US009051349B2
(12) United States Patent (10) Patent No.: US 9,051,349 B2 Callens et al. (45) Date of Patent: Jun. 9, 2015
(54) LARAZOTIDEACETATE COMPOSITIONS FOREIGN PATENT DOCUMENTS EP O184243 6, 1986 (71) Applicant: Alba Therapeutics Corp., Baltimore, JP 61-268.700 11, 1986 MD (US) JP 2003034653 2, 2003 WO WO96,37196 11, 1996 (72) Inventors: Roland Callens, Grimbergen (BE): WO WOOOO7609 2, 2000 WO WOO1,895.51 11, 2001 Georges Blondeel, Aalst (BE); Thierry WO WOO3,055900 T 2003 Delplanche, Mont-St-Guibert (BE) WO WO 2005/063800 7/2005 WO WO 2005/121164 12/2005 (73) Assignee: Alba Therapeutics Corporation, WO WO 2006/034056 3, 2006 WO WO 2006/041945 4/2006 Baltimore, MD (US) WO WO 2006/119388 11, 2006 WO WO 2006,135811 12/2006 (*) Notice: Subject to any disclaimer, the term of this WO WO 2007/095092 8, 2007 patent is extended or adjusted under 35 WO WO 2009/065836 5, 2009 U.S.C. 154(b) by 0 days. WO WO 2009/065949 5, 2009 (21) Appl. No.: 13/832,820 OTHER PUBLICATIONS Kolpuru et al., “Analysis of binding affinity of the Zonulin agonist (22) Filed: Mar 15, 2013 (AT1002) and antagonist (AT1001) to the Zonulin intestinal recep tor.' North American Society for pediatric gastroenterology, hepatol (65) Prior Publication Data ogy, and nutritional annual meeting, J. Ped. Gastroent. Nutr., 43(4): US 2013/0281384 A1 Oct. 24, 2013 E14-E76, p. E52, poster #121; Oct. 21, 2006).* DiPierro et al., "Zonula occludens toxin structure-function analysis: identification of the fragment biologically active on tight junctions and of the Zonulin receptor binding domain.” J. Biol. Chem. Related U.S. Application Data 276:19160-165 (2001)).* (63) Continuation-in-part of application No. 12/743,411, Singhal et al. "Drug polymorphism and dosage form design: a prac filed as application No. PCT/EP2008/066037 on Nov. tical perspective” Advanced Drug Delivery Reviews, 2004, 56,335 21, 2008. 347.* Brittain et al. “Physical Characterization of Pharmaceutical Solids.” (60) Provisional application No. 61/014,938, filed on Dec. Pharmaceutical Research, vol. 8, No. 8, 1991, pp.963-973.* 19, 2007, provisional application No. 61/073,843, Raw et al. “Regulatory considerations of pharmaceutical Solid filed on Jun. 19, 2008, provisional application No. polymorphism in Abbreviated New Drug Applications (ANDAs).” 61/103,289, filed on Oct. 7, 2008. Advanced Drug Delivery Reviews 56 (2004)397-414.* Shah et al. "Analytical Techniques for Quantification of Amorphous/ Crystalline Phases in Pharmaceutical Solids,” Journal of Pharmaceu (30) Foreign Application Priority Data tical Sciences, vol. 95, No. 8, Aug. 2006, pp. 1641-1665.* Newman et al. “Solid-state analysis of the active pharmaceutical Nov. 21, 2007 (EP) ...... O7121207 ingredient in drug products.” Drug Discovery Today vol. 8, No. 19 Oct. 2003, pp. 898-905.* (51) Int. Cl. Bis et al. “Defining & Addressing Solid-State Risks. After the Proof C07K 7/06 (2006.01) of-Concept Stage of Pharmaceutical Development.” Drug Develop C07C 53/10 (2006.01) ment & Delivery, Apr. 2011, pp. 32-34.* A6 IK38/08 (2006.01) Andersson, L. et al., "Large-scale synthesis of peptides.” (52) U.S. Cl. Biopolymers, 55(3):227-250 (2000). CPC. C07K 7/06 (2013.01); C07C 53/10 (2013.01); (Continued) A6 IK38/08 (2013.01) (58) Field of Classification Search None Primary Examiner — Christina Bradley See application file for complete search history. (74) Attorney, Agent, or Firm — Morgan, Lewis & Bockius LLP (56) References Cited U.S. PATENT DOCUMENTS (57) ABSTRACT 3,960,830 A 6/1976 Bayer et al. The invention provides crystalline forms of the peptide Gly 4,725,645 A 2, 1988 Anteunis et al. Gly-Val-Leu-Val-Gln-Pro-Gly (SEQ ID NO 1), and salts of 6,884,345 B1 4/2005 Irgum et al. the peptide, which may further have associated water mol 7,026,294 B2 4/2006 Fasano et al. ecules. These salts and hydrated salts of the peptide and 8,546,530 B2 10/2013 Callens et al. 2002/0115825 A1* 8/2002 Fasano ...... 530/324 compositions comprising these materials have advantageous 2005, OO65074 A1 3/2005 Fasano et al. pharmaceutical properties. 2005/O1652.15 A1 7/2005 Bigelow et al. 2008/O103100 A1* 5/2008 Fasano et al...... 514, 16 2010/028O221 A1 11/2010 Callens et al. 8 Claims, 11 Drawing Sheets US 9,051,349 B2 Page 2
(56) References Cited Wisniewski, R., “Principles of the design and operational consider ations of large scale high performance liquid chromatography OTHER PUBLICATIONS (HPLC) systems for proteins and peptides purification. BioSepara tion, 3(2-3):77-143 (1992). von Bahr-Lindstrom, H. et al., “Ion-exchange high-performance liq Di Pierro, M. et al., "Zonula occludens toxin structure-function uid-chromatography steps in peptide purifications. Bioscience analysis. Identification of the fragment biologically active on tight Reports, 2(10):803-811 (1982). junctions and of the Zonulin receptor binding domain.” J. Biol. Herraiz, T., "Sample preparation and reversed phase-high perfor Chem., 276(22): 19160-19165 (2001). mance liquid chromatography analysis of food-derived peptides.” Analytica Chemica Acta, 352(1): 119-139 (1997). Wang, W. et al. “Human Zonulin, a potential modulator of intestinal Pedroso, E. et al., “Reversed-phase high-performance liquid chroma tight junctions,” Journal of Cell Science, 113(Pt. 24):4435-4440 tography of protected peptide segments.” Journal of Chromatogra (2000). phy, 409:281-290 (1987). Mostafavi. H. et al., “Synthesis of phospho-urodilatin by combina “Integrilin, Eptifibatide solution for injection.” online). tion of global phosphorylation with the segment coupling approach.” XP002400963, Retrieved from the Internet:
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U.S. Patent Jun. 9, 2015 Sheet 2 of 11 US 9,051,349 B2
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US 9,051,349 B2 1. 2 LARAZOTDEACETATE COMPOSITIONS peptide and pharmaceutical compositions comprising the same. In one embodiment of the present invention, the salt of PRIORITY the peptide is crystalline (or highly organized). In a second embodiment, the crystalline form of the salt of the peptide This Application is a continuation-in-part of U.S. applica- 5 may further comprise (stoichiometric and non-stoichiomet tion Ser. No. 12/743,411 filed May 18, 2010, which is a ric) waters of hydration (a “hydrate'). The present invention national stage Application of PCT/EP2008/066037 filed Nov. additionally provides pharmaceutical compositions compris 21, 2008, which claims priority to U.S. Provisional Applica ing a crystalline Stoichiometric or non-stoichiometric hydrate tion No. 61/103,289 filed Oct. 7, 2008, U.S. Provisional of a salt of the peptide. It is well recognized that pharmaceu Application No. 61/073,843 filed Jun. 19, 2008, U.S. Provi- 0 tical Solids can exist in more than one solid State form (i.e., sional Application No. 61/014,938 filed Dec. 19, 2007, and crystalline, noncrystalline/amorphous, quasicrystalline/orga EP 07121207.0 filed Nov. 21, 2007, all of which are hereby nized aggregate). Polymorphism is defined as the ability of a incorporated by reference in their entireties. Solid compound to exist in more than one crystalline form with the same covalent chemical structure, but different DESCRIPTION OF THE TEXT FILE SUBMITTED 15 Supra-molecular structures and ordered arrangements of mol ELECTRONICALLY ecules within the crystalline lattice. In addition to exhibiting polymorphism, many pharmaceutical Solids form hydrates The contents of the text file submitted electronically here and organic Solvates, which themselves can be crystalline and with are incorporated herein by reference in their entirety: A exhibit polymorphism. Hydrates can be stoichiometric or computer readable format copy of the Sequence Listing (file- 20 non-stoichiometric. In a stoichiometric hydrate the water aC. ALBA 062 00US SeqList ST25.txt, date molecules are tightly associated with or bound to the phar recorded: May 22, 2013, file size 8 kilobytes). maceutical compound as well as to other water molecules and as a result are integral to the crystal lattice. In contrast, the TECHNICAL FIELD water molecules of a non-stoichiometric hydrate (sometimes 25 referred to as a variable hydrate) are loosely associated with The present invention relates to a process for the manufac the pharmaceutical compound and the crystal lattice. For ture of a certain octapeptide ("LaraZotide'), and in particular example, in certain systems the water molecules of a variable to Such a process comprising a purification step. The inven hydrate reside in channels in the crystal lattice (Vogt et al. J tion further relates to salts of the peptide, and in particular to Pharm Biomed Anal 40 (2006) 1080-1088). Generally speak Solid state forms of Such salts including crystalline (or highly 30 ing, the amount of water present in a non-stoichiometric or organized) forms that may also incorporate water (stoichio variable hydrate can be typically a function of the relative metric and non-stoichiometric hydrates of such salts). humidity (RH) environment of the sample. Hydrates, in par ticular non-stoichiometric hydrates, can be a difficult pros BACKGROUND pect for development because of the additional effort needed 35 to investigate and characterize the different hydration states Zonula occludens toxin (“Zot') is an enterotoxin produced of the pharmaceutical compound. At the same time, the by Vibrio cholerae. Zot increases permeability by reversibly manufacturing process has to be scrutinized to determine affecting the structure of tight junctions as described in WO what conditions are needed to ensure that the API contains a 1996/37196. The comparison of its sequence with a potential predictable composition. human analogue (“Zonulin') revealed an 8-amino acid shared 40 It is well recognized that different solid state forms of the motif (GXXXVGXG, SEQ ID NO 23), described by D1 same compound can exhibit significantly different chemical PIERRO, et al. Zonula Occludens Toxin Structure—Function and physical properties including color, morphology, stabil Analysis. J. Biol. Chem. 276(22): 19160-19165 (2001). ity, solubility, dissolution and bioavailability. As with all The octapeptide of the following sequence: Gly-Gly-Val pharmaceutical compounds and compositions, the chemical Leu-Val-Gln-Pro-Gly (SEQID NO 1) has been described as 45 and physical properties of a particular Solid state form of a a peptide antagonist of Zonulin by Fasano in WO 0007609 and compound are important to its commercial development. by WANG, et al. Human Zonulin, a Potential Modulator of These properties include, but are not limited to: (1) packing Intestinal Tight Junctions. Journal of Cell Science. 2000, properties such as molar volume, density and hygroscopicity; 113:4435-4440 (2000). Fasano discloses its application in (2) thermodynamic properties such as melting temperature, methods for treatment of gastrointestinal inflammation as 50 vapor pressure and solubility; (3) kinetic properties such as well as of conditions associated with breakdown of the blood dissolution rate and stability (including stability at ambient brain barrier. Fasano et al. also describe in U.S. Pat. No. conditions, especially to moisture, and under storage condi 7,026,294 its use in a method for delay of onset of diabetes. tions); (4) Surface properties such as Surface area, wettability, Fasano describes delivery of the peptide to, inter alia, the interfacial tension and shape; (5) mechanical properties Such Small intestine via peptide-coated beads having a gastroresis- 55 as hardness, tensile strength, compactability, handling, flow tant coating. and blend; and (6) filtration properties. These properties can This octapeptide ("LaraZotide') is a promising treatment affect, for example, processing and storage of pharmaceutical of various diseases that involve disordered intercellular com compositions, sometimes referred to as drug product and/or munication, which include developmental and intestinal dis of the active pharmaceutical ingredient (API), sometimes orders leading to autoimmune disease (coeliac disease and 60 referred to as drug substance. As mentioned above, different type 1 diabetes), tissue inflammation, malignant transforma solid state forms of the API can have different rates of solu tion, and metastasis. None of the above references describe bility which can translate into differences in bioavailability in any process for the synthesis of this octapeptide. The present vivo. invention now makes available a process for its manufacture. In general, the Solid State form of a compound can be The present invention also makes available various salts of the 65 distinguished from another solid state form of the same com peptide Suitable for pharmaceutical use. The present inven pound by one or more of the following techniques: X-ray tion further provides solid state forms of such salts of the powder diffraction (XRPD), thermal techniques including US 9,051,349 B2 3 4 thermogravimetric analysis (TGA) and differential scanning In a first particular aspect, the process according to the calorimetry (DSC), as well as Infrared (IR), Raman and/or present invention comprises coupling a peptide of formula: solid state NMR (ssNMR) spectroscopy. Val-Gln-Pro-Gly-Y (SEQID NO2); wherein the C-terminal The Applicant describes here a process for the synthesis of amino acid is protected by a carboxylic acid protecting group the octapeptide Gly-Gly-Val-Leu-Val-Gln-Pro-Gly (SEQID 5 Y; with a Leucine or a C-terminal Leucine peptide, preferably NO 1), which allows for an efficient production of said selected from formulae: X-Gly-Gly-Val-Leu (SEQIDNO3), octapeptide with a good yield and a high quality and purity X-Gly-Val-Leu and X-Val-Leu; and wherein said Leucine or level while presenting advantages in terms of productivity C-terminal Leucine peptide is optionally activated by a car and required manufacturing equipment. boxylic acid activating agent. 10 The carboxylic acid protecting group Y is preferably DESCRIPTION OF THE FIGURES selected from alkyl esters, aryl esters, aralkyl esters and silyl groups. Y is more preferably selected from alkyl esters and FIG. 1 illustrates a scheme 1 for synthesizing the octapep silyl groups. Excellent results were obtained with alkyl esters tide of SEQID NO 1. and in particular with the tert-butyl ester of the Val-Gln-Pro FIG. 2 illustrates scheme 2, which is an embodiment of 15 Gly (SEQID NO 4) peptide. scheme 1 for synthesizing the octapeptide of SEQID NO 1. On the other hand, the amino protecting group X is pref FIG. 3 illustrates a scheme 3 for synthesizing the octapep erably selected from allyloxycarbonyl groups, tert-butyloxy tide of SEQID NO 1. carbonyl (BOC), benzyloxycarbonyl (Z), 9-fluorenylmethy FIG. 4 illustrates a scheme 4 for synthesizing the octapep loxycarbonyl (Fmoc), 4-nitrobenzenesulfonyl (Nosyl), tide of SEQID NO 1. 2-nitrobenzenesulfenyl (Nps) and optionally substituted FIG. 5 illustrates scheme 5, which is an embodiment of derivatives thereof. Excellent results were obtained with the scheme 3 for synthesizing the octapeptide of SEQID NO 1. tert-butyloxycarbonyl (BOC) group. FIG. 6 illustrates scheme 6, which is an embodiment of For the purpose of the present invention, the term "peptide' scheme 3 for synthesizing the octapeptide of SEQID NO 1. refers to a polymer in which the monomers are amino acids FIG. 7 shows exemplary XRPD profiles of various solid 25 covalently attached together through amide bonds. state forms of Larazotide acetate. Peptides are two or often more amino acids monomers FIG. 8 shows an exemplary moisture sorption analysis for long. In addition, all peptide sequences are represented by crystalline Larazotide acetate. formulae whose left to right orientation is in the conventional FIG. 9 shows an exemplary moisture sorption analysis for direction of amino-terminus to carboxy-terminus. amorphous LaraZotide acetate. 30 For the purpose of the present invention, the term “amino FIG. 10 shows the results of a Larazotide Acetate stability acid' is intended to denote any compound comprising at least study at 40° C. and 75% RH (SEQID NO 24). one NR1R2 group, preferably NH group, and at least one FIG. 11 shows an exemplary X-ray powder diffraction carboxyl group. The amino acids of this invention can be (XRPD) for Larazotide acetate. naturally occurring or synthetic. The natural amino acids, FIG. 12 shows an exemplary differential scanning calorim 35 with exception of glycine, contain a chiral carbon atom. etry (DSC) and thermogravimetry (TG) of crystalline lara Unless otherwise specifically indicated, the compounds con Zotide acetate. taining natural amino acids with the L-configuration are pre FIG. 13 shows exemplary moisture sorption/desorption ferred. Amino acids residues are abbreviated as follows data for crystalline Larazotide acetate. throughout the application: Glycine is Gly or G; Valine is Val FIG. 14 compares exemplary XRPD patterns of Larazotide 40 or V: Leucine is Leu or L: Glutamine is Gln or Q: Proline is acetate Form A (top) and Form A (bottom). Pro or P: Pyroglutamic acid (or pyrrolidone carboxylic acid) is Glp. DETAILED DESCRIPTION OF INVENTION For the purpose of the present invention, the term “C-ter minal of a peptide is the end of the amino acid chain termi The present invention concerns a process for the synthesis 45 nated by a free carboxyl group (-COOH). On the other hand, of a Gly-Gly-Val-Leu-Val-Gln-Pro-Gly octapeptide (SEQID the term “N-terminal refers to the end of a peptide termi NO 1) comprising at least one peptide coupling step carried nated by an amino acid with a free amine group (-NH). out in Solution. For the purpose of the present invention, the term “cou The process according to the present invention allows the pling” refers to the reaction between the carboxyl group of an use of a convergent synthetic strategy, with a limited number 50 amino acid or the C-terminus of a first peptide to the amino of steps, and avoids Successive protection/deprotection reac group of another amino acid or the N-terminus of a second tions. peptide. In other words, during coupling, two peptide inter The process according to the invention provides the mediate fragments, or a peptide intermediate fragment and a octapeptide in an industrial scale without Substantial forma reactive amino acid, are coupled, generally, in an appropriate tion of by-products or racemisation. Moreover, the by-prod 55 Solvent, and usually in the presence of additional reagents that ucts possibly formed during the process according to the promote the efficiency and quality of the coupling reaction. present invention are readily separable from the final octapep The peptide intermediate fragments are reactively arranged tide by a specific purification process. The present invention so the N-terminus of one fragment becomes coupled to the allows the synthesis of peptides containing both L- and C-terminus of the other fragment, or vice versa. D-amino acid configurations. Moreover it has been found that 60 For the purposes of the present invention, the term “crys intermediates and products of the process according to the talline” refers to a solid state form of a compound in which the invention can be isolated and purified easily by solid/liquid constituent atoms are organized into a repeating motif and separation techniques such as precipitation or crystallization. produces a characteristic X-ray powder diffraction (XRPD) The process according to the invention even allows, if desired, pattern. Seegenerally, USP35, NF 30,<941> (May 1, 2012) to Substantially avoid any time consuming purification steps 65 pp. 427 et seq. Such as chromatography. This is unusual and unexpected in In a further particular aspect, the process according to the the framework of the synthesis of an octapeptide. present invention comprises coupling a peptide of formula US 9,051,349 B2 5 6 Val-Gln-Pro-Gly-Y (SEQID NO2) with a peptide of formula The term “carboxylic acid protecting group Y” refers to X-Gly-Gly-Val-Leu (SEQID NO3). protecting groups which can be used in the present invention In the present invention, the protecting group is any sort of to replace the acidic proton of a carboxylic acid. Preferred group that can prevent the atom or moiety to which it is groups are selected from optionally Substituted alkyl, aryl, attached, e.g., oxygen or nitrogen, from participating in aralkyl and, preferably, silyl groups. Trialkylsilyl groups are undesired reactions during processing and synthesis. Protect still more particularly preferred. Examples of Such groups ing groups include side chain protecting groups and C- or include methoxymethyl, methylthiomethyl, 2.2.2-trichloro N-terminal protecting groups. Protecting groups can also pre ethyl, 2-haloethyl 2-(trimethylsilyl)ethyl, t-butyl, aryl, alkyl, vent reaction or bonding of carboxylic acids, thiols and the aralkyl, allyl, benzyl, triphenylmethyl (trity1), benzhydryl, 10 p-nitrobenzyl, p-methoxybenzyl, and trialkylsilyl groups like. such as trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, The term "amino protecting group X' refers to protecting i-propyl-dimethylsilyl. A trimethylsilyl group is more par groups which can be used in the present invention to replace ticularly preferred. In the process according to the present an acidic proton of an amino group in order to reduce its invention, the Val-Gln-Pro-Gly (SEQ ID NO 4) peptide is nucleophilicity. As it will be illustrated herein below, the 15 more preferably protected by a group selected from a persi amino protecting group X can be removed, if appropriate, in lylated derivative and an alkyl ester. Good results were a deprotection reaction prior to possible Subsequent addition obtained by using MSA (N-Methyl-N-trimethylsilylaceta of a next amino acid. mide). The persilylation of an amino acid or of a peptide can The amino protecting group X is preferably sterically hin be carried out, for example, according to the method dering. The term “sterically hindering” is intended to denote described in Patent Application EP 184243 B (SOLVAY). in particular a Substituent comprising at least 3 carbon atoms, Excellent results were also obtained by using the Val-Gln in particular at least 4 carbon atoms, including at least one Pro-Gly-Y (SEQ ID NO 2) peptide where Y is a tert-butyl secondary, tertiary or quaternary carbon atom. The sterically ester group. hindering group often comprises at most 100, preferably at The carboxylic acid protecting groups Y may be introduced most 50 carbon atoms. 25 by various methods including esterification and silylation. On By way of non-limiting examples of Suitable amino pro the other hand, the removal of carboxylic acid protecting tecting groups represented herein by X, which can be used in groupSY may, for example, be effected by hydrolysis, Saponi the process according to the present invention, mention may fication, acidolysis, hydrogenolysis or enzymatic hydrolysis. in particular be made of substituted or unsubstituted groups of It will be appreciated that the intermediate peptides, i.e. acyl type, Such as the formyl, acrylyl (Acr), benzoyl (BZ). 30 shorter peptides than the octapeptide having appropriate acetyl (Ac), trifluoroacetyl, substituted or unsubstituted sequence of amino acids, to be coupled by the process accord groups of aralkyloxycarbonyl type, such as the benzyloxy ing to the present invention may, if desired, be prepared using carbonyl (Z), p-chlorobenzyloxycarbonyl, p-bromobenzy the Solid-phase method of peptide synthesis. In Sucha method loxycarbonyl, p-nitrobenzyloxycarbonyl, p-methoxybenzy the carboxylic acid protecting group of the C-terminal amino loxycarbonyl, benzhydryloxycarbonyl, 2-(p-biphenylyl) 35 acid is usually bound to a resin. isopropyloxycarbonyl, 2-(3,5-dimethoxyphenyl) In another particular aspect of the present invention, the isopropyloxycarbonyl, p-phenylaZobenzyloxycarbonyl, process as above described may thus be carried out in the triphenylphosphonoethyloxycarbonyl or 9-fluorenylmethy presence of a carboxylic acid activating agent. loxycarbonyl group (Fmoc), Substituted or unsubstituted For the purposes of the present invention, the term “car groups of alkyloxycarbonyl type, such as the tert-butyloxy 40 boxylic acid activating agent, also referred to as "coupling carbonyl (BOC), tert-amyloxycarbonyl, diisopropylmethy agent, is a reagent that replaces the hydroxyl group of a loxycarbonyl, isopropyloxycarbonyl, ethyloxycarbonyl, carboxylic acid with a suitable leaving group which is Sus allyloxycarbonyl, 2-methylsulphonylethyloxycarbonyl or ceptible to nucleophilic displacement, allowing the coupling 2.2.2-trichloroethyloxycarbonyl group, groups of cycloalky of an amino acid or peptide free carboxy group with a free loxycarbonyl type. Such as the cyclopentyloxycarbonyl, 45 amino group of another amino acid or peptide to form an cyclohexyloxycarbonyl, adamantyloxycarbonyl or isoborny amide bond between the reactants. loxycarbonyl group, and groups containing a hetero atom, Examples of carboxylic acid activating agent and activated Such as the benzenesulphonyl, p-toluenesulphonyl, mesityle groups which are useful in the present invention include car neSulphonyl, methoxytrimethylphenyl-Sulphonyl, 2-ni bodiimides, carbonyldiimidazoles, carbonyl halides, in par trobenzenesulfonyl, 2-nitrobenzenesulfenyl, 4-nitrobenzene 50 ticular acyl halides or haloformiates, azides, phosphonium Sulfonyl or 4-nitrobenzenesulfenyl group. Among these salts and uronium or guanidinium salts, symmetric or mixed groups X, those comprising a carbonyl, a Sulfenyl or a Sul anhydrides or active ester. Such carboxylic acid activating phonyl group are preferred. An amino protecting group X is agent may be used before the coupling step in order to isolate preferably selected from allyloxycarbonyl groups, tert-buty the activated peptide derivative or used in situ prior to the loxycarbonyl (BOC), benzyloxycarbonyl (Z), 9-fluorenylm 55 introduction of the free amino peptide derivative. ethyloxycarbonyl (Fmoc), 4-nitrobenzenesulfonyl (Nosyl), Non-limitative particular examples of such carboxylic acid 2-nitrobenzenesulfenyl (Nps) and substituted derivatives. activating agent include carbodiimide reagents such as N,N'- More preferably, the amino protecting group X is tert-buty dicyclohexylcarbodiimide (DCC), N-Ethyl-N'-(3-dimethy loxycarbonyl (BOC). laminopropyl)carbodiimide (EDC), 1-(3-dimethylaminopro Amino protecting groups X may be introduced by various 60 pyl)-3-ethylcarbodiimide hydrochloride (EDCI also referred methods e.g. by reaction with Suitable acid halides Such as to as “WSC), carbodiimidazoles reagents such as 1,1'-car carbobenzoxyl chloride or acid anhydrides Such as acetic bonyldiimidazole (CDI), diisopropylcarbodiimide anhydride. On the other hand, amino protecting groups X (DIPCDI), diisopropylcarbodiimide (DIC) or derivatives may be removed, for example, by acidolysis, hydrogenolysis, thereof: phosphonium salts such as (benzotriazol-1-yloxy) treatment with dilute ammonium hydroxide, treatment with 65 tris-(dimethylamino)phosphonium (BOP), benzotriazol-1- Sodium, treatment with sodium amide, treatment with hydra yl-oxytripyrrolidinophosphonium hexafluorophosphate (Py Zine, or enzymatic hydrolysis. BOP), (7-AZabenzotriazol-1-yloxy) US 9,051,349 B2 7 8 tripyrrolidinophosphonium hexafluorophosphate (PyAOP), ethylamine (DIPEA) or mixtures thereof. More preferably, it bromo-tris-pyrrolidino phosphoniumhexafluorophosphate is chosen from N-methylmorpholine and diisopropylethy (PyBroP), chloro-tris-pyrrolidino phosphoniumhexafluoro lamine. phosphate (PyCloP) or derivatives thereof; uronium or guani In another particular aspect of the present invention, the dinium salts such as o-benzotriazole-N.N.N',N'-tetramethyl peptide coupling as above described is carried out in a polar uronium-hexafluorophosphate (HBTU), o-(benzotriazol-1- organic solvent. In a particular preferred embodiment, the yl)-N.N.N',N'-tetramethyluronium tetrafluoroborate polar organic solvent allows for particularly efficient control (TBTU), 2-(7-aza-1-H-benzotriazole-1-yl)-1,1,3,3-tetram of racemization of the peptide bond formed, the solubility of ethyluronium hexafluorophosphate (HATU), O-(7-azaben the peptide and/or peptide fragments, and the coupling reac Zotriazol-1-yl)-1,1,3,3 bis(tetramethylene)uronium 10 tion rate. The polar organic solvent is preferably selected from amide type solvents such as N,N-dimethylacetamide hexafluorophosphate (HAPyU) or derivatives thereof; acyl (DMA), N,N-dimethylformamide (DMF), N-methylpyrroli halides such as isobutyl chloroformate (iBCF), pivaloyl chlo done (NMP), dimethylsulfoxide (DMSO), ethyl acetate ride (Pivol), t-butylchloroformate (TBCF), ethyl chlorofor (AcOEt), dichloromethane (DCM), methylene chloride, pyri mate (ECF) or derivatives thereof; esterificating agent such as 15 dine, chloroform, acetonitrile (ACN or CHCN), dimethoxy pentafluorophenol (PfP), N-hydroxysuccinimide (NHS) or ethane, dioxane, tetrahydrofuran (THF) or mixtures thereof. derivatives thereof. azidination agent Such as diphenylphos More preferably, it is selected from N,N-dimethylacetamide phoryl azide (DPPA) or derivatives thereof. Preactivated (DMA), N-methylpyrrolidone (NMP) and N,N-dimethylfor amino acids or under the form of N-carboxyanhydrides, and mamide (DMF). The most preferably, the polar organic sol in particular urethane-N-carboxyanhydrides (UNCA's) are vent is N,N-dimethylacetamide (DMA). also good examples of carboxylic acid activating agents. In the present invention, the coupling reaction is generally The carboxylic acid activating agent is preferably chosen carried out at a temperature of greater than or equal to -45°C. from carbodiimides, carbonyldiimidazoles, acyl halides, Often, the reaction is carried out at a temperature greater than phosphonium salts and uronium or guanidinium salts and or equal to -25°C. Preferably, the temperature is greater than more preferably from isobutyl chloroformate and pivaloyl 25 or equal to -20°C. In the process according to the invention, chloride. Carbonyl halide, more particularly acyl halide, in the reaction is generally carried out at a temperature of less particular acyl chloride coupling agents as described above than or equal to +45° C. Often, the reaction is carried out at a are preferred. Tertiary acyl halides are more particularly pre temperature of less than or equal to +5° C. Preferably, the ferred. Examples of tertiary acyl halides are interalia 1-ada temperature is less than or equal to 0°C. mantoyl chloride, 2,2-dimethylbutyroyl chloride and pivaloyl 30 In another particular aspect of the present invention, the chloride. Pivaloyl chloride is more particularly preferred as Solution, which is generally the Solution in which a coupling carboxylic acid activating agent. has taken place, can suitably be treated after the coupling step It has been found, Surprisingly, that it is possible, in par with an aqueous phase so as to provide a solution of coupled ticular, by carefully selecting the carboxylic acid activating product in the aqueous phase and then, the coupled product is agent to Substantially or completely avoid racemisation, in 35 extracted from the aqueous phase into an organic solvent. In particular of any Leu group when coupling Leu or Leu-C- this case, the pH value of the aqueous phase is preferably terminal fragments as described herein before. Moreover controlled to be greater than or equal to 1. More preferably, it coupling conditions have been identified which are described is greater than or equal to 1.5. Still more preferably, it is hereafter, which allow for high overall high productivity and greater than or equal to 2. On the other hand, the pH value of yield in particular of desired octapeptide while maintaining 40 the aqueous phase is preferably controlled to be less than or excellent optical purity. equal to 9. In some embodiments this pH value is preferably Good results are often obtained when using additional controlled to be less than or equal to 5. More preferably, in reagents which reduce side reactions and/or increase reaction this embodiment, it is less than or equal to 3.5. Still more efficiency. For example, phosphonium and uronium salts can, preferably, it is less than or equal to 3. Excellent results were in the presence of a tertiary base, for example, diisopropyl 45 obtained with a pH value of the aqueous phase of about 2.5. ethylamine (DIPEA) and triethylamine (TEA), convert pro It has been found that in the process according to the tected amino acids into activated species (for example, BOP invention it is possible by carrying out washing operations at PyBOP, HBTU, and TBTU all generate HOBtesters). Other a pH as contemplated above, to eliminate acidic or basic reagents which help prevent racemization include carbodiim impurities while maintaining high product quality of coupled ides (for example, DCC or WSCDI) with an added auxiliary 50 product in particular with regard to sensitive groups such as nucleophile (for example, 1-hydroxy-benzotriazole (HOBt), the Glin moiety or protective groups optionally required for 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine further coupling steps such as Boc or tRu group. (HOOBT), 1-hydroxy-azabenzotriazole (HOAt), or HOSu) In still another particular aspect of the present invention, or derivatives thereof. TBTU may also be used. The mixed the solution, which is generally the solution in which the anhydride method, using isobutyl chloroformate, with or 55 coupling has taken place, in particular to produce, in particu without an added auxiliary nucleophile, is also used, as is the lar protected, X-Gly-Gly-Val-Leu-Val-Gln-Pro-Gly-Y azide method, due to the low racemization obtained. These octapeptide (SEQID NO 5), may be directly poured into an types of compounds can also increase the rate of carbodiim aqueous solvent in order to precipitate the desired product. In ide-mediated couplings, as well as prevent dehydration of another embodiment, an aqueous solvent may be added to the ASn and Glin residues. 60 solution in particular of protected X-Gly-Gly-Val-Leu-Val When Such carboxylic acid activating agents are used, the Gln-Pro-Gly-Yoctapeptide (SEQID NO5) in order to crys coupling reaction is often carried out in the presence of a base tallize the desired product. Such aqueous solvent may be, for as additional reagent. In another particular aspect of the example, water, salt water or any other aqueous mineral salt present invention, the coupling reaction is thus carried out in solution. The water pH value is preferably greater than or the presence of a base. The base is preferably chosen from 65 equal to 1.5, more preferably, greater than or equal to 2. On tertiary and heteroaromatic amines such as N-methylmorpho the other hand, the pH value of the aqueous phase is prefer line (NMM), pyridine, triethylamine (TEA), diisopropyl ably controlled to be less than or equal to 10. More preferably, US 9,051,349 B2 10 it is less than or equal to 9. Still more preferably, less than or Substantially anhydrous, containing less than 2% by weight equal to 8. Suitable salts to be used in above mentioned salt relative to the total weight of the reaction medium of water. water solutions include alkali or earth alkali chlorides, in Preferably this content is equal to or less than 1% wt, in particular sodium chloride alkali or earth alkali Sulphates, in particular equal to or less than 0.5% wt. particular potassium Sulphate alkali or earth alkali hydro 5 In a preferred embodiment Boc-Gly-Gly-Val-Leu-Val genocarbonates, in particular sodium hydrogenocarbonate. A Gln-Pro-Gly-OtBuoctapeptide (SEQID NO 6) which is pref preferred aqueous phase consists of deionized water. erably obtained by crystallization as described above, is dis In a preferred embodiment Boc-Gly-Gly-Val-Leu-Val Solved in a solvent comprising or, preferably, consisting of Gln-Pro-Gly-OtBu octapeptide (SEQ ID NO 6) is obtained glacial acetic acid. A solution of HCl in glacial acetic acid is by coupling. In some embodiments, an amide type solvent, 10 more preferably in DMA, and an aqueous phase, preferably added and deprotection is carried out at a temperature as comprising or consisting of water, Such as deionized water, in described here before. In this most preferred embodiment, particular GMP quality water having controlled quality, hav generally from 3 to 12 equivalents of HCl per molecule of ing a pH of about 7 is added to the solution of the protected protected peptide are used, preferably, from 5 to 10, more peptide. The aqueous phase has generally a temperature of 15 preferably from 6 to 8 equivalents of HCl per molecule of from 20°C to 70°C, preferably from 40° C. to 50° C. protected peptide are used. Using about 7 equivalents of HCl In another embodiment, the organic solution of Boc-Gly per molecule of protected peptide is more particularly pre Gly-Val-Leu-Val-Gln-Pro-Gly-OtBu octapeptide (SEQ ID ferred. In this preferred embodiment, the reaction medium of NO 6) is added to the aqueous phase. the deprotection reaction is preferably substantially anhy It has been found that this preferred embodiment allows for drous as described above. particularly high purity and easy separation of the protected It has been found that in particular the preferred embodi peptide which can be isolated in high yield as a purified solid. ment here before allows for efficient deprotection while Usually, the reaction product after the coupling step con avoiding potential desamidation of Gln moiety and providing tains one or more protecting group(s). An example of Such a solid deprotected peptide salt which can be recovered easily coupling product is a peptide derivative of formula X-Gly 25 from the reaction medium of the deprotection step. Gly-Val-Leu-Val-Gln-Pro-Gly-Y (SEQID NO 5), wherein X In the process according to the invention, at least one and Y are as defined above. If desirable, the protecting groups peptide coupling step is carried out in Solution. Preferably, at can be removed, for example in a selective way. Thus it is least 2, for example 2, 3 or 4 and more preferably at least 5 possible to remove only certain protecting groups, keeping peptide coupling steps for example 5, 6 or 7 coupling steps are others intact during the Subsequent reaction(s). 30 carried out in solution. Still more preferably, all coupling In a preferred aspect of the process according to the inven steps are carried out in solution. Particular solution phase tion, the peptide derivative of formula X-Gly-Gly-Val-Leu coupling steps which are useful in the process according to Val-Gln-Pro-Gly-Y (SEQID NO5) is further deprotected of the invention are apparent from the synthesis in the schemes the amino protecting group X and of the acid protecting group hereafter. Y to provide the free Gly-Gly-Val-Leu-Val-Gln-Pro-Gly 35 octapeptide (SEQID NO 1). A first synthetic approach is detailed in the scheme 1 shown In a particular aspect of the invention, a deprotection pro in FIG. 1. cess is provided which comprises deprotecting at least the A particular embodiment of the process according to amino protecting group X in the peptide derivative of formula scheme 1 is in accordance with scheme 2, shown in FIG. 2. X-Gly-Gly-Val-Leu-Val-Gln-Pro-Gly-Y (SEQ ID NO 5) 40 Good results were obtained when the fragments 3-4, 6-7, wherein Y is an optional protecting group for the carboxyl 6-8 and 5-8 of scheme 1 or 2 were under their persilylated function. Preferably Y is a free carboxyl group or an acid form. In another preferred embodiment, the 8-Gly in scheme labile carboxyl protecting group, for example, a tert. Butyl 1 is protected as carboxylic ester, in particular a tert-butyl ester. In this embodiment, X is preferably an acid labile pro ester. tecting group, in particular a Boc group. Deprotection can be 45 A further synthetic approach is detailed in scheme 3 shown carried out by means of an organic acid or a mineral acid. The in FIG. 3, wherein X and Y are as generally defined above. organic acid can for example be selected from trifluoroacetic Good results are obtained when the 8-Gly in scheme 2 is acid (TFA), trifluoromethylsulfonic acid, formic acid, p-tolu protected as carboxylic ester, in particular as tert.-butyl ester. ene Sulfonic acid and methanesulphonic acid. Deprotection is In the process according to the invention, the pressure in preferably carried out by means of a mineral acid, in particu 50 the solution phase coupling step is generally chosen so as to lar HCl, preferably dissolved in an organic solvent. Good maintain the Solution in the liquid State. Atmospheric pressure results are obtained by providing a solution of the amino (approximately 101.3 kPa) and Superatmospheric pressures protected peptide in a solvent comprising a carboxylic acid, are very suitable. preferably glacial acetic acid and adding a solution of mineral The reaction products can be isolated and purified by puri acid, preferably HCl in a polar organic solvent. In a first 55 fication methods, such as for example extraction, crystalliza embodiment, the Solution of the amino protected peptide is tion, lyophilisation, spray-drying, precipitation or chroma provided by adding organic solvent to a solution of the pep tography (e.g. thin layer or column). Isolation and tide in another solvent, for example, a workup solution from purification by precipitation or crystallization is preferred. In a coupling step. In a second embodiment, the amino protected one embodiment, at least one intermediate peptide or the final peptide is in a first step precipitated or crystallized, filtrated 60 product is isolated and purified by precipitation or crystalli and optionally washed and, in a second step, dissolved in the zation. In a particularly preferred embodiment of the process Solvent, for example in glacial acetic acid. Ethers, in particu according to the invention, all intermediates and final prod lar dioxane can also be used as polar organic solvent or ucts are isolated and purified, if desired, by precipitation or co-solvent. The deprotection step is generally carried out at a crystallization. Intermediates and the end products may, for temperature of from 0°C. to about 45° C., preferably from 65 example, be characterized by chromatographic parameters 30° C. to less than about 45° C., most preferably about 30° C. (purity control), optical rotation and possibly spectroscopic Generally, the reaction medium of the deprotection step is data. US 9,051,349 B2 11 12 The X-Val-Gln-Pro-Gly peptide (SEQ ID NO 7) may be The above described couplings are preferably carried out in obtained by various synthetic approaches. Excellent results Solution. In this case, the pressure is chosen so as to maintain were obtained with the two approaches below, namely the the Solution in the liquid state. Atmospheric pressure (ap 1+3 and the 2+2 approaches. proximately 101.3 kPa) and Superatmospheric pressures are In still another particular aspect of the present invention, very Suitable. When this coupling according to the present the process as above described may comprise the manufac invention is carried out in solution, said solution may suitably ture of X-Val-Gln-Pro-Glypeptide (SEQID NO 7) whereinX comprise acetonitrile (ChhcN) and/or an aqueous medium. is an amino protecting group as described above by coupling In another embodiment, this coupling is carried out in an of X-Val with Gln-Pro-Glypeptide (1+3 approach). organic solvent which is preferably chosen from alkyl ester Such tetrapeptide manufacture preferably comprises acti 10 solvents such as ethyl acetate (AcOEt), chlorinated solvents such as dichloromethane (DCM), and amide-type solvents Vating the carboxylic acid function of X-Val e.g. in an acti such as N,N-dimethylacetamide (DMA) and N,N-dimethyl vated ester form of the X-Val, preferably with N-hydroxysuc formamide (DMF). In this embodiment, in particular when an cinimide. In particular, X may be benzyloxycarbonyl (Z). amide type solvent is used, it is possible to use the tetrapeptide In still another particular aspect of the present invention, 15 Solution after optional separation, in particular filtration of the process as above described may comprise the manufac optionally present solids such as hydrogenation catalyst, ture of X-Val-Gln-Pro-Glypeptide (SEQID NO 7) whereinX without isolation of the tetrapeptide in a further coupling step is an amino protecting group as described above by coupling as described above, in particular in accordance with Scheme 2 of X-Val-Gln with Pro-Glypeptide (2+2 approach). or 3. The X-Val-Gln dipeptide is preferably obtained by the The reaction medium may then be suitably treated after the activation of the carboxylic acid function of X-Val e.g. in an coupling step with an aqueous phase so as to provide a solu activated ester form of the X-Val, preferably with N-hydrox tion of coupled product in the aqueous phase and then, the ySuccinimide. In particular, X may be benzyloxycarbonyl X-Val-Gln-Pro-Gly-Y peptide (SEQ ID NO 8) is extracted (Z). On the other hand, the X-Pro-Gly-Y is preferably from the aqueous phase into an organic solvent. obtained by the reaction between X-Pro with Gly-Y, wherein 25 The intermediate fragments and the X-Val-Gln-Pro-Gly-Y X is preferably a benzyloxycarbonyl (Z) group and Y is pref peptide (SEQ ID NO 8) may be recovered by precipitation erably a tert-butyl ester. Such reaction may be performed and/or crystallization. In some cases, the X-Val-Gln-Pro under classical activation conditions as above described, in Gly-Y peptide (SEQID NO 8) is generally provided as solu particular by using carbodiimides and N-hydroxysuccinim tion in a first solvent and then precipitated by addition to a ide reagents. 30 second solvent wherein the peptide is less soluble than in the The obtained X-Pro-Gly-Y peptide may be deprotected first solvent. In other cases, the X-Val-Gln-Pro-Gly-Y peptide from its X group by acidolysis, hydrogenolysis, treatment (SEQ ID NO 8) is generally provided as solution in a first with dilute ammonium hydroxide, treatment with sodium, solvent and then crystallized by the addition of a second treatment with sodium amide, treatment with hydrazine, or solvent wherein the peptide is less soluble than in the first enzymatic hydrolysis. It is preferably removed by hydro 35 solvent. genolysis. The first solvent is advantageously selected from the group The coupling between the X-Val-Gln and Pro-Gly-Y may consisting of ethyl acetate, tetrahydrofuran, dichlo be also performed under various conditions. Carbonylhalide, romethane, dioxane, methanol, n-butanol, isobutanol, 2-bu more particularly acyl halide, in particular acyl chloride cou tanol, 2-propanol, diisopropyl ether, diethyl ether, methylter pling agents as described above are preferred. Acyl halide, in 40 butylether, N,N-dimethylacetamide (DMA), N.N- particular acyl chloride coupling agents as described above dimethylformamide (DMF), and mixtures thereof. Good are preferred. Tertiary acyl halides are more particularly pre results were obtained with a dichloromethane/isobutanol ferred. Examples of tertiary acyl halides are interalia 1-ada mixture. mantoyl chloride, 2,2-dimethylbutyroyl chloride and pivaloyl The second solvent advantageously comprises at least one chloride. Excellent results were obtained while using pivaloyl 45 Solvent chosen from water, diisopropyl ether, acetonitrile, chloride, isobutyl chloroformate is also a very suitable cou diethyl ether, methylterbutylether, ethyl acetate, isopropyl pling agent. acetate, acetone, tetrahydrofuran, dichloromethane or diox It has been found that it is possible, in particular, by care ane. Good results were obtained with diisopropyl ether. fully selecting the carboxylic acid activating agent and in In still another particular aspect of the present invention, particular with tertiary acyl halides to substantially or com 50 the process as above described may comprise the manufac pletely avoid racemisation, in particular of any Gln group ture of Val-Gln-Pro-Gly tetrapeptide (SEQID NO4), wherein when coupling X-Val-Gln or Pro-Gly-Y fragments as the manufacture of the X-Val-Gln-Pro-Gly-Y (SEQID NO 8) described herein before. More particularly, it is possible to peptide further comprises the deprotection of the N-terminal Substantially avoid undesired side-reactions on the side chain amino protecting group X. In particular, X may be benzyloxy of Gln and of the Pro moiety. Moreover coupling conditions 55 carbonyl (Z). have been identified which are described here after, which Such amino protecting groups X may be removed by aci allow for high overall high productivity and yield in particular dolysis, hydrogenolysis, treatment with dilute ammonium of desired tetrapeptide while maintaining excellent optical hydroxide, treatment with sodium, treatment with sodium purity. amide, treatment with hydrazine, or enzymatic hydrolysis. It The above described couplings are generally carried out at 60 is preferably removed by hydrogenolysis. a temperature of greater than or equal to -30°C. Often, the In still another particular aspect of the present invention, reaction is carried out at a temperature greater than or equal to the process as above described comprises in addition the -10°C. Preferably, the temperature is greater than or equal to manufacture of X-Gln-Pro-Gly-Y tripeptide, wherein X is an -5°C. This reaction is generally carried out at a temperature amino protecting group, by ring opening of X-Glp-Pro of less than or equal to +45° C. Often, the reaction is carried 65 Gly-Y tripeptide with ammonia. out at a temperature of less than or equal to +30° C. Prefer In still another particular aspect of the present invention, ably, the temperature is less than or equal to +25°C. the process as above described comprises in addition the US 9,051,349 B2 13 14 manufacture of Gly-Gly-Val-Leu tetrapeptide (SEQ ID NO The coupling steps as above described of the present inven 9) by coupling of Val-Leu dipeptide with X-Gly-Gly or tion may be carried out under persilylation conditions. In X-Gly. Generally, this coupling is carried out in the presence other words, the amino acids or peptides used in the process of a carboxylic acid activating agent. In one embodiment, according to the present invention may be protected under carbodiimides, acyl halides, phosphonium salts and uronium their persilylated form. They are preferably protected under or guanidinium salts are generally preferred as carboxylic their persilylated form. acid activating agent. Carbonylhalide, more particularly acyl Another aspect of the present invention is related to a halide, in particular acyl chloride coupling agents as tripeptide of the formula Glp-Pro-Gly or protected peptide of described above are preferred. Tertiary acyl halides are more the formula X-Glp-Pro-Gly, wherein X is an amino protecting particularly preferred. Examples of tertiary acyl halides are 10 group, or protected peptide of the formula X-Glp-Pro-Gly-Y. interalia 1-adamantoyl chloride, 2,2-dimethylbutyroyl chlo wherein X is an amino protecting group and Y is a carboxylic ride and pivaloyl chloride. More preferably, the coupling acid protecting group, to a tripeptide of the formula Gln-Pro agent is chosen from isobutyl chloroformate and pivaloyl Gly or protected peptide of the formula X-Gln-Pro-Gly, chloride. wherein X is an amino protecting group, to a tetrapeptide of In another embodiment, the coupling is preferably carried 15 the formula Val-Gln-Pro-Gly (SEQ ID NO 4) or protected out with an activated ester form of the X-Gly-Gly, preferably peptide of the formula X-Val-Gln-Pro-Gly (SEQ ID NO 7), with N-hydroxysuccinimide. wherein X is an amino protecting group and in particular When acyl halides are used as carboxylic acid activating when X is benzyloxycarbonyl, to a pentapeptide of the for agents, this coupling is generally carried out in the presence mula Leu-Val-Gln-Pro-Gly (SEQID NO 10), to an hexapep of a base as additional reagent. It is preferably chosen from tide of the formula Val-Leu-Val-Gln-Pro-Gly (SEQ ID NO N-methylmorpholine (NMM), pyridine, diisopropylethy 11) and to an heptapeptide of the formula Gly-Val-Leu-Val lamine (DIPEA) or triethylamine (TEA). More preferably it Gln-Pro-Gly (SEQ ID NO 12), which may be obtained, as is N-methylmorpholine (NMM). Such or under their protected form, as intermediates during a The coupling of a Val-Leu dipeptide with X-Gly-Gly or process according to the present invention. X-Gly is preferably carried out in solution. In this case, the 25 Besides, the present invention also relates to a dodecapep pressure is chosen so as to maintain the Solution in the liquid tide of the formula Gly-Gly-Val-Leu-Val-Gln-Pro-Gly-Val state. Atmospheric pressure (approximately 101.3 kPa) and Gln-Pro-Gly (SEQID NO 13), to an hexakaidecapeptide of Superatmospheric pressures are very Suitable. The Solution the formula Gly-Gly-Val-Leu-Val-Gln-Pro-Gly-Gly-Gly generally comprises a polar organic solvent. Preferably, the Val-Leu-Val-Gln-Pro-Gly (SEQID NO 14) and to a modified polar organic solvent is selected from N,N-dimethylaceta 30 peptide of the formula CHC(=O) NH-Gly-Gly-Val-Leu mide, N,N-dimethylformamide, N-methylpyrrolidone, dim Val-Gln-Pro-Gly (SEQ ID NO 15), which may be obtained ethylsulfoxide, ethyl acetate, dichloromethane or mixtures during a process according to the present invention. thereof. More preferably, the solution comprises N,N-dim The present invention also relates to the use of the follow ethylacetamide or ethyl acetate. ing peptides as intermediates in peptide synthesis: Tripeptide The Glycine amino acid or dipeptide Gly-Gly is generally 35 of the formula Glp-Pro-Gly; Tetrapeptide of the formula Val Gln-Pro-Gly (SEQ ID NO 7); pentapeptide of the formula protected by an amino protecting group X. The amino pro Leu-Val-Gln-Pro-Gly (SEQ ID NO 10); hexapeptide of the tecting group X is preferably selected from tert-butyloxycar formula Val-Leu-Val-Gln-Pro-Gly (SEQ ID NO 11); hep bonyl, benzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, tapeptide of the formula Gly-Val-Leu-Val-Gln-Pro-Gly (SEQ 2-nitrobenzenesulfonyl, 2-nitrobenzenesulfenyl, and substi ID NO 12), as such or under their protected form. tuted derivatives. More preferably, the amino protecting 40 One of the major issues in peptide synthesis is related to the group X is tert-butyloxycarbonyl (BOC). isolation and purification of the peptides, which are often the The Val-Leu dipeptide is generally protected by a carboxy cause of a reduction in the yield of the final peptide product. lic acid protecting group Y. In consequence, the invention also relates to the purifica Preferred carboxylic acid protecting group Y are alkyl, aryl tion of the above described peptides, as such or under their and silylated derivatives. In a particularly preferred variant, 45 protected form. The purification according to the invention the Val-Leu peptide derivative is a persilylated derivative. The allows in particular meeting specifications concerning puri persilylation of Val-Leu dipeptide can be carried out, for fication related to organic impurities Such as for example example, according to the method described in Patent Appli organic solvents, such as acetonitrile and is industrializable. cation EP-A-184243. It is preferably carried out with MSA. The purification process according to the present invention The coupling of a Val-Leu dipeptide with a X-Gly-Gly or 50 allows an efficient and low cost production of said purified X-Gly is generally carried out at a temperature of greater than octapeptide. or equal to -45° C. Often, the reaction is carried out at a It is thus also an object of the present invention to provide temperature greater than or equal to -25° C. Preferably, the a process for the purification of Gly-Gly-Val-Leu-Val-Gln temperature is greater than or equal to -20°C. In the method Pro-Glypeptide (SEQID NO 1) or anyone of the peptides of according to the invention, the coupling is generally carried 55 the formula Val-Gln-Pro-Gly (SEQ ID NO 4) or X-Val-Gln Pro-Gly (SEQ ID NO 7), wherein X is an amino protecting out at a temperature of less than or equal to +45°C. Often, this group, Leu-Val-Gln-Pro-Gly (SEQID NO 10), Val-Leu-Val reaction is carried out at a temperature of less than or equal to Gln-Pro-Gly (SEQ ID NO 11), Gly-Val-Leu-Val-Gln-Pro +5° C. Preferably, the temperature is less than or equal to 0° Gly (SEQ ID NO 12), Gly-Gly-Val-Leu-Val-Gln-Pro-Gly C. Val-Gln-Pro-Gly (SEQ ID NO 13), Gly-Gly-Val-Leu-Val The tetrapeptide obtained as above described may be fur 60 Gln-Pro-Gly-Gly-Gly-Val-Leu-Val-Gln-Pro-Gly (SEQ ID ther deprotected of the amino protecting group X and of the NO 14), and CHC(=O) NH-Gly-Gly-Val-Leu-Val-Gln acid protecting group Y to provide the free Gly-Gly-Val-Leu Pro-Gly (SEQID NO 15), as such or under a protected form tetrapeptide (SEQID NO 9). wherein the peptide is dissolved in a first solvent and then The reaction products can then be isolated and purified by precipitated or crystallized. The precipitation is performed by purification methods, such as for example extraction, crystal 65 the addition of the solution of the peptide in the first solvent lization, lyophilisation, spray-drying, precipitation or chro into a second solvent wherein the peptide is less soluble than matography (e.g. thin layer or column). in the first solvent. The crystallization is performed by the US 9,051,349 B2 15 16 addition to the solution of the peptide in the first solvent of a least one polar organic Solvent while the second solvent is second solvent wherein the peptide is less soluble than in the preferably an aqueous medium. first solvent. The organic solvent is preferably chosen from isopropyl ether (IPE), acetonitrile (CHCN), methylterbutylether The peptide may be dissolved by the addition of a first (MTBE), ethyl acetate (AcOEt), isopropyl acetate (AcOiPr), solvent or may be directly obtained after the work-up as a acetone, tetrahydrofurane (THF), dichloromethane (DCM), Solution in a first solvent. In this latter case, the solution may dioxane, methanol, tert-butanol, isopropanol, ethanol, acetic be concentrated under vacuum before the addition of the acid, N,N-dimethylacetamide (DMA), N,N-dimethylforma second solvent. mide (DMF) and the like or mixtures thereof. Good results The nature of the first and second solvent depends on the were obtained with isopropyl ether and/or acetonitrile. nature of the peptide, on its isoelectric point value and on its 10 In one particular aspect, a polar organic solvent, preferably protected or unprotected form. The peptide should be more selected from acetonitrile (CH3CN), ethyl acetate (AcOEt), soluble in the first solvent than in the second solvent. isopropyl acetate (AcOiPr), acetone, tetrahydrofurane In one aspect, when the peptide is unprotected, the first (THF), dichloromethane (DCM), dioxane, methanol, tert Solvent is preferably an aqueous medium and the second butanol, isopropanol, ethanol, acetic acid, N,N-dimethylac Solvent preferably comprises at least one polar organic Sol 15 etamide (DMA), N.N dimethylformamide (DMF) and the vent. The pH of the aqueous medium may in some cases like or mixtures thereof. preferably be controlled. On the other hand, in the case of a Tables 1 and 2 give exemplary combinations of first and protected peptide, the first solvent comprises preferably at second solvents for the different fragments. TABLE 1.
Solvent Combinations
Peptide First solvent Second solvent H-Wal-Leu-OH methanol/water isopropanol
Z-Glp-Pro-OH water/acetonitrile Water Z-Glp-Pro-Gly-OH water Adichloromethane Aqueous KHSO solution
H-Gln-Pro-Gly-OH Water ethanol Z-Val-Gln-Pro-Gly-OH Isobutanol/dichloromethane diisopropylether (SEQ ID NO 16)
H-Val-Gln-Pro-Gly-OH methanol acetonitrile (SEQ ID NO 4) HCl-H-Gly-Gly-Val-Leu-Val Acetic acid/dioxane diisopropylether Gln-Pro-Gly-OH (SEQ ID NO 1) CH3COO-H-Gly-Gly-Val-Leu Ammonium acetate aqueous acetonitrile Val-Gln-Pro-Gly-OH louffer (SEQ ID NO 1)
TABLE 2
Alternative Solvent Combinations
Peptide First solvent Second solvent
H-Wal-Leu-OH isopropanol/water isopropanol Boc-Gly-Gly-Val-Leu-OH ACOEt diisopropylether (SEO ID NO : 17)
Isobutanol AcOiPr
H-Val-Gln-Pro-Gly-OtBu ACOEt diisopropylether (SEQ ID NO 18)
Boc-Gly-Gly-Val-Leu-Val DMA Water Gln-Pro-Gly-OtBu (SEQ ID NO 19) HCl-H-Gly-Gly-Val-Leu-Val Acetic acid/dioxane MeCN/diisopropylether Gln-Pro-Gly-OH (SEQ ID NO 1) Ammonium acetate aqueous EtOH Leu-Val-Gln-Pro-Gly-OH louffer (SEQ ID NO 1) US 9,051,349 B2 17 18 It may be advantageous to isolate and purify the desired Leu-Val-Gln-Pro-Gly (SEQ ID NO 10), Val-Leu-Val-Gln peptide product by Salt formation (e.g. hydrochloride, Pro-Gly (SEQ ID NO 11), Gly-Val-Leu-Val-Gln-Pro-Gly acetate, dicyclohexyl ammonium, cyclohexyl ammonium or (SEQ ID NO 12), Gly-Gly-Val-Leu-Val-Gln-Pro-Gly-Val trifluoroacetate salt formation) or by Zwitterion formation. As Gln-Pro-Gly (SEQ ID NO 13), Gly-Gly-Val-Leu-Val-Gln used herein, particularly with reference to the peptide having Pro-Gly-Gly-Gly-Val-Leu-Val-Gln-Pro-Gly (SEQ ID NO the amino acid sequence Gly-Gly-Val-Leu-Val-Gln-Pro-Gly 14), and CHC(=O) NH-Gly-Gly-Val-Leu-Val-Gln-Pro (SEQID NO 1), the term “salt(s) of the peptide' includes and Gly (SEQID NO 15), as such or under a protected form, in a encompasses stoichiometric and non-stoichiometric salts. As Solvent mixture comprising water and apolar organic solvent. stated above, these salts may also have associated water mol The above described solution according to the invention ecules (hydrates) and may further be crystalline materials. 10 preferably comprises acetonitrile and alcohols such as metha The peptide can also be separated from a solution for nol, ethanol, propanol and the like. Such solution may be used example by spray-drying, filtration or decantation and dried in a purification operation. before optionally being Submitted to further processing steps Another preferred aspect of the present invention is related Such as combining with other ingredients, lyophilization, to providing acetate-containing Gly-Gly-Val-Leu-Val-Gln spray-drying, packaging and/or storage. 15 Pro-Gly octapeptide (SEQID NO 1) having different acetate According to one Suitable approach, the peptide is col content. It has been found that, depending on the isolation lected via filtering and optionally washed, in particular to method used, different molar contents of acetate in the final reduce possible salt content, and then dried. peptide can be achieved, which may present certain advan A further particular aspect of the present invention is tages as to their stability. It should be understood that the related to a process for purifying Gly-Gly-Val-Leu-Val-Gln present invention includes Stoichiometric and non-stoichio Pro-Glypeptide (SEQID NO 1), or anyone of the peptides of metric salts of the peptide having the amino acid sequence the formula Val-Gln-Pro-Gly (SEQ ID NO 4) or X-Val-Gln Gly-Gly-Val-Leu-Val-Gln-Pro-Gly (SEQID NO 1) and that Pro-Gly (SEQID NO 7), wherein X is an amino protecting the general terms “salt(s) and “salt(s) of the peptide' are to group, Leu-Val-Gln-Pro-Gly (SEQID NO 10), Val-Leu-Val be understood as referring to and including Stoichiometric Gln-Pro-Gly (SEQ ID NO 11), Gly-Val-Leu-Val-Gln-Pro 25 and non-stoichiometric salts. It should also be understood that Gly (SEQ ID NO 12), Gly-Gly-Val-Leu-Val-Gln-Pro-Gly the phrase "acetate containing peptide also refers to and Val-Gln-Pro-Gly (SEQ ID NO 13), Gly-Gly-Val-Leu-Val includes Stoichiometric and non-stoichiometric acetate salts. Gln-Pro-Gly-Gly-Gly-Val-Leu-Val-Gln-Pro-Gly (SEQ ID In addition, as used herein, the peptide (or octapeptide) hav NO 14), and CHC(=O) NH-Gly-Gly-Val-Leu-Val-Gln ing the sequence Gly-Gly-Val-Leu-Val-Gln-Pro-Gly (SEQ Pro-Gly (SEQID NO 15) as such or under a protected form, 30 ID NO 1) may also be referred to by the USAN/INN name which comprises Subjecting a crude peptide of any of the “larazotide'. It has also been found that, depending on the aforesaid sequences to a chromatography operation. isolation method used, the acetate salt of the peptide may have In the present invention, the chromatography is preferably varying amounts of water also associated with it; thus these chosen from medium pressure liquid chromatography acetate salts can be classified as (Stoichiometric as well as (MPLC) and high pressure liquid chromatography (HPLC). 35 non-stoichiometric) hydrates (or “hydrated acetate salts'). As In this aspect, the chromatography operation may take used herein, unless expressly stated to the contrary, the terms place prior or after an optional precipitation or crystallization “larazotide acetate' or “acetate-containing peptide' or of the peptide. “acetate-containing octapeptide' refers to all solid state The chromatography operation may be processed for forms of the acetate-containing peptide having the sequence example on columns with a continuous bed (monolithic col 40 Gly-Gly-Val-Leu-Val-Gln-Pro-Gly (SEQID NO 1) irrespec umns). In that case, normal phase stationary phases can be tive of the amounts of acetate and/or water associated with the used, for example silica or alumina. In that case apolar mobile peptide. phases are generally used. Reverse phase stationary phases In a first embodiment, the acetate-containing peptide is Such as hydrophobically modified inorganic Supports, typi isolated from an aqueous peptidic solution by lyophilization. cally silica grafted with organic hydrophobic compounds are 45 Lyophilization is intended to denote a means of drying a preferably used. In that case polar mobile phases are gener desired substance, achieved by freezing an aqueous medium ally used, for example aqueous mobile phases containing an containing said Substance and causing ice to Sublime directly organic co-solvent, in particular a polar organic co-solvent, to vapor by exposing it to a low partial pressure of water Such as methanol, ethanol, isopropanol, acetonitrile or diox vapor. In this case, the acetate-containing peptide has gener ane. Medium polar such as silica with bonded diol, propyl 50 ally a concentration of acetate of at least or equal to 60 mol cyano or amino groups may also be used in particular with %/mole of peptide. moderately polar mobile phases such as buffered aqueous/ In a second embodiment, the acetate-containing peptide is organic mobile phases. precipitated from the liquid medium by concentrating the The chromatography operation may be a medium pressure liquid medium e.g. by evaporation. In this case, the acetate liquid chromatography (MPLC). In Such a chromatography 55 containing peptide has generally a concentration of acetate of operation, the eluent may comprise water, acetonitrile from 30 mol% to 60 mol% preferably about 50 mol%/mole (CHCN), alcohols such as methanol, ethanol, propanol and of peptide. A Suitable starting solution can be obtained, for the like. Preferably, it comprises water (HO) and/or aceto example, by chromatography, in particular MPLC, of a crude nitrile (CHCN). The eluent may also comprise a certain product obtained from a deprotection step as described above. amount of salts to maintain its pH value to a certain area 60 For example, a crude product obtained in particular by depro (buffer solution). Good results were obtained when an aque tection with HCl can be subjected to a chromatography opera ous solution of ammonium acetate was used as eluent. tion in particular as described above. The solution containing A further particular aspect of the present invention is acetate-containing peptide and a polar organic solvent, in related to a solution of Gly-Gly-Val-Leu-Val-Gln-Pro-Gly particular acetonitrile, which solution is obtained from the peptide (SEQ ID NO 1) or anyone of the peptides of the 65 chromatography operation can be concentrated for example formula Val-Gln-Pro-Gly (SEQID NO 4) or X-Val-Gln-Pro by evaporation, preferably under reduced pressure, prefer Gly (SEQID NO 7), wherein X is an amino protecting group, ably at a temperature of from 20 to 50° C. As the solution is US 9,051,349 B2 19 20 concentrated, acetate-containing peptide starts to precipitate ID No 1) is crystalline and comprises less than about 7 mol% and may be recovered by filtration. Precipitation efficiency water. In another embodiment, the acetate-containing may be enhanced, for example by cooling down the concen octapeptide Gly-Gly-Val-Leu-Val-Gln-Pro-Gly (SEQID No trated solution, typically to a temperature below 10°C. 1) is crystalline and comprises less than about 5 mol% water. In a third embodiment, the acetate-containing peptide is In a further embodiment, the acetate-containing octapeptide crystallized from the liquid medium by exchanging the chlo Gly-Gly-Val-Leu-Val-Gln-Pro-Gly (SEQID No 1) is crystal ride counter-ion of the chloride salt of the octapeptide Gly line and comprises less than about 3 mol % water. In a yet Gly-Val-Leu-Val-Gln-Pro-Gly (SEQID NO 1) by an acetate further embodiment, the acetate-containing octapeptide Gly ion. In this case, the acetate-containing peptide (laraZotide Gly-Val-Leu-Val-Gln-Pro-Gly (SEQ ID No 1) is crystalline acetate) has generally a concentration of acetate from more 10 than 0 to less than 50 mole %/mole of peptide, preferably and comprises between about 1 mol % to about 2 mol % from 20 to 30 mole %/mole of peptide. In another embodi Water. ment, the acetate-containing octapeptide Gly-Gly-Val-Leu The invention concerns also the manufacture of said Val-Gln-Pro-Gly (SEQID NO 1) is crystalline and comprises acetate-containing peptides by the methods indicated. less than about 20 mole % acetate. In still another embodi 15 The chloride salt introduced into a salt exchange step of the ment, the acetate-containing octapeptide Gly-Gly-Val-Leu Gly-Gly-Val-Leu-Val-Gln-Pro-Gly octapeptide (SEQID NO Val-Gln-Pro-Gly (SEQID NO 1) is crystalline and comprises 1) has preferably a purity of at least 98.5% by HPLC. less than about 15 mole % of acetate. In a yet further embodi ment, the acetate-containing octapeptide Gly-Gly-Val-Leu EXAMPLES Val-Gln-Pro-Gly (SEQID NO 1) is crystalline and comprises less than about 10 mole % of acetate, preferably between The following examples are intended to illustrate the about 4 to about 8 mol % of acetate. It should be understood invention without, however, limiting its scope. that any of the foregoing references to crystalline acetate In these examples and throughout this specification the containing octapeptide Gly-Gly-Val-Leu-Val-Gln-Pro-Gly abbreviations employed are defined as follows: (SEQ ID NO 1) may further include associated water mol 25 AcOH is acetic acid, AcOEt is ethyl acetate, AcOiPr is ecules, i.e., crystalline hydrated acetate salts of the peptide, as isopropyl acetate, Boc is t-butoxycarbonyl, n-BuOH is n-bu further described below. tanol, Cbz is benzyloxycarbonyl, DCC is 1.3 dicyclohexyl In a particularly advantageous aspect, it is possible to crys carbodiimide, DCM is dichloromethane, DIC is 1,3-diisopro tallize the acetate-containing peptide by adding a source of pylcarbodiimide, DIPEA is N,N-diisopropylethylamine, acetate ions to a liquid medium obtained by dissolving chlo 30 DMAPA is 3-dimethylaminopropylannine, DMF is N.N- ride salt of the Gly-Gly-Val-Leu-Val-Gln-Pro-Gly octapep dimethylformamide, DMA is N,N-dimethylacetamide, Fmoc tide (SEQ ID NO 1) obtained from a deprotection step as is fluorenylmethyloxycarbonyl, HBTU is N.N.N',N'-tetram described above in water. Suitable sources of acetate include ethyl-O-(1H-benzotriazol-1-yl)uronium-hexafluororphos acetate salts, for example sodium acetate, potassium acetate phate), HOBT is 1-hydroxybenzotriazole, HOOBT is 3,4- or ammonium acetate. Ammonium acetate has given good 35 dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, HPW is high results. Preferably, the pH during crystallization is controlled purity water IBCF is isobutyl chloroformate, Pivoclis Pivaloyl in a range from 2.5 to 7.5. A pH of from 3.5 to 6.5 is more chloride, i-BuOH is isobutanol, IPE is diisopropylether, particularly preferred. A pH of about 4.5 has given good MeCN or ACN is acetonitrile, MeOH is methanol, NMM is results. In this embodiment the initial concentration of the N-methylmorpholine, NMP is 1-methyl-2-pyrrolidone, THF chloride salt of the Gly-Gly-Val-Leu-Val-Gln-Pro-Gly 40 is tetrahydrofuran, MSA is N-Methyl-N trimethylsilylaceta octapeptide (SEQ ID NO 1) determined as free peptide is mide, Tos is tosyl, MTBE is Methyl-tert-butylether. generally from 2 to 20% wt relative, to the total weight of the liquid medium containing said chloride Salt and the source of Examples 1 to 11 acetate ions, and, if necessary, the pH adjusting agent, for example a base Such as ammonia, all preferably dissolved in 45 Scheme 4 (FIG. 4) represents a first general synthetic water. Preferably, this initial concentration is from 10 to 15% approach of the Gly-Gly-Val-Leu-Val-Gln-Pro-Gly (SEQID by weight. The temperature during crystallization is generally NO 1) octapeptide which will be detailed in the following from 5° C. to 35° C., preferably from 20° C. to 30° C. examples. In a fourth embodiment, it is possible to prepare an acetate free peptide, preferably in Zwitterionic form, by crystallizing 50 Example 1 from the liquid medium by adjusting the pH of an aqueous solution of the chloride salt of the Gly-Gly-Val-Leu-Val-Gln Synthesis of H-Val-Leu-OH Pro-Gly octapeptide (SEQID NO 1) to the isoelectric point of the peptide, which is about 6.0 to 7.0, more particularly about Leucine (1.2 eq.) was silylated in pure MSA at maximum 6.5. When the free peptide is desired, it is preferred to avoid 55 50° C. until complete dissolution and then diluted with the presence of Supplementary counter-ions such as acetate. AcOEt. The leucine solution was transferred to a Z-Val-OSu The invention also concerns said acetate-containing pep solution under stirring at 35°C. The reaction was quenched tides. It has been found, surprisingly, that the stability of the with water, diluted with AcOEt and the organic phase was peptide is improved when the acetate content is reduced and washed with KHSO and NaCl. The solvent was removed also on account of its manufacturing process. An acetate 60 under vacuum and replaced with MeOH. Water was then containing peptide obtained by crystallization or precipita added to the methanolic solution followed by the addition of tion as described above is more stable than a lyophilized the palladium catalyst. The deprotection of the Z group took peptide. It has also been found that the amount of water place with the introduction of gaseous hydrogen at 30° C. associated with the acetate-containing octapeptide Gly-Gly Once the reaction completed, the catalyst was filtrated and Val-Leu-Val-Gln-Pro-Gly (SEQID No 1) also varies with the 65 washed with a 50/50 mixture of methanol and water. The method of preparation. In one embodiment the acetate-con solvent was evaporated and the mixture diluted with isopro taining octapeptide Gly-Gly-Val-Leu-Val-Gln-Pro-Gly (SEQ panol to precipitate the dipeptide. The dipeptide was then US 9,051,349 B2 21 22 recovered by filtration, washed with isopropanol at room 25° C. over at least 4 h. The excess of DCC was neutralized temperature and then dried. The dipeptide was isolated with a with AcOH (0.05 eq.) and the suspension was cooled to 10°C. yield of 85%. before filtering the DCU precipitated which was then washed with MeCN. The resulting solution was warmed to 25°C. Example 2 5 H-Pro-OH (2.0 eq.) was added to a solution of MeCN and (1.9 eq.) of MSA. The suspension was heated to 45° C. and left stirring until a clear Solution was obtained. The Solution was Synthesis of H-Val-Leu-OH then cooled to 25°C. The silylated proline solution was added to the solution of Z-Glp-OSu and left to stir for at least 3 hat Leucine (1.2 eq.) was silylated in pure MSA at at most 50° 25° C. The coupling solution was diluted with water and C. until complete dissolution and then diluted with AcOEt. 10 MeCN was evaporated in vacuo at a maximum temperature of The leucine solution was transferred to a Z-Val-OSu Solution 65°C. The remaining slurry was then diluted with water and under stirring at 35°C. The unreacted ZValOSu was neutral the precipitate was left stirring for at least 10 h at 5°C. The ized with DMAPA (0.05 eq.) and the reaction was quenched solid was filtered, washed with water. After drying under with water, diluted with AcOEt and the organic phase was vacuum 80% of Z-Glp-Pro-OH was recovered. washed with KHSO and NaCl. The solvent was removed 15 under vacuum and replaced with iPrCH until the AcOEt Example 5 content in the evaporates was <5% weight. Water was then added to the peptidic solution followed by the addition of the Synthesis of Z-Glp-Pro-Gly-OH palladium catalyst. The deprotection of the Z group took place with the introduction of gaseous hydrogen at about 35° 20 Two Solutions were prepared before the peptide coupling. C. Once the reaction completed, the catalyst was filtered and Solution A: H-Gly-OH (1.2 eq.) was dissolved in MSA (3.0 washed with water. The filtrates were collected, diluted with eq.) at maximum 60°C. The Suspension was cooled down to 25° C., diluted with DCM and stirred for at least 8 hours isopropanol and cooled to +5°C. to precipitate the dipeptide before being cooled to -15°C. In solution B, the carboxylic which was then recovered by filtration, washed with isopro function of the Z-Glp-Pro-OH was dissolved with DCM and panol and MeCN at room temperature and then dried. The 25 NMM (1.05 eq.). The solution was cooled to -15° C. The dipeptide was isolated with a yield of 85%. carboxylic acid was activated with IBCF (1.05 eq.) and the silylated solution A was then added to the slurry. The slurry Example 3 was stirred for at least 0.5 h and left to warm to 25°C. The mixture was quenched with water and the addition of a solu Synthesis of Boc-Gly-Gly-Val-Leu-OH (SEQ ID NO 30 tion of KHSO under stirring precipitated the peptide. The 19) solid was filtered and washed with water. After drying under vacuum, 80% of Z-Glp-Pro-Gly-OH was recovered. On one hand, H-Val-Leu-OH (1 eq.) was added to a solu tion of MSA (2.72 eq.) in AcOEt. The slurry was stirred at 25° Example 6 C. until a solution was obtained. The solution was then cooled 35 to -15° C. On the other hand, Boc-Gly-Gly-OH (1.05 eq., Synthesis of H-Gln-Pro-Gly-OH commercially available) was added together with NMM (1.0 eq.), AcOEt and DMF. The slurry was stirred until complete Z-Glp-Pro-Gly-OH (1 eq.) was dissolved under stirring in dissolution and then cooled to -25°C. IBCF (1.0 eq.) was DMA at 25° C. and NH-OH 25% (6 eq.) was added in such added to the Boc-Gly-Gly-OH solution to activate the car- 40 way that the temperature did not rise above 30°C. The mix boxylic function. The silylated Val-Leu was then added and ture was stirred for at least 4 h at 25°C. The solution was left to stir at least 30 min. The reaction mixture was condi concentrated under vacuum till the pH was s3. The concen trate was diluted with a NaCl aqueous solution and the pH tioned to 25° C. before being quenched by the addition of was adjusted to 2.5 with a solution of KHSO. The resulting water. The mixture was then diluted with AcOEt and washed aqueous solution was washed twice with IPE and then with a solution of KHSO under stirring. The aqueous phase 45 extracted three times with n-BuOH at 25°C. The organic was discarded and the organic layer was washed again with a layers were combined and washed with water. The resulting solution of NaCl. The organic phase was finally concentrated organic Solution was concentrated under reduced pressure. and the tetrapeptide crystallized under gentle stirring for at The concentrated solution was diluted with ethanol and water least 8 h at 5°C. The solid was recovered by filtration, once at 20° C. and Pd/C (0.02 eq.) was added to the peptide solu washed with cold AcOEtat 5°C. After drying under vacuum, 50 tion. The solution was stirred at 20°C. followed by the intro 85% of Boc-Gly-Gly-Val-Leu-OH (SEQ ID NO 19) was duction of hydrogen under pressure (0.3 bar). The solution recovered. was stirred for at least 2 hours and the completion of the reaction was checked by HPLC. The solution was filtered to Example 4 remove the catalyst and, after one washing with deminera 55 lised water, the pH of the solution was adjusted to Synthesis of Z-Glp-Pro-OH 6.0 SEQUENCE LISTING <16 Os NUMBER OF SEO ID NOS: 24 <21 Os SEQ ID NO 1 &211s LENGTH: 8 212s. TYPE: PRT <213> ORGANISM: Artificial Sequence US 9,051,349 B2 35 36 - Continued FEATURE: OTHER INFORMATION: peptide antagonist of zonulin SEQUENCE: 1 Gly Gly Val Lieu Val Glin Pro Gly 1. 5 SEQ ID NO 2 LENGTH: 4 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: partial peptide antagonist of zonulin FEATURE: NAME/KEY: MISC FEATURE LOCATION: (4) ... (4) OTHER INFORMATION: May be modified with a carboxylic acid protecting group SEQUENCE: 2 Val Glin Pro Gly 1. SEQ ID NO 3 LENGTH: 4 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: partial peptide antagonist of zonulin FEATURE: NAME/KEY: MISC FEATURE LOCATION: (1) ... (1) OTHER INFORMATION: May be modified with an amino protecting group < 4 OOs SEQUENCE: 3 Gly Gly Val Lieu 1. SEQ ID NO 4 LENGTH: 4 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: partial peptide antagonist of zonulin SEQUENCE: 4 Val Glin Pro Gly 1. SEO ID NO 5 LENGTH: 8 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: peptide antagonist of zonulin FEATURE: NAME/KEY: MISC FEATURE LOCATION: (1) ... (1) OTHER INFORMATION: May be modified with an amino protecting group FEATURE: NAME/KEY: MISC FEATURE LOCATION: (8) ... (8) OTHER INFORMATION: May be modified with a carboxylic acid protecting group SEQUENCE: 5 Gly Gly Val Lieu Val Glin Pro Gly 1. 5 US 9,051,349 B2 37 38 - Continued <210s, SEQ ID NO 6 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: peptide antagonist of zonulin 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: May be modified with a tert-butyloxycarbonyl amino protecting group 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: May be modified with a tert-butyl ester carboxy protecting group <4 OOs, SEQUENCE: 6 Gly Gly Val Lieu Val Glin Pro Gly 1. 5 <210s, SEQ ID NO 7 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: partial peptide antagonist of zonulin 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: May be modified with an amino protecting group <4 OO > SEQUENCE: 7 Val Glin Pro Gly 1. <210s, SEQ ID NO 8 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: partial peptide antagonist of zonulin 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: May be modified with an amino protecting group 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: May be modified with a carboxylic acid protecting group <4 OOs, SEQUENCE: 8 Val Glin Pro Gly 1. <210s, SEQ ID NO 9 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: partial peptide antagonist of zonulin <4 OOs, SEQUENCE: 9 Gly Gly Val Lieu 1. <210s, SEQ ID NO 10 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence US 9,051,349 B2 39 40 - Continued 22 Os. FEATURE: <223> OTHER INFORMATION: partial peptide antagonist of zonulin <4 OOs, SEQUENCE: 10 Lieu Val Glin Pro Gly 1. 5 <210s, SEQ ID NO 11 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: partial peptide antagonist of zonulin <4 OOs, SEQUENCE: 11 Val Lieu Val Glin Pro Gly 1. 5 <210s, SEQ ID NO 12 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: partial peptide antagonist of zonulin <4 OOs, SEQUENCE: 12 Gly Val Lieu Val Glin Pro Gly 1. 5 <210s, SEQ ID NO 13 211 LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: peptide antagonist of zonulin <4 OOs, SEQUENCE: 13 Gly Gly Val Lieu Val Glin Pro Gly Val Glin Pro Gly 1. 5 1O <210s, SEQ ID NO 14 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: partial peptide antagonist of zonulin <4 OOs, SEQUENCE: 14 Gly Gly Val Lieu Val Glin Pro Gly Gly Gly Val Lieu Val Glin Pro Gly 1. 5 1O 15 <210s, SEQ ID NO 15 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: partial peptide antagonist of zonulin 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: May be modified with an acetamide amino protecting group <4 OOs, SEQUENCE: 15 Gly Gly Val Lieu Val Glin Pro Gly 1. 5 US 9,051,349 B2 41 42 - Continued SEQ ID NO 16 LENGTH: 4 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: partial peptide antagonist of zonulin FEATURE: NAME/KEY: MISC FEATURE LOCATION: (1) ... (1) OTHER INFORMATION: May be modified with a benzyloxycarbonyl amino protecting group SEQUENCE: 16 Val Glin Pro Gly 1. SEO ID NO 17 LENGTH: 4 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: partial peptide antagonist of zonulin FEATURE: NAME/KEY: MISC FEATURE LOCATION: (1) ... (1) OTHER INFORMATION: May be modified with a tert-butyloxycarbonyl amino protecting group SEQUENCE: 17 Gly Gly Val Lieu 1. SEQ ID NO 18 LENGTH: 4 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: partial peptide antagonist of zonulin FEATURE: NAME/KEY: MISC FEATURE LOCATION: (4) ... (4) OTHER INFORMATION: May be modified with a tert-butyl ester carboxy protecting group SEQUENCE: 18 Val Glin Pro Gly 1. SEQ ID NO 19 LENGTH: 8 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: peptide antagonist of zonulin FEATURE: NAME/KEY: MISC FEATURE LOCATION: (1) ... (1) OTHER INFORMATION: May be modified with a tert-butyloxycarbonyl amino protecting group FEATURE: NAME/KEY: MISC FEATURE LOCATION: (8) ... (8) OTHER INFORMATION: May be modified with a tert-butyl ester carboxy protecting group SEQUENCE: 19 Gly Gly Val Lieu Val Glin Pro Gly 1. 5 US 9,051,349 B2 43 44 - Continued SEQ ID NO 2 O LENGTH: 8 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: peptide antagonist of zonulin FEATURE: NAME/KEY: MISC FEATURE LOCATION: (1) ... (1) OTHER INFORMATION: May be modified with a tert-butyloxycarbonyl amino protecting group SEQUENCE: 2O Gly Gly Val Lieu Val Glin Pro Gly 1. 5 SEQ ID NO 21 LENGTH: 4 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: partial peptide antagonist of zonulin FEATURE: NAME/KEY: MISC FEATURE LOCATION: (1) ... (1) OTHER INFORMATION: May be modified with a benzyloxycarbonyl amino protecting group FEATURE: NAME/KEY: MISC FEATURE LOCATION: (4) ... (4) OTHER INFORMATION: May be modified with a tert-butyl ester carboxy protecting group SEQUENCE: 21 Val Glin Pro Gly 1. SEQ ID NO 22 LENGTH: 4 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: partial peptide antagonist of zonulin FEATURE: NAME/KEY: MISC FEATURE LOCATION: (1) ... (1) OTHER INFORMATION: May be modified with a benzyloxycarbonyl amino protecting group FEATURE: NAME/KEY: MISC FEATURE LOCATION: (4) ... (4) OTHER INFORMATION: May be modified with a tert-butyl ester carboxy protecting group SEQUENCE: 22 Val Glin Pro Gly 1. SEQ ID NO 23 LENGTH: 8 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: peptide antagonist of zonulin FEATURE: NAMEAKEY: misc feature LOCATION: (2) ... (4) OTHER INFORMATION: Xaa can be any naturally occurring amino acid FEATURE: NAMEAKEY: misc feature LOCATION: (7) . . (7) OTHER INFORMATION: Xaa can be any naturally occurring amino acid US 9,051,349 B2 45 46 - Continued <4 OOs, SEQUENCE: 23 Gly Xaa Xala Xaa Val Gly Xaa Gly 1. 5 SEQ ID NO 24 LENGTH: 5 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: partial peptide antagonist of zonulin <4 OOs, SEQUENCE: 24 Gly Gly Val Lieu Val 1. 5 The invention claimed is: 5. The crystalline acetate salt of claim 1, wherein the 1. A crystalline acetate salt of the peptide Gly-Gly-Val- 20 acetate is present at a concentration of from about 20 to 30 Leu-Val-Gln-Pro-Gly (SEQID NO:1) having an XRPD pat mole %/mole of peptide. tern comprising peaks at 9.08+0.2 degrees 20, 9.52+0.2 6. The crystalline acetate salt of claim 5, wherein the degrees 20, 10.34+0.2 degrees 20, and 14.24+0.2 degrees 20. acetate is presentata concentration of less than about 15 mole 2. A pharmaceutical composition comprising or prepared %. with the crystalline peptide salt of claim 1. 25 7. The crystalline acetate salt of claim 6, wherein the 3. The crystalline acetate salt of claim 1 further comprising acetate is presentata concentration of less than about 10 mole one or more of the following peaks: 18.23+0.2 degrees 20, %. 19.08+0.2 degrees 20 and/or 21.31+0.2 degrees 20. 8. The crystalline acetate salt of claim 7, wherein the 4. The crystalline acetate salt of claim 3 having an XRPD acetate is present at a concentration of between about 4 to pattern comprising peaks at 14.24-0.2 degrees 20 and 30 about 8 mole %. 18.23+0.2 degrees 20.