Process for the preparation of a x-beta-ala-glu(OTBU)-gly tetramer by LPPS and its use in SPPS synthesis

Abstract

The invention relates to a process for preparing a tetramer of the formula (I), or salts thereof, wherein R1is hydrogen or is a link to a solid support and PROT is an ester protecting group, to a crystalline polymorph of the tetramer of formula I and its preparation and to its use as intermediate in the preparation of a GLP-1R / GIPR agonist.

Description

[0001] P39746

[0002] Process for the preparation of a X-beta-Ala-Glu(OtBu)-Gly tetramer by LPPS and its use in SPPS synthesis

[0003] FIELD OF THE INVENTION

[0004] The invention relates to a process for the preparation of the tetramer compound of formula I, or salts thereof, wherein

[0005] PROT is an ester protecting group, to a crystalline polymorph of the tetramer compound of formula I and to the use of the tetramer compound produced according to this process in the manufacturing of the peptide of formula XX, or of a pharmaceutically acceptable salt or ester thereof X-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16- Ile17-Ala18-Gln19-Lys20(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala21-Phe22-Val23- Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38- Ser39-NH2

[0006] (XX) wherein X is

[0007] AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetyl.

[0008] The peptide of formula XX has the potential to function as GLP-1R / GIPR agonist.

[0009] BACKGROUND

[0010] Glucagon-like peptide- 1 (GLP-1) and gastric inhibitory polypeptide (GIP) are primary incretin hormones secreted from small intestinal L cells and K cells, respectively, on ingestion of glucose or nutrients to stimulate insulin secretion from pancreatic cells. The actions of GIP and GLP-1 are believed to be mediated by their receptors, the GIP receptor (GIPR) and the GLP-1 receptor (GLP-1R), respectively, which both belong to the G-protein coupled receptor family and are expressed in pancreatic cells, as well as in various tissues and organs. GLP- 1R / GIPR agonists are compounds that mimic the action of the naturally occurring hormones GLP-1 and GIP. These hormones play a crucial role in regulating blood sugar levels by enhancing insulin secretion in response to meals, inhibiting glucagon release, and slowing gastric emptying. GLP-1R / GIPR agonists are thus useful medicaments in the treatment of type 2 diabetes mellitus to improve glycemic control. Additionally, they have been shown to promote weight loss, which can be beneficial for patients with obesity or those who are overweight. GLP-1R / GIPR agonists are effective in reducing HbAlc levels and have a favorable impact on cardiovascular outcomes in diabetic or overweight patients. Obesity is the most prevalent chronic disease worldwide and is associated with many other diseases.

[0011] Particularly, the peptide of formula XX is a dual GLP-1R / GIPR agonist, which potently activates production of cyclic adenosine monophosphate (cAMP), but has no or minimal activity on the P-arrestin signaling pathways on either GLP-1R or GIPR. That is, the agonist is fully biased towards cAMP activation, as opposed to being partially biased (i.e., with some P-arrestin activity) or unbiased (i.e., with full P-arrestin activity), on both GLP-1R and GIPR. P-Arrestin activates kinase signaling pathways but also causes the GLP-1R and GIPR to be turned off and internalized. The peptide of formula XX does not cause internalization and consequently, desensitization of either GLP-1R or GIPR, and thus has enhanced signaling efficacy. Object of the invention was to facilitate the synthesis of the peptide of formula XX and to provide a novel process for a tetramer, which acts as a building block allowing easy integration into a solid phase peptide synthesis (SPPS) reaction set up. In addition, the facilitation should aim at improving the impurity profile and shortening the overall production time by reducing the number of cycles.

[0012] SUMMARY OF THE INVENTION

[0013] It was found that the object of the invention could be reached with the tetramer compound of formula I, in particular a process for its preparation or of a salt thereof wherein PROT is an ester protecting group, comprising, coupling, glycine (Gly), glutamic acid (Glu) with the y-carboxylic acid protected with PROT, P-alanine (Ala) and 2-(3-cyano-5-fluorophenyl)-2-methyl-propionic acid of the formula II, under liquid phase peptide synthesis (LPPS) conditions. The invention further relates to a crystalline polymorph of the tetramer compound of formula I and to the use of the tetramer compound produced according to this process in the manufacturing of the peptide of formula XX, or of a pharmaceutically acceptable salt or ester thereof X-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-

[0014] Ile17-Ala18-Gln19-Lys20(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-

[0015] Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-

[0016] Ser39-NH2

[0017] (XX) wherein X is

[0018] AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetyl.

[0019] DETAILED DESCRIPTION OF THE INVENTION

[0020] The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein.

[0021] The term "pharmaceutically acceptable salt" refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable. The salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, in particular hydrochloric acid, and organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N- acetylcysteine and the like. In addition, these salts may be prepared by addition of an inorganic base or an organic base to the free acid. Salts derived from an inorganic base include but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium salts, and the like. Salts derived from organic bases include but are not limited to salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, A-ethylpiperidine, piperidine, polyimine resins and the like. The term “salt” in the context of the present invention encompasses typical salts of carboxylic acids, which can be formed with inorganic bases such as with alkali hydroxide, like sodium hydroxide or with organic bases such as with amines, like ammonia. Further viable examples can be found in the definition of the term "pharmaceutically acceptable salt".

[0022] The term “alkyl” stands for a linear or branched alkyl group, usually of 1 to 6 C-atoms. Representatives are methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl or t-butyl, pentyl and its isomers and hexyl and its isomers. In the context of the present invention lower alkyl groups of 1 to 4 C-atoms are preferred.

[0023] The term “ester protecting group” refers to protecting groups of the carboxylic acid functionalities of the amino acid, which typically can be cleaved under acidic conditions. Commonly used is tert-butyl (tBu) which is cleavable e.g., with trifluoroacetic acid. Alternatively, 3-methyl-pent-3-yl (Mpe) can be used.

[0024] The term “solid support” in the context of the present invention encompasses solid supports for SPPS which are insoluble, chemically inert materials that serve as anchoring points for the growing peptide chain during synthesis. These supports are designed to withstand the repeated cycles of coupling, deprotection, and washing steps involved in peptide assembly. They provide a physical matrix that allows for easy separation of the peptide from reagents and by-products through simple filtration.

[0025] The term “solid phase peptide synthesis (SPPS) conditions” refers to specific parameters and reagents for assembling peptides on a solid support. These conditions are tailored to ensure efficient coupling, minimize side reactions, and yield high-purity peptides, varying based on the peptide sequence, scale, and desired synthesis outcome. Key components include, but are not limited to the solid support, suitable protecting groups for the amino acids, suitable coupling agents and solvents, procedures to eliminate by-products and suitable cleavage conditions to remove the peptide from the resin and deprotect side chains.

[0026] The term "liquid phase peptide synthesis (LPPS) conditions" refers to specific parameters and reagents for assembling peptides in solution. These conditions are tailored to ensure efficient coupling, minimize side reactions, and yield high-purity peptides, varying based on the peptide sequence, scale, and desired synthesis outcome. Key components include but are not limited to suitable protecting groups for the amino acids, suitable coupling agents and solvents, procedures to eliminate by-products, typically involving isolation steps after each coupling and deprotection reaction, suitable deprotection conditions and final cleavage conditions. In one aspect of the invention, the process for the preparation of a tetramer compound or a salt thereof, of the formula I wherein PROT is an ester protecting group, comprises, coupling, glycine (Gly), glutamic acid (Glu) with the y-carboxylic acid protected with

[0027] PROT, P-alanine (Ala) and 2-(3-cyano-5-fluorophenyl)-2-methyl-propionic acid of the formula II, under liquid phase peptide synthesis (LPPS) conditions. In one further aspect of the invention, the process for the preparation of a tetramer compound or a salt thereof, of formula I wherein PROT is an ester protecting group, comprises, coupling, in the direction of the peptide synthesis, glycine (Gly), glutamic acid (Glu), 0- alanine (Ala) and 2-(3-cyano-5-fluorophenyl)-2-methyl-propionic acid of the formula II, under liquid phase peptide synthesis (LPPS) conditions.

[0028] PROT preferably is tert-butyl (tBu).

[0029] In a further aspect, the invention relates to a process, wherein a) glycine is coupled with an amino group protected and activated glutamic acid of formula III

[0030] Rla-Glu(OPROT)-O-R2a(III) wherein,

[0031] Rlais an amino protecting group,

[0032] R2ais an activated ester and

[0033] PROT is an ester protecting group, in the presence of a coupling additive to form the dipeptide of the formula IV

[0034] Rla-Glu(OPROT)-Gly-OH (IV) b) the amino protecting group Rlais removed to form the dipeptide of formula V,

[0035] H-Glu(OPROT)-Gly-OH (V) c) the dipeptide of formula V is coupled to an amino group protected and activated 0- alanine of formula VI Rlb-P-Ala-O-R2b(VI) wherein,

[0036] Rlbis an amino protecting group,

[0037] R2bis an activated ester, in the presence of a coupling additive to form the tripeptide of the formula VII Rlb-P-Ala-Glu(OPROT)-Gly-OH (VII), wherein

[0038] Rlbis an amino protecting group, and

[0039] PROT is an ester protecting group, d) the amino protecting group Rlbis removed to form the tripeptide of formula VIII

[0040] H-P-Ala-Glu(OPROT)-Gly-OH (VIII), wherein PROT is an ester protecting group, e) the tripeptide of formula VIII is coupled to an activated 2-(3-cyano-5-fluorophenyl)- 2 -methyl-propionic acid of the formula Ila

[0041] Ila wherein R3is an activated ester, or, alternatively, the tripeptide of formula VIII coupled to an acid halide of 2-(3-cyano- 5-fluorophenyl)-2-methyl-propionic acid of the formula lib lib wherein X is a halogen, in the presence of a coupling additive, to form the tetramer compound of formula I.

[0042] As a general principle, the conditions for every step in the peptide synthesis, such as for the coupling, deprotection and isolation of the peptide intermediates may vary. Accordingly, amino protecting groups, ester protecting groups or coupling agents or additives can be chosen individually for each step of the synthesis. Also, the amino group protected and activated amino acids can be isolated before coupling with the next peptide fragment, or the amino group protected and activated amino acids may not be isolated and can be further reacted in solution without prior isolation.

[0043] As used herein, the term glutamic acid (Glu) means a glutamic acid wherein the y- carboxylic acid is protected with an ester protecting group PROT. In the formulae an abbreviated terminology Glu(OPROT) is applied.

[0044] Suitable amino protecting group for Glu and P-Ala are selected from benzyloxycarbonyl (Cbz), Fluorenylmethoxycarbonyl (Fmoc) or tert-butoxycarbonyl (Boc), preferably benzyloxycarbonyl (Cbz) or tert-butoxycarbonyl (Boc).

[0045] The isolated activated ester for Glu, P-Ala, and 2-(3-cyano-5-fluorophenyl)-2-methyl- propionic acid is usually the pentafluorophenyl ester (OPfp) or the A-hydroxysuccinimide (OSu) ester.

[0046] The activated ester may be selected independently for each coupling step.

[0047] These activated esters are typically prepared using a coupling agent well known to the skilled person in the art, e.g., EDCI or DSC.

[0048] Alternatively, the activated ester is not isolated, in which case it may be formed in situ using e.g. benzotriazol- l-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), l-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), A, A'-Diisopropylcarbodiimide (DIC) in combination with 2- hydroxypyridi ne-A-oxide (HOPO), O-(7-azabenzotriazol- 1 -yl )-A, A, A', A'-tetramethyluronium hexafluorophosphate (HATU), l,l’-carbonyl-di -imidazole (CDI), l,l'-carbonyl-di-( 1,2,4- triazole) (CDT), pivaloylchloride (PivCl), isobutylchloroformate (IBCF) or propanephosphonic acid anhydride (T3P). A summary of useful coupling agents is also discussed and disclosed in F. Albericio et al. Org. Process Res. Dev. 2018, 22, 7, 760-772.

[0049] The coupling additive, as used herein, usually is bis(trimethylsilyl)acetamide (BSA). BSA supports the solubilization of zwitterionic amino acids and acts through silylation in the temporary protection of the C-terminal carboxylic acid.

[0050] Thus, in one aspect, provided is a process as defined above, wherein in step a)

[0051] Rlais an amino protecting group selected from benzyloxycarbonyl (Cbz) or tertbutoxycarbonyl (Boc),

[0052] R2ais the pentafluorophenyl ester (OPfp) or the A-hydroxysuccinimide ester (OSu), in case the amino group protected and activated glutamic acid of the formula III is isolated, or

[0053] R2ais an activated ester formed in situ with a coupling agent selected from benzotriazol-l-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), l-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino- carbenium hexafluorophosphate (COMU), AA'-Diisopropylcarbodiimide (DIC) in combination with 2-hydroxypyri di ne-A-oxide (HOPO), O-(7-azabenzotriazol- l-yl)-A, A, A', A'-tetramethyluronium hexafluorophosphate (HATU), 1,1’ -carbonyldi -imidazole (CDI), l,l'-carbonyl-di-(l,2,4-triazole) (CDT), pivaloylchloride (PivCl), isobutylchloroformate (IBCF) or propanephosphonic acid anhydride (T3P) in case the amino group protected and activated glutamic acid of the formula III is not isolated,

[0054] PROT is tert, butyl and the coupling additive is (trimethyl silyl)acetamide (BSA).

[0055] In another aspect, provided is a process as defined above, wherein in step c)

[0056] Rlbis an amino protecting group selected from benzyloxycarbonyl (Cbz) or tert- butoxycarbonyl (Boc), R2bis the pentafluorophenyl ester (OPfp) or / ' / -hydroxysuccinimide ester (OSu) in case the amino group protected and activated P-alanine of formula VI is isolated, or

[0057] R2bis an activated ester formed in situ with a coupling agent selected from benzotriazol-l-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), l-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino- carbenium hexafluorophosphate (COMU), V V'-Diisopropylcarbodiimide (DIC) in combination with 2-hydroxypyridine-V-oxide (HOPO), O-(7-azabenzotriazol- l-yl)-V,V,V'V'-tetramethyluronium hexafluorophosphate (HATU), 1,1’ -carbonyldi -imidazole (CD I), l,l'-carbonyl-di-(l,2,4-triazole) (CDT), pivaloylchloride (PivCl), isobutylchloroformate (IBCF) or propanephosphonic acid anhydride (T3P) in case the amino group protected and activated P-alanine of formula VI is not isolated,

[0058] PROT is tert, butyl and the coupling additive is (trimethyl silyl)acetamide (BSA).

[0059] In another aspect, provided is a process as defined above, wherein in step e)

[0060] R3is pentafluorophenyl ester (OPfp) or V-hydroxysuccinimide ester (OSu) in case the activated 2-(3-cyano-5-fluorophenyl)-2-methyl-propionic acid of the formula Ila is isolated, or

[0061] R3is an activated ester formed in situ with a coupling agent selected from benzotriazol-l-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), l-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino- carbenium hexafluorophosphate (COMU), isobutylchloroformate (IBCF), N,N'~ diisopropylcarbodiimide (DIC) in combination with 2-hydroxypyridine-N-oxide (HOPO), pivaloylchloride (PivCl), O-(7-azabenzotriazol- l -yl)-V,V,A',V'- tetramethyluronium hexafluorophosphate (HATU) or propanephosphonic acid anhydride (T3P) in case the activated 2-(3-cyano-5-fluorophenyl)-2-methyl- propionic acid of the formula Ila is not isolated,

[0062] X is chlorine, and the coupling additive is bis(trimethylsilyl)acetamide (BSA).

[0063] The coupling in steps a), c) and e) is expediently performed in the presence of a polar aprotic solvent, selected from tetrahydrofuran (THF), 2-methyltetrahydrofuran, acetonitrile, dichloromethane, V, V-dimethylformamide or ethyl acetate at a reaction temperature from -20°C to 40°C.

[0064] The acid halide of the 2-(3-cyano-5-fluorophenyl)-2-methyl-propionic acid of the formula lib can be prepared by treating 2-(3-cyano-5-fluorophenyl)-2-methyl-propionic acid with a suitable halogenating agent such as with thionyl chloride in the presence of a tertiary amine. The subsequent coupling of the acid halide in accordance with step e) can be performed in the presence of a suitable polar aprotic solvent, such as with N-methyl pyrrolidone at a reaction temperature of -20°C to 40°C.

[0065] The isolation of the dipeptide of formula IV or of the tripeptide of formula VII from the reaction mixture can be accomplished by an aqueous wash, by separating the organic layer and concentrating.

[0066] The deprotection of the amino protecting group in step b) and d) is performed by standard methods. For the preferred Cbz protecting group, hydrogenation with hydrogen in the presence of Pd / C is the method of choice.

[0067] Isolation of the V-deprotected dipeptide of formula V or the deprotected tripeptide of formula VIII can be accomplished by catalyst removal and aqueous work-up, followed by crystallization from an organic solvent such as MTBE.

[0068] The amino group protected and activated glutamic acid of the formula III and the protected and activated P-alanine of formula VI are as a rule formed from the amino group protected precursors by activating the carboxylic acid with a suitable activating agent selected from e.g. l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N'~ diisopropylcarbodiimide (DIC), V,V'-di cyclohexylcarbodiimide (DCC), preferably with 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and by forming the ester with the respective alcohol selected from pentafluorophenol (PfpOH) or V-hydroxsuccinimide (HOSu), preferably N-hydroxsuccinimide (HOSu).

[0069] Alternatively, the OSu-ester of the activated glutamic acid of the formula III or the protected and activated P-alanine of formula VI can be formed by a reaction with the combination of N, V'-disuccinimidyl carbonate (DSC) and 4-dimethylaminopyridine (DMAP) as catalyst.

[0070] The activation reaction can typically happen in an organic solvent, selected from dichloromethane, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, or ethyl acetate at a reaction temperature between -10 °C and 30 °C. Isolation of the amino group protected and activated glutamic acid of the formula III and P-alanine of formula VI from the reaction mixture can happen by an aqueous wash, separating and drying the organic layer and by precipitating with a suitable organic solvent, such as with n-heptane. The same work-up can be applied for isolating the tetramer compound of formula I after performing step e).

[0071] In another aspect of the invention, provided is a tripeptide of the formula VII

[0072] Rlb-P-Ala-Glu(OPROT)-Gly-OH (VII) wherein, Rlbis an amino protecting group, and

[0073] PROT is an ester protecting group.

[0074] In a further aspect, provided is a tripeptide of formula VIII

[0075] H-P-Ala-Glu(OPROT)-Gly-OH (VIII) wherein PROT is an ester protecting group. In a still further aspect, provided is a tetramer compound of formula I wherein PROT is an ester protecting group, preferably tert-butyl (tBu).

[0076] In a still further aspect of the present invention, there is provided a crystalline polymorph of the tetramer compound of the formula la

[0077] which is characterized by an X-ray powder diffraction pattern having characteristic peaks expressed in angle 2-theta at approximately The crystalline polymorph of the tetramer compound of the formula la is characterized by further peaks in the X-ray powder diffraction pattern expressed in angle 2-theta at approximately In a further aspect of the present invention, the crystalline polymorph of the tetramer compound of the formula la is characterized by the IR absorption spectrum having characteristic peaks expressed in cm'1at approximately

[0078] As a further characteristic the crystalline polymorph has a melting point of 116°C to 117°C.

[0079] The present invention in a further aspect also comprises a process for the preparation of crystalline polymorph of the tetramer of the formula la.

[0080] The process comprises the steps of

[0081] • providing a solution of the tetramer compound of formula I in isopropyl acetate (iPrOAc),

[0082] • measuring the water content with the KF method in %,

[0083] • lowering the water content to KF<1.0 %, by repeated concentration under reduced pressure and elevated temperature and iPrOAc addition,

[0084] • stirring the iPrOAc solution at elevated temperature,

[0085] • dropwise adding MTBE,

[0086] • stirring the solution at elevated temperature,

[0087] • cooling the solution,

[0088] • precipitating the solid polymorph,

[0089] • filtering and drying under vacuo.

[0090] In a preferred embodiment the process comprises:

[0091] • providing a solution of the tetramer compound of formula I in isopropyl acetate (iPrOAc),

[0092] • measuring the water content with the KF method in %, • lowering the water content to < 0.5 % by repeated concentration under reduced pressure of <100 mbar, preferably < 50 mbar, and at a temperature of 30°C to 70°C, preferably of 40°C to 60°C, and addition of iPrOAc,

[0093] • stirring the iPrOAc solution at a temperature of 30°C to 70°C, preferably of 40°C to 60°C, at a concentration of about 3 V to 5V,

[0094] • dropwise adding MTBE (usually, two to four times the amount of MTBE is added to the initial iPrOAc solution),

[0095] • stirring the solution for about 1 h to 6 h at a temperature of 30°C to 70°C, preferably of 40 °C to 60 °C,

[0096] • slowly cooling the solution down to a temperature of 10°C to 30°C, preferably of 15 °C to 25 °C, within 2h to 8h, preferably within 3h to 6h,

[0097] • precipitating the solid polymorph and stirring for 1 h to 6 h at temperature of 10°C to 30°C, preferably of 15 °C to 25 °C,

[0098] • filtering and drying under vacuo.

[0099] Fig- 1 further illustrates the X-ray powder diffractogram of the polymorph of the tetramer compound of formula la obtained from the process as outlined above.

[0100] Fig- 2 shows the IR absorption spectrum of the polymorph of the tetramer compound of formula la obtained from the process as outlined above.

[0101] The crystalline tetramer compound of the formula la typically contains a very low residual solvent of <1.%.

[0102] Accordingly, the crystalline tetramer compound is favored for its use in the subsequent preparation of the peptide of formula III via SPPS.

[0103] In a further aspect of the present invention, the tetramer compound of formula I, wherein R1is hydrogen, or a salt thereof, can be used for the preparation of the peptide of formula XX, or of a pharmaceutically acceptable salt or ester thereof:

[0104] X-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15- Lys16-Ile17-Ala18-Gln19-Lys20(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala21-Phe22- Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37- Pro38-Ser39-NH2

[0105] (XX) wherein X is

[0106] AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetyl.

[0107] In a further aspect of the invention, the tetramer compound of formula I, wherein R1is hydrogen, or a salt thereof, can be used for the preparation of the peptide of formula Xia, or of a functionalized derivative thereof,

[0108] X1-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15- Lys16-Ile17-Ala18-Gln19-Lys20-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30- Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH-linker-solid support

[0109] (Xia) wherein X is

[0110] The term “functionalized derivative” in this and the following context means that the reactive sites of respective amino acids are protected with suitable protecting groups, e.g., Thr Ser, Tyr and Asp with tBu, Trp or Lys16with Boc, Gin with Trt and Lys20with Mtt or ivDde.

[0111] The tetramer compound of formula I, wherein R1is hydrogen, or a salt thereof, can in a further aspect of the present invention particularly be used for the preparation of the functionalized peptide derivative of Xllb,

[0112] X1-P-Ala2-Glu(tBu)3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10- Ser(tBu)11-Ile12-Aib13-Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys20(Mtt / ivDde)- Ala21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser(tBu)32- Ser(tBu)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(tBu)39-NH-linker-solid support

[0113] (Xllb), wherein X is as above.

[0114] In a still further aspect of the invention, there is provided a process for the preparation of the peptide of formula XX, or of a pharmaceutically acceptable salt or ester thereof

[0115] X-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15- Lys16-Ile17-Ala18-Gln19-Lys20(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala21-Phe22- Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37- Pro38-Ser39-NH2

[0116] (XX) wherein X is

[0117] AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetyl. comprising the steps a) solid phase synthesis of the peptide of formula XX on a resin, comprising coupling the tetramer compound of formula I to a functionalized peptide fragment of formula Xia

[0118] H-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18- Gln19-Lys20-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33- Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH-linker-solid support

[0119] (Xia) and forming the functionalized peptide fragment of formula Xlla

[0120] X1-p-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-He12-Aib13-Leu14-Asp15- Lys16-Ile17-Ala18-Gln19-Lys20-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30- Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH-linker-solid support

[0121] (Xlla) b) deprotection of the Lys20amino protecting group, c) coupling the deprotected amino group of Lys20with the moiety

[0122] PROT-O-20-oxoicosanoyl-L-Glu (AEEAc-AEEAc-OH)-O-PROT

[0123] (Xllla), wherein AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetyl and PROT is an ester protecting group, and forming the peptide of formula XX, bound to the resin; d) cleavage of the peptide of formula XX from the resin and global deprotection with an acid and e) precipitation of the peptide formula XX and optionally f) purification / isolation.

[0124] Whereas the term “functionalized derivative” has the meaning as outlined above, Lys20is ideally functionalized with an ivDde (l-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)-3- methylbutyl) or a MTT (4-methyltrityl) protecting group. Preferred functionalization of Lys20is Mtt.

[0125] Thus, in one aspect, the protecting group is MTT and the functionalized peptide derivative of formula Xllb has the formula Xllbi

[0126] X1-P-Ala2-Glu(tBu)3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10- Ser(tBu)11-Ile12-Aib13-Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys20(Mtt)-Ala21- Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser(tBu)32-Ser(tBu)33- Gly34-Ala35-Pro36-Pro37-Pro38-Ser(tBu)39-NH-linker-solid support

[0127] (Xllbi), wherein X is as above.

[0128] In another aspect, the protecting group is ivDde and the functionalized peptide derivative of formula Xllb has the formula Xllbii

[0129] X1-P-Ala2-Glu(tBu)3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10- Ser(tBu)11-Ile12-Aib13-Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys20(ivDde)- Ala21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser(tBu)32- Ser(tBu)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(tBu)39-NH-linker-solid support

[0130] (Xllbii), wherein X is as above.

[0131] In a preferred aspect of the invention the functionalized peptide derivative of Xllb has the formula Xllbi, comprising Lys20functionalized with Mtt.

[0132] In a further particular aspect of the present invention, the process comprises the steps a) solid phase synthesis of the peptide of formula XX on a resin, comprising coupling the tetramer compound of formula I to a functionalized peptide fragment of formula Xlb

[0133] H-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10-Ser(tBu)11-Ile12-Aib13- Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys20(Mtt / ivDde)-Ala21-Phe22-Val23- Gln(Trt)24-Trp(Boc)25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser(tBu)32-Ser(tBu)33-Gly34-Ala35- Pro36-Pro37-Pro38-Ser(tBu)39-NH-linker-solid support

[0134] (Xlb) and forming the functionalized peptide fragment of formula Xllb

[0135] X1-P-Ala2-Glu3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10- Ser(tBu)11-Ile12-Aib13-Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys20(ivDde)- Ala21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser(tBu)32- Ser(tBu)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(tBu)39-NH-linker-solid support

[0136] (Xllb) b) deprotection of the Lys20s-amino protecting group Mtt or ivDde, c) acylation of the s-amino group of Lys20with the moiety tBu-O-20-oxoicosanoyl-L-Glu(AEEAc-AEEAc-OH)-OtBu

[0137] (Xlllb), wherein AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetyl, and forming the peptide of formula XX, bound to the resin; d) cleavage of the peptide of formula XX from the resin and global deprotection with an acid and e) precipitation of the peptide formula XX and optionally f) purification / isolation.

[0138] In a further particular aspect of the present invention Lys20in the peptide fragments of formula Xlb and Xllb are functionalized with Mtt.

[0139] Step a)

[0140] Step a) as outlined above requires the solid phase synthesis of the peptide of formula XX on a resin, comprising coupling the tetramer of formula I to the functionalized peptide fragment of formula Xia or Xlb and forming the functionalized peptide fragment of formula Xlla or XIIb.

[0141] The general SPPS method is well known in the art and is described for instance in W.C. Chan and P.D. White, Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press Inc., 1999.

[0142] However, the reaction conditions of the SPPS must be adapted for every peptide. For the solid phase synthesis of the peptide fragment of formula Va or Vb the following key conditions and reagents were found to be suitable.

[0143] The Sieber amide resin with Fmoc protection was found to be well suited for this SPPS.

[0144] Fmoc was found to be the amino protecting group of choice and for its deprotection a common 20% v / v piperidine solution in DMF has been applied.

[0145] For the coupling, a combination of the coupling agents DIC and OxymaPure (ethyl 2- cyano-2-(hydroxyimino)acetate) or the combination PyBOP / collidine was found to be favorable for its properties to reduce epimerization during activation / coupling.

[0146] Step b)

[0147] In step b) the Lys20amino protecting group is removed. For the deprotection of the ivDde (l-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)-3- m ethylbutyl) protecting group a 2 to 10% v / v solution of hydrazine monohydrate in DMF can be applied.

[0148] For the deprotection of the Mtt (4-methyltrityl) protecting group a 1% v / v TFA solution in DCM or alternatively a mixture of hexafluoroisopropanol (HFIP) and scavengers like trisopropylsilane (TIPS), di thioerythrol (DTT) or A -acetyl cy stein (NAC) in an organic solvent like DCM or toluene can be used. Preferably, the MTT deprotection is conducted with a mixture of hexafluoroisopropanol (HFIP) with the scavenger trisopropylsilane (TIPS) in toluene.

[0149] Step c

[0150] In step c) the s-amino group of Lys20is acylated with the moiety of formula Xllla or Xlllb

[0151] PROT-O-20-oxoicosanoyl-L-Glu(AEEAc-AEEAc-OH)-O-PROT

[0152] (Xllla) or tBu-O-20-oxoicosanoyl-L-Glu(AEEAc-AEEAc-OH)-OtBu

[0153] (Xlllb) wherein AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetyl and PROT is an ester protecting groups, whereby the peptide of formula XX, bound to the resin, is formed.

[0154] The coupling conditions are essentially the same as described for step a).

[0155] Step d)

[0156] In step d) the peptide of formula XX, bound to the resin, is subjected to cleavage from the resin and to global deprotection, whereby the desired peptide of formula XX is obtained.

[0157] Cleavage can be accomplished with trifluoroacetic acid. The cleavage cocktail may include water, DTT (dithiothreitol) and TIS (triisopropylsilane) as scavengers.

[0158] Step e) Precipitation peptide of formula XX precipitated in step e) in a suitable organic solvent such as methyl tert-butyl ether and dried.

[0159] Optionally a further purification step f) may be added.

[0160] The term (9-20-oxoicosanoyl refers to the moiety

[0161] An alternative term for this moiety is 19-carboxy-nonadecanoyl.

[0162] EXAMPLES

[0163] Abbreviations:

[0164] BSA bis(trimethylsilyl)acetamide

[0165] Cbz b enzy 1 oxy carb ony 1

[0166] DCM di chloromethane

[0167] DIC AA'-diisopropylcarbodiimide

[0168] DIPEA diisopropylethylamine

[0169] DMF A,A-di methyl form am ide

[0170] DMSO dimethylsulfoxide

[0171] DSC A,A'-Disuccinimidyl carbonate

[0172] DTT dithiothreitol

[0173] EDC l-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0174] EtOAc ethyl acetate

[0175] Fmoc 9-fluorenylmethoxycarbonyl

[0176] HOSu A-hydroxy succinimide

[0177] HPLC high pressure liquid chromatography iPrOAc / .w-propyl acetate ivDde 1 -(4, 4-dimethyl-2,6-di oxocyclohex- 1 -ylidene)-3 - methylbutyl

[0178] KF Water content measured in % by the Karl Fischer (KF) method

[0179] NMP A-methylpyrrolidine

[0180] MeOH methanol

[0181] MTBE / methyl tert. -butyl ether TMBE

[0182] OPfp O-pentafluorophenyl OxymaPure ethyl 2-cyano-2-(hydroxyimino)acetate

[0183] PfpOH pentafluorophenol

[0184] PyBOP benzotriazol- 1 -yloxytripyrrolidinophosphonium hexafluorophosphate rt room temperature tBu tert, -butyl

[0185] TFA trifluoroacetic acid

[0186] THF tetrahydrofuran

[0187] TIS triisopropylsilane

[0188] LPPS experimental procedure for the preparation of the X-B-Ala-Glu(OtBu)-Gly tetramer Example 1

[0189] X-p-Ala-Glu(OtBu)-Gly-OH (X=2-(3-cyano-5-fluorophenyl)-2-methylpropanoyl-);

[0190] 2-[[(2S)-2-[3-[[2-(3-cyano-5-fluorophenyl)-2-methylpropanoyl]amino]propanoylamino]-5- [(2-methylpropan-2-yl)oxy]-5-oxopentanoyl]amino]acetic acid

[0191] Step 1: 2

[0192] Step la: Cbz-Glu(OtBu)-OPfp

[0193] Dichloromethane (1000 mL) is added to Cbz-Glu(tBu)-OH (100 g) and pentafluorophenol (PfpOH) (1.2 equiv). The mixture is cooled to 0°C and EDCI (1.2 equiv) is added portionwise. After the end of the addition, the mixture is allowed to warm up to room temperature and stirred until judged complete by HPLC (~3 h). The mixture is washed sequentially with 5% aq. H3PO4 (3 x 500 mL) and brine (500 mL). The organic layer is dried over ISfeSCU and filtered. Following solvent swap to n-heptane, Cbz-Glu(OtBu)-OPfp is isolated as a solid after filtration and drying (134 g).

[0194] 1H NMR (400 MHz, DMSO-d6) 5 8.16 (d, J = 7.3 Hz, 1H), 7.48 - 7.21 (m, 5H), 5.10 (s, 2H), 4.60 - 4.50 (m, 1H), 2.49 - 2.38 (m, 2H), 2.22 - 2.12 (m, 1H), 2.07 - 1.92 (m, 1H), 1.41 (s, 9H).

[0195] Step lb: Cbz-Glu(OtBu)-Gly-OH

[0196] Glycine (21.3 g) is dissolved in dry THF (105 mL) and BSA (1.5 equiv) is added at 10 - 20°C. After stirring for 1 h at room temperature, the mixture is cooled to 0°C and a solution of Cbz-Glu(OtBu)-OPfp (130 g) in THF (105 mL) is added dropwise. Stir at room temperature until judged complete by HPLC (~15 h).

[0197] Water (80 mL) and EtOAc (80mL) are added. After stirring for 15 minutes, the layers are separated, and the organic layer is sequentially washed with 5% aq. H3PO4 (3 x 100 mL) and brine (100 mL). The organic layer is dried over Na2SO4, filtered, and concentrated to dryness. 97.6 g of Cbz-Glu(OtBu)-Gly-OH are obtained.

[0198] 1H NMR (400 MHz, DMSO-d6) 5 8.20 (t, J = 5.9 Hz, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.42 - 7.28 (m, 5H), 5.11 - 4.98 (m, 2H), 4.10 - 4.02 (m, 1H), 3.87 - 3.72 (m, 2H), 2.33 - 2.23 (m, 2H), 1.97 - 1.85 (m, 1H), 1.81 - 1.68 (m, 2H), 1.39 (s, 9H).

[0199] MS (ESI) calculated for C19H27N2O7 [M+H]+, 395.17; found, 395.16.

[0200] Step 2: H-Glu(OtBu)-Gly-OH

[0201] Cbz-Glu(OtBu)-Gly-OH (92 g), Pd / C (0.2 g / g) and THF (920 mL) were mixed in an autoclave and reacted at room temperature under 0.5 MPa H2 atmosphere until judged complete by HPLC (~3 h).

[0202] The mixture was filtered, and the filter cake was washed with water (180 mL). The aqueous solution was concentrated several times with toluene / MeOH 1 : 1 (920 mL), whereupon a solid precipitated. MTBE (5 x 460 mL) was added, and the suspension was concentrated. Upon completion of the concentration, MTBE (920 mL) was added, and the resulting slurry was stirred for 30 minutes. H-Glu(OtBu)-Gly-OH was obtained by filtration followed by drying under reduced pressure.

[0203] 1H NMR (400 MHz, D2O) 5 3.99 (t, J = 6.6 Hz, 1H), 3.93 (d, J = 1.0 Hz, 2H), 2.42 - 2.34 (m, 2H), 2.08 - 1.96 (m 2H), 1.32 (s, 9H).

[0204] MS (ESI) calculated for C11H21N2O5 [M+H]+, 261.15; found, 261.09.

[0205] Step 3:

[0206] Step 3a: Cbz-p-Ala-OPfp

[0207] Dichloromethane (600 mL) is added to Cbz-P-Ala-OH (60.0 g) and pentafluorophenol (PfpOH) (1.2 equiv). The mixture is cooled to 0°C and EDCI (1.2 equiv) is added portionwise. After the end of the addition, the mixture is allowed to warm up to room temperature and stirred until judged complete by HPLC (~3 h).

[0208] The mixture is washed sequentially with 5% aq. H3PO4 (3 x 300 mL) and brine (300 mL). The organic layer is dried over Na2SO4 and filtered. Following solvent swap to n-heptane, Cbz-P-Ala-OPfp is isolated as a solid by filtration and drying (101 g).

[0209] 1H NMR (400 MHz, DMSO-d6) 5 7.51 (t, J = 5.6 Hz, 1H), 7.48 - 7.17 (m, 5H), 5.05 (s, 2H), 3.41 (q, J = 6.4 Hz, 2H), 2.98 (t, J = 6.6 Hz, 2H).

[0210] MS (ESI) calculated for C17H13F5NO4 [M+H]+, 390.08; found, 389.7.

[0211] Step 3b: Cbz-p-Ala-Glu(OtBu)-Gly-OH

[0212] H-Glu(OtBu)-Gly-OH (21.3 g) is dissolved in dry THF (150 mL) and BSA (1.5 equiv) is added at 10 - 20°C. After stirring for 1 h at room temperature, the mixture is cooled to 0°C and a solution of Cbz-P-Ala-OPfp (1.0 equiv, 30 g) in THF (105 mL) is added dropwise. Stir at room temperature until judged complete by HPLC (~2 h).

[0213] Water (120 mL) and EtOAc (120 mL) are added. After stirring for 15 minutes, the layers are separated, and the organic layer is sequentially washed with 5% aq. H3PO4 (2 x 150 mL) and brine (150 mL). The organic layer is dried over Na2SO4, filtered, and concentrated to dryness. 19.1 g of Cbz-P-Ala-Glu(OtBu)-Gly-OH are obtained.

[0214] 1H NMR (400 MHz, DMSO-d6) 5 8.20 (t, J = 5.9 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.42 - 7.28 (m, 5H), 7.19 (t, J = 5.7 Hz, 1H), 5.02 (s, 2H), 4.38 - 4.28 (m, 1H), 3.80 - 3.73 (m, 2H), 3.25 (q, J = 6.8 Hz, 2H), 2.37 (t, J = 7.2 Hz, 2H), 2.28 (t, J = 8.0 Hz, 2H), 1.97 - 1.85 (m, 1H), 1.78 - 1.66 (m, 1H), 1.39 (s, 9H).

[0215] MS (ESI) calculated for C22H32N3O8 [M+H]+, 466.22; found, 466.0.

[0216] Step 4: H-p-Ala-Glu(OtBu)-Gly-OH

[0217] Cbz-P-Ala-Glu(OtBu)-Gly-OH (29 g), Pd / C (0.2 g / g), THF (260 mL) and water (29 mL) were mixed in an autoclave and reacted at room temperature under 0.5 MPa H2 atmosphere until judged complete by HPLC (~3 h).

[0218] The mixture was filtered, and the filter cake was washed with water (60 mL). THF was removed by concentration under reduced pressure, and the residue was washed with MTBE (3 x 30 mL). The aqueous solution was concentrated several times with toluene / MeOH 1 : 1 (10 V), whereupon a solid precipitated. Following solvent swap to methanol, MTBE was added, leading to the precipitation. After filtration and drying, 19.1 g of H-P-Ala-Glu(OtBu)-Gly-OH are obtained.

[0219] 1H NMR (400 MHz, D2O) 5 4.28 (dd, J = 8.8, 5.6 Hz, 1H), 3.89 (d, J = 1.9 Hz, 2H), 3.17 (t, J = 6.7 Hz, 2H), 2.65 (t, J = 6.7 Hz, 2H), 2.37 - 2.23 (m, 2H), 2.05 - 1.97 (m, 1H), 1.90 - 1.75 (m, 1H), 1.33 (s, 9H).

[0220] MS (ESI) calculated for C14H26N3O6 [M+H]+, 332.18; found, 332.43. Step 5:

[0221] Step 5a: (2,5-dioxopyrrolidin-l-yl) 2-(3-cyano-5-fluorophenyl)-2-methylpropanoate

[0222] Dichloromethane (100 mL) is added to 2-(3-cyano-5-fluorophenyl)-2-methylpropanoic acid (10 g) and N-hydroxysuccinimide (HOSu) (1.2 equiv). The mixture is cooled to 0°C and EDCI (1.2 equiv) is added portionwise. After the end of the addition, the mixture is allowed to warm up to room temperature and stirred until judged complete by HPLC (~2 h).

[0223] The mixture is washed sequentially with 5% aq. H3PO4 (3 x 50 mL) and brine (50 mL). The organic layer is dried over Na2SO4 and filtered. Following solvent swap to n-heptane, (2,5- dioxopyrrolidin-l-yl) 2-(3-cyano-5-fluorophenyl)-2-methylpropanoate is isolated as a solid by filtration and drying (6.8 g).

[0224] 1H NMR (400 MHz, DMSO-d6) 5 7.87 - 7.81 (m, 2H), 7.73 (dt, J = 10.4, 2.2 Hz, 1H), 2.90 - 2.70 (m, 4H), 1.70 (s, 6H).

[0225] Step 5b: X-p-Ala-Glu(OtBu)-Gly-OH (X=2-(3-cyano-5-fluorophenyl)-2-methylpropanoyl-)

[0226] H-P-Ala-Glu(OtBu)-Gly-OH (5.5 g, 1.2 equiv) is dissolved in dry THF (50 mL) and BSA (2.0 equiv) is added at room temperature. A solution of (2,5-dioxopyrrolidin-l-yl) 2-(3-cyano-5- fluorophenyl)-2-methylpropanoate (5 g. 1.0 equiv) in THF (25 mL) is added dropwise. Stir at room temperature until judged complete by HPLC (~3 h).

[0227] Water (30 mL) is added, and the mixture is concentrated under reduced pressure to remove most of the THF. EtOAc (50 mL) is added. After stirring for 15 minutes, the layers are separated, and the organic layer is sequentially washed with 5% aq. H3PO4 (2 x 25 mL) and brine (25 mL). The organic layer is dried over Na2SO4, filtered, and concentrated to a residual volume of 15 mL. The mixture was added dropwise to n-heptane (150 mL) at -10°C and stirred for 3 h after the end of the addition. The suspension is filtered and the solid is dried under reduced pressure. 6.8 g X-P-Ala-Glu(OtBu)-Gly-OH are obtained as amorphous powder.

[0228] 1H NMR (400 MHz, DMSO-d6) 5 12.54 (s, 1H), 8.18 (t, J = 5.9 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1H), 7.72 - 7.67 (m, 1H), 7.62 - 7.58 (m, 1H), 7.53 (t, J = 5.6 Hz, 1H), 7.49 - 7.44 (m, 1H), 4.34 - 4.25 (m, 1H), 3.77 - 3.70 (m, 2H), 3.27 (q, J = 6.7 Hz, 2H), 2.31 (t, J = 7.0 Hz, 2H), 2.25 (t, J = 8.1 Hz, 2H), 1.96 - 1.85 (m, 1H), 1.78 - 1.66 (m, 1H), 1.47 (s, 6H), 1.39 (s, 9H).

[0229] MS (ESI) calculated for C25H34FN4O7 [M+H]+, 521.24; found, 521.18.

[0230] Example 2

[0231] X-p-Ala-Glu(OtBu)-Gly-OH (X=2-(3-cyano-5-fluorophenyl)-2-methylpropanoyl-); 2-[[(2S)-2-[3-[[2-(3-cyano-5-fluorophenyl)-2-methylpropanoyl]amino]propanoylarnino]-5- [(2-methylpropan-2-yl)oxy]-5-oxopentanoyl]amino]acetic acid

[0232] Step la: Cbz-Glu(OtBu)-OSu

[0233] 0DMAP / DSC0

[0234] CbzHN^ g n _ _ CbzHN^AQgy Step 1a

[0235] CO2tBu CO2tBu

[0236] Cbz-Glu(OtBu)-OH Cbz-Glu(OtBu)-OSu

[0237] A solution of Cbz-Glu(OtBu)-OH (500 g, 1.48 mol, 1.00 equiv.) and DMAP (0.1 equiv.) in 2- MeTHF (3 V) and MeCN (0.2 V) is maintained at 20-30 °C. N,N'-Disuccinimidyl carbonate (DSC) (1.3 equiv.) is added in three portions. The resulting mixture is stirred at 20-30 °C until the starting Cbz-Glu(OtBu)-OH material is <1.0% as determined by derivation with benzylamine. The reaction mixture is filtered at 20-30 °C to remove the solid, and the solid is rinsed with 2-Me-THF (0.5 V). The filtrate is charged back to the reactor. ^-Heptane (2 V) is added dropwise to the reactor at 20-30 °C and stirred for 1-2 h. An additional ^-heptane (8 V) is subsequently added, and the mixture is stirred for 1-2 h. The resulting solid is filtered, rinsed with n-heptane (2 V), and dried with nitrogen at room temperature. Cbz-Glu(OtBu)- OSu is isolated as white solid (774 g, 91.4% yield, 97.8% a / a, 75.9% w / w).

[0238] 1H NMR (400 MHz, DMSO-d6) 5 8.11 (d, J= 7.9 Hz, 1H), 7.42 - 7.29 (m, 5H), 5.15 - 5.00 (m, 2H), 4.60 - 4.48 (m, 2H), 2.82 (s, 4H), 2.48 - 2.38 (m, 2H), 2.16 - 2.04 (m, 1H), 2.00 - 1.88 (m, 1H), 1.40 (s, 9H).

[0239] MS (ESI) calculated for C21H27N2O8 [M+H]+, 435.18; found, 435.42. Step lb: Cbz-Glu(OtBu)-Gly-OH

[0240] Step 1b

[0241] H-Gly-OH Cbz-Glu(OtBu)-Gly-OH

[0242] A suspension of glycine (57.0, 1.1 equiv.) and N,O-Bis(trimethylsilyl)acetamide (BSA) (2.5 equiv.) in DMF (5 V) is held in Reactor 1 at 10-20 °C and stirred for 2 hours. In Reactor 2, Cbz-Glu(OtBu)-OSu (300 g assay corrected, 0.69 mol, 1.0 equiv.) is added to DMF (2 V). This mixture is cooled to 0-10 °C and stirred until the solid is completely dissolved. The solution from Reactor 2 is added to Reactor 1 over the course of 1 h, maintaining the temperature at 10-20 °C. The resulting mixture is allowed to react until the remaining Cbz- Glu(OtBu)-OSu was <0.5%. zPrOAc (5 V) and 10% NaCl solution (6 V) are added to Reactor 1 at 15 °C and stirred for about 30 minutes. The phases are separated, and the aqueous layer is extracted with zPrOAc (5 V). The organic phases are combined and washed with 10% NaCl solution (5 V). The combined organic phase is then concentrated under reduced pressure to yield a solution of Cbz-Glu(OtBu)-Gly-OH (1.51 kg, 96.4 yield, 17.4% w / w, 99.2% a / a) that is telescoped to the next step directly.

[0243] Step 2: H-Glu(OtBu)-Gly-OH

[0244] O CbzHN^H ^ OH H2, Pd-C O

[0245] CO2tBu Step 2

[0246] H-Glu(OtBu)-Gly-OH

[0247] The zPrOAc solution of Cbz-Glu(OtBu)-Gly-OH (containing 200 g assay corrected, 0.51 mol, 1.0 equiv.) is charged to the reactor and maintained at 25 °C. 5 w / w% Pd / C (10% content) and water (2 V) are added to the reactor, maintaining the temperature at 25 °C. The reactor is evacuated and purged with hydrogen three times. The mixture is allowed to react under hydrogen at 20-30 °C until the conversion is judged complete. The mixture is filtered at 25 °C, and the solid is rinsed with water (0.5 V). The filtrate and rinse solution are combined. Acetonitrile (30 V) is added dropwise to the combined aqueous solution at 25 °C and stirred for about 2 hours. The resulting solid is filtered, rinsed with acetonitrile (2 V), and dried with nitrogen at room temperature. H-Glu(OtBu)-Gly-OH is obtained as white powder (118.6 g, 89.2% yield, 99.4% a / a, 99.2% w / w).

[0248] Step 1 / 2 (telescoped procedure)

[0249] A solution of Cbz-Glu(OtBu)-OH (500 g, 1.48 mol, 1.00 equiv.) and DMAP (0.1 equiv.) in THF (3 V) is maintained at 20-30 °C. N,N'-Disuccinimidyl carbonate (1.05 equiv.) is added in three portions. The resulting mixture is stirred at 20-30 °C until the starting Cbz-Glu(OtBu)- OH material is <1.0% as determined by derivation with benzylamine. The mixture is filtered at 20-30 °C to remove the solid, and the solid is rinsed with THF (0.5 V).

[0250] In a second reactor, a suspension of glycine (122.3 g, 1.1 equiv.) and N,O- Bis(trimethylsilyl)acetamide (BSA) (3.0 equiv.) in DMF (5 V) is held in Reactor 1 at 10- 20 °C and stirred for 2 hours. A solution of Cbz-Glu(OtBu)-OSu is added over the course of 1 h, maintaining the temperature at 10-20 °C. The resulting mixture is allowed to react until the remaining raw material (OSu-ester) was <0.5%. zPrOAc (5 V) and 10% NaCl solution (6 V) are added to Reactor 1 at 15 °C and stirred for about 30 minutes. The phases are separated, and the aqueous layer is extracted with zPrOAc (5 V). The organic phases are combined and washed with 10% NaCl solution (5 V). The combined organic phase is then concentrated under reduced pressure to yield a solution of Cbz-Glu(OtBu)-Gly-OH (2.13 kg, 92.7% yield, 25.4% w / w, 98.3% a / a) that is telescoped to the next step directly.

[0251] A part of the zPrOAc solution of Cbz-Glu(OtBu)-Gly-OH (1.31 kg, 25.4% w / w, containing 332.7 g assay corrected, 0.84 mol, 1.0 equiv.) is charged to the reactor and maintained at 25 °C. 5 w / w% Pd / C (10% content) and water (2 V) are added to the reactor, maintaining the temperature at 25 °C. The reactor is evacuated and purged with hydrogen three times. The mixture is allowed to react under hydrogen at 20-30 °C until the conversion is judged complete. The mixture is filtered at 25 °C, and the solid is rinsed with water (0.5 V). The filtrate and rinse solution are combined. Acetonitrile (30 V) is added dropwise to the combined aqueous solution at 25 °C and stirred for about 2 hours. The resulting solid is filtered, rinsed with acetonitrile (2 V), and dried with nitrogen at room temperature. H- Glu(OtBu)-Gly-OH is obtained as white powder (201.2 g, 91.2% yield, 99.5% a / a, 99.5% w / w). Step 3a: Cbz-p-Ala-OSu

[0252] DMAP / DSC s ep a

[0253] Cbz-P-Ala-OH (10 g, 44.8 mmol, 1.0 equiv.) and DMAP (0.1 equiv.) are charged to the reactor along with THF (3 V), maintaining the temperature at 20-30 °C. N,N'-Disuccinimidyl carbonate (1.05 equiv.) is added portion wise. The mixture is stirred and allowed to react until the starting raw material (Cbz-P-Ala-OH) is less than 0.5%. The reaction mixture containing Cbz-P-Ala-OSu is telescoped into step 3b without isolation. A yield of 100% is assumed.

[0254] 1H NMR (400 MHz, DMSO-d6) 5 7.47 (t, J = 5.7 Hz, 1H), 7.41 - 7.29 (m, 5H), 5.05 (s, 2H), 3.39 - 3.31 (m, 2H), 2.91 - 2.85 (m, 2H), 2.82 (s, 4H).

[0255] MS (ESI) calculated for C15H17N2O6 [M+H]+, 321.11; found, 321.36.

[0256] Step 3b: Cbz-p-Ala-Glu(OtBu)-Gly-OH

[0257] Step 3b

[0258] H-Glu(OtBu)-Gly-OH (12.83 g Assay-corrected, 0.049 mol, 1.0 eq), N,O- Bis(trimethylsilyl)acetamide (2.5 eq) and THF (3 V) are charged to the reactor, and the mixture is stirred for 1-3 hours at 25 °C. To this, the OSu-ester mixture from Step 3a is added. The reaction is monitored until conversion is judged completed. zPrOAc (5 V) and 10% NaCl (6 V) solution are added to the reactor and stirred for approximately 30 minutes. The phases are separated, and the organic phase is subsequently washed with 10% NaCl solution (6 V). The organic phase is concentrated to approximately 3 V of a solution containing Cbz-P-Ala-Glu(OtBu)-Gly-OH (49.2 g, 93.0% yield, 39.4% w / w, 99.0% a / a) that is telescoped to the next synthetic step.

[0259] Step 4: H-p-Ala-Glu(OtBu)-Gly-OH The zPrOAc solution of Cbz- P -Ala-Glu(OtBu)-Gly-OH (20.0 g Assay-corrected, containing 1.0 equiv.) is charged to the reactor and maintained at 20-30 °C. 5 w / w% Pd / C (10% content) and water (2 V) are added to the reactor, maintaining the temperature at 20-30 °C. The reactor is evacuated and purged three times with hydrogen. The mixture is allowed to react under hydrogen at 20-30 °C until the conversion is judged complete. The mixture is filtered at 20- 30 °C, and the solid is rinsed with water (0.5 V). The mixture is separated, and the aqueous solution, which contains the desired product, is collected. Acetonitrile (30 V) is added to the reactor, then add the aqueous solution dropwise to the reactor and stirred for about 2 hours. The resulting solid is filtered, rinsed with acetonitrile (2 V), and dried with nitrogen at room temperature. H- P -Ala-Glu(OtBu)-Gly-OH is obtained as white powder (12.8 g, 88.9% yield, 99.1% w / w, 99.5% a / a).

[0260] Step 5a: (X-OSu) (X= 2-(3-cyano-5-fluorophenyl)-2-methylpropanoyl) EDCI, HOSu OSu

[0261] Step 5a l J O CN

[0262] 2-(3-cyano-5-fluorophenyl)-2-methylpropanoic acid (1.15 kg, 5.55 mol, 1.0 equiv.) and N- Hydroxysuccinimide (1.2 equiv.) are charged to the reactor along with MeCN (8 V) and cooled to -10 °C. EDCI (1.2 equiv.) is added portionwise. The mixture is stirred at -10 to 0 °C until the conversion is judged completed. zPrOAc (5 V), 5% phosphoric acid aqueous solution (3 V), and brine (4 V) are added to the reactor at -10 to 0 °C and stirred for 1 h. The phases are separated. 5% phosphoric acid aqueous solution (3 V) and brine (4 V) are added to the organic phase, stirred for 1 h, and this operation is repeated once. Brine (5 V) is added to the reactor at 15 to 25 °C, stirred for 1 h, and the phases are separated to collect the organic phase. The organic phase is concentrated to 5 V. iPrOAc (5 V) is added, and the solution is concentrated again to 5 V. n-Heptane (10 V) is then added. The mixture is stirred at 20 °C for 1 h. The resulting solid is filtered, rinsed with / / -heptane (1 V), and dried to obtain l-(4- cyano-3-fluorophenyl)-l-methyl-l-[(2,5-dioxopyrrolidin-l-yl)oxycarbonyl]ethyl-isobutyrate (OSu) as solid (1.52 kg, 90.1% yield, 99.8% w / w, 99.7% a / a).

[0263] Step 5b 1: X-p-Ala-Glu(OtBu)-Gly-OH (X=(2,5-dioxopyrrolidin-l-yl) 2-(3-cyano-5- fluorophenyl)-2-methylpropanoyl-)

[0264] Step 5b X1-p-Ala-Glu(OtBu)-Gly-OH

[0265] The reactor is charged with 10 V of THF, l-(4-cyano-3-fhiorophenyl)-l-methyl-l-[(2,5- dioxopyrrolidin-l-yl)oxycarbonyl]ethyl-isobutyrate (OSu) (1.25 kg, 4.1 mol, 1.0 equiv.) and H-P-Ala-Glu(OtBu)-Gly-OH (Trimer) (1.63 kg, 4.93 mol, 1.2 eq), maintaining the temperature at 15-25 °C. The mixture is stirred for 30 minutes. The mixture is then cooled to -5°C to 5 °C. Keeping the temperature at -5 to 5 °C, N,O-Bis(trimethylsilyl)acetamide (2.0 equiv.) is added to the reactor dropwise. The reaction is allowed to proceed until the raw material 1 -(4-cyano-3 -fluorophenyl)- 1 -methyl- 1 -[(2, 5-dioxopyrrolidin- 1 - yl)oxycarbonyl]ethyl-isobutyrate is less than 0.1%. zPrOAc (10 V), 10% phosphoric acid aqueous solution (6 V) and brine (6 V) are added to the reactor and stirred for 30 min. The phases are separated. 10% HiPCU-solution (6 V) and brine (6 V) are added to the organic phase, stirred for 30 min, and this wash operation is repeated twice. The temperature is maintained at 15 to 25 °C. Water (10 V) is added to the reactor, stirred for 30 min, and the phases are separated to collect the organic phase. The organic phase is concentrated to 3 V. zPrOAc (10 V) is added, and the mixture is concentrated again to 3 V. This concentration / addition operation is repeated until the KF<0.1%. The solution is added into a mixture of zz-Heptane (27 V) and MTBE (7 V) at 0 °C The resulting suspension is stirred at 0 °C for 1 hour. The solid is filtered, rinsed with zz-heptane (2 V), and dried to yield amorphous tetramer X-P-Ala-Glu(OtBu)-Gly-OH (2.35 kg, 92.2% yield, 85.9% w / w, 99.3%a / a)

[0266] Step 5b 2: X-p-Ala-Glu(OtBu)-Gly-OH (X=(2,5-dioxopyrrolidin-l-yl) 2-(3-cyano-5- fluorophenyl)-2-methylpropanoyl-)

[0267] The reactor is charged with THF (10 vol), l-(4-cyano-3-fluorophenyl)-l-methyl-l-[(2,5- dioxopyrrolidin-l-yl)oxycarbonyl]ethyl-isobutyrate (30.0 g, 98.7 mmol, 1.0 equiv.) and H-P- Ala-Glu(OtBu)-Gly-OH (Trimer) (39.2 g, 118.4 mmol, 1.2 equiv.), maintaining the temperature at 15 to 25 °C. The mixture is stirred for 30 minutes. The mixture is then cooled to -5 °C to 5 °C. Keeping the temperature at -5 °C to 5 °C, N,O-Bis(trimethylsilyl)acetamide (2.0 equiv.) is added to the reactor dropwise. The reaction is allowed to proceed until the raw material is less than 0.1%. zPrOAc (10 vol), 10% phosphoric acid aqueous solution (6 vol) and brine (6 vol) are added to the reactor and stirred for 30 min. The phases are separated. 10% HaPCU-solution (6 vol) and brine (6 vol) are added to the organic phase, stirred for 30 min, and this wash operation is repeated twice. The temperature is maintained at 15-25 °C. Water (10 vol) is added to the reactor, stirred for 30 min, and the phases are separated to collect the organic phase. The organic phase is concentrated to 5 V. zPrOAc (10 vol) is added, and the mixture is concentrated under reduced pressure (<20 mmHg) at 45-55°C to 3 V. This concentration / addition operation is repeated until the KF value is <0.5%. The resulting iPrOAc solution (approx. 3 V) was stirred at 50 °C for 1 h, then MTBE (15 vol) was added dropwise at 45-55 °C within 2 h. The mixture was stirred at 45-55 °C (Target 50 °C) for another Ih, the reaction mixture then was slowly cooled down to 15-25 °C (target 20 °C) within 4 h and aged for another 2 h at 20 °C. The solid is filtered at 20 °C, rinsed with MTBE (2 V), and dried under reduced pressure at 15-25 °C to yield the crystalline tetramer X P -Ala- Glu(OtBu)-Gly-OH (45.8 g, 89.1% yield, 100.5% w / w, 99.68% a / a).

[0268] The X-Ray Powder Diffractogram is shown in Fig. 1, and the IR absorption spectrum is shown in Fig. 2.

[0269] The table below illustrates that the crystalline tetramer obtained in Step 5b2 contains a very low content of solvent residue and accordingly a high assay value compared to the amorphous tetramer obtained in Step 5b 1.

[0270] Example 3

[0271] H-p-Ala-Glu(OtBu)-Gly-OH (Trimer)

[0272] H-p-Ala-Glu(OtBu)-Gly-OH (Trimer)

[0273] Step 3a: Cbz-p-Ala-OPfp EDCI, PFPOH

[0274] Cbz-p-Ala-OH Cbz-p-Ala-OPFP

[0275] Cbz-P-Ala-OH (5.0 kg, 22.4 mol, 1.0 equiv.) and pentafluorophenol (PfpOH) (1.2 equiv.) are added to the reactor containing DCM (8 V) at 20 to 30 °C. EDCI (1.2 equiv.) is added portion wise. The mixture is stirred at 20-30 °C for 2-4 hours and tracked until the starting material is consumed. At 20 to 30 °C, 20% sodium chloride aqueous solution (20 L) is added to the reactor, stirred for 1 h, and the phases are separated to collect the organic phase. This washing operation is repeated three times. Following solvent swap to n-heptane, Cbz-P-Ala-OPfp is isolated as a solid by filtration and drying (8.02 kg, 87.5 yield, 95.2% w / w, 96.08% a / a containing 3.75% PfpOH).

[0276] 1H NMR (400 MHz, DMSO-d6) 5 7.51 (t, J = 5.6 Hz, 1H), 7.48 - 7.17 (m, 5H), 5.05 (s, 2H), 3.41 (q, J = 6.4 Hz, 2H), 2.98 (t, J = 6.6 Hz, 2H).

[0277] MS (ESI) calculated for Ci7Hi3F5NO4 [M+H]+, 390.08; found, 389.7.

[0278] Step 3b: Cbz-p-Ala-Glu(OtBu)-Gly-OH

[0279] CO2fBu Step 3b CO2tBu

[0280] H-Glu(OtBu)-Gly-OH Cbz-p-Ala-Glu(OtBu)-Gly-OH (Dimer) (Cbz-trimer)

[0281] H-Glu(OtBu)-Gly-OH (4.4 kg, 17.0 mol, 1.1 equiv.) and BSA (2.0 equiv.) are charged to Reactor 1 containing THF (75 L) at 20 to 30 °C, and the mixture is stirred for 1 hour. In a separate reactor, THF (75 L) and Cbz-b-Ala-OPFP (6.0 kg Assay corrected, 15.4 mol, 1.0 equiv.) are added at 20-30 °C, and the mixture is stirred for 1 hour. At -5°C to 5 °C, the Cbz- P-Ala-OPfp-solution is added dropwise over 1 hour to Reactor 1. The mixture is warmed to 20 °C to 30 °C and stirred for 2-4 hours until conversion is judged complete. At 20 °C to 30 °C, 20% sodium chloride aqueous solution (24 L) and z-PrOAc (24 L) are added to the reactor. The mixture is stirred for 1 h, and the phases are separated to collect the organic phase. The organic phase is returned to the reactor and washed with 20% sodium chloride aqueous solution (24 L). The mixture is stirred for 1 h, and the phases are separated to collect the organic phase. The organic phase is concentrated to 2-3 V at 30 °C to 40 °C. 2-MeTHF (30 L) is added, and the solution is concentrated again to 2-3 V. This operation is repeated twice. Finally, 2-MeTHF (30 L) is added, and Cbz-P-Ala-Glu(OtBu)-Gly-OH is isolated as solution (40.8 kg, 96.7% yield, 17.0% w / w, 60.1% a / a containing 38.6% PfpOH).

[0282] 1H NMR (400 MHz, DMSO-d6) 5 8.20 (t, J = 5.9 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.42 - 7.28 (m, 5H), 7.19 (t, J = 5.7 Hz, 1H), 5.02 (s, 2H), 4.38 - 4.28 (m, 1H), 3.80 - 3.73 (m, 2H), 3.25 (q, J = 6.8 Hz, 2H), 2.37 (t, J = 7.2 Hz, 2H), 2.28 (t, J = 8.0 Hz, 2H), 1.97 - 1.85 (m, 1H), 1.78 - 1.66 (m, 1H), 1.39 (s, 9H).

[0283] MS (ESI) calculated for C22H32N3O8 [M+H]+, 466.22; found, 466.0.

[0284] Step 4: H-p-Ala-Glu(OtBu)-Gly-OH

[0285] CO2tBu CO2ffiu

[0286] Step 4

[0287] Cbz-p-Ala-Glu(OtBu)-Gly-OH H-p-Ala-Glu(OtBu)-Gly-OH

[0288] (Cbz-trimer) (Trimer)

[0289] A solution of Cbz-P-Ala-Glu(OtBu)-Gly-OH in 2-MeTHF (40.3 kg, 17.0% w / w, 14.7 mol, 1.0 equiv.), 2-MeTHF (5 V), H2O (1 V), and 10% Pd / C (0.05 g / g) are charged into a flask. The mixture is stirred at 25°C and 5 bar H2 pressure. The reaction progress is monitored by HPLC. After conversion is judged complete, the reaction mixture is filtered, and the filter cake is washed with H2O (3.5 L). The filtrate is separated, and the aqueous layer is washed twice with MTBE (5 V each wash). At 20-30 °C, acetonitrile (30 V) is added dropwise to the flask, and the mixture is stirred for approximately 16 hours. The solution is filtered, and the filter cake is washed with MeCN (1 V). The resulting solid is collected and dried to obtain H- P-Ala-Glu(OtBu)-Gly-OH as white solid (3.54 kg, 72.6% yield, 99.8% w / w, 99.1% a / a).

[0290] 1H NMR (400 MHz, D2O) 5 4.28 (dd, J = 8.8, 5.6 Hz, 1H), 3.89 (d, J = 1.9 Hz, 2H), 3.17 (t,J = 6.7 Hz, 2H), 2.65 (t, J = 6.7 Hz, 2H), 2.37 - 2.23 (m, 2H), 2.05 - 1.97 (m, 1H), 1.90 - 1.75 (m, 1H), 1.33 (s, 9H).

[0291] MS (ESI) calculated for C14H26N3O6 [M+H]+, 332.18; found, 332.43. Example 4

[0292] H-p-Ala-Glu(OtBu)-Gly-OH (Trimer)

[0293] H-Gly-OH Step 1b CC^fBu

[0294] Cbz-Glu(OtBu)-Gly-OH

[0295] Cbz-dimer

[0296] Pivaloyl chloride (1.97 g, 1.1 equiv.) and MeCN (6 V) are added at room temperature and cooled to -5 °C to 0 °C. Cbz-Glu(OtBu)-OH (5.0 g, 1.0 equiv.), N-methylmorpholine (1.2 equiv.), and MeCN (4 V) are mixed and added dropwise into the reaction. The reaction is stirred at -5 to 0 °C and monitored by derivation using BnNFh. The reaction mixture is cooled to -25 °C to -20 °C, and glycine (1.2 equiv.) is added. The mixture is stirred at -25 to -20 °C for 2 hours. N,O-Bis(trimethylsilyl)acetamide (1.5 equiv.) is then added, and the mixture is stirred at room temperature. The mixture is quenched with 20% aqueous NaCl (5 V) and 5% H3PO4 (5 V) at room temperature, stirred for 0.5 hours, and the phases are separated. The organic phase is washed twice with 10% aqueous NaCl (5 V per wash). The organic phase is evaporated, and solvent exchange is performed with 2-MeTHF (5 V x 5) to afford a 2-

[0297] MeTHF solution of Cbz-Glu(OtBu)-Gly-OH (19.2 g, 93.7% yield, 28.5% w / w, 94.6% a / a). Step 2: H-Glu(OtBu)-Gly-OH

[0298] Cbz-Glu(OtBu)-Gly-OH H-Glu(OtBu)-Gly-OH Cbz-

[0299] Cbz-dimer Dimer

[0300] A solution of Cbz-Glu(OtBu)-Gly-OH in 2-MeTHF (35.0g, 88.7 mol, 1.0 equiv.) is dissolved in 2-MeTHF (6 V) and water (2 V). 10% Pd / C (0.1 g / g) is added, and the mixture is stirred under a hydrogen atmosphere at room temperature overnight. The mixture is filtered, and the filter cake is washed with water (2.5 V). The phases are separated. MeCN (30 V) is added to the aqueous phase at room temperature, and the mixture is stirred at room temperature for at least 6 hours. The solution is filtered, and the filter cake is washed with MeCN (1 V). The solid is collected and dried to obtain H-Glu(OtBu)-Gly-OH (22.2 g, 90.3% yield, 94.0% w / w, 98.68% a / a).

[0301] Step 3a+b: Cbz-p-Ala-Glu(OtBu)-Gly-OH Cbz-Trimer

[0302] Pivaloyl chloride (11.34 g, 1.05 equiv.) and 2-MeTHF (5 V) are added at room temperature and cooled to -5 °C to 0 °C. Cbz-P-Ala-OH (20.0 g, 1.0 equiv.), N-methylmorpholine (1.1 equiv.), and 2-MeTHF (3 V) are mixed and added dropwise into the reaction. The reaction is stirred at -5 °C to 0 °C and monitored by derivation using BnNH2. In another flask, H- Glu(OtBu)-Gly-OH (25.6 g, 1.1 equiv.) was suspended in 2-MeTHF (6 V) at room temperature, followed by the addition of N,O-Bis(trimethylsilyl)acetamide (1.5 equiv.), stirred at room temperature for 2 h to afford a clear mixture, the added dropwise to the mix- anhydride solution at -5 °C to 0 °C. The mixture is stirred at room temperature for 4 h, then quenched with 20% aqueous NaCl (5 V) and 5% H3PO4 (5 V) at room temperature. After stirring for 0.5 hours the phases are separated. The organic phase is washed with 10% aqueous NaCl (5 V), and then partially evaporated to yield a 2-MeTHF solution of Cbz-P-Ala- Glu(OtBu)-Gly-OH (86.2 g, 93.8% yield, 45.2% w / w, 91.6% a / a, containing 4.1% Cbz-P-Ala- OH).

[0303] Step 4: H-p-Ala-Glu(OtBu)-Gly-OH

[0304] Cbz-p-Ala-Glu(OtBu)-Gly-OH

[0305] Cbz-Trimer

[0306] Cbz- P-Ala-Glu(OtBu)-Gly-OH (39.0 g, 1.0 equiv.) is dissolved in 2-MeTHF (6 V) and water (1 V). 10% Pd / C (0.1 g / g) is added, and the mixture is stirred under a hydrogen atmosphere at room temperature overnight. The mixture is filtered, and the filter cake is washed with water (1 V). The phases are then separated. The aqueous phase is added to MeCN (30 V), and the mixture is stirred overnight. The mixture is filtered, and the filter cake is washed with MeCN (1 V). The filter cake is added to water (0.5 V) and heated to 45 °C to 50 °C. At this temperature, ethanol (20 V) is added dropwise, and the mixture is stirred at 45 °C to 50 °C for 1 hour. The mixture is then cooled to room temperature and stirred overnight, followed by stirring at 0 °C to 5 °C for 2 hours. The solid is filtered, washed with MeCN and dried to obtain H P-Ala-Glu(OtBu)-Gly-OH (25.6 g, 90.4% yield, 98.2% w / w, 98.7% a / a).

[0307] Step 5a / b: X-p-Ala-Glu(OtBu)-Gly-OH (X=2-(3-cyano-5-fluorophenyl)-2- methylpropanoyl-)

[0308] Step 5b X1-p-Ala2-Glu(OtBu)3-Gly4-OH In the first flask, 2-(3-cyano-5-fluorophenyl)-2 -methylpropanoic acid (3.0 g, 1.0 equiv.), TEA (0.1 equiv.), and toluene (8 V) are added at room temperature and warmed to 60 °C. SOCh (1.5 equiv.) is added dropwise at 60 °C. The mixture is stirred overnight. The mixture is concentrated to approximately 5 V, then diluted to approximately 10 V with toluene. This dilution-concentration process is repeated three times, and the mixture is finally concentrated to approximately 5 V. In another flask, H-b-Ala-Glu(OtBu)-Gly-OH (1.2 equiv.), and N,O- Bis(trimethylsilyl)acetamide (2.0 equiv.) are added to NMP (6 V) and stirred for 1 hour. The mixture is cooled to 0 °C to 5 °C, and the acyl chloride solution is added dropwise. The mixture is stirred at room temperature overnight. zPrOAc (10 V), 20% aqueous NaCl (6 V), and 5% H3PO4 (6 V) are sequentially added, and the mixture is stirred at room temperature for 10 minutes. The phases are then separated. The organic phase is washed with 20% aqueous NaCl (6 V), then evaporated to 3 V. The solvent is exchanged to zPrOAc and cooled to 0 °C. This solution is added into a pre-cooled mixture of zz-Heptane (27 V) and MTBE (7 V). The suspension is stirred at 0 °C for 1 hour. The product is then filtered and collected to obtain amorphous X-P-Ala-Glu(OtBu)-Gly-OH as a white solid (7.26 g, 82.4% yield, 85.6% w / w, 98.36% a / a).

[0309] Solid-Phase Peptide Synthesis using X-B-Ala-Glu(OtBu)-Gly tetramer compound as building block

[0310] X-P-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-

[0311] Ly s(AEEAc-AEEAc-y-Glu- 19-carboxynonadecanoyl)-Ala-Phe-

[0312] Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NEk TFA

[0313] X-|3-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-lle-Aib-Leu-Asp-Lys-lle-Ala-Gln-Lys(AEEAc-AEEAc-Y-Glu-19-carboxynonadecanoyl)-Ala-Phe-

[0314] Val-GIn-Trp-Leu-lle-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NI-^ TFA

[0315] Synthetic Procedure: SPPS

[0316] Preparation of the Resin

[0317] Fmoc Sieber Resin (25 g, loading 0.75 mmol / g, 18.75 mmol) was charged into a 0.5 L SPPS reactor. The resin was swelled with DMF (226 mL, 8.52 g / g resin) and stirred for 2 h at 23 °C.

[0318] The reactor was then drained and the resin was washed twice with DMF (2 x 226 mL, 8.52 g / g resin).

[0319] General Synthetic Procedure

[0320] Washing steps were designed to remove remaining deprotection solution, coupling reagents, and additives between process steps. Before each wash, the reactor was completely drained

[0321] (i.e., until the flow into the waste accumulation vessel has stopped). The wash solvent was added to the reactor and the mixture was stirred for 5 min (starting after solvent addition is complete and stirring is started). The stirrer was then stopped and the reactor was drained completely.

[0322] Fmoc deprotection was performed by stirring the resin in Fmoc deprotection solution. Deprotections in cycles 1-7 were performed with a stirring time of 15 min, while deprotection cycles 8-32 were performed with a stirring time of 30 min. Time starts after solvent addition is complete and stirring is started. The Fmoc deprotection solution consists of a 20% v / v piperidine solution in DMF.

[0323] In cycle 33, ivDde deprotection was performed by 8 treatments with a 3% v / v solution of hydrazine monohydrate in DMF (6x 30 min, lx 15 h).

[0324] For all couplings, a DIC / OxymaPure procedure was used. The protected amino acid or building block and OxymaPure were dissolved together in DMF. As the resin volume increases with the progressing SPPS build, the later coupling steps were conducted under more diluted conditions. A solution of DIC in DMF was then added and the resulting mixture was stirred at ambient temperature for 5 min to pre-activate the amino acid.

[0325] Once pre-activation was complete, the amino acid solution was added to the reactor. The reaction mixture was then stirred. The reaction time for the coupling was 1.5-6 h. Recoupling was performed for cycle 24 using the same procedure as the coupling.

[0326] In-process monitoring was conducted following selected deprotections and couplings. To this end, the fragment was cleaved from the resin and conversion was assessed by HPLC.

[0327] Synthetic Procedure

[0328] Starting with the Fmoc-deprotection of the protected resin, the peptide backbone, and the side chain on Lys20were built using the general conditions for each amino acid coupling and deprotection outlined in Table 1.

[0329] Table 1 : Conditions used for the SPPS build on 0.75 mmol scale.

[0330] The detailed coupling sequence and step conditions in the various cycles are listed in Table 2.

[0331] Table 2: Detailed conditions for the individual cycles in the SPPS build on 460 mmol scale.

[0332] Synthetic Procedure: Cleavage / Global Deprotection and Precipitation

[0333] The resin bound material, obtained after the SPPS step, was subjected to cleavage / global deprotection to afford the crude peptide.

[0334] The cleavage cocktail was prepared as follows: TFA (90 mL, 9 mL / g of resin bound material) was cooled to 0 °C. Water (2.5 mL, 0.25 mL / g of resin bound material) was added, followed by DTT (5 g, 0.5 g / g of resin bound material) and TIS (2.5 mL, 0.25 mL / g of resin bound material).

[0335] Resin bound material (10 g) was charged into a 250 L jacketed reactor equipped with a frit.

[0336] The pre-cooled (0 °C) cleavage cocktail was pumped into the reactor and the resulting mixture was warmed to 20 °C and stirred at this temperature for 90 min. The mixture was then filtered and the filtrate was collected in a 2 L jacketed reactor. The spent resin was washed with TFA (2x 10 mL) and the washing liquid was combined with the filtrate. The resulting mixture was cooled to -15 C. Then, pre-cooled (-15 °C) TBME (660 mL, 66 mL / g of resin bound material) was added slowly to the filtrate over 65 min while keeping IT <-5 °C. After the addition, the suspension was stirred at -5 °C for 30 min before it was warmed to 20 °C over 30 min. The suspension was filtered and the filter cake was washed with TBME

[0337] (3x 100 mL). The isolated solid was dried at 5 mbar and 22 °C for 16 h.

[0338] The crude peptide was isolated as an off-white powder (5.94 g, purity 72.4 area %).

[0339] Solid-Phase Peptide Synthesis using X-B-Ala-Glu(OtBu)-Gly tetramer and Fmoc-L-

[0340] Lys(MTT)-OH as building block

[0341] X-p-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-lle-Aib-Leu-Asp-Lys-lle-Ala-Gln-Lys(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala-

[0342] Phe-Val-Gln-Trp-Leu-lle-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2-TFA

[0343] Synthetic Procedure: SPPS

[0344] Preparation of the Resin

[0345] Sieber resin (25 g, loading 0.77 mmol / g, 19.25 mmol) was charged into a 0.5-L SPPS reactor (fully automated). The resin was swelled with DMF (226 mL, 8.52 g / g resin) and stirred for 2 h at 23 °C. The reactor was then drained, and the resin was washed twice with DMF (2 x 226 mL, 8.52 g / g resin).

[0346] General Synthetic Procedure

[0347] Washing steps were designed to remove the remaining deprotection solution, coupling reagents, and additives between process steps. Before each wash, the reactor was completely drained (i.e., until the flow into the waste accumulation vessel has stopped). The wash solvent was added to the reactor and the mixture was stirred for 5 min (starting after solvent addition is complete and stirring is started). The stirrer was then stopped, and the reactor was drained completely.

[0348] All Fmoc deprotections were performed by 1-2 treatments with piperidine / DMF (20% v / v) with a stirring time of 20-30 min per cycle. After Fmoc removal, the resin was washed with DMF.

[0349] In cycle 35, Mtt deprotection was performed by 3 treatments with a HFIP / toluene / TIPS (29.5 / 69.5 / 1 v / v / v). Subsequently, the resin was washed with DMF.

[0350] For all couplings in cycle 1-33 and 35, a DIC / Oxyma Pure procedure was used. The protected amino acid or building block and Oxyma Pure were dissolved together in DMF. A solution of DIC in DMF was then added and the resulting mixture was typically stirred at 20 °C for 10 - 60 min to pre-activate the building block.

[0351] For the coupling in cycle 34, PyBOP / 2,4,6-trimethylpyridine was used. The tetramer building block (X1-p-Ala2-Glu3-Gly4) was dissolved in DMF together with PyBOP. A solution of 2,4,6-trimethylpyridine in DMF was then added and the resulting mixture was typically stirred at 20 °C for 5 min to pre-activate the building block.

[0352] Once the pre-activation was complete, the protected amino acid or building block solution was added to the resin. The reaction mixture was then stirred. The reaction time for the coupling was 1.5-24 h.

[0353] In-process monitoring was conducted following selected deprotections and couplings. To this end, the fragment was cleaved from the resin and conversion was assessed by HPLC.

[0354] A capping step was performed after coupling in cycle 11, 12, 18, 25 and 26. Following the post coupling washes with DMF the peptide resin was treated with a mixture of Ac2O / pyridine / DMF (1 / 1 / 98, v / v / v) for 10 min. Subsequently, the peptide was washed with DMF.

[0355] After completion of the solid phase synthesis, the resin was washed several times with DMF, 2 PrOH and MTBE before discharging. The resin was dried under reduced pressure for 48 h. Table 3: Conditions used for the linear SPPS using Fmoc-Lys(Mtt)-OH.

[0356] The detailed coupling sequence and step conditions in the various cycles are listed in Table 4.

[0357] Table 4: Detailed conditions for the linear SPPS using Fmoc-Lys(Mtt)-OH and the tetramer building block.

[0358] TFA-mediated cleavage from the solid support with concomitant global deprotection

[0359] The resin bound material, obtained after the SPPS step, was subjected to cleavage / global deprotection to afford the crude peptide. The resin bound peptide was added to the cleavage cocktail (TFA / H2O / TIS 90.0 / 5.0 / 5 (v / v / v); 10 mL per g resin). The reaction mixture was stirred for 1.5 h at 20°C before it was filtered. The filtrate was transferred into a jacketed reactor and cooled to -15 °C. Pre-cooled TBME (- 15°C, 54 ml / g of resin bound material) was then added slowly while maintaining the internal temperature below -5°C.

[0360] The resulting suspension was then warmed to 20 °C and aged at this temperature for 30 min. The crude precipitate was filtered, and the filter cake was washed with TBME (3x 10 mL / g of resin bound material for each wash). The isolated solid was dried at 1-3 mbar and 22 °C for about 16 h. The crude peptide was isolated as an off-white powder (6.68 g, purity 69.8 area %).

[0361] ***

Claims

CLAIMS1. Process for the preparation of a tetramer compound of formula I, or a salt thereof,wherein PROT is an ester protecting group, comprising, coupling, glycine (Gly), glutamic acid (Glu) with the y-carboxylic acid protected with PROT, P-alanine (Ala) and 2-(3-cyano-5-fluorophenyl)-2-methyl-propionic acid of the formula II,under liquid phase peptides synthesis (LPPS) conditions.

2. The process of claim 1, wherein PROT is tert-butyl (tBu).

3. The process of claim 1 or 2, wherein a) glycine is coupled with an amino group protected and activated glutamic acid of the formula IIIRlaGlu(OPROT)-O-R2a(III) wherein, Rlais an amino protecting group,R2ais an activated ester andPROT is an ester protecting group, in the presence of a coupling additive to form the dipeptide of the formula IVRlaGlu(OPROT)-Gly-OH (IV) b) the amino protecting group Rlais removed to form the dipeptide of formula V,H-Glu(OPROT)-Gly-OH (V) c) the dipeptide of formula V is coupled to an amino group protected and activated P- alanine of formula VIRlbP-Ala-O-R2b(VI) wherein,Rlbis an amino protecting group,R2bis an activated ester, in the presence of a coupling additive to form the tripeptide of the formula VIIRlbP-Ala-Glu(OPROT)-Gly-OH (VII) wherein,Rlbis an amino protecting group, andPROT is an ester protecting group, d) the amino protecting group Rlbis removed to form the tripeptide of formula VIIIH-P-Ala-Glu(OPROT)-Gly-OH (VIII) ) e) the tripeptide of formula VIII is coupled to an activated 2-(3-cyano-5-fluorophenyl)- 2 -methyl-propionic acid of the formula Ila,Ila wherein,R3is an activated ester, or coupled to an acid halide of 2-(3-cyano-5-fluorophenyl)-2-methyl-propionic acid of the formula lib,lib wherein,X is a halogen, in the presence of a coupling additive to form the tetramer of formula I.

4. The process of any one of claims 1 to 3, wherein in step a)Rlais an amino protecting group selected from benzyloxycarbonyl (Cbz) or tertbutoxycarbonyl (Boc),R2ais pentafluorophenyl ester (OPfp) or / f-hydroxysuccinimide ester (OSu) in case the amino group protected and activated glutamic acid of the formula III is isolated, or R2ais formed in situ with a coupling agent selected from benzotriazol- 1-yloxy- tris(dimethylamino)phosphonium hexafluorophosphate (BOP), l-cyano-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), M / f'-Diisopropylcarbodiimide (DIC) in combination with 2- hydroxypyridine-7V-oxide (HOPO), O-(7-azabenzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (HATU), l,l’-carbonyl-di -imidazole(CDI), l,l'-carbonyl-di-(l,2,4-triazole) (CDT), pivaloylchloride (PivCl), isobutylchloroformate (IBCF) or propanephosphonic acid anhydride (T3P) in case the amino group protected and activated glutamic acid of the formula III is not isolated,PROT is tert. Butyl, and the coupling additive is (trimethylsilyl)acetamide (BSA).

5. The process of any one of claims 1 to 4, wherein the reaction in step a) is performed in the presence of a polar aprotic solvent, selected from tetrahydrofuran (THF), 2- methyltetrahydrofuran, acetonitrile, di chloromethane, V, V-dimehylformamide or ethyl acetate at a reaction temperature from -20°C to 40°C.

6. The process of any one of claims 1 to 5, wherein in step b) the amino protecting group Rlais removed by hydrogenation with hydrogen in the presence of a hydrogenation catalyst.

7. The process of any one of claims 1 to 6, wherein in step c)Rlbis an amino protecting group selected from benzyloxycarbonyl (Cbz) or tertbutoxycarbonyl (Boc),R2bis pentafluorophenyl ester (OPfp) or V-hydroxysuccinimide ester (OSu) in case the amino group protected and activated P-alanine of formula VI is isolated, or R2bis formed in situ with a coupling agent selected from benzotriazol- 1-yloxy- tris(dimethylamino)phosphonium hexafluorophosphate (BOP), l-cyano-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), V, V'-Diisopropylcarbodiimide (DIC) in combination with 2- hydroxypyridine-N-oxide (HOPO), O-(7-azabenzotriazol- 1 -yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (HATU), l,l’-carbonyl-di -imidazole (CDI), l,l'-carbonyl-di-(l,2,4-triazole) (CDT), pivaloylchloride (PivCl), isobutylchloroformate (IBCF) or propanephosphonic acid anhydride (T3P) in case the amino group protected and activated P-alanine of formula VI is not isolated,PROT is tert, butyl and the coupling additive is (trimethyl silyl)acetamide (BSA).

8. The process of any one of claims 1 to 7, wherein the reaction in step c) is performed in the presence of a polar aprotic solvent, selected from, tetrahydrofuran (THF), acetonitrile, di chloromethane or ethyl acetate at a reaction temperature from -10°C to 40°C.

9. The process of any one of claims 1 to 8, wherein in step d) the amino protecting group Rlbis removed by hydrogenation in the presence of a hydrogenation catalyst.

10. The process of any one of claims 1 to 9 wherein in step e)R3is pentafluorophenyl ester (OPfp) or A -hydroxy succinimide ester (OSu) in case the activated 2-(3-cyano-5-fluorophenyl)-2-methyl-propionic acid of the formula Ila is isolated, orR3is formed in situ with a coupling agent selected from benzotriazol- 1-yloxy- tris(dimethylamino)phosphonium hexafluorophosphate (BOP), l-cyano-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), M / f'-Diisopropylcarbodiimide (DIC) in combination with 2- hydroxypyridine-A-oxide (HOPO), O-(7-azabenzotriazol- l -yl)-A,A,A',A'- tetramethyluronium hexafluorophosphate (HATU), l,l’-carbonyl-di -imidazole (CDI), l,l'-carbonyl-di-(l,2,4-triazole) (CDT), pivaloylchloride (PivCl), isobutylchloroformate (IBCF) or propanephosphonic acid anhydride (T3P) in case the activated 2-(3-cyano-5-fluorophenyl)-2-methyl-propionic acid of the formula Ila is not isolated,X is chlorine, and the coupling additive is bis(trimethylsilyl)acetamide (BSA).

11. The process of any one of claims 1 to 10, wherein in step e) the coupling to an activated 2-(3-cyano-5-fluorophenyl)-2-m ethyl -propionic acid of the formula Ila or to an acid halide of 2-(3-cyano-5-fluorophenyl)-2-methyl-propionic acid of the formula lib is performed in the presence of a polar aprotic solvent selected from tetrahydrofuran (THF), 2-methyltetrahydrofuran, acetonitrile, dichloromethane, N,N- dimehylformamide, A-methylpyrrolidone or ethyl acetate at a reaction temperature from -20°C to 40°C.

12. Crystalline polymorph of the tetramer compound of the formula lacharacterized by an X-ray powder diffraction pattern having characteristic peaks expressed in angle 2-theta at13. Crystalline polymorph of claim 12, characterized by the X-Ray powder diffractogram of Fig. 1.

14. Crystalline polymorph of the tetramer compound of the formula la, characterized by the IR absorption spectrum having characteristic peaks expressed in cm'1at15. Crystalline polymorph of claim 13, characterized by the IR absorption spectrum of Fig. 2.

16. Process for the preparation of a crystalline polymorph of any one of claims 12 to 15 comprising the steps of• providing a solution of the tetramer compound of formula I in isopropyl acetate (iPrOAc),• measuring the water content with the KF method in %,• lowering the water content to KF<1.0 %, by repeated concentration under reduced pressure and elevated temperature and iPrOAc addition,• stirring the iPrOAc solution at elevated temperature,• dropwise adding MTBE,• stirring the solution at elevated temperature,• cooling the solution,• precipitating the solid polymorph,• filtering and drying under vacuo.

17. Use of the tetramer compound of formula I, or a salt thereof, prepared in accordance with the process of claims 1 to 11 for the preparation of the peptide of formula XX, or of a pharmaceutically acceptable salt or ester thereofX-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15- Lys16-Ile17-Ala18-Gln19-Lys20(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala21-Phe22- Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37- Pro38-Ser39-NH2(XX) wherein X isAEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetyl.

18. Use of the crystalline polymorph of the tetramer compound of the formula la for the preparation of the peptide of formula Xlla, or of a functionalized derivative thereof,X1-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15- Lys16-Ile17-Ala18-Gln19-Lys20-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30- Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH-linker-solid support(Xlla) wherein X is19. The use of claim 18, wherein the functionalized derivative has the formula Xllb,X1-P-Ala2-Glu3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10-Ser(tBu)11-Ile12-Aib13-Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys20-Ala21-Phe22- Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser(tBu)32-Ser(tBu)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(tBu)39-NH-linker-solid support(Xllb), wherein X is as above.

20. Process for the preparation of the peptide of formula XX, or of a pharmaceutically acceptable salt or ester thereofX-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-He12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-Gln19-Lys20(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37- Pro38-Ser39-NH2(XX) wherein X isAEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetyl. comprising the steps of a) solid phase synthesis of the peptide of formula XX on a resin, comprising coupling the crystalline polymorph of the tetramer compound of the formula la to a functionalized peptide fragment of formula XiaH-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18- Gln19-Lys20-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33- Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH-linker-solid support(Xia) and forming the functionalized peptide fragment of formula XllaX1-p-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-He12-Aib13-Leu14-Asp15- Lys16-Ile17-Ala18-Gln19-Lys20-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30- Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH-linker-solid support(Xlla) b) deprotection of the Lys20s-amino protecting group, c) acylation of the s-amino group of Lys20with the moietyPROT-O-20-oxoicosanoyl-L-Glu(AEEAc-AEEAc-OH)-O-PROT(Xllla), wherein AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetyl and PROT is an ester protecting group, and forming the peptide of formula XX, bound to the resin; d) cleavage of the peptide of formula XX from the resin and global deprotection with an acid and e) precipitation of the peptide formula XX and optionally f) purification / i solation.

21. The process of claim 20, comprising the steps of a) solid phase synthesis of the peptide of formula XX on a resin, comprising coupling the crystalline polymorph of the tetramer compound of the formula la to a functionalized peptide fragment of formula IVbH-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10-Ser(tBu)11-Ile12-Aib13- Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys20(Mtt / ivDde)-Ala21-Phe22-Val23- Gln(Trt)24-Trp(Boc)25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser(tBu)32-Ser(tBu)33-Gly34-Ala35- Pro36-Pro37-Pro38-Ser(tBu)39-NH-linker-solid support(Xlb) and forming the functionalized peptide fragment of formula VbX1-P-Ala2-Glu3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10- Ser(tBu)11-Ile12-Aib13-Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys20(Mtt / ivDde)- Ala21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser(tBu)32- Ser(tBu)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(tBu)39-NH-linker-solid support(Xllb) b) deprotection of the Lys20s-amino protecting group Mtt or ivDde, c) acylation of the s-amino group of Lys20-70- with the moiety tBu-O-20-oxoicosanoyl-L-Glu(AEEAc-AEEAc-OH)-OtBu(Xlllb), wherein AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetyl, and forming the peptide of formula XX, bound to the resin; d) cleavage of the peptide of formula XX from the resin and global deprotection with an acid and e) precipitation of the peptide formula XX and optionally f) purification / isolation.

22. The process of claim 21, wherein Lys20in the derivative of formula Xllb is functionalized with Mtt.

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