Title of Invention : PROCESS FOR THE PREPARATION OF LINACLOTIDE

Technical Field

[1] The present invention relates to peptide synthesis. In particular, it relates to a process for the preparation of linaclotide. More specifically, it relates to a method of oxidative folding of the linear linaclotide to obtain the cyclic linaclotide.

Background Art

[2] Linaclotide is a cyclic peptide which was originally derived from the bacterial heat-stable enterotoxin. It gained clinical interest when it was found that it acts as a potent guanylate cyclase 2C agonist for the treatment of the irritable bowel syndrome (IBS) with predominant constipation. Linaclotide binds to the guanylate cyclase-C (GC-C) receptor in the small bowel mucosa and induces the increase of intracellular and extracellular cyclic guanosine monophosphate levels, thereby mimicking the prosecretory action of paracrine hormones guanylin and uroguanylin (Chandar A. K. Int. J. Gen. Med. 2017, 10, 385-393; Bassotti G. et al. Expert Opin. Pharmacother. 2018, 19, 1261-1266).

[3] The peptide sequence of Linaclotide (I) consists of 14 amino acids with three disulfide bonds Cys ¹ -Cys ⁶ , Cys ² -Cys ¹⁰ and Cys ⁵ -Cys ¹³ , which provide high enzymatic and acidic stability of the peptide. Linaclotide can be represented by the following Formula I:

[4] Linaclotide (CAS No. 851199-59-2) is currently marketed as the acetate salt (CAS No. 851199-60-5) with the brand name Constella (EU) or Linzess (US).

[5] Linaclotide was first disclosed in W02004069165 (Microbia, Inc.), where it was obtained either by a recombinant method or by Boc solid phase synthesis using acetamidomethyl and 4-methylbenzyl protections for Cys with following deprotection by hydrogen fluoride and consecutive cyclizations by treatment with dimethylsulfoxide and iodine. In the following years, several alternative synthetic processes were proposed. In general, these can be divided in two groups, where (i) disulfide bonds are formed stepwise by consecutive removal of different orthogonal protective groups for cysteine residues, or where (ii) the oxidative folding of the linear linaclotide (II, SEQ ID NO: 1), defined below, is carried out in the presence of an oxidant with simultaneous formation of all three disulfide bonds.

H-Cys ¹ -Cys ² -Glu ³ -Tyr ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ -Tyr ¹⁴ -OH II [6] As to the first method a number of different combinations of orthogonal protections of cysteine residues for stepwise disulfide bond formation were disclosed. For instance, CN 103626849 disclosed the use of three protective groups, in particular, Hqm, Trt and Acm for Cys in the positions 1-6, 2-10 and 5-13, respectively. A disadvantage of methods comprising consecutive formation of the appropriate disulfide bonds is the incomplete or premature removal of the protective groups leading to the accumulation of impurities. In addition, the use of different reagents for the deprotection and oxidation noticeably complicates the synthesis.

[7] As to the second method, the oxidative folding of the linear linaclotide precursor with simultaneous formation of the three disulfide bonds has a number of advantages. It is less laborious, cheaper and requires reagents and solvents commonly used in peptide synthesis. Various oxidants were proposed for such method. For instance, CN 102875655 disclosed a process for the synthesis of linaclotide, wherein Mmt protection was used for all Cys residues with subsequent oxidation with a mixture of reduced and oxidized glutathione. In WO2017134687, the addition of alcohols, such as methanol, ethanol or isopropanol, allowed to increase the concentration of the linear peptide during the cyclization.

[8] It is known that the removal of acid labile side-chain protective groups, which are used to prepare the linear linaclotide precursor, can be achieved by treatment with trifluoroacetic acid (TFA) in the presence of scavengers, which are able to sequester and neutralize the forming carbocations thereby preventing the irreversible damage of the amino acid sidechains. Several deprotection mixtures based on TFA solutions are described in the prior art to prepare linear linaclotide. The reported concentration of TFA is ranging from 80 to 95%. Scavengers, like triisopropylsilane (TIS), water, phenol, ethanedithiol, dithiothreitol, anisole, thioanisole and mercaptopropionic acid, were added as the remaining 5-20% amount in the mixture (WO2016038497 and W02014188011). In WO2017134687 a 25% solution of TFA in DCM with addition of TIS was used to completely deprotect the peptide.

[9] The preparation of peptides comprising several sulfhydryl groups, i.e. several Cys residues, such as linear linaclotide (II), is challenging because of the formation of a number of side-products at the step of cleavage from the solid support and at the deprotection step. For example, impurities can be formed due to the reaction with t-buty I trifluoroacetate, which is present in the cleavage mixture (Lundt et al. Int. J. Pept. Protein Res. 1978, 12, 258-268; Guy et al. Methods Enzymol. 1997, 289, 67-83). The reactions of S-alkylation and trifluoracetylation, as well as incomplete deprotection, noticeably decrease the purity and yield of the peptide. [10] Another problem encountered in the synthesis of linaclotide is the presence of a significant amount of impurities closely eluting with linaclotide in the crude peptide, such as, for example, misfolded peptides. The efforts performed to remove impurities like these, as well as peptide polymers derived from intermolecular disulfide bond formation, usually result in a decrease of the product yield. Thus, there remains a need to develop an efficient, simple and industrially viable synthetic process for the preparation of linaclotide which can lessen the drawbacks of the prior art and allow its preparation with high yield and a favorable impurity profile.

[11] The present invention has the purpose of solving the above problems or at least improving the product yield and lowering the amount of impurities in the crude linaclotide.

Summary of Invention

[12] 2,2,2-trifluoroethanol (TFE) has been used as co-solvent in circular dichroism and NMR studies, where it was observed that the population of folded structures in secondary- structure-forming peptides increased (Roccatano et al. PNAS 2002, 99, 12179-12184). The mechanism of this secondary structure stabilization is still unclear. TFE or hexafluoroisopropanol (HFIP) were also used as solvents for the removal of peptides sidechain protections by 0.1 M HCI (Palladino et al. Org. Lett. 2012, 14, 6346-6349).

[13] It has been surprisingly found that the use of fluorinated alcohols in the synthesis of linaclotide can remarkably improve the yield of the target peptide and its impurity profile. As a matter of fact, the addition of a fluorinated alcohol to the reaction mixture is able to decrease the amount of impurities formed during the deprotection step and of misfolded peptide and peptide polymers during the oxidative folding step. The result is a higher yield arising from the decrease of undesired species.

[14] Moreover, the employment of fluorinated alcohols, in particular in the deprotection step, allows to avoid the aggressive, corrosive and toxic trifluoroacetic acid (TFA), commonly used at this step, thus making the production of linaclotide more environmentally friendly.

[15] The oxidative folding of the linear linaclotide (II) may be carried out immediately after deprotection without isolation of the linear peptide by only increasing the pH to basic values.

[16] Accordingly, the present invention provides a process for the preparation of linaclotide (I), or a pharmaceutically acceptable salt thereof, comprising a step of: oxidative folding of the linear linaclotide (II, SEQ ID NO: 1):

H-Cys ¹ -Cys ² -Glu ³ -Tyr ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ -Tyr ¹⁴ -OH II to obtain linaclotide (I), which is carried out in the presence of at least one oxidant in an aqueous solution comprising a fluorinated alcohol.

Description of Embodiments

[17] The term “about” is used herein to include plus/minus 10% of the value it refers to.

[18] The term “process for the preparation”, as used herein, refers to any method suitable for the synthesis of linaclotide (I).

[19] The term “oxidative folding” or “oxidative folding step”, as used herein, refers to a step wherein the linear linaclotide (II, SEQ ID NO: 1) is converted into linaclotide (I) by formation of the three disulfide bonds Cys ¹ -Cys ⁶ , Cys ² -Cys ¹⁰ and Cys ⁵ -Cys ¹³ between the respective sulfhydryl groups of Cys amino acids side-chains. Such folding comprising disulfide bonds formation allows the creation of the correct 3D secondary structure.

[20] The term “oxidant”, as used herein, refers to an agent required to perform the oxidative folding step and which is present in the oxidative folding step reaction mixture, which comprises the substrate linear linaclotide (II, SEQ ID NO: 1) and a solvent.

[21] The term “aqueous solution”, as used herein, refers to a solvent comprising water. In the process for the preparation of linaclotide (I) herein disclosed, an aqueous solution is used both in the oxidative folding and in the deprotection step.

[22] The term “fluorinated alcohol”, as used herein, refers to an alcohol wherein one or more hydrogen atoms, except the hydrogen in the hydroxyl group, are replaced by corresponding fluorine atoms. Examples of fluorinated alcohols are 2,2,2-trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP). A mixture HFIP/TFE refers to mixed HFIP and TFE in variable proportions.

[23] The term “acid labile protective group”, as used herein, refers to a suitable protective group, attached to the amino acids side chain functional groups or the C-terminal end of a peptide, which is removable under acidic conditions. According to the invention these groups are referred to as (A), (B), (C), ... to (K) or as Y. According to the invention such groups are used in the linear side-chain protected linaclotide of Formula III:

H-Cys(A) ¹ -Cys(B) ² -Glu(C) ³ -Tyr(D) ⁴ -Cys(E) ⁵ -Cys(F) ⁶ -Asn(G) ⁷ -Pro ⁸ -Ala ⁹ -

Cys(H) ¹⁰ -Thr(l) ¹¹ -Gly ¹² -Cys(J) ¹³ -Tyr(K) ¹⁴ -OY III

[24] The term “deprotection” or “deprotecting step”, as used herein, refers to a step wherein the side-chain protective groups (such as A to K) in a side-chain protected peptide, such as linaclotide (III), as defined above, are removed by acidic treatment.

[25] The term “cleavage” or “cleaving step”, as used herein, refers to a step wherein a peptide is removed from a solid support. According to the invention, the cleaving step refers to the step wherein the linear side-chain protected linaclotide (III) as defined above, wherein Y is a solid support, is removed from such solid support by acidic treatment.

[26] The term “solid support”, as used herein, refers to a resin which is a functionalized insoluble polymer to which an amino acid or a peptide can be attached and which is suitable for the synthesis of peptides by stepwise amino acids elongation, or fragmentbased elongation, until the full desired sequence is obtained. The so-called solid phase peptide synthesis (SPPS) which employs such kind of solid supports is well known in the art.

[27] The term “acidic treatment”, as used herein, refers to a treatment with a solution comprising at least one acid, typically a mixture of solvents comprising at least one acid, i.e. an acidic mixture.

[28] The terms “simultaneously” or “simultaneous”, are used herein to describe the fact, that the deprotecting step and the cleaving step are performed at the same time, i.e. in the same acidic mixture. Such mixture has to be strong enough to allow both removal of the side-chain protective groups and detachment of the linear linaclotide from the solid support.

[29] The term “concentration” is used herein when describing both the amount (vol%) of the fluorinated alcohol in a solution and the amount (g/L) of the linear linaclotide (II, SEQ ID NO: 1) in the aqueous solution used in the oxidative folding step.

[30] The term “purification”, as used herein, refers to any step performed aiming at increasing the % purity value of linaclotide (I) after the oxidative folding step, typically measured as HPLC % purity. [31] In a preferred embodiment, the present invention provides a process for the preparation of linaclotide (I), or a pharmaceutically acceptable salt thereof, comprising a step of: oxidative folding of the linear linaclotide (II, SEQ ID NO: 1):

H-Cys ¹ -Cys ² -Glu ³ -Tyr ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ -Tyr ¹⁴ -OH II to obtain the linaclotide (I), which is carried out in the presence of at least one oxidant in an aqueous solution comprising a fluorinated alcohol, wherein the fluorinated alcohol is 2,2,2-trifluoroethanol (TFE), hexafluoroisopropanol (HFIP) or a mixture thereof, wherein the amount of HFIP can vary from 0 vol% to 100 vol%.

Preferably, the fluorinated alcohol is hexafluoroisopropanol (HFIP) or a mixture of hexafluoroisopropanol and 2,2,2-trifluoroethanol (TFE).

Preferably, a mixture is used wherein the HFIP vol% is higher than 0 vol%, more preferably it is 50 vol%.

[32] In a further preferred embodiment, the concentration of the fluorinated alcohol in the aqueous solution used in the oxidative folding step is in the range 5-90 vol%, preferably 20-60 vol%, more preferably 30 vol%.

If the fluorinated alcohol is a mixture of HFIP and TFE, it is preferred that the aqueous solution contains 15% HFIP and 15% TFE.

[33] The oxidative folding step requires the presence of at least one oxidant. Such oxidant may simply be the air oxygen which diffuses into the reaction mixture from the atmosphere, facilitated by the stirring of such mixture. The at least one oxidant may also be another oxidant selected from the group consisting of cysteine, a cysteine/cystine mixture, reduced glutathione, a reduced/oxidized glutathione mixture, and N-halosuccinimide. Preferably, the oxidant is selected from the group consisting of air oxygen, cysteine, a cysteine/cystine mixture and a combination thereof.

[34] In another preferred embodiment, the oxidative folding step is carried out by controlling the pH of the aqueous solution. This can be obtained for instance by addition of an aqueous base, such as, for example, diluted NaOH, to the aqueous solution comprising the fluorinated alcohol or a mixture thereof. Accordingly, the oxidative folding step is preferably carried out at a pH from about 7 to about 10, more preferably at a pH about 8. [35] In yet another preferred embodiment, in the oxidative folding step the concentration of the linear linaclotide (II, SEQ ID NO: 1) as defined above is in the range of 0.1 - 10 g/L, preferably in the range of 0.5 - 5 g/L, more preferably in the range of 2 - 5 g/L, most preferably about 2 g/L.

[36] In the oxidative folding step, features like the fluorinated alcohol, its concentration in the aqueous solution, the at least one oxidant, the pH and the concentration of the linear linaclotide (II, SEQ ID NO: 1) are to be considered independently in their preferred embodiments and may be combined with each other in more preferred embodiments of the invention.

[37] For instance, a particularly preferred embodiment of the invention is a process for the preparation of linaclotide (I), or a pharmaceutically acceptable salt thereof, comprising a step of oxidative folding of the linear linaclotide (II, SEQ ID NO: 1) to obtain the linaclotide (I), which is carried out in the presence of at least one oxidant in an aqueous solution comprising a fluorinated alcohol, wherein the fluorinated alcohol is a mixture HFIP/TFE, the oxidant is air oxygen and cysteine, the pH is about 8 and the concentration of the linear linaclotide (II) is about 2 g/L.

[38] The linear linaclotide (II, SEQ ID NO: 1) which undergoes the process of the invention as defined above may be prepared by any method known to the person skilled in the art. Preferred methods encompass SPPS, LPPS (Liquid Phase Peptide Synthesis) and a combination thereof. SPPS is the most preferred, either as a stepwise elongation with amino acids added one-by-one to a growing peptide chain or as a stepwise condensation of peptide fragments.

[39] Regardless of the method of preparation selected to provide the linear linaclotide (II, SEQ ID NO: 1), protective groups at the amino acids side-chains may be useful in the synthesis. Typically, these protective groups are acid labile protective groups, i.e. groups which are stable during the reactions forming the amide bonds and the reactions removing the alpha-amino protecting groups, which are generally performed in basic conditions, in both SPPS and LPPS.

[40] According to the invention it is preferred that the acid labile protective groups at the linear linaclotide are tert-butyloxycarbonyl (Boc), tert-butyl (indicated as tBu or OtBu), trityl (Trt), and the like. Trt for Cys and Asn, and tBu for Glu, Thr and Tyr are more preferred, but other acid labile protective groups may be efficiently used as it is apparent to the person skilled in the art.

[41] When SPPS is used for the synthesis of the linear linaclotide (II), preferred solid supports are 2-chloro-trityl chloride (2-CTC) resin and trityl chloride (TC) resin. Such solid supports allow obtaining the target peptide (II) with C-terminal carboxylic group after the cleavage step. Such solid supports may be cleaved from the peptides which have been grown onto them by acidic treatment, as it will be described hereinbelow.

[42] When SPPS is used, and acid labile solid supports are employed, the deprotecting step may be performed either after the cleaving step or simultaneously with the cleaving step. Generally, the acidic conditions required for the deprotection are harsher than the acidic conditions required for the cleavage, thus deprotection is not possible without cleaving the substrate from the solid support. The cleavage step may, however, be performed before the deprotection step.

[43] Accordingly, the process of the invention preferably further comprises a step, preceding the oxidative folding step, of: deprotecting a linear side-chain protected linaclotide of Formula III:

H-Cys(A) ¹ -Cys(B) ² -Glu(C) ³ -Tyr(D) ⁴ -Cys(E) ⁵ -Cys(F) ⁶ -Asn(G) ⁷ -Pro ⁸ -Ala ⁹ -

Cys(H) ¹⁰ -Thr(l) ¹¹ -Gly ¹² -Cys(J) ¹³ -Tyr(K) ¹⁴ -OY III wherein

(A) to (K) are acid labile side-chain protective groups, and

Y is H, an acid labile protective group or a solid support, by acidic treatment to obtain the linear linaclotide (II, SEQ ID NO: 1) as defined above.

[44] For example, a linear side-chain protected linaclotide (III) may be prepared starting from the loading of Fmoc-Tyr(tBu)-OH, onto a 2-chloro-trityl chloride resin (2-CTC resin), as described in Example 1 of the present disclosure. Alternatively, other protecting groups can be used for the side-protection, and different solid supports may be used.

[45] Accordingly, a preferred linear side-chain protected linaclotide (III) is compound 3:

Fmoc-Cys(T rt)-Cys(T rt)-Glu(OtBu)-Tyr(tBu)-Cys(T rt)-Cys(T rt)-Asn(T rt)-Pro-Ala-

Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-O-CTC resin compound 3.

[46] As explained above, the deprotecting step precedes the oxidative folding step and may be performed on a linear side-chain protected linaclotide (III) either being attached to a solid support (wherein Y is a solid support), or not being attached to a solid support (wherein Y is H or an acid labile protective group).

[47] In a preferred embodiment, the process according to the invention comprises a deprotecting step which is preceded by a step of cleaving the linear side-chain protected linaclotide (III), wherein Y is a solid support, from said solid support by acidic treatment to obtain the linear side-chain protected linaclotide (III) wherein Y is H.

[48] In another preferred embodiment, the steps of cleaving and deprotecting are performed simultaneously. Accordingly, in the process the step of deprotecting is performed simultaneously with a step of cleaving the linear side-chain protected linaclotide (III), wherein Y is a solid support, from said solid support by acidic treatment to obtain the linear linaclotide (II).

[49] The cleavage of linear linaclotide from the solid support and the deprotection step may result in the formation of a number of impurities, such as linear S-f-butyl-linaclotide and linear trifluoroacetyl-linaclotide. The addition of scavengers, such as thiols or water, cannot completely eliminate these impurities, which may be due to the presence of f-butyl trifluoroacetate in the cleavage mixture. These impurities, as well as those caused by incomplete removal of the protective groups, noticeably decrease the yield of the target peptide.

[50] The prior art describes the use of diluted HCI in HFIP or TFE for different substrates (0.1 M HCI, Palladino et al. Org. Lett. 2012, 14, 6346-6349). This approach however did not result in convincing yields of deprotected linaclotide. The treatment time required to gain sufficient deprotection rates resulted in increased degradation rates of the peptide. However, it was surprisingly found that the increase of the concentration of HCI to 1M afforded complete deprotection of linaclotide within 1 h. Only traces of S-f-butyl-linaclotide were observed (see Example 4).

[51] Accordingly, a preferred embodiment of the present invention is a process for the preparation of linaclotide of Formula I which comprises a deprotecting step, or a simultaneous cleaving and deprotecting step, which is carried out by treatment of the linear side-chain protected linaclotide (III) as defined above with 0.5-3M HCI in a fluorinated alcohol, preferably with about 1M HCI.

[52] An even more preferred embodiment is a process according to the invention which comprises a deprotecting step, or a simultaneous cleaving and deprotecting step, which is performed in the presence of a fluorinated alcohol, being HFIP or a mixture of HFIP and TFE, wherein preferably the HFIP vol% varies from about 50 vol% to 100 vol%. Most preferably the fluorinated alcohol is HFIP (100 vol%).

[53] Optionally, the deprotecting step or the simultaneous cleaving and deprotecting step is carried out in the presence of one or more scavengers. Preferably, such scavengers are selected from the group consisting of water, thiols, phenols, and trialkylsilanes. The concentration of the scavenger depends on which scavenger is used and may be easily selected from the person skilled in the art, for instance, in a range of 1 to20 vol%. More preferred scavengers are selected from the group consisting of triisopropylsilane (TIS), triethylsilane (TES) and ethanedithiol (EDT). Most preferably, the deprotecting step is carried out in the presence of TIS, which may be employed in a concentration varying from about 5 vol% to about 10 vol%.

[54] The crude linear linaclotide (II, SEQ ID NO: 1) obtained by the deprotecting step or by the simultaneous cleaving and deprotecting step may be optionally purified, for instance, by preparative RP-HPLC before the oxidative folding step, or the oxidative folding can be carried out on the crude linear linaclotide (II). In a preferred embodiment, the crude linear linaclotide (II) is not purified prior to oxidative folding.

[55] In a particularly preferred embodiment, the oxidative folding of the linear linaclotide (II) is carried out immediately after the deprotection, or after the simultaneous cleavage and deprotection, without isolating the linear linaclotide from the previous step. Accordingly, the deprotection crude mixture, or the simultaneous cleavage and deprotection mixture, may be diluted and the pH adjusted to neutral-basic values, such as to allow the oxidative folding occurrence.

[56] Therefore, a more preferred embodiment of the present invention is a process for the preparation of linaclotide (I), comprising a step of oxidative folding of the linear linaclotide (II, SEQ ID NO: 1) to obtain linaclotide (I), which is preceded by a simultaneous cleaving and deprotecting step, and wherein the oxidative folding is carried out without isolating the linear linaclotide from the previous step, i.e. directly onto the crude filtered solution by simple dilution with an aqueous solution comprising a fluorinated alcohol and the addition of a base until pH in the range 7-10 is reached.

[57] Accordingly, an even more preferred embodiment of the present invention is a process comprising the steps of: simultaneously cleaving and deprotecting a linear side-chain protected linaclotide (III):

H-Cys(A) ¹ -Cys(B) ² -Glu(C) ³ -Tyr(D) ⁴ -Cys(E) ⁵ -Cys(F) ⁶ -Asn(G) ⁷ -Pro ⁸ -Ala ⁹ -

Cys(H) ¹⁰ -Thr(l) ¹¹ -Gly ¹² -Cys(J) ¹³ -Tyr(K) ¹⁴ -OY III wherein

(A) to (K) are acid labile side-chain protective groups, and

Y is an acid labile protective group or a solid support, by acidic treatment with 1M HCI in a fluorinated alcohol, preferably HFIP or a mixture of HFIP/TFE, to obtain the linear linaclotide of Formula II: H-Cys ¹ -Cys ² -Glu ³ -Tyr ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -

Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ -Tyr ¹⁴ -OH II ; and oxidative folding of the linear linaclotide (II) by diluting the acidic solution obtained from the deprotecting step and adjusting the pH to 7-10, preferably pH 8, to afford linaclotide (I), or a pharmaceutically acceptable salt thereof.

[58] The most preferred embodiment of the present invention is a process comprising the steps of: simultaneously cleaving and deprotecting the linear side-chain protected linaclotide (compound 3):

Fmoc-Cys(T rt)-Cys(T rt)-Glu(OtBu)-Tyr(tBu)-Cys(T rt)-Cys(T rt)-Asn(T rt)-Pro-Ala-

Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-O-CTC resin by acidic treatment with 1 M HCI in HFIP, to obtain the linear linaclotide of Formula II:

H-Cys ¹ -Cys ² -Glu ³ -Tyr ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -

Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ -Tyr ¹⁴ -OH II ; and oxidative folding of the linear linaclotide (II) by diluting the acidic solution obtained from the deprotecting step and adjusting to pH 8, to afford linaclotide (I), or a pharmaceutically acceptable salt thereof.

[59] Optionally, the process according to the invention may comprise isolating and purifying linaclotide (I). A preferred purification method involves the use of preparative RP-HPLC. To this aim, a solution of the linaclotide (I) may be loaded onto a HPLC column with a suitable stationary phase, preferably C18 or C8 modified silica, and an aqueous mobile phase comprising an organic solvent, preferably acetonitrile or methanol, is passed through the column. A gradient of the mobile phase is applied, if necessary. The peptide with desired purity is collected and optionally lyophilized.

[60] The experimental details are described in the following Examples section.

Examples

[61] Abbreviations h hour min minutes eq equivalents RT Room Temperature

RP-HPLC Reverse Phase HPLC

SPPS Solid phase peptide synthesis

LPPS Liquid phase peptide synthesis

TC resin trityl chloride resin

2-CTC resin 2-chloro-trityl chloride resin

Fmoc-Pro-OH 9-Fluorenylmethyloxycarbonyl-L-proline

Fmoc-Thr(fBu)-OH 9-Fluorenylmethyloxycarbonyl-O-f-butyl-L- threonine

Fmoc-Cys(Trt)-OH 9-Fluorenylmethyloxycarbonyl-S-trityl-L-cysteine

Fmoc-Glu(OfBu)-OH 9-Fluorenylmethyloxycarbonyl-L-glutamic acid y- f-butyl ester

Fmoc-Tyr(fBu)-OH 9-Fluorenylmethyloxycarbonyl-O-f-butyl-L- tyrosine

Fmoc-Asn(Trt)-OH 9-Fluorenylmethyloxycarbonyl-N-Y-trityl-L- asparagine

Fmoc-Ala-OH 9-Fluorenylmethyloxycarbonyl-L-alanine

Fmoc-Gly-OH 9-Fluorenylmethyloxycarbonyl-glycine

Fmoc 9-Fluorenylmethyloxycarbonyl

Boc t-Butyloxycarbonyl

HPLC High performance liquid chromatography

DIPEA Diisopropylethylamine

TFA Trifluoroacetic acid

DMF N,N-dimethylformamide

DCM Dichloromethane

MeOH Methanol

DIG Diisopropyl carbodiimide

TIS triisopropylsilane

HFIP Hexafluoro-2-propanol TFE 2,2,2-trifluoroethanol

OxymaPure® Ethyl 2-cyano-2-hydroxyimino-acetate

[62] Detailed experimental procedures for the synthesis of linaclotide described below are illustrative and do not limit the present invention to the embodiments herein exemplified.

[63] All solvents and reagents were obtained from commercial suppliers, of the best grade, and used without further purification, unless specified otherwise.

Example 1 : Solid phase synthesis of linear side-chain protected linaclotide of Formula III

[64] Solid phase synthesis of the peptide was carried out on 2-CTC resin (5 g, 1 .6 mmol/g) using a Biotage MultiSynTech synthesizer. The resin was swollen in 20 mL DCM for 30 min. A mixture of Fmoc-Tyr(tBu)-OH (1 eq, 3.67 g) and DIPEA (3 eq, 4.18 mL) was prepared in DCM (21 ml) and added to the resin. The reaction mixture was stirred for 1 h at RT. Then the resin was washed with DCM (3 x 20 mL). The unreacted peptide chains were capped with a mixture of DIPEA (3 eq, 4.18 mL) and MeOH (3 eq, 0.97 mL) in DCM (20 mL) for 15 min at RT. The resin was washed with DCM (3 x 30 mL) and treated with a mixture of DI PEA (3 eq, 4.18 mL) and acetic anhydride (3 eq, 2.27 mL) in DCM (20 mL) for 15 min at RT. The resin was then washed with DCM (3 x 30 mL) and DMF (3 x 30 mL). For estimating the attachment of the first residue, the Fmoc-loading test was performed. Fmoc group was cleaved from the solid support with 30 mL of 20% solution of piperidine in DMF (two cycles for 5 min and 20 min, respectively). The filtered solutions were collected and combined with those resulting from the washing of the resin with DMF (5 x 30 mL). The concentration of any liberated dibenzofulvene in the resulting solution was measured by UV spectroscopy. Fmoc quantitation gave a loading value of 1.2 mmol/g. The solid phase synthesis was carried out using 3 eq of protected amino acids that were preactivated for 5 min with 3 eq of OxymaPure® and 3 eq of DIC in 30 ml of DMF. After addition to the resin, the reaction mixture was stirred for 1 h at RT. The cycles of coupling and deprotection were continued to incorporate the following Fmoc-amino acids: Fmoc- Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(OtBu)-OH. At the end of the synthesis the resin was washed with DMF (3 x 30 mL), DCM (3 x 30 mL) and dried under vacuum. of linear side-chain linaclotide (III) by cone. ethanol to WO [65] Compound 3 (1 .46 g, corresponding to 0.25 g of the resin) was treated with the mixture TFA/TIS/DCM (25:15:60 v/v/v) and the reaction mixture was stirred for 4 h at RT. The crude peptide was precipitated in 30 mL of cold diethyl ether, washed with diethyl ether (3x) and dried under vacuum. The obtained crude linear linaclotide (II, SEQ ID NO: 1) was dissolved in 30% or 50% ethanol in water (90 mL) at the concentration of 5 g/L. Cysteine (20 mg) was added to the mixture and the pH was adjusted to 8 using 5M NaOH. The reaction mixture was stirred at RT for 24 h.

[66] HPLC purity: 30% ethanol - 71%; 50% ethanol - 72%. The crude linaclotide was purified by RP-HPLC and lyophilized. HPLC purity > 99%. Yield: 30% ethanol - 27%; 50% ethanol - 25% (Table 3, Entries 1 , 2).

Example 3: Simultaneous cleavage/deprotection of linear side-chain protected linaclotide of Formula III by cone. TFA and oxidative folding in fluorinated alcohol

[67] Compound 3 (1 .46 g, corresponding to 0.25 g of the resin) was treated with the mixture TFA/TIS/DCM (25:15:60 v/v/v) and the reaction mixture was stirred for 4 h at RT. The crude peptide was precipitated in 30 mL of cold diethyl ether, washed with diethyl ether (3x) and dried under vacuum. The crude linear linaclotide (II) was dissolved in 30% HFIP/TFE (1/1 v/v) in water (90 mL) at the concentration of 5 g/L. Cysteine (20 mg) was added to the mixture and the pH was adjusted to pH 8 using 5M NaOH. The reaction mixture was stirred at RT for 24 h. HPLC purity 74%. The crude linaclotide was purified by RP-HPLC and lyophilized. HPLC purity > 99%. Yield: 34% (Table 3, Entry 3).

Example 4: Cleavage of linear side-chain protected linaclotide of Formula III (compound 3) with diluted TFA and deprotection in the presence of HFIP, HFIP/TFE, or TFE.

[68] Compound 3 (1 .46 g, corresponding to 0.25 g of the resin) was treated with 2% TFA in DCM (5 x 10 ml x 2 min). The mixture was completely evaporated and the linear side- chain_protected linaclotide (III, Y=H) was treated with 1M HCI in HFIP, HFIP/TFE (v/v 1 :1) or TFE (4 mL of a solution prepared by mixing 8 mL HCI cone, with 92 mL of respective fluorinated alcohol or mixture thereof), with addition of 5 vol% TIS at RT under stirring (Table 1).

[69] The experimental results show that when a mixture of HFIP and TFE is used, the reaction is complete within 2.5 h. On the contrary, 100 vol% TFE does not allow to obtain the linear peptide even with the concentrated hydrochloric acid and several linear protected linaclotide derivatives are present in the reaction mixture. [70] Table 1. Deprotection of linear side-chain protected linaclotide (III, Y=H) with 1 M HCI in fluorinated alcohols

* The residual 14% linear protected linaclotide consists of a mixture of trityl-protected derivatives, which all convert to the desired linaclotide during the oxidation step. Thus, the presence of such trityl-protected derivatives in the mixture does not affect the quality and yield of final product.

** The residual 82% linear protected linaclotide is a mixture of trityl-protected derivatives and other derivatives (e.g. tertbutyl-protected derivatives), which do not lead to the desired product during the oxidation step.

Example 5: Oxidative folding at different concentrations of linear linaclotide (II, SEQ ID NO: 1) with aqueous HFIP.

[71] The linear linaclotide (II) obtained in Example 4 was dissolved at different concentrations in 20 vol% or 30 vol% HFIP in water. Cysteine (20 mg) was added to the mixture and the pH was adjusted to 8 using 5M NaOH. The reaction mixture was stirred at RT for 24 h (Table 2). Conversion was calculated based on the HPLC peak area.

[72] Table 2. Oxidative folding at different concentrations (C, g/L) of linear linaclotide (II) in the presence of HFIP (vol%)

Example 6: 2-step cleavage/deprotection of linear side-chain protected linaclotide (III, Y = solid support) and oxidative folding in aqueous fluorinated alcohol

[73] Compound 3 (1.46 g, corresponding to 0.25 g of the resin) was treated with 2%TFA in DCM (5 x 10 ml x 2 min). The mixture was completely evaporated and the protected linear linaclotide of Formula III (Y=H) was dissolved in 1 M HCI in HFIP (16 ml) solution containing 5 vol% TIS. The reaction mixture was stirred for 1 h at RT. Then the solution was diluted by addition of TFE (11 ml) and water (61 ml) to obtain 5 g/L concentration of the peptide, resulting in a mixed solution of HFIP and TFE in water. Cysteine (20 mg) was added and the pH was adjusted to 8 using 5M NaOH. The reaction mixture was stirred at RT for 24 h.

[74] HPLC purity: 80%. The crude linaclotide was purified by RP-HPLC and lyophilized. HPLC purity > 99%. Yield: 35% (Table 3, Entry 4).

Example 7: 1-step cleavage/deprotection and oxidative folding in aqueous fluorinated alcohol

[75] Compound 3 (1.46 g, corresponding to 0.25 g of the resin) was treated with 1 M HCI in HFIP (prepared as described in Example 4, 4 ml * 4 x 2 min) and 5 vol% TIS was added to the solution. The reaction mixture was stirred for 1 h at RT. Then the mixture was diluted by addition of TFE (11 ml) and water (61 mL) to obtain 5 g/L concentration of the peptide, resulting in a mixture of HFIP and TFE in water. Cysteine (20 mg) was added and the pH was adjusted to 8 with 5 M NaOH. The reaction mixture was stirred at RT for 24 h.

[76] HPLC purity: 80%. The crude linaclotide was purified by reverse phase HPLC and lyophilized. HPLC purity > 99%. Yield: 37% (Table 3, Entry 5).

[77] Table 3. Summary of preparations of linaclotide

Example 8: 1-step cleavaqe/deprotection and oxidative folding in aqueous TFE

[78] Compound 3 (1.46 g, corresponding to 0.25 g of the resin) was treated with 1 M HCI in TFE (prepared as described in Example 4, 4 times x 2 min each) and 5 vol% TIS was added to the solution. The reaction mixture was stirred for 1 h at RT. Then the mixture was diluted by addition of water (71 mL) to obtain 5 g/L concentration of the peptide, resulting in a mixture of TFE in water. Cysteine (20 mg) was added and the pH was adjusted to 8 with 5 M NaOH. The reaction mixture was stirred at RT for 24 h.

[79] HPLC purity: 20%. The crude linaclotide was purified by reverse phase HPLC and lyophilized. HPLC purity > 99%. Yield: 7%.

Sequence Listing Free Text

[80] SEQ ID NO: 1 - linear linaclotide

CCEYCCNPAC TGCY

Citation List

Patent Literature

[81] PTL1: W02004069165

[82] PTL 2: CN 103626849

[83] PTL 3: CN 102875655

[84] PTL 4: WO2017134687

[85] PTL 5: WO2016038497

[86] PTL 6: W02014188011

Non Patent Literature

[87] NPL1 : Chandar A. K. Int. J. Gen. Med. 2017, 10, 385-393

[88] NPL 2: Bassotti G. et al. Expert Opin. Pharmacother. 2018, 19, 1261-1266

[89] NPL 3: Lundt et al. Int. J. Pept. Protein Res. 1978, 12, 258-268

[90] NPL 4: Guy et al. Methods Enzymol. 1997, 289, 67-83

[91] NPL 5: Roccatano et al. PNAS 2002, 99, 12179-12184

[92] NPL 6: Palladino et al. Org. Lett. 2012, 14, 6346-6349