# US 2022/0033440 A1 · An improved process for the preparation of plecanatide

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- Number: US 2022/0033440 A1
- Document ID: US20220033440A1
- Status: Published application
- Kind: A1
- Application number: 17/311,789
- Filed: 2019-04-01
- Published: 2022-02-03
- Earliest claimed: 2018-12-08
- Assignees: Enzene Biosciences Limited
- Applicants: Enzene Biosciences Limited
- Inventors: Sandip ADAK; Manoj BONTE; Chandrakant KULKARNI; Veeranarayana SWAMY; Nivrutti JOGDAND; Himanshu GADGIL
- Web page: https://patentgrep.com/patent/US20220033440A1

## CPC

- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- C07K 1/003: {by transforming the C-terminal amino acid to amides}
- C07K 1/047: {Simultaneous synthesis of different peptide species; Peptide libraries}
- C07K 1/061: {using protecting groups}
- C07K 1/1075: {by covalent attachment of amino acids or peptide residues}
- C07K 1/36: by a combination of two or more processes of different types
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

A process for the preparation of plecanatide. Specifically, an improved process for preparation of plecanatide using a non-linear solid phase peptide synthesis. In particular, the process for preparation of plecanatide involves solid phase synthesis of peptide fragments of five amino acid units using 2-chlorotrityl chloride (2-ClTrt) resin and eleven amino acid unit using Wang resin.

## Claims

### Claim at position 1

1-16. (canceled)

### Claim 17

A process for preparing plecanatide comprising the steps of:
(a) Monocyclizing of a linear chain of formula (I), H2N-Asn1-Asp2-Glu3-Cys4-Glu5-Leu6-Cys(Acm)7-Val8-Asn9-Val10-Ala11-Cys12-Thr13-Gly14-Cys(Acm)15-Leu16-OH with a suitable reagent to obtain a monocyclic peptide of formula (VI),
(b) purifying the monocyclic peptide of formula (VI) obtained in step (a) using reverse phase column chromatography,
(c) dicyclizing the monocyclic peptide of formula (VI) obtained in step (b) with suitable reagents to obtain dicyclic peptide of formula (VII),
and
(d) purifying and desalting the dicyclic peptide of formula (VII) obtained in step (c) using reverse phase column chromatography to obtain pure plecanatide.

### Claim 18 (depends on 17)

The process for preparing plecanatide as claimed in claim 17, wherein the linear chain of formula (I) instep (a) is obtained by a process comprising the steps of:
(a) coupling fragment A with Wang resin bound fragment B using coupling reagent to obtain a Wang resin bound protected peptide fragment;
(b) cleaving and deprotecting the Wang resin bound protected peptide fragment obtained in step (a) using reagents, trifluoroacetic acid/triisopropylsilane/DODT/Water to obtain the linear chain of formula (I), H2N-Asn1-Asp2-Glu3-Cys4-Glu5-Leu6-Cys(Acm)7-Val8-Asn9-Val10-Ala11-Cys12-Thr13-Gly14-Cys(Acm)15-Leu16-OH.

### Claim 19 (depends on 17)

The process for preparing plecanatide as claimed in claim 17, wherein the reverse phase column chromatography for purifying monocyclic peptide has column specification: 250×80 mm, media specification: C-18, 10μ, 100 A°, mobile phase A: 0.02M triethyl ammonium phosphate in water of pH 6.5 and mobile phase B: acetonitrile.

### Claim 20 (depends on 17)

The process for preparing plecanatide as claimed in claim 17, wherein the reverse phase column chromatography for purifying the dicyclic peptide of formula (VII) has column specification: 250×80 mm, media specification: C-18, 7-10μ, 100 A°, mobile phase A: 0.02 M triethyl ammonium phosphate in water of pH 6.5 and mobile phase B: acetonitrile.

### Claim 21 (depends on 17)

The process for preparing plecanatide as claimed in claim 17, wherein the reverse phase column chromatography for desalting the dicyclic peptide of formula (VII) has column specification: 250×80 mm, media specification: C-18, 10μ, 100 A°, mobile phase A: 0.025% ammonium hydroxide, mobile phase B: acetonitrile, mobile phase C: 0.1-0.2M ammonium acetate in water and mobile phase D: 0.1% trifluoroacetic acid in water.

### Claim 22 (depends on 17)

The process for preparing plecanatide as claimed in claim 17, wherein the reverse phase column chromatography for purifying the dicyclic peptide of formula (VII) has column specification: 250×80 mm, media specification: C-18, 10μ, 100 A°, mobile phase A: 0.1-0.2% trifluoroacetic acid in water and mobile phase B: acetonitrile.

### Claim 23 (depends on 17)

The process for preparing plecanatide as claimed in claim 17, wherein the suitable reagent in monocyclizing step is 1-2% H2O2 solution.

### Claim 24 (depends on 17)

The process for preparing plecanatide as claimed in claim 17, wherein the suitable reagent in dicyclizing step is 2% iodine solution.

### Claim 25 (depends on 18)

The process for preparing plecanatide as claimed in claim 18, wherein the fragment A is Fmoc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-OH or Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-OH.

### Claim 26 (depends on 18)

The process for preparing plecanatide as claimed in claim 18, wherein the Wang resin bound fragment B is H2N-Leu-Cys(Acm)-Val-Asn(Trt)-Val-Ala-Cys(Trt)-Thr(OtBu)-Gly-Cys(Acm)-Leu-O-Wang resin.

### Claim 27 (depends on 18)

The process for preparing plecanatide as claimed in claim 18, wherein the fragment A is not more than 5 amino acid units in length.

### Claim 28 (depends on 18)

The process for preparing plecanatide as claimed in claim 18, wherein the Wang resin bound fragment B is not more than 11 amino acid units in length.

### Claim 29 (depends on 18)

The process for preparing plecanatide as claimed in claim 18, wherein the fragment A and fragment B are prepared using solid-phase peptide synthesis.

### Claim 30 (depends on 18)

The process for preparing plecanatide as claimed in claim 18, wherein the mixture of trifluoroacetic acid/triisopropylsilane/3,6-dioxa-1,8-octanedithiol/water is in the ratio of 9:0.5:0.25:0.25 v/v respectively.

### Claim 31 (depends on 18)

The process for preparing plecanatide as claimed in claim 18, wherein the Wang resin bound fragment B, H2N-Leu-Cys(Acm)-Val-Asn(Trt)-Val-Ala-Cys(Trt)-Thr(OtBu)-Gly-Cys(Acm)-Leu-O-Wang resin is prepared by coupling amino acids at the temperature in the range of 25° C. to 65° C. under stirring for 20 minutes to 3 hours.

### Claim 32 (depends on 17)

The process for preparing plecanatide as claimed in claim 17, wherein the linear chain of formula (I) in step (a) is obtained by a process comprising coupling fragment A with Wang resin bound fragment B using coupling reagent to obtain a Wang resin bound protected peptide fragment at the temperature in the range of 25° C. to 65° C. under stirring for 45 minutes to 5 hours.