WO2015022575A2 - A process for the preparation of gc-c agonist - Google Patents

A process for the preparation of gc-c agonist Download PDF

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Publication number
WO2015022575A2
WO2015022575A2 PCT/IB2014/001570 IB2014001570W WO2015022575A2 WO 2015022575 A2 WO2015022575 A2 WO 2015022575A2 IB 2014001570 W IB2014001570 W IB 2014001570W WO 2015022575 A2 WO2015022575 A2 WO 2015022575A2
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WIPO (PCT)
Prior art keywords
cys
formula
resin
linaclotide
trt
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PCT/IB2014/001570
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French (fr)
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WO2015022575A3 (en
Inventor
Agasaladinni NAGANA GOUD
Vadlamani SURESH KUMAR
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Auro Peptides Ltd
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Publication of WO2015022575A3 publication Critical patent/WO2015022575A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system

Definitions

  • the present invention relates to a process for the preparation of Linaclotide of formula I.
  • Linaclotide is a guanylate cyclase-C (GC-C) agonist.
  • Guanylate cyclase C refers to a transmembrane form of guanylate cyclase that acts as the intestinal receptor for the heat-stable toxin (ST) peptides secreted by enteric bacteria.
  • ST heat-stable toxin
  • Guanylate cyclase C is also the receptor for the naturally occurring peptides guanylin and uroguanylin.
  • Linaclotide and its active metabolite bind to GC-C and act locally on the luminal surface of the intestinal epithelium. Activation of GC-C results in an increase in both intracellular and extracellular concentrations of cyclic guanosine monophosphate (cGMP). Elevation in intracellular cGMP stimulates secretion of chloride and bicarbonate into the intestinal lumen, mainly through activation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel, resulting in increased intestinal fluid and accelerated transit. In animal models, Linaclotide has been shown to both accelerate GI transit and reduce intestinal pain.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Linaclotide-induced reduction in visceral pain in animals is thought to be mediated by increased extracellular cGMP, which was shown to decrease the activity of pain-sensing nerves.
  • Linaclotide is a peptide having 14 amino acids, with the sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr. This molecule is cyclical by forming three disulfide bonds between Cysi and Cys 6 , between Cys 2 and Cysi 0 and between Cys 5 and Cys 13 .
  • Linaclotide is marketed in USA under the trade name LINZESS in the form of capsules having dosage forms 145 meg and 290 meg for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.
  • Linaclotide for first time disclosed in US 7,304,036 discloses two different methods for the preparation of Linaclotide either by solid phase synthesis or by recombinant DNA technology.
  • Solid-phase synthesis is carried out by sequential addition of amino acids (Boc/Fmoc strategy) using an automated peptide synthesizer such as Cyc(4-CH2Bxl)-OCH2-4-(oxymethyl)-phenylacetamidomethyl resin to yield linear protected Linaclotide, which is deprotected and cleaved from resin using hydrogen fluoride, dimethyl sulfide, anisole and p-thiocresol. Thereafter obtained linear Linaclotide is oxidized, then purified using RP-HPLC and lyophilized to obtain Linaclotide in 10-20% yield.
  • Biopolymers, Issue 96, Volume 1 , Pages 69-80 also discloses synthesis of Linaclotide by solid phase synthesis, following sequential addition of amino acids to the supported resin (Wang or 2-chlorotrityl resin) and thereafter cleaved from resin and de-protection is carried out in two steps.
  • the above processes disclose synthesis of Linaclotide by sequential addition of amino acids to a solid support resin.
  • the disadvantage of this process is that the final compound is obtained with inconsistent yields, because of premature loss of peptide during synthesis.
  • WO 2012/1 18972 discloses a process for the preparation of Linaclotide by coupling the two fragments in solution phase in presence of a coupling agents HBTU, Cl-HOBt, DIPEA, DMF to obtain linear protected Linaclotide, which is deprotected in presence of TFA:EDT:TIS:H 2 0 and oxidation in presence of sodium bicarbonate and glutathione hydrochloride, followed by purification using preparative RP-HPLC and lyophilization.
  • the disadvantage of the above process is that the end product will be contaminated with many impurities which are difficult to remove latter.
  • the present inventors have found a new process of making Linaclotide, which is simple and industrially scalable with consistent yields. Further, the Linaclotide obtained by the process of the present invention results in higher yield and purity.
  • An objective of the present invention is to provide a process for preparing Linaclotide, which is simple, industrially applicable and economically viable.
  • Another objective of the present invention is to provide a process for preparing Linaclotide, which is having high purity.
  • the present invention relates to a process for the preparation of Linaclotide of formula I.
  • FIGURES Figure 1 Synthesis of Linaclotide by coupling two fragments on solid phase peptide synthesis.
  • the present invention relates to a process for the preparation of Linaclotide by coupling of two protected suitable fragments in presence of a coupling agent, cleavage and de- protection of peptide, oxidation and isolation of Linaclotide.
  • Linaclotide The use of two or three suitable fragments to prepare Linaclotide according to the present invention leads to a better quality product, particularly with low level of impurities. Further, such process is easier to use and less time-consuming; furthermore, such process leads to a higher yield of Linaclotide.
  • the yield of the crude Linaclotide (protected) by fragment coupling method was greater than 80% when cleaved it from the insoluble support, whereas in straight-through synthesis there was inconsistency in the yields and loss of peptide due to premature cleavage during straight through synthesis.
  • the Linaclotide prepared using the process of the present invention results in substantially high yield.
  • the present invention relates to the preparation of Linaclotide of high purity.
  • one or more of the different factors relating to the agents and their quantities which are used in carrying out the various steps to prepare Linaclotide; the manner in which the steps are carried out; the methods used; and the various process parameters such as the temperature, pH, concentration, etc. are optimized and controlled in proper manner so as to obtain the desired product in a consistent manner.
  • the present invention relates to a process for the preparation of Linaclotide by coupling of two protected suitable fragments by solid phase synthesis in presence of a coupling agent, cleavage and de-protection of peptide, oxidation and isolation of Linaclotide.
  • the present invention relates to a process for the preparation of Linaclotide by coupling of two protected suitable fragments by solid phase synthesis in the presence of a coupling agent.
  • the coupling agent are selected from the group comprising of HOBt, TBTU, DCC, DIC, HBTU, BOP, PyBOP, PyBrOP, PyClOP, Oxyma Pure, TCTU, EEDQ, COMU, DEPBT and the like, and mixtures thereof.
  • the coupling takes place in one of the solvents selected from the group comprising of DMF, DCM, THF, NMP, DMAC or mixtures thereof.
  • the solid phase synthesis is carried out on an insoluble polymer which is acid sensitive.
  • An acid sensitive resin is selected from a group comprising CTC, Sasrin, Wang Resin, 4-methytrityl chloride, TentaGel S and TentaGel TGA.
  • the linear protected peptide is de- protected with a mixture of reagents selected from the group comprising of TFA, TIS, DTT, EDT, ammonium iodide, 2,2'-(ethylenedioxy)diethane and acetyl cystein, DMS, phenol, cresol and thiocresol.
  • a mixture of reagents selected from the group comprising of TFA, TIS, DTT, EDT, ammonium iodide, 2,2'-(ethylenedioxy)diethane and acetyl cystein, DMS, phenol, cresol and thiocresol.
  • the oxidation step in the process of preparation of Linaclotide comprises the use of an oxidizing agent.
  • the oxidizing agent is selected from a group comprising of hydrogen peroxide, dimethyl sulfoxide (DMSO), glutathione, and the like, and a mixture thereof.
  • the oxidation is carried out in a buffer.
  • the buffer is selected from a group comprising of ammonium acetate, sodium carbonate, ammonium bicarbonate, water, and the like, and a mixture thereof.
  • the oxidation is carried out in a buffer solution at a pH range of about 7 to about 9.
  • the final isolation of Linaclotide is carried out by lyophilization.
  • the lyophilization is earned out in a controlled manner such that the stability of Linaclotide is not affected.
  • purification of Linaclotide is carried out by Reverse Phase HPLC and size exclusion chromatography.
  • purification of Linaclotide is carried out by Reverse Phase HPLC using ammonium acetate and a mixture of solvents comprising TFA, water, acetic acid and acetonitrile.
  • the purity of Linaclotide achieved by the fragment coupling was greater than 97%, preferably 99% (by analytical HPLC and size exclusion chromatography) and it was free from dimer and multimer impurities.
  • the suitable fragments for the preparation of Linaclotide are as follows:
  • a process for the preparation of Linaclotide comprises of the following steps:
  • Another embodiment of the present invention relates to synthesis of fragment of Formula II by sequential addition of amino acids to a solid support.
  • synthesis of fragment of Formula III either by sequential addition of amino acids to a solid support or coupling of two suitable fragments using solid phase synthesis.
  • Yet another embodiment of the present invention relates to the synthesis of fragment of Formula III, by coupling the fragments Formula IV and Formula V using solid phase synthesis.
  • Z represents thiol protecting group
  • X represents carboxyl, phenolic and alcoholic protecting groups
  • Y represents amino protecting group
  • ⁇ represents resin
  • the thiol protecting groups are selected from but not limited to a group comprising Tit, Acm, StBu, Tmob, Tacm, MMT, and the like.
  • the carboxyl, phenolic and alcoholic groups are protected with groups selected from but not limited to a group comprising DMT, MMT, Trt, tert-butyl, t-butoxy carbonyl, and the like.
  • the amino protecting groups are selected from but not limited to a group comprising Fmoc, Boc, Cbz, Bpoc, and the like.
  • CTC Resin 2-ChloroTrityl Resin (150 g) (1.6mmol/g) was transferred to a glass reaction vessel containing a sintered disk.
  • Anhydrous dichloromethane (1 100 ml) was added to the glass vessel and drained after two min.
  • a clear solution of Fmoc-Pro-OH (56 g, 1.2 eqv) dissolved in dry dichloromethane (785 ml) and N,N di-isopropyl ethylamine (1 15 ml) was added.
  • the reaction mixture was stirred mechanically for 3 hrs and the solution was drained out, and the resin was washed with 1% DIPEA in DCM (800 ml).
  • the peptide resin was washed with a mixture of 1 : 1 [10% DIPEA: Methanol; 600 ml]; 1%DIPEA in DCM and with 0.5% DIPEA in MTBE and dried under vacuum.
  • Step (2) Coupling of Fmoc-Asn (Trt) to Pro-CTC-Resin:
  • Fmoc-Pro-Resin from step (1 ) was swelled in dichloromethane (700 ml) for 20 min and dimethylformamide (DMF) (700 ml) for 20 min. 20% piperidine in DMF (750 ml) (5+15 min) was added to the Fmoc-Pro-Resin and the resin was washed with DMF (750 ml), Isopropylalcohol (IPA) (450 ml) and DMF (750 ml). Thereafter resin beads were taken out and checked for Kaiser Test (positive) and chloranil test (positive).
  • DMF dimethylformamide
  • Step (3) Coupling of Fmoc-Cys(Trt)-OH to Asn (Trt)-Pro-Resin.
  • Step (5) Coupling of Fmoc-Tyr(OtBu)-OH to Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro- Resin.
  • Step (6) Coupling of Fmoc-Glu(OtBu)-OH to Tyr(OtBu)-Cys(Trt)-Cys(Trt)- Asn(Trt)-Pro-Resin.
  • Step (7) Coupling of Fmoc-Cys(Trt)-OH to Glu(OtBu)-Tyr(OtBu)-Cys(Trt)- Cys(Trt)-Asn(Trt)-Pro-Resin.
  • Step (8) Coupling of Fmoc-Cys(Trt)-OH to Cys(Trt)-Glu(OtBu)-Tyr(OtBu)- Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Resin. 20% piperidine in DMF (750 ml) (5+15 min) was added to Fmoc-Cys(Trt)-Glu(OfBu)- Tyr(OtBu)-Cys (Trt)-Cys(Trt)-Asn(Trt)-Pro-Resin.
  • Step (9) Cleavage of protected peptide from the resin
  • the protected octapeptide (450 g) was cleaved from the solid support by using 0.8% TFA in DCM (4 X 800 ml) and the resulting solution was neutralized by using 10% DIPEA in DCM (4 X 200 ml). The fractions which were found to be UV positive were collected, combined and evaporated.
  • the crude was dissolved in ethyl acetate (2.5 L) and the organic layer was washed with water (500 ml); and 0.1 M NaCl Solution (700 ml). The organic layer was dried over sodiumsulphate, filtered and evaporated to a solid. The solid was treated with MTBE, filtered and dried under vacuum for 16 hrs. Weight: 262 g
  • CTC Resin 2-ChloroTrityl Resin (CTC Resin) (65 g) (1.6mmol/g) was transferred to a glass reaction vessel containing a sintered disk. Anhydrous dichloromethane (400 ml) was added to the glass vessel and drained after two min. A clear solution of Fmoc- Tyr(OtBu)-OH (29 g, 1.1 eqv.) dissolved in dry DCM (300 ml) and N.N di- isopropylethylamine (41 ml) was added. The reaction mixture was stirred mechanically for 3 hrs and the solution was drained out, and the resin was washed with 1 % DIPEA in DCM (400 ml).
  • the peptide resin was washed with a mixture of 1 : 1 [ 10% DIPEA: Methanol; 300 ml], 1% DIPEA in DCM (400 ml), 0.5% DIPEA in MTBE (300 ml) and dried under vacuum.
  • Step (2) Coupling of Fmoc-Cys (Trt) to Tyr (OtBu)-CTC-Resin
  • Fmoc-Tyr(OtBu)-Resin from step (1) was swelled in dichloromethane (250 ml) for 20 min and dimethylformamide (DMF) (250 ml) for 20 min. 20% piperidine in DMF (300 ml) (5+15 min) was added to the Fmoc-Tyr (OtBu)-Resin and the resin was washed with DMF (300 ml), Isopropylalcohol (IP A) (200 ml) and DMF (300 ml). Thereafter resin beads were taken out and checked for Kaiser Test (positive) and chloranil test (positive).
  • Step (3) Coupling of Fmoc-Gly-OH to Cys(Trt)-Tyr-(OtBu)-Resin
  • Step (5) Coupling of Fmoc-Cys(Trt)-OH to Thr(OtBu)-Gly-Cys(Trt)-Tyr(OtBu)- Resin
  • Step (6) Coupling of Fmoc-Ala-OH to Cys-(Trt)-Thr-(OtBu)-Gly-Cys-(Trt)-Tyr- (OtBu)-Resin
  • the experiment conducted for cleavage of Linaclotide (protected) from the peptide resin (190 g) is carried out by using 1% TFA in DCM (4X300 ml) and the resulting solution was neutralized by using 10% DIPEA in DCM (4X100 ml). The fractions which were found to be UV positive were collected, combined and evaporated.
  • the crude was dissolved in ethyl acetate (1.5 L) and the organic layer was washed with water (250 ml); and 0.1 M NaCl Solution (250 ml). The organic layer was dried over sodium sulphate, filtered and evaporated to a solid. The solid was treated with MTBE, filtered and dried under vacuum for 16 hrs.
  • PROCESS FOR PREPARING LINACLOTIDE Linear Linaclotide (10 g) was dissolved in degassed 0.01 M Ammonium bicarbonate (10 L) (pH 8.1 ). After dissolving the compound slowly bubbled the compressed air and added the hydrogen peroxide ( 1 ml). After 18 hrs the completion of the reaction was monitored by reverse phase analytical HPLC. The oxidized product was purified by reverse phase HPLC.
  • the oxidized crude peptide solution obtained from the example 5 was passed through Novasep lab system RP-HPLC system, wavelength 220nm, 1 10 X 700mm column of C I 8 reverse phase column, mobile phase: A : 0.01 M ammonium acetate ; mobile phase B: acetonitrile flow rate: 400 ml / min, peaks of fractions were collected and pooled the fractions having the purity greater than 80%.
  • stage-1 The main pool obtained from the stage-1 were diluted with equal amount of water and passed through Novasep lab system RP-HPLC system, wavelength 220nm, 1 10 X 700mm column of C I 8 reverse phase column, mobile phase: A: 0.1 % TFA in water; mobile phase B: acetonitrile flow rate: 400 ml / min, fractions collected and analyzed by RPHPLC and SEC the fractions having the purity greater than 98% were taken and pooled and taken to the next stage.
  • Stage-3 The main pool obtained from the stage-1 were diluted with equal amount of water and passed through Novasep lab system RP-HPLC system, wavelength 220nm, 1 10 X 700mm column of C I 8 reverse phase column, mobile phase: A: 0.1 % TFA in water; mobile phase B: acetonitrile flow rate: 400 ml / min, fractions collected and analyzed by RPHPLC and SEC the fractions having the purity greater than 98% were taken and pooled and taken to the next
  • the main pool obtained from the stage-2 were diluted with equal amount of water and passed through Novasep lab system RP-HPLC system, wavelength 220nm, 1 10 X 700mm column of C I 8 reverse phase column, mobile phase: A: 0.1% acetic acid in water; mobile phase B: acetonitrile flow rate: 400 ml / min, fractions collected and analyzed by RPHPLC and SEC the fractions having the purity greater than 99% were collected, pooled together, concentrated using rotary evaporation and lyophilized to give pure Linaclotide.

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Abstract

The present invention relates to a process for the preparation of Linaclotide by coupling of two or three suitable fragments by solid phase synthesis.

Description

A PROCESS FOR THE PREPARATION OF GC-C AGONIST
FIELD OF THE INVENTION
The present invention relates to a process for the preparation of Linaclotide of formula I.
H- Cys- Cys- Glu- Tyr- Cys- Cys- Asn - Pro- Ala- Cys- Thr- Gly - Cys - Tyr- OH
s s s
: S I Formula
S
BACKGROUND OF THE INVENTION
Linaclotide is a guanylate cyclase-C (GC-C) agonist. Guanylate cyclase C refers to a transmembrane form of guanylate cyclase that acts as the intestinal receptor for the heat-stable toxin (ST) peptides secreted by enteric bacteria. Guanylate cyclase C is also the receptor for the naturally occurring peptides guanylin and uroguanylin.
Both Linaclotide and its active metabolite bind to GC-C and act locally on the luminal surface of the intestinal epithelium. Activation of GC-C results in an increase in both intracellular and extracellular concentrations of cyclic guanosine monophosphate (cGMP). Elevation in intracellular cGMP stimulates secretion of chloride and bicarbonate into the intestinal lumen, mainly through activation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel, resulting in increased intestinal fluid and accelerated transit. In animal models, Linaclotide has been shown to both accelerate GI transit and reduce intestinal pain. The Linaclotide-induced reduction in visceral pain in animals is thought to be mediated by increased extracellular cGMP, which was shown to decrease the activity of pain-sensing nerves. Linaclotide is a peptide having 14 amino acids, with the sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr. This molecule is cyclical by forming three disulfide bonds between Cysi and Cys6, between Cys2 and Cysi0 and between Cys5 and Cys13.
Figure imgf000003_0001
Linaclotide is marketed in USA under the trade name LINZESS in the form of capsules having dosage forms 145 meg and 290 meg for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.
Linaclotide for first time disclosed in US 7,304,036. This patent discloses two different methods for the preparation of Linaclotide either by solid phase synthesis or by recombinant DNA technology. Solid-phase synthesis is carried out by sequential addition of amino acids (Boc/Fmoc strategy) using an automated peptide synthesizer such as Cyc(4-CH2Bxl)-OCH2-4-(oxymethyl)-phenylacetamidomethyl resin to yield linear protected Linaclotide, which is deprotected and cleaved from resin using hydrogen fluoride, dimethyl sulfide, anisole and p-thiocresol. Thereafter obtained linear Linaclotide is oxidized, then purified using RP-HPLC and lyophilized to obtain Linaclotide in 10-20% yield.
Biopolymers, Issue 96, Volume 1 , Pages 69-80 (201 1) also discloses synthesis of Linaclotide by solid phase synthesis, following sequential addition of amino acids to the supported resin (Wang or 2-chlorotrityl resin) and thereafter cleaved from resin and de-protection is carried out in two steps. The above processes disclose synthesis of Linaclotide by sequential addition of amino acids to a solid support resin. The disadvantage of this process is that the final compound is obtained with inconsistent yields, because of premature loss of peptide during synthesis.
WO 2012/1 18972 discloses a process for the preparation of Linaclotide by coupling the two fragments in solution phase in presence of a coupling agents HBTU, Cl-HOBt, DIPEA, DMF to obtain linear protected Linaclotide, which is deprotected in presence of TFA:EDT:TIS:H20 and oxidation in presence of sodium bicarbonate and glutathione hydrochloride, followed by purification using preparative RP-HPLC and lyophilization.
The disadvantage of the above process is that the end product will be contaminated with many impurities which are difficult to remove latter. The present inventors have found a new process of making Linaclotide, which is simple and industrially scalable with consistent yields. Further, the Linaclotide obtained by the process of the present invention results in higher yield and purity.
OBJECTIVE
An objective of the present invention is to provide a process for preparing Linaclotide, which is simple, industrially applicable and economically viable.
Another objective of the present invention is to provide a process for preparing Linaclotide, which is having high purity.
Yet another objective of the present invention is to provide a process for preparing Linaclotide, which result in high yield. Yet another objective of the present invention is to provide a process for preparing Linaclotide, which results in consistent yields.
SUMMARY OF THE INVENTION
The present invention relates to a process for the preparation of Linaclotide of formula I.
H- Cys- Cys- Olu- Tyr- Cys- Cys- Asn - Pro- Ala- Cys- Thr- Gly- Cys- Tyr- OH Formula 1 s= |=i 1 I
s s
which comprises the following steps:
a) preparing two or three suitable fragments by solid phase peptide synthesis;
b) coupling of the fragments by solid phase synthesis to obtain a protected peptide; c) concurrent cleaving the protected peptide from the peptide resin and de- protecting the peptide;
d) oxidizing the deprotected peptide to obtain Linaclotide; and
e) isolation of Linaclotide.
BRIEF DESCRIPTION OF THE FIGURES Figure 1 : Synthesis of Linaclotide by coupling two fragments on solid phase peptide synthesis.
Figure 2: Synthesis of Fragment of formula II
Figure 3: Synthesis of Fragment of formula III by sequential addition of amino acids Figure 4: Synthesis of Fragment of formula III by coupling of two fragments
BRIEF DESCRIPTION OF ABBREVIATIONS
HBTU - O-Benzotriazole-N.N.N' N-tetramethyluroniumhexafluoro phosphate
Cl-HOBt - 6-chloro 1 -hydroxy-benzotriazole
HOBt - Hydroxy benzotriazole
TBTU - 0-(benzotriazol-l -yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, DCC - 1 ,3-dicyclohexylcarbodiimide DIC - Diisopropylcarbodiimide
HBTU - 0-(benzotriazol-l-yl)-l , l ,3,3-tetramethyluronium hexafluorophosphate
BOP - Benzotriazol- l -yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate
PyBOP - Benzotriazol- 1 -yloxy tri(pyrrolidino)phosphonium hexafluorophosphate
PyBrOP - Bromotri(pyrrolidino)phosphonium hexafluorophosphate
PyClOP - Chlorotri(pyrrolidino)phosphonium hexafluorophosphate (PyClOP),
Oxyma - Ethyl-2-cyano-2-(hydroxyimino) acetate (Oxyma Pure),
TCTU - 0-(6-Chlorobenzotriazol-l -yl)-l , l ,3,3-tetramethyluronium tetrafluoroborate
EEDQ - Ethyl 1 ,2- dihydro-2-ethoxyquinoline-l -carboxylate
COMU - 1 -Cyano-2-ethoxy-2-oxoethyHdenaminooxy)dimethylaminomorpholino carbenium hexafluorophosphate
DIPEA - iV.jV-diisopropylethylamine
DMF - N,N-dimethylformamide
DCM - Dichloromethane
THF - Tetrahydrofuran
NMP - N-Methyl pyrrolidine
DMAC - Dimethylacetamide
TFA - Trifluoro acetic acid
EDT - Ethanedithiol
TIS - Triisopropyl silane
DTT - Diothreitol
DMS - Dimethyl sulfide
DMSO - Dimethyl sulfoxide
MTBE - Methyl tert-butylether
MeOH - Methanol
IPA - Isopropyl alcohol
CTC - Chlorotrityl chloride
Trt - Trityl
Acm - Acetamidomethyl
StBu - S-tert-butylmercapto
Tmob - Trimethoxybenzyl
DMT - dimethoxy trityl
MMT- Methoxytrityl
Fmoc - 9-fluorenyln ethoxycarbonyl
Boc- tert-butoxycarbonyl
Cbz - Benzyloxycarbonyl Bpco - 2-(4-biphenyl)-2-propyloxycarbonyl
TACM - S-Trimethylacetamidomethyl
DEPBT - 3-(Diethoxy-phosphoryloxy)-3H-benzo[d][l ,2,3] triazin-4- one DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a process for the preparation of Linaclotide by coupling of two protected suitable fragments in presence of a coupling agent, cleavage and de- protection of peptide, oxidation and isolation of Linaclotide.
The use of two or three suitable fragments to prepare Linaclotide according to the present invention leads to a better quality product, particularly with low level of impurities. Further, such process is easier to use and less time-consuming; furthermore, such process leads to a higher yield of Linaclotide.
The yield of the crude Linaclotide (protected) by fragment coupling method was greater than 80% when cleaved it from the insoluble support, whereas in straight-through synthesis there was inconsistency in the yields and loss of peptide due to premature cleavage during straight through synthesis.
The Linaclotide prepared using the process of the present invention results in substantially high yield. Particularly, the present invention relates to the preparation of Linaclotide of high purity. According to the present invention, one or more of the different factors relating to the agents and their quantities which are used in carrying out the various steps to prepare Linaclotide; the manner in which the steps are carried out; the methods used; and the various process parameters such as the temperature, pH, concentration, etc. are optimized and controlled in proper manner so as to obtain the desired product in a consistent manner.
In an embodiment, the present invention relates to a process for the preparation of Linaclotide by coupling of two protected suitable fragments by solid phase synthesis in presence of a coupling agent, cleavage and de-protection of peptide, oxidation and isolation of Linaclotide.
In an embodiment, the present invention relates to a process for the preparation of Linaclotide by coupling of two protected suitable fragments by solid phase synthesis in the presence of a coupling agent.
In another embodiment of the present invention the coupling agent are selected from the group comprising of HOBt, TBTU, DCC, DIC, HBTU, BOP, PyBOP, PyBrOP, PyClOP, Oxyma Pure, TCTU, EEDQ, COMU, DEPBT and the like, and mixtures thereof.
In another embodiment of the present invention the coupling takes place in one of the solvents selected from the group comprising of DMF, DCM, THF, NMP, DMAC or mixtures thereof.
In yet another embodiment of the present invention, the solid phase synthesis is carried out on an insoluble polymer which is acid sensitive. An acid sensitive resin is selected from a group comprising CTC, Sasrin, Wang Resin, 4-methytrityl chloride, TentaGel S and TentaGel TGA.
In yet another embodiment of the present invention, the linear protected peptide is de- protected with a mixture of reagents selected from the group comprising of TFA, TIS, DTT, EDT, ammonium iodide, 2,2'-(ethylenedioxy)diethane and acetyl cystein, DMS, phenol, cresol and thiocresol.
In an embodiment of the present invention, the oxidation step in the process of preparation of Linaclotide comprises the use of an oxidizing agent.
In another embodiment of the present invention, the oxidizing agent is selected from a group comprising of hydrogen peroxide, dimethyl sulfoxide (DMSO), glutathione, and the like, and a mixture thereof.
In yet another embodiment of the present invention, the oxidation is carried out in a buffer.
In yet another embodiment of the present invention, the buffer is selected from a group comprising of ammonium acetate, sodium carbonate, ammonium bicarbonate, water, and the like, and a mixture thereof.
In yet another embodiment of the present invention, the oxidation is carried out in a buffer solution at a pH range of about 7 to about 9.
In an embodiment of the present invention, the final isolation of Linaclotide is carried out by lyophilization.
In an embodiment, the lyophilization is earned out in a controlled manner such that the stability of Linaclotide is not affected.
In another embodiment of the present invention, purification of Linaclotide is carried out by Reverse Phase HPLC and size exclusion chromatography. In yet another embodiment of the present invention, purification of Linaclotide is carried out by Reverse Phase HPLC using ammonium acetate and a mixture of solvents comprising TFA, water, acetic acid and acetonitrile. The purity of Linaclotide achieved by the fragment coupling was greater than 97%, preferably 99% (by analytical HPLC and size exclusion chromatography) and it was free from dimer and multimer impurities.
In yet another embodiment of the present invention, the suitable fragments for the preparation of Linaclotide are as follows:
Y- Cys(Z>- Cys(Z)- G lu(X>- Tyr(X>- Cys(Z)- Cys(Z)- Asn(Y)- Pro- OH Formula II
AIa- Cys(Z)- Thr(X>- Gly— Cys(Z)- Tyr(XHg) Formula III wherein Z represents thiol protecting group; X represents carboxyl, phenolic and alcoholic protecting groups; Y represents amino protecting group and © represents resin. In an embodiment of the present invention, a process for the preparation of Linaclotide comprises of the following steps:
a) preparing fragments of Formula II and Formula III by a solid phase synthesis; b) coupling of fragments of Formula II and Formula III by a solid phase synthesis to obtain a protected peptide;
c) concurrently cleaving the protected peptide from the peptide resin and de- protecting the peptide;
d) oxidizing the deprotected peptide to obtain Linaclotide; and
e) isolation of Linaclotide. Another embodiment of the present invention relates to synthesis of fragment of Formula II by sequential addition of amino acids to a solid support. In yet another embodiment of the present invention relates to the synthesis of fragment of Formula III either by sequential addition of amino acids to a solid support or coupling of two suitable fragments using solid phase synthesis. Yet another embodiment of the present invention relates to the synthesis of fragment of Formula III, by coupling the fragments Formula IV and Formula V using solid phase synthesis.
Y- Ala- Cys(Z)- Thr(X)- Gly- OH Formula IV
H:\- Cys(Z) - Tyr(X)- Q Formula V
wherein Z represents thiol protecting group; X represents carboxyl, phenolic and alcoholic protecting groups; Y represents amino protecting group and © represents resin.
In yet another embodiment of the present invention, the thiol protecting groups are selected from but not limited to a group comprising Tit, Acm, StBu, Tmob, Tacm, MMT, and the like.
In yet another embodiment of the present invention the carboxyl, phenolic and alcoholic groups are protected with groups selected from but not limited to a group comprising DMT, MMT, Trt, tert-butyl, t-butoxy carbonyl, and the like. In yet another embodiment of the present invention, the amino protecting groups are selected from but not limited to a group comprising Fmoc, Boc, Cbz, Bpoc, and the like. The invention is illustrated with the following examples, which are provided by way of illustration only and should not be construed to limit the scope of the invention in any manner whatsoever. EXAMPLE !
PROCESS FOR PREPARING PROTECTED Fmoc-Cys(Trt)-Cys(Trt)- G!u(OtBu)-Tyr(OtBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-OH on 2-ChloroTrityl Resin (N-terminal octapeptide fragment on CTC-Resin)
Step (1): Synthesis of Fmoc-Pro- Chloro Trity! Resin:
2-ChloroTrityl Resin (CTC Resin) (150 g) (1.6mmol/g) was transferred to a glass reaction vessel containing a sintered disk. Anhydrous dichloromethane (1 100 ml) was added to the glass vessel and drained after two min. A clear solution of Fmoc-Pro-OH (56 g, 1.2 eqv) dissolved in dry dichloromethane (785 ml) and N,N di-isopropyl ethylamine (1 15 ml) was added. The reaction mixture was stirred mechanically for 3 hrs and the solution was drained out, and the resin was washed with 1% DIPEA in DCM (800 ml). The peptide resin was washed with a mixture of 1 : 1 [10% DIPEA: Methanol; 600 ml]; 1%DIPEA in DCM and with 0.5% DIPEA in MTBE and dried under vacuum.
Weight: 190 g
Step (2): Coupling of Fmoc-Asn (Trt) to Pro-CTC-Resin:
Fmoc-Pro-Resin from step (1 ) was swelled in dichloromethane (700 ml) for 20 min and dimethylformamide (DMF) (700 ml) for 20 min. 20% piperidine in DMF (750 ml) (5+15 min) was added to the Fmoc-Pro-Resin and the resin was washed with DMF (750 ml), Isopropylalcohol (IPA) (450 ml) and DMF (750 ml). Thereafter resin beads were taken out and checked for Kaiser Test (positive) and chloranil test (positive). To this resin a solution consisting of Fmoc-Asn(Trt)-OH (140 g; 2 eqv); HOBT (36 g; 2 eqv) dissolved in DMF (600 ml) and DIC (62 ml; 3eqv) was added and the reaction was allowed at 25°C for 2 hrs, followed by washing with DMF (700 ml) to obtain Fmoc-Asn (Trt)-Pro-Resin.
Step (3): Coupling of Fmoc-Cys(Trt)-OH to Asn (Trt)-Pro-Resin.
20% piperidine in DMF (750 ml) (5+15 min) was added to the Fmoc-Asn(Trt)-Pro- Resin. The resin was washed with DMF (750 ml), IPA (450 ml) and DMF (750 ml) thereafter resin beads were taken out and checked for Kaiser Test (positive). To this resin a solution consisting of Fmoc-Cys(Trt)-OH (155 g; 2 eqv); HOBT (36 g; 2 eqv) dissolved in DMF (600 ml) and DIC (62 ml; 3eqv) was added and the reaction was allowed at 25°C for 2 hrs, followed by washing with DMF (700 ml) to obtain Fmoc- Cys(Trt)-Asn(Trt)-Pro-Resin. Step (4): Coupling of Fmoc-Cys(Trt)-OH to Cys(Trt)-Asn(Trt)-Pro-Resin
20% piperidine in DMF (750 ml) (5+15 min) was added to the Fmoc-Cys (Trt)-Asn (Trt)-Pro-Resin. The resin was washed with DMF (750 ml), IPA (450 ml) and DMF (750 ml) thereafter resin beads were taken out and checked for Kaiser Test (positive). To this resin a solution consisting of Fmoc-Cys(Trt)-OH (155 g; 2 eqv); HOBT (36 g; 2 eqv) dissolved in DMF (700 ml) and DIC (62 ml; 3eqv) was added and the reaction was allowed at 25°C for 2 hrs, followed by washing with DMF (700 ml) to obtain Fmoc- Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Resin.
Step (5): Coupling of Fmoc-Tyr(OtBu)-OH to Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro- Resin.
20% piperidine in DMF (750 ml) (5+15 min) was added to the Fmoc-Cys(Trt)- Cys(Trt)-Asn(Trt)-Pro-Resin. The resin was washed with DMF (750 ml), IPA (450 ml) and DMF (750 ml) thereafter resin beads were taken out and checked for Kaiser Test (positive). To this resin a solution consisting of Fmoc-Tyr(OtBu)-OH (120 g; 2 eqv); HOBT (36 g; 2 eqv) dissolved in DMF (700 ml) and DIC (62 ml; 3eqv) was added and the reaction was allowed at 25°C for 2 hrs, followed by washing with DMF (700 ml) to obtain Fmoc-Tyr(OtBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Resin.
Step (6): Coupling of Fmoc-Glu(OtBu)-OH to Tyr(OtBu)-Cys(Trt)-Cys(Trt)- Asn(Trt)-Pro-Resin.
20% piperidine in DMF (750 ml) (5+15 min) was added to the Fmoc-Tyr(OtBu)- Cys(Trt)-Cys(Trt)-Asn (Trt)-Pro-Resin. The resin was washed with DMF (800 ml), IPA (600 ml) and DMF (800 ml) thereafter resin beads were taken out and checked for Kaiser Test (positive). To this resin a solution consisting of Fmoc-Glu(OtBu)-OH (1 10 g; 2 eqv); HOBT (36 g; 2 eqv) dissolved in DMF (700 ml) and DIC (62 ml; 3eqv) was added and the reaction was allowed at 25°C for 2 hrs, followed by washing with DMF (700 ml) to obtain Fmoc-Glu(OtBu)-Tyr(OtBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Resin.
Step (7): Coupling of Fmoc-Cys(Trt)-OH to Glu(OtBu)-Tyr(OtBu)-Cys(Trt)- Cys(Trt)-Asn(Trt)-Pro-Resin.
20% piperidine in DMF (750 ml) (5+15 min) was added to Fmoc-Glu(OtBu)- Tyr(OtBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Resin. The resin was washed with DMF (800 ml), IPA (600ml) and DMF (800 ml) thereafter resin beads were taken out and checked for Kaiser Test (positive). To this resin a solution consisting of Fmoc- Cys(Trt)-OH (155 g; 2 eqv); HOBT (36 g; 2 eqv) dissolved in DMF (700 ml) and DIC (62 ml; 3eqv) was added and the reaction was allowed at 25°C for 2 hrs, followed by washing with DMF (850 ml) to obtain Fmoc-Cys (Trt)-Glu (OtBu)-Tyr (OtBu)-Cys Trt)-Cys (Trt)-Asn (Trt)-Pro-Resin.
Step (8) Coupling of Fmoc-Cys(Trt)-OH to Cys(Trt)-Glu(OtBu)-Tyr(OtBu)- Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Resin. 20% piperidine in DMF (750 ml) (5+15 min) was added to Fmoc-Cys(Trt)-Glu(OfBu)- Tyr(OtBu)-Cys (Trt)-Cys(Trt)-Asn(Trt)-Pro-Resin. The resin was washed with DMF (800 ml), IPA (600ml) and DMF (800 ml) thereafter resin beads were taken out and checked for Kaiser Test (positive). To this resin a solution consisting of Fmoc- Cys(Trt)-OH (155 g; 2 eqv); HOBT (36 g; 2 eqv) dissolved in DMF (700 ml) and DIC (62 ml; 3eqv) was added and the reaction was allowed at 25°C for 2 hrs, followed by washing with DMF (850 ml) to obtain Fmoc-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(OtBu)- Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Resin. The obtained peptide-resin was washed with DCM (600 ml), MeOH (600 ml) and MTBE (500 ml), and dried under vacuum.
Weight: 467 g
Step (9): Cleavage of protected peptide from the resin
The protected octapeptide (450 g) was cleaved from the solid support by using 0.8% TFA in DCM (4 X 800 ml) and the resulting solution was neutralized by using 10% DIPEA in DCM (4 X 200 ml). The fractions which were found to be UV positive were collected, combined and evaporated. The crude was dissolved in ethyl acetate (2.5 L) and the organic layer was washed with water (500 ml); and 0.1 M NaCl Solution (700 ml). The organic layer was dried over sodiumsulphate, filtered and evaporated to a solid. The solid was treated with MTBE, filtered and dried under vacuum for 16 hrs. Weight: 262 g
Analyzed for mass spectrum as Fmoc-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(OtBu)- Cys(Trt)-Cys(trt)-Asn(Trt)-Pro-OH by LCMS. Mol. Wt. (calculated mass 2478; Observed mass: 2478.8872 (M+). EXAMPLE-2
PROCESS FOR PREPARING NH2-Ala-Cys(Trt)-Thr(Ot-Bu)-GIy-Cys(Trt)- Tyr(Ot-Bu)-CTC-Resin (III) (C-terminal hexapeptide fragment on CTC-Resin) Step (1): Preparation of Fmoc-Tyr (Ot-Bu)-Chloro Trityl Resin
2-ChloroTrityl Resin (CTC Resin) (65 g) (1.6mmol/g) was transferred to a glass reaction vessel containing a sintered disk. Anhydrous dichloromethane (400 ml) was added to the glass vessel and drained after two min. A clear solution of Fmoc- Tyr(OtBu)-OH (29 g, 1.1 eqv.) dissolved in dry DCM (300 ml) and N.N di- isopropylethylamine (41 ml) was added. The reaction mixture was stirred mechanically for 3 hrs and the solution was drained out, and the resin was washed with 1 % DIPEA in DCM (400 ml). The peptide resin was washed with a mixture of 1 : 1 [ 10% DIPEA: Methanol; 300 ml], 1% DIPEA in DCM (400 ml), 0.5% DIPEA in MTBE (300 ml) and dried under vacuum.
Weight: 91 g
Step (2): Coupling of Fmoc-Cys (Trt) to Tyr (OtBu)-CTC-Resin
Fmoc-Tyr(OtBu)-Resin from step (1), was swelled in dichloromethane (250 ml) for 20 min and dimethylformamide (DMF) (250 ml) for 20 min. 20% piperidine in DMF (300 ml) (5+15 min) was added to the Fmoc-Tyr (OtBu)-Resin and the resin was washed with DMF (300 ml), Isopropylalcohol (IP A) (200 ml) and DMF (300 ml). Thereafter resin beads were taken out and checked for Kaiser Test (positive) and chloranil test (positive). To this resin a solution consisting of Fmoc-Cys(Trt)-OH (68 g; 2 eqv); HOBT (16 g; 2 eqv) dissolved in DMF (300 ml) and DIC (27 ml; 3eqv) was added and the reaction was allowed at 25°C for 2 hrs, followed by washing with DMF (300 ml) to obtain Fmoc-Cys(Trt)-Tyr(OtBu)-Resin.
Step (3): Coupling of Fmoc-Gly-OH to Cys(Trt)-Tyr-(OtBu)-Resin
20%) piperidine in DMF (300 ml) (5+15 min) was added to the Fmoc-Cys(Trt)- Tyr(OtBu)-Resin. The resin was washed with DMF (300 ml), IPA (200 ml) and DMF (300 ml) thereafter resin beads were taken out and checked for Kaiser Test (positive). To this resin a solution consisting of Fmoc-Gly-OH (35 g; 2 eqv); HOBT (16 g; 2 eqv) dissolved in DMF (300 ml) and DIC (27ml; 3eqv) was added and the reaction was allowed at 25°C for 2 hrs, followed by washing with DMF (300 ml) to obtain Fmoc- Gly-Cys(Trt)-Tyr(OtBu)-Resin. Step (4): Coupling of Fmoc-Thr(OtBu)-OH to GIy-Cys(Trt)-Tyr(OtBu)-Resin
20% piperidine in DMF (300 ml) (5+15 min) was added to the Fmoc-Gly-Cys(Trt)- Tyr(OtBu)-Resin. The resin was washed with DMF (300 ml), IPA (200 ml) and DMF (300 ml) thereafter resin beads were taken out and checked for Kaiser Test (positive). To this resin a solution consisting of Fmoc-Thr(OtBu)-OH (46 g; 2 eqv); HOBT ( 16 g; 2 eqv) dissolved in DMF (300 ml) and DIC (27ml; 3eqv) was added and the reaction was allowed at 25°C for 2 hrs, followed by washing with DMF (300 ml) to obtain Fmoc-Thr(OtBu)-Gly-Cys(Trt)-Tyr(OtBu)-Resin.
Step (5): Coupling of Fmoc-Cys(Trt)-OH to Thr(OtBu)-Gly-Cys(Trt)-Tyr(OtBu)- Resin
20% piperidine in DMF (300 ml) (5+15 min) was added to the Fmoc-Thr(OtBu)-Gly- Cys(Trt)-Tyr(OtBu)-Resin. The resin was washed with DMF (300 ml), IPA (200 ml) and DMF (300 ml) thereafter resin beads were taken out and checked for Kaiser Test (positive). To this resin a solution consisting of Fmoc-Cys(Trt)-OH (68 g; 2 eqv); HOBT ( 16 g; 2 eqv) dissolved in DMF (300 ml) and DIC (27ml; 3eqv) was added and the reaction was allowed at 25°C for 2 hrs, followed by washing with DMF (300 ml) to obtain Fmoc-Cys(Trt)-Thr(OtBu)-Gly-Cys(Trt)-Tyr(OtBu)-Resin.
Step (6): Coupling of Fmoc-Ala-OH to Cys-(Trt)-Thr-(OtBu)-Gly-Cys-(Trt)-Tyr- (OtBu)-Resin
20% piperidine in DMF (300 ml) (5+15 min) was added to the Fmoc-Cys(Trt)- Thr(OtBu)-Gly-Cys (Trt)-Tyr(OtBu)-Resin. The resin was washed with DMF (300 ml), IPA (200 ml) and DMF (300 ml) thereafter resin beads were taken out and checked for Kaiser Test (positive). To this resin a solution consisting of Fmoc-Ala-OH (37 g; 2 eqv); HOBT (16 g; 2 eqv) dissolved in DMF (300 ml) and DIC (27ml; 3eqv) was added and the reaction was allowed at 25°C for 2 hrs, followed by washing with DMF (300 ml) to obtain Fmoc-Ala-Cys(Trt)-Thr(OtBu)-Gly-Cys(Trt)-Tyr(OtBu)-Resin.
EXAMPLE-3
PROCESS FOR PREPARING LINEAR PROTECTED LINACLOTIDE
Coupling of Fmoc-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(OtBu)-Cys(Trt)-Cys(Trt)- Asn(Trt)-Pro to NH2-Ala-Cys(Trt)-Thr(OtBu)-Gly-Cys(Trt)-Tyr(OtBu)-CTC- Resin
20% piperidine in DMF (300 ml) (5+15 min) was added to the Fmoc-Ala-Cys(Trt)- Thr(OtBu)-Gly-Cys (Trt)-Tyr(OtBu)-Resin obtained from the Example-2, step (6). The resin was washed with DMF (300 ml), IPA (200 ml) and DMF (300 ml) thereafter resin beads were taken out and checked for Kaiser Test (positive). To this resin a solution consisting of Protected Octa Peptide fragment obtained from Example- 1 , step (9) (215 g; 1.5 eqv); HOBT (12 g; 1.5 eqv) was dissolved in DMF (800 ml) and DIC (23 ml; 2.5 eqv) added stirred for two min. The solution was added to the peptide resin, and the reaction was allowed at 25°C for 16 hrs, followed by washed with DMF (900 ml) Fmoc-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(OtBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala- Cys(Trt)-Thr(OtBu)-Gly-Cys(Trt)-Tyr(OtBu)-Resin. After washing of the peptidyl resin the Fmoc group of Cys(Trt) was deprotected by using 20% piperidine in DMF (800 ml) (5+15 min). The resin was washed with DMF (800 ml), IPA (600 ml) and DMF (800 ml), thereafter a small amount of resin beads were taken out and checked for Kaiser Test (positive). The obtained peptide-resin was washed with DCM (600 ml), MeOH (600 ml) and MTBE (500 ml) and dried under vacuum for 16 hrs.
Weight: 279 g EXAMPLE-4
PROCESS FOR PREPARING LINEAR LINACLOTIDE Step (1): Cleavage of Protected Linaclotide from the peptide resin
The experiment conducted for cleavage of Linaclotide (protected) from the peptide resin (190 g) is carried out by using 1% TFA in DCM (4X300 ml) and the resulting solution was neutralized by using 10% DIPEA in DCM (4X100 ml). The fractions which were found to be UV positive were collected, combined and evaporated. The crude was dissolved in ethyl acetate (1.5 L) and the organic layer was washed with water (250 ml); and 0.1 M NaCl Solution (250 ml). The organic layer was dried over sodium sulphate, filtered and evaporated to a solid. The solid was treated with MTBE, filtered and dried under vacuum for 16 hrs.
Weight: 145 g
Step (2): Deprotection of Protected Linaclotide
50 g of protected peptide obtained above was added to the reactor containing cold solution (500 ml) of 80% TFA (400 ml), 5% TIS (25 ml), 10% H20 (50 ml), 5% EDT (25 ml); stirred the coack tail for 30 min at 0-5°C and filtered the solid and the TFA coack tail was stirred for 2 hours at room temperature. After two hours the solution was filtered and precipitated by the addition of 10 volumes of MTBE (5 L). The obtained product was filtered and washed with MTBE and dried under vacuum to obtain linear crude product.
Yield: 24 g
HPLC Purity: 81%. EXAMPLE-5
PROCESS FOR PREPARING LINACLOTIDE Linear Linaclotide (10 g) was dissolved in degassed 0.01 M Ammonium bicarbonate (10 L) (pH 8.1 ). After dissolving the compound slowly bubbled the compressed air and added the hydrogen peroxide ( 1 ml). After 18 hrs the completion of the reaction was monitored by reverse phase analytical HPLC. The oxidized product was purified by reverse phase HPLC.
Oxidized Product Purity: 74 %
EXAMPLE-6
PURIFICATION OF CRUDE LINACLOTIDE
Stage-1
The oxidized crude peptide solution obtained from the example 5 was passed through Novasep lab system RP-HPLC system, wavelength 220nm, 1 10 X 700mm column of C I 8 reverse phase column, mobile phase: A : 0.01 M ammonium acetate ; mobile phase B: acetonitrile flow rate: 400 ml / min, peaks of fractions were collected and pooled the fractions having the purity greater than 80%.
Stage-2
The main pool obtained from the stage-1 were diluted with equal amount of water and passed through Novasep lab system RP-HPLC system, wavelength 220nm, 1 10 X 700mm column of C I 8 reverse phase column, mobile phase: A: 0.1 % TFA in water; mobile phase B: acetonitrile flow rate: 400 ml / min, fractions collected and analyzed by RPHPLC and SEC the fractions having the purity greater than 98% were taken and pooled and taken to the next stage. Stage-3
The main pool obtained from the stage-2 were diluted with equal amount of water and passed through Novasep lab system RP-HPLC system, wavelength 220nm, 1 10 X 700mm column of C I 8 reverse phase column, mobile phase: A: 0.1% acetic acid in water; mobile phase B: acetonitrile flow rate: 400 ml / min, fractions collected and analyzed by RPHPLC and SEC the fractions having the purity greater than 99% were collected, pooled together, concentrated using rotary evaporation and lyophilized to give pure Linaclotide.
Yield: 1.5 g
HPLC purity: 98.9%

Claims

We claim
1 ) A process for the preparation of Linaclotide of formula I,
Formula I
Figure imgf000022_0001
which comprises the following steps:
a) preparing two or three suitable fragments by solid phase peptide synthesis; b) coupling of the fragments by solid phase synthesis to obtain a protected peptide;
c) concurrently cleaving the protected peptide from the peptide resin and de- protecting the peptide;
d oxidizing the deprotected peptide to obtain Linaclotide; and
e) isolation of Linaclotide.
2) The process according to claim 1 , wherein the suitable fragments used in step (a) are selected from compound of formula II and compound of formula III.
Y- Cys(Z)-'Cys(Z)- Glu(X)- Tyr(X>- Cys(Z)- Cys(Z)- Asn(Y)- Pro- OH Formula II Ala- Cys(Z)- Thr(X)- Gly— Cys(Z)- Tyr(X)→ | Formula III wherein, Z represents thiol protecting group; X represents carboxyl, phenolic and alcoholic protecting groups; Y represents amino protecting group and ® represents resin.
3) The process according to claim 1 , wherein the suitable fragments used in step (a) are selected from the compound of formula II, compound of formula IV and compound of formula V.
Y- Cys(Z)- Cys(Z)- Glu(X)- Tyr(X>- Cys(Z)- Cys(Z)- Asn(Y)- Pro- OH Formula II Y- Ala- Cys(Z)- Thr(X)- Gly- OH Formula IV
H2\'~ Cys(Z) - Tyr(X)— O Formula V wherein, Z represents thiol protecting group; X represents carboxyl, phenolic and alcoholic protecting groups; Y represents amino protecting group and © represents resin.
4) A process for the preparation of Linaclotide of formula I, comprising the following steps:
a) preparing fragments of Formula II and formula III by a solid phase synthesis; b) coupling of fragments of Formula II and Formula III by a solid phase synthesis to obtain a protected peptide;
c) concurrently cleaving the protected peptide from the peptide resin and de- protecting the peptide;
d) oxidizing the deprotected peptide to obtain Linaclotide; and
e) isolating the Linaclotide.
5) A process for the preparation of fragment of Formula III, which comprises the following steps,
a) preparing fragments of formula IV and formula V by a solid phase synthesis; and
b) coupling of fragments of Formula IV and Formula V by a solid phase synthesis.
6) A process for the preparation of fragment of Formula IV, of claim 5, which comprises by sequential addition of corresponding amino acids using a solid phase.
7) A process for the preparation of fragment of Formula V, of claim 5, which comprises by sequential addition of corresponding amino acids using a solid phase. 8) The process according to claims 1 , 4 and 5 wherein the coupling is carried out in presence of coupling agent(s).
9) The coupling agent (s) according to claim 8, is selected from the group comprising of HOBt, TBTU, DCC, DIC, HBTU, BOP, PyBOP, PyBrOP, PyClOP, Oxyma
Pure, TCTU, EEDQ, COMU, DEPBT and the like or mixtures thereof.
10) The process according to claims 1 and 4, wherein the de-protection of peptide is carried out using a mixture of reagents selected from the group comprising of TFA, TIS, DTT, EDT, ammonium iodide, 2,2'-(ethylenedioxy)diethane, acetyl cystein, DMS, phenol, cresol and thiocresol.
1 1) The process according to claims 1 and 4, wherein the oxidation is carried by using oxidizing agents.
12) The oxidizing agents according to claim 1 1 , is selected from the group comprising of hydrogen peroxide, dimethyl sulfoxide (DMSO), glutathione and the like or mixtures thereof. 13) Linaclotide prepared according to the claims 1 and 4, having purity of greater than 97%.
14) Linaclotide prepared according to the claims 1 and 4, wherein the said Linaclotide is substantially free from dimer impurities and multimer impurities.
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US10501498B2 (en) * 2014-12-26 2019-12-10 Bgi Shenzhen Conotoxin peptide κ-CPTx-BTL01, preparation method therefor, and uses thereof
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