X-β-ala-glu(OTBU)-gly tetramer and its use in SPPS synthesis

Abstract

The invention relates to a tetramer of the formula (I), or salts thereof, wherein R1 is hydrogen or is a link to a solid support and PROT is an ester protecting group or hydrogen, to a process for the preparation of a tetramer of the formula I and to its use as intermediate in the preparation of a GLP-1R / GIPR agonist.

Description

[0001] P39745

[0002] X-0-Ala-Glu(OtBu)-Gly tetramer and its use in SPPS synthesis

[0003] The invention relates to a tetramer of the formula I, or salts thereof, wherein

[0004] R1is hydrogen or is a link to a solid support and

[0005] PROT is an ester protecting group or hydrogen, and to a process for the preparation of a tetramer of the formula I and to its use for the manufacturing of the peptide of formula III, or of a pharmaceutically acceptable salt or ester thereof

[0006] X-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-He12-Aib13-Leu14-Asp15-Lys16-

[0007] Ile17-Ala18-Gln19-Lys20(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-

[0008] Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-

[0009] Ser39-NH2

[0010] (III) wherein X is

[0011] 02.11.2025

[0012] AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid

[0013] The peptide of formula III has the potential to act as GLP-1R / GIPR agonist.

[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 (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.

[0015] Particularly the peptide of formula III 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 III does not cause internalization and consequently, desensitization of either GLP-1R or GIPR, and thus has enhanced signaling efficacy.

[0016] Object of the invention was to facilitate the synthesis of the peptide of formula III and to provide a building block which can be easily integrated into a solid phase peptide synthesis (SPPS) reaction set up. In addition the facilitation should aim at improving the impurity profile, and shortening the overall production time by reducing the number of SPPS cycles. It was found that the object of the invention could be reached with the tetramer of the formula I, a process for its preparation and the use of the tetramer of formula I in the manufacturing of the peptide of formula III, or of a pharmaceutically acceptable salt or ester thereof

[0017] X-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-He12-Aib13-Leu14-Asp15-Lys16- Ile17-Ala18-Gln19-Lys20(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala21-Phe22-Val23- Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38- Ser39-NH2

[0018] (III) wherein X is

[0019] AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid.

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

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

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

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

[0024] The term “solid support” in the context of the present invention encompasses solid supports for SPPS which are insoluble, chemically inert materials that serve as anchoring points for the growing peptide chain during synthesis. These supports are designed to withstand the repeated cycles of coupling, deprotection, and washing steps involved in peptide assembly. They provide a physical matrix that allows for easy separation of the peptide from reagents and by-products through simple filtration. Typically, the solid supports are polystyrene resins like the 2-chlorotrityl chloride (CTC) resin, which is an acid-labile resin that allows for mild cleavage conditions, e.g. with trifluoroacetic acid 1% in dichloromethane. Furthermore, the term includes resins which are composed of the polymeric solid support and linked permanently to a linker (bifunctional spacer, or handle) that facilitate temporary anchoring of the first amino acid to the polymeric solid support. Depending on the type of linker, the C-terminus of the first amino acid is anchored to the solid support as an amide, ester, thioester, O- substituted oxime, or hydrazide. For instance, benzhydrylamine resins, such as the Rink amide resin or xanthenyl linker resins, such as the Sieber amide resin can be used. Alternatively, the Ramage amide resin or a tricyclic amide linker resin can be applied. All these resins will form an amide with the first amino acid. After cleavage from solid support, the peptide will comprise a C-terminal amide group.

[0025] The term “link to a solid support” which has been used as a substituent for R1means “connected to a solid support” via a single bond. This single bond establishes the connection to the solid support according to the definition above. The connection to the solid support can be made via a bifunctional spacer, such as the chlorotrityl group in the CTC resin. The term “solid phase peptide synthesis (SPPS) conditions” refers to specific parameters and reagents for assembling peptides on a solid support. These conditions are tailored to ensure efficient coupling, minimize side reactions, and yield high-purity peptides, varying based on the peptide sequence, scale, and desired synthesis outcome. Key components include, but are not limited to the solid support, suitable protecting groups for the amino acids, suitable coupling agents and solvents, procedures to eliminate by-products and suitable cleavage conditions to remove the peptide from the resin and deprotect side chains.

[0026] In one aspect, the invention relates to the tetramer compound of the formula I, or salts thereof wherein

[0027] R1is hydrogen or is a link to a solid support and

[0028] PROT is an ester protecting group or hydrogen.

[0029] PROT preferably is an ester protecting group, more preferably tert-butyl (tBu).

[0030] In one aspect, R1is hydrogen and the tetramer of formula I is the free acid.

[0031] In another aspect, R1is a link to a solid support, preferably a link to an acid labile solid support. More preferably, “link to a solid support” means that R1is the CTC resin of the formula:

[0032] In a further aspect, the invention relates to the preparation of the tetramer compound of formula I, wherein, in the direction of the peptide synthesis, a) glycine, glutamic acid, 0-alanine and 2-(3-cyano-5-fluorophenyl)-2-methyl- propionic acid of the formula II are coupled on a solid support under solid phase peptides synthesis (SPPS) conditions, b) the solid phase bound tetramer is cleaved from the solid support under acidic conditions.

[0033] Step a)

[0034] In general, glycine, glutamic acid and P-alanine are applied in a form, wherein their amino group is protected by an amino protecting group. The y-carboxylic acid of glutamic acid is protected by an ester protecting group, typically by tert-butyl (tBu). The common amino protecting group is ((9H-fluoren-9-yl)methoxy)carbonylamino (Fmoc).

[0035] As a rule, the peptide synthesis is performed in the presence of a coupling agent, 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-[(l H-benzotriazol-1- yl)(dimethylamino)methylene]-A-methylmethanaminium tetrafluoroborate A-oxide (TBTU), 2-(lH-benzotriazole-l-yl)-l,l,3,3-tetramethylaminium hexafluorophosphate (HBTU), 1- [bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU or A-[(dimethylamino)-lH-l,2,3-triazolo-[4,5-b]pyridin-l- ylmethylene]-A-methylmethanaminium hexafluorophosphate A-oxide), carbonyldiimidazole (CDI), dicyclohexylcarbodiimide (DCC), diisoopropylcarbodiimide (DIC), or A-(3- dimethylaminopropyl)-A’-ethylcarbodiimide (EDC). DCC, DIC, and EDC may be used in combination of an additive like 1 -hydroxy -benzotriazole (HOBt), l-hydroxy-7-aza- benzotriazole (HO At), or ethyl 2-cyano-2-(hydroxyimino)acetate (OxymaPure).

[0036] Preferably the coupling agent is PyBOP or diisoopropylcarbodiimide (DIC) in combination with ethyl 2-cyano-2-(hydroxyimino)acetate (OxymaPure). For the coupling reaction with BOP, PyBOP, PyBrOP, PyAOP, TBTU, HBTU, or HATU, an organic base, such as a trialkylamine selected from triethylamine, A-methyl morpholine or diisopropylethylamine (DIPEA) or collidine, but preferably diisopropylethylamine (DIPEA) or collidine is used. Preferred collidine is 2,4,6-collidine.

[0037] In addition an organic solvent selected from a polar, aprotic solvent such as N’N- dimethylformamide (DMF), A'-alkyl pyrrolidones, like A-butyl pyrrolidone, dimethyl sulfoxide (DMSO), ethyl acetate or mixtures thereof, are expediently present.

[0038] The loading of glycine to the CTC resin in the first sub step can take place with the amino group of the glycine protected with an amino protecting group, typically Fmoc, in the presence of a trialkylamine, typically DIPEA and in a polar, aprotic solvent, typically in DMF.

[0039] Further repetitive washes of the resin with the polar, aprotic solvent can be applied to remove excess of reagents, by products and any unreacted material.

[0040] Deprotection of the amino protecting group, typically Fmoc, and exposing the amino group for the next coupling step, can be accomplished by reaction with piperidine in the polar, aprotic solvent. As a rule, solutions of 10 to 30% piperidine in DMF are used.

[0041] The reaction temperature for the activation of the acid typically takes place between 0 °C and 20 °C, while the subsequent coupling reaction takes place in a temperature range between 10 °C and 40 °C, preferably at ambient temperature.

[0042] The solid phase bound tetramer can finally be subjected to repetitive washes with an organic solvent. Optionally, these repetitive washes may be performed using a solvent that leads to a shrinking of the resin (such as tert-butyl ether) followed by a solvent that leads to a swelling of the resin (such as dichloromethane) in an alternating way. These repetitive washes result in an effective removal of the polar aprotic solvent, such as DMF. Thereafter the resin can be isolated and dried.

[0043] Step b)

[0044] The solid phase bound tetramer can be cleaved from the solid support under acidic conditions which have to be selected in a manner that the acid protection group PROT of Glu remains unaffected. Cleavage therefore happens under soft conditions, typically with a low concentrated solution, preferably 1% solution, of trifluoroacetic acid (TFA) in an organic solvent. The common solvent is dichloromethane. However, alternative solvents can be used as described in Alhassan et al., Green Chem. 2020, 22, 2840-2845, https: / / doi.org / 10.1039 / D0GC00834F. Trifluoroethanol or hexafluoroisopropanol (HFIP) can be used as an alternative to TFA.

[0045] The isolation of the free acid of the tetramer of formula I can happen by filtration of the reaction solution, neutralization with e.g. pyridine, followed by a solvent swap from e.g. dichloromethane to isopropyl acetate, aqueous wash and precipitation from isopropyl acetate and methyl tert-butyl ether / n-heptane.

[0046] Filtration and drying delivers a tetramer of formula I, in the form of the free acid (R1is hydrogen) in yields of about 75%.

[0047] In a further aspect of the present invention, the tetramer of formula I, wherein R1is hydrogen, or of a salt thereof can be used for the preparation of the peptide of formula III, or of a pharmaceutically acceptable salt or ester thereof

[0048] X-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-He12-Aib13-Leu14-Asp15-

[0049] Lys16-Ile17-Ala18-Gln19-Lys20(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala21-Phe22-

[0050] Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-

[0051] Pro38-Ser39-NH2

[0052] (III) wherein X is

[0053] AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid.

[0054] The tetramer of formula I, wherein R1is hydrogen, or a salt thereof, can in a still further aspect of the present invention be used for the preparation of the peptide of formula Va, or of a functionalized derivative thereof,

[0055] X1-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15- Lys16-Ile17-Ala18-Gln19-Lys20-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30- Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH-linker-solid support

[0056] (Va) wherein X is

[0057] The term “functionalized derivative” in this and the following contexts means that reactive sites of respective amino acids are protected with suitable protecting groups, e.g. Thr, Ser, Glu, Asp or Tyr with tBu, Trp or Lys16with Boc, Gin with Trt and Lys20with Mtt or ivDde.

[0058] The tetramer of formula I, wherein R1is hydrogen, or a salt thereof, can in a further aspect of the present invention particularly be used for the preparation of the functionalized peptide derivative of Vb,

[0059] X1-P-Ala2-Glu(tBu)3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10- Ser(tBu)11-Ile12-Aib13-Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys20(Mtt / ivDde)- Ala21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-He27-Ala28-Gly29-Gly30-Pro31-Ser(tBu)32- Ser(tBu)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(tBu)39-NH-linker-solid support

[0060] (Vb), wherein X is as above.

[0061] In a still further aspect of the invention, there is provided a process for the preparation of the peptide of formula III, or of a pharmaceutically acceptable salt or ester thereof

[0062] X-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Sern-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

[0063] (III) wherein X is AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid. comprising the steps a) solid phase synthesis of the peptide of formula III on a resin, comprising coupling the tetramer of formula I to a functionalized peptide fragment of formula IVa

[0064] H-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18- Gln19-Lys20-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33- Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH-linker-solid support

[0065] (IVa) and forming the functionalized peptide fragment of formula Va

[0066] X1-p-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-He12-Aib13-Leu14-Asp15- Lys16-Ile17-Ala18-Gln19-Lys20-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30- Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH-linker-solid support

[0067] (Va) b) deprotection of the Lys20s-amino group protecting group, c) acylation of the s-amino group of Lys20with the moiety

[0068] PROT-O-20-oxoicosanoyl-L-Glu (AEEAc-AEEAc-OH)-O-PROT

[0069] (Via), wherein AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid and PROT is an ester protecting group, and forming the peptide of formula III, bound to the resin; d) cleavage of the peptide of formula III from the resin and global deprotection with an acid and e) precipitation of the peptide formula III and optionally f) purification / isolation. Whereas the term “functionalized derivative” has the meaning as outlined above, Lys20is ideally functionalized with an ivDde (l-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)- 3 -methylbutyl) or a Mtt (4-methyltrityl) protecting group. Preferred functionalization of Lys20is Mtt.

[0070] Thus, in one aspect of the invention, the functionalized peptide derivative of Vb has the formula Vbi

[0071] X1-P-Ala2-Glu(tBu)3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10- Ser(tBu)11-Ile12-Aib13-Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys20(Mtt)-Ala21- Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-He27-Ala28-Gly29-Gly30-Pro31-Ser(tBu)32-Ser(tBu)33- Gly34-Ala35-Pro36-Pro37-Pro38-Ser(tBu)39-NH-linker-solid support

[0072] (Vbi), wherein X is as above.

[0073] In another aspect, the functionalized peptide derivative of Vb has the formula Vbii

[0074] X1-P-Ala2-Glu(tBu)3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10- Ser(tBu)11-Ile12-Aib13-Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys20(ivDde)- Ala21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-He27-Ala28-Gly29-Gly30-Pro31-Ser(tBu)32- Ser(tBu)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(tBu)39-NH-linker-solid support

[0075] (Vbii), wherein X is as above.

[0076] In a preferred aspect of the invention, the functionalized peptide derivative of Vb has the formula Vbi, comprising Lys20functionalized with Mtt.

[0077] In a further particular aspect of the present invention the process comprises the steps a) solid phase synthesis of the peptide of formula III on a resin, comprising coupling the tetramer of formula I to a functionalized peptide fragment of formula IVb

[0078] H-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10-Ser(tBu)n-Ile12-Aib13- Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys20(Mtt / ivDde)-Ala21-Phe22-Val23- Gln(Trt)24-Trp(Boc)25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser(tBu)32-Ser(tBu)33-Gly34-Ala35- Pro36-Pro37-Pro38-Ser(tBu)39-NH-linker-solid support

[0079] (IVb) and forming the functionalized peptide fragment of formula Vb

[0080] X1-P-Ala2-Glu3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10- Ser(tBu)11-Ile12-Aib13-Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys20(Mtt / ivDde)- Ala21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-He27-Ala28-Gly29-Gly30-Pro31-Ser(tBu)32- Ser(tBu)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(tBu)39-NH-linker-solid support,

[0081] (Vb) b) deprotection of the Lys20s-amino protecting group Mtt or ivDde, c) acylation of the s-amino group of Lys20with the moiety tBu-O-20-oxoicosanoyl-L-Glu(AEEAc-AEEAc-OH)-OtBu

[0082] (VIb), wherein AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid, and forming the peptide of formula III, bound to the resin; d) cleavage of the peptide of formula III from the resin and global deprotection with an acid and e) precipitation of the peptide formula III and optionally f) purification / isolation.

[0083] In a further particular aspect of the present invention, Lys20is functionalized with Mtt in the peptide fragments of formula IVb and Vb.

[0084] Step a)

[0085] Step a) as outlined above requires the solid phase synthesis of the peptide of formula III on a resin, comprising coupling the tetramer of formula I to the functionalized peptide fragment of formula IVa or IVb and forming the functionalized peptide fragment of formula Va or Vb.

[0086] The general SPPS method is well known in the art and is described for instance in W.C. Chan and P.D. White, Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press Inc., 1999. However, the reaction conditions of the SPPS need to be adapted for every peptide. For the solid phase synthesis of the peptide fragment of formula Va or Vb, the following key conditions and reagents were found to be suitable.

[0087] The Sieber amide resin with Fmoc protection was found to be well suited for this SPPS.

[0088] Fmoc was found to be the amino protecting group of choice and for its deprotection a common 20% v / v piperidine solution in DMF has been applied.

[0089] For the coupling, a combination of the coupling agents DIC and OxymaPure (ethyl 2- cyano-2-(hydroxyimino)acetate) or the combination PyBOP / collidine was found to be favorable for its properties to reduce epimerization during activation / coupling.

[0090] Step b)

[0091] In step b) the Lys20s-amino protecting group is removed.

[0092] For the deprotection of the ivDde (l-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)-3- methylbutyl) protecting group a 2 to 10% v / v solution of hydrazine monohydrate in DMF can be applied.

[0093] For the deprotection of the Mtt (4-methyltrityl) protecting group a 1% v / v TFA solution in DCM or alternatively a mixture of hexafluoroisopropanol (HFIP) and scavengers like trisopropylsilane (TIPS), di thioerythrol (DTT) or V-acety Icy stein (NAC) in an organic solvent like DCM or toluene can be used. Preferably the MTT deprotection is conducted with a mixture of hexafluoroisopropanol (HFIP) with the scavenger trisopropylsilane (TIPS) in toluene.

[0094] Step c

[0095] In step c) the s-amino group of Lys20is acylated with the moiety of formula Via or VIb

[0096] PROT-O-20-oxoicosanoyl-L-Glu(AEEAc-AEEAc-OH)-O-PROT

[0097] (Via) or tBu-O-20-oxoicosanoyl-L-Glu(AEEAc-AEEAc-OH)-OtBu

[0098] (VIb) wherein AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid and PROT is an ester protecting group, whereby the peptide of formula III, bound to the resin, is formed.

[0099] The coupling conditions are essentially the same as described in step a).

[0100] Step d)

[0101] In step d) the peptide of formula III, bound to the resin, is subjected to cleavage from the resin and to global deprotection, whereby the desired peptide of formula III is obtained.

[0102] Cleavage can be accomplished with trifluoroacetic acid. The cleavage cocktail may include water, DTT (dithiothreitol) and TIPS (triisopropylsilane) as scavengers.

[0103] Step e)

[0104] Precipitation peptide of formula III precipitated in step e) in a suitable organic solvent such as methyl tert-butyl ether and dried.

[0105] Optionally a further purification and isolation step f) may be added.

[0106] Examples

[0107] Abbreviations:

[0108] CTC Resin 2-chlorotrityl chloride resin

[0109] DCM di chloromethane

[0110] DIC V,7V'-diisopropylcarbodiimide

[0111] DIPEA diisopropylethylamine

[0112] DMF Vf-dimethylform amide

[0113] DMSO dimethylsulfoxide

[0114] DTT dithiothreitol

[0115] EtOAc ethyl acetate

[0116] Fmoc 9-fluorenylmethoxycarbonyl

[0117] HPLC high pressure liquid chromatography iPrOAc / .w-propyl acetate ivDde 1 -(4, 4-dimethyl-2,6-di oxocyclohex- 1 -ylidene)-3 - methylbutyl

[0118] MTBE / methyl tert, -butyl ether TMBE

[0119] NAC V-acetyl cystein

[0120] OxymaPure ethyl 2-cyano-2-(hydroxyimino)acetate

[0121] PyBOP benzotriazol- 1 -yloxytripyrrolidinophosphonium hexafluorophosphate rt room temperature tBu tert. -butyl

[0122] TFA trifluoroacetic acid

[0123] THF tetrahydrofuran

[0124] TIPS triisopropylsilane

[0125] V Volume equivalent SPPS experimental procedure of the X-B-Ala-Glu(OtBu)-Gly tetramer

[0126] 2-[[(2S)-2-[3-[[2-(3-cyano-5-fluorophenyl)-2-methylpropanoyl]amino]propanoylamino]-5- [(2-methylpropan-2-yl)oxy]-5-oxopentanoyl]amino]acetic acid

[0127] Under a nitrogen atmosphere, CTC resin (303 g, 1.10 mmol / g) was added to a jacketed glass peptide synthesis reaction vessel with sintered glass filter frit and DMF (8 V). The suspension was stirred gently for 30 minutes, and the solvent was drained.

[0128] Fmoc-Gly-OH (198 g, 2.0 equiv.) and DIPEA (344g, 8.0 equiv.) were dissolved in DMF (8 V) and the solution was added into the reaction vessel. The suspension was stirred gently for 2 h. Methanol (500 mL) was added, and gentle stirring continued for 30 minutes before the solvent was drained. The resin was washed 8 times with DMF (8 V).

[0129] The resin was treated twice for 15 minutes with 20% piperidine in DMF (8 V) followed by solvent draining and 8 wash steps with DMF (8 V).

[0130] Fmoc-Glu(OtBu)-OH (443 g, 3.0 equiv.), PyBOP (520 g, 3.0 equiv.) and DIPEA (258 g, 6.0 equiv.) were dissolved in DMF (8 V) and the solution was stirred gently at 5°C for 10 min before being added into the reaction vessel. The suspension was stirred gently for 4 h. The solvent was drained, and the resin was washed 8 times with DMF (8 V). The resin was treated twice for 15 minutes with 20% piperidine in DMF (8 V) followed by solvent draining and 8 wash steps with DMF (8 V).

[0131] Fmoc-P-Ala-OH (207g, 2.0 equiv), PyBOP (347 g, 2.0 equiv.) and DIPEA (172 g, 4.0 equiv.) were dissolved in DMF (8 V) and the solution was stirred at 5°C for 10 min before being added into the reaction vessel. The suspension was stirred for 4 h. The solvent was drained, and the resin was washed 8 times with DMF (8 V).

[0132] The resin was treated twice for 15 minutes with 20% piperidine in DMF (8 V) followed by solvent draining and 8 wash steps with DMF (8 V).

[0133] 2-(3-cyano-5-fluorophenyl)-2-methyl-propionic acid (69.0 g, 1.0 equiv.), PyBOP (173 g, 1.0 equiv.) and DIPEA (86.1 g, 2.0 equiv.) were dissolved in DMF (8 V) and the solution was stirred at 5 °C for 10 min before being added into the reaction vessel. The suspension was stirred for 4 h. The solvent was drained, and the resin was washed 8 times with DMF (8 V).

[0134] The resin was washed sequentially with 2 x 8 V MTBE, 2 x 8 V DCM and finally 2 x 8 V MTBE. The resin was dried by N2 flow in the reaction vessel.

[0135] The resin was swelled twice with DCM (7 V) for each 30 min. 1% TFA / DCM (3.5 V) was charged to the reactor and the resulting suspension was stirred for 15 min under nitrogen maintaining 25°C. The liquids were filtered off and the filtrate was neutralized by slow addition of pyridine (27.7 g, 1.05 equiv.). The resin treatment with 1% TFA / DCM followed by filtrate neutralization was repeated four more times. The resin was washed three times with DCM (3.5 V) by stirring for 10 min. All the filtrates and washes were combined.

[0136] The combined solution was concentrated under reduced pressure to 1.5 V at T<35°C. 2-PrOAc (2.5 V) was added to the solution and the volatiles were distilled off under reduced pressure to 1.5 V at T<40°C.

[0137] The addition of 2-PrOAc and distillation under reduced pressure were repeated. The residue was dissolved with MTBE (3.5 V) to give the crude product solution.

[0138] The solution was washed three times with saturated aq. NaCl solution (I V) and the layers were separated. The organic layer was evaporated to dryness under reduced pressure at T<40°C.

[0139] The residue was dissolved in DCM (2.5 V), filtered, and concentrated under reduced pressure 1.5 V at T<35°C. The residue was dissolved in 2-PrOAc (2.5 V) and concentrated under reduced pressure to 1.5 V at T<35 °C. The residue was dissolved in 2-PrOAc (2 V) and the solution was added to the mixture of MTBE (2 V) and n-heptane (7.5 V) dr op wise at 0 - 10 °C.

[0140] The resultant slurry was stirred for 1 h and then filtered. The filter cake was washed with n- heptane (3 V) and dried under nitrogen to give the target product in 75% overall yield.

[0141] 1H NMR (500 MHz, DMSO-d6) 8 ppm 10.70 - 14.06 (m, 1 H), 8.06 (d, J=8.1 Hz, 1 H), 7.99

[0142] - 8.04 (m, 1 H), 7.69 - 7.73 (m, 1 H), 7.60 (d, J=1.4 Hz, 1 H), 7.56 - 7.59 (m, 1 H), 7.45 - 7.48 (m, 1 H), 4.20 - 4.32 (m, 1 H), 3.58 - 3.74 (m, 2 H), 3.23 - 3.28 (m, 2H), 2.26 - 2.34 (m, 2 H), 2.20 - 2.26 (m, 2 H), 1.83 - 1.97 (m, 1 H), 1.64 - 1.77 (m, 1 H), 1.46 (s, 6 H), 1.39 (s, 9 H).

[0143] 13C NMR (126 MHz, DMSO-d6): 6 ppm 174.9, 172.2, 171.7, 171.4, 171.1, 162.0 (d, J=247 Hz), 151.3 (d, J=8 Hz), 127.0, 119.4 (d, J=23 Hz), 118.3, 117.6 (d, J=25 Hz), 113.1, 80.1, 52.0, 46.9, 41.5, 36.5, 35.4, 31.7, 28.2, 27.7, 26.8.

[0144] 19F NMR (471 MHz, DMSO-d6) 6 = -110.56 (d, J = 8.5 Hz, IF).

[0145] MS (ESI) calculated for C25H33FN4O7, 520.2333; found, 520.2334.

[0146] Solid-Phase Peptide Synthesis using X-B-Ala-Glu(OtBu)-Gly tetramer as building block

[0147] X-P-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-

[0148] Ly s(AEEAc-AEEAc-y-Glu- 19-carboxynonadecanoyl)-Ala-Phe-

[0149] Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NTE TFA ,

[0150] 5 dry at 22°C at 5 mbar

[0151] X-B-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-lle-Aib-Leu-Asp-Lys-lle-Ala-Gln-Lys(AEEAc-AEEAc-Y-Glu-19-carboxynonadecanoyl)-Ala-Phe-

[0152] Val-GIn-Trp-Leu-lle-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2TFA

[0153] Synthetic Procedure: SPPS

[0154] Preparation of the Resin

[0155] Fmoc Sieber Resin (25 g, loading 0.75 mmol / g, 18.75 mmol) was charged into a 0.5 L SPPS reactor. The resin was swelled with DMF (226 mL, 8.52 g / g resin) and stirred for 2 h at 23 °C. The reactor was then drained, and the resin was washed twice with DMF (2 x 226 mL, 8.52 g / g resin).

[0156] General Synthetic Procedure

[0157] Washing steps were designed to remove the remaining deprotection solution, coupling reagents, and additives between process steps. Before each wash, the reactor was completely drained (i.e., until the flow into the waste accumulation vessel has stopped). The wash solvent was added to the reactor and the mixture was stirred for 5 min (starting after solvent addition is complete and stirring is started). The stirrer was then stopped, and the reactor was drained completely.

[0158] Fmoc deprotection was performed by stirring the resin in Fmoc deprotection solution. Deprotections in cycles 1-7 were performed with a stirring time of 15 min, while deprotection cycles 8-32 were performed with a stirring time of 30 min. Time starts after solvent addition is complete and stirring is started. The Fmoc deprotection solution consists of a 20% v / v piperidine solution in DMF.

[0159] In cycle 33, ivDde deprotection was performed by 8 treatments with a 3% v / v solution of hydrazine monohydrate in DMF (6x 30 min, lx 15 h).

[0160] For all couplings, a DIC / OxymaPure procedure was used. The protected amino acid or building block and OxymaPure were dissolved together in DMF. As the resin volume increases with the progressing SPPS build, the later coupling steps were carried out under more diluted conditions. A solution of DIC in DMF was then added and the resulting mixture was stirred at ambient temperature for 5 min to pre-activate the amino acid.

[0161] Once pre-activation was complete, the amino acid solution was added to the reactor. The reaction mixture was then stirred. The reaction time for the coupling was 1.5-6 h. Recoupling was performed for cycle 24 using the same procedure as the coupling.

[0162] In-process monitoring was carried out following selected deprotections and couplings. To this end, the fragment was cleaved from the resin and conversion was assessed by HPLC.

[0163] Synthetic Procedure

[0164] Starting with the Fmoc-deprotection of the protected resin, the peptide backbone and the side chain on Lys20were built using the general conditions for each amino acid coupling and deprotection outlined in Table 1.

[0165] Table 1 : Conditions used for the SPPS build on 0.75 mmol scale.

[0166]

[0167] The detailed coupling sequence and step conditions in the various cycles are listed in Table 2.

[0168] Table 2: Detailed conditions for the individual cycles in the SPPS build on 460 mmol scale.

[0169] Synthetic Procedure: Cleavage / Global Deprotection and Precipitation

[0170] The resin bound material, obtained after the SPPS step, was subjected to cleavage / global deprotection to afford the crude peptide.

[0171] The cleavage cocktail was prepared as follows: TFA (90 mL, 9 mL / g of resin bound material) was cooled to 0 °C. Water (2.5 mL, 0.25 mL / g of resin bound material) was added, followed by DTT (5 g, 0.5 g / g of resin bound material) and TIPS (2.5 mL, 0.25 mL / g of resin bound material). Resin bound material (10 g) was charged into a 250 L jacketed reactor equipped with a frit. The pre-cooled (0 °C) cleavage cocktail was pumped into the reactor and the resulting mixture was warmed to 20 °C and stirred at this temperature for 90 min. The mixture was then filtered, and the filtrate was collected in a 2 L jacketed reactor. The spent resin was washed with TFA (2x 10 mL) and the washing liquid was combined with the filtrate. The resulting mixture was cooled to -15 ° C. Then, pre-cooled (-15 °C) TBME (660 mL, 66 mL / g of resin bound material) was added slowly to the filtrate over 65 min while keeping IT <-5 °C. After the addition, the suspension was stirred at -5 °C for 30 min before it was warmed to 20 °C during 30 min. The suspension was filtered, and the filter cake was washed with TBME (3x 100 mL). The isolated solid was dried at 5 mbar and 22 °C for 16 h.

[0172] The crude peptide was isolated as an off-white powder (5.94 g, purity 72.4 area %).

[0173] Solid-Phase Peptide Synthesis using X-B-Ala-Glu(OtBu)-Gly tetramer and Fmoc-L- Lys(MTT)-OH as building block

[0174] X-p-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-lle-Aib-Leu-Asp-Lys-lle-Ala-Gln-Lys(AEEAc-AEEAc-y-Glu-19-cart>oxynonadecanoyl)-Ala-

[0175] Phe-Val-Gln-Trp-Leu-lle-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2-TFA

[0176] Synthetic Procedure: SPPS

[0177] Preparation of the Resin

[0178] Sieber resin (25 g, loading 0.77 mmol / g, 19.25 mmol) was charged into a 0.5-L SPPS reactor (fully automated). The resin was swelled with DMF (226 mL, 8.52 g / g resin) and stirred for 2 h at 23 °C. The reactor was then drained, and the resin was washed twice with DMF (2 x 226 mL, 8.52 g / g resin).

[0179] General Synthetic Procedure

[0180] Washing steps were designed to remove the remaining deprotection solution, coupling reagents, and additives between process steps. Before each wash, the reactor was completely drained (i.e., until the flow into the waste accumulation vessel has stopped). The wash solvent was added to the reactor and the mixture was stirred for 5 min (starting after solvent addition is complete and stirring is started). The stirrer was then stopped, and the reactor was drained completely.

[0181] All Fmoc deprotections were performed by 1-2 treatments with piperidine / DMF (20% v / v) with a stirring time of 20-30 min per cycle. After Fmoc removal, the resin was washed with DMF.

[0182] In cycle 35, Mtt deprotection was performed by 3 treatments with a HFIP / toluene / TIPS (29.5 / 69.5 / 1 v / v / v). Subsequently, the resin was washed with DMF.

[0183] For all couplings in cycle 1-33 and 35, a DIC / Oxyma Pure procedure was used. The protected amino acid or building block and Oxyma Pure were dissolved together in DMF. A solution of DIC in DMF was then added and the resulting mixture was typically stirred at 20 °C for 10 - 60 min to pre-activate the building block.

[0184] For the coupling in cycle 34, PyBOP / 2,4,6-trimethylpyridine was used. The tetramer building block (X1-p-Ala2-Glu3-Gly4) was dissolved in DMF together with PyBOP. A solution of 2,4,6-trimethylpyridine in DMF was then added and the resulting mixture was typically stirred at 20 °C for 5 min to pre-activate the building block.

[0185] Once the pre-activation was complete, the protected amino acid or building block solution was added to the resin. The reaction mixture was then stirred. The reaction time for the coupling was 1.5-24 h.

[0186] In-process monitoring was carried out following selected deprotections and couplings. To this end, the fragment was cleaved from the resin and conversion was assessed by HPLC.

[0187] A capping step was performed after coupling in cycle 11, 12, 18, 25 and 26. Following the post coupling washes with DMF the peptide resin was treated with a mixture of Ac2O / pyridine / DMF (1 / 1 / 98, v / v / v) for 10 min. Subsequently, the peptide was washed with DMF.

[0188] After completion of the solid phase synthesis, the resin was washed several times with DMF, 2 PrOH and MTBE before discharging. The resin was dried under reduced pressure for 48 h.

[0189] Table 3: Conditions used for the linear SPPS using Fmoc-Lys(Mtt)-OH.

[0190]

[0191]

[0192] The detailed coupling sequence and step conditions in the various cycles are listed in Table 4.

[0193] Table 4: Detailed conditions for the linear SPPS using Fmoc-Lys(Mtt)-OH and the tetramer building block.

[0194] TFA-mediated cleavage from the solid support with concomitant global deprotection

[0195] The resin bound material, obtained after the SPPS step, was subjected to cleavage / global deprotection to afford the crude peptide.

[0196] The resin bound peptide was added to the cleavage cocktail (TFA / H2O / TIS 90.0 / 5.0 / 5 (v / v / v); 10 mL per g resin). The reaction mixture was stirred for 1.5 h at 20°C before it was filtered. The filtrate was transferred into a jacketed reactor and cooled to -15 °C. Pre-cooled TBME (-15°C, 54 ml / g of resin bound material) was then added slowly while maintaining the internal temperature below -5°C.

[0197] The resulting suspension was then warmed to 20 °C and aged at this temperature for 30 min. The crude precipitate was filtered, and the filter cake was washed with TBME (3x 10 mL / g of resin bound material for each wash). The isolated solid was dried at 1-3 mbar and 22 °C for about 16 h.

[0198] The crude peptide was isolated as an off-white powder (6.68 g, purity 69.8 area %).

[0199] ***

Claims

Claims:

1. Tetramer compound or a salt thereof, of formula IwhereinR1is hydrogen or is a link to a solid support andPROT is an ester protecting group or hydrogen.

2. The tetramer compound of claim 1, wherein PROT is an ester protecting group.

3. The tetramer compound of claim 1 or 2, wherein PROT is tert-butyl (tBu).

4. The tetramer compound of claim 1, wherein PROT is hydrogen.

5. The tetramer compound of any one of claims 1 to 4, wherein R1is hydrogen.

6. The tetramer compound of any one of claims 1 to 4, wherein R1is a link to a solid support of the formula:

7. Process for the preparation of the tetramer compound of formula I of any one of claims 1 to 6, wherein, in the direction of the peptide synthesis,a) glycine, glutamic acid, 0-alanine and 2-(3-cyano-5-fluorophenyl)-2-methyl-propionic acid of the formula IIare coupled on a solid support under solid phase peptide synthesis (SPPS) conditions, and b) the solid phase bound tetramer is cleaved from the solid support under acidic conditions.

8. The process of claim 7, wherein the solid phase support is an acid-labile solid support.

9. The process of claim 8, wherein the 2-Chlorotrityl chloride (CTC) resin is used.

10. The process of any one of claims 7 to 9, wherein the solid phase bound tetramer is cleaved from the solid support under acidic conditions and isolated as a solid.

11. The process of any one of claims 7 to 10, wherein glycine, glutamic acid and P-alanine are applied with their amino group protected by an amino protecting and glutamic acid with its y-carboxylic acid protected by an ester protecting group.

12. The process of claim 11, wherein the amino protecting group is ((9H-fluoren-9- yl)methoxy)-carbonylamino (Fmoc).

13. The process of any one of claims 7 to 12, wherein the peptide synthesis is performed in the presence of a coupling agent, selected from benzotriazol- 1- yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol- 1- yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP), (7-azabenzotriazol-l- yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), N-[(lH-benzotriazol-l- yl)(dimethylamino)methylene]-N-methylmethanaminium tetrafluorob orate N-oxide (TBTU), 2-(lH-benzotriazole-l-yl)-l,l,3,3-tetramethylaminium hexafluorophosphate (HBTU), 1- [bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), carbonyldiimidazole (CDI), dicyclohexylcarbodiimide (DCC), diisoopropylcarbodiimide (DIC), or N-(3-dimethylaminopropyl)-N’ -ethylcarbodiimide (EDC).

14. The process of any one of claims 7 to 13, wherein the peptide synthesis is performed in a polar aprotic solvent.

15. The process of any one of claims 7 to 14, wherein the deprotection of the amino protecting group is performed with a solution of piperidine in a polar aprotic solvent.

16. The process of any one of claims 7 to 15, wherein the acidic conditions for cleaving the solid phase bound tetramer from the solid support comprises a solution of trifluoroacetic acid (TFA), trifluoroethanol or hexafluoroisopropanol (HFIP), preferably with trifluoroacetic acid (TFA) solutions in an organic solvent.

17. Use of the tetramer of formula I of claim 1, wherein R1is hydrogen, or of a salt thereof, for the preparation of the peptide of formula III, or of a pharmaceutically acceptable salt or ester thereofX-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-He12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-Gln19-Lys20(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2(III) wherein X isAEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid.

18. Use of the tetramer of formula I of claim 1, wherein R1is hydrogen, or of a salt thereof, for the preparation of the peptide of formula Va, or of a functionalized derivative thereof,X1-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15- Lys16-Ile17-Ala18-Gln19-Lys20-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30- Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH-linker-solid support(Va) wherein X is19. The use of claim 18, wherein the functionalized derivative has the formula Vb,X1-P-Ala2-Glu3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10- Ser(tBu)11-Ile12-Aib13-Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys20(Mtt / ivDde)- Ala21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-He27-Ala28-Gly29-Gly30-Pro31-Ser(tBu)32- Ser(tBu)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(tBu)39-NH-linker-solid support(Vb), wherein X is as above.

20. The use of claim 18, wherein Lys20in the derivative of formula Vb is functionalized with Mtt.

21. Process for the preparation of the peptide of formula III, or of a pharmaceutically acceptable salt or ester thereofX-P-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Sern-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(III) wherein X isAEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid. comprising the stepsa) solid phase synthesis of the peptide of formula III on a resin, comprising coupling the tetramer of formula I of claim 1 to a functionalized peptide fragment of formula IVaH-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18- Gln19-Lys20-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33- Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH-linker-solid support(IVa) and forming the functionalized peptide fragment of formula VaX1-p-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-He12-Aib13-Leu14-Asp15- Lys16-Ile17-Ala18-Gln19-Lys20-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30- Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH-linker-solid support(Va) b) deprotection of the Lys20s-amino protecting group, c) acylation of the s-amino group of Lys20with the moietyPROT-O-20-oxoicosanoyl-L-Glu(AEEAc-AEEAc-OH)-O-PROT(Via), wherein AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid and PROT is an ester protecting group, and forming the peptide of formula III, bound to the resin; d) cleavage of the peptide of formula III from the resin and global deprotection with an acid and e) precipitation of the peptide formula III and optionally f) purification / isolation.

22. The process of claim 21, comprising the stepsa) solid phase synthesis of the peptide of formula III on a resin, comprising coupling the tetramer of formula I to a functionalized peptide fragment of formula IVbH-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10-Ser(tBu)n-Ile12-Aib13- Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys20(Mtt / ivDde)-Ala21-Phe22-Val23- Gln(Trt)24-Trp(Boc)25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser(tBu)32-Ser(tBu)33-Gly34-Ala35- Pro36-Pro37-Pro38-Ser(tBu)39-NH-linker-solid support(IVb) and forming the functionalized peptide fragment of formula VbX1-P-Ala2-Glu3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10- Ser(tBu)11-Ile12-Aib13-Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys20(Mtt / ivDde)- Ala21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-He27-Ala28-Gly29-Gly30-Pro31-Ser(tBu)32- Ser(tBu)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(tBu)39-NH-linker-solid support(Vb) b) deprotection of the Lys20s-amino protecting group Mtt or ivDde, c) acylation of the s-amino group of Lys20with the moiety tBu-O-20-oxoicosanoyl-L-Glu(AEEAc-AEEAc-OH)-OtBu (VIb), wherein AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid, and forming the peptide of formula III, bound to the resin; d) cleavage of the peptide of formula III from the resin and global deprotection with an acid and e) precipitation of the peptide formula III and optionally f) purification / isolation.

23. The process of claim 22, wherein Lys20in the peptide fragments of formula IVb and Vb is functionalized with Mtt.***

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