Preparation method of simeglutide side chain deprotection active ester

Abstract

The invention discloses a preparation method of stemeglutide side chain deprotection activated ester, and belongs to the technical field of chemical pharmacy. The preparation method comprises the following steps: carrying out condensation on octadecanedioic acid monobenzyl ester active ester and alpha-glutamic acid benzyl ester to obtain an intermediate I (BnO-Ste-Glu-OBn), carrying out condensation on the intermediate I and AEEA-AEEA to obtain an intermediate II (BnO-Ste-Glu (AEEA-AEEA-OH)-OBn), activating the intermediate II to obtain an intermediate III (BnO-Ste-Glu (AEEA-AEEA-OR)-OBn), and carrying out hydrogenation deprotection on the intermediate III to obtain the semeglutide side chain deprotection active ester (HO-Ste-Glu (AEEA-AEEA-OR)-OH). The total reaction yield is about 80%, and the HPLC purity is greater than or equal to 99.0%.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a method for preparing a semaglutide side chain deprotected active ester. Background Art

[0002] Semaglutide is a new type of dual receptor agonist for glucagon-like peptide-1 receptor (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP, also known as gastric inhibitory polypeptide), which is administered once a week. The lysine position at the 26th position of the peptide chain of semaglutide is connected to an 18-carbon fatty diacid side chain, and PEG modification is added to promote the binding of semaglutide to albumin and reduce renal clearance. Compared with liraglutide with a C16 side chain, the affinity of the semaglutide side chain for albumin is enhanced, and the binding with albumin increases the molecular weight of this product, increases its molecular size, and it can also carry a large number of water molecules, so a PEG-protein increases by 5 to 10 times.

[0003] The synthesis of the side chain Ste-Glu-AEEA-AEEA-OSU of semaglutide is extremely important in the preparation process. In the prior art, solid phase synthesis is used for coupling, and then obtained by cutting, such as patent US2019309040A1. The disadvantages of solid phase synthesis are poor atom economy, relatively expensive raw materials for side chains, and low yields, resulting in high costs, difficult quality control, and high preparation costs. Another example: Patent CN 117924142 A uses tert-butyl as a carboxyl protecting group. During the reaction, intermediate 1 is connected to AEEA-AEEA and then to an active ester, which is an oily substance and is not easy to purify. After deprotection with trifluoroacetic acid, it is difficult to purify because it is not very stable. Trifluoroacetic acid has an impact on the environment, and the EU has begun to restrict its use in recent years. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for preparing a side chain deprotected active ester of semaglutide, which has the advantages of simple operation, high yield and high purity.

[0005] The technical solution adopted by the present invention to solve the technical problem is: a method for preparing a semaglutide side chain deprotected active ester, comprising the following steps:

[0006] Step 1), condensing the active ester of octadecane dioic acid monobenzyl ester with α-glutamic acid benzyl ester to obtain the intermediate IBnO-Ste-Glu-OBn;

[0007]

[0008] Step 2), intermediate I is condensed with AEEA-AEEA to obtain intermediate II BnO-Ste-Glu(AEEA-AEEA-OH)-OBn;

[0009]

[0010] Step 3), intermediate II is activated by an activator to obtain intermediate III BnO-Ste-Glu (AEEA-AEEA-OR)-OBn;

[0011]

[0012] Step 4), intermediate III is hydrogenated and deprotected to obtain the side chain deprotected active ester of semaglutide HO-Ste-Glu(AEEA-AEEA-OR)-OH;

[0013]

[0014] Furthermore, in the step 1), the molar ratio of octadecane dioic acid monobenzyl ester to α-glutamic acid benzyl ester is 1:1 to 1:1.5; in the step 2), the molar ratio of intermediate I to AEEA-AEEA is 1:1 to 1:1.5; in the step 3), the molar ratio of intermediate II to the activator is 1:1 to 1:1.5.

[0015] In the step 1) and the step 2), the condensing agent is selected from any one or more of N,N'-diisopropylcarbodiimide (DIC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl), and N,N'-dicyclohexylcarbodiimide (EDCl).

[0016] In the steps 1) to 4), the reaction solvent is one or more of DMF, acetonitrile, dioxane, DMSO, DMAC, THF, dichloromethane, chloroform, isopropanol, ethanol, methanol, and ethyl acetate.

[0017] The base used in the reaction is selected from an organic weak base or an inorganic base, including one or more of N,N-diisopropylethylamine (DIEA), triethylamine (Et3N), N-methylmorpholine, pyridine, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, and lithium hydroxide.

[0018] In the step 3), the activator includes one or more of hydroxy-N-succinimide (HOSu), hydroxy-N-phthalimide (PHT), pentafluorophenol (C6F5OH), hydroxy-N-5-norbornene-2,3-dicarboximide (HONB), 1-hydroxy-benzotriazole (HOBt), 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) and HO-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate; the active group R in the activator is one or more of succinimidyl (-Su), phthalimide (-PHT), pentafluorophenyl (-C6F5), 5-norbornene-2,3-dicarboximide (-NB), 1-benzotriazole (-Bt), and 7-azobenzotriazole (-At).

[0019] In the step 4), the benzyl group is removed by hydrogenation reaction, and the catalyst is Pd / C, platinum oxide, and Raney nickel; the reaction pressure is normal pressure to 10 MPa. The present invention uses normal pressure catalytic hydrogenation to remove the protecting group, and the catalyst can be reused.

[0020] The total reaction yield is about 80%, and the HPLC purity is ≥99%.

[0021] Octadecanedioic acid monobenzyl ester active ester and α-glutamic acid benzyl ester and other reagents are sourced from the market.

[0022] The beneficial effects of the present invention are as follows: compared with the prior art, the preparation method of the semaglutide side chain deprotection active ester provided by the present invention uses hydrogenation deprotection, is easy to operate, avoids the use of strong acids such as trifluoroacetic acid, the catalyst can be reused multiple times, and the process is environmentally friendly. The product has high purity and high yield, which is conducive to industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The liquid chromatogram of the semaglutide side chain provided in Example 1 of the present invention;

[0024] Figure 2 The NMR spectrum of the semaglutide side chain provided in Example 1 of the present invention;

[0025] Figure 3 This is the mass spectrum of the semaglutide side chain provided in Example 1 of the present invention.

[0026] Figure 4 This is a liquid chromatogram of the semaglutide side chain provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0027] The present invention is further described below by specific examples, but these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0028] Example 1 Preparation of Deprotected Active Ester of Semaglutide Side Chain

[0029] Step 1), at 15-25°C, add acetonitrile (525mL, 5V) and tetrahydrofuran (420mL, 4V) to the reaction bottle 1, and then add octadecane dioic acid monobenzyl ester (105g, 0.27mol), HOSU (37.3g, 0.32mol) and EDCI (62.1g, 0.32mol) in sequence, stir at 15-25°C under nitrogen protection for 8h, and monitor the completion of the reaction by TLC spot plate (PE:EA=4:1).

[0030] At 15-25°C, add 10% Na2CO3 aqueous solution (315mL, 3V) to reaction flask 2, then add α-glutamic acid benzyl ester (76.9g, 0.32mol), and stir at 15-25°C for 30mins. Transfer the solution in reaction flask 2 to reaction flask 1 by constant dropwise addition. Stir for 4h under nitrogen protection at 15-25°C. After the reaction is completed, monitor the reaction with a TLC spot plate (PE:EA=2:1), filter, concentrate the filtrate to remove acetonitrile and tetrahydrofuran, then add ethyl acetate (500mL) to extract the liquid, wash the organic phase with 1M hydrochloric acid (100mL x 2) and saturated brine (100mL x 2), dry over anhydrous sodium sulfate, filter and concentrate to obtain intermediate I (155.9g, yield 95.1%).

[0031] Step 2), at 15-25°C, add acetonitrile (1.56L, 10V) to the reaction bottle 3, then add intermediate I (155.9g, 0.26mol), HOSU (35.9g, 0.31mol) and EDCI (59.8g, 0.31mol) in sequence, and stir for 2h under nitrogen protection at 15-25°C. After the reaction is completed, filter the TLC plate (PE:EA=0:1) and set aside.

[0032] At 15-25°C, add 10% Na2CO3 aqueous solution (468mL, 3V) to reaction flask 4. Then add AEEA-AEEA (96.2g, 0.31mol) and stir at 15-25°C for 30mins. Transfer the solution in reaction flask 4 to reaction flask 3 through a constant pressure dropping funnel. Stir for 3h under nitrogen protection at 15-25°C. TLC spot plate (EA:MeOH=20:1) After the reaction is completed, filter, concentrate the filtrate to remove acetonitrile, then add ethyl acetate (500mL) to extract the liquid, wash the organic phase with 1M hydrochloric acid (300mLx 2) and saturated brine (200mL x 2), dry over anhydrous sodium sulfate, filter and concentrate to obtain intermediate 2 (214.5g, yield 93.2%).

[0033] Step 3), at 15-25°C, tetrahydrofuran (1.07L, 5V) was added to the reaction bottle 5, and then intermediate II (214.5g, 0.24mol), TSTU (87.3g, 0.29mol) and DIPEA (37.5g, 0.29mol) were added in sequence, and stirred for 2h under nitrogen protection at 15-25°C. After the reaction was completed, the filtrate was concentrated to remove tetrahydrofuran, and then ethyl acetate (500mL) was added to extract the liquid, and the organic phase was washed with 1M hydrochloric acid (300mL x2) and saturated brine (200mL x2), dried over anhydrous sodium sulfate, and concentrated at 40°C to obtain intermediate III (223.6g, yield 94.1%).

[0034] Step 4), place intermediate III (223.6 g, 0.22 mol) in a reactor, add isopropanol (4.47 L, 20 V), add 1 g Pd / C, stir, introduce nitrogen for replacement 3 times, introduce hydrogen, and maintain positive pressure. Stir at room temperature for 3 hours. TLC detection shows that the reaction is complete. Filter Pd / C, concentrate the mother liquor to obtain a white solid, and slurry with n-hexane to obtain semaglutide side chain (179 g, yield 96.1%). HPLC purity is 99.6%, and the specific liquid phase spectrum is as follows Figure 1 As shown, its NMR Figure 2 As shown. MS: 831.4656, its mass spectrum is shown Figure 3 shown.

[0035] Example 2 Preparation of Deprotected Active Ester of Semaglutide Side Chain

[0036] Step 1), at 15-25°C, add acetonitrile (525mL, 5V) and tetrahydrofuran (420mL, 4V) to the reaction bottle 1, and then add octadecane dioic acid monobenzyl ester (105g, 0.27mol) and HOSU (37.3g, 0.32mol) in sequence. Maintain the system at 20-25°C, control the temperature at 20-25°C, and add DIC (36.5g, 0.32mol) dropwise. After the dropwise addition, raise the temperature of the system to 15-25°C, and stir for 12h under nitrogen protection. Monitor the reaction by TLC spot plate (PE:EA=4:1), filter after completion, and set aside the filtrate.

[0037] At 15-25°C, add 10% Na2CO3 aqueous solution (315mL, 3V) to reaction flask 2, then add α-glutamic acid benzyl ester (76.9g, 0.32mol) and stir at 15-25°C for 30mins. Transfer the solution in reaction flask 2 to reaction flask 1 by constant dropwise addition. Stir for 4h under nitrogen protection at 15-25°C. After the reaction is completed, monitor the reaction on a TLC spot plate (PE:EA=2:1), filter, concentrate the filtrate to remove acetonitrile and tetrahydrofuran, then add ethyl acetate (500mL) to extract the liquid, wash the organic phase with 1M hydrochloric acid (100mL x 2) and saturated brine (100mL x 2), dry over anhydrous sodium sulfate, filter and concentrate to obtain intermediate I (151g, yield 92.3%).

[0038] Step 2), at 15-25°C, add acetonitrile (1.51L, 10V) to the reaction bottle 3, then add intermediate I (151g, 0.25mol) and HOSU (34.5g, 0.30mol) in sequence. Maintain the system at 20-25°C, control the temperature at 20-25°C, drop DIC (34.3g, 0.30mol), raise the temperature of the system to 15-25°C after dropping, and stir for 6h under nitrogen protection. TLC spot plate (PE:EA=0:1) After the reaction is completed, filter and set aside the filtrate.

[0039] At 15-25°C, add 10% Na2CO3 aqueous solution (468mL, 3V) to reaction flask 4. Then add AEEA-AEEA (96.2g, 0.31mol) and stir at 15-25°C for 30mins. Transfer the solution in reaction flask 4 to reaction flask 3 by constant dropwise addition. Stir for 8h under nitrogen protection at 15-25°C. TLC spot plate (EA:MeOH=20:1) After the reaction is completed, filter, concentrate the filtrate to remove acetonitrile, then add ethyl acetate (500mL) to extract the liquid, wash the organic phase with 1M hydrochloric acid (300mL x 2) and saturated brine (200mL x 2), dry over anhydrous sodium sulfate, filter and concentrate to obtain intermediate II (197g, yield 88.2%).

[0040] Step 3), at 15-25°C, tetrahydrofuran (985mL, 5V) was added to the reaction bottle 5, and then intermediate II (197g, 0.22mol), TSTU (78.3g, 0.26mol) and DIPEA (33.6g, 0.26mol) were added in sequence, and stirred for 2h under nitrogen protection at 15-25°C. After the reaction was completed, the filtrate was concentrated to remove tetrahydrofuran, and then ethyl acetate (500mL) was added to extract the liquid, and the organic phase was washed with 1M hydrochloric acid (300mL x 2) and saturated brine (200mL x2), dried over anhydrous sodium sulfate, and concentrated at 40°C to obtain intermediate III (205g, yield 93.8%).

[0041] Step 4), place intermediate III (205g, 0.21mol) in a reactor, add isopropanol (4.10L, 20V), add 1g Pd / C, stir, replace with nitrogen 3 times, and introduce hydrogen. Maintain positive pressure. Stir at room temperature for 3h. TLC detection, the reaction is complete. Filter Pd / C, concentrate the mother liquor to obtain a white solid, and slurry with n-hexane to obtain semaglutide side chain (164g, yield 95.8%). HPLC purity is 99.7%, the specific liquid phase spectrum is as follows Figure 4 shown.

[0042] The above implementation modes are only used to illustrate the present invention, but not to limit the present invention. Ordinary technicians in the relevant technical field can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention. The patent protection scope of the present invention should be defined by the claims.

Claims

1. A method for preparing a semaglutide side chain deprotected active ester, characterized in that: The steps include: Step 1), condensing the active ester of octadecane dioic acid monobenzyl ester with α-glutamic acid benzyl ester to obtain the intermediate IBnO-Ste-Glu-OBn; Step 2), intermediate I is condensed with AEEA-AEEA to obtain intermediate II BnO-Ste-Glu(AEEA-AEEA-OH)-OBn; Step 3), intermediate II is activated by an activator to obtain intermediate III BnO-Ste-Glu (AEEA-AEEA-OR)-OBn; Step 4), intermediate III is hydrogenated and deprotected to obtain the side chain deprotected active ester of semaglutide HO-Ste-Glu(AEEA-AEEA-OR)-OH; 2. The method for preparing a semaglutide side chain deprotected active ester according to claim 1, characterized in that: In the step 1), the molar ratio of octadecane dioic acid monobenzyl ester to α-glutamic acid benzyl ester is 1:1 to 1:1.5; in the step 2), the molar ratio of intermediate I to AEEA-AEEA is 1:1 to 1:1.5; in the step 3), the molar ratio of intermediate II to the activator is 1:1 to 1:1.

5.

3. The method for preparing a semaglutide side chain deprotected active ester according to claim 1, characterized in that: In the step 1) and the step 2), the condensing agent is selected from any one or more of N,N'-diisopropylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N,N'-dicyclohexylcarbodiimide.

4. The method for preparing a semaglutide side chain deprotected active ester according to claim 1, characterized in that: In the steps 1) to 4), the reaction solvent is one or more of DMF, acetonitrile, dioxane, DMSO, DMAC, THF, dichloromethane, chloroform, isopropanol, ethanol, methanol, and ethyl acetate.

5. The method for preparing a semaglutide side chain deprotected active ester according to claim 1, characterized in that: The base used in the reaction is selected from an organic weak base or an inorganic base, including one or more of N,N-diisopropylethylamine, triethylamine, N-methylmorpholine, pyridine, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, and lithium hydroxide.

6. The method for preparing a semaglutide side chain deprotected active ester according to claim 1, characterized in that: In the step 3), the activator includes one or more of hydroxy-N-succinimide, hydroxy-N-phthalimide, pentafluorophenol, hydroxy-N-5-norbornene-2,3-dicarboximide, 1-hydroxy-benzotriazole, 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate and HO-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate; the active group R in the activator is one or more of succinimidyl, phthalimide, pentafluorophenyl, 5-norbornene-2,3-dicarboximide, 1-benzotriazole, and 7-azobenzotriazole.

7. The method for preparing a semaglutide side chain deprotected active ester according to claim 1, characterized in that: In the step 4), debenzylation is performed by hydrogenation reaction, the catalyst is Pd / C, platinum oxide, Raney nickel; the reaction pressure is from normal pressure to 10 MPa.

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