Process of preparing a GLP-1r / GIPR agonist by various fragment approaches

Abstract

The invention relates to the synthesis of a peptide having the potential to act as GLP- 1R / GIPR agonist via the coupling of suitable functionalized peptide fragments.

Description

[0001] Case P60005

[0002] Process of preparing a GLP-1R / GIPR agonist by various fragment approaches

[0003] Field of the Invention

[0004] The invention relates to the synthesis of a peptide having the potential to act as GLP-1R / GIPR agonist via the coupling of suitable functionalized peptide fragments.

[0005] In particular, the invention relates to a novel process for the preparation of a peptide of formula I (SEQ ID NO:1), or of a pharmaceutically acceptable salt or ester thereof

[0006] X1-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

[0007] (I)

[0008] wherein X is

[0009]

[0010] GNanci

[0011] AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid.

[0012] The peptide of formula I has the potential to act as GLP-1R / GIPR agonist.

[0013] Background

[0014] 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

[0015] 05.03.2026(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.

[0016] Particularly the peptide of formula I 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 I does not cause internalization and consequently, desensitization of either GLP-1R or GIPR, and thus has enhanced signaling efficacy.

[0017] Object of the invention was to provide a novel synthesis approach involving the combination of suitable peptide fragments.

[0018] Summary of the Invention

[0019] It was found that the object of the invention could be reached with the new process for the preparation of the peptide of formula I (SEQ ID NO: 1), or of a pharmaceutically acceptable salt or ester thereof

[0020] X1-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

[0021] (I)

[0022] wherein X is

[0023]

[0024] GNanci

[0025] AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid;

[0026] comprising

[0027] a) coupling of a functionalized peptide fragment Fl

[0028] H2N-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

[0029] (Fl) (SEQ ID NO:2)

[0030] with a functionalized peptide fragment F2

[0031] X1-β-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-OH

[0032] (F2) (SEQ ID NO:3)

[0033] wherein X and AEEAc are as defined above,

[0034] b) cleaving off the protecting groups,

[0035] c) precipitation of the peptide of formula I and optionally

[0036] d) purification / isolation.

[0037] In one further aspect, the invention relates to the following functionalized peptide fragments:

[0038] H2N- Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-Y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2

[0039] (Fla) (SEQ ID NO:4);Fmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-OH

[0040] (F3a) (SEQ ID NO: 5);

[0041] H2N-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2

[0042] (F4a) (SEQ ID NO: 6);

[0043] Fmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-Y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-OH

[0044] (F5a) (SEQ ID NO:7);

[0045] H2N-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2

[0046] (F6a) (SEQ ID NO:8);or

[0047] X1-β-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)-Tyr10-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-OH

[0048] (F2a) (SEQ ID NO:9),

[0049] wherein X is

[0050]

[0051] CN

[0052] In one additional aspect, the invention relates to the following functionalized peptide fragment that are bound to the resin:

[0053] Fmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-Y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-O-2-chlorotrityl resin

[0054] (F3b) (SEQ ID NO: 10), orFmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-O-2-chlorotrityl resin

[0055] (F5b) (SEQ IDNO:11), or

[0056] X1-β-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)-Tyr10-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-O-2-chlorotrityl resin

[0057] (F2b) (SEQ ID NO: 12),

[0058] wherein X is

[0059]

[0060] CN

[0061] Brief Description of the Figures:

[0062] FIG. 1 shows the synthesis of the functionalized peptide fragment of formula (F4a) in the liquid phase.

[0063] FIG. 2 shows the solid-phase synthesis of the functionalized peptide fragment of formula (F3b) using 2-chlorotrityl chloride resin.

[0064] FIG. 3 shows the synthesis of the functionalized peptide fragment of formula (F3a) from the functionalized peptide fragment of formula (F3b) under soft cleavage conditions.

[0065] In FIG. 4 it is shown the solid-phase synthesis of the functionalized peptide fragment of formula (F2b) using 2-chlorotrityl chloride resin.

[0066] FIG. 5 is a reaction scheme showing the release of the functionalized peptide fragment (F2a) by soft cleavage from the resin.

[0067] FIG. 6 shows the liquid phase synthesis (LPPS) of the functionalized peptide fragment of formula (Fla) from fragments (F3a) and (F4a).

[0068] In FIG. 7 the liquid phase synthesis (LPPS) of the functionalized peptide of formula (I) from fragments (Fla) and (F2a) is shown.FIG. 8 shows the solid-phase synthesis (SPPS) of the functionalized peptide fragment of formula (F5b) using 2-chlorotrityl chloride resin.

[0069] FIG. 9 is a reaction scheme showing the release of the functionalized peptide fragment (F5a) by soft cleavage from the resin.

[0070] FIG. 10 shows the coupling procedure of the functionalized peptide of formula (F5a) with the functionalized peptide fragment of formula (F6a) to produce the functionalized peptide fragment (Fla) by liquid phase synthesis.

[0071] Detailed Description of the Invention:

[0072] The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein.

[0073] 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-acetyl cysteine 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, N-ethylpiperidine, piperidine, polyimine resins and the like.

[0074] 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".

[0075] 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.

[0076] 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 ester protecting groups are tert-butyl (tBu) or 3 -methyl-pent-3 -yl (Mpe) which are cleavable with a strong acid, such as e.g. with trifluoroacetic acid.

[0077] The term “solid phase peptide synthesis (SPPS) conditions” in the context of the present invention refers to specific parameters and reagents for assembling the peptide fragments 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.

[0078] The solid support typically consists of a polymeric resin, most commonly low crosslinked polystyrene beads, which is equipped with reactive groups to enable covalent binding between the carboxyl group of the first amino acid of the nascent peptide chain and the resin through a linker. The most common polymeric solid support used is a resin composed of a 1-2% divinylbenzene - cross-linked polystyrene. Furthermore, a resin is composed of the polymeric solid support linked permanently to a linker (bifunctional spacer, or handle) that facilitates temporary anchoring of the first amino acid to the polymeric solid support. For the peptide fragments used in the present invention, which comprise a C-terminal acid, the 2-chlorotrityl resin is the solid support of choice. The 2 -chlorotrityl resin together with the first amino acid of the peptide fragment forms an ester. The cleavage of the peptide fragments from the 2-chlorotrityl resin can be accomplished with TFA in combination with water and at least one scavenger, selected from EDT (ethane- 1,2-dithiol), TIS (triisopropylsilane) and DTT (dithiothreitol).

[0079] Each amino acid to be coupled to the peptide chain / f-terminus must bear an appropriate protective group on its a-amino group and potentially on its side chain. The base-labile fluorenylm ethyl oxy carbonyl (Fmoc) group is commonly used to protect the a-amino group of the incoming amino acid. The repeated cycles involve alternate deprotection and coupling reactions.Deprotection of Fmoc, and exposing the amino group for the next coupling step, can be accomplished by a reaction with an organic base such as with di ethylamine or piperidine in a polar, aprotic solvent. Solutions of 10 to 30% (v / v) piperidine in DMF are a typical deprotection agent.

[0080] The coupling under SPPS conditions can be accomplished with coupling agents selected from benzotriazol-l-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol- 1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP), (7-azabenzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), A-[(777-benzotriazol-l-yl)(dimethylamino)methylene]-A-methylmethanaminium tetrafluoroborate A-oxide (TBTU), 2-(7A-benzotriazole- 1 -yl)- 1, 1,3,3 -tetramethylaminium hexafluorophosphate (HBTU), hydroxybenzotriazole (HOBt), l-[bis(dimethylamino)methylene]-7A-l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, A-[(dimethylamino)-3A-l,2,3-triazolo-[4,5-b]pyridin-l-ylmethylene]-A-methylmethanaminium hexafluorophosphate A -oxi de (HATU), propanephosphonic acid anhydride (T3P), O-(2-oxo-l(2H)pyridyl)-N, N, N', N'-tetramethyluronium tetrafluoroborate (TPTU), l-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyOXIM), pentafluorophenyl diphenylphosphinate (FDPP), Diphenylphosphoryl azide (DPP A), 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) or from a combination of AA'-diisopropylcarbodiimide (DIC) with A-hydroxysduccinimide, with 4-(dimethylamino)pyridine (DMAP), with 2-hydroxypyridine-A-oxide (HOPO) or with (ethyl-cyano (hydroximino)acetate) (OxymaPure), or from a combination of (7-azabenzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) with (ethyl-cyano (hydroximino)acetate) (OxymaPure), or from a combination of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) with 2-hydroxypyridine-A-oxide (HOPO).

[0081] The coupling reaction under SPPS conditions takes place in the presence of an organic solvent which can be selected from dimethylacetamide, AA-dimethylformamide, toluene, methylene chloride, acetonitrile, A-butylpyrrolidone (NBP), dimethylsulfoxide (DMSO), 1,3-dimethyl-2-imidazolidinone (DMI) or mixtures thereof.

[0082] Where appropriate, an organic base which can be selected from A, A-diisopropylethylamine, A-methylmorpholine (NMM), A-methylimidazole (NMI), 2-6-lutidine, 2,4,6-collidine or from triethylamine may be present.At the end of the synthesis, the crude peptides can be cleaved from the solid support while simultaneously removing all protecting groups, typically using an acidic reagent such as trifluoroacetic acid, and the crude peptide can be precipitated and optionally purified.

[0083] In one preferred aspect of the invention, the crude peptides can be softly cleaved from the solid support while keeping all protecting groups intact. Soft cleavage can be achieved with an acidic reagent such as a diluted trifluoroacetic acid solution in an organic solvent and a scavenger. In one preferred aspect, the peptide is cleaved form the solid support with TFA in combination with water and at least one scavenger, selected from EDT (ethane- 1,2-dithiol), TIS (triisopropylsilane) and DTT (dithiothreitol), e.g. with a solution of 2% TFA and 1% TIS in CH2CI2.

[0084] While the optimum reaction conditions of the SPPS have to be adapted for every peptide, the general SPPS method is well known in the art and is described for instance in detail in W. C. Chan and P. D. White, Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press Inc., 1999.

[0085] 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.

[0086] The coupling under LPPS conditions can in principle be accomplished with the same coupling agents as used for the SPPS. Thus, the coupling agents can be selected from benzotriazol- 1 -yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol- 1 -yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP), (7-azabenzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), A-[( / A-benzotriazol-l-yl)(dimethylamino)methylene]-A-methylmethanaminium tetrafluorob orate A-oxide (TBTU), 2-( / A-benzotriazol e- 1 -yl)- 1, 1,3,3 -tetramethylaminium hexafluorophosphate (HBTU), hydroxybenzotriazole (HOBt), l-[bis(dimethylamino)methylene]-7A-l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, A-[(dimethylamino)-3A-l,2,3-triazolo-[4,5-b]pyridin-l-ylmethylene]-A-methylmethanaminium hexafluorophosphate A-oxide (HATU),propanephosphonic acid anhydride (T3P), O-(2-oxo-l(2H)pyridyl)-N, N, N', N'-tetramethyluronium tetrafluoroborate (TPTU), l-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyOXIM), pentafluorophenyl diphenylphosphinate (FDPP), Diphenylphosphoryl azide (DPP A), 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) or from a combination of N, A'-diisopropylcarbodiimide (DIC) with A-hydroxysduccinimide, with 4-(dimethylamino)pyridine (DMAP), with 2-hydroxypyridine-A-oxide (HOPO) or with (ethyl-cyano (hydroximino)acetate) (OxymaPure), or from a combination of (7-azabenzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) with (ethyl-cyano (hydroximino)acetate) (OxymaPure), or from a combination of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) with 2-hydroxypyridine-A-oxide (HOPO).

[0087] The coupling reaction under LPPS conditions takes place in the presence of organic solvent which can be selected from dimethylacetamide, AA-dimethylformamide, toluene, methylene chloride, acetonitrile, A-butylpyrrolidone (NBP), dimethylsulfoxide (DMSO), 1,3-dimethyl-2-imidazolidinone (DMI) or mixtures thereof and an organic base which can usually be selected from AA-diisopropylethylamine, A-methylmorpholine (NMM), A-methylimidazole (NMI), 2-6-lutidine, 2,4,6-collidine or from triethylamine may be present.

[0088] At the end of the synthesis, the protecting groups can be removed with TFA in combination with water and at least one scavenger, selected from EDT (ethane- 1,2-dithiol), TIS (triisopropylsilane) and DTT (dithiothreitol).

[0089] In one aspect, the invention relates to a process for the preparation of the peptide of formula I, or of a pharmaceutically acceptable salt or ester thereof

[0090] X1-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

[0091] (I) (SEQ ID NO:1)

[0092] wherein X is

[0093]

[0094] GNanci

[0095] AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid;

[0096] comprising

[0097] a) coupling of a functionalized peptide fragment Fl

[0098] H2N-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

[0099] (Fl) (SEQ ID NO:2)

[0100] with a functionalized peptide fragment F2

[0101] X1-β-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-OH

[0102] (F2) (SEQ ID NO:3)

[0103] wherein X and AEEAc are as defined above,

[0104] b) cleaving off the protecting groups,

[0105] c) precipitation of the peptide of formula I and optionally

[0106] d) purification / isolation.

[0107] In general, the coupling of the peptide fragments is performed in the presence of a coupling agent, an organic base and an organic solvent as outlined above.

[0108] The preferred coupling agent for this synthesis is pentafluorophenyl diphenylphosphinate (FDPP).

[0109] Preferably, the organic base is A, A-di isopropyl ethyl amine and the preferred organic solvent is dimethylacetamide.The reaction temperature can be chosen between -30° C and 20°C, typically the process is run at 0°C.

[0110] The term “functionalized peptide” in this and the following contexts means that functional groups of respective amino acids are protected with suitable protecting groups, e.g. Thr, Ser, Tyr, Glu, Asp with OtBu, Trp or Lys with Boc, Gin with Trt.

[0111] In one particular aspect of the invention, the functionalized peptide fragment Fl has the formula Fla

[0112] H2N- Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-Y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2

[0113] (Fla) (SEQ ID NO:4), and

[0114] the functionalized peptide fragment F2 has the formula F2a

[0115] X1-β-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)-Tyr10-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-OH

[0116] (F2a) (SEQ ID NO:9),

[0117] wherein X and AEEAc are as defined above.

[0118] In a preferred aspect of the invention the process comprises coupling the functionalized peptide fragment of formula Fla

[0119] H2N- Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-Y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2

[0120] (Fla) (SEQ ID NO:4),

[0121] with a functionalized peptide fragment of formula F2a

[0122] X1-β-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)-T

[0123]

[0124] yr10-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-OH

[0125] (F2a) (SEQ ID NO:9),wherein X and AEEAc are as defined above.

[0126] The protecting groups are usually cleaved with TFA in combination with water and at least one scavenger.

[0127] Particularly preferred is a cleavage cocktail containing TFA, water and one or more scavenger compounds selected from EDT (ethane- 1,2-dithiol), TIS (triisopropylsilane) and DTT (dithiothreitol).

[0128] The following composition TFA / H2O / TIS: 90 / 5 / 5 (v / v / v) with 0.35g DTT was found to be suitable, although it is within the skilled person to vary the composition and the values as is considered appropriate.

[0129] The precipitation of the resulting peptide of formula I in step c) is accomplished with a suitable organic solvent such as diisopropyl ether (IPE), methyl tert-butyl ether (MTBE), cyclopentyl methylether (CPME), n-heptane or diethylether, but preferably methyl tert-butyl ether (MTBE).

[0130] The isolation in step d) can be carried out by methods known in the art, such as by filtration.

[0131] Preparation of Peptide Fragment Fl or Fla

[0132] In a further aspect of the invention, the peptide fragment Fl is prepared according to two different routes

[0133] Route 1 comprises the coupling of a functionalized peptide fragment F3

[0134] PROT-HN-Ala18-Gln19-Lys20(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24-OH

[0135] (F3) (SEQ ID NO: 13)

[0136] wherein PROT is a protecting group;

[0137] with a functionalized peptide fragment F4

[0138] H2N-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2(F4) (SEQ ID NO: 14)

[0139] and subsequent removal of the protecting group PROT.

[0140] Route 2 comprises the coupling of a functionalized peptide fragment F5

[0141] PROT-HN-Ala18-Gln19-Lys20(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-OH

[0142] (F5) (SEQ ID NO: 15)

[0143] wherein PROT is a protecting group;

[0144] with a functionalized peptide fragment F6

[0145] H2N-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2

[0146] (F6) (SEQ ID NO: 16)

[0147] and subsequent removal of the protecting group PROT.

[0148] Preparation of Peptide Fragment Fl or Fla according to Route 1:

[0149] The coupling of the functionalized peptide fragment F3

[0150] PROT-HN-Ala18-Gln19-Lys20(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24-OH

[0151] (F3) (SEQ ID NO: 13)

[0152] wherein PROT is a protecting group;

[0153] with a functionalized peptide fragment F4

[0154] H2N-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2

[0155] (F4) (SEQ ID NO: 14)

[0156] is in general performed in the presence of a coupling agent, an organic base and an organic solvent.

[0157] Suitable coupling agents are selected from those listed above. The combination of (7-azabenzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) with (ethyl-cyano (hydroximino)acetate) (OxymaPure) was found to be a favorable coupling agent.

[0158] The organic base is usually selected from those mentioned above. Preferably, the organic base is A, A-diisopropylethylamine and the preferred organic solvent is N, N-dimethylformamide.

[0159] The reaction temperature can be chosen between -30° C and 20°C, typically the process is run at -20 °C.

[0160] The protecting group PROT is an amino protecting group. Typically the Fmoc ((9H-fluoren-9-yl)methoxy)carbonylamino) protecting group is used. The removal of Fmoc is accomplished with an organic base, such as with piperidine or diethylamine, preferably diethylamine.

[0161] In a preferred aspect, the peptide fragment has the formula Fla

[0162] H2N- Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-Y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2

[0163] (Fla) (SEQ ID NO:4),

[0164] and the preparation comprises coupling of a functionalized peptide fragment F3a

[0165] Fmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-OH

[0166] (F3a) (SEQ ID NO: 5)

[0167] with a functionalized peptide fragment F4a

[0168] H2N-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2

[0169] (F4a) (SEQ ID NO: 6)

[0170] and the subsequent removal of the Fmoc protecting group as described above.

[0171] Preparation of Peptide Fragment F3 or F3aThe peptide fragment F3, in particular or functionalized peptide fragment F3a

[0172] Fmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-OH

[0173] (F3a) (SEQ ID NO: 5)

[0174] is prepared under common Fmoc-solid phase peptide synthesis conditions on a suitable resin, preferably on the 2-chlorotrityl resins (2-CTC-resins).

[0175] The combination of benzotriazol- 1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) with (ethyl-cyano (hydroximino)acetate) (OxymaPure) for cycle 2 and the combination A, A'-diisopropylcarbodiimide (DIC) with (ethyl-cyano (hydroximino)acetate) (OxymaPure) for the other cycles has been proved successful as coupling agents.

[0176] Except for cycle 2 no organic base is required for the assembly of peptide fragment F3 or F3a. For cycle 2 an organic base, usually selected from those mentioned above, preferably, A, A-diisopropylethylamine can be used.

[0177] Preferred organic solvent is A, A-dimethylformamide.

[0178] The coupling temperature is chosen between 0° C and 40°C, typically the process is run at 30°C.

[0179] The preparation of functionalized peptide fragment F3, in particular fragment F3a, comprises sequential coupling of the following functionalized building blocks on a resin, particularly on a 2-chlorotrityl resin.

[0180] Aminoacid # Cycle# Building Block

[0181] 2-chlorotrityl resin

[0182] 24 1 Fmoc-L-Gln(Trt)-OH

[0183] 23 2 Fmoc-L-Val-OH

[0184] 22 3 Fmoc-L-Phe-OH

[0185] 21 4 Fmoc-L-Ala-OH H2O

[0186] 20 5 Fmoc-L-Lys(sc)-OH

[0187] 19 6 Fmoc-L-Gln(Trt)-OH

[0188]

[0189] 18 7 Fmoc-L-Ala-OH H2OFmoc-L-Lys(sc)-OH is a compound wherein sc stands for the side chain (AEEAc-AEEAc-y-Glu(OtBu)-tBu-O- 19-carboxynonadecanoyl).

[0190] The cleavage of the peptide fragment F3 or F3a from the resin can be accomplished with a diluted TFA in combination with water and at least one scavenger.

[0191] A solution of 2% TFA and 1% TIS in CH2Cl2was found to be suitable, although it is within the skilled person in the art to vary the composition and the values as is considered appropriate.

[0192] Preparation of Peptide Fragment F4 or F4a:

[0193] The peptide fragment F4, in particular the functionalized peptide fragment F4a

[0194] H2N-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34- Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2

[0195] (F4a) (SEQ ID NO: 6)

[0196] can be prepared according to the scheme below.

[0197] Oxyma Pure EDCI, DIPEA DMAc

[0198] HNEt2DMAc

[0199]

[0200] The Fmoc-Trp25- / / -Gly29-OH fragment is coupled with the H-Gly30- / / -Ser39-NH2 fragment under LPPS conditions using the combination of (ethyl-cyano (hydroximino)acetate) (OxymaPure) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC1) as coupling agent. The reaction is performed in the presence of N, N-diisopropylethylamine as organic base and dimethylacetamide as organic solvent. The Fmoc group can then be removed with diethylamine in the presence of dimethylacetamide as organic solvent. The resulting peptide fragment F4a can be isolated form the reaction mixture by filtration.

[0201] Preparation of Peptide Fragment Fl or Fla according to Route 2

[0202] The coupling of the functionalized peptide fragment F5

[0203] PROT-HN-Ala18-Gln19-Lys20(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-OH

[0204] (F5) (SEQ ID NO:15)

[0205] wherein PROT is a protecting group;

[0206] with a functionalized peptide fragment F6

[0207] H2N-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2

[0208] (F6) (SEQ ID NO:16)

[0209] and the subsequent removal of the protecting group PROT is in general performed in the presence of a coupling agent, an organic base and an organic solvent.

[0210] Suitable coupling agents can be selected from those listed above. Pentafluorophenyl diphenylphosphinate (FDPP) was found to be a favorable coupling agent.

[0211] The organic base is usually selected from those mentioned above. Preferably, the organic base is A, A-diisopropylethylamine and the preferred organic solvent is N, N-dimethylacetmaide.

[0212] The reaction temperature can be chosen between -10°C and 30°C, typically the process is run at ambient temperature.The protecting group PROT is an amino protecting group. Typically the Fmoc ((9H-fluoren-9-yl)methoxy)carbonylamino) protecting group is used.

[0213] The removal of Fmoc can be accomplished with an organic base, such as with piperidine or diethylamine, preferably diethylamine.

[0214] In a preferred aspect, the peptide fragment Fl has the formula Fla

[0215] H2N- Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2

[0216] (Fla) (SEQ ID NO:4),

[0217] and the preparation comprises

[0218] coupling of a functionalized peptide fragment F5a

[0219] Fmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-OH

[0220] (F5a) (SEQ ID NO:7)

[0221] with a functionalized peptide fragment F6a

[0222] H2N-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2

[0223] (F6a) (SEQ ID NO: 8)

[0224] and the subsequent removal of the Fmoc protecting group.

[0225] Preparation of Peptide Fragment F5 or F5a

[0226] The peptide fragment F5, in particular the functionalized peptide fragment F5a

[0227] Fmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-OH

[0228] (F5a) (SEQ ID NO:7)is prepared under common Fmoc-solid phase peptide synthesis conditions on a suitable resin, preferably on the 2-chlorotrityl resins (2-CTC-resins).

[0229] The combination of benzotriazol- 1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) with (ethyl-cyano (hydroximino)acetate) (OxymaPure) for cycle 2 and the combination of A, A'-diisopropylcarbodiimide (DIC) with (ethyl-cyano (hydroximino)acetate) (OxymaPure) for the other cycles has been proven successful as coupling agents.

[0230] Except for cycle 2 no organic base is required for the assembly of peptide fragment F5 or F5a. For cycle 2 an organic base, usually selected from those mentioned above, preferably, A, A-diisopropylethylamine can be used.

[0231] Preferred organic solvent is N,N-dimethylformamide.

[0232] The coupling temperature is chosen between 0° C and 40°C, typically the process is run at 30°C.

[0233] The preparation of functionalized peptide fragment F5, in particular fragment F5a, comprises sequential coupling of the following functionalized building blocks on a resin, particularly on a 2-chlorotrityl resin.

[0234] Aminoacid # Cycle# Building Block

[0235] 2-chlorotrityl resin

[0236] 28 1 Fmoc-L-Ala-OH·H2O

[0237] 27 2 Fmoc-L-Ile-OH

[0238] 26 3 Fmoc-L-Leu-OH

[0239] 25 4 Fmoc-L-Trp(Boc)-OH

[0240] 24 5 Fmoc-L-Gln(Trt)-OH

[0241] 23 6 Fmoc-L-Val-OH

[0242] 22 7 Fmoc-L-Phe-OH

[0243] 21 8 Fmoc-L-Ala-OH H2O

[0244] 20 9 Fmoc-L-Lys(sc)-OH

[0245] 19 10 Fmoc-L-Gln(Trt)-OH

[0246]

[0247] 18 11 Fmoc-L-Ala-OH H2O

[0248] The cleavage of the peptide fragment F5 or F5a from the resin can be accomplished with a diluted TFA in combination with water and at least one scavenger.A solution of 2% TFA and 1% TIS in CH2Cl2was found to be suitable, although it is within the skilled person in the art to vary the composition and the values as is considered appropriate.

[0249] Preparation of Peptide Fragment F6 or F6a:

[0250] The peptide fragment F6, in particular the functionalized peptide fragment F6a

[0251] H2N-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38- Ser39(OtBu)-NH2

[0252] (F6a) (SEQ ID NO: 8)

[0253] is be prepared according to the scheme below:

[0254] HOBt, EDCI DIPEA DMAc

[0255] o

[0256] H2, Pd / CCH2Cl2v O

[0257]

[0258] F6aThe Cbz-Gly29- / / -Gly34-OH fragment is coupled with a H-Ala35- / / -Ser39-NH2 fragment under LPPS conditions using the combination hydroxybenzotriazole (HOBt) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC1) as coupling agent. The reaction is performed in the presence of A, A-diisopropylethylamine as organic base and dimethylacetamide as organic solvent. The Cbz group can then be removed by hydrogenation with hydrogen in the presence of a Pd / C catalyst at a hydrogen pressure of about 5 bar and at ambient temperature in methylene chloride / dimethylacetamide as organic solvent. The resulting peptide fragment can be isolated form the reaction mixture by filtration.

[0259] Preparation of Peptide Fragment F2 of F2a

[0260] The peptide fragment F2, in particular the functionalized peptide fragment F2a

[0261] X1-β-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)-Tyr10-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-OH

[0262] (F2a) (SEQ ID NO: 9)

[0263] is prepared under common Fmoc-solid phase peptide synthesis conditions on a suitable resin, preferably on the 2-chlorotrityl resins (2-CTC-resins).

[0264] The combination of benzotriazol- 1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) with (ethyl-cyano (hydroximino)acetate) (OxymaPure) for cycle 2 and the combination of A, A'-diisopropylcarbodiimide (DIC) with (ethyl-cyano (hydroximino)acetate) (OxymaPure) for the other cycles has been proven successful as coupling agents.

[0265] Except for cycle 2 no organic base is required for the assembly of peptide fragment F2 or F2a. For cycle 2 an organic base, usually selected from those mentioned above, preferably, A, A-diisopropylethylamine can be used.

[0266] Preferred organic solvent is A, A-dimethylformamide.

[0267] The coupling temperature is chosen between 0° C and 40°C, typically the process is run at 30°C.

[0268] The preparation of functionalized peptide fragment F2, in particular fragment F2a, comprises sequential coupling of the following functionalized building blocks on a resin, particularly on a 2-chlorotrityl resin.Aminoacid # Cycle# Building Block

[0269] 2-chlorotrityl resin

[0270] 17 1 Fmoc-L-Ile-OH

[0271] 16 2 Fmoc-L-Lys(Boc)-OH

[0272] 15 3 Fmoc-L-Asp(OtBu)-OH

[0273] 14 4 Fmoc-L-Leu-OH

[0274] 12-13 5 Fmoc-Ile-Aib-OH

[0275] 11 6 Fmoc-L-Ser(OtBu)-OH

[0276] 10 7 Fmoc-L-Tyr(OtBu)-OH

[0277] 9 8 Fmoc-L-Asp(OtBu)-OH

[0278] 8 9 Fmoc-L-Ser(OtBu)-OH

[0279] 7 10 Fmoc-L-Thr(OtBu)-OH

[0280] 5-6 11 Fmoc-Thr-Phe-OH

[0281] 1-4 X-P-Ala-Glu-Gly-OH, wherein

[0282]

[0283] CN

[0284] The cleavage of the peptide fragment F2 or F2a from the resin can be accomplished with a diluted TFA in combination with water and at least one scavenger.

[0285] A solution of 2% TFA and 1% TIS in CH2Cl2was found to be suitable, although it is within the skilled person in the art to vary the composition and the values as is considered appropriate.

[0286] For purification the precipitated crude peptide can be dissolved in water / AcOH / MeCN for instance in a ratio of 60:35:5 and at a peptide net concentration of e.g. 20 g / L. If needed, the solution can be pre-treated by stirring it at ambient conditions for 20 ± 4 h to facilitate decarboxylation at Trp25. Prior to loading, the peptide solution can be filtered over a filter with a suitable pore size, e.g. 0.45 pm.

[0287] Purification can then be performed using preparative reversed-phase HPLC using suitable stationary phases such as for instance the Kromasil-100-10-C18 ) in a multiple sequential chromatographic approach. 2 to 4 sequences, preferably 3 sequences were found to be appropriate to achieve a high purity peptide product.The purification process is usually run on an automated pump skid controlling the composition of the mobile phase mixture and enabling manual fractionation (UV-based) and collecting fractions by defined time intervals.

[0288] The product can be eluted using a linear gradient, step gradient or a combination of both with increasing or partially constant organic content. The peptide can be eluted from the column and the effluent is fractionated and assayed for purity using reversed phase HPLC-UV analysis. The fractions can finally be pooled after analysis based on their purity.

[0289] Fractions not meeting the pooling criteria may be combined for recycling injections.

[0290] In one further aspect, the invention relates to the following functionalized peptide fragments:

[0291] H2N- Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-Y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2

[0292] (Fla) (SEQ ID NO:4),

[0293] Fmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-OH

[0294] (F3a) (SEQ ID NO: 5),

[0295] H2N-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2

[0296] (F4a) (SEQ ID NO:6),

[0297] Fmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28OH

[0298] (F5a) (SEQ ID NO:7),

[0299] H2N-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2

[0300] (F6a) (SEQ ID NO: 8), and / orX1-β-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)-Tyr10-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-OH

[0301] (F2a) (SEQ ID N0:9),

[0302] wherein X is

[0303]

[0304] In still one further aspect, the invention relates to the following functionalized peptide fragments on the resin. These are the following:

[0305] Fmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-O-2-chlorotrityl resin

[0306] (F3b) (SEQ ID NO: 10),

[0307] Fmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-O-2-chlorotrityl resin

[0308] (F5b) (SEQ ID NO: 11),

[0309] X1-β-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)-Tyr10-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-O-2-chlorotrityl resin

[0310] (F2b) (SEQ ID NO: 12),

[0311] wherein X is

[0312]

[0313] CNExamples

[0314] Abbreviations:

[0315] Boc tert-butoxy carbonyl

[0316] DIC N, N'-diisopropylcarbodiimide

[0317] DIPEA diisopropylethylamine

[0318] DMAc dimethylacetamide

[0319] DMF N, N-dimethylformamide

[0320] EtOAc ethyl acetate

[0321] Fmoc 9-fluorenylmethoxycarbonyl

[0322] IPE diisopropylether

[0323] ivDde 1 -(4, 4-dimethyl-2,6-di oxocyclohex- 1 -ylidene)- 3 -methylbutyl

[0324] MTBE methyl tert, -butyl ether

[0325] Mpe 3-methyl-pentyl

[0326] OxymaPure ethyl 2-cyano-2-(hydroxyimino)acetate

[0327] 2-PrOH 2-propanol

[0328] tBu tert, -butyl

[0329] sc side chain (AEEAc-AEEAc-y-Glu(OtBu)-tBu- O- 19-carboxynonadecanoyl) TFA trifluoroacetic acid

[0330] TIS triisopropylsilane

[0331] Trt trityl1. Fragment Approach Routes

[0332] Route 1

[0333] sidechain F4

[0334] 1. PyAOP, Oxyma Pure, DMF 2.then Fmoc deprotection

[0335] sidechain FDPP, DIPEA - P X1 -ZZ-Ser39— \ F1

[0336] DMAc X NH2- / ° sidechain

[0337]

[0338] X1- / / -lle17— - OH

[0339] F2

[0340] Route 2

[0341]

[0342] sidechain F6

[0343] F5

[0344] 1. FDPP, DIPEA, DMAc

[0345] 2. then Fmoc deprotection

[0346]

[0347] ,0

[0348]

[0349] NH

[0350] sidechain FDPP, DIPEA - Xl-ZZ-Ser

[0351]

[0352] 39

[0353] DMAc - side -chainNH2 - P

[0354]

[0355] X1-ZZ-lle17—

[0356] OH

[0357] F2Fragments Fl, F2, F3, F4, F5 and F6 are functionalized peptide fragments, meaning that functional groups of respective amino acids are protected with suitable protecting groups.

[0358] 2. Route 1

[0359] 2,1 LPPS of Fragment H-Trp25- / / -Ser39-NH2 (Fmoc Route)

[0360] 2.1.1 Synthesis of H-Pro37-Pro33-Ser' (tBu)-NPh

[0361] o isobutyl chloroformate O O NMM, THF CbzHN. A HC2, Pd / CbzHNY^OH - ► NH2

[0362] then aq. NH3THF ^O'BuXOfBu

[0363] 1 M LiOH HOBt,

[0364] EDCI THF DIPEA DMAc H2, Pd / C, TsOH·H2O DMAc

[0365]

[0366] Cbz-Ser(tBu)-OH (50.0 g, 0.17 mol, 1.0 equiv) was dissolved in THF (500 mL, 10 vol.) and the resulting solution was cooled to -15 °C. NMM (17.2 g, 0.17 mol, 1.0 equiv) was added at this temperature, followed by isobutyl chloroformate (23.1 g, 0.17 mol, 1.0 equiv). The resulting mixture was stirred for 15 min before aq. NH3 solution (28 w%, 15.4 g, 0.25 mol, 1.5 equiv) was added dropwise. The reaction mixture was allowed to warm to ambient temperature. EtOAc (500 mL, 10 vol) and water (750 mL, 15 vol.) were added, the mixture was stirred for 15 min and the phases were separated. The organic layer was sequentially washed with 8% aq. NaHCCL solution (500 mL, 10 vol.) and 15% aq. NaCl solution

[0367] (250 mL, 5 vol.), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was slurried in MTBE (150 mL, 3 vol.), filtered and dried to afford Cbz-Ser(tBu)-NH2 (46.4 g, 99.2% content by NMR, 92.3% yield, purity 99.8% area%) as a white solid.

[0368] 'H NMR (400 MHz, DMSO-t / 6): d 7.42-7.26 (m, 6H), 7.12-7.07 (m, 2H), 5.04 (d, J= 2.4 Hz, 2H), 4.08-4.03 (m, 1H), 3.56-3.41 (m, 2H), 1.11 (s, 9H).

[0369] Cbz-Ser(tBu)-NH2 (27.6 g, 0.10 mol, 1.0 equiv) and 10% wet Pd / C (5.52 g, 20 w%) were taken up in DMAc (110 mL, 4 vol.) and the resulting mixture was stirred under a 0.5 MPa H2atmosphere at ambient temperature for 2 h. The mixture was then filtered, and the filter cake was washed with DMAc (28 mL, 1 vol.). The filtrate containing H-Ser(tBu)-NH2 as a solution in DMAc was directly used in the following transformation (100% yield assumed).

[0370] Cbz-Pro-OH (50.0 g, 200.6 mmol, 1.0 equiv) and H-Pro-OMe (39.9 g, 240.7 mmol,

[0371] 1.2 equiv) were taken up in DMAc (200 mL, 4 vol.). HOBt (32.5 g, 240.7 mmol, 1.2 equiv) was added and the mixture was cooled to 0 °C before EDCI (46.2 g, 240.7 mmol, 1.2 equiv) was added in portions. DIPEA (31.1 g, 240.7 mmol, 1.2 equiv) was added dropwise, and the resulting reaction mixture was allowed to warm to ambient temperature. Upon completion of the reaction, 5% aq. H3PO4 solution (100 mL, 2 vol.), water (500 mL, 10 vol.) and CH2Cl2(200 mL, 4 vol.) were added, the mixture was stirred for 15 min, and the phases were separated. The aqueous phase was extracted with CH2Cl2(250 mL, 5 vol.). The combined organic phases were sequentially washed with 8% aq. NaHCCL solution (200 mL, 4 vol.) and 15% aq. NaCl solution (200 mL, 4 vol.), dried over ISfeSCL, filtered, and concentrated under reduced pressure to afford Cbz-Pro-Pro-OMe (122.3 g, 57.4% content by NMR, 97.1% yield, purity 96.9 area%) as a white solid.

[0372] 1H NMR (400 MHz, CDCl3): d 7.29-7.15 (m, 5H), 5.11-4.90 (m, 2H), 4.52-4.46 (m, 1H), 4.38-4.27 (m, 1H), 3.76-3.71 (m, 1H), 3.64-3.38 (m, 6H), 2.15-2.01 (m, 3H), 1.83-1.71 (m, 5H).

[0373] Cbz-Pro-Pro-OMe (25.0 g, 69.4 mmol, 1.0 equiv) was taken up in THF (125 mL, 5 vol.) and water (125 mL, 5 vol.) and the mixture was cooled to 0 °C. 1 M LiOH (128 mL, 128 mmol, 1.85 equiv) was added dropwise and the reaction mixture was then allowed to warm to ambient temperature and was stirred at this temperature. Upon completion of the reaction, 5% aq. H3PO4 solution (125 mL, 5 vol.) was added to adjust the pH to 1-2. The resulting suspension was filtered and the filtrate was extracted with EtOAc (2 x 125 mL, 2 x 5 vol.). The combined organic phases were concentrated under reduced pressure and the residue was slurried in TBME (250 mL, 10 vol.). The suspension was filtered and the filter cake was slurried in MeCN (250 mL, 10 vol.). The suspension was filtered and the filter cake was dried to afford Cbz-Pro-Pro-OH (19.4 g, 96.4% content by NMR, 77.9% yield, purity 99.4 area%) as a white solid.

[0374] 'HNMR (400 MHz, DMSO-t / 6): d 7.44-7.18 (m, 5H), 5.10-4.87 (m, 2H), 4.53-4.49 (m, 1H), 4.26-4.15 (m, 1H), 4.26-4.19 (m, 4H), 1.94-1.61 (m, 8H) (COO / / not visible).

[0375] LC-MS (ESI+): mass calculated for C18H23N2O5 [(M+H)+] 347.16, found 347.27.A solution of H-Ser(tBu)-NH2 in DMAc (135 mmol, 1.2 equiv) was added to Cbz-Pro-Pro-OH (39.0 g, 113 mmol, 1.0 equiv). HOBt (18.3 g, 135 mmol, 1.2 equiv) was added, and the mixture was cooled to 0 °C before EDCI (25.9 g, 135 mmol, 1.2 equiv) was added in portions. DIPEA (34.9 g, 270 mmol, 2.4 equiv) was added dropwise and the reaction mixture was then allowed to warm to ambient temperature and was stirred at this temperature. Upon completion of the reaction, CH2Cl2(390 mL, 10 vol.) and 8% aq. NaHCCL solution (390 mL, 10 vol.) were added, and the mixture was stirred for 15 min. The phases were separated, and the aqueous phase was extracted with CH2CI2 (2 x 195 mL, 2 x 5 vol.). The combined organic phases were sequentially washed with 8% aq. NaHCCL solution (390 mL, 10 vol.), 5% aq. H3PO4 solution (390 mL, 10 vol.) and 15% aq. NaCl solution (195 mL, 5 vol.), dried over Na2SO4, filtered, and concentrated under reduced pressure to afford Cbz-Pro-Pro-Ser(tBu)-NH2 (58.1 g, 72.8% content by NMR, 76.9% yield, purity 98.4 area%) as a colorless oil.

[0376] 1H NMR (400 MHz, DMSO-6): d 7.55 (d, J= 7.8 Hz, 1H), 7.42-7.08 (m, 9H), 5.07-4.88 (m, 2H), 4.54-4.52 (m, 1H), 4.39-4.31 (m, 1H), 4.48-4.14 (m, 1H), 3.57-3.41 (m, 6H), 1.91-1.78 (m, 6H), 1.11 (s, 9H).

[0377] LC-MS (ESI+): mass calculated for C25H37N4O6 [(M+H)+] 489.27, found 489.44.

[0378] Cbz-Pro-Pro-Ser(tBu)-NH2 (31.3 g, 64.1 mmol, 1.0 equiv) was taken up in DMAc (125 mL, 4 vol.). TsOH H2O (12.8 g, 67.3 mmol, 1.05 equiv) and 10% wetPd / C (6.26 g, 20 w%) were added and the mixture was stirred at ambient temperature for 2 h under a 0.5 MPa

[0379] H2 atmosphere. Upon completion of the reaction, the mixture was filtered, and the filter cake was washed with DMAc (31 mL, 1 vol.). The filtrate containing H-Pro-Pro-Ser(tBu)-NH2 as solution in DMAc was directly used in the following transformation (100% yield assumed).

[0380] 2.1.2 Synthesis of Cbz-Ala35-Pro36-OH

[0381] nH-Pro-OMe HCI o

[0382] II HOBt, EDCI, DIPEA ll< I CbzHN^ Jlu! - - CbzHN^XX

[0383] OH E DMAc Q

[0384] 1 M LiOH THF / H2O

[0385] CbzHN

[0386]

[0387] OH-Pro-OMe HC1 (44.5 g, 269 mmol, 1.2 equiv) and Cbz-Ala-OH (50.0 g, 224 mmol, 1.0 equiv) were taken up in DMAc (200 mL, 4 vol.). HOBt (36.3 g, 269 mmol, 1.2 equiv) was added and the mixture was cooled to 0 °C. EDCI (51.5 g, 269 mmol, 1.2 equiv) was added in portions, followed by the dropwise addition of DIPEA (34.7 g, 269 mmol, 1.2 equiv). The reaction mixture was allowed to warm to ambient temperature. Upon completion of the reaction, 5% aq. H3PO4 solution (100 mL, 2 vol.) was added to the reaction mixture to adjust the pH to 6. CH2CI2 (200 mL, 4 vol.) and water (500 mL, 10 vol.) were added, the mixture was stirred for 15 min and the phases were separated. The aqueous phase was extracted with CH2Cl2(200 mL, 4 vol.) and the combined organic layers were sequentially washed with 8% aq. NaHCO3 solution (200 mL, 4 vol.) and sat. aq. NaCl solution (200 mL, 4 vol.), dried over Na2SO4, filtered, and concentrated under reduced pressure to afford Cbz-Ala-Pro-OMe (103.2 g, 68.0% content by NMR, 93.8% yield, purity 95.7 area%) as a colorless oil.

[0388] ¹H NMR (400 MHz, CDCl3): δ 7.32-7.28 (m, 6H), 5.07 (s, 2H), 4.55-4.48 (m, 2H), 3.70 (s, 3H), 3.65-3.58 (m, 2H), 2.27-2.10 (m, 1H), 2.05-1.88 (m, 3H), 1.38 (d, J= 6.9 Hz, 3H).

[0389] LC-MS (ESI+): mass calculated for C17H23N2O5 [(M+H)+] 335.16, found 335.33.

[0390] Cbz-Ala-Pro-OMe (58.8 g, 120 mmol, 1.0 equiv) was dissolved in THF (180 mL, 3 vol.) at ambient temperature. Water (180 mL, 3 vol.) was added and the resulting mixture was cooled to 0 °C. 1 M LiOH (220 mL, 220 mmol, 1.85 equiv) was added dropwise and the resulting reaction mixture was stirred at this temperature for 2 h. Upon completion of the reaction, 5% aq. H3PO4 solution was added at 0 °C to adjust the pH to 2-3, during which the mixture turned into a white suspension. The mixture was extracted with EtOAc (2 x 300 mL, 2 x 5 vol.), the phases were separated, and the organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to afford an off-white gum. This residue was slurried in MeCN (600 mL, 10 vol) at ambient temperature for 1 h. The suspension was filtered, and the filter cake was dried under a stream of nitrogen for 16 h to afford the Cbz-Ala-Pro-OH (29.8 g, 98.2% content by NMR, 75.0% yield, purity 99.8 area%) as a white solid.

[0391] ¹H NMR (400 MHz, DMSO-d6): δ 12.47 (s, 1H), 7.51 (d, J = 7.5 Hz, 1H), 7.44-7.27 (m, 5H), 5.10-4.96 (m, 2H), 4.39-4.14 (m, 2H), 3.67-3.29 (m, 2H), 2.21-2.06 (m, 1H), 1.99-1.78 (m, 3H), 1.23-1.14 (d, J = 8.0 Hz, 3H).

[0392] LC-MS (ESI+): mass calculated for C16H21N2O5 [(M+H)+] 321.14, found 321.28.2.1.3 Synthesis of Fragment H-Ala35- / / -Ser39-NH2

[0393] EDCI, Oxyma Pure

[0394] DIPEA, DMAc

[0395] H2, Pd / C

[0396] TsOH·H2O, DMAc

[0397]

[0398] Cbz-Ala-Pro-OH (14.9 g, 46.4 mmol, 1.0 equiv) and the above solution of H-Pro-Pro-Ser(tBu)-NH2 in DMAc (55.7 mmol, 1.2 equiv) were combined and diluted with DMAc (60 mL, 4 vol.). Oxyma Pure (9.90 g, 69.7 mmol, 1.5 equiv) was added and the mixture was cooled to 0 °C. EDCI (29.9 g, 69.7 mmol, 1.5 equiv) was added in portions. DIPEA (7.20 g, 55.7 mmol, 1.2 equiv) was added and the reaction mixture was allowed to warm to ambient temperature. Upon completion of the reaction, CH2Cl2 (600 mL, 10 vol.) was added, followed by 8% aq. NaHCO3 solution (600 mL, 10 vol.), and the mixture was stirred for 15 min before separating the phases. The aqueous phase was extracted with CH2Cl2(2 x 300 mL, 2 x 5 vol.). The combined organic phases were sequentially washed with 8% aq NaHCCL solution (600 mL, 10 vol.), 5% aq. H3PO4 solution (600 mL, 10 vol.), 8% aq. NaHCCh solution (600 mL, 10 vol.), and 15%. aq. NaCl solution (300 mL, 5 vol.), then dried over Na2SO4, filtered, and concentrated under reduced pressure to afford a colorless oil. This residue was dissolved in THF (180 mL, 3 vol.) and the resulting solution was added dropwise to heptane (420 mL, 7vol.). The resulting suspension was stirred for at ambient temperature 1 h before it was filtered and the filter cake was dried to afford Cbz-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 (26.9 g, 90.2% content by NMR, 79.7% yield, purity 98.8 area%) as a solid.

[0399] ¹H NMR (400 MHz, DMSO-d6): δ 7.55 (d, J = 8.0 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.41-7.27 (m, 6H), 7.24-7.03 (m, 2H), 5.09-4.94 (m, 2H), 4.64-4.51 (m, 2H), 4.39-4.26 (m, 2H), 4.17-4.13 (m, 1H), 3.65 -3.44 (m, 9H), 2.19-2.12 (m, 2H), 1.91-1.76 (m, 8H), 1.16 (d, J = 6.9 Hz, 3H), 1.11 (s, 9H).

[0400] LC-MS (ESI+): mass calculated for C33H49N6O8 [(M+H)+] 657.36, found 657.52.

[0401] Cbz-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 (24.4 g, 37.1 mmol, 1.0 equiv) was taken up in DMAc (97 mL, 4 vol.). 10% wet Pd / C (4.87 g, 20 w%) was added. The mixture was stirred at ambient temperature for 2 h under a 0.5 MPa H2 atmosphere. Upon completion of the reaction, the mixture was filtered and the filter cake was washed with DMAc (73 mL, 3 vol.). The filtrate was partially concentrated and the resulting DMAc solution containing H-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 was directly used in the following transformation (100% yield assumed).

[0402] 2.1.4 Synthesis of H-Ser32(tBu)-Ser33(tBu)-Gly34-OMe

[0403] H-Gly-OMe·HCl, HBTU, DIPEA, CH2Cl2 / DMAc / MTBE, H2 Pd / C, TsOH·H2O

[0404] Cbz-Ser(tBu)-OH, HBTU, DIPEA, DMAc

[0405] H2, Pd / C, TsOH·H2O

[0406] DMAc

[0407]

[0408] Cbz-Ser(tBu)-OH (100 g, 339 mmol, 1.0 equiv) and H-Gly-OMe (51.0 g, 406 mmol, 1.2 equiv) were taken up in CH2Cl2 (800 mL, 8 vol.). The mixture was cooled to 0 °C and HBTU (155 g, 407 mmol, 1.2 equiv) was added in portions. DIPEA (109 g, 847 mmol, 2.5 equiv) was added dropwise, and the resulting reaction mixture was allowed to warm to ambient temperature and was stirred at this temperature for 5 h. Upon completion of the reaction, 5% aq. H3PO4 solution (400 mL, 4 vol.) was added and the mixture was stirred for 20 min, during which precipitation was observed. The solid was removed by filtration and the phases were separated. The organic phase was sequentially washed with 5% aq. H3PO4 solution (500 mL, 5 vol.), 8% aq. NaHCO3 solution (3 x 300 mL, 3 x 3 vol.) and 15% aq. NaCl solution (500 mL, 5 vol.), dried over Na2SO4, filtered, and concentrated under reduced pressure to afford Cbz-Ser(tBu)-Gly-OMe (221 g, 52.9% content by NMR, 94.2% yield, purity 98.2 area%) as a syrup.

[0409] ¹H NMR (400 MHz, DMSO-d6): δ 8.36-8.33 (m, 1H), 7.38-7.26 (m, 6H), 5.09-5.01 (m, 2H), 4.20-4.15 (m, 1H), 3.93-3.78 (m, 2H), 3.63 (s, 3H), 3.57-3.42 (m, 2H), 1.12 (s, 9H).

[0410] Cbz-Ser(tBu)-Gly-OMe (74.0 g, 201 mmol, 1.0 equiv) was taken up in DMAc (148 mL, 2 vol.) and MTBE (148 mL, 2 vol.). TsOH H2O (36.4 g, 211 mmol, 1.05 equiv) and 10% wet Pd / C (14.8 g, 20 w%) were added. The resulting reaction mixture was stirred at ambient temperature under a 0.5 MPa H2 atmosphere. Upon completion of the reaction, the mixture was filtered and the filter cake was washed with DMAc (74 mL, 1 vol.). The filtrate was partially concentrated. The resulting DMAc solution containing H-Ser(tBu)-Gly-OMe was directly used in the following transformation (100% yield assumed).

[0411] LC-MS (ESI+): mass calculated for C10H21N2O4 [(M+H)+] 233.15, found 233.25.

[0412] Cbz-Ser(tBu)-OH (45.0 g, 152 mmol, 1.0 equiv) and the above solution of H-Ser(tBu)-Gly-OMe in DMAc (183 mmol, 1.2 equiv) were combined. The mixture was cooled to 0 °C and HBTU (69.3 g, 183 mmol, 1.2 equiv) was added. DIPEA (49.2 g, 381 mmol, 2.5 equiv) was added dropwise and the reaction mixture was allowed to warm to ambient temperature and was stirred at this temperature for 15 h. Upon completion of the reaction, 5% aq.

[0413] H3PO4 solution (180 mL, 4 vol.) and water (360 mL, 8 vol.) were added and the mixture was stirred for 20 min, during which precipitation was observed. The solid was collected by filtration and slurried with 8% aq. NaHCO3 solution (2 x 450 mL, 2 x 10 vol.). The solid was again collected by filtration and washed with water (135 mL, 3 vol.) before it was dissolved in MeCN (360 mL, 8 vol.) and filtered to obtain a clear solution. Water (1080 mL, 24 vol.) was added dropwise to this solution and the mixture was stirred for 2 h to precipitate the product. The solid was isolated by filtration and dried under a stream of nitrogen to afford theCbz-Ser(tBu)-Ser(tBu)-Gly-OMe (67.9 g, 97.9% content by NMR, 85.9% yield, purity 99.2 area%) as a white solid.

[0414] ¹H NMR (400 MHz, DMSO-d6): δ 8.30 (t, J = 5.9 Hz, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.46-7.24 (m, 6H), 5.06 (s, 2H), 4.44-4.39 (m, 1H), 4.18-4.16 (m, 1H), 3.88 (d, J= 5.8 Hz, 2H), 3.63 (s, 3H), 3.52-3.44 (m, 4H), 1.12 (d, J = 4.0 Hz, 18H).

[0415] Cbz-Ser(tBu)-Ser(tBu)-Gly-OMe (63.7 g, 125 mmol, 1.0 equiv) and TsOH H2O (12.7 g, 131 mmol, 1.05 equiv) were taken up in DMAc (268 mL, 4 vol.). 10% wet Pd / C (12.8 g, 20 w%,) was added and the resulting reaction mixture was stirred at 30 °C for 4 h under a 0.5 MPa H2 atmosphere. Upon completion of the reaction, the mixture was filtered and the filter cake was washed with DMAc (64 mL, 1 vol.). The filtrate was partially concentrated. The DMAc solution containing H-Ser(tBu)-Ser(tBu)-Gly-OMe was directly used in the following transformation (100% yield assumed).

[0416] LC-MS (ESI+): mass calculated for C17H34N3O6 [(M+H)+] 376.24, found 376.42.

[0417] 2.1.5 Synthesis of Cbz-Gly30-Pro31-OH

[0418] Pfp-OH, EDCI, CH2Cl2; H-Pro-OH, BSA, THF

[0419]

[0420] Cbz-Gly-OH (50.0 g, 239 mmol, 1.0 equiv) and pentafluorophenol (Pfp-OH, 44.0 g, 287 mmol, 1.2 equiv) were dissolved in CH2CI2 (500 mL, 10 vol.). The solution was cooled to 0 °C and EDCI (55.0 g, 287 mmol, 1.2 equiv) was added in portions at this temperature. The resulting reaction mixture was allowed to warm to ambient temperature and was stirred at this temperature for 3 h. Upon completion of the reaction, the mixture was sequentially washed with 5% aq. H3PO4 solution (3 x 250 mL, 3 x 5 vol.) and sat. aq. NaCl solution (250 mL, 5 vol.), dried over Na2SO4, filtered, and concentrated under reduced pressure to afford Cbz-Gly-OPfp (106.0 g, 83.3% content by NMR, 98.5% yield, purity 98.7 area%) as a white solid.

[0421] ¹H NMR (400 MHz, DMSO-d6): δ 8.04 (t, J = 6.0 Hz, 1H), 7.47-7.26 (m, 5H), 5.10 (s, 2H), 4.30 (d, J = 6.0 Hz, 2H).

[0422] L-Proline (41.3 g, 353 mmol, 1.5 equiv) and BSA (97.2 g, 470 mmol, 2.0 equiv) were taken up in THF (880 mL, 10 vol.). The resulting mixture was stirred at 20 °C for 1.5 h before itwas cooled to 0 °C. Cbz-Gly-OPfp (88.3 g, 235 mmol, 1.0 equiv) was added in portions and the resulting reaction mixture was allowed to warm to ambient temperature and was stirred at this temperature. Upon completion of the reaction, water (880 mL, 10 vol.) was added, and the mixture was stirred for 15 min before the phases were separated. The aqueous phase was extracted with CH2Cl2(2 x 880 mL, 2 x 10 vol.). The combined organic phases were washed with 15% aq. NaCl solution (440 mL, 5 vol.), dried over Na2SO4, filtered, and concentrated under reduced pressure. EtOAc (440 mL, 5 vol.) was added to the residue and the mixture was concentrated to 1-2 vol. This procedure was repeated 5 times. Then, EtOAc (440 mL, 5 vol.) was added and the mixture was slurried for 30 minutes. The resulting solid was collected by filtration and dried under a stream of nitrogen to afford Cbz-Gly-Pro-OH (63.1 g, 99.4% content by NMR, 87.1% yield, purity 99.4 area%) as a solid.

[0423] ¹H NMR (400 MHz, DMSO-d6): δ 12.62 (s, 1H), 7.45-7.25 (m, 6H), 5.05 (d, J= 2.4 Hz, 2H), 4.25 (dd, J= 8.8, 3.3 Hz, 1H), 3.95-3.75 (m, 2H), 3.59-3.31 (m, 2H), 2.28-2.01 (m, 1H), 1.98-1.63 (m, 3H).

[0424] LC-MS (ESI+): mass calculated for C15H19N2O5 [(M+H)+] 307.13, found 307.23.

[0425] 2.1.6 Synthesis of Fragment Cbz-Gly30- / / -Gly34-OH

[0426] Oxyma Pure, EDCI, DIPEA, DMAc

[0427] LiOH·H2O, THF / H2O

[0428]

[0429] Cbz-Gly-Pro-OH (30.0 g, 98 mmol, 1.0 equiv) and the above solution of H-Ser(tBu)-Ser(tBu)-Gly-OMe in DMAc (118 mmol, 1.2 equiv) were combined and diluted with DMAc (120 mL, 4 vol.). Oxyma Pure (16.7 g, 118 mmol, 1.2 equiv) was added, and the mixture was cooled to 0 °C. EDCI (22.53 g, 118 mmol, 1.2 equiv) was added in portions, followed by the dropwise addition of DIPEA (15.2 g, 118 mmol, 1.2 equiv). The resulting reaction mixturewas allowed to warm to ambient temperature and was stirred at this temperature for 3 h. Upon completion of the reaction, 5% aq. H3PO4 solution (240 ml, 8 vol.) and heptane (240 ml, 8 vol.) were added, the phases were separated and the organic phase was discarded. The aqueous phase was extracted with CH2Cl2(2 x 210 mL, 2 x 7 vol.). The combined organic phases were sequentially washed with 5% aq. H3PO4 solution (150 mL, 5 vol.), 8% aq.

[0430] NaHCO3 solution (3 x 150 mL, 3 x 5 vol.), and 15% aq. NaCl solution (150 mL, 5 vol.), dried over Na2SO4, filtered, and concentrated under reduced pressure to afford fragment Cbz-Gly30- / / -Gly34-OMe (84.0 g, 69.9% content by NMR, 90.3% yield, purity 98.6 area%) as a colorless oil.

[0431] ¹H NMR (400 MHz, DMSO-d6): δ 8.29 (m, 1H), 8.20 (t, J = 5.9 Hz, 1H), 8.09 (d, J= 7.6 Hz, 1H), 7.59 (d, J= 8.0 Hz, 1H), 7.43-7.26 (m, 5H), 5.09-4.99 (m, 2H), 4.60-4.26 (m, 3H), 3.91-3.84 (m, 2H), 3.62 (s, 3H), 3.59-3.30 (m, 8H), 1.93-1.71 (m, 4H), 1.17-1.08 (m, 18H).

[0432] Cbz-Gly30- / / -Gly34-OMe (55.0 g, 83.0 mmol, 1.0 equiv) was dissolved in THF (275 mL, 5 vol.) and water (165 mL, 3 vol.) and the resulting mixture was cooled to 0 °C. A solution of LiOH·H2O (3.47 g, 83.0 mmol, 1.0 equiv) in water (110 mL, 2 vol.) was added dropwise and the resulting reaction mixture was stirred at this temperature for 5 h. Upon completion of the reaction, 3 M HCl was added to adjust the pH to 1-2 (approximately 55 mL, 1 vol.). The mixture was extracted with CH2Cl2 (2 x 275 mL, 2 x 5 vol.) and the combined organic phases were sequentially washed with 5% aq. H3PO4 solution (275 mL, 5 vol.) and 15% aq. NaCl solution (275 mL, 5 vol.), dried over Na2SO4, filtered, and concentrated to 1-2 vol. MTBE (275 mL, 5 vol.) was added and the mixture was again concentrated to 1-2 vol. This process was repeated four times before the mixture was concentrated to dryness. The resulting residue was slurried in EtOAc / MTBE (1:9 v / v, 550 mL, 10 vol.) for 1 h. The resulting solid was isolated by filtration and dried to afford fragment Cbz-Gly30- / / -Gly34-OH (51.1 g, 92.5% content by NMR, 87.9% yield, purity 99.4 area%) as a white solid.

[0433] ¹H NMR (400 MHz, DMSO-d6): δ 12.60 (s, 1H), 8.14-7.98 (m, 2H), 7.61 (d, J= 8.0 Hz, 1H), 7.41-7.23 (m, 6H), 5.08-4.99 (m, 2H), 4.60-4.23 (m, 3H), 3.83-3.77 (m, 2H), 3.58-3.24 (m, 8H), 2.08-1.72 (m, 4H), 1.12 (s, 9H), 1.11 (s, 9H).

[0434] LC-MS (ESI+): mass calculated for C31H48N5O10 [(M+H)+] 650.34, found 650.51.2.1.7 Synthesis of Fragment Fmoc-Trp25- / / -Gly29-OH

[0435] H-Gly-OMe-HCI

[0436] Oxyma Pure, EDCI, DIPEA, DMAc / MTBE; H2, Pd / C, TsOH·H2O, DMAc

[0437] Cbz-Ile-OH, Oxyma Pure, EDCI, DIPEA, DMAc / MTBE; H2, Pd / C, TsOH·H2O, DMAc Cbz-Leu-OH Oxyma Pure EDCI, DIPEA H2, Pd / C, TsOH·H2O DMAc / MTBE DMAc

[0438] Fmoc-Trp(Boc)-OH, HATU, DIPEA, DMAc / DMSO

[0439]

[0440] Cbz-Ala-OH (50.0 g, 224 mmol, 1.0 equiv) and H-Gly-OMe HC1 (34.1 g, 269 mmol, 1.2 equiv) were taken up in DMAc (200 mL, 4 vol.) and MTBE (200 mL, 4 vol.) and the resulting mixture was cooled to 0 °C. Oxyma Pure (38.2 g, 269 mmol, 1.2 equiv) and EDCI (51.6 g, 269 mmol, 1.2 equiv) were added at this temperature, followed by the dropwise addition of DIPEA (34.7 g, 269 mmol, 1.2 equiv). The reaction mixture was then allowed to warm to 20 °C and was stirred at this temperature for 3 h. Upon completion of the reaction, MTBE was removed under reduced pressure at 30-35 °C, and the mixture was diluted with EtOAc (400 mL, 8 vol.) and water (400 mL, 8 vol.). The phases were separated and the aqueous phase was extracted with EtOAc (2 x 200 mL, 2 x 4 vol.). The combined organic phases were sequentially washed with 8% aq. NaHCOs solution (500 mL, 10 vol.), 5% aq. H3PO4 solution (500 mL, 10 vol.), 8% aq. NaHCOs solution (500 mL, 10 vol.) and 24% aq.NaCl solution (250 mL, 5 vol.), dried over Na? SO4, filtered, and concentrated under reduced pressure to afford Cbz-Ala-Gly-OMe (61.4 g, 97.5% content by NMR, 91.2% yield, purity 99.9 area%) as a yellow solid.

[0441] ¹H NMR (400 MHz, CDCl3): δ 7.39-7.22 (m, 5H), 7.09 (d, J = 6.2 Hz, 1H), 5.84 (d, J= 7.8 Hz, 1H), 5.10 (q, J= 12.2 Hz, 2H), 4.36 (m, 1H), 3.99 (d, J= 5.5 Hz, 2H), 3.71 (s, 3H), 1.39 (d, J = 7.4 Hz, 3H).

[0442] Cbz-Ala-Gly-OMe (30.0 g, 101 mmol, 1.0 eq) was taken up in DMAc (120 mL, 4 vol.).

[0443] TsOH H2O (20.3 g, 107 mmol, 1.05 eq) and 10% wet Pd / C (6.0 g, 20 w%) were added and the resulting reaction mixture was stirred at 20 °C under a H2 atmosphere at ambient pressure for 4 h. Upon completion of the reaction, the mixture was filtered over celite and the filter cake was washed with DMAc (2 x 30 mL, 2 x 1 vol.). The resulting DMAc solution containing H-Ala-Gly-OMe was directly used in the following transformation (100% yield assumed).

[0444] LC-MS (ESI+): mass calculated for C6H13N2O3 [(M+H)+] 161.09, found 161.15.

[0445] Cbz-IIe-OH (45.4 g, 169 mmol, 1.0 equiv) and a solution of H-Ala-Gly-OMe in DMAc (203 mmol, 1.2 eq) and were taken up in DMAc (360 mL, 8 vol.) and MTBE (180 mL, 4 vol.) at 25 °C. The mixture was cooled to 0 °C. Oxyma Pure (28.9 g, 203 mmol, 1.2 equiv) and EDCI (35.7 g, 187 mmol, 1.1 equiv) were added at this temperature, followed by the dropwise addition of DIPEA (26.2 g, 203 mmol, 1.2 eq). The resulting reaction mixture was stirred at 0 °C for 4 h. Upon completion of the reaction, MTBE (540 mL, 12 vol.) was added and the mixture was stirred at 5 °C for 1 h. The resulting suspension was filtered, and the filter cake was washed with MTBE (2 x 90 mL, 2 x 2 vol.) and dried under a stream of nitrogen. The crude product was then slurried in MeCN (450 mL, 10 vol.) at 0 °C for 1 h and the resulting solid was isolated by filtration, washed with MeCN (2 x 90 mL, 2 x 2 vol.), and dried to afford Cbz-Ile-Ala-Gly-OMe (52.7 g, 99.2% content by NMR, 75.8% yield, purity 99.7 area%) as a white solid.

[0446] ¹H NMR (400 MHz, DMSO-d6): δ 8.32 (t, J = 5.9 Hz, 1H), 8.00 (d, J = 7.5 Hz, 1H), 7.41-7.21 (m, 6H), 5.03 (s, 2H), 4.40-4.30 (m, 1H), 3.96-3.76 (m, 3H), 3.62 (s, 3H), 1.78-1.63 (m, 1H), 1.48-1.33 (m, 1H), 1.22 (d, J = 7.0 Hz, 3H), 1.18-1.00 (m, 1H), 0.94-0.73 (m, 6H).

[0447] Cbz-Ile-Ala-Gly-OMe (20.0 g, 48.9 mmol, 1.0 equiv) was taken up in DMAc (80 mL, 4 vol.). TsOH·H2O (9.8 g, 51 mmol, 1.05 equiv) and 10% wet Pd / C (4.0 g, 20 w%) were added and the resulting reaction mixture was stirred at 20 °C under ambient H2 pressure for 4 h. Uponcompletion of the reaction, the mixture was filtered over celite and the filter cake was washed with DMAc (2 x 20 mL, 2 x 1 vol.). The resulting DMAc solution containing H-Ile-Ala-Gly-OMe was directly used in the following transformation (100% yield assumed).

[0448] Cbz-Leu-OH (10.8 g, 40.6 mmol, 1.0 equiv) and the above solution of H-Ile-Ala-Gly-OMe in DMAc (48.7 mmol, 1.2 equiv) were taken up in MTBE (40 mL, 4 vol.) at ambient temperature. The resulting mixture was cooled to 0 °C and Oxyma Pure (6.9 g, 49 mmol, 1.2 equiv) and EDCI (8.6 g, 45 mmol, 1.1 equiv) were added. DIPEA (6.3 g, 49 mmol, 1.2 equiv) was then added dropwise at 0 °C and the resulting reaction mixture was stirred at this temperature for 4 h. Upon completion of the reaction, MTBE (200 mL, 20 vol.) was added and the mixture was stirred at 0 °C for 1 h. The resulting suspension was filtered, and the filter cake was washed with MTBE (2 x 20 mL, 2 x 2 vol.) and dried. The resulting solid was then slurried in MeCN (150 mL, 15 vol.) at 0 °C for 1 h and the solid was isolated by filtration, washed with MeCN (2 x 30 mL, 2 x 3 vol.), and dried to afford Cbz-Leu-Ile-Ala-Gly-OMe (16.4 g, 97.3% content by NMR, 75.8% yield, purity >99.9 area%) as a white solid.

[0449] ¹H NMR (400 MHz, DMSO-d6): δ 8.30 (t, J = 5.9 Hz, 1H), 8.06 (d, J = 7.4 Hz, 1H), 7.74 (d, J= 8.8 Hz, 1H), 7.52 (d, J= 8.5 Hz, 1H), 7.40-7.25 (m, 5H), 5.12-4.95 (m, 2H), 4.37-4.27 (m, 1H), 4.21 (dd, J= 8.8, 7.3 Hz, 1H), 4.13-4.06 (m, 1H), 3.93-3.77 (m, 2H), 3.64 (s, 3H), 1.81-1.55 (m, 2H), 1.52-1.33 (m, 3H), 1.23 (d, J= 7.1 Hz, 3H), 1.15-0.95 (m, 1H), 0.91-0.70 (m, 12H).

[0450] Cbz-Leu-Ile-Ala-Gly-OMe (16.0 g, 30.4 mmol, 1.0 equiv) was taken up in DMAc (160 mL, 10 vol.). TsOH·H2O (6.60 g, 31.9 mmol, 1.05 equiv) and 10% wet Pd / C (3.2 g, 20 w%) were added and the resulting reaction mixture was stirred at 20 °C under ambient H2 pressure for 4 h. Upon completion of the reaction, DMAc (128 mL, 8 vol.) and DMSO (48 mL, 3 vol.) were added, and the mixture was filtered over celite. The filter cake was washed with DMAc (2 x 56 mL, 2 x 3.5 vol.) and DMSO (2 x 16 mL, 2 x 1 vol.) and the resulting solution containing H-Leu-Ile-Ala-Gly-OMe was directly used in the following transformation (100% yield assumed).

[0451] Fmoc-Trp(Boc)-OH (24.1 g, 45.8 mmol, 1.0 equiv) was added to a solution of H-Leu-Ile-Ala-Gly-OMe (59.5 mmol, 1.3 equiv) in DMAc and DMSO and the resulting mixture was cooled to 0 °C. HATU (20.9 g, 54.9 mmol, 1.2 equiv) was added, followed by the slow addition of DIPEA (14.8 g, 114 mmol, 2.5 equiv). The resulting reaction mixture was stirred at 0 °C for 4 h. Upon completion of the reaction, water (495 mL, 20 vol.) was slowly added over 1.5 h, maintaining the temperature below -5°C. The resulting suspension was then stirred for 1 hour at 0 °C before it was filtered. The filter cake was washed with water (2 x 22mL, 2 x 1 vol.) and dried to afford Fmoc-Trp(Boc)-Leu-Ile-Ala-Gly-OMe (38.9 g, 95.8% content by NMR, 90.9% yield, purity 98.5 area%) as a white solid.

[0452] ¹H NMR (400 MHz, DMSO-d₆): δ 8.27 (d, J = 7.0 Hz, 2H), 8.00 (d, J= 7.5 Hz, 2H), 7.88-7.70 (m, 5H), 7.58 (dd, J= 14.2, 6.8 Hz, 3H), 7.40-7.15 (m, 6H), 4.44-4.28 (m, 2H), 4.20 (t, J= 7.9 Hz, 1H), 4.16-4.07 (m, 2H), 3.91-3.72 (m, 2H), 3.59 (s, 3H), 3.14-2.85 (m, 3H), 1.79-1.34 (m, 15H), 1.20 (d, J= 7.0 Hz, 3H), 1.12-0.96 (m, 1H), 0.93-0.72 (m, 12H).

[0453] Fmoc-Trp(Boc)-Leu-Ile-Ala-Gly-OMe (10.0 g, 10.7 mmol, 1.0 equiv) was taken up in THF (300 mL, 30 vol.) and H₂O (70 mL, 7 vol.) and the resulting mixture was cooled to -5 °C. A solution of CaBr₂ (7.5 g, 37.6 mmol, 3.5 equiv) in water (70 mL, 7 vol.) was added at this temperature, followed by the addition of 1.0 MNaOH (31.3 mL, 31.3 mmol, 2.9 equiv). The resulting reaction mixture was stirred at -5 °C for 20 h. Upon completion of the reaction, the mixture was neutralized to pH 7 with 5% aq. H3PO4 solution (28 mL, 2.8 vol.). The resulting mixture was partially concentrated under reduced pressure at 20 °C to remove THF. MeCN (20 mL, 2 vol.) was added, and the mixture was acidified to pH 2-3 with 5% aq. H3PO4 solution (20 mL, 2 vol.) and stirred at 0 °C for 1 h. The resulting suspension was filtered and the filter cake was washed with water (2 x 20 mL, 2 x 2 vol.) and dried to afford Fmoc-Trp(Boc)-Leu-Ile-Ala-Gly-OH (9.83 g, 87.1% content by NMR, 90.8% yield, purity 98.5 area%) as a white solid.

[0454] ¹H NMR (400 MHz, DMSO-d₆): δ 8.34–8.21 (m, 2H), 8.03 (t, J= 7.2 Hz, 2H), 7.91-7.71 (m, 5H), 7.60 (app dd, J= 14.0, 6.7 Hz, 3H), 7.41-7.18 (m, 6H), 4.48-4.30 (m, 3H), 4.27-4.20 (m, 1H), 4.18-4.11 (m, 2H), 3.93-3.75 (m, 2H), 3.61 (s, 3H), 3.14-2.95 (m, 1H), 1.80-1.36 (m, 13H), 1.23 (d, J= 7.0 Hz, 3H), 1.12-0.96 (m, 1H), 0.93-0.74 (m, 12H

[0455]

[0456] ) (COO / / not visible).

[0457] LC-MS (ESI+): mass calculated for C₄₈H₆₁N₆O₁₀ [(M+H)⁺] 881.44, found 881.02.2.1.8 Synthesis of Fragment H-Gly30- / / -Ser39-NH 2

[0458] H2, Pd / C

[0459] CH2CI2

[0460]

[0461] Cbz-Gly30- / / -Gly34-OH (23.5 g, 36.2 mmol, 1.0 equiv) and a solution of H-Ala35- / / -Ser39-NH2 in DMAc (43.4 mmol, 1.2 equiv) were combined. HOBt (5.86 g, 43.4 mmol, 1.2 equiv) was added and the mixture was cooled to 0 °C. EDCI (8.32 g, 43.4 mmol, 1.2 equiv) was added in portions. DIPEA (5.61 g, 43.4 mmol, 1.2 equiv) was added dropwise and the reaction mixture was allowed to warm to ambient temperature and was stirred at this temperature for 3 h. Upon completion of the reaction, 5% aq. H3PO4 solution (188 mL, 8 vol.) and heptane (188 mL, 8 vol.) were added, the phases were separated, and the organic phase was discarded. The aqueous phase was extracted with CH2Cl2(2 x 188 mL, 2 x 8 vol.). The combined organic phases were sequentially washed with 5% aq. H3PO4 solution (188 mL, 8 vol.), 8% aq.

[0462] NaHCO₃ solution (118 mL, 5 vol.) and sat. aq. NaCl solution (118 mL, 5 vol.), dried over Na2SO4, filtered, and concentrated under reduced pressure to afford fragment Cbz-Gly30- / / -Ser39-NH2 (47.5 g, 77.0% content by NMR, 87.6% yield, purity 98.7 area%).¹H NMR (400 MHz, DMSO-d₆): δ 8.24 (d, J = 8 Hz, 1H), 8.08-7.90 (m, 3H), 7.79 (d, J= 8 Hz, 1H), 7.56 (d, J= 8 Hz, 1H), 7.22 (s, 1H), 7.11 (s, 1H), 4.62-4.31 (m, 8H), 4.18-4.14 (m, 1H), 3.90-3.73 (m, 5H), 3.65-3.37 (m, 17H), 2.16-1.82 (m, 18H), 1.17 (d, J= 8.0 Hz, 3H), 1.14-1.06 (m, 27H).

[0463] Cbz-Gly30- / / -Ser39-NH2 (30.0 g, 26.0 mmol, 1.0 equiv) was dissolved in CH2CI2 (240 mL, 8 vol.). 10% wet Pd / C (6.0 g, 20 w%) was added, and the resulting reaction mixture was stirred under a 0.5 MPa H2 atmosphere at ambient temperature for 5 h. Upon completion of the reaction, the mixture was filtered, and the filter cake was washed with CH2Cl2(60 mL, 2 vol.). The filtrate was concentrated to 2-3 vol. before it was added dropwise to MTBE (600 mL, 20 vol.) at ambient temperature. The resulting solid was isolated by filtration and the filter cake was washed with MTBE (2 x 60 mL, 2 x 2 vol.) and dried to afford fragment H-Gly30- / / -Ser39-NH2 (29.2 g, 78.0% content by NMR, 85.8% yield, purity 98.7 area%) as a solid.

[0464] LC-MS (ESI+): mass calculated for C48H82N11O13 [(M+H)+] 1020.86, found 1020.61.

[0465] 2.1.9 Synthesis of Fragment H-Trp25- / / -Ser39-NH

[0466] The reaction scheme is shown in FIG.l.

[0467] Fragment H-Gly30- / / -Ser39-NH2 (6.09 g, 5.97 mmol, 1.0 equiv) and fragment Fmoc-Trp25- / / -Gly29-OH (6.84 g, 7.76 mmol, 1.3 equiv) were taken up in DMAc (90 mL, 15 vol.). Oxyma Pure (1.32 g, 9.31 mmol, 1.56 equiv) was added and the resulting mixture was cooled to 0 °C. EDCI (1.79 g, 9.31 mmol, 1.56 equiv) was added in portions, followed by the dropwise addition of DIPEA (1.20 g, 9.31 mmol, 1.56 equiv). The resulting reaction mixture was allowed to warm to ambient temperature and was stirred at this temperature for 2 h. Upon completion of the reaction, the mixture was added dropwise to MTBE (450 mL, 75 V) and the resulting suspension stirred at ambient temperature for 30 min. The mixture was filtered, and the filter cake was washed with MTBE (24 mL, 4 vol.). The filter cake was slurried in 5% H3PO4 solution (60 mL, 10 V) for 1 h. The suspension was filtered and the filter cake was washed with water (3 x 12 mL, 3 x 2 vol.) and dried under a stream of nitrogen to afford fragment Fmoc-Trp25- / / -Ser39-NH2 (10.9 g, >99% yield, purity 95.9 area%) as a solid.

[0468] Fragment Fmoc-Trp25- / / -Ser39-NH2 (10.9 g, 5.97 mmol, 1.0 equiv) was taken up in DMAc (165 mL, 15 vol.). Diethylamine (3.49 g, 47.8 mmol, 8.0 equiv) was added dropwise and the resulting reaction mixture was stirred at ambient temperature for 7 h. Upon completion of the reaction, the mixture was added dropwise to MTBE (825 mL, 75 vol) and the resultingsuspension was stirred at ambient temperature for 30 min. The solid was isolated by filtration and the filter cake was washed with MTBE (3 x 55 mL, 3 x 5 vol.) and dried under a stream of nitrogen to afford fragment H-Trp25- / / -Ser39-NH2 (9.12 g, 85.1% content by NMR, 78.3% yield, purity 95.2 area%) as a solid.

[0469] LC-MS (ESI+): mass calculated for C77H121N17O20 [M+] 1659.9600, found [(M+2H)2+] 831.79.

[0470] 2,2 LPPS of Fragment H-Trp25- / / -Ser39-NH2 (Cbz Route)

[0471] 2.2.1 Synthesis of Fragment Cbz-Trp25- / / -Gly29-OH

[0472] O o A CbzHN^AOHCbz-OSu, NaHCO3uYJ^BOC THF / H2O NBoc

[0473] HATU, DIPEA, DMAc / DMSO

[0474] 1 M LiOH THF / H2O

[0475]

[0476] H-Trp(Boc)-OH (40.0 g, 131 mmol, 1.0 equiv) was taken up in water (400 mL, 10 vol.) and THF (400 mL, 10 vol.). The resulting mixture was cooled to 0 °C and NaHCOs (11.6 g, 138 mmol, 1.05 equiv) was added in portions. Cbz-OSu (34.4 g, 138 mmol, 1.05 equiv) was then added in portions at 0 °C and the resulting reaction mixture was stirred at this temperature for20 h. Upon completion of the reaction, 5% aq. H3PO4 solution (150 mL, 3.8 vol.) was added. The mixture was concentrated under reduced pressure at 20-25 °C to remove most of the THF. EtOAc (400 mL, 10 vol.) was added to the mixture, the phases were separated, and the aqueous phase was extracted with EtOAc (2 x 200 mL, 2 x 5 vol.). The combined organic phases were washed with 24% aq. NaCl solution (400 mL, 10 vol.) and concentrated under reduced pressure to afford Cbz-Trp(Boc)-OH (69.8 g, 82.2% content by NMR, 99.6% yield, purity 97.3 area%) as a yellow solid.

[0477] ¹H NMR (400 MHz, CDCl₃): δ 8.30–8.07 (m, 2H), 7.55-7.45 (m, 3H), 7.40-7.11 (m, 6H), 5.47 (d, J= 7.5 Hz, 1H), 5.10 (d, J= 6.4 Hz, 2H), 4.77 (d, J= 7.1 Hz, 1H), 3.46-3.16 (m, 1H), 1.65 (s, 9H) (COOH not visible).

[0478] Cbz-Trp(Boc)-OH (11.2 g, 25.5 mmol, 1.0 equiv) and the above solution of H-Leu-Ile-Ala-Gly-OMe (30.5 mmol, 1.2 equiv) in DMAc were taken up in DMAc (400 mL, 35.7 vol.) and DMSO (80 mL, 7.1 vol.). The resulting mixture was cooled to -5 °C before HATU (11.6 g, 30.5 mmol, 1.2 equiv) was added, followed by the dropwise addition of DIPEA (8.2 g, 63.6 mmol, 2.5 equiv). The resulting reaction mixture was stirred at -5 °C for 4 h. Upon completion of the reaction, water (495 mL, 44 vol.) was added over 1.5 h at -5 °C and the resulting suspension was stirred at 0 °C for 1 h. The mixture was filtered and the filter cake was washed with water (2 x 22 mL, 2 x 2 vol.) and dried to afford a solid which was then slurried in MeCN (280 mL, 25 vol.) at 75 °C for 1 h. The mixture was slowly cooled to ambient temperature and filtered and the filter cake washed with MeCN (2 x 22 mL, 2 x 2 vol.) and dried under a stream of nitrogen to afford fragment Cbz-Trp25- / / - Gly29-OMe (20.6 g, 90.7% content by NMR, 91.2% yield, purity 98.5 area%) as a white solid.

[0479] ¹H NMR (400 MHz, DMSO-d₆): δ 8.27 (dd, J = 7.3, 4.2 Hz, 2H), 8.02 (dd, J = 11.4, 7.8 Hz, 2H), 7.76 (t, J= 8.6 Hz, 2H), 7.66-7.53 (m, 2H), 7.36-7.12 (m, 7H), 4.94 (s, 2H), 4.58-4.11 (m, 4H), 4.00-3.73 (m, 2H), 3.60 (s, 3H), 3.06 (dd, J= 15.1, 3.7 Hz, 1H), 2.96-2.85 (m, 1H), 2.53 (s, 1H), 1.77-1.67 (m, 1H), 1.60 (s, 9H), 1.56-1.30 (m, 3H), 1.22 (d, J= 7.0 Hz, 3H), 1.14-1.09 (m, 1H), 0.90-0.73 (m, 12H).

[0480] Fragment Cbz-Trp25- / / -Gly29-OMe (18.3 g, 22.7 mmol, 1.0 equiv) was taken up in THF (1100 mL, 60 vol.) and water (220 mL, 12 vol.) and the resulting mixture was cooled to 0 °C. 1 M LiOH (24.9 mL, 24.9 mmol, 1.1 equiv) was added, and the resulting reaction mixture was stirred at 0 °C for 4 h. Upon completion of the reaction, 5% aq. H3PO4 solution (28 mL, 1.4 vol.) was added and THF was removed under reduced pressure at 20-25 °C. Another portion of 5% aq. H3PO4 solution (20 mL, 1 vol.) was added to adjust the pH to 2-3, and the resulting suspension was stirred at 0 °C for 1 h. The solid was isolated by filtration and thefilter cake was washed with water (2 x 22 mL, 2 x 1.2 vol.) and dried to afford fragment Cbz-Trp25- / / -Gly29-OH (17.2 g, 96.0% content by NMR, 92.2% yield, purity 98.2 area%) as a white solid.

[0481] LC-MS (ESI+): mass calculated for C₄₁H₅₇N₆O₁₀ [(M+H)⁺] 793.41, found [(M+2H)2+] 793.92.

[0482] 2.2.2 Synthesis of Fragment H-Trp25- / / -Ser39-NH 2

[0483]

[0484] Fragment Cbz-Trp25- / / -Gly29-OH (15.9 g, 20.0 mmol, 1.0 equiv) and fragment H-Gly30- / / -Ser39-NH2 (21.5 g, 21.1 mmol, 1.05 equiv) were taken up in DMAc (240 mL, 15 vol.).

[0485] Oxyma Pure (4.00 g, 28.1 mmol, 1.2 equiv) was added and the resulting mixture was cooled to 0 °C. EDCI (5.39 g, 28.1 mmol, 1.2 equiv) was added in portions, followed by the dropwise addition of DIPEA (3.63 g, 28.1 mmol, 1.2 equiv). The resulting reaction mixture was allowed to warm to ambient temperature and was stirred at this temperature for 20 h. Upon completion of the reaction, the mixture was added dropwise to MTBE (1200 mL, 75 vol.), the resulting suspension was filtered, and the filter cake was washed with MTBE (64 mL, 4 vol.). The resulting solid was slurried in 5% aq. H3PO4 solution (160 mL, 10 vol.) at ambient temperature for 1 h before the suspension was again filtered. The filter cake was washed with water (3 x 32 mL, 3 x 2 vol.) and dried under a stream of nitrogen to afford fragment Cbz-Trp25- / / -Ser39-NH2 (34.2 g, 95.4% content by NMR, 90.5% yield, purity 94.5 area%) as a solid.Fragment Cbz-Trp25- / / -Ser39-NH2 (31.0 g, 17.3 mmol, 1.0 equiv) was taken up in CH2CI2 (248 mL, 8 vol.) and DMAc (62 mL, 2 vol.). 10% wet Pd / C (6.2 g, 20 w%) was added and the resulting reaction mixture was stirred under a 0.5 MPa H2 atmosphere at ambient temperature for 15 h. Upon completion of the reaction, the mixture was filtered, and the filter cake was washed with DMAc (62 mL, 2 vol.). The filtrate was concentrated under reduced pressure to 4 vol. and the resulting solution was added dropwise to MTBE (2170 mL, 70 vol.) at ambient temperature. The resulting suspension was filtered, and the filter cake was washed with MTBE (2 x 62 mL, 2 x 2 vol.) and dried to afford fragment H-Trp25- / / -Ser39-NH2 (26.4 g, 92.0% yield, purity 97.0 area%) as a solid.

[0486] LC-MS (ESI+): mass calculated for C77H121N17O20 [M+] 1659.9600, found [(M+2H)2+] 831.9896.

[0487] 2,3 SPPS of Fragment Fmoc- Alai 8- / / -Lys20(sc)- / / -Gln24-resin

[0488] The reaction scheme is shown in FIG.2.

[0489] Preparation of the Resin

[0490] 2-Chlorotrityl chloride resin (50.0 g, loading 1.0 mmol / g; after the coupling of the first amino acid, the loading was 0.77 mmol / g and the weight of the resin was 55.4 g) was charged into a 2 L SPPS reactor. The resin was swelled twice with DMF (6 mL / g resin) and stirred for 8 min at ambient temperature. The reactor was then drained.

[0491] General Synthetic Procedure

[0492] Washing steps were designed to remove remaining deprotection solution, coupling / capping 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 ceased). The wash solvent was added to the reactor and the mixture was first bubbled with nitrogen for 3 min, then stirred for 5-10 min (starting after solvent addition is complete and stirring is started). The stirrer was then stopped and the reactor was completely drained.

[0493] Fmoc deprotections were performed by stirring the resin with piperidine / DMF (20% v / v) containing 2% Oxyma Pure. In all cycles, two treatments were performed for deprotection. Stirring times with piperidine / DMF / Oxyma Pure were 10 minutes for the first treatment and 30 minutes for the second treatment in all cycles. After Fmoc removal, the resin was washed with DMF.Clcey

[0494] In cycle 1 Aiid,mno ac the resin was loaded using the protected amino acid and DIPEA with DMF as the solvent. For all couplings except in cycle 1 and 2, a DIC / Oxyma Pure procedure was used. The mixture of protected amino acid or building block, Oxyma Pure and DIC in DMF was stirred at 5 °C for 20 Aiittcaonv min to pre-activate the amino acid. In cycle 2, the coupling was

[0495] iidt conons

[0496] performed using PyBOP / Oxyma Pure / DIPEA.

[0497] After this time, the amino acid Aiittcaonv solution was added to the resin. The resulting reaction mixture °C [] ttemperareu

[0498] was first bubbled with nitrogen for 3 min, then stirred. The reaction time for the coupling was 3-20 h and the coupling temperat Aiittucaonvre was 30 °C. After coupling, the reactor was drained and the resin was washed with DMF. ii [] tmemn

[0499] Cilopngu

[0500] After completion of the solid phase synthesis, th°C [] tteemperareu resin was washed sequentially with CH2Cl2and MTBE before discharging. The resin was dried Ciil u topngmeunder reduced pressure at 25 °C for 12 h.

[0501] i []mn

[0502] Synthetic Procedure

[0503] DMF

[0504] Cilhopngasesu w

[0505] l 66ox v.

[0506] The synthesis was carried out on 42.7 mmol scale, resulting in the isolation of fragment Fmoc-Ala18- / / -Lys20(sc)- / / Gln24-resin (122.4 g).

[0507] DFemoc- Table 1: Conditions used for the SPPS build of fragment Fmoc-Ala18- / / -Lys20(sc)- / / Gln24-resin on 42.7 mmol scale. DFemoc- i (i) tmemn

[0508] i Dtteproecon 2-CTC resin, 0.77 mmol / g Swelling: DMF, 2x6 vol., 8 min per swell, RThases w

[0509] Fmoc-L- DIPEA

[0510] Gln(Trt)- (6.0 equiv)

[0511] 1 OH 5 20 30 180 — — —

[0512] DMF

[0513] (2.0

[0514] (6 vol.)

[0515]

[0516] equiv)2-CTC resin, 0.77 mmol / g Swelling: DMF, 2x6 vol., 8 min per swell, RT PyBOP

[0517] (2.0 equiv)

[0518] Oxyma 20% Fmoc-L- Pure pip / DMF DMF Val-OH

[0519] (2.0 equiv) 5 20 30 180 +2% 10 / 30 6 x6 (2.0

[0520] DIPEA Oxyma vol. equiv)

[0521] (3.0 equiv) Pure DMF

[0522] (6 vol.)

[0523] Oxyma

[0524] Pure 20% Fmoc-L- (2.0 equiv) pip / DMF DMF Phe-OH 180- DIC 5 20 30 +2% 10 / 30 6 x6 (2.0 240

[0525] (2.2 equiv) Oxyma vol. equiv)

[0526] DMF Pure (6 vol.)

[0527] Oxyma

[0528] Fmoc-L- Pure 20%

[0529] DMF DMF DMF DMF DMF DMF

[0530] Ala- (2.0 equiv) pip / DMF DMF 180- 666666666666oooooox vx vx vx vx vx v......

[0531] OH H2O DIC 5 20 30 +2% 10 / 30 6 x6

[0532] 240

[0533] (2.0 (2.2 equiv) Oxyma vol. equiv) DMF Pure

[0534] (6 vol.)

[0535] Oxyma

[0536] Pure 20% Fmoc-L- (3.0 equiv) pip / DMF DMF Lys(sc)- DIC 25 20 30 1180 +2% 10 / 30 6 x6 OH (1.5

[0537] (3.3 equiv) Oxyma vol. equiv)

[0538] DMF Pure (6 vol.)

[0539] Oxyma

[0540] Fmoc-L- Pure 20%

[0541] Gln(Trt)- (2.0 equiv) pip / DMF DMF 180- OH DIC 25 20 30 +2% 10 / 30 6 x6

[0542] 240

[0543] (2.0 (2.2 equiv) Oxyma vol. equiv) DMF Pure

[0544] (6 vol.)

[0545] DMF

[0546] Oxyma 6 x6 Fmoc-L- Pure 20% vol. Ala- (2.0 equiv) pip / DMF CH2CI2

[0547] 180- OH H2O DIC 5 20 30 +2% 10 / 30 4 x6

[0548] 240

[0549] (2.0 (2.2 equiv) Oxyma vol. equiv) DMF Pure MTBE (6 vol.) 4 x6

[0550]

[0551] vol.2.4 Soft Cleavage of Fragment Fmoc-Ala18- / / -Lys20(sc)- / / Gln24-OH

[0552] The reaction scheme is shown in FIG.3.

[0553] Fragment Fmoc-Ala18- / / -Lys20(sc)- / / Gln24-resin (122.4 g) was swelled using CH2Cl2(720 mL) at 20 °C for 30 min before it was filtered. Pre-cooled (5 °C) TFA / TIS / CH2CI2 (1:2:97 NININ, 1.2 L) was charged into the reactor and the resulting suspension was stirred for 15 min at 15 °C. The suspension was filtered and the filtrate was neutralized immediately by the slow addition of a solution of pyridine (15.3 g) in DMSO (120 mL). The resulting mixture was stirred at 10 °C for 30-60 min, then set aside. This sequence was repeated with the same batch of resin two more times. The three neutralized mixtures were then combined and concentrated under reduced pressure to 600 mL. The resulting solution was added to precooled (2 °C) MeCN / water (1.2 L, 1:4 v / v) at 2 °C over 40-60 min and the resulting suspension was stirred at 15 °C for 40 min. The solid was isolated by filtration and the wet cake was washed with MeCN / water (4 x 600 mL, 1:4 v / v) at 15 °C. The solid was dried under reduced pressure with a flow of nitrogen at T < 25 °C until the water content was below 2% as determined by KF titration. Fragment Fmoc-Ala18- / / -Lys20(sc)- / / Gln24-OH was isolated as a solid (52.6 g, purity 99.2 area%, 52.4% yield).

[0554] LC-MS (ESI+): mass calculated for C134H177N13O24 [M+] 2352.30, found [(M+2H]2+] 1177.16.

[0555] 2.5 SPPS of Fragment X'-ZZ-Ile^-resin

[0556]

[0557] The reaction scheme is shown in FIG.4.

[0558] Preparation of the Resin

[0559] 2-Chlorotrityl chloride resin (50.0 g, loading 1.0 mmol / g; after the coupling of the first amino acid, the loading was 0.79 mmol / g and the weight of the resin was 54.1 g) was charged into a 2 L SPPS reactor. The resin was swelled twice with DMF (6 mL / g resin) and stirred for 8 min at ambient temperature. The reactor was then drained.

[0560] General Synthetic Procedure

[0561] Washing steps were designed to remove remaining deprotection solution, coupling / capping 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 ceased). The wash solvent was added to the reactor and the mixture was first bubbled with nitrogen for 3 min, thenstirred for 5-10 min (starting after solvent addition is complete and stirring is started). The stirrer was then stopped and the reactor was drained completely.

[0562] Fmoc deprotections were performed by stirring the resin with piperidine / DMF (20% v / v) containing 2% Oxyma Pure. In all cycles, two treatments were performed for deprotection. Stirring times with piperidine / DMF / Oxyma Pure were 10 minutes for the first treatment and 30 minutes for the second treatment in all cycles. After Fmoc removal, the resin was washed with DMF.

[0563] In cycle 1, the resin was loaded using the protected amino acid and DIPEA with DMF as the solvent. For all couplings except in cycle 1 and 2, a DIC / Oxyma Pure procedure was used. The mixture of protected amino acid or building block, Oxyma Pure and DIC in DMF was stirred at 5-25 °C for 20-60 min to pre-activate the amino acid. In cycle 2, the coupling was performed using PyBOP / Oxyma Pure / DIPEA.

[0564] After this time, the amino acid solution was added to the resin. The resulting reaction mixture was first bubbled with nitrogen for 3 min, then stirred. The reaction time for the coupling was 3-9 h and the coupling temperature was 30 °C. After coupling, the reactor was drained and the resin was washed with DMF.

[0565] After completion of the solid phase synthesis, the resin was washed sequentially with CH2Cl2and MTBE before discharging. The resin was dried under reduced pressure at 25 °C for 12 h.

[0566] Synthetic Procedure

[0567] The synthesis was carried out on 42.8 mmol scale, resulting in the isolation of fragment X1- / / -Ile17-resin (145.6 g).

[0568] Table 2: Conditions used for the SPPS build of fragment X^ / AHe^-resin on 42.8 mmol scale.o> LA 4^ LJ I—* Cycle

[0569] g pg,,,, Swe: vo.e sweCCes.o / llin DMF 2x6l 8 minrll RT 2T rin 079 mml- Amino acid

[0570] LJ LJ 73 73 73 73 73 73 _ 73

[0571] u woo O O MQo OOMQo O C o 2 o -□ O O'Z?

[0572] < S' CD _ C, £

[0573] o < S' cc JS o c >> Activation conditions F- — Ffl * X 3i ^ X3 3 B 3 O!— a -Ci a a O!— - aSrj -c ci " -c £i C aDs a B O 2-g „-O g -gg

[0574] rj (S " (S ° B

[0575] P<’ <’P<■ <■p< < <

[0576] Activation tx) LA LA LA LA LA LA

[0577] temperature [°C] DIPEA

[0578] oc FmL DMF-- q (). euv60i

[0579] bJ bJ bJ bJ Os ActivationeO IlH RT 1806x6- O O O O O O DMF

[0580] q (). euv vo.20il time [min]

[0581] () vo.6l

[0582] Coupling temperature LJ LJ LJ LJ LJ

[0583] o O O O O

[0584] [°C]

[0585] oc FmL-- DMF Coupling time [min] DMFy()soc LB- / 1806x6 1030

[0586] OH vo. 6x6l

[0587] vo.l

[0588] q (). euv20i

[0589] Coupling washes

[0590] o o o o o as + as K> as + as K> aS + aS as ~ as s + ~ ’’-i

[0591] 1 g an Sa g X an Sa g X an Sa g X an Sa g X ar| Sa De-Fmoc B L4 \OJFJ \© B B x,oJrj \© o 3 ©\ H ©\ o 3 ©^ H ©\ o 3 ©^ H ©\ o 3 ©^ H ©\oc FmL-- P s. P s. P s. P s. P s. DMF

[0592] DMFp()sOtu 180 AB- / 6x6 1030

[0593] OH 240 - vo. 6x6l De-Fmoc vo.l 1

[0594] q (). euv20i time (min)

[0595] 1 Deprotection washes oc FmL DMF-- DMF 180-

[0596]

[0597] euO / LH 6x6 1030- 240 vo. 6x6l

[0598] q (). euv vo.20il

[0599] DMFoce FmIl-- DMF

[0600] bO / AiH 5406x6 1030- vo. 6x6l

[0601] q (). euv vo.20il

[0602] y Oaxm

[0603] oc FmL--ue Pr

[0604] DMF DMFq() () SeOtu. euv 180rB25i-- / 6x6 1030

[0605] OH 240C vo. DI 6x6l

[0606] vo.l

[0607] q (. euv275iq (). euv25i

[0608] )DMF

[0609] (6 vol.)

[0610] Oxyma

[0611] 20%

[0612] Pure

[0613] Fmoc-L- pip / DM

[0614] (2.0 equiv) DMF

[0615] Tyr(OtBu)- 180- F DMF DIC 5 20 30 6x 6 10 / 30

[0616] OH 240 +2% 6x 6 vol.

[0617] (2.2 equiv) vol.

[0618] (2.0 equiv) Oxyma

[0619] DMF

[0620] Pure

[0621] (6 vol.)

[0622] 20%

[0623] Oxyma pip / DM Pure F

[0624] Fmoc-L- (2.0 equiv) DMF 20%

[0625] Asp(OtBu) 180- DMF DIC 5 20 30 6x 6 pip / DM 10 / 30

[0626] -OH 240 6x 6 vol.

[0627] (2.2 equiv) vol. F

[0628] (2.0 equiv)

[0629] DMF +2%

[0630] (6 vol.) Oxyma

[0631] Pure

[0632] Oxyma

[0633] Pure 20%

[0634] Fmoc-L- (2.5 equiv) pip / DM

[0635] DMF

[0636] Ser(OtBu)- DIC 180- F DMF 5 20 30 6x 6 10 / 30

[0637] OH (2.75 equiv 240 +2% 6x 6 vol.

[0638] vol.

[0639] (2.5 equiv) ) Oxyma

[0640] DMF Pure

[0641] (6 vol.)

[0642] Oxyma

[0643] 20%

[0644] Pure

[0645] Fmoc-L- pip / DM

[0646] (2.0 equiv) DMF

[0647] Thr(OtBu)- 180- F DMF DIC 5 20 30 6x 6 10 / 30

[0648] OH 240 +2% 6x 6 vol.

[0649] (2.2 equiv) vol.

[0650] (2.0 equiv) Oxyma

[0651] DMF

[0652] Pure

[0653] (6 vol.)

[0654] Oxyma

[0655] Pure 20%

[0656] (2.5 equiv) pip / DM Fmoc-Thr- DMF

[0657] DIC 180- F DMF Phe-OH 5 20 30 6x 6 10 / 30

[0658] (2.75 equiv 240 +2% 6x 6 vol. (2.5 equiv) vol.

[0659] ) Oxyma

[0660] DMF Pure

[0661] (6 vol.)

[0662] X-p-Ala- Glu-Gly- Oxyma

[0663] 20% DMF OH Pure

[0664] pip / DM 6x 6 vol. (1.5 equiv) (3.0 equiv) DMF

[0665] 360- F CH2CI2 DIC 25 60 30 6x 6 10 / 30

[0666] 540 +2% 4x 6 vol. (3.3 equiv) vol.

[0667] Oxyma MTBE DMF

[0668] Pure 4x 6 vol. (6 vol.)

[0669]

[0670] 2.6 Soft Cleavage of Fragment X^ZZ-Ile^-OH

[0671]

[0672] The reaction scheme is shown in FIG.5.

[0673] Fragment X1- / / -Ile17-resin (145.6 g) was swelled using CH2Cl2(870 mL) at 20 °C for 30 min before it was filtered. Pre-cooled (5 °C) TFA / TIS / CH₂Cl₂ (1:2:97 v / v / v, 1.47 L) was charged into the reactor and the resulting suspension was stirred for 15 min at 15 °C. The suspension was filtered and the filtrate was neutralized immediately by the slow addition of a solution of pyridine (19.2 g) in DMSO (150 mL). The neutralized mixture was stirred at 10 °C for 30-60 min, then set aside. This sequence was repeated two more times. The three neutralized mixtures were then combined and concentrated under reduced pressure to 750 mL. The resulting solution was added to pre-cooled (2 °C) MeCN / water (1.5 L, 1:4 v / v) at 2 °C over 40-60 min and the resulting suspension was stirred at 15 °C for 40 min. The solid was isolated by filtration and the wet cake was washed with MeCN / water (4 x 750 mL, 1:4 v / v) at 15 °C. The solid was dried under reduced pressure with a flow of nitrogen at T < 25 °C until the water content was below 2% as determined by KF titration. Fragment X1- / / -Ile17-OH was isolated as a solid (42.1 g, purity 92.4 area%, 41.9% yield).

[0674] LC-MS (ESI+): mass calculated for C126H201FN18O31[M+] 2481.4689, found [(M+2H)2+] 1241.7518.

[0675] 2.7 LPPS of Fragment H-Ala18-ZZ-Lys20(sc)-ZZ-Ser39-NH2

[0676] The reaction scheme is shown in Fig. 6.

[0677] Fragment Fmoc-Ala18- / / -Lys20(sc)- / / Gln24-OH (21.1 g, 8.96 mmol, 1.0 equiv) and fragment H-Trp25- / / -Ser39-NH2(16.4 g, 9.86 mmol, 1.1 equiv) were taken up in DMF (530 mL, 25 vol.) at ambient temperature. Once the solution became clear, it was cooled to -20 °C and a solution of Oxyma Pure (2.00 g, 14.3 mmol, 1.6 equiv) in DMF (42 mL, 2 vol.) was added dropwise over 1-2 min, followed by 5 min of stirring. Then, a solution of PyAOP (7.50 g, 14.3 mmol, 1.6 equiv) in DMF (63 mL, 3 vol.) was added over 1-2 minutes, followed by the dropwise addition of DIPEA (4.60 g, 35.8 mmol, 4.0 equiv). The reaction mixture was stirred at -20 °C for 3 h. Upon completion of the reaction, the mixture was warmed to 0 °C, water (10.6 mL, 0.5 vol.) was added dropwise, and the resulting mixture was stirred at this temperature for 30 min. Then, diethylamine (12.7 g, 179 mmol, 20.0 equiv) was added over 1-2 min at 0 °C. The resulting reaction mixture was allowed to warm to ambient temperature and was stirred at this temperature for 2 h. Upon completion of the deprotection reaction, the mixture was added dropwise to pre-cooled (0 °C) 7.5% aq. NaCl solution (3.2 L, 150 vol.).The resulting suspension was stirred at 0 °C for 1 h before it was filtered and the filter cake was washed with water (3 x 210 mL, 3 x 10 vol) and dried under a stream of nitrogen. The solid was then dissolved in CH2CI2 (420 mL, 20 vol.) at ambient temperature. This solution was concentrated under reduced pressure at 25-35 °C to 5-10 vol. before it was cooled to 0 °C. Pre-cooled (0 °C) MTBE (2.2 L, 100 vol.) was added dropwise to the CH2CI2 solution and the resulting suspension was stirred at 0 °C for 30 min. The solid was isolated by filtration and filter cake was washed with MTBE (3 x 60 mL, 3 x 3 vol) and dried under a stream of nitrogen to afford fragment H-Ala18- / / -Lys20(sc)- / / -Ser39-NH2(34.0 g, peptide content 85.0%, 85.6% yield, purity 84.5 area%) as a white solid.

[0678] LC-MS (ESI+) for Fmoc-protected fragment: mass calculated for C215H304N30O43[M+] 3996.2591, found [(M+3H)3+] 1333.0935.

[0679] LC-MS (ESI+) for fully deprotected fragment: mass calculated for C137H218N30O39[M+] 2907.5997, found [(M+2H)2+] 1454.8153.

[0680] 2.8 LPPS of X1- / / -Lys20(sc)- / / -Ser39-NH2and Global Deprotection

[0681] The reaction scheme is shown in FIG.7.

[0682] Fragment H-Ala18- / / -Lys20(sc)- / / -Ser39-NH2 (25.5 g, 6.76 mmol, 1.0 equiv) and fragment X1- / / -Ile17-OH (18.5 g, 7.43 mmol, 1.1 equiv) were dissolved in DMAc (460 mL, 18 vol.) at ambient temperature. The resulting solution was cooled to 0 °C and a solution of FDPP (6.70 g, 17.6 mmol, 2.6 equiv) in DMAc (50 mL, 2 vol.) was added dropwise over 1-2 min.

[0683] DIPEA (5.20 g, 40.5 mmol, 7.2 mL, 6.0 equiv) was then added dropwise over 1-2 min and the resulting reaction mixture was stirred at 0 °C for 3 h. Upon completion of the reaction, the mixture was added dropwise to 15% aq. NaCl solution (3.8 L, 150 vol.) at 0 °C. The resulting suspension was stirred at 0 °C for 2 h before the solid was isolated by filtration and the filter cake was washed with water (3 x 250 mL, 3 x 10 vol.) and dried under a stream of nitrogen to afford X1- / / -Lys20(sc)- / / -Ser39-NH2 (51.3 g, peptide content 64.2%, 78.1% yield, purity 64.2 area%) as a white solid.

[0684] TFA (250 mL, 8.5 vol.), water (13.9 mL, 0.47 vol.), DTT (10.4 g, 0.35 g / g), and TIS (13.9 mL, 0.47 vol.) were combined at ambient temperature and the mixture was then cooled to 0 °C. X1- / / -Lys20(sc)- / / -Ser39-NH2 (29.6 g, 4.74 mmol, 1.0 equiv) was added in portions over 20 min while the mixture was stirred vigorously and the temperature was maintained at <10 °C. The resulting reaction mixture was then allowed to warm to 25 °C over 30 min and was stirred at this temperature for 3 h. The mixture was cooled to 0 °C and pre-cooled (-20 °C)MTBE (1.6 L, 54 vol.) was slowly added (300 mL over 10 min, then 1.3 L over 5 min). The resulting suspension was stirred at 0°C for 1 h before it was filtered and the filter cake was washed with MTBE (3 x 90 mL, 3 x 3 vol.) and dried under a stream of nitrogen to afford the crude API (40.4 g, peptide content 39.0%, 68.8% yield, purity 58.6 area%) as a white solid.

[0685] LC-MS (ESI+): mass calculated for C226H345FN48O67[M+] 4824.5116, found [(M+3H)3+] 1608.4845.

[0686] 3. Route 2

[0687] 3.1 LPPS of Fragment H-Gly29- / / -Ser39-NH2

[0688] 3.1.1 Synthesis of Cbz-Gly29-Gly30-Pro31-OH

[0689] O

[0690] Pfp-OH CbzHN^ANCbzHN^A OHEDC' >H

[0691] H o CH2Cl2

[0692] F

[0693]

[0694] Cbz-Gly-Gly-OH (30.0 g, 113 mmol, 1.0 equiv) and Pfp-OH (24.9 g, 135 mmol, 1.2 equiv) were dissolved in CH2Cl2(300 mL, 10 vol.). The mixture was cooled to 0 °C and EDCI (25.9 g, 135 mmol, 1.2 equiv) was added in portions. The reaction mixture was allowed to warm to ambient temperature and was stirred at this temperature for 8 h. Upon completion of the reaction, the mixture was sequentially washed with 5% aq. H3PO4 solution (3 x 150 mL, 3 x 5 vol.), sat. aq. NaCl solution (150 mL, 5 vol.), dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was slurried in heptane (300 mL, 10 vol.) for 30 minutes. The solid was collected by filtration and dried under a stream of nitrogen to afford Cbz-Gly-Gly-OPfp (42.6 g, 98.3% content by NMR, 86.1% yield, purity 97.6 area%) as a solid.

[0695] 1H NMR (400 MHz, CDCl3): d 7.35-7.33 (m, 5H), 7.02 (s, 1H), 5.68 (t, J= 6.0 Hz, 1H), 5.14 (s, 2H), 4.40 (d, J= 5.7 Hz, 2H), 3.98 (d, J= 6.2 Hz, 2H).L-Proline (14.5 g, 126 mmol, 1.5 equiv) and BSA (34.23 g, 168 mmol, 2.0 equiv) were taken up in THF (360 mL, 10 vol.) and the resulting mixture was stirred at 20 °C for 1.5 h. The mixture was then cooled to 0 °C and Cbz-Gly-Gly-OPfp (36.4 g, 84 mmol, 1.0 equiv) was added in portions. The reaction mixture was allowed to warm to 20 °C and was stirred at this temperature for 2 h. Upon completion of the reaction, water (360 mL, 10 vol.) was added, the mixture was stirred for 15 min and the phases were separated. The aqueous phase was extracted with EtOAc (2 x 144 mL, 2 x 4 vol.). The combined organic layers were washed with 15% aq. NaCl solution (180 mL, 5 vol.), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was taken up in EtOAc (180 mL, 5 vol.) and the mixture was concentrated to 36-72 mL (1-2 vol.). This process was repeated 5 times. EtOAc (180 mL, 5 vol.) was added and the resulting suspension was slurried for 30 min. The product was collected by filtration and dried under a stream of nitrogen to afford Cbz-Gly-Gly-Pro-OH (28.4 g, 97.2% content by NMR, 90.1% yield, purity 99.4 area%) as a solid.

[0696] 'H NMR (400 MHz, CDC13): d 9.05 (s, br, 1H), 7.78-7.58 (m, 1H), 7.37-7.28 (m, 5H), 5.11 (s, 2H), 4.44-4.43 (m, 1H), 4.09-3.92 (m, 4H), 3.60-3.42 (m, 2H), 2.27-1.86 (m, 5H).

[0697] LC-MS (ESI+): mass calculated for C17H22N3O6 [(M+H)+] 364.15, found 364.26.

[0698] 3.1.2 Synthesis of Fragment Cbz-Gly29- / / -Gly34-OH

[0699] LiOH·H2O, THF / H2O

[0700]

[0701] Oxyma Pure (2.46 g, 17.3 mmol, 1.2 equiv) was dissolved in DMAc (20 mL, 4 vol.) and MTBE (20 mL, 4 vol.). The mixture was cooled to 0 °C and EDCI (3.32 g, 17.3 mmol, 1.2 equiv) was added in portions. The above solution of H-Ser(tBu)-Ser(tBu)-Gly-OMe in DMAc(17.3 mmol, 1.2 equiv) and Cbz-Gly-Gly-Pro-OH (6.50 g, 14.4 mmol, 1.0 equiv) were added to the mixture. DIPEA (2.24 g, 17.3 mmol, 1.2 equiv) was added dropwise and the reaction mixture was allowed to warm to ambient temperature and stirred at this temperature. Upon completion of the reaction, 5% aq. H3PO4 solution (40 mL, 8 vol.) and heptane (40 mL, 8 vol.) were added, the phases were separated and the organic phase was discarded. The aqueous phase was extracted with CH2Cl2(2 x 35 mL, 2 x 7 vol.). The combined organic phases were washed successively with 5% aq. H3PO4 solution (25 mL, 5 vol.), 8% aq.

[0702] NaHCO3solution (3 x 25 mL, 3 x 5 vol.), and 15% aq. NaCl solution (25 mL, 5 vol.), dried over Na2SO4, filtered, and concentrated under reduced pressure to afford Cbz-Gly29- / / -Gly34-OMe (16.1 g, 59.8% content by NMR, 92.7% yield, purity 99.7 area%) as a colorless oil.

[0703] 1H NMR (400 MHz, DMSO-6): d 8.35-8.10 (m, 2H), 8.08 (d, J= 8.0 Hz, 1H), 7.91-7.82 (m, 1H), 7.60-7.50 (m, 1H), 7.40-7.27 (m, 5H), 5.05 (s, 2H), 4.57-4.30 (m, 4H), 4.02-3.99 (m, 1H), 3.90-3.86 (m, 4H), 3.63 (s, 3H), 3.54-3.44(m, 8H), 1.92-1.88 (m, 2H), 1.18 (s, 18H).

[0704] Cbz-Gly29- / / -Gly34-OMe (8.74 g, 12.1 mmol, 1.0 equiv) was dissolved in THF (43 mL, 5 vol.) and water (26 mL, 3 vol.). The mixture was cooled to 0 °C. A solution of LiOH·H2O (0.66 g, 15.8 mmol, 1.3 equiv) in water (17 mL, 2vol.) was added dropwise at this temperature and stirring was continued. Upon completion of the reaction, 3 M HCl was added to adjust the pH to 2-3. The mixture was extracted with CH2Cl2(2 x 43 mL, 2 x 5 vol.). The combined organic layers were sequentially washed with 5% aq. H3PO4 solution (43 mL, 5 vol.) and 15% aq. NaCl solution (43 mL, 5 vol.), dried over Na2SO4, filtered, and concentrated to 1-2 vol. MTBE (43 mL, 5 vol.) was added, and the mixture was concentrated to 1-2 vol. This process was repeated 4 times before the mixture was concentrated to dryness to afford Cbz-Gly29- / / -Gly34-OH (9.30 g, 91.6% content by NMR, 99.4% yield, purity 99.2 area%).

[0705] LC-MS (ESI+): mass calculated for C33H51N6O11[(M+H)+] 707.36, found 707.53.3.1.3 Synthesis of Fragment H-Gly29- / / -Ser39-NH2

[0706] o

[0707] HOBt, EDCI DIPEA, DMAc

[0708] H2, Pd / C

[0709] CH2CI2

[0710]

[0711] Fragment Cbz-Gly29- / / -Gly34-OH (8.2 g, 11.7 mmol, 1.0 equiv) and a solution of fragment H-Ala35- / / -Ser39-NH2 in DMAc (14.0 mmol, 1.2 equiv, DMAc solution) were combined and diluted with DMAc (33 mL, 4 vol.). HOBt (1.9 g, 14.0 mmol, 1.2 equiv) was added and the mixture was cooled to 0 °C. EDCI (2.7 g, 14.0 mmol, 1.2 equiv) was added in portions, followed by the dropwise addition of DIPEA (1.8 g, 14.0 mmol, 1.2 equiv). The reaction mixture was allowed to warm to ambient temperature and stirred at this temperature for 3 h. Upon completion of the reaction, 5% aq. H3PO4 solution (33 mL, 4 vol.) and heptane (66 mL, 8 vol.) were added, the phases were separated and the organic phase was discarded.

[0712] CH2Cl2(41 mL, 5 vol.) and 8% aq. NaHCCL solution (41 mL, 5 vol.) were added, the mixture was stirred for 15 min and the phases were separated. The aqueous phase was extracted with CH2Cl2(2 x 41 mL, 2 x 5 vol.). The combined organic layers were sequentially washed with 5% aq. H3PO4 solution (41 mL, 5 vol.), 8% aq. NaHCO3solution (3 x 41 mL, 3 x 5 vol.) and 15% aq. NaCl solution (41 mL, 5 vol.), dried over Na2SO4, filtered, and concentrated under reduced pressure to afford fragment Cbz-Gly29- / / -Ser39-NH2(13.3 g, 94.2% content by NMR, 88.4% yield, purity 96.7 area%) as a colorless oil.Fragment Cbz-Gly29- / / -Ser39-NH2 (12.5 g, 10.3 mmol, 1.0 equiv) was dissolved in CH2Cl2(75 mL, 6 vol.). 10% wet Pd / C (2.5 g, 20 w%) was added. The mixture was stirred at ambient temperature for 2 h under a 0.5 MPa H2 atmosphere. Upon completion of the reaction, the mixture was filtered and the filter cake was washed with CH2CI2 (25 mL, 2 vol.). The filtrate was concentrated to 25 mL (2 vol.). The resulting CH2Cl2solution was added dropwise to MTBE (250 mL, 20 vol.) at ambient temperature. The resulting solid was collected by filtration and the filter cake was washed with MTBE (2 x 12.5 mL, 2 x 1 vol.) and dried under a stream of nitrogen to afford fragment H-Gly29- / / -Ser39-NH2(10.6 g, 95.7% yield, purity 96.8 area%) as a solid.

[0713] LC-MS (ESI+): mass calculated for C50H85N12O14[(M+H)+] 1077.63, found 1077.86.

[0714] 3.2 SPPS of Fragment Fmoc-Ala18- / / -Lys20(sc)- / / -Ala28-resin

[0715] The reaction scheme is shown in FIG.8.

[0716] Preparation of the Resin

[0717] 2-Chlorotrityl chloride resin (10.0 g, loading 1.0 mmol / g; after the coupling of the first amino acid, the loading was 0.83 mmol / g and the weight of the resin was 11.5 g) was charged into a SPPS reactor. The resin was swelled twice with DMF (6 mL / g resin) and stirred for 8 min at ambient temperature. The reactor was then drained.

[0718] General Synthetic Procedure

[0719] Washing steps were designed to remove remaining deprotection solution, coupling / capping 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 ceased). The wash solvent was added to the reactor and the mixture was first bubbled with nitrogen for 3 min, then stirred for 5-10 min (starting after solvent addition is complete and stirring is started). The stirrer was then stopped and the reactor was completely drained.

[0720] Fmoc deprotections were performed by stirring the resin with piperidine / DMF (20% v / v). In all cycles, two treatments were performed for deprotection. Stirring times with piperidine / DMF were 10 minutes for the first treatment and 30 minutes for the second treatment in all cycles. After Fmoc removal, the resin was washed with DMF.

[0721] In cycle 1, the resin was loaded using the protected amino acid and DIPEA with DMF as the solvent. For all couplings except in cycle 1 and 2, a DIC / Oxyma Pure procedure was used. Themixture of protected amino acid or building block, Oxyma Pure and DIC in DMF was stirred at 5 °C for 20 Aiidmno ac min to pre-activate the amino acid. In cycle 2, the coupling was performed using PyBOP / Oxyma Pure / DIPEA.

[0722] After this time, the amino acid solution was added to the resin. The resulting reaction mixture Aiittcaonv

[0723] was first bubbled with nitroiidt cononsgen for 3 min, then stirred. The reaction time for the coupling was 3-20 h and the coupling temperature was 30 °C. After coupling, the reactor was drained and the resin was washed with DMF. Aiittcaonv

[0724] °C [] ttemperareu

[0725] After completion of the solid phase synthesis, the resin was washed sequentially with CH2CI2

[0726] Aiittcaonv

[0727] and MTBE before discharging. The resin waii [] tsmemn dried under reduced pressure at 25 °C for 12 h.

[0728] Synthetic Procedure Cilopngu

[0729] o^ r / iX _. _.- _ _. - _ _

[0730] Ciil topngmeu

[0731] The synthesis was carried out on 9.5 mmol scale, resultini []gmn in the isolation of fragment Fmoc-Ala18- / / -Lys20(sc)- / / Ala28-resin (33.0 g).

[0732] Cilhopngasesu w

[0733] Table 3: Conditions used for the SPPS build of fragment Fmoc-Ala18- / / -Lys20(sc)- / / Ala28-resin on 9.5 mmol scale. DFemoc- 2-CTC resin, 0.83 mmol / g Swelling: DMF, 2x6 vol., 8 min per swell, RT DFemoc- i (i) tmemn

[0734] i Dtteproecon Cycl hases w e

[0735] DIPEA

[0736] Fmoc-L- DM

[0737] (6.0 equiv

[0738] Ala H R

[0739] 12O F

[0740] ( ) 5 20 180 — — — 2.0 equiv T 6x6

[0741] DMF

[0742] ) vol.

[0743] (6 vol.)

[0744] PyBOP

[0745] (2.0 equiv

[0746] )

[0747] Oxyma

[0748] Fmoc-L- DM

[0749] Pure 20% DMF Ile-OH F

[0750] 2 (2.0 equiv 5 20 30 180 pip / DM 10 / 30 8 x6 (2.0 equiv 6x6

[0751] ) DIPEA F vol. ) vol.

[0752] (3.0 equiv

[0753] )

[0754] DMF

[0755]

[0756] (6 vol.)2-CTC resin, 0.83 mmol / g Swelling: DMF, 2x6 vol., 8 min per swell, RT Oxyma

[0757] Pure

[0758] Fmoc-L- (2.0 equiv DM

[0759] 180 20% DMF Leu-OH ) DIC F

[0760] 5 20 30 pip / DM 10 / 30 8 x6 (2.0 (2.2 equiv 6 x6

[0761] 240 F vol. equiv) ) vol.

[0762] DMF

[0763] (6 vol.)

[0764] Oxyma

[0765] Pure

[0766] Fmoc-L- (2.0 equiv DM

[0767] Tryp(Boc- 180 20% DMF ) DIC F

[0768] OH 5 20 30 pip / DM 10 / 30 8 x6

[0769] (2.2 equiv 6 x6

[0770] (2.0 equiv 240 F vol.

[0771] ) vol.

[0772] ) DMF

[0773] (6 vol.)

[0774] Oxyma

[0775] Pure

[0776] Fmoc-L- (2.0 equiv DM

[0777] Gln(Trt)- 180 20% DMF ) DIC F

[0778] OH 5 20 30 pip / DM 10 / 30 8 x6

[0779] (2.2 equiv 6 x6

[0780] (2.0 240 F vol. i ) ) vol.

[0781] equ v

[0782] DMF

[0783] (6 vol.)

[0784] Oxyma

[0785] Pure

[0786] Fmoc-L- (2.0 equiv DM

[0787] 20% DMF Val-OH ) DIC F

[0788] 5 20 30 180 pip / DM 10 / 30 8 x6 (2.0 (2.2 equiv 6 x6

[0789] F vol. equiv) ) vol.

[0790] DMF

[0791] (6 vol.)

[0792] Oxyma

[0793] Pure

[0794] Fmoc-L- (2.0 equiv DM

[0795] 180 20% DMF Phe-OH ) DIC F

[0796] 5 20 30 pip / DM 10 / 30 8 x6 (2.0 (2.2 equiv 6 x6

[0797] 240 F vol. equiv) ) vol.

[0798] DMF

[0799] (6 vol.)

[0800] Oxyma

[0801] Pure

[0802] Fmoc-L- (2.0 equiv DM

[0803] Ala- 20% DMF ) DIC F

[0804] OH H2O 5 20 30 180 pip / DM 10 / 30 8 x6

[0805] (2.2 equiv 6 x6

[0806] (2.0 F vol.

[0807] ) vol.

[0808] equiv)

[0809] DMF

[0810]

[0811] (6 vol.)2-CTC resin, 0.83 mmol / g Swelling: DMF, 2x6 vol., 8 min per swell, RT Oxyma

[0812] Pure

[0813] Fmoc-L- (3.0 equiv DM

[0814] 20% DMF Lys(sc)- ) DIC 118 F

[0815] 9 5 20 30 pip / DM 10 / 30 8 x6 OH (1.5 (3.3 equiv 0 6 x6

[0816] F vol. equiv) ) vol.

[0817] DMF

[0818] (6 vol.)

[0819] Oxyma

[0820] Pure

[0821] Fmoc-L- (2.0 equiv DM

[0822] Gln(Trt)- 180 20% DMF ) DIC F

[0823] 10 OH 5 20 30 pip / DM 10 / 30 8 x6

[0824] (2.2 equiv 6 x6

[0825] (2.0 240 F vol.

[0826] ) vol.

[0827] equiv)

[0828] DMF

[0829] (6 vol.)

[0830] DMF

[0831] Oxyma 8 x6 Pure vol. Fmoc-L- (2.0 equiv DM CH2CI Ala- 20%

[0832] ) DIC F 2 11 OH H2O 5 20 30 180 pip / DM 10 / 30

[0833] (2.2 equiv 6 x6 4 x6 (2.0 F

[0834] ) vol. vol. equiv)

[0835] DMF MTBE

[0836] (6 vol.) 4 x6

[0837]

[0838] vol.

[0839] 3,3 Soft Cleavage of Fragment Fmoc-Ala18- / / -Lys20(sc)- / / -Ala28-OH

[0840] The reaction scheme is shown in FIG.9.

[0841] Fragment Fmoc-Ala18- / / -Lys20(sc)- / / -Ala28-resin (33.0 g) was swelled using CH2Cl2(200 mL) at 20 °C for 30 min before it was filtered. Pre-cooled (5 °C) TFA / TIS / CH2Cl2(1:2:97 v / v / v, 330 mL) was charged into the reactor and the resulting suspension was stirred for 15 min at 15° C. The suspension was filtered and the filtrate was cooled to 10 °C and neutralized immediately by the slow addition of a solution of pyridine (4.2 g) in DMSO (33 mL). The resulting mixture was stirred at 10 °C for 30-60 min, then set aside. This sequence was repeated with the same batch of resin two more times. The three neutralized mixtures were then combined and concentrated under reduced pressure to 190 mL. The resulting solution was added to pre-cooled (2 °C) water (1.0 L) at 2 °C over 40-60 min and the resulting suspension was stirred at 2 °C for 40 min. The solid was isolated by filtration and the wet cake was washed with water (4 x 165 mL) at 2 °C. The solid was dried under reduced pressure with a flow of nitrogen at T < 25 °C until the water content was below 2% asdetermined by KF titration. Fragment Fmoc-Ala18- / / -Lys20(sc)- / / Ala28-OH was isolated as a solid (15.8 g, purity 97.6 area%, 56.2% yield).

[0842] LC-MS (ESI+): mass calculated for C165H222N18O30 [M]+2935.64, found [(M+2H)2+] 1469.7.

[0843] 3,4 LPPS of Fragment H-Ala18- / / -Lys20(sc)- / / -Ser39-NH2

[0844] The reaction scheme is shown in FIG.10.

[0845] Fragment Fmoc-Ala18- / / -Lys20(sc)- / / -Ala28-OH (5.00 g, 1.70 mmol, 1.0 equiv) and fragment H-Gly29- / / -Ser39-NH2 (2.0 g, 1.87 mmol, 1.1 equiv) were taken up in DMAc (90 mL, 18 vol.). The resulting mixture was cooled to 0 °C and a solution of FDPP (1.0 g, 2.72 mmol, 1.6 equiv) in DMAc (10 mL, 2 vol.) was added dropwise over 1-2 min. DIPEA (879 mg, 6.80 mmol, 4.0 equiv) was then added dropwise over 1-2 min and the resulting reaction mixture was stirred at 0 °C for 16 h. Upon completion of the reaction, water (2.5 mL, 0.5 vol.) was added dropwise over 1-2 min and the mixture was stirred for 30 min. Diethylamine (2.50 g, 34.0 mmol, 20.0 equiv) was added dropwise over 1-2 min at 0 °C and the resulting reaction mixture was allowed to warm to ambient temperature and was stirred for 2 h. Upon completion of the deprotection reaction, the mixture was added dropwise into 7.5% aq. NaCl solution (750 mL, 150 vol.) at 0 °C. The resulting suspension was stirred at this temperature for 2 h before it was filtered and the filter cake was washed with water (3 x 50 mL, 3 x 10 vol.) and dried under a stream of nitrogen. The resulting solid was dissolved in CH2CI2 (100 mL, 20 vol.) at ambient temperature. This solution was concentrated under reduced pressure at 25-35 °C to 5-10 vol.. The resulting solution was cooled to 0 °C and pre-cooled (0 °C) MTBE (500 mL, 100 vol.) was added dropwise. The resulting suspension was stirred at 0 °C for 30 min before it was filtered and the filter cake was washed with MTBE (3 x 15 mL, 3 x 5 vol.) and dried under a stream of nitrogen to afford fragment H-Ala18- / / -Lys20(sc)- / / -Ser39-NH2 (5.8 g, purity 75.2 area%) as a white solid.

[0846] LC-MS (ESI+) for Fmoc-protected fragment: mass calculated for C215H304N30O43 [M+] 3996.2591, found [(m+3H)3+] 1333.0919.

[0847] LC-MS (ESI+) for fully deprotected fragment: mass calculated for C137H218N30O39[M+] 2907.5997, found [(M+2H)2+] 1454.8153.

[0848] 4. Purification

[0849] The precipitated crude peptide was dissolved in water / AcOH / MeCN (60:35:5) at a peptide net concentration of 20 g / L. The solution was pre-treated by stirring it at ambient conditionsfor 20 ± 4 h to facilitate decarboxylation at Trp25. Prior to loading, the peptide solution was filtered (0.45 pm).

[0850] Purification was performed using preparative reversed-phase HPLC (stationary phase:

[0851] Kromasil-100-10-C18) in a multiple (3 times) sequential chromatographic approach. The purification process was run on an automated pump skid controlling the composition of the mobile phase mixture and enabling manual fractionation (UV-based) and collecting fractions by defined time intervals.

[0852] The product was eluted using a linear gradient, step gradient or a combination of both with increasing or partially constant organic content. The peptide was eluted from the column and the effluent was fractionated and assayed for purity using reversed phase HPLC-UV analysis. The fractions were pooled after analysis based on their purity. Fractions not meeting the pooling criteria were combined for recycling injections.

[0853] 4,1 1st, Preparative chromatography

[0854] In the 1st purification step, the peptide solution was purified using the process parameters listed in Table 4. After combination of qualified fractions, a main pool purity of 93.1% was obtained

[0855] Table 4. Process parameters used for the 1stpurification step.

[0856] I'1puril'icalion step

[0857] Column dimensions DAC-150 (150x280 mm, CV = 4945.5 mL)

[0858] Stationary phase Kromasil 100-10-C18

[0859] Column loading 4.63 g / mLcv (22.92 g net peptide)

[0860] Mobile phase A Water / MeCN 90: 10 + 0.1% TFA (v / v)

[0861] Mobile phase B MeCN / water 80:20 + 0.1% TFA (v / v)

[0862] Detection wavelength 254 nm (monitor wavelength 280 nm)

[0863] Flow rate 504 mL / min

[0864] Gradient t [min| 0 20 26 120 140 142 160

[0865] B [%1 31.4 31.4 45.7 51.4 51.4 94.3 94.3 Collection strategy Manual, UV based1

[0866] Fraction duration 2 min

[0867] Fraction pooling criteria HPLC purity > 90.0% (< 90.0 to > 80.0% for recycling; < 80.0%

[0868] waste)

[0869]

[0870] Storage temperature 2-8°C (fractions and pool)

[0871] 1Collection was performed from 90.89 to 112.89 min, 22 min collection time.

[0872] 4,22nd, Preparative chromatography

[0873] In the 2ndpurification step, the peptide solution was purified using the process parameters listed in Table 5. After combination of qualified fractions, a main pool purity of 97.5% was obtained.Table 5. Process parameters used for the 2ndpurification step.

[0874] 2'"1purilicalion step

[0875] Column dimensions DAC-150 (150x280 mm, CV = 4945.5 mL)

[0876] Stationary phase Kromasil 100-10-C18

[0877] Column loading 4.54 g / mL CV (22.46 g net peptide)

[0878] Pool diluent Water / AcOH 80:20 (v / v)

[0879] Pool dilution 2: 1 (Pool diluent: 1stpass pool, 3 volumetric equivalents)

[0880] Mobile phase A 50 mM NH4OAc(aa / MeCN 90: 10 (v / v, adjusted to pH 8.0-8.5)1Mobile phase B MeCN

[0881] Detection wavelength 254 nm (monitor wavelength 280 nm)

[0882] Flow rate 504 mL / min

[0883] Gradient t [min| 0 2 5 94 95 109

[0884] B [%] 0 0 20 38.9 72.2 72.2 Collection strategy manual, UV-based2

[0885] Fraction duration 2 min

[0886] Fraction pooling criteria HPLC purity > 95.0% (< 95.0 to > 80.0% for recycling; < 80.0% waste)

[0887]

[0888] Storage temperature 2-8°C (fractions and pool)

[0889] 1pH adjustment is done using ammonia.

[0890] 2Collection was performed from 39.1 to 49.1 min, 10 min collection time.

[0891] 4,3 3rd, Preparative chromatography

[0892] In the 3rdpurification step, the peptide solution was purified using the process parameters listed in Table 6. After combination of qualified fractions, a main pool purity of 98.6% was obtained.

[0893] Table 6. Process parameters used for the 3rdpurification step.

[0894] 3"1purification step

[0895] Column diemnsions DAC-150 (150x280 mm, CV = 4945.5 mL)

[0896] Stationary phase Kromasil 100-10-C18

[0897] Column loading 3.76 g / mL CV (18.58 g net peptide)

[0898] Pool diluent Water / AcOH 70:30 (v / v)

[0899] Pool dilution 2: 1 (Pool diluent: 2ndpass pool, 3 volumetric equivalents)

[0900] Mobile phase A Water

[0901] Mobile phase B EtOH

[0902] Detection wavelength 254 nm (monitor wavelength 280 nm)

[0903] Flow rate 252 mL / min

[0904] Gradient t [min] 0 5 30 115

[0905] B [%] 5 5 30 70 Collection strategy manual, UV-based1

[0906] Fraction duration 1 min

[0907] Fraction pooling criteria HPLC purity > 97.0% (< 97.0% waste)

[0908]

[0909] Storage temperature 2-8°C (fractions and pool)

[0910] 1Collection was performed from 86.50 to 93.60 min, 7.10 min collection time.

[0911] ***

Claims

1. Claims:

1. Process for the preparation of the peptide of formula I, or of a pharmaceutically acceptable salt or ester thereofX1-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(I)wherein X isCNand AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid,comprisinga) coupling of a functionalized peptide fragment FlH2N-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(Fl)with a functionalized peptide fragment F2X1-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15- Lys16-Ile17-OH(F2),wherein X and AEEAc are as defined above,b) cleaving off the protecting groups,c) precipitation of the peptide of formula I, and optionallyd) purification / isolation.

2. Process of claim 1, wherein the coupling is performed in the presence of a coupling agent, an organic base and an organic solvent.

3. Process of claims 1 or 2, wherein the coupling agent is selected from benzotriazol- 1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol- 1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP), (7-azabenzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), A-[(777-benzotriazol-l-yl)(dimethylamino)methylene]-A-methylmethanaminium tetrafluoroborate A-oxide (TBTU), 2-(7A-benzotriazole- 1 -yl)- 1, 1,3,3 -tetramethylaminium hexafluorophosphate (HBTU), hydroxybenzotriazole (HOBt), l-[bis(dimethylamino)methylene]-7A-l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, A-[(dimethylamino)-3A-l,2,3-triazolo-[4,5-b]pyridin-l-ylmethylene]-A-methylmethanaminium hexafluorophosphate A -oxi de (HATU), propanephosphonic acid anhydride (T3P), O-(2-oxo-l(2H)pyridyl)-N, N, N', N'-tetramethyluronium tetrafluoroborate (TPTU), l-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyOXIM), pentafluorophenyl diphenylphosphinate (FDPP), Diphenylphosphoryl azide (DPP A), 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) or from a combination of AA'-diisopropylcarbodiimide (DIC) with A-hydroxysduccinimide, with 4-(dimethylamino)pyridine (DMAP), with 2-hydroxypyridine-A-oxide (HOPO) or with (ethyl-cyano (hydroximino)acetate) (OxymaPure), or from a combination of (7-azabenzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) with (ethyl-cyano (hydroximino)acetate) (OxymaPure), or from a combination of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) with 2-hydroxypyridine-A-oxide (HOPO).

4. Process of any one of claims 1 to 3, wherein the organic base is selected from A, A-diisopropylethylamine, A-methylmorpholine (NMM), A-methylimidazole (NMI), 2-6-lutidine, 2,4,6-collidine or from triethylamine.

5. Process of any one of claims 1 to 4, wherein the organic solvent is selected from dimethylacetamide, AA-dimethylformamide, toluene, methylene chloride, acetonitrile or mixtures thereof.

6. Process of any one of claims 1 to 5, wherein the functionalized peptide fragment Fl has the formula FlaH2N- Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-Y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2(Fla),and the functionalized peptide fragment F2 has the formula F2aX1-β-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)-Tyr10-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-OH (F2a),wherein X and AEEAc are as defined above.

7. Process of any one of claims 1 to 6, wherein the process comprises coupling the functionalized peptide fragment of formula FlaH2N- Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-Y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2(Fla),with a functionalized peptide fragment of formula F2aX1-β-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)-Tyr10-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-OH(F2a),wherein X and AEEAc are as defined above.

8. Process of any one of claims 1 to 7, wherein the protecting groups are cleaved with TFA in combination with water and at least one scavenger.

9. Process of any one of claims 1 to 8, wherein the preparation of peptide fragment Fl comprises(A) coupling of a functionalized peptide fragment F3PROT-HN-Ala18-Gln19-Lys20(AEEAc-AEEAc-Y-Glu-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24-OHwherein PROT is a protecting group,with a functionalized peptide fragment F4H2N-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2(F4)and subsequent removal of the protecting group PROT; or(B) coupling of a functionalized peptide fragment F5PROT-HN-Ala18-Gln19-Lys20(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-OH(F5)wherein PROT is a protecting group;with a functionalized peptide fragment F6H2N-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2(F6)and subsequent removal of the protecting group PROT.

10. Process of claim 9, wherein the peptide fragment Fl has the formula FlaH2N- Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-Y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2(Fla),and the preparation comprises,(A) coupling of a functionalized peptide fragment F3aFmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-OH(F3a)with a functionalized peptide fragment F4aH2N-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2and the subsequent removal of the Fmoc protecting group; or(B) coupling of a functionalized peptide fragment F5aFmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28OH (F5a)with a functionalized peptide fragment F6aH2N-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38- Ser39(OtBu)-NH2(F6a)and the subsequent removal of the Fmoc protecting group.

11. Process of any one of claims 1 to 10, wherein the preparation of peptide fragment F2 or F2a comprises sequential coupling of the following functionalized building blocks on a resin under solid phase peptide synthesis (SPPS) conditions:Aminoacid # Cycle# Building Block2-chlorotrityl-resin17 1 Fmoc-L-Ile-OH16 2 Fmoc-L-Lys(Boc)-OH)15 3 Fmoc-L-Asp(OtBu)-OH14 4 Fmoc-L-Leu-OH12-13 5 Fmoc-Ile-Aib-OH11 6 Fmoc-L-S er(OtBu)-OH10 7 Fmoc-L-Tyr(OtBu)-OH9 8 Fmoc-L-Asp(OtBu)-OH8 9 Fmoc-L-S er(OtBu)-OH7 10 Fmoc-L-Thr(OtBu)-OH5-6 11 Fmoc-Thr-Phe-OH1-4 12 X-P-Ala-Glu-Gly-OHXT XX = CN12. Process of claim 11, wherein the peptide fragment F2 or F2a is cleaved from the resin with TFA in combination with an organic solvent and at least one scavenger.

13. A functionalized peptide fragment of the formula:H2N- Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-Y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2(Fla), orFmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-OH(F3a), orH2N-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2(F4a), orFmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28OH (F5a), orH2N-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2(F6a), orX1-β-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)-Tyr10-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-OH(F2a),wherein X isTJ ICN, orFmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-O-2-chlorotrityl resin(F3b), orFmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-O-2-chlorotrityl resin(F5b), orX1-β-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)-Tyr10-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-O-2-chlorotrityl resin(F2b),wherein X isCN14. A functionalized peptide fragment of formulaX1-β-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)-Tyr10-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-OH(F2a),wherein X isCN15. A functionalized peptide fragment of formulaH2N- Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-Y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29- Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2(Fla).

16. A functionalized peptide fragment of formulaFmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-OH(F3a).

17. A functionalized peptide fragment of formulaH2N-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34- Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH2(F4a).

18. A functionalized peptide fragment of formulaFmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28OH(F5a).

19. A functionalized peptide fragment of formulaH2N-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38- Ser39(OtBu)-NH220. A functionalized peptide fragment of formulaFmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-O-2-chlorotrityl resin(F3b).

21. A functionalized peptide fragment of formulaFmoc-HN-Ala18-Gln19(Trt)-Lys20(AEEAc-AEEAc-y-Glu(OtBu)-tBu-O-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-O-2-chlorotrityl resin(F5b).

22. A functionalized peptide fragment of formulaXJ-P-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)- Ser8(OtBu)-Asp9(OtBu)-Tyr10-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-O-2-chlorotrityl resin(F2b),wherein X isCN***

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