed solution contains 20% to 30% (V / V) piperidine, the amount of the deprotection reagent is 1.2 to 1.5 times the volume of the peptide resin, the deprotection number is 1, and the deprotection time is 20 to 30 minutes; 8. The method for preparing Retatrutide according to claim 1, characterized in that: In step 1, the coupled Sieber amide resin has a resin substitution degree ranging from 0.3 to 0.8 mmol / g; In step 2, the coupled CTC resin has a resin substitution degree ranging from 0.5 to 0.8 mmol / g.

9. The method for preparing Retatrutide according to claim 1, characterized in that: In step three, the cracking agent is a mixed solvent of trifluoroacetic acid, 1,2-ethanedithiol, triisopropylsilane and water; the mixed solvent contains TFA 90% to 95%, EDT 1% to 4% and TIS 1% to 4%, the amount of the cracking agent consumed per gram of intermediate M3 is 5 to 10 ml, and the cracking time is 2 to 3 hours at room temperature.

10. The method for preparing Retatrutide according to claim 1, characterized in that: The crude solution of retatrutide is purified by high performance liquid chromatography, wherein the purification chromatographic column is reverse octadecyl bonded silica gel; and the mobile phases are ammonium acetate solution and acetonitrile solution respectively.

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