Preparation of Liraglutide by Continuous Flow Solid Phase Reaction

Abstract

The invention discloses a method for preparing liraglutide by combination of a continuous flow solid phase synthesis system. A main chain adopts Boc-His(x)-Ala-Glu(OtBu)-Gly,Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Psi(me.me)Pro)-OH,H-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Palm-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(y)-Gly-Arg(y)-Gly- resin fragments, and besides, a continuous flow solid phase is adopted for synthesis of a growth peptide chain.

Description

technical field

[0001] The invention belongs to the field of polypeptide drug synthesis, and in particular relates to a method for preparing liraglutide combined with a continuous-flow solid-phase synthesis system. Background technique

[0002] Liraglutide is a GLP-1 receptor agonist, suitable for the control of blood sugar in adult patients with type 2 diabetes; it is suitable for patients whose blood sugar is still poorly controlled after treatment with metformin or sulfonylureas alone. Combined use of metformin or sulfonylureas. Liraglutide is the drug with the best hypoglycemic effect among GLP-1 receptor agonists such as exenatide, albiglutide, lixisenatide, and benaglutide. Liraglutide was as effective as dulaglutide. Liraglutide is effective in reducing body weight. Compared with traditional hypoglycemic drugs, GLP-1 can also delay gastric emptying, and act on the central fullness center to reduce food intake, thereby producing weight loss effects by suppressing ap...

Examples

Embodiment 1

[0109] Step 1: Preparation of Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Psi(me.me)Pro)-OH Fragment 1

[0110]

[0111] 1) Fmoc-Val-Ser(Psi(me.me)Pro)-2-CTC-resin

[0112] 10g of 2-CTC-resin (substitution degree=0.72mmol / g) was added to the solid phase reactor, the resin was swollen with 120mL of dichloromethane for 5 minutes, and then Fmoc-Val-Ser(Psi(me.me)Pro) was added sequentially -OH 1g (2.16mmol), 10.2mL DIEA, stirred for 2 hours. Drain the solvent, add 100mL of 10:90 methanol / dichloromethane solution, stir for half an hour to block unreacted complete chloromethyl. Wash 3 times with 100 mL of dichloromethane each time. After vacuum drying, the substitution degree of Fmoc-Val-Ser(Psi(me.me)Pro)-2-CTC-resin was measured to be 0.61 mmol / g.

[0113] Fmoc-Val-Ser(Psi(me.me)Pro)-2-CTC-resin was removed with 100mL 2%DBU / 2%1-octylthiol and DMF solution for 12 minutes at room temperature, and H- Val-Ser(Psi(me.me)Pro)-2-CTC-resin.

[0114] 2) Fmoc-Thr(tBu)-Phe...

Embodiment 2

[0146] Step 1: Fmoc-Gly-HMPA-PEGA resin synthesis

[0147]

[0148] Weigh 10 g of HMPA-PEGA resin (degree of substitution = 0.44 mmol / g) and add it to a solid-phase reactor, swell the resin with 100 mL of dichloromethane for 20 minutes, and filter with suction. Then, 5.0 g (17 mmol) of Fmoc-Gly-OH, 2.6 mL (34 mmol) of pyridine, and 2.3 mL (17 mmol) of 2,6-dichlorobenzoyl chloride were added in sequence, and stirred for 2 hours. Drain the solvent, add 100mL acetic anhydride:pyridine=1:1 to carry out resin blocking reaction solution, stir for half an hour to block unreacted complete methylol. Wash 3 times with 100 mL of dichloromethane each time. After vacuum drying, the degree of substitution of the Fmoc-Gly-HMPA-PEGA resin was measured to be 0.36 mmol / g.

[0149] Step 2: H-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Palm-Glu-OtBu)-Glu(OtBu)-Phe-Ile -Ala-Trp(Boc)-Leu-Val-Arg(Boc) 2 -Gly-Arg(Boc) 2 -Gly-HMPA-PEGA resin fragment 3 synthesis

[0150] The Fmoc...

Embodiment 3

[0165] Step 1: Fmoc-Gly-HMPA-Thio PEG-UREA-PS resin synthesis

[0166]

[0167] Weigh 15g of HMPA-Thio PEG-UREA-PS resin (degree of substitution = 0.41mmol / g) and add it to a solid-phase reactor, swell the resin with 100mL of dichloromethane for 5 minutes, then add 5.4g of Fmoc-Gly-OH, 7.7 mLDIEA, stirred for 3 hours. Drain the solvent, add 100mL of 10:90 methanol / dichloromethane solution, and stir for half an hour to block unreacted triphenylchloromethyl. Wash 3 times with 100 mL of dichloromethane each time. After vacuum drying, the degree of substitution of the Fmoc-Gly-Trt-Thio PEG-AM-PS resin was measured to be 0.33 mmol / g.

[0168] Step 2: H-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Palm-Glu-OtBu)-Glu(OtBu)-Phe-Ile -Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-HMPA-Thio PEG-UREA-PS resin 3 synthesis

[0169] The Fmoc-Gly-HMPA-Thio PEG-UREA-PS resin synthesized in step 1 was used for liraglutide resin synthesis. Weigh 10 g of Fmoc-Gly-HMPA-Thio PE...

technical field

[0001] The invention belongs to the field of polypeptide drug synthesis, and in particular relates to a method for preparing liraglutide combined with a continuous-flow solid-phase synthesis system. Background technique

[0002] Liraglutide is a GLP-1 receptor agonist, suitable for the control of blood sugar in adult patients with type 2 diabetes; it is suitable for patients whose blood sugar is still poorly controlled after treatment with metformin or sulfonylureas alone. Combined use of metformin or sulfonylureas. Liraglutide is the drug with the best hypoglycemic effect among GLP-1 receptor agonists such as exenatide, albiglutide, lixisenatide, and benaglutide. Liraglutide was as effective as dulaglutide. Liraglutide is effective in reducing body weight. Compared with traditional hypoglycemic drugs, GLP-1 can also delay gastric emptying, and act on the central fullness center to reduce food intake, thereby producing weight loss effects by suppressing ap...

Examples

Embodiment 1

[0109] Step 1: Preparation of Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Psi(me.me)Pro)-OH Fragment 1

[0110]

[0111] 1) Fmoc-Val-Ser(Psi(me.me)Pro)-2-CTC-resin

[0112] 10g of 2-CTC-resin (substitution degree=0.72mmol / g) was added to the solid phase reactor, the resin was swollen with 120mL of dichloromethane for 5 minutes, and then Fmoc-Val-Ser(Psi(me.me)Pro) was added sequentially -OH 1g (2.16mmol), 10.2mL DIEA, stirred for 2 hours. Drain the solvent, add 100mL of 10:90 methanol / dichloromethane solution, stir for half an hour to block unreacted complete chloromethyl. Wash 3 times with 100 mL of dichloromethane each time. After vacuum drying, the substitution degree of Fmoc-Val-Ser(Psi(me.me)Pro)-2-CTC-resin was measured to be 0.61 mmol / g.

[0113] Fmoc-Val-Ser(Psi(me.me)Pro)-2-CTC-resin was removed with 100mL 2%DBU / 2%1-octylthiol and DMF solution for 12 minutes at room temperature, and H- Val-Ser(Psi(me.me)Pro)-2-CTC-resin.

[0114] 2) Fmoc-Thr(tBu)-Phe...

Embodiment 2

[0146] Step 1: Fmoc-Gly-HMPA-PEGA resin synthesis

[0147]

[0148] Weigh 10 g of HMPA-PEGA resin (degree of substitution = 0.44 mmol / g) and add it to a solid-phase reactor, swell the resin with 100 mL of dichloromethane for 20 minutes, and filter with suction. Then, 5.0 g (17 mmol) of Fmoc-Gly-OH, 2.6 mL (34 mmol) of pyridine, and 2.3 mL (17 mmol) of 2,6-dichlorobenzoyl chloride were added in sequence, and stirred for 2 hours. Drain the solvent, add 100mL acetic anhydride:pyridine=1:1 to carry out resin blocking reaction solution, stir for half an hour to block unreacted complete methylol. Wash 3 times with 100 mL of dichloromethane each time. After vacuum drying, the degree of substitution of the Fmoc-Gly-HMPA-PEGA resin was measured to be 0.36 mmol / g.

[0149] Step 2: H-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Palm-Glu-OtBu)-Glu(OtBu)-Phe-Ile -Ala-Trp(Boc)-Leu-Val-Arg(Boc) 2 -Gly-Arg(Boc) 2 -Gly-HMPA-PEGA resin fragment 3 synthesis

[0150] The Fmoc...

Embodiment 3

[0165] Step 1: Fmoc-Gly-HMPA-Thio PEG-UREA-PS resin synthesis

[0166]

[0167] Weigh 15g of HMPA-Thio PEG-UREA-PS resin (degree of substitution = 0.41mmol / g) and add it to a solid-phase reactor, swell the resin with 100mL of dichloromethane for 5 minutes, then add 5.4g of Fmoc-Gly-OH, 7.7 mLDIEA, stirred for 3 hours. Drain the solvent, add 100mL of 10:90 methanol / dichloromethane solution, and stir for half an hour to block unreacted triphenylchloromethyl. Wash 3 times with 100 mL of dichloromethane each time. After vacuum drying, the degree of substitution of the Fmoc-Gly-Trt-Thio PEG-AM-PS resin was measured to be 0.33 mmol / g.

[0168] Step 2: H-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Palm-Glu-OtBu)-Glu(OtBu)-Phe-Ile -Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-HMPA-Thio PEG-UREA-PS resin 3 synthesis

[0169] The Fmoc-Gly-HMPA-Thio PEG-UREA-PS resin synthesized in step 1 was used for liraglutide resin synthesis. Weigh 10 g of Fmoc-Gly-HMPA-Thio PE...

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