SPPS synthesis of a peptide comprising a fatty acid side chain

Abstract

The invention relates to the SPPS synthesis of a peptide of formula (I) comprising a fatty acid side chain, wherein in the final synthesis step the fatty acid side chain is coupled to the peptide.

Description

[0001] P39804

[0002] SPPS Synthesis of a Peptide comprising a Fatty Acid Side chain

[0003] The invention relates to a novel process for the preparation of a peptide of formula I, or of a pharmaceutically acceptable salt or ester thereof

[0004] 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

[0005] (I)

[0006] wherein X is

[0007]

[0008] CN and

[0009] AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid.

[0010] The peptide of formula I has the potential to act as GLP-1R / GIPR agonist.

[0011] 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

[0012] 24.11.2025 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.

[0013] 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 III does not cause internalization and consequently, desensitization of either GLP-1R or GIPR, and thus has enhanced signaling efficacy.

[0014] Object of the invention was to provide improved processes for producing the peptide of formula I, which are applicable on a technical scale. The processes are characterized by including the fatty acid side chain in the final steps of the synthesis and by using N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N6-[(4-methylphenyl)diphenylmethyl]-L-lysine (Fmoc-L-Lys(Mtt)-OH) as a building block.

[0015] It was found that the object could be reached with the new process manufacturing of the peptide of formula I, or of a pharmaceutically acceptable salt or ester thereof

[0016] 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

[0017] (I)

[0018] wherein X is

[0019]

[0020] and AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid.

[0021] The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein.

[0022] 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.

[0023] 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".

[0024] 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.

[0025] 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.

[0026] Alternatively, 3 -methyl-pent-3 -yl (Mpe) can be used.

[0027] 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.

[0028] The SPPS is characterized by the assembly of the desired peptide chain on a solid support. 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.

[0029] 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, the Rink amide resin comprises benzhydrylamine and the Sieber amide resin comprises xanthenylamine. Alternatively, the Ramage amide resin (2-(5-(9-fluorenylmethyloxy carbonyl)amino-10, 11 -dihydro-5 / / -dibenzo[a,d]cycloheptenyl-2-oxy)actamido polystyrene) or the tricyclic amide linker resin (5-Fmoc-amino-10,l l-dihydro-5J7-dibenzo[a,d]-cycloheptenyl-2-oxyacetyl- / J / .-Nle-4-methyl-benzhydrylamide resin) can be used. 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.

[0030] Each amino acid to be coupled to the peptide chain A-terminus must bear an appropriate protective group on its a-amino group and potentially on its side chain. The base-labile fluorenylmethyloxy 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.

[0031] Deprotection of Fmoc, and exposing the amino group for the next coupling step, can be accomplished by a reaction with the basic piperidine in a polar, aprotic solvent. As a rule solutions of 10 to 30% (v / v) piperidine in DMF are used.

[0032] The coupling reactions as a rule require activation of the carboxylic acid moiety with coupling agents or activators such as AA-diisopropylcarbodiimide (DIC) to support an effective amide bond formation. Conditions of a coupling reaction determine the acylation rate, as well as the extent of side reactions, such as racemization. Racemization is suppressed with 'racemization suppressing' additives such as 1-hydroxy -benzotriazole (HOBt), l-hydroxy-7-aza-benzotriazole (HO At) and ethyl cyano(hydroxyimino)acetate (OxymaPure). OxymaPure has been developed as an efficient additive for carbodiimide-based coupling.

[0033] The combination A, A-diisopropylcarbodiimide (DIC) and ethyl cyano(hydroxyimino)acetate (OxymaPure) has been proved to be the coupling agent / additive combination of choice. Typically the activation process takes place in the presence of a polar aprotic solvent, such as in DMF.

[0034] The use of a -amino group and side chain protecting groups is essential during peptide synthesis to avoid undesirable side reactions, such as self-coupling of the activated amino acid. Two principle protecting group schemes are typically used in solid phase peptide synthesis. The t-butyloxycarbonyl (Boc) / benzyl (Bzl) strategy utilizes TFA-labile TV-terminal Boc protection alongside side chain protection that is removed simultaneously with cleavage of the peptide from the solid support using anhydrous hydrogen fluoride (HF) during the final cleavage step. The fluorenylmethyloxycarbonyl (Fmoc) / / -butyl (tBu) strategy uses base-labile Fmoc TV-terminal protection whereas the side chain protection and the resin linkage are acid-labile and the final acidic cleavage is carried out with an acid such as TFA.

[0035] The term “functionalized derivative” or “functionalized peptide” in this and the following context means that reactive sites of respective amino acids are protected with suitable protecting groups, e.g. Thr, Ser, Glu, or Tyr with OtBu, Asp with tBu or OMpe, Trp or Lys16with Boc, Gin with Trt and Lys20in particular with Mtt (4-methyltrityl). In one aspect, functionalized peptide means a peptide, wherein Thr, Ser, Glu or Tyr are protected with OtBu, Trp or Lys16are protected with Boc, Gin is protected with Trt, Asp is protected with OtBu or OMpe, and Lys20is protected with Mtt.

[0036] At the end of the synthesis, the crude peptide can be cleaved from the solid support while simultaneously removing all protecting groups, typically using an acidic reagent such as trifluoroacetic acid, and optionally purified.

[0037] While the 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.

[0038] Herein provided is a process for the preparation of the peptide of formula I, or of a pharmaceutically acceptable salt or ester thereof 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

[0039] (I)

[0040] wherein X is

[0041]

[0042] CN and

[0043] AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid,

[0044] which is comprising the steps

[0045] a) solid phase synthesis of a functionalized peptide fragment of formula Ila on a resin

[0046] 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

[0047] (Ila),

[0048] wherein X is

[0049]

[0050] CN

[0051] Lys20is functionalized with Mtt

[0052] and the solid support is an amide resin,

[0053] b) deprotection of the Lys20(Mtt),

[0054] c) adding the fatty acid side chain to the deprotected Lys20either by sequential coupling of the side chain fragments in the order c1) R1-AEEA-OH c2) R1-AEEA-OH c3) R1-Glu-O-PROT

[0055] c4) (19-carboxynonadecanoyl-O-PROT)-OH,

[0056] wherein R1is an amino protection group and PROT is an ester protecting group,

[0057] or, alternatively, by coupling the whole side chain of formula IV

[0058] PROT-O- 19-carboxy-nonadecanoyl-L-Glu (AEEAc-AEEAc-OH)-O-PROT

[0059] (IV),

[0060] wherein AEEAc and PROT are as above, to the deprotected Lys20,

[0061] and forming the functionalized peptide of formula III, bound to the resin;

[0062] 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-NH-solid support

[0063] (III),

[0064] wherein X and the solid support are as above,

[0065] d) cleavage of the functionalized peptide of formula III from the resin and global deprotection with an acid and

[0066] e) precipitation of the peptide of formula I and optionally

[0067] f) purification / isolation.

[0068] In one aspect, provided is a process for the preparation of the peptide of formula I, or of a pharmaceutically acceptable salt or ester thereof 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

[0069] (I)

[0070] wherein X is

[0071]

[0072] CN and

[0073] AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid.

[0074] which is comprising the steps

[0075] a) solid phase synthesis of a functionalized peptide fragment of formula Ila on a resin

[0076] 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

[0077] (Ila),

[0078] wherein X is

[0079]

[0080] CN

[0081] Lys20is functionalized with Mtt

[0082] and the solid support is an amide resin,

[0083] b) deprotection of the Lys20(Mtt),

[0084] c) sequential coupling to the deprotected Lys20the side chain fragments in the order c1) R1-AEEA-OH c2) R1-AEEA-OH c3) R1-Glu-O-PROT

[0085] c4) (19-carboxynonadecanoyl-O-PROT)-OH,

[0086] wherein R1is an amino protection group and PROT is an ester protecting group,

[0087] and forming the functionalized peptide of formula III, bound to the resin;

[0088] 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-NH-solid support

[0089] (III),

[0090] wherein X and the solid support are as above,

[0091] d) cleavage of the functionalized peptide of formula III from the resin and global deprotection with an acid and

[0092] e) precipitation of the peptide of formula I and optionally

[0093] f) purification / isolation.

[0094] In a particular aspect of the process of the present invention, step a) provides the functionalized peptide fragment of formula lib

[0095] X1-P-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)-Tyr10(OtBu)-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-Ala18-Gln19(Trt)-Lys20(Mtt)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH linker-solid support

[0096] (IIb),

[0097] wherein X is

[0098]

[0099] CN

[0100] and the solid support is an amide resin.

[0101] The amide resin is typically selected from the group consisting of Rink amide resin, Sieber amide resin, and Ramage amide resin, preferably the Sieber resin is used.

[0102] The process in step a) is particularly characterized in the sequential coupling of the following

[0103] 1 Fmoc-L-Ser(OtBu)-OH

[0104] 2 Fmoc-L-Pro-OH·H2O

[0105] 3 Fmoc-L-Pro-L-Pro-OH

[0106] 4 Fmoc-L-Ala-OH

[0107] 5 Fmoc-Gly-OH

[0108] 6 Fmoc-L-Ser(OtBu)-OH

[0109] 7 Fmoc-L-Ser(OtBu)-OH

[0110] 8 Fmoc-L-Pro-OH·H2O

[0111] 9 Fmoc-Gly-Gly-OH

[0112] 10 Fmoc-L-Ala-OH·H2O

[0113] 11 Fmoc-L-Ile-OH

[0114] 12 Fmoc-L-Leu-OH

[0115] 13 Fmoc-L-Trp(Boc)-OH

[0116] 14 Fmoc-L-Gln(Trt)-OH

[0117] 15 Fmoc-L-Val-OH

[0118] 16 Fmoc-L-Phe-OH

[0119]

[0120] 17 Fmoc-L-Ala-OH·H2O 18 Fmoc-L-Lys(Mtt)-OH

[0121] 19 Fmoc-L-Gln(Trt)-OH

[0122] 20 Fmoc-L-Ala-OH-H2O

[0123] 21 Fmoc-L-Ile-OH

[0124] 22 Fmoc-L-Lys(Boc)-OH

[0125] 23 Fmoc-L-Asp(OtBu)-OH

[0126] 24 Fmoc-L-Leu-OH

[0127] 25 Fmoc-Aib-OH

[0128] 26 Fmoc-L-Ile-OH

[0129] 27 Fmoc-L-Ser(OtBu)-OH

[0130] 28 Fmoc-L-Tyr(tBu)-OH

[0131] 29 Fmoc-L-Asp(OtBu)-OH

[0132] 30 Fmoc-L-Ser(OtBu)-OH

[0133] 31 Fmoc-L-Thr(OtBu)-OH

[0134] 32 Fmoc-L-Phe-OH

[0135] 33 Fmoc-L-Thr(OtBu)-OH

[0136] 34 Fmoc-Gly-OH

[0137] 35 Fmoc-L-Glu(OtBu)- OH H2O

[0138] 36 Fmoc-P-Ala-OH

[0139] 37 X = 2-(3-cyano-5- fluorophenyl)-2-

[0140]

[0141] methylpropanoic acid

[0142] Fmoc-L-Lys(Mtt)-OH stands for N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N6-[(4-methylphenyl)diphenylmethyl]-L-lysine, wherein the amino group in the amino acid side chain of lysine is protected with Mtt (4-m ethyltrityl). Step b) requires deprotection of Lys20(Mtt), which can be accomplished with a mixture of hexafluoroisopropanol (HFIP) and triisopropylsilane (TIPS) in an organic solvent. In a preferred aspect, the deprotection of Mtt is performed with a mixture of hexafluoroisopropanol (HFIP) and trisopropylsilane (TIPS) in dichloromethane (DCM), suitably in a v / v / v ratio of HFIP / DCM / TIPS of 20 to 40 / 80 to 60 / 1, preferably 29.5 / 69.5 / 1.

[0143] Complete deprotection of Lys20(Mtt) can conveniently be achieved with more than one treatment with the mixture of hexafluoroisopropanol (HFIP) and triisopropylsilane (TIPS) in dichloromethane, usually the functionalized peptide is treated 2 to 5 times with the mixture of hexafluoroisopropanol (HFIP) and triisopropylsilane (TIPS) in di chloromethane and afterwards washed 6 to 8 times with dichloromethane (DCM) and 6 to 8 times with dimethyl formamide (DMF).

[0144] Step c) is particularly characterized in the sequential coupling of the side chain fragments in the order

[0145] cl) Fmoc-AEEA-OH

[0146] c2) Fmoc-AEEA-OH

[0147] c3) Fmoc-Glu-O-tBu

[0148] c4) (19-carboxynonadecanoyl-O-tBu)-OH.

[0149] The functionalized peptide of formula III is in step d) subjected to cleavage from the resin and to global deprotection with an acid. In a preferred embodiment the cleavage and global deprotection is accomplished with TFA in combination with at least one scavenger. Particularly preferred is a cleavage cocktail containing TFA, water, TIS (triisopropylsilane), and optionally a thiol scavenger, e.g. EDT (ethane- 1,2-dithiol) DTT (dithiothreitol), DTE (dithioerythritol), DODT (3,6-dioxa-1,8-octanedithiol), or 1,4-BDMT (1,4-benzenedimethanethiol). Preferred thiol scavengers are EDT and DTT.

[0150] The precipitation of the functionalized peptide of formula I in step e) is typically performed in a suitable organic solvent such as in tert-butyl methyl ether (TBME) and dried.

[0151] Optionally a further purification and isolation step f) may be added.

[0152] The functionalized peptide fragment of formula Ila 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,

[0153] (Ila),

[0154] wherein X is

[0155]

[0156] CN

[0157] Lys20is functionalized with Mtt

[0158] and the solid support is an amide resin;

[0159] and the functionalized peptide fragment of formula lib

[0160] X1-P-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)-Tyr10(OtBu)-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-Ala18-Gln19(Trt)-Lys20(Mtt)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH linker-solid support

[0161] (IIb)

[0162] wherein X is

[0163]

[0164] CN

[0165] and the solid support is an amide resin,

[0166] are novel compounds, so far not disclosed in the state of the art and therefore constitute a further aspect of the present invention.

[0167] The functionalized peptide fragment of formula lie X1-P-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)-Tyr10(OtBu)-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-Ala18-Gln19(Trt)-Lys20(Mtt)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH linker-Sieber amide resin

[0168] (IIc)

[0169] wherein X is as defined above,

[0170] is particularly preferred.

[0171] In another aspect, provided is a process for the preparation of the peptide of formula I, or of a pharmaceutically acceptable salt or ester thereof

[0172] 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

[0173] (I)

[0174] wherein X is

[0175]

[0176] CN and

[0177] AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid,

[0178] which is comprising the steps

[0179] a) solid phase synthesis of a functionalized peptide fragment of formula Ila on a resin

[0180] 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 wherein X is

[0181]

[0182] Lys20is functionalized with Mtt

[0183] and the solid support is an amide resin,

[0184] b) deprotection of the Lys20(Mtt),

[0185] c) coupling the whole fatty acid side chain of formula IV

[0186] PROT-O- 19-carboxy-nonadecanoyl-L-Glu (AEEAc-AEEAc-OH)-O-PROT

[0187] (IV),

[0188] wherein AEEAc and PROT are as above, to the deprotected Lys20

[0189] and forming the functionalized peptide of formula III, bound to the resin;

[0190] 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-NH-solid support

[0191] (III),

[0192] wherein X and the solid support are as above,

[0193] d) cleavage of the functionalized peptide of formula III from the resin and global deprotection with an acid and

[0194] e) precipitation of the peptide of formula I and optionally

[0195] f) purification / isolation.

[0196] For PROT the protecting group tert-butyl (tBu) is preferred. In one particular aspect, the compound of formula IV corresponds to the compound of formula IVa tBu-O- 19-carboxy-nonadecanoyl-L-Glu(AEEAc-AEEAc-OH)-O-tBu

[0197] (IVa),

[0198] wherein AEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid, and is described in WO 2009 / 115469 A1

[0199] The term 19-carboxy-nonadecanoyl refers to the moiety

[0200]

[0201] An alternative term for this moiety is O-20-oxoicosanoyl. Examples

[0202] Abbreviations:

[0203] BDMT benzenedimethanethiol

[0204] DCM di chloromethane

[0205] DIC N,N'-diisopropylcarbodiimide DIPEA diisopropylethylamine

[0206] DMF N,N-dimethylformamide

[0207] DODT 3,6-dioxa-1,8-octanedithiol

[0208] DTE dithioerythritol

[0209] DTT dithiothreitol

[0210] EtOAc ethyl acetate

[0211] Fmoc 9-fluorenylmethoxycarbonyl

[0212] HFIP hexafluoroisopropanol

[0213] HPLC high pressure liquid chromatography TBME tert.-butyl methyl ether

[0214] Mpe 3-methyl-pentyl

[0215] Mtt 4-methyltrityl

[0216] OxymaPure ethyl 2-cyano-2-(hydroxyimino)acetate 2-PrOH 2-propanol

[0217] tBu tert.-butyl

[0218] TFA trifluoroacetic acid

[0219] TIPS triisopropylsilane Example 1: Linear SPPS using Fmoc-Lys(Mtt)-OH as building block and sequential coupling of the fatty acid side chain

[0220] X-β-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys(AEEAc-AEEAc-γ-Glu-19-carboxynonadecanoyl)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2·TFA

[0221] Resin swelling DMF, 2 h Cycles 1-37 and 39-41 1. piperidine (20% v / v in DMF) 2. post-deprotection washes (DMF) 3. Fmoc-AA-OH or building block, Oxyma Pure, DIC, DMF, RT 4. coupling wash Cycle 38 1. HFIP / DCM / TIPS (29.5 / 69.5 / 1 v / v / v) 2. post-deprotection washes (DCM and DMF) 3. building block, Oxyma Pure, DIC, DMF, RT 4 post-coupling washes (DMF) Cycle 41 1 deswelling washes 2-PrOH and TBME Global deprotection and resin cleavage 1. TFA / TIS / H2O 90:5:5, 0°C to 20°C 2 Filtration 3 TBME, 2 - 8°C

[0222]

[0223] 4. Filtration, wash TBME 5. dry at 24°C at 7 mbar

[0224] X-β-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys(AEEAc-AEEAc-γ-Glu-19-carboxynonadecanoyl)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2·TFA

[0225] \nAmino acid / building block coupled

[0226] 1 2 3 4 5 6 Ser39Pro38Pro37-Pro36Ala35Gly34Ser337 8 9 10 11 12 Ser32Pro31Gly30-Gly29Ala28Ile27Leu2613 14 15 16 17 18 Trp25Gln24Val23Phe22Ala21Lys20(Mtt) 19 20 21 22 23 24 Gln19Ala18Ile17Lys16Asp15Leu1426 27 28 29 30 Aib13Ile12Ser11Tyr10Asp9Ser8

[0227]

[0228] 31 32 33 34 35 36 Thr7Phe6Thr5Gly4Glu3β-Ala237 38 39 40 41 X1= 2-(3-cyano-5-fluorophenyl)-2- AEEAc20.1AEEAc20.2γ-Glu20.3Eicosanedioic

[0229]

[0230] methylpropanoic acid acid204

[0231] Synthetic Procedure: SPPS

[0232] Preparation of the Resin

[0233] Sieber resin (6.25 g, loading 0.77 mmol / g, 4.81 mmol) was charged into a 0.5-L SPPS reactor (fully automated). The resin was swelled with DMF (53.3 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 53.3 mL, 8.52 g / g resin).

[0234] General Synthetic Procedure

[0235] Washing steps were designed to remove 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.

[0236] All Fmoc deprotections were performed by 3 treatments with piperidine / DMF (20% v / v) with a stirring time of 10 min per cycle. After Fmoc removal, the resin was washed with DMF.

[0237] In cycle 38, Mtt deprotection was performed by a 5 treatments with a HFIP / DCM / TIPS (29.5 / 69.5 / 1 v / v / v) for 30 min each. Subsequently, the resin was washed with DCM followed by DMF.

[0238] For all couplings, 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 ambient temperature for 5 min to pre-activate the amino acid. Once the pre-activation was complete, the amino acid solution was added to the resin. The reaction mixture was then stirred. The reaction time for the coupling was 1.5-24 h. A recoupling was performed for cycle 26.

[0239] 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.

[0240] After completion of the solid phase synthesis, the resin was washed several times with DMF and 2-PrOH before discharging. The resin was dried under reduced pressure for 48 h.

[0241] Table 1: Conditions used for the linear SPPS using Fmoc-Lys(Mtt)-OH.

[0242] Entry Condition / Parameter 19.25 mmol Build

[0243] 1 Initial Sieber resin loading 0.77 mmol / g

[0244] 2 Amount DMF for each post-coupling wash 8.52 g / g resin

[0245] 3 Amount piperidine in DMF (20% v / v) for each 8.38 g / g resin

[0246] Fmoc deprotection treatment

[0247] 4 Amount HFIP / DCM / TIPS (29.5 / 69.5 / 1 v / v / v) 11 mL / g resin

[0248] for each Mtt deprotection treatment

[0249] 5 Amount DMF for each post-deprotection wash 8.52 g / g resin

[0250] 6 Amount DCM for each post Mtt-deprotection 9.0 mL g / g resin

[0251] wash

[0252] 7 Amount 2-PrOH for each de-swelling wash 7.9 g / g resin

[0253] 8 Amount TBME for each de-swelling wash 7.4 g / g resin

[0254] 9 Pre-activation time for all couplings 5 min

[0255]

[0256] Entry Condition / Parameter 19.25 mmol Build

[0257] 10 Reagents used for coupling cycles 2 equiv Fmoc-AA-OH or building block

[0258] 2 equiv Oxyma Pure

[0259] 3 equiv DIC

[0260] DMF solvent

[0261] 10 Volume of coupling solution 1-32 5.69 mL / g of initial resin charge excluding Oxyma Pure and

[0262] building blocks used for (103 mb DMF used for dissolving coupling in cycles the protected amino acid or building block and Oxyma Pure, 15.4 mb DMF and 8.25 mb DIC used to make the DIC / DMF solution, 15.4 mb DMF directly charged into the reactor)

[0263] 33-37 6.06 mL / g of initial resin charge (112 mb DMF used for dissolving the protected amino acid or building block and Oxyma Pure, 15.4 mb DMF and 8.25 mb DIC used to make the DIC / DMF solution, 15.4 mb DMF directly charged into the reactor)

[0264] 38-40 6.58 mL / g of initial resin charge (125 mb DMF used for dissolving the protected amino acid or building block and Oxyma Pure, 15.4 mb DMF and 8.25 mb DIC used to make the DIC / DMF solution, 15.4 mb DMF directly charged into the reactor)

[0265] 41 8.00 mL / g of initial resin charge (161 mb DMF used for dissolving the building block and Oxyma Pure, 15.4 mb DMF and 8.25 mb DIC used to make the DIC / DMF solution, 15.4 mb DMF directly

[0266]

[0267] charged into the reactor) Entry Condition / Parameter 19.25 mmol Build

[0268] 11 Resin bound drying temperature 23 °C

[0269] 12 Resin bound drying vacuum conditions 1-3 mbar

[0270] 13 Resin bound drying duration 15 h

[0271] 14 Mass of the dried and discharged resin bound 26.0 g (5.3 g lost due to a broken intermediate frit at the bottom of the reactor)

[0272] Molecular weight increase from the initial Sieber resin to the protected peptide on resin = 6000.52 g / mol

[0273] 15 Theoretical weight gain of the protected 27.8 g

[0274] peptide on resin

[0275] 16 Actual weight gain 21.2 g

[0276] 17 Yield of SPPS step 76.3%

[0277]

[0278] The detailed coupling sequence and step conditions in the various cycles are listed in Table 2. Table 2: Detailed conditions for the linear SPPS using Fmoc-Lys(Mtt)-OH.

[0279] AA # Cycle Amino Acid / Number and PostCoupling Number # Building Block Duration of deprotection / Stir and Deprotection washes Time [h] Duration Treatments of PostCoupling DMF

[0280] Washes - - Sieber amide - 2.0

[0281] resin (swell) 39 1 Fmoc-L- 3 x 10 min 8 x DMF 2:59 4 x 5 min

[0282]

[0283] Ser(OtBu)-OH AA # Cycle Amino Acid / Number and PostCoupling Number # Building Block Duration of deprotection / Stir and Deprotection washes Time [h] Duration Treatments of PostCoupling DMF

[0284] Washes 38 2 Fmoc-L-Pro- 3 x 10 min 8 x DMF 2:14 4 x 5 min OH H2O

[0285] 37-36 3 Fmoc-L-Pro-L- 3 x 10 min 8 x DMF 3:29 4 x 5 min Pro-OH

[0286] 35 4 Fmoc-L-Ala-OH 3 x 10 min 8 x DMF 3:25 4 x 5 min 34 5 Fmoc-Gly-OH 3 x 10 min 8 x DMF 2:59 4 x 5 min 33 6 Fmoc-L- 3 x 10 min 8 x DMF 3 4 x 5 min Ser(OtBu)-OH

[0287] 32 7 Fmoc-L- 3 x 10 min 8 x DMF 2:17 4 x 5 min Ser(OtBu)-OH

[0288] 31 8 Fmoc-L-Pro- 3 x 10 min 8 x DMF 2:42 4 x 5 min OH·H2O

[0289] 30-29 9 Fmoc-Gly-Gly- 3 x 10 min 8 x DMF 3:37 4 x 5 min OH

[0290] 28 10 Fmoc-L-Ala- 3 x 10 min 8 x DMF 2:46 4 x 5 min OH·H2O

[0291] 27 11 Fmoc-L-Ile-OH 3 x 10 min 8 x DMF 2:21 4 x 5 min 26 12 Fmoc-L-Leu-OH 3 x 10 min 8 x DMF 3:25 4 x 5 min 25 13 Fmoc-L- 3 x 10 min 8 x DMF 2:30 4 x 5 min Trp(Boc)-OH

[0292] 24 14 Fmoc-L-Gln(Trt)- 3 x 10 min 8 x DMF 2:22 4 x 5 min OH

[0293] 23 15 Fmoc-L-Val-OH 3 x 10 min 8 x DMF 5:20 4 x 5 min 22 16 Fmoc-L-Phe-OH 3 x 10 min 8 x DMF 2:41 4 x 5 min 21 17 Fmoc-L-Ala- 3 x 10 min 8 x DMF 2:39 4 x 5 min OH·H2O

[0294] 20 18 Fmoc-L- 3 x 10 min 8 x DMF 24 4 x 5 min

[0295]

[0296] Lys(Mtt)-OH AA # Cycle Amino Acid / Number and PostCoupling Number # Building Block Duration of deprotection / Stir and Deprotection washes Time [h] Duration Treatments of PostCoupling DMF

[0297] Washes 19 19 Fmoc-L-Gln(Trt)- 3 x 10 min 8 x DMF 20 4 x 5 min OH

[0298] 18 20 Fmoc-L-Ala-OH- 3 x 10 min 8 x DMF 2:36 4 x 5 min H2O

[0299] 17 21 Fmoc-L-Ile-OH 3 x 10 min 8 x DMF 3:58 4 x 5 min 16 22 Fmoc-L- 3 x 10 min 8 x DMF 3:37 4 x 5 min Lys(Boc)-OH

[0300] 15 23 Fmoc-L- 3 x 10 min 8 x DMF 2:34 4 x 5 min Asp(OtBu)-OH

[0301] 14 24 Fmoc-L-Leu-OH 3 x 10 min 8 x DMF 3:04 4 x 5 min 13 25 Fmoc-Aib-OH 3 x 10 min 8 x DMF 5:37 4 x 5 min 12 26 Fmoc-L-Ile-OH 3 x 10 min 8 x DMF 7:19 4 x 5 min - - Recoupling of - 8 x DMF 11:17 4 x 5 min Fmoc-L-Ile-OH

[0302] 11 27 Fmoc-L- 3 x 10 min 9 x DMF 3 4 x 5 min Ser(OtBu)-OH

[0303] 10 28 Fmoc-L- 3 x 10 min 9 x DMF 3 4 x 5 min Tyr(tBu)-OH

[0304] 9 29 Fmoc-L- 3 x 10 min 9 x DMF 2:33 4 x 5 min Asp(OtBu)-OH

[0305] 8 30 Fmoc-L- 3 x 10 min 9 x DMF 2:37 4 x 5 min Ser(OtBu)-OH

[0306] 7 31 Fmoc-L- 3 x 10 min 10 x DMF 2:29 4 x 5 min Thr(OtBu)-OH

[0307] 6 32 Fmoc-L-Phe-OH 3 x 10 min 10 x DMF 10:55 4 x 5 min 5 33 Fmoc-L- 3 x 10 min 10 x DMF 3 4 x 5 min Thr(OtBu)-OH

[0308]

[0309] 4 34 Fmoc-Gly-OH 3 x 10 min 10 x DMF 3 4 x 5 min AA# Cycle Amino Acid / Number and PostCoupling Number # Building Block Duration of deprotection / Stir and Deprotection washes Time [h] Duration Treatments of PostCoupling DMF

[0310] Washes 3 35 Fmoc-L- 3 x 10 min 10 x DMF 10:51 4 x 5 min Glu(OtBu)- OH·H2O

[0311] 2 36 Fmoc-β-Ala-OH 3 x 10 min 10 x DMF 10:51 4 x 5 min 1 37 2-(3-cyano-5- 3 x 10 min 10 x DMF 10:51 8 x 5 min fluorophenyl)-2- methylpropanoic 8x DCM

[0312] acid

[0313] - - Deprotection of 5x 30 min 8x DCM - Mtt with

[0314] HFIP / DCM / TIPS 5x DMF

[0315] (29.5 / 69.5 / 1

[0316] v / v / v)

[0317] 20.1 38 Fmoc-AEEA-OH 3 x 10 min 10 x DMF 5 4 x 5 min 20.2 39 Fmoc-AEEA-OH 3 x 10 min 10 x DMF 5 4 x 5 min 20.3 40 Fmoc-Glu-OtBu 3 x 10 min 10 x DMF 14 4 x 5 min 20.4 41 Eicosanedioic - 10 x DMF 24 4 x 5 min acid mono-tert- butyl ester

[0318] - - Final washes 4x 2-PrOH

[0319] 4x MTBE

[0320]

[0321] TFA-mediated cleavage from solid support with concomitant global deprotection The resin bound material, obtained after the SPPS step, was subjected to cleavage / global deprotection to afford the crude peptide.

[0322] The cleavage cocktail was prepared as follows: TFA (86 mL, 8.6 mL / g of resin bound material) was cooled to 0 °C. DTT (4.8 g, 0.48 g / g of resin bound material was added, followed by Water (2.4 mL, 0.24 mL / g of resin bound material) and TIS (2.4 mL, 0.24 mL / g of resin bound material).

[0323] Resin bound material (10 g) was charged into a 250 mL 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 18 °C over 160 min and subsequently stirred for an additional 40 min. The mixture was then filtered and the filtrate was collected in a 2 L jacketed reactor. The spent resin was washed with TFA (1 x 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 (530 mL, 53 mL / g of resin bound material) was added slowly to the filtrate over 70 min while keeping IT < -5 °C. After the addition, the suspension was warmed to 22 °C over 100 min and stirred for an additional 30 min. The suspension was filtered and the filter cake was washed with TBME (2 x 88 mL). The isolated solid was dried under vacuum and ambient temperature for 46 h.

[0324] The crude peptide was isolated as an off-white powder (5.70 g, purity 77.4 area%).

[0325] Example 2: SPPS using Fmoc-Lys(Mtf)-OH as building block and coupling of the whole fatty acid side chain

[0326] X-P-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln- Ly s(AEEAc-AEEAc-y-Glu- 19-carboxynonadecanoyl)-Ala-Phe- Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NFE TFA

[0327] Resin swelling 1. DMF, 2 h 2 post-swelling washes (DMF) Cycles 1-37 1 piperidine (20% v / v in DMF) 2 post-deprotection washes (DMF) 3 Fmoc-AA-OH or building block, Oxyma Pure, DIC, DMF, 23°C 4 coupling wash Cycle 38 1 solvent swap to toluene 2 HFIP / toluene / TIS (29.5 / 69.5 / 1 v / v / v) 3 post-deprotection washes (toluene and DMF) 4 building block, Oxyma Pure, DIC, DMF, 23°C 5 post-coupling washes (DMF), deswelling washes 2-PrOH and TBME Global deprotection and resin cleavage 1 TFA / TIS / H2O 90:25:2.5 v / v / v, DTT (0.5 g / g peptide resin) 0" C to 20”C 2 filtration, wash TFA, 20“C 3 TBME, 0" C 4 filtration, wash TBME 5. dry at 22“C at 1-3 mbar

[0328]

[0329] X-p-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-lle-Aib-Leu-Asp-Lys-lle-Ala-Gln-Lys(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-Ala-Phe- Val-Gln-Trp-Leu-lle-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2TFA Amino acid building block coupled

[0330] 1 2 3 4 5 6 Ser30Pro38Pro37-Pro36Ala35Glv34Scr33

[0331] 7 8 9 10 11 12 Ser32Pro31Gly30-Gly29Ala28He2-T.eu26

[0332] 13 14 15 16 17 18 Trp25(illl21Vai23Phe22Ala21I.\ s2"(Mll) 19 20 21 22 23

[0333] Cdn1'' Ala18lie1' I. vs1'' Asp15I. Cll1 1

[0334] 26 27 28 29 30 Aib13lie12Ser11Tvr10Asp” Scr831 32 33 34 35 36 Thr7Phe6Thr5Gly4Glu3P-Ala2

[0335] 37 38

[0336] X1= 2-(3-cyano-5-fluorophenyl)-2-methylpropanoic C20acid20.4-γ-Glu20.3-AEEAc20.2-AEEAc20.1

[0337]

[0338] acid

[0339] Synthetic Procedure: SPPS

[0340] Preparation of the Resin

[0341] 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, 9mL / 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, 9 mL / g resin).

[0342] General Synthetic Procedure

[0343] Washing steps were designed to remove 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.

[0344] All Fmoc deprotections were performed by 3 treatments with piperidine / DMF (20% v / v) with a stirring time of 10 min per cycle. After Fmoc removal, the resin was washed with DMF.

[0345] In cycle 38, Mtt deprotection was performed by 5 treatments with a HFIP / toluene / TIPS (29.5 / 69.5 / 1 v / v / v) for 60 min each. Subsequently, the resin was washed with toluene followed by DMF.

[0346] For all couplings, 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 ambient temperature for 5 min to pre-activate the amino acid.

[0347] Once the pre-activation was complete, the amino acid solution was added to the resin. The reaction mixture was then stirred. The reaction time for the coupling was 1.5-24 h. A recoupling was performed for cycle 26.

[0348] 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.

[0349] After completion of the solid phase synthesis, the resin was washed several times with DMF and 2-PrOH before discharging. The resin was dried under reduced pressure for 48 h.

[0350] Table 3: Conditions used for the linear SPPS using Fmoc-Lys(Mtt)-OH.

[0351] Entry Condition / Parameter 19.25 mmol Build

[0352] 1 Initial Sieber resin loading 0.77 mmol / g

[0353] 2 Amount DMF for each post-coupling wash 9 mL / g of resin charged

[0354] 3 Amount piperidine in DMF (20% v / v) for each Fmoc 9 mL / g of resin charged deprotection treatment

[0355] 4 Amount HFIP / toluene / TIPS (29.5 / 69.5 / 1 v / v / v) for 11 mL / g of resin charged

[0356] each Mtt deprotection treatmet

[0357]

[0358] Entry Condition / Parameter 19.25 mmol Build

[0359] 5 Amount DMF for each post-deprotection wash 9 mL / g of resin charged

[0360] 6 Amount toluene and DMF for each post Mtt- 11 mL / g of resin charged deprotection wash

[0361] 7 Amount 2-PrOH for each de-swelling wash 9 mL / g of resin charged

[0362] 8 Amount TBME for each de-swelling wash 9 mL / g of resin charged

[0363] 9 Pre-activation time for all couplings 5 min

[0364] 10 Coupling Chemistry Building block / Oxyma / DIC (ratio 1 / 1 / 1.5) in DMF

[0365] 11 Amount of building block per coupling Standard: 2.0

[0366] Cycle 38: 1.25

[0367] Volume of coupling solution

[0368] 12 excluding Oxyma Pure and 1-24 5.61 mL DMF / g of resin charged building blocks used for

[0369] coupling in cycles

[0370] 25 5.93 mL DMF / g of resin charged

[0371] 26–31 6.23 mL DMF / g of resin charged

[0372] 32–38 6.75 mL DMF / g of resin charged

[0373] 13 Resin bound drying temperature ambient

[0374] 14 Resin bound drying vacuum conditions 10 mbar

[0375] 15 Resin bound drying duration 60 h

[0376] 16 Mass of the dried and discharged resin bound 128 g

[0377] intermediate

[0378]

[0379] Entry Condition / Parameter 19.25 mmol Build

[0380] Molecular weight increase from the initial Sieber resin to the protected peptide on resin = 6000.52 g / mol

[0381] 15 Theoretical weight gain of the protected peptide on 116 g

[0382] resin

[0383] 16 Actual weight gain 103 g

[0384] 17 Yield of SPPS step 89%

[0385]

[0386] The detailed coupling sequence and step conditions in the various cycles are listed in Table 4. Table 4: Detailed conditions for the linear SPPS using Fmoc-Lys(Mtt)-OH and tBu-O-19-carboxy-nonadecanoyl-L-Glu (AEEAc-AEEAc-OH)-O-tBu.

[0387] Number and Number and Coupling

[0388] PostDuration AA Cycle Amino Acid / Building Duration of / Stir deprotection of Post# # Block Deprotection Time

[0389] washes Coupling Treatments [min]

[0390] DMF Washes 120

[0391] - - Ramage AM resin - (swell)

[0392] 39 1 Fmoc-L-Ser(OtBu)-OH 3 x 10 min 8 x DMF 90 4 x 5 min 38 2 Fmoc-L-Pro-OH·H2O 3 x 10 min 8 x DMF 90 4 x 5 min 37- 3 Fmoc-L-Pro-L-Pro-OH 3 x 10 min 8 x DMF 90 4 x 5 min 36

[0393] 35 4 Fmoc-L-Ala-OH 3 x 10 min 8 x DMF 90 4 x 5 min 34 5 Fmoc-Gly-OH 3 x 10 min 8 x DMF 90 4 x 5 min 33 6 Fmoc-L-Ser(OtBu)-OH 3 x 10 min 8 x DMF 90 4 x 5 min

[0394]

[0395] 32 7 Fmoc-L-Ser(OtBu)-OH 3 x 10 min 8 x DMF 90 4 x 5 min Number and Number and Coupling

[0396] PostDuration AA Cycle Amino Acid / Building Duration of / Stir deprotection of Post# # Block Deprotection Time

[0397] washes Coupling Treatments [min]

[0398] DMF

[0399] Washes 31 8 Fmoc-L-Pro-OH H2O 3 x 10 min 8 x DMF 90 4 x 5 min 30- 29 9 Fmoc-Gly-Gly-OH 3 x 10 min 8 x DMF 90 4 x 5 min 29

[0400] 28 10 Fmoc-L-Ala-OH·H2O 3 x 10 min 8 x DMF 90 4 x 5 min 27 11 Fmoc-L-Ile-OH 3 x 10 min 8 x DMF 90 4 x 5 min 26 12 Fmoc-L-Leu-OH 3 x 10 min 8 x DMF 150 4 x 5 min 25 13 Fmoc-L-Trp(Boc)-OH 3 x 10 min 8 x DMF 90 4 x 5 min 24 14 Fmoc-L-Gln(Trt)-OH 3 x 10 min 8 x DMF 90 4 x 5 min 23 15 Fmoc-L-Val-OH 3 x 10 min 8 x DMF 150 4 x 5 min 22 16 Fmoc-L-Phe-OH 3 x 10 min 8 x DMF 90 4 x 5 min 21 17 Fmoc-L-Ala-OH H2O 3 x 10 min 8 x DMF 90 4 x 5 min 20 18 Fmoc-L-Lys(Mtt)-OH 3 x 10 min 8 x DMF 1440 7 x 5 min 19 19 Fmoc-L-Gln(Trt)-OH 3 x 10 min 8 x DMF 360 4 x 5 min 18 20 Fmoc-L-Ala-OH-H2O 3 x 10 min 8 x DMF 120 4 x 5 min 17 21 Fmoc-L-Ile-OH 3 x 10 min 8 x DMF 180 4 x 5 min 16 22 Fmoc-L-Lys(Boc)-OH 3 x 10 min 8 x DMF 360 4 x 5 min 15 23 Fmoc-L-Asp(OtBu)-OH 3 x 10 min 8 x DMF 360 4 x 5 min 14 24 Fmoc-L-Leu-OH 3 x 10 min 8 x DMF 180 4 x 5 min 13 25 Fmoc-Aib-OH 3 x 10 min 8 x DMF 360 4 x 5 min 12 26 Fmoc-L-Ile-OH 3 x 10 min 9 x DMF 720 4 x 5 min Recoupling of Fmoc-L- - - - - 720 4 x 5 min Ile-OH

[0401]

[0402] 11 27 Fmoc-L-Ser(OtBu)-OH 3 x 10 min 9 x DMF 180 4 x 5 min Number and Number and Coupling

[0403] PostDuration AA Cycle Amino Acid / Building Duration of / Stir deprotection of Post# # Block Deprotection Time

[0404] washes Coupling Treatments [min]

[0405] DMF

[0406] Washes 10 28 Fmoc-L-Tyr(ItBu)-OH 3 x 10 min 9 x DMF 180 4 x 5 min 9 29 Fmoc-L-Asp(OtBu)-OH 3 x 10 min 9 x DMF 90 4 x 5 min 8 30 Fmoc-L-Ser(OtBu)-OH 3 x 10 min 9 x DMF 90 4 x 5 min 7 31 Fmoc-L-Thr(OtBu)-OH 3 x 10 min 10 x DMF 90 7 x 5 min 6 32 Fmoc-L-Phe-OH 3 x 10 min 10 x DMF 360 7 x 5 min 5 33 Fmoc-L-Thr(OtBu)-OH 3 x 10 min 12 x DMF 90 7 x 5 min 4 34 Fmoc-Gly-OH 3 x 10 min 12 x DMF 90 7 x 5 min Fmoc-L-Glu(OtBu)- 3 35 3 x 10 min 12 x DMF 360 7 x 5 min OH H2O

[0407] 2 36 Fmoc-P-Ala-OH 3 x 10 min 12 x DMF 360 7 x 5 min 7 x 5 min X1= 2-(3-cyano-5- DMF 1 37 fluorophenyl)-2- 3 x 10 min 12 x DMF 360

[0408] methylpropanoic acid 8x 5 min toluene Deprotection of Mtt 4x toluene

[0409] - 38 with HFIP / toluene / TIPS 5x 60 min - - (29.5 / 69.5 / 1 v / v / v) 8x DMF

[0410] Side Chain building

[0411] block

[0412] 20.1- 38 tBu-O- 19-carboxy- 1.25 12 x DMF 720 8 x 5 min 20.4

[0413] nonadecanoyl-L-Glu

[0414] (AEEAc-AEEAc- OH)-O- / Bu

[0415] 4x 2-PrOH

[0416] - - Final washes

[0417] 4x MTBE

[0418]

[0419] TFA-mediated cleavage from solid support with concomitant global deprotection

[0420] The resin bound material, obtained after the SPPS step, was subjected to cleavage / global deprotection to afford the crude peptide.

[0421] 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.

[0422] 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 18 h.

[0423] The crude peptide was isolated as an off-white powder (6.56 g, 58% crude yield).

[0424] ***

Claims

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 isCN andAEEAc stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid.comprising the stepsa) solid phase synthesis of a functionalized peptide fragment of formula Ila on a resinX1-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(Ila),wherein X isCNLys20is functionalized with Mttand the solid support is an amide resin,b) deprotection of the Lys20(Mtt),c) sequential coupling to the deprotected Lys20the side chain fragments in the orderc1) R1-AEEA-OHc2) R1-AEEA-OHc3) R1-Glu-O-PROTc4) (19-carboxynonadecanoyl-O-PROT)-OH,wherein R1is an amino protection group and PROT is an ester protecting group,and forming the functionalized peptide of formula III, bound to the resin;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-NH-solid support(III),wherein X and the solid support are as above,d) cleavage of the peptide of formula III from the resin and global deprotection with an acid ande) precipitation of the peptide of formula I and optionallyf) purification / isolation.

2. Process of claim 1, wherein the functionalized peptide fragment of formula Ila has the formula libX1-P-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)-Tyr10(OtBu)-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-Ala18-Gln19(Trt)-Lys20(Mtt)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH linker-solid support(IIb),wherein X isCNand the solid support is an amide resin.

3. Process of claim 1 or 2, wherein the amide resin is selected from the group consisting of Rink amide resin, Sieber amide resin, and Ramage amide resin.

4. Process of any one of claims 1 to 3, wherein the solid phase synthesis of the functionalized peptide fragment of formula Ila or lib in step a) comprises sequential coupling of the following1 Fmoc-L-Ser(OtBu)-OH2 Fmoc-L-Pro-OH·H2O3 Fmoc-L-Pro-L-Pro-OH4 Fmoc-L-Ala-OH5 Fmoc-Gly-OH6 Fmoc-L-Ser(OtBu)-OH7 Fmoc-L-Ser(OtBu)-OH8 Fmoc-L-Pro-OH·H2O9 Fmoc-Gly-Gly-OH10 Fmoc-L-Ala-OH·H2O11 Fmoc-L-Ile-OH12 Fmoc-L-Leu-OH13 Fmoc-L-Trp(Boc)-OH14 Fmoc-L-Gln(Trt)-OH15 Fmoc-L-Val-OHFmoc-L-Phe-OH Fmoc-L-Ala-OH H2O Fmoc-L-Lys(Mtt)-OH Fmoc-L-Gln(Trt)-OH Fmoc-L-Ala-OH-H2O Fmoc-L-Ile-OH Fmoc-L-Lys(Boc)-OH Fmoc-L-Asp(OtBu)-OH Fmoc-L-Leu-OH Fmoc-Aib-OHFmoc-L-Ile-OH Fmoc-L-Ser(OtBu)-OH Fmoc-L-Tyr(ItBu)-OH Fmoc-L-Asp(OtBu)-OH Fmoc-L-Ser(OtBu)-OH Fmoc-L-Thr(OtBu)-OH Fmoc-L-Phe-OH Fmoc-L-Thr(OtBu)-OH Fmoc-Gly-OHFmoc-L-Glu(OtBu)- OH H2OFmoc-P-Ala-OHX = 2-(3-cyano-5- fluorophenyl)-2-methylpropanoic acid5. Process of any one of claims 1 to 4, wherein the deprotection of Lys20(Mtt) in step b) is accomplished with a mixture of hexafluoroisopropanol (HFIP) and trisopropylsilane (TIPS) in an organic solvent.

6. Process of any one of claims 1 to 5, wherein in step c) a sequential coupling to the deprotected Lys20takes place of the side chain fragments in the ordercl) R'-AEEA-OHc2) R'-AEEA-OHc3) RkGlu-O-PROTc4) (19-carboxynonadecanoyl-O-PROT)-OH,wherein R1is an amino protection group and PROT is an ester protecting group.

7. Process of any one of claims 1 to 6, wherein in step c) the sequential coupling of the side chain fragments in the ordercl) Fmoc-AEEA-OHc2) Fmoc-AEEA-OHc3) Fmoc-Glu-O-tBuc4) (19-carboxynonadecanoyl-O-tBu)-OH,takes place.

8. Process of any one of claims 1 to 5, wherein in step c) coupling of the whole side chain of formula IVPROT-O- 19-carboxy-nonadecanoyl-L-Glu (AEEAc-AEEAc-OH)-O-PROT(IV),wherein AEEAc and PROT is an ester protecting group, to the deprotected Lys20takes place.

9. Process of any one of claims 1 to 5 or 8, wherein in step c) coupling of the whole side chain of formula IVaZBu-O- 19-carboxy-nonadecanoyl-L-Glu ( AEE Ac- AEE Ac-OH)-O- / Bu(IVa),to the deprotected Lys20takes place.

10. Process of any one of claims 1 to 9, wherein the cleavage of the functionalized peptide of formula III from the resin and global deprotection is accomplished with TFA in combination with a scavenger.

11. The process of any one of claims 1 to 10, wherein the peptide of formula I, in step e) is precipitated in an organic solvent, preferably, in t-butyl methyl ether (TBME).

12. Functionalized peptide fragment of formulae IlaX1-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(Ila),wherein X isCNLys20is functionalized with Mttand the solid support is an amide resin.

13. Functionalized peptide fragment of formula libX1-P-Ala2-Glu3(OtBu)-Gly4-Thr5(OtBu)-Phe6-Thr7(OtBu)-Ser8(OtBu)-Asp9(OtBu)- Tyr10(OtBu)-Ser11(OtBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-Ala18-Gln19(Trt)-Lys20(Mtt)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31- Ser32(OtBu)-Ser33(OtBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(OtBu)-NH linker-solid support (lib),wherein X isCNand the solid support is an amide resin.***

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