WO2014188011A2

WO2014188011A2 - Method for preparation of linaclotide

Info

Publication number: WO2014188011A2
Application number: PCT/EP2014/070577
Authority: WO
Inventors: Ulf ALTENHOENER
Original assignee: Lonza Ltd
Priority date: 2014-09-25
Filing date: 2014-09-25
Publication date: 2014-11-27
Also published as: WO2014188011A3

Abstract

The invention discloses a method for the preparation of linaclotide with CAS 851199-59-2 with solid phase peptide synthesis.

Description

METHOD FOR PREPARATION OF LINACLOTIDE

BACKGROUND OF THE INVENTION

Linaclotide is an peptide agonist of guanylate cyclase 2C. It was approved by the FDA for the treatment of chronic idiopathic constipation and to treat irritable bowel syndrome with constipation (IBS-C) in adults. Linaclotide is a peptide consisting of 14 amino acids.

Meanings of abbreviations, which are used, if not otherwise stated:

AA Amino acid

CTC resin 2-Chlorotrityl Resin

DCI Diisopropylcarbodiimide

DCM Dichloromethane

DIPCDI N,N'-Diisopropylcarbodiimide

DIPE Diisopropyl ether

DIPEA N,N-Diisopropylethylamine

DMF Dimethylformamide

HOBt 1-Hydroxybenzotriazole

Fmoc Fluorenylmethyloxycarbonyl

Mtt Methyltrityl

tBu tert-Butyl

TFA Trifluoroactic acid

TIS Triisopropylsilane

TOTU [(1 -Cyano- 1 -ethoxycarbonyl-methyleneaminooxy)-dimethylamino-methylene] - dimethyl-ammonium tetrafluoro borate

Trt Trityl; triphenylmethyl SUMMARY OF THE INVENTION

Subject of the invention is a method for the preparation of linaclotide by elongation with solid phase peptide synthesis, global deprotection and oxidation, followed by purification and drying. DETAILED DESCRIPTION OF THE INVENTION

FIG 1 illustrates the method.

In FIG 1 the following annotations are used: ii) Fmoc-Cys(Trt)-OH

Fmoc-Gly-OH

Fmoc-Thr(tBu)

Fmoc-Cys(Trt)-OH

Fmoc-Ala-OHxH20

Fmoc-Pro-OH

Fmoc-Asn(Mtt)-OH

Fmoc-Cys(Trt)-OH

Fmoc-Tyr(tBu)-OH

Fmoc-Glu(OtBu)-OH

Fmoc-Cys(Trt)-OH

Special point to be considered:

• instead of Fmoc-Asn(Trt) also Fmoc-Asn(Mtt) can be used

• Activation in each coupling step ii) can be done via Oxyma pure (Cnoxim-Et) and DCI or via HOBt

• Cys 13 can also be activated with TOTU/DIPEA

• cocktails such as TFA - H20- Trt-OH - TIS or TFA - H20- tert-butanol - TIS or TFA - H20 - TIS can be used for global deprotection

Purification and Drying:

Step 1 : Primary purification on CI 8 HPLC column with AcOH/Acetonitrile/water gradient Step 2: Secondary purification on CI 8 HPLC column with ammonia

acetate/ Acetonitrile/water gradient

Step 3: Desalting and concentration on CI 8 HPLC column (tert-Butanol/water) Then follows as Option 1 :

Step a: destillative removal of H₂0 and switch to > 95% tert-BuOH

Step b: destillative solvent switch to heptane

Step c: precipitate on filter flushed with wet nitrogen

Step d: drying of the water wet cake into specs

Or Option 2 follows:

Step a: freeze drying of the tert-Butanol-water solution

Step b: displacement of residual tert-Butanol by wet nitrogen flush on filter

Step c: drying of the water wet cake into specs

Provided is pure Linaclotide.

Examples

Amberchrom is a registered trader mark of The Dow Chemical Company

Kromasil^® is a registered trademark of EKA Chemicals AB

Oxyma Pure is a product of Merck KGaA

A) Synthesis of Fmoc-Tyr(tBu)-CTC resin

CTC resin (15 g, 24 mmol, 1.6 mmol/g) in DCM (90 mL) was placed into a solid phase reactor at 20°C. Then DCM (60 mL) was added to wash the resin to swell for 30 min at 20°C. After swelling, DCM (45 mL) was added to the CTC resin, followed by adding

Fmoc-Tyr(tBu)-OH (5.18 g, 11.28 mmol, 0.47 equiv) and DIPEA (3.93 mL, 2.0 equiv) in DCM (24 mL), and the mixture was stirred for 4 h at 20°C. After the reaction was complete, the resin was capped by adding DMF (45 mL), DIPEA (4.18 mL) and MeOH (7.29 mL) for 30 min at 10°C. Then the resin was washed with DMF (5 x 75 mL) and DCM (3 x 50 mL).

B) Synthesis of H-Cys(Trt)-Cys(Trt)-Glu(tBu)-Tyr(tBu)-Cys(Trt)-Asn(Mtt)-Pro-Ala- Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-CTC resin

Cleavage of Fmoc group was effected by treating Fmoc-Tyr(tBu)-CTC resin with 40% (w/w) piperidine in DMF (2 x 110 mL x 15 min), followed by washing the resin with DMF (5 x 75 mL). Fmoc-Cys(Trt)-OH (9.91 g, 1.5 equiv), Oxyma Pure (2.4 g, 1.5 equiv) and DIPCDI (2.62 g, 1.5 equiv) were dissolved in DMF (45 mL) and DCM (56 mL), stirred for 10 min, added to the resin, and the coupling reaction was stirred for 2 h. After the reaction was complete, the resin was washed with DMF (5 x 75 mL) and DCM (3 x 50 mL).

By following this procedure, the following Fmoc-AAs (1.5 equiv) were sequentially incorporated with DIPCDI (1.5 equiv) and Oxyma Pure (1.5 equiv) in DMF. Cleavage of

Fmoc group was effected by treatment with 40% (w/w) piperidine in DMF (2 x 110 mL x 15 min) (Note: AAs = Fmoc-Cys(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc- Asn(Mtt)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH H₂0, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH) C) Synthesis of H-Cys-Cys-Glu-Tyr-Cys- Asn-Pro- Ala-Cys-Thr-Gly-Cys-Tyr-OH · TF A (crude linear linaclotide-TFA)

After finishing the elongation, the side-chain protected peptide was cleaved from resin using 1% TFA in DCM (5 x 57 mL x 10 min) at 0°C, the protected peptide was washed with DCM (5 x 75 mL x 10 min), concentrated and extracted three times with 1%> (w/w) aqueous KHS0₄ (173 mL) and EtOH (43 mL). The organic phase was concentrated, precipitated with heptane (526 mL) under stirring for 2 h at 20°C. The filter cake containing the protected peptide was washed with heptane (3 x 88 mL) and dried for 24 h at 30°C.

The protected peptide (4.8 g, 0.001 mmol) was treated with a mixture of TFA-TIS-H₂0 (80:15:5) (25 mL, v/v) at 20°C for 3 h. After addition of TIS at -5 to 0°C, the mixture was stirred 45 min and 127 mL toluene added at 20°C. The mixture was precipitated with DIPE (143 mL), filtered and washed with DIPE (3 x 47 mL). The filter cake was dried in vacuum at 30°C to obtain crude linear linaclotide-TFA. D) Synthesis of crude linaclotide

The crude linear linaclotide-TFA (1 g, 0.001 mmol) was subjected to DMSO (300 mL) oxidation in NaH₂P0₄-Na₂HP0₄-EDTA buffer (700 mL, pH=7.5) for 24 h at 30°C with constant air/nitrogen flushing. The crude peptide was then purified by RP-chromatography. E) Purification of linaclotide

The crude solution of linaclotide is loaded on a RP-HPLC-column packed e.g. with

Kromasil^®-100-10-C18 for primary purification. The column is equilibrated with aqueous buffer (0.5% acetic acid, 5% acetonitrile) before loading. The pH of the crude solution is lowered to pH 5.0 and an amount equivalent to 10 g linaclotide per 1 column volume is loaded. The product and impurities are eluted from the column by an increasing acetonitrile gradient. The UV-trace of a typical large scale chromatographic run is depicted below.

Fractions from the main peak are selected in order to achieve a pool purity > 90 area-% by RP-HPLC. The pooled fractions from all primary purification runs of one batch are diluted with purified water in the ratio 1 : 1 and loaded on a RP-HPLC-column packed e.g. with Kromasil^®-100-10- C18 in an amount equivalent to 10 g linaclotide per 1 column volume for secondary purification. The column is equilibrated with aqueous buffer (10 mM ammonia acetate, 5% acetonitrile) before loading. The product and impurities are eluted from the column by an increasing acetonitrile gradient. Fractions from the main peak are selected in order to achieve a pool purity > 97.0 area-% by RP-HPLC.

F) Isolation of linaclotide The assembled fraction pools of all secondary purification runs of one batch are assembled and diluted 1 to 1 with purified water. A chromatographic column is charged with a reversed phase resin (e.g. Amberchrom™ CG300M) and the column is equilibrated with 5% t-butanol in water. The diluted pool is charged to the column with a load equivalent to 25 g linaclotide per 1 column volume. Residual buffer and excess counter ions are washed from the column before the product is eluted with 50% tert-Butanol in water. The concentrated product solution is then lyophilized under reduced pressure. The achieved intermediate product is a dry powder with a residual content of t-butanol.

By purging the powder with wet nitrogen at ambient temperature the residual tert-Butanol (and other residual solvents) are stripped from the product. During this process step the product may pick up water, which is removed by conventional drying at reduced pressure and increased temperature.

Claims

WO 2014/188011 PCT/EP2014/070577 7 Claims

1. Method for the preparation of linaclotide by elongation with solid phase peptide synthesis, global deprotection and oxidation, followed by purification and drying.