Method for producing peptides having lysine derivatives

Abstract

The present invention relates to a method for preparing a peptide P, which comprises the step of condensing an alpha amino acid derivative S-am having one unprotected alpha amino group or one unprotected alpha imino group with a protected alpha amino acid derivative of formula (Pr-L), wherein the amino protecting group R L-N-1 The present invention also relates to a method for preparing an alpha amino acid derivative of formula (Pr-L) and the associated lysine derivative having its alpha amino group protected by 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl (Bsmoc). [Formula 1] TIFF2025533940000114.tif88131

Description

[Technical Field]

[0001] The present invention relates to a method for producing peptides containing a lysine residue having a fatty acid conjugation motif covalently bound to its epsilon-nitrogen atom, comprising condensing an alpha amino acid derivative with a lysine derivative that retains a fatty acid conjugation motif covalently bound to its epsilon-nitrogen atom and that retains a specific amino-protecting group, i.e., 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl, at its alpha-nitrogen atom. The present invention further relates to lysine derivatives and methods for producing the lysine derivatives. The present invention also relates to lysine precursors that retain a portion of the fatty acid conjugation motif covalently bound to their epsilon-nitrogen atom and that retain a specific amino-protecting group at their alpha-nitrogen atom, and methods for producing the lysine precursors. The present invention further relates to methods for producing lysine intermediates that retain a specific amino-protecting group at their alpha-nitrogen atom. [Background technology]

[0002] Semaglutide (CAS number 910463-68-2) is an active pharmaceutical ingredient known as a glucagon-like peptide-1 receptor agonist. Semaglutide, when written as the three-letter peptide code, has the following formula: H-His 1 -Aib-Glu-Gly-Thr 5 -Phe-Thr-Ser-Asp-Val 10 -Ser-Ser-Tyr-Leu-Glu 15 -Gly-Gln-Ala-Ala-Lys 20 (HO-CO-(CH2) 16 -CO-gamma-Glu-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])-Glu-Phe-Ile-Ala-Trp 25 -Leu-Val-Arg-Gly-Arg 30 -Gly-OH (SEQ ID NO: 5)

[0003] Thus, semaglutide has a linear 31-mer peptide backbone, the lysines of which 20 The epsilon nitrogen atom of lysine holds the fatty acid side chain. 20 The epsilon nitrogen atom of HO-CO-(CH2) 16 -CO-gamma-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl].

[0004] Patent Document 1 discloses the peptide semaglutide in Example 4. Semaglutide is a peptide derived from lysine 20 The peptide is already synthesized in a complete linear peptide backbone by acylation of the lysine nitrogen atom. 20 The fatty acid side chains at are introduced after the complete linear peptide backbone of semaglutide has been synthesised.

[0005] Patent Document 2 discloses the synthesis of semaglutide using an Fmoc-protected lysine derivative that already bears a fatty acid side chain as a building block. 20 is introduced during the synthesis of the linear peptide backbone of semaglutide. The Fmoc-protected lysine derivative (CAS number 1662688-20-1) used is shown below.

[0006] [ka]

[0007] In Example 1 of Patent Document 3, solid phase peptide synthesis of the aforementioned Fmoc-protected lysine derivative (CAS No. 1662688-20-1) via solid phase conjugated Alloc-protected lysine derivative (CAS No. 2721349-46-6) is disclosed, which is shown below.

[0008] [ka]

[0009] In Example 4 of Patent Document 4, Boc-Lys(tert-BuO-CO-(CH)) is synthesized via a Boc-protected lysine derivative (CAS number 2921565-73-1). 16 The synthesis of —CO-gamma-Glu-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])-Ala-OAll (CAS number 2921565-74-2) has been disclosed and is shown below.

[0010] [ka]

[0011] Patent document 5 discloses the Nsc group of 2-(4-nitrophenylsulfonyl)ethoxycarbonyl as an amino protecting group in solid-phase peptide chemistry, and states on page 18 that the Nsc group is completely resistant to the action of acidic reagents that are commonly used for cleaving tert-butyl type protecting groups.

[0012] Non-Patent Document 1 discloses a comparative study of Fmoc and Nsc groups in automated solid-phase peptide synthesis. It concludes that the comparative synthesis of three test peptides resulted in similar HPLC-UV chromatograms. On page 312, it states that analytical HPLC using a C18 column and a gradient of 0.1% aqueous trifluoroacetic acid / acetonitrile showed a retention time of 16 minutes for Nsc-Phe-OH, while that of Fmoc-Phe-OH was 24 minutes. Considering the single retention time, it appears that decomposition of Nsc-Phe-OH did not occur.

[0013] Non-Patent Document 2, p. 7822, discloses the preparation of Nsc-Lys(Boc)-OH via its trimethylsilyl derivative. A yield of 83% is reported for the homogeneous product after recrystallization. During workup, exposure to 5% aqueous NaHCO3 occurs during extraction.

[0014] Non-Patent Document 3 discloses solid-phase peptide synthesis using alpha-azido-protected amino acids, and in Table 2 it states that alpha-azido acids outperform comparative Fmoc-amino acids in several test peptide sequences.

[0015] Non-Patent Document 4 discloses the Bsmoc group, which is an abbreviation for 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl, as an amino-protecting group in peptide synthesis.

[0016] Non-Patent Document 5 discloses the orthogonal protection of the epsilon amino group of lysine linked with a Boc group, i.e., the respective Psc group of 2-(phenyl-sulfonyl)ethoxycarbonyl for Boc-Lys(Psc)-OH, and its use in solution-phase peptide synthesis.

[0017] Patent Document 6 discloses in Example 11 the peptide Psc-D-Phe-Cys(Bzm)-Phe-D-Trp(For)-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol(Psc)2 (SEQ ID NO: 12), where Psc stands for 2-(phenylsulfonyl)ethoxycarbonyl. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] WO2006-097537A2 [Patent Document 2] CN104356224A [Patent Document 3] CN113461801A [Patent Document 4] CN115677827A [Patent Document 5] WO96-25394 [Patent Document 6] RU2196144C1 [Non-patent literature]

[0019] [Non-Patent Document 1] Protein and Peptide Letters (1997), Volume 4, No. 5, pp. 307-312 [Non-patent document 2] Tetrahedron Letters (1994), Volume 35, No. 42, pp. 7821-7824 [Non-patent document 3] Organic Letters (2001), Volume 3, No. 5, pp. 781-783 [Non-patent document 4] Journal of American Chemical Society (1997), Vol. 119, pp. 9915-9916 [Non-patent document 5] Bulletin of Korean Chemical Society (1998), Volume 19, No. 6, pp. 696-698 Summary of the Invention [Problem to be solved by the invention]

[0020] There remains a need for further improvements. Improving the yield of condensation reactions is desirable, especially when the condensation reaction is part of a multistep reaction scheme for larger molecules, e.g., molecules with molecular weights exceeding 1000 g / mol, as is often the case with (poly)peptides. This is because it is often difficult to remove one or more by-products, which are relatively large molecules generated due to ineffective condensation reactions, from the target molecule. This is often due to the overall similar physical behavior, which, in the case of (poly)peptides, often requires purification by preparative high-performance liquid chromatography. Therefore, the initial purity of the raw materials containing the target larger molecule is important and contributes to the effectiveness of the condensation reaction. Furthermore, in the field of peptide synthesis, it is beneficial if the applied starting material has sufficient, at least temporary, stability against decomposition upon contact with base or acid, e.g., trifluoroacetic acid. The term "at least temporary" refers to sufficient stability under typical conditions, such as room temperature, exposure times of up to 1 hour, and the presence of water. On the one hand, this allows for convenient synthesis of the normally applied starting material, e.g., during aqueous workup. Here too, the initial purity of the starting materials, including the starting materials, and thus the avoidance of preparative liquid chromatography, are important. On the other hand, the possibility of analytical monitoring of the starting materials using typical acid-analytical reversed-phase high-performance liquid chromatography allows for deeper insight into and therefore manipulation of the condensation reaction in the case of (poly)peptides. Another desirable aspect is that, in the case of (poly)peptides, the condensation reaction can be limited to a base-catalyzed deprotection scheme of the amino protecting group for further condensation reactions. Thus, new reactive amine and imine groups can be generated for further condensation reactions to form amide bonds with amino acids already condensed in existing peptides without endangering the acid-sensitive linking groups attached to the resin or the acid-sensitive protecting groups at the respective side chains. Furthermore, condensation reactions that do not result in the presence of heavy metals during the subsequent deprotection of the amino protecting groups may be desirable. [Means for solving the problem]

[0021] Peptide P is preferably a compound of formula Pr-LS. It will be understood that the term "peptide P" is a designation for the peptide to be synthesized. The designation "P" may be omitted or written in parentheses without changing the meaning of the peptide defined by its structural features. The term "Pr-LS" describes the structure as defined in the structure shown. It is intended to indicate the structural elements described below. This designation "Pr-LS" may be omitted or written in parentheses without changing the meaning of the peptide defined by the structure shown.

[0022] Currently, there is provided a method for producing peptide P, comprising the steps of: (c) an alpha amino acid derivative S-am having one unprotected alpha amino group or one unprotected alpha imino group, Compound of formula Pr-L [ka] (In the formula, R L-O-1 and R L-O-2 are each independently a carboxylic acid protecting group. to obtain peptide Pr-LS. Contains R L-N-1 is an amino protecting group of formula Bsmoc [ka] (In the formula, * indicates a bond to a nitrogen atom) (Bsmoc) A method has been found, characterized in that:

[0023] To condense one unprotected alpha-amino group or one unprotected alpha-amino group of the alpha amino acid derivative S-am with a compound of formula Pr-L, the carboxylic acid group of the compound of formula Pr-L is typically activated with a condensing agent in step (c). The condensing agent in step (c) results in an activated compound of formula Pr-L, which contains a leaving group instead of a hydroxy group at the carboxylic acid group, enhancing the reactivity of the activated compound of formula Pr-L toward the unprotected alpha-amino group or unprotected alpha-amino group of the alpha amino acid derivative S-am. Depending on the specific condensing agent in step (c), the activated compound of formula Pr-L may or may not be an intermediate suitable for isolation at room temperature. The condensing agent in step (c) is, for example, a carbodiimide derivative. Examples of carbodiimide derivatives include diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1-tert-butyl-3-ethylcarbodiimide, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Preferably, the carbodiimide derivative is diisopropylcarbodiimide, dicyclohexylcarbodiimide, or 1-tert-butyl-3-ethylcarbodiimide. More preferably, the carbodiimide derivative is diisopropylcarbodiimide or 1-tert-butyl-3-ethylcarbodiimide. Highly preferably, the carbodiimide derivative is diisopropylcarbodiimide. Preferably, the carbodiimide is applied together with the coupling additive in step (c). The coupling additive in step (c) is, for example, cyano-hydroxy-imino-acetic acid ethyl ester, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, 5-hydroxy-imino-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione, ethyl 1-hydroxy-1,2,3-triazole-4-carboxylate, 2-hydroxypyridine N-oxide, or a mixture thereof. Preferably, the coupling additive in step (c) is cyano-hydroxyimino-acetic acid ethyl ester, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzo-triazole or 2-hydroxypyridine N-oxide.More preferably, the coupling additive in step (c) is cyano-hydroxyimino-acetic acid ethyl ester or 1-hydroxybenzotriazole. Highly preferably, the coupling additive in step (c) is cyano-hydroxyimino-acetic acid ethyl ester. If the alpha amino acid derivative S-am contains an unprotected carboxylic acid group, activation of the compound of formula Pr-L must occur before contact with the alpha amino acid derivative S-am. If the alpha amino acid derivative S-am possesses a functional group that interferes with the condensation in step (c) and is different from one unprotected alpha amino group or one unprotected alpha imino group, the functional group is protected with an appropriate protecting group. It is preferred that the alpha amino acid derivative S-am does not have an unprotected guanidino group or an unprotected mercapto group.

[0024] Preferred is the process wherein in step (c) the compound of formula Pr-L is activated by a condensing agent in step (c).

[0025] In step (c), a process in which a coupling additive of step (c) is present is preferred.

[0026] Preferred is a process wherein in step (c) the compound of formula Pr-L is activated with a condensing agent of step (c) which is a carbodiimide derivative, and in the presence of a coupling additive of step (c) which is a cyano-hydroxyimino-acetic acid ethyl ester.

[0027] Step (c) is preferably carried out in the presence of a solvent for step (c). The solvent for step (c) dissolves the compound of formula Pr-L. In the case of a resin covalently bonded to an alpha amino acid derivative S-am, the solvent for step (c) preferably swells the resin. Examples of solvents for step (c) include N,N-dimethylformamide, N-methyl-2-pyrrolidone, N-butyl-pyrrolidone, dimethylisosorbide, gamma-valerolactone, dihydrolevo-glucosenone (e.g., commercially available Silene™), dimethyl sulfoxide, tetrahydropyran, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, 1,3-dioxolane, ethyl acetate, dichloromethane, acetonitrile, toluene, water, and mixtures thereof. Preferably, the solvent for step (c) comprises N,N-dimethylformamide. More preferably, the solvent in step (c) comprises at least 20 vol.%, very preferably 40 vol.%, in particular 70 vol.%, more in particular 90 vol.%, and very in particular more than 95 vol.% N,N-dimethylformamide. Preferably, the solvent in step (c) is N,N-dimethylformamide.

[0028] Preferred is a process wherein step (c) is carried out in a solvent for step (c), wherein the solvent for step (c) comprises N,N-dimethylformamide.

[0029] Preferably, step (c) is carried out at a temperature between 5° C. and 50° C., more preferably between 10° C. and 40° C., very preferably between 15° C. and 35° C., particularly between 18° C. and 30° C., more particularly between 19° C. and 28° C., very particularly between 20° C. and 27° C., and especially between 22° C. and 25° C., which is defined herein as room temperature.

[0030] A method in which step (c) is carried out at a temperature between 15°C and 35°C is preferred.

[0031] Preferably, the compound of formula Pr-L is applied in step (c) in molar excess relative to the alpha amino acid derivative S-am. The molar excess is, for example, a molar amount of the compound of formula Pr-L, which is 1.05 to 3.5 times the molar amount of the alpha amino acid derivative S-am. Preferably, the molar amount is 1.3 to 3.0 times, more preferably 1.5 to 2.5 times, very preferably 1.7 to 2.3 times, particularly 1.8 to 2.2 times, and even more particularly 1.9 to 2.1 times.

[0032] Preferably, the condensing agent of step (c) is added in molar excess relative to the compound of formula Pr-L in step (c). The molar excess is, for example, a molar amount of the condensing agent of step (c), 1.05 to 3 times the molar amount of the compound of formula Pr-L. Preferably, the molar amount is 1.3 to 2.5 times, more preferably 1.5 to 2.3 times, very preferably 1.7 to 2.2 times, specifically 1.8 to 2.2 times, and even more specifically 1.9 to 2.0 times. The total amount of the condensing agent of step (c) is added to the compound of formula Pr-L, for example, in one or more portions. A portion of the condensing agent of step (c), for example, 50 to 70 wt.% of the total amount of the condensing agent of step (c), is added to the compound of formula Pr-L dissolved in the solvent of step (c), for example. The resulting solution is stirred, for example, for 5 to 30 minutes, and then added to the alpha amino acid derivative S-am.

[0033] Preferably, the coupling additive in step (c) is applied in a molar amount relative to the compound of formula Pr-L that is 0.5 to 3 times the molar amount of the compound of formula Pr-L. This molar amount is, for example, 0.8 to 2.5 times the molar amount of the compound of formula Pr-L. Preferably, this molar amount is 0.9 to 2.2 times, more preferably 1.1 to 2.0 times, very preferably 1.3 to 1.7 times, and in particular 1.4 to 1.6 times the molar amount of the compound of formula Pr-L.

[0034] A peptide bond is herein understood to mean a covalent bond between the alpha amino group or alpha imino group of a first alpha amino acid residue and the alpha carboxylic acid group of a second alpha amino acid residue. An alpha amino acid derivative S is herein understood to mean either a compound lacking a peptide bond or a compound having at least one peptide bond. In the latter case, the alpha amino acid derivative S is, for example, a dipeptide derivative, tripeptide derivative, or tetrapeptide derivative. In both compounds, the N-terminal alpha amino group or the carboxamide group of the C-terminal amino acid residue, which may also be the alpha imino group of the N-terminal amino acid residue and the C-terminal carboxylic acid group, are present. In compounds lacking a peptide bond, the N-terminal alpha amino group, which may also be the alpha imino group, and the C-terminal carboxylic acid group or carboxamide group are each substituents of the same alpha carbon atom belonging to the same alpha amino acid residue. The alpha amino acid derivative S-am is, for example, bound to a resin. The resin comprises a linking group and a polymeric support. The linking group is covalently bound to the polymeric support. The linking group serves to covalently bond to the alpha amino acid residue, where the covalent bond of the alpha amino acid residue is cleavable. Typically, the C-terminal alpha amino acid residue is covalently bonded to the resin. The resin is, for example, Resin-1 or Resin-2. Resin-1 carries a linking group particularly suitable for covalent bonding of the oxygen atom of a carboxylic acid group. The linking group of Resin-1, designated as a di-yl substituent, is, for example, 2-chlorotrityl-p-amidomethyl, 2-chlorotrityl-yl, or 4-(methyleneoxy)benzyl. Resin-2 carries a linking group particularly suitable for covalent bonding of the nitrogen atom of a carboxylic acid amide group. The linking group of Resin-2, designated as a di-yl substituent, is, for example, xanthene-3-(oxymethylene)-9-yl or alpha-(2,4-dimethoxyphenyl)-alpha-(4-(N-methyleneamido-methyleneoxy)phenyl)methyl. The polymeric support of resin-1 or resin-2 is, for example, polystyrene, a copolymer containing polymerized styrene units and polymerized ethylene oxide units, or polyethylene oxide. The alpha amino acid derivative S-am is, for example, a single molecule. Preferably, the alpha amino acid derivative S-Am is covalently bound to the resin.Preferably, the alpha amino acid derivative S-Am is a single molecule. Preferably, the alpha amino acid derivative S-Am does not have an unprotected carboxylic acid group.

[0035] The peptide P has the formula L [ka] (In the formula, # and ## indicates a covalent bond) (L) The structural elements of R L-O-1 and R L-O-2 are each independently H or a carboxylic acid protecting group.

[0036] The covalent bond of formula L, indicated by #, is directed to the alpha nitrogen atom of the alpha amino acid residue that is the N-terminal alpha amino acid residue of the alpha amino acid derivative S-am. The covalent bond of formula L, indicated by ##, is directed to the hydrogen atom, amino protecting group or alpha carbonyl group of the alpha amino acid residue that is, for example, the C-terminal alpha amino acid residue of a further alpha amino acid derivative.

[0037] The alpha amino acid derivatives S-am having one unprotected alpha amino group or one unprotected alpha imino group preferably have the formula S-I-am or S-II-am [ka] is an alpha amino acid derivative of During the ceremony, R SI-1 AA nx ) m -O-[Resin-1], -(AA nx ) m -N-[Resin-2], -O-[Resin-1], -N-[Resin-2], -(AA nx ) m -OR SI-1-1 , OR SI-1-1 or NH2, RSI-2 is H, C 1-6 Alkyl or OR SI-2-1 , S.R. SI-2-2 , SCH3, NR SI-2-3 R SI-2-4 , CO-OR SI-2-5 , CO-NR SI-2-6 R SI-2-7 , N'-R SI-2-8 -N”-R SI-2-9 -guanidino, phenyl, para-(R SI-2-10 O)-phenyl, 1-R SI-2-11 -imidazol-4-yl or 1-R SI-2-12 -indol-3-yl monosubstituted C 1-6 is alkyl, -(AA nx ) m - m condensed alpha amino acid residues AA nx wherein each x is an integer and x varies from 1 to m; Each condensed alpha amino acid residue AA nx are independently selected, and when a side chain bearing a functional group is present, the functional group is unprotected or protected by a protecting group, provided that interfering functional groups are protected; m is an integer from 1 to 30; R SI-1-1 is H or a carboxylic acid protecting group, R SI-2-1 is H or a hydroxy protecting group, R SI-2-2 is a thiol protecting group, R SI-2-3 and R SI-2-4 are H, an amino protecting group, or when neither is H, together form an amino protecting group; R SI-2-5 is H or a carboxylic acid protecting group, R SI-2-6 and R SI-2-7 is H or an amide protecting group, R SI-2-8 and R SI-2-9 is H or, if both are not H, a guanidino protecting group; R SI-2-10is a protecting group for an aromatic hydroxy group, R SI-2-11 is H or a protecting group for the imidazole nitrogen atom, R SI-2-12 is H or a protecting group for the indole nitrogen atom, R SII-1 is R SI-1 As defined in

[0038] The carboxylic acid protecting group is, for example, tert-butyl, benzyl, phenacyl, 2-phenyl-isoprop-2-yl or methyl, preferably tert-butyl. Preferably, the carboxylic acid protecting group is tert-butyl or 3-methuryl-pent-3-yl. More preferably, the carboxylic acid protecting group is tert-butyl. Preferably, R L-O-1 and R L-O-2 is a carboxylic acid protecting group that is removed under acidic conditions, more preferably under exposure to trifluoroacetic acid. L-O-1 and R L-O-2 are each independently tert-butyl or 3-methyl-pent-3-yl. More preferably, R L-O-1 and R L-O-2 are the same and are tert-butyl or 3-methyl-pent-3-yl. Highly preferably, R L-O-1 and R L-O-2is tert-butyl. Hydroxy-protecting groups are, for example, tert-butyl, benzyl, 2-bromobenzyloxycarbonyl, trityl, or 2-chlorotrityl, preferably tert-butyl. In the case of an alpha amino acid residue containing a hydroxy group at the beta position, the hydroxy-protecting group can also be, for example, a 1,1-dimethyldiyl group, which forms an oxazolidine ring together with the alpha nitrogen atom, the alpha carbon atom, and the oxygen atom of the hydroxy group at the beta position (so-called pseudoproline). Thiol-protecting groups are, for example, tert-butyl, 4-methylbenzyl, acetamidomethyl, trityl, tert-butylsulfanyl, tetrahydropyran-2-yl, diphenylmethyl, or 2,4,6-trimethoxybenzyl. Amino-protecting groups are, for example, tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, or allyloxycarbonyl. Amide protecting groups include, for example, 9-H-xanthen-9-yl, 2,4,6-trimethoxybenzyl, dimethylcyclopropylmethyl, trityl, or 3-methylpent-3-yl. Guanidino protecting groups include, for example, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-ylsulfonyl (=Pbf), 2,2,5,7,8-pentamethylchroman-6-ylsulfonyl, mesitylsulfonyl, tosyl, trityl, or methoxytrityl, preferably 2,2,4,6,7-pentamethyldihydrobenzofuran-5-ylsulfonyl. Protecting groups for aromatic hydroxy groups include, for example, tert-butyl, benzyl, 2-chlorotrityl, or 2-bromogenzyloxycarbonyl. Protecting groups for imidazole nitrogen include, for example, tert-butyloxycarbonyl, 2,4-dinitrophenyl, benzyloxymethyl, trityl, or methoxytrityl, preferably tert-butyloxycarbonyl. A protecting group for the indole nitrogen is for example tert-butyloxycarbonyl or formyl, preferably tert-butyloxycarbonyl.

[0039] AA n1has the nitrogen atom of its alpha amino group covalently bonded to the carbon atom of a carbonyl group of formula SI-am or S-II-am. nm The carbonyl group covalently bonds the carbon atom of the carbonyl group to an oxygen or nitrogen atom. nm Preferably, the alpha carbonyl group of the formula (I) is an alpha carbonyl group of the formula (II). An example of a side chain functional group that interferes with the condensation in step (c) is an amino group. Therefore, the amino group is protected by a protecting group.

[0040] AA nx are, for example, alpha amino acid residues, glycine, alanine, serine, serine with its side chain protected by tert-butyl, threonine, threonine with its side chain protected by trityl, cysteine ​​with its side chain protected by tert-butyl, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine with its side chain protected by tert-butyl, tryptophan with its side chain protected by tert-butyloxycarbonyl, and cysteine ​​with its side chain protected by tert-butyl. and condensed forms thereof as aspartic acid protected at its side chain with tert-butyl, glutamic acid protected at its side chain with asparagine, asparagine protected at its side chain with trityl, glutamine, glutamine protected at its side chain with dimethylcyclopropylmethyl, histidine protected at its side chain with trityl, lysine protected at its side chain with allyloxycarbonyl, or arginine protected at its side chain with 2,2,4,6,7-pentamethyldihydrobenzofuran-5-ylsulfonyl.

[0041] m=3, AA n1 = Gly, AA n2 = Arg(Pbf) and AA n3 = Gly substituent -(AA nx ) m An example for - is shown below: -(Gly-Arg(Pbf)-Gly)- [ka] (In the formula, * and **indicates a covalent bond) is.

[0042] C 1-6 Alkyl is, for example, methyl, 1-methylethyl, 1-methylpropyl or 2-methylpropyl. SI-2-1 Monosubstituted C 1-6 Alkyl is, for example, hydroxymethyl, R SI-2-1 hydroxymethyl, 1-hydroxyethyl or R SI-2-1 SR is a 1-hydroxyethyl protected amine. SI-2-2 Monosubstituted C 1-6 Alkyl is, for example, R SI-2-2 protected mercaptomethyl. SCH3 monosubstituted C 1-6 Alkyl is, for example, 2-(methylthio)ethyl. NR SI-2-3 R SI-2-4 Monosubstituted C 1-6 Alkyl is, for example, R SI-2-3 , R SI-2-4 or R SI-2-3 and R SI-2-4 CO-OR is a 4-aminobutyl protected SI-2-5 Monosubstituted C 1-6 Alkyl is, for example, R SI-2-5 protected carboxymethyl or R SI-2-5 2-carboxyethyl protected by CO-NR SI-2-6 R SI-2-7 Monosubstituted C 1-6 Alkyl is, for example, 2-amino-2-oxoethyl or R SI-2-6 , R SI-2-7 Or R SI-2-6 and R SI-2-7 and 2-amino-2-oxoethyl protected by CO-NR SI-2-6 R SI-2-7 Monosubstituted C 1-6 Alkyl can also be used, for example, 3-amino-3-oxopropyl or R SI-2-6 , R SI-2-7 Or R SI-2-6 and R SI-2-7 N'-R is 3-amino-3-oxopropyl protected bySI-2-8 -N”-R SI-2-9 -guanidino monosubstituted C 1-6 Alkyl is, for example, R SI-2-8 , R SI-2-9 or R SI-2-8 and R SI-2-9 phenyl-monosubstituted C 1-6 Alkyl is, for example, benzyl. SI-2-10 O)-phenyl monosubstituted C 1-6 Alkyl is, for example, R SI-2-10 1-R is 4-hydroxybenzyl protected by SI-2-11 -imidazol-4-yl monosubstituted C 1-6 Alkyl can be substituted, for example, by imidazol-4-ylmethyl or by R at the 1-position of the imidazole ring. SI-2-11 1-R is an imidazol-4-ylmethyl protected by SI-2-12 -indol-3-yl monosubstituted C 1-6 Alkyl can be, for example, indol-3-ylmethyl or R at position 1 of the indole ring. SI-2-12 is indol-3-ylmethyl protected by the formula:

[0043] Preferably, R SI-1 AA nx ) m -O-[Resin-1], -(AA nx ) m -N-[resin-2], -O-[resin-1], -N-[resin-2] or NH2. More preferably, R SI-1 AA nx ) m -O-[Resin-1], -(AA nx ) m -N-[resin-2], -O-[resin-1] or -N-[resin-2].

[0044] Preferably, R SI-1-1 is a carboxylic acid protecting group. Preferably, R SI-2-5 is a carboxylic acid protecting group. Preferably, all AA nx More preferably, RSI-1-1 and R SI-2-5 are, independently of each other, carboxylic acid protecting groups, and all AAs nx has no unprotected carboxylic acid groups. More preferably, the alpha amino acid derivatives of formula SI-am or S-II-am have no unprotected carboxylic acid groups.

[0045] Preferably, R SI-2 is different from H. More preferably, R SI-2 is C 1-6 Alkyl or OR SI-2-1 , S.R. SI-2-2 , SCH3, NR SI-2-3 R SI-2-4 , CO-OR SI-2-5 , CO-NR SI-2-6 R SI-2-7 , N'-R SI-2-8 -N”-R SI-2-9 -guanidino, phenyl, para-(R SI-2-10 O)-phenyl, 1-R SI-2-11 -imidazol-4-yl or 1-R SI-2-12 -indol-3-yl monosubstituted C 1-6 Highly preferably, R SI-2 is OR SI-2-1 , S.R. SI-2-2 , SCH3, NR SI-2-3 R SI-2-4 , CO-OR SI-2-5 , CO-NR SI-2-6 R SI-2-7 , N'-R SI-2-8 -N”-R SI-2-9 -guanidino, phenyl, para-(R SI-2-10 O)-phenyl, 1-R SI-2-11 -imidazol-4-yl or 1-R SI-2-12 -indol-3-yl monosubstituted C 1-6 Specifically, R SI-2 CO-OR SI-2-5 or CO-NR SI-2-6 R SI-2-7 Monosubstituted C 1-6 It is alkyl.

[0046] Preferably, m is an integer from 1 to 25, more preferably from 1 to 20, very preferably from 1 to 15, particularly from 2 to 12, more particularly from 3 to 11, and very particularly from 4 to 10.

[0047] Preferably, R SI-2 The alpha carbon atom of the compound of formula SI-am substituted by mR SI-2 is different from H. Preferably, R SII-1 The alpha carbon atom in the compound of formula S-II-am that is substituted by a carbonyl group covalently bonded to is in the S configuration.

[0048] If the alpha amino acid derivative S-am in step (c) is a compound of formula S-I-am or S-II-am, the peptide Pr-LS obtained in step (c) is a compound of formula Pr-LS-I or Pr-LS-II [ka] is a compound of During the ceremony, R L-O-1 , R L-O-2 and R L-N-1 is as defined in formula Pr-L, R SI-1 , R SI-2 and R SII-1 is as defined in formula SI-am or S-II-am.

[0049] Preferred is a process wherein the alpha amino acid derivative S-am is a compound of formula SI-am or S-II-am and the peptide Pr-LS obtained in step (c) is a compound of formula Pr-LS1 or Pr-LS-II.

[0050] The alpha amino acid derivative S-am in step (c) is R SI-1 But-(AA nx ) 10 -O-[resin-1] is a derivative of formula SI-am, n1 is Phe and AA n2 is Ile and AAn3 is Ala, AA n4 is Trp with a protected side chain, and AA n5 is Leu and AA n6 is Val and AA n7 is Arg with a protected side chain, and AA n8 is Gly and AA n9 is Arg with a protected side chain, and AA n10 is Gly, resin-1 is 2-chlorotritylamido-methyl resin, R SI-2 is preferably 2-(tert-butyloxycarbonyl)ethyl.

[0051] The alpha amino acid derivative S-am in step (c) is R SI-1 But-(AA nx ) 10 -O-[resin-1] is a derivative of formula SI-am, n1 is Phe and AA n2 is Ile and AA n3 is Ala, AA n4 is Trp with a protected side chain, and AA n5 is Leu and AA n6 is Val and AA n7 is Arg with a protected side chain, and AA n8 is Gly and AA n9 is Arg with a protected side chain, and AA n10 is Gly, resin-1 is 2-chlorotrityl resin, and R SI-2 is preferably 2-(tert-butyloxycarbonyl)ethyl.

[0052] R L-O-1 and R L-O-2 are, independently of each other, tert-butyl or 3-methyl-pent-3-yl.

[0053] A preferred method is where the compound of formula Pr-L is compound 508. Compound 508 is shown in Example B-16-1.

[0054] The chemical name for the molecular structure of formula Bsmoc is 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl, also commonly referred to as benzo[b]thiophenesulfone-2-methyloxy-carbonyl.

[0055] Preferably, the method comprises the step of removing an amino protecting group R from a compound of formula Pr-LS1 or Pr-LS-II. L-N-1 Remove the formula LSI-am or LS-II-am [ka] (In the formula, R L-O-1 , R L-O-2 , R SI-1 , R SI-2 and R SII-2 is as defined in formula Pr-LS1 or Pr-LS-II) and step (d) obtaining a compound of formula (I).

[0056] Amino protecting group R L-N-1 The removal of is carried out using a deprotection composition in step (d). The deprotection composition in step (d) is, for example, 20 vol.% piperidine in N,N-dimethylformamide. Appropriate selection of the other protecting group in the compound of formula Pr-LSI or Pr-LS-II leads to a compound of formula LSI-am or LS-II-am, which contains a deprotected alpha amino group and no other unprotected amino or imino groups, and preferably no unprotected carboxylic acid groups.

[0057] Process (d) (d) a compound of formula Pr-LS1 or Pr-LS-II, wherein the amino protecting group R L-N-1 to obtain a compound of formula LSI-am or LS-II-am A method comprising the steps of:

[0058] Preferably, the method comprises reacting a compound of formula LSI-am or LS-II-am with an alpha-amino acid derivative of formula T R T-N-1 -(AA py ) q -OH (T) (In the formula, R T-N-1 is an amino protecting group, -(AA py ) q - q condensed alpha amino acid residues AA py wherein each y is an integer and y varies from 1 to q; Each condensed alpha amino acid residue AA py are independently selected, and if they possess a side chain bearing a functional group, that functional group is unprotected or protected by a protecting group, provided that interfering functional groups are protected; AA pq has its alpha carbonyl carbon atom covalently bonded to a hydroxy group, q is an integer from 1 to 30. to form a compound of formula TLSI or TLS-II [ka] (In the formula, R L-O-1 , R L-O-2 , R SI-1 , R SI-2 and R SII-2 is as defined in formula LSI-am or LS-II-am, R T-N-1 and -(AA py ) q - is as defined in formula T) and step (e) of obtaining:

[0059] AA p1 The alpha amino group of R T-N-1 Protected by AA pq covalently bonds the carbon atom of its alpha carbonyl group to the indicated hydroxy group of formula T.pq The carbon atom of its alpha carbonyl group is covalently bonded to the nitrogen atom of the alpha nitrogen atom of formula TLSI or TLS-II. The compound of formula T contains one unprotected carboxylic acid group. The one unprotected carboxylic acid group is attached to the amino acid residue AA. pq and the hydroxy group represented by formula T. An example of a side chain functional group that interferes with the condensation in step (e) is an amino group. Therefore, the amino group is protected by a protecting group. AA py An example of this is AA in step (c). nx The following are listed:

[0060] q=1, R T-N-1 = 9-Fluorenylmethyloxycarbonyl and AA p1 An example of a compound of formula T where = alanine residue is e.g. Fmoc-Ala-OH as shown below. [ka]

[0061] Preferably, q is an integer from 1 to 25, more preferably from 1 to 20, very preferably from 1 to 15, particularly from 1 to 10, more particularly from 1 to 8, very particularly from 1 to 5, especially from 1 to 3, especially from 1 to 2, and very particularly q is 1.

[0062] To condense with one of the unprotected alpha amino groups of the compound of formula LSI-am or LS-II-am, the carboxylic acid group of the compound of formula T is typically activated with a condensing agent in step (e). The condensing agent in step (e) results in an activated compound of formula T containing a leaving group instead of a hydroxy group in the carboxylic acid group, making the activated compound of formula T more reactive toward the unprotected alpha amino group of the compound of formula LSI-am or LS-II-am. Depending on the specific condensing agent in step (e), the activated compound of formula T may or may not be an intermediate suitable for isolation at room temperature. When the compound of formula LSI-am or LS-II-am contains an unprotected carboxylic acid group, activation of the compound of formula T must occur before contacting it with the compound of formula LSI-am or LS-II-am. The condensing agent in step (e) is, for example, a carbodiimide. The carbodiimide is, for example, diisopropylcarbodiimide. Preferably, the carbodiimide is applied together with a coupling additive in step (e), such as cyano-hydroxyimino-acetic acid ethyl ester.

[0063] Process (e) (e) condensing the compound of formula LSI-am or LS-II-am with an alpha-amino acid derivative of formula T to obtain a compound of formula TLSI or TLS-II A method comprising the steps of:

[0064] Preferably, after the condensation in step (c), this method includes one or more additional condensation cycles to elongate the peptide Pr-LS obtained in step (c) with one or more additional alpha amino acid residues. The condensation cycle includes a first step in which the amino-protecting group of the alpha amino group of the N-terminal alpha amino acid residue of the peptide to be elongated is removed to obtain one unprotected amino group in the peptide to be elongated. The amino group of the N-terminal alpha amino acid residue also refers to an imino group, and in the case of proline, it is the N-terminal alpha amino acid residue of the peptide to be elongated. The condensation cycle also includes a second step in which one unprotected amino acid group from the first step is condensed with an alpha amino acid derivative having an alpha amino group protected by an amino-protecting group at its N-terminal amino acid residue and one unprotected alpha carboxylic acid group located at its C-terminal amino acid residue to obtain the peptide obtained from the condensation cycle. An example of the first step is step (d). An example of the second step is step (e).

[0065] Optionally, the condensation step is followed by a capping step, i.e., a step of treating the reaction mixture containing the resulting peptide of the condensation step with acetic anhydride. The capping step precedes the removal of the amino protecting group as part of the condensation cycle, e.g., the first step of removing the amino protecting group. Potential remaining amino or imino groups that did not react in the condensation step are thus acetylated and are not suitable for the next condensation step.

[0066] Preferred are processes comprising one or more further condensation cycles applied to compounds of formula TLSI or TLS-II, each of which comprises: an amino-protecting group R of the compound of formula TLSI or TLS-II, which is the amino-protecting group of the alpha amino group of the N-terminal amino acid residue of the peptide resulting from the previous condensation cycle; T-N-1 a first step in which each of the following is removed to give the relevant peptide with one unprotected amino group; a second step in which the unprotected amino group of the related peptide obtained in the first step is condensed with an alpha amino acid derivative having an alpha amino group protected by an amino-protecting group at its N-terminal amino acid residue and one unprotected alpha carboxylic acid group located at its C-terminal amino acid residue to obtain the peptide resulting from the condensation cycle; Includes.

[0067] If the alpha amino acid derivative S-am is covalently bound to the resin, the peptide Pr-LS obtained in step (c) is also covalently bound to the resin. This also applies to the extended peptide obtained in one or more further condensation cycles. The peptide obtained in the last condensation step can be cleaved from the resin with a cleavage composition to obtain the cleaved peptide. Whether the protecting groups of the extended peptide are retained, partially removed, or completely removed in the cleaved peptide depends, inter alia, on the cleavage composition, reaction conditions, and the specific protecting groups.

[0068] Preferably, the alpha amino acid derivative S-am is covalently attached to the resin in step (c) and the method further comprises step (x) (x) cleaving the peptide obtained from the last condensation step from the resin with a cleavage composition to obtain the cleaved peptide. Includes.

[0069] If the alpha amino acid derivative S-am is covalently attached to the resin in step (c) via the carbonyl group of its C-terminal alpha amino acid residue, the cleaved peptide will contain a carboxylic acid group at its C-terminal alpha amino acid residue, which has an unprotected alpha carboxylic acid group or an unprotected alpha carboxamide group.

[0070] A method is preferred in which the alpha amino acid derivative S-am is covalently attached to the resin in step (c), the method further comprising step (x) (x) cleaving the peptide obtained from the last condensation step from the resin with a cleavage composition to obtain the cleaved peptide. Includes.

[0071] Preferably, the method further comprises step (y) (y) removing any remaining protecting groups from the peptide resulting from the last condensation step to obtain a peptide free of protecting groups; Includes.

[0072] The removal of the protecting groups occurs with a deprotection composition. If all of the protecting groups of the peptide resulting from the last condensation step can be removed with the same deprotection composition, for example, if all of the protecting groups are labile to trifluoroacetic acid, then only one deprotection composition of step (y) is required for removal. If the peptide resulting from the last condensation step is covalently bound to a resin, step (y) and step (x) can occur simultaneously, and the cleavage composition of step (x) also serves as the deprotection composition of step (y) for all of the protecting groups of the peptide resulting from the last condensation step, for example, if all of the protecting groups are labile to trifluoroacetic acid and the covalent bond to the linking group of the resin is also labile to trifluoroacetic acid.

[0073] Process (y) (y) removing any remaining protecting groups from the peptide resulting from the last condensation step to obtain a peptide free of protecting groups; A method comprising the steps of:

[0074] An example of a peptide P without protecting groups is semaglutide (CAS number 910463-68-2), which is shown in the three letter code of the alpha amino acid residues (excluding the fused L-lysine residue with its fatty acid conjugation motif covalently bound to its epsilon nitrogen atom, shown as a structural element of formula L), using the numbering of the alpha amino acid residues starting with 1 for the N-terminal fused L-histidine residue and ending with 31 for the C-terminal fused glycine residue (corresponding to SEQ ID NO: 5): [ka]

[0075] The explanations and preferences given above regarding the method and steps for producing peptide P apply equally to further embodiments of the present invention.

[0076] A further embodiment of the present invention is a compound of formula Pr-L [ka] and During the ceremony, R L-O-1 and R L-O-2 are each independently a carboxylic acid protecting group, R L-N-1 is an amino protecting group of formula Bsmoc [ka] (In the formula, * indicates a bond to a nitrogen atom) (Bsmoc) is.

[0077] Preferred are compounds of formula Pr-L, where R L-O-1 and R L-O-2 are independently tert-butyl or 3-methyl-pent-3-yl.

[0078] Preferred are compounds of formula Pr-L, where R L-O-1 and R L-O-2 is tert-butyl.

[0079] The compound of formula Pr-L is preferred, which is compound 508. Compound 508 is shown in Example B-16-1.

[0080] A further embodiment of the present invention comprises: In the synthesis of peptide P Compound of formula Pr-L [ka] The use of During the ceremony, R L-O-1 and R L-O-2are each independently a carboxylic acid protecting group, R L-N-1 is an amino protecting group of formula Bsmoc [ka] (In the formula, * indicates a bond to a nitrogen atom) (Bsmoc) is.

[0081] A further embodiment of the present invention is a compound of formula Pr-L [ka] A method for producing During the ceremony, R L-O-1 and R L-O-2 are each independently a carboxylic acid protecting group, R L-N-1 is an amino protecting group of formula Bsmoc [ka] (In the formula, * indicates a bond to a nitrogen atom) (Bsmoc) is.

[0082] This method comprises the following steps (prl-b) and (prl-c): (prl-b) Peptide of formula Pr-A [ka] (In the formula, R A-N-1 is R L-N-1 is an amino protecting group different from R L-N-1 is as defined in formula Pr-L) The amino protecting group R in A-N-1 Remove the Compound of formula Pr-A-am [ka] (In the formula, R L-N-1 is as defined in formula Pr-L) obtaining a step of (prl-c) Compound of formula Pr-A-am with compound of formula B [ka] (In the formula, R L-O-1 and R L-O-2 is as defined in formula Pr-L) to obtain a compound of formula Pr-L Includes.

[0083] A method for preparing a compound of formula Pr-L is preferred, wherein in step (prl-c) a compound of formula Pr-A-am is persilylated to obtain a persilylated intermediate compound, a compound of formula B is activated with a condensing agent in step (prl-c) to obtain an activated compound of formula B, and the persilylated compound and the activated compound are combined and reacted with each other.

[0084] Preferably, the process for producing peptide P uses a compound of formula Pr-L obtained from a process for producing a compound of formula Pr-L.

[0085] A further embodiment of the present invention is a compound of formula Pr-A [ka] and During the ceremony, R A-N-1 is R L-N-1 , an amino-protecting group different from 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl and 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl; R L-N-1 is an amino protecting group of formula Bsmoc [ka] (In the formula,* indicates a bond to a nitrogen atom) (Bsmoc) is.

[0086] Preferably, R A-N-1 is tert-butyloxycarbonyl.

[0087] Preferably, in step (c) of the method for producing peptide P, a compound of formula Pr-L is used which is obtained by using a compound of formula Pr-A.

[0088] A further embodiment of the present invention is a compound of formula Pr-A [ka] (In the formula, R A-N-1 is R L-N-1 is an amino protecting group different from R L-N-1 is an amino protecting group of formula Bsmoc [ka] (In the formula, * indicates a bond to a nitrogen atom) (Bsmoc) is) This is a method for manufacturing the above.

[0089] This method comprises the following steps (pra-b) and (pra-c): (pra-b) Compound of formula Pr-Int-A [ka] (In the formula, R IntA-N-1 is R L-N-1 is an amino protecting group different from R L-N-1 is as defined in formula Pr-A) and an amino protecting group R IntA-N-1 Remove the Compound of formula Pr-Int-A-ep-am [ka] (In the formula, R L-N-1 is as defined in formula Pr-A) obtaining a step of (pra-c) A compound of formula Pr-Int-A-ep-am is treated with a compound of formula E [ka] (In the formula, R A-N-1 is as defined in formula Pr-A) to obtain a compound of formula Pr-A Includes.

[0090] Preferably, in step (c) of the method for producing peptide P, a compound of formula Pr-L is used which is obtained by using a compound of formula Pr-A which is obtained from a method for producing a compound of formula Pr-A.

[0091] Preferably, the method for producing peptide P uses, in step (c), a compound of formula Pr-L obtained from a method for producing a compound of formula Pr-L, and in step (prl-b), a compound of formula Pr-A obtained from a method for producing a compound of formula Pr-A.

[0092] A further embodiment of the invention is a compound of formula Pr-Int-A [ka] (In the formula, R IntA-N-1 is R L-N-1 is an amino protecting group different from R L-N-1 is an amino protecting group of formula Bsmoc [ka] (In the formula, * indicates a bond to a nitrogen atom) (Bsmoc) is) This is a method for manufacturing the above.

[0093] This method is (c-int) Compound of formula Int-A-am [ka] (In the formula, R IntA-N-1 is as defined in formula Pr-Int-A) with a compound of formula Pr-F [ka] (In the formula, R F-1 is p-nitrophenoxy, (4,6-dimethylpyrimidin-2-yl)-mercaptoyl or phthalimido-N-oxy) to obtain a compound of formula Pr-Int-A. Includes.

[0094] Preferably, in step (c) of the method for producing peptide P, a compound of formula Pr-L obtained by using a compound of formula Pr-A obtained from a method for producing a compound of formula Pr-A is used, and in step (pra-b) of the method for producing peptide P, a compound of formula Pr-Int-A is used.

[0095] Preferably, the method for producing peptide P uses, in step (c), a compound of formula Pr-L obtained from a method for producing a compound of formula Pr-L, in step (prl-b) a compound of formula Pr-A obtained from a method for producing a compound of formula Pr-A, and in step (pra-b) a compound of formula Pr-Int-A obtained from a method for producing a compound of formula Pr-Int-A. [Brief explanation of the drawings]

[0096] [Figure 1]Figure 1 shows the complete HPLC-UV chromatogram from D-02-1. One of the two peaks represents compound 701, the other is a degradation product. Both peaks are marked and their area percentages are shown. [Figure 2] An excerpt from approximately 14 to 20 minutes of the HPLC-UV chromatogram from D-02-1 in Figure 1 is shown. Two peaks, one representing compound 701, are marked and their area percentages are shown. [Figure 3] The complete HPLC-UV chromatogram from D-02-2 is shown. The peak representing compound 702 is marked and its area percentage is indicated. [Figure 4] An excerpt from approximately 10 to 22 minutes of the HPLC-UV chromatogram from D-02-2 in Figure 3 is shown. The peak representing compound 702 is marked and its area percentage is indicated. [Figure 5] Figure 1 shows the complete HPLC-UV chromatogram from D-02-3. The peak representing compound 702 is marked and its area percentage is indicated. [Figure 6] An excerpt from approximately 14 to 24 minutes of the HPLC-UV chromatogram from D-02-3 in Figure 5 is shown. The peak representing compound 702 is marked and its area percentage is indicated. [Figure 7] The complete HPLC-UV chromatogram from D-02-4 is shown. The peak representing compound 702 is marked and its area percentage is indicated. [Figure 8] An excerpt from approximately 11 to 22 minutes of the HPLC-UV chromatogram from D-02-4 in Figure 7 is shown. The peak representing compound 702 is marked and its area percentage is indicated. [Figure 9] The complete HPLC-UV chromatogram from D-02-5 is shown. The peak representing compound 702 is marked and its area percentage is indicated. [Figure 10] An excerpt from approximately 14 to 20 minutes of the HPLC-UV chromatogram from D-02-5 is shown in Figure 9. The peak representing compound 702 is marked and its area percentage is indicated. [Figure 11]The complete HPLC-UV chromatogram from D-02-6 is shown. The peak representing compound 702 is marked and its area percentage is indicated. [Figure 12] An excerpt from approximately 14 to 20 minutes of the HPLC-UV chromatogram from D-02-6 is shown in Figure 11. The peak representing compound 702 is marked and its area percentage is indicated. [Figure 13] The complete HPLC-UV chromatogram from D-04-2 is shown. The peak representing compound 704 is marked and its area percentage is indicated. [Figure 14] An excerpt from approximately 10 to 22 minutes of the HPLC-UV chromatogram from D-04-2 in Figure 13 is shown. The peak representing compound 704 is marked and its area percentage is indicated. [Figure 15] Figure 1 shows the complete HPLC-UV chromatogram from D-04-3. The peak representing compound 704 is marked and its area percentage is indicated. [Figure 16] An excerpt from approximately 14 to 24 minutes of the HPLC-UV chromatogram from D-04-3 in Figure 15 is shown. The peak representing compound 704 is marked and its area percentage is indicated. [Figure 17] Figure 1 shows the complete HPLC-UV chromatogram from D-04-4. The peak representing compound 704 is marked and its area percentage is indicated. [Figure 18] An excerpt from approximately 11 to 22 minutes of the HPLC-UV chromatogram from D-04-4 in Figure 17 is shown. The peak representing compound 704 is marked and its area percentage is indicated. [Figure 19] The complete HPLC-UV chromatogram from D-04-5 is shown. The peak representing compound 704 is marked and its area percentage is indicated. [Figure 20] An excerpt from approximately 14 to 20 minutes of the HPLC-UV chromatogram from D-04-5 in Figure 19 is shown. The peak representing compound 704 is marked and its area percentage is indicated. [Figure 21] The complete HPLC-UV chromatogram from D-04-6 is shown. The peak representing compound 704 is marked and its area percentage is indicated. [Figure 22] An excerpt from approximately 14 to 22 minutes of the HPLC-UV chromatogram from D-04-6 in Figure 21 is shown. The peak representing compound 704 is marked and its area percentage is indicated. [Figure 23] Figure 1 shows the complete HPLC-UV chromatogram from D-06-1. The peak representing compound 706 is marked and its area percentage is indicated. [Figure 24] An excerpt from approximately 9 to 14 minutes of the HPLC-UV chromatogram from D-06-1 in Figure 23 is shown. The peak representing compound 706 is marked and its area percentage is indicated. [Figure 25] Figure 1 shows the complete HPLC-UV chromatogram from D-07-1. The peak representing compound 707 is marked and its area percentage is indicated. [Figure 26] An excerpt from approximately 10 to 14 minutes of the HPLC-UV chromatogram from D-07-1 in Figure 25 is shown. The peak representing compound 707 is marked and its area percentage is indicated. [Figure 27] Figure 1 shows the complete HPLC-UV chromatogram from D-08-1. The peak representing compound 708 is marked and its area percentage is indicated. [Figure 28] An excerpt from approximately 10 to 22 minutes of the HPLC-UV chromatogram from D-08-1 in Figure 27 is shown. The peak representing compound 708 is marked and its area percentage is indicated. [Figure 29] Figure 1 shows the complete HPLC-UV chromatogram from D-09-1. The peak representing compound 709 is marked and its area percentage is indicated. [Figure 30] An excerpt from approximately 14 to 24 minutes of the HPLC-UV chromatogram from D-09-1 in Figure 29 is shown. The peak representing compound 709 is marked and its area percentage is indicated. [Figure 31] Figure 1 shows the complete HPLC-UV chromatogram from D-10-1. The peak representing compound 710 is marked and its area percentage is indicated. [Figure 32]An excerpt from approximately 11 to 22 minutes of the HPLC-UV chromatogram from D-10-1 in Figure 31 is shown. The peak representing compound 710 is marked and its area percentage is indicated. [Figure 33] Figure 1 shows the complete HPLC-UV chromatogram from D-11-1. The peak representing compound 711 is marked and its area percentage is indicated. [Figure 34] An excerpt from approximately 14 to 22 minutes of the HPLC-UV chromatogram from D-11-1 in Figure 33 is shown. The peak representing compound 711 is marked and its area percentage is indicated. DETAILED DESCRIPTION OF THE INVENTION

[0097] Sequence Listing Compound 701 KEFIAWLVRGRG (SEQ ID NO: 1) K at position 1 = 2-(p-nitrophenylsulfonyl)ethoxycarbonyl-lysine (17-carboxy-heptadecacarbonyl-gamma-Glu-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])

[0098] Compound 702 KEFIAWLVRGRG (SEQ ID NO: 2) K at position 1 = Lysine (17-carboxy-heptadecacarbonyl-gamma-Glu-2-[2-(2-aminoethoxy)-ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])

[0099] Compound 703 AKEFIAWLVRGRG (SEQ ID NO: 3) A at position 1 = 9-fluorenylmethyloxycarbonyl-alanine K at position 2 = Lysine (17-carboxy-heptadecacarbonyl-gamma-Glu-2-[2-(2-aminoethoxy)-ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])

[0100] Compound 704 AKEFIAWLVRGRG (SEQ ID NO: 4) K at position 2 = Lysine (17-carboxy-heptadecacarbonyl-gamma-Glu-2-[2-(2-aminoethoxy)-ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])

[0101] Compound 705 (Semaglutide) HXEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 5) 2nd place: X=Aib K at position 20 = Lysine (17-carboxy-heptadecacarbonyl-gamma-Glu-2-[2-(2-aminoethoxy)-ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])

[0102] compound 706 FIAWLVRGRG (SEQ ID NO: 6)

[0103] Compound 707 EFIAWLVRGRG (SEQ ID NO: 7)

[0104] Compound 708 KEFIAWLVRGRG (SEQ ID NO: 8) K at position 1 = alpha-azido-lysine (17-carboxy-heptadecacarbonyl-gamma-Glu-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])

[0105] Compound 709 KEFIAWLVRGRG (SEQ ID NO: 9) K=9-Fluorenylmethyloxycarbonyl-lysine (17-carboxy-heptadecacarbonyl-gamma-Glu-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl]) at position 1

[0106] compound 710 KEFIAWLVRGRG (SEQ ID NO: 10) K at position 1 = 2-(phenylsulfonyl)ethoxycarbonyl-lysine (17-carboxy-heptadecacarbonyl-gamma-Glu-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])

[0107] Compound 711 KEFIAWLVRGRG (SEQ ID NO: 11) K at position 1 = 1,1-dioxobenzo[b]thiophen-2-yl-methyloxycarbonyl-lysine (17-carboxy-hepta-decacarbonyl-gamma-Glu-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxy-acetyl]) [Example]

[0108] A) General A-1) Abbreviation % Unless otherwise stated, percentage values ​​refer to percentages by weight ACN Acetonitrile Aib 2-aminoisobutyric acid Bsmoc 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl (also known as benzo[b]thiophenesulfone-2-methyloxycarbonyl) Boc tert-butyloxycarbonyl DBU 1,8-diazabicyclo(5.4.0)undec-7-ene DEPBT 3-(diethoxyphosphoryloxy)-3H-benzo[d][1,2,3]triazin-4-one DIC Diisopropylcarbodiimide DIPEA Diisopropylethylamine DMF N,N-dimethylformamide EDT Ethane-1,2-dithiol Fmoc 9-Fluorenylmethyloxycarbonyl HPLC High Performance Liquid Chromatography IPA Isopropanol Mpe 3-methylpent-3-yl MTBE Methyl tert-butyl ether N3 Azide NMP N-methylpyrrolidone Nsc 2-(p-nitrophenylsulfonyl)ethoxycarbonyl OxymaPure™ Cyano-hydroxyimino-acetic acid ethyl ester Pbf 2,2,4,6,7-pentamethyl-2,3-dihydrobenzfuran-5-ylsulfonyl Psc 2-(phenylsulfonyl)ethoxycarbonyl Psi(Me,Me)pro 1,1-dimethylmethadiyl (pseudoproline) covalently bound to the beta oxygen atom of serine or threonine and covalently bound to the alpha nitrogen of serine or threonine RT room temperature TBTU (Benzotriazolyl)tetramethyluronium tetrafluoroborate tBu tert-butyl TFA trifluoroacetic acid TIS Triisopropylsilane Trt Trityl UV ultraviolet light vol.% Volume percentage

[0109] Unless otherwise specified, alpha amino acids other than glycine are in the L-form. Room temperature, as used herein, means a temperature between 22°C and 25°C.

[0110] A-2) Chemicals All reagents and solvents are obtained from standard suppliers of raw materials for peptide synthesis and are used as received.

[0111] A-3) Analysis method A-3-1: HPLC method 1 HPLC Method 1 is analytical HPLC performed on a Dionex™ Ultimate™ 3000 RS UHPLC system using an ACQUITY™ UPLC BEH 130 C18 (1.7 μm, 2.1 × 50 mm) column with a flow rate of 0.4 mL / min, UV or CAD detection at 220 nm, and an oven temperature of 25° C. Buffer A is 0.05 vol.% TFA in ACN / HO (1:99 v / v), and Buffer B is 0.05 vol.% TFA in ACN.

[0112] A-3-2: HPLC method 2 HPLC Method 2 is analytical HPLC performed on a Thermo Scientific Vanquish UHPLC system or a Dionex Ultimate 3000 RS UHPLC system using an ACQUITY UPLC BEH 130 C18 (1.7 μm, 2.1 x 150 mm) column at a flow rate of 0.4 mL / min, UV detection at 220 nm, and an oven temperature of 50 °C. For mass spectrometry, MS analysis is performed on a UHPLC coupled to a Bruker maXis II spectrometer using ultra-high-resolution QTOF technology with electron transfer dissociation (ETD) capability. Buffer A is 0.05 vol.% TFA in ACN / HO (1:99 v / v), and Buffer B is 0.05 vol.% TFA in ACN.

[0113] Gradient program for HPLC method 2 [Table 1]

[0114] A-4) General synthesis procedure A-4-1: General procedure 1 for condensing lysine derivatives 200 mg (0.22 mmol / g, 0.044 mmol) of compound 612-SP (Example C-06-1) was pre-swollen twice in DMF (10 mL per g of resin) for 15 min each at RT. Each lysine derivative (0.088 mmol, 2.0 equiv.) and OxymaPure (19.4 mg, 0.136 mmol, 3.1 equiv.) were dissolved in DMF (10 mL per g of resin). To this solution, 17.7 μL (0.114 mmol, 2.6 equiv.) of DIC was added. The solution was stirred at RT for 15 min and then added to the reaction mixture. After a 20-min reaction time at RT, an additional 8.8 μL (0.057–0.143 mmol, 1.3 equiv.) of DIC was added to the reaction mixture, which was then stirred at normal RT for 24 h. The liquid portion was removed, and the resin material was washed three times with DMF / IPA alternatingly and three times with IPA (10 mL per 1 g of resin for each washing step). The washed peptide material was dried under high vacuum at RT for 24 h to obtain the respective products in which the lysine derivative was condensed with compound 612-SP.

[0115] For analytical purposes, small scale test cleavages could be performed.

[0116] A-4-2: General Procedure for Test Cleavage 2 The peptide resin was treated with 0.5–1.0 mL of a cleavage cocktail (TFA / TIS / HO = 90 / 5 / 5 by volume) for 2 h at RT to cleave the peptide from the resin in a 2 mL plastic syringe (50–100 mg of peptide resin). The cleaved peptide in solution was then precipitated with 5–10 mL of cold diisopropyl ether (<-15 °C), centrifuged, and washed two more times with cold diisopropyl ether. The resulting crude peptide, which is usually a solid, was dried under high vacuum.

[0117] A-4-3: General Procedure 3 for Condensation of Fmoc-Ala-OH and Subsequent Removal of Fmoc The procedure is described using 200 mg of compound 614-SP as the starting material. The procedure can be adjusted for smaller or larger amounts of starting material.

[0118] 200 mg (0.21 mmol per gram of resin) of compound 614-SP shown in Example C-08-1 was pre-swollen twice in DMF (10 mL per gram of resin) for 15 minutes each at room temperature. If compound 614-SP is a resin material already contacted with DMF, pre-swelling can be omitted. 27.7 mg (0.084 mmol, 2.0 equiv.) of Fmoc-Ala-OH and 18.5 mg (0.130 mmol, 3.1 equiv.) of OxymaPure were dissolved in 2.0 mL of DMF. 16.9 μL (0.109 mmol, 2.6 equiv.) of DIC was added to this solution, and the solution was stirred at room temperature for 15 minutes before being added to each of the pre-swollen DMF-contacted compound 614-SP. After 20 min of reaction time at RT, an additional 8.5 μL (0.059 mmol, 1.3 equiv.) of DIC was added to the reaction mixture, which was then stirred at normal temperature for 17 h. The liquid portion was removed, and the resin material was washed three times with alternating DMF / IPA and three times with IPA (10 mL per g of resin per wash step) to give compound 615-SP, shown in Example C-09-1.

[0119] Compound 615-SP was treated with 20 vol.% piperidine in DMF (10 mL per gram of resin) twice, the first treatment for 20 minutes and the second treatment for 60 minutes. The resin material was then washed three times with alternating DMF / IPA and three times with IPA (10 mL per gram of resin for each wash). The washed resin material could be used in further reactions without drying or after drying. A portion of the washed resin material was dried under high vacuum at RT for 24 hours to obtain compound 616-SP.

[0120] For analytical purposes, a small scale test cleavage could be carried out to give compound 704 shown in Example D-04-1.

[0121] B) Synthesis of Lysine Derivatives and Some of Their Starting Materials Example B-01-1: Synthesis of Compound 101 [ka]

[0122] Compound 101 is By analogy to Molecule II in International Journal Peptide Research (1975), Vol. 7, pp. 295-305, For example, compound 102 (CAS number 21386-32-3) [ka] and compound 103 (CAS number 7693-46-1) [ka] It could be obtained by condensation reaction with

[0123] Example B-02-1: Synthesis of Compound 104 [ka]

[0124] Compound 104 is By analogy with the preparation of molecule 10 of US 3,791,830, For example, compound 105 (CAS number 102093-85-6) [ka] and compound 106 (CAS number 22325-27-5) [ka] It could be obtained by condensation reaction with

[0125] Example B-02-2: Synthesis of Compound 104 Compound 104 is By analogy with Example 5 of US3936452, For example, compound 102 (CAS number 21386-32-3) [ka] and compound 107 (CAS number 41840-26-0). [ka] It could be obtained by condensation reaction with

[0126] Example B-03-1: Synthesis of Compound 108 [ka]

[0127] Compound 108 could be obtained, for example, by condensation reaction of compound 105 (CAS No. 102093-85-6) with N-hydroxyphthalimide, in analogy to molecule IVb in International Journal Peptide Research (1975), Vol. 7, pp. 295-305.

[0128] Example B-04-1: Synthesis of Compound 109 in the form of hydrochloride [ka]

[0129] Compound 109 in the form of its hydrochloride salt (WO2002-098903A1, Table 1 / entry 19) can be prepared, for example, by Compound 110 (CAS number 160422-23-1) using HCl in dioxane [ka] It could be obtained by deprotection reaction of

[0130] Example B-05-1: Synthesis of Compound 110 [ka]

[0131] Compound 110 (CAS No. 160422-23-1) can be prepared, for example, as described in Example 3f of Tetrahedron Letters (1994), Vol. 35, No. 42, pp. 7821-7824, i.e. Using chlorotrimethylsilane as shown in Scheme 1 of Tetrahedron Letters (1994), Vol. 35, No. 42, pp. 7821-7824. Compound 111 (CAS number 2418-95-3) [ka] Compound 112 was obtained by the protection reaction of [ka] Obtained, Then, it could be obtained by reacting with compound 105 (CAS number 102093-85-6).

[0132] Example B-05-2: Synthesis of Compound 110 Compound 110 (CAS No. 160422-23-1) can be obtained, for example, by the protection reaction of compound 111 (CAS No. 2418-95-3) with compound 101, by analogy with page 302 / General Procedure B of International Journal Peptide Research (1975), Vol. 7, pp. 295-305.

[0133] Example B-05-3: Synthesis of Compound 110 Compound 110 (CAS No. 160422-23-1) can be obtained by protection reaction of compound 111 (CAS No. 2418-95-3) with compound 104, for example, by analogy with Example 26 of US Pat. No. 3,936,452.

[0134] Example B-05-4: Synthesis of Compound 110 Compound 110 (CAS No. 160422-23-1) can be obtained by the protection reaction of compound 111 (CAS No. 2418-95-3) with compound 108, for example, in analogy to the solid phase synthesis of Example 7 of WO2004-065412.

[0135] Example B-06-1: Synthesis of Compound 201 [ka]

[0136] Compound 201 is By analogy with Example 3c) of CN113121627A, For example, compound 202 (CAS number 1069067-08-8) [ka] It was obtained by condensation reaction of N-hydroxysuccinimide supported on dicyclohexylcarbodiimide with N-hydroxysuccinimide supported on dicyclohexylcarbodiimide.

[0137] Example B-07-1: Synthesis of Compound 301 [ka]

[0138] Compound 301 can be prepared by the protection reaction of compound 109 with, for example, chlorotrimethylsilane to give compound 113, in analogy to step 3b of EP 3819308 A1. [ka] Obtained, Thereafter, by analogy with step 3b of EP 3819308 A1, reaction with compound 201 This was achieved by:

[0139] Example B-08-1: Synthesis of Compound 302 in the form of hydrochloride [ka]

[0140] Compound 302 in the form of the hydrochloride salt could be obtained by deprotection reaction of compound 301 using, for example, HCl in dioxane.

[0141] Example B-09-1: Synthesis of Compound 303 in the form of hydrochloride [ka]

[0142] Compound 303 (CAS number 1662688-19-8) in the form of its hydrochloride salt can be used e.g. Using HCl in dioxane Compound 304 (CAS number 1662688-18-7) [ka] It could be obtained by deprotection reaction of

[0143] Example B-10-1: Synthesis of Compound 501 [ka]

[0144] Compound 501 (CAS No. 1662688-20-1) can be prepared, for example, as described in Example 9 of CN104356224A, i.e., compound 303 (CAS No. 1662688-19-8) [ka] and compound 203 (CAS number 1188328-22-4) [ka] It could be obtained by coupling reaction with

[0145] Example B-10-2: Synthesis of Compound 501 Compound 501 (CAS No. 1662688-20-1) can be obtained by a protection reaction of compound 303 (CAS No. 1662688-19-8) in the form of its hydrochloride salt with, for example, chlorotrimethylsilane to give compound 305. [ka] Obtained, Then, by analogy with step 3b of EP 3819308 A1, reacting with compound 203 (CAS number 1188328-22-4) This was achieved by:

[0146] Example B-10-3: Synthesis of Compound 501 Compound 501 (CAS number 1662688-20-1) can be obtained, for example, as described in Example 6 of WO2021-205388A2.

[0147] Example B-11-1: Synthesis of Compound 502 [ka]

[0148] Compound 502 (CAS No. 2682856-38-6) can be prepared, for example, by the synthesis of compound 306 (CAS No. 1118767-16-0) as described for molecule 10 in Scheme 3 in Organic Process Research and Development (2021), 25(7), pp. 1598-1611. [ka] and compound 114 (CAS number 21512-99-2) [ka] Compound 503 (CAS number 2682856-37-5) was obtained by coupling reaction with [ka] and then treating with tetrasodium ethylenediaminetetraacetate for dechelation. This was achieved by:

[0149] Example B-11-2: Synthesis of Compound 502 Compound 502 (CAS number 2682856-38-6) could be obtained by deprotection reaction of compound 501 (CAS number 1662688-20-1) using, for example, piperidine.

[0150] Example B-11-3: Synthesis of Compound 502 100 g (84 mmol) of compound 501 was dissolved in 600 mL of ACN at 40 °C, and the clear solution was cooled to RT. 33 mL of piperidine was added to this solution, and the resulting suspension was stirred for 30 minutes. Then, another 100 mL of ACN was added, and stirring was continued for 1 hour. The suspension was filtered through a suction filter, and the filter cake was washed five times with 200 mL of ACN and twice with 300 mL of ACN. The solid was dried overnight under vacuum at 35 °C. The obtained crude product (78 g) was dissolved in 600 mL of ACN and 85 mL of water at 50 °C. From the resulting solution, 200 mL was evaporated under reduced pressure using a rotary evaporator. To the formed emulsion, 400 mL of ACN was added, and the same amount was evaporated under reduced pressure. The addition / evaporation cycle was repeated two more times. The resulting suspension was then diluted with another 400 mL of ACN and filtered through a vacuum filter. The filter cake was washed three times with 300 mL of ACN and dried under vacuum at 35° C. for 15 hours to give 73.6 g (76 mmol, 90%) of compound 502 as a white solid.

[0151] HPLC Method 1 with CAD detection: 98.7 area %, retention time 6.19 min. Gradient program for HPLC method 1 [Table 2]

[0152] Example B-12-1: Synthesis of Compound 504 [ka]

[0153] Compound 504 can be obtained by the protection reaction of compound 502 (CAS No. 2682856-38-6) with compound 105 (CAS No. 102093-85-6), for example, by analogy with Example 1 of US Pat. No. 5,616,788.

[0154] Example B-12-2: Synthesis of Compound 504 Compound 504 can be prepared by a protection reaction of compound 502 (CAS No. 2682856-38-6) with, for example, chlorotrimethylsilane to give compound 505. [ka] Obtained, Then, by analogy with Example 3 of US Pat. No. 6,165,590, reacting with compound 105 (CAS No. 102093-85-6) This was achieved by:

[0155] Example B-12-3: Synthesis of Compound 504 Compound 504 is By analogy with the solid phase synthesis of Example 7 of WO2004-065412, For example, compound 115 (CAS number 122865-54-7) [ka] Compound 502 (CAS No. 2682856-38-6) could be obtained by the protection reaction using

[0156] Example B-12-4: Synthesis of Compound 504 Compound 504 can be obtained, for example, by the protection reaction of compound 502 (CAS number 2682856-38-6) with compound 101, by analogy with page 302 / General Procedure B of International Journal Peptide Research (1975), Vol. 7, pp. 295-305.

[0157] Example B-12-5: Synthesis of Compound 504 Compound 504 can be obtained by protection reaction of compound 502 (CAS number 2682856-38-6) with compound 104, for example, by analogy with Example 26 of US Pat. No. 3,936,452.

[0158] Example B-12-6: Synthesis of Compound 504 Compound 504 can be obtained by a protection reaction of compound 502 (CAS number 2682856-38-6) with compound 108, for example, in analogy to the solid phase synthesis of Example 7 of WO2004-065412.

[0159] Example B-12-7: Synthesis of Compound 504 Compound 504 can be prepared by a protection reaction of compound 302 in the form of its hydrochloride salt with, for example, chlorotrimethylsilane, to give compound 307, in analogy to step 3b of EP 3819308 A1. [ka] Obtained, Then, by analogy with step 3b of EP 3819308 A1, reacting with compound 203 (CAS number 1188328-22-4) This was achieved by:

[0160] Example B-12-8: Synthesis of Compound 504 19.5 g (20 mmol, 1.0 equiv.) of compound 502 was suspended in 60 mL of ACN, followed by the addition of 8.1 mL (50 mmol, 2.5 equiv.) of N-methyl-N-trimethylsilylacetamide. The mixture was heated to 40° C. to form a clear solution and then cooled to RT. 300 mL of DMF and 1.6 mL (20 mmol, 1.0 equiv.) of pyridine were added to the solution, followed by the addition of 8.5 g (20 mmol, 1.0 equiv.) of compound 101 over 30 minutes. After 5 hours, an additional 0.8 mL (10 mmol, 0.5 equiv.) of pyridine and 8.5 g (20 mmol, 1.0 equiv.) of compound 101 were added, and stirring was continued for 17 hours. 120 mL of 0.25 N aqueous hydrochloric acid was added to the mixture, and the solvent was evaporated under reduced pressure at 45° C. The resulting thin oil was dissolved in 250 mL of ethyl acetate and transferred to a separatory funnel. 50 mL of water was added, and the pH was adjusted to 1-2 with 5.5 N aqueous hydrochloric acid. The phases were separated, and the organic phase was washed three times with 50 mL of water and then concentrated under reduced pressure to yield a viscous oil. The purity of the viscous oil was 88.1 area % according to HPLC Method 1 and the gradient program described below. The desired extraction with aqueous sodium bicarbonate resulted in even poorer purity; the latter was omitted. It was hypothesized that the fundamental sensitivity of the Nsc protecting group in the present molecular structure was relatively high, which was already activated during contact with pyridine and enhanced during initial trial extractions with aqueous sodium bicarbonate. Purification of the viscous oil by a first preparative reverse-phase HPLC run (C4 column, starting from 3 vol.% ACN in 0.1% TFA-water, with a gradient to 100 vol.% ACN) resulted in partial decomposition during lyophilization of the collected and combined fractions. It was hypothesized that the TFA content was deleterious. The lyophilized material was purified by a second preparative HPLC without added TFA (C4 column, no TFA - starting from 3 vol.% ACN in water, gradient to 100 vol.% ACN). Lyophilization of the collected combined fractions gave 2.1 g (1.7 mmol, 9% yield) of compound 504.

[0161] HPLC Method 1 with CAD detection: 99.4 area %, retention time 5.14 min. Gradient program for HPLC method 1 [Table 3]

[0162] Example B-13-1: Synthesis of Compound 117 [ka]

[0163] Compound 117 is By analogy to Molecule II in International Journal Peptide Research (1975), Vol. 7, pp. 295-305, For example, compound 116 (CAS number 20611-21-6) [ka] and compound 103 (CAS number 7693-46-1) [ka] It can be obtained by a condensation reaction with

[0164] Example B-14-1: Synthesis of Compound 506 [ka]

[0165] Preparation of azide reagent 23.35 g (359 mmol, 10 equiv.) of sodium azide was dissolved in 82 mL of water and cooled in an ice bath. A solution of 12.1 mL (72 mmol, 2.0 equiv.) of trifluoromethanesulfonic anhydride in 135 mL of cyclohexane was added slowly over 35 minutes to the cooled solution, maintaining the internal temperature below 5°C. The reaction mixture was stirred for 2 hours while cooling in the ice bath and then transferred to a separatory funnel. The phases were separated. The aqueous phase was extracted twice with 120 mL of cyclohexane. The combined organic phase was used as the azide reagent in the subsequent azide formation.

[0166] Azide formation Compound 502 (35.0 g, 36 mmol, 1.0 equiv.) and potassium bicarbonate (8.99 g, 90 mmol, 2.5 equiv.) were suspended in water (65 mL) and MeOH (130 mL). The mixture was heated to 35 °C until the remaining solid dissolved. CuSO4·5H2O (90 mg, 0.4 mmol, 0.01 equiv.) was added at 20 °C, followed by the addition of the azide reagent solution over 30 min. The resulting emulsion was diluted with dioxane (56 mL) and stirred for 20 h. Cyclohexane (130 mL) and dioxane (50 mL) were added, the phases were separated in a separatory funnel, and the aqueous phase was extracted three times with diisopropyl ether (240 mL) and three times with MTBE (250 mL). To the aqueous phase, 5-methyltetrahydrofuran (400 mL) and 5% aqueous sodium bicarbonate (200 mL) were added. The phases were separated, and the organic phase was extracted repeatedly with 5% aqueous sodium bicarbonate, dilute aqueous hydrochloric acid, and water. The organic phase was evaporated under reduced pressure to a white oil, which was dissolved in 250 mL of 5-methyltetrahydrofuran and evaporated again under reduced pressure. This procedure was repeated four times to give a yellow viscous oil, which was dried under vacuum overnight. 29.5 g (29.5 mmol, 82% yield) of compound 506 was obtained as a yellow viscous oil.

[0167] HPLC Method 1 with CAD detection: 94.8 area %, retention time 7.40 min. Gradient program for HPLC method 1 [Table 4]

[0168] Example B-15-1: Synthesis of Compound 507 [ka]

[0169] In analogy with compound 508 in Example B-16-1, compound 507 was prepared from 19.5 g of compound 502, 9.6 mL (60 mmol, 3.0 equivalents) of N-methyl-N-trimethylsilylacetamide, and 8.3 g (20 mmol, 1.0 equivalents) of compound 117. 22.8 g (19.2 mmol, 96% yield) of compound 507 was obtained as a viscous oil.

[0170] HPLC Method 1 with CAD detection: 99.3 area %, retention time 7.11 min. Gradient program for HPLC method 1 [Table 5]

[0171] Example B-16-1: Synthesis of Compound 508 [ka]

[0172] 15.4 g (15.8 mmol, 1.0 equiv.) of compound 502 was suspended in 47 mL of ACN, followed by the addition of 6.3 mL (39.5 mmol, 2.5 equiv.) of N-methyl-N-trimethylsilylacetamide. The mixture was heated to 40° C. for 2 hours to form a clear solution, then cooled to RT. 107 mL of ACN and 2.5 mL (36.3 mmol, 2.3 equiv.) of pyridine were added to the solution, and the mixture was cooled to 0° C. While maintaining the internal temperature at 0° C., a solution of 4.71 g (18.2 mmol, 1.15 equiv.) of 1,1-dioxobenzo[b]thiophen-2-yl-methyloxycarbonyl chloride in 178 mL of ACN was added over 30 minutes. A solution of 0.6 g of 1,1-dioxobenzo[b]thiophen-2-yl-methyloxycarbonyl chloride (CAS No. 135204-19-2, 2.3 mmol, 0.15 equiv.) in 0.4 mL of additional pyridine (5 mmol, 0.3 equiv.) and 26 mL of ACN was then added, and stirring was continued at 0 °C for 15 h. The reaction mixture was then transferred to a separatory funnel, and 1400 mL of MTBE and 320 mL of water were added, followed by acidification with 1 N aqueous hydrochloric acid. The phases were separated, and the organic phase was washed three times with 350 mL of dilute aqueous hydrochloric acid and 320 mL of water. The organic phase was concentrated under reduced pressure to give the crude product as a viscous oil. The crude product was dissolved in a mixture of 170 mL of water, 170 mL of ACN, and 170 mL of diisopropyl ether at 35 °C and transferred to a separatory funnel. 31 mL of 10% aqueous sodium bicarbonate solution was added, and the phases were separated. The aqueous phase was washed with 340 mL of a mixture of diisopropyl ether and ACN, the phases were separated, and the aqueous phase was acidified by the addition of 5.5 N aqueous hydrochloric acid and then extracted with 1200 mL of MTBE. The phases were separated, and the organic phase was washed with 250 mL of dilute aqueous hydrochloric acid and 250 mL of water. The organic phase was evaporated under reduced pressure, and the resulting oil was dried under vacuum to give 10.6 g (8.8 mmol, 56% yield) of compound 508 as a very viscous oil.

[0173] HPLC Method 1 with CAD detection: 97.7 area %, retention time 7.16 min. Gradient program for HPLC method 1 [Table 6]

[0174] C) Synthesis of solid-phase conjugate compounds Example C-01-1: Synthesis of solid-phase conjugated compound 601-SP Fmoc-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotritylamidomethyl resin] (SEQ ID NO: 13) (601-SP)

[0175] Compound 601-SP can be, for example, compound 602-SP H-Gly-[2-chlorotritylamidomethyl resin] (602-SP) It was obtained by solid phase synthesis starting from

[0176] The coupling cycle of compound 602-SP with Fmoc-Arg(Pbf)-OH was followed by coupling cycles of Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, and Fmoc-Glu(OtBu)-OH.

[0177] Unless the N-terminal alpha-amino group was not already protected, the coupling cycle included a deprotection reaction of the N-terminal alpha-amino group of the solid-phase conjugated intermediate. The deprotection reaction for Fmoc was typically carried out using 20 vol.% piperidine in DMF or, if desired, 20 vol.% piperidine in NMP, typically for a time range of 0.5 to 4.0 hours. The coupling cycle also included a coupling reaction of the solid-phase conjugated intermediate with its unprotected N-terminal alpha-amino group, i.e., acylation of the unprotected N-terminal alpha-amino group with the activated alpha-carboxylic acid group of each amino acid derivative, activated with either DIC / OxymaPure, TBTU / DIPEA, or DEPBT / DIPEA. The typical time range for the acylation reaction was 1.5 to 24 hours. The coupling cycle also included one or more washes of the solid phase after the acylation reaction. DMF or IPA was used as the solvent for the one or more washes. The coupling cycle also optionally included an acetylation reaction using acetic anhydride after the acylation reaction. The coupling cycle also optionally included one or more washes after the acetylation reaction with acetic anhydride. DMF or IPA was used as the solvent for the optional one or more washes after the acetylation reaction with acetic anhydride.

[0178] Example C-02-1: Synthesis of solid-phase conjugated compound 603-SP [ka]

[0179] Compound 603-SP(=Nsc-Lys(tBuO-CO-(CH2) 16-CO-gamma-Glu(OtBu)-2-[2-(2-aminoethoxy)-ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxy]acetyl])-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotritylamidomethyl resin]) (SEQ ID NO: 26) can be obtained by, for example, applying a coupling cycle of compound 504 to compound 601-SP, i.e., by deprotection of compound 601-SP using 20 vol.% piperidine in DMF to compound 604-SP. [ka] Obtained, Then, react with compound 504 and DIC / OxymaPure. This was achieved by:

[0180] As used herein, the residue tBu-O-CO-(CH) 16 —CO-gamma-Glu-2-[2-(2-aminoethoxy)-ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl]) may also be referred to as O-tert-butyl-17-carboxy-heptadecacarbonyl-gamma-Glu-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl].

[0181] As used herein, the residue HO-CO-(CH) 16 —CO-gamma-Glu-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl]) may also be referred to as 17-carboxy-heptadecacarbonyl-gamma-Glu-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl].

[0182] Example C-03-1: Synthesis of solid-phase conjugated compound 605-SP [ka]

[0183] Compound 605-SP(=Fmoc-Ala-Lys(tBuO-CO-(CH2) 16 —CO-gamma-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotritylamidomethyl resin]) (SEQ ID NO: 15) can be obtained by, for example, applying Fmoc-Ala-OH to compound 603-SP in a coupling cycle, i.e., deprotection of compound 603-SP with 20 vol.% piperidine in DMF to compound 606-SP. [ka] Obtained, Then, react with Fmoc-Ala-OH and DIC / OxymaPure. This was achieved by:

[0184] Example C-04-1: Synthesis of solid-phase conjugate compound 607-SP Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Psi(Me,Me)pro)-Ser(tBu)-Asp(OMpe)- Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(tBuO-CO-(CH2) 16 -CO-gamma-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotritylamidomethyl resin] (SEQ ID NO: 16) (607-SP)

[0185] Compound 607-SP (=protected solid-phase conjugated derivative of semaglutide (CAS number 910463-68-2)) can be obtained by several coupling cycles, starting with a coupling cycle in which Fmoc-Ala-OH is applied to compound 605-SP, followed by deprotection of compound 605-SP with 20 vol.% piperidine in DMF to give compound 608-SP. [ka] Obtained, Then, react with Fmoc-Ala-OH and DIC / OxymaPure. This was achieved by:

[0186] The following sequences were used: Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OMpe)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Phe-Thr(Psi(Me,Me))pro)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, and Boc-His(l-Trt)-Aib-OH, and coupling cycles were continued within the sequence.

[0187] The coupling cycle was carried out as described in Example C-01-1.

[0188] Example C-05-1: Synthesis of solid-phase conjugated compound 611-SP [ka]

[0189] Compound 611-SP (=H-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-2-chlorotrityl resin) (SEQ ID NO: 17) was synthesized on a 30 mmol scale using an automated synthesizer (Sonata, Gyros Protein Technologies). Compound 610-SP (=H-Gly-2-chlorotrityl resin, 66.7 g, 0.45 mmol per gram of resin, 30 mmol), i.e. H-Gly-[2-chlorotrityl resin] (610-SP) A compound represented by The resin was pre-swollen in DMF for two 30-minute cycles (10 mL per g of resin). The first Fmoc-protected amino acid, i.e., Fmoc-Arg(Pbf)-OH, was pre-activated with TBTU / DIPEA (1.8 equiv / 3.0 equiv) for 3 minutes and added to the resin. The reaction mixture was stirred at room temperature for 2 hours. The following Fmoc-protected amino acid derivative (2.0 equiv) was pre-activated for 15 minutes using DIC / OxymaPure (2.6 equiv / 3.1 equiv, Fmoc-Arg(Pbf)-OH was pre-activated for 5 minutes) and added to the resin. After a 20-minute reaction time, additional DIC (1.3 equiv) was added to the reaction mixture (total reaction volume is 670 mL), and the coupling step was continued. The total reaction times for the coupling steps were 1.5 h for Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, and Fmoc-Ala-OH, 2 h for Fmoc-Arg(Pbf)-OH, and 5 h for Fmoc-Ile-OH and Fmoc-Phe-OH. At the end of each coupling step, the resin was drained and acetylated using a solution of 0.05 M acetic anhydride, 17 mM 2,4,6-collidine, and 1.6 mM OxymaPure in DMF (670 mL). This acetylation reaction acetylated any remaining unreacted amino groups, preventing their reaction (i.e., capping) in the following coupling step. The resin was then filtered, washed with DMF, and Fmoc deprotection was carried out using two or three 670 mL portions of 20 vol.% piperidine in DMF (5 min duration for the first portion and 10 min duration for the second portion prior to coupling of Fmoc-Trp(Boc)-OH and Fmoc-Ala-OH; 5 min duration for the first portion, 10 min duration for the second portion, and 15 min duration for the third portion prior to coupling of Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, and Fmoc-Phe-OH). After Fmoc deprotection, the resin was washed twice with 670 mL of DMF, 670 mL of IPA, and two 670 mL portions of DMF.After coupling of Fmoc-Phe-OH, acetylation, and Fmoc deprotection, the resin material was washed alternately with 670 mL of DMF, 670 mL of IPA three times, and 670 mL of IPA three times. The washed resin material was dried under vacuum at RT for 3 days to give 132 g of dry compound 611-SP.

[0190] For analytical purposes, a small-scale test cleavage from compound 611-SP could be performed, as described in Example D-06-1.

[0191] Example C-06-1: Synthesis of solid-phase conjugate compound 612-SP [ka]

[0192] Compound 611-SP (10.00 g, 2.2 mmol, 0.22 mmol / g) was pre-swollen twice in 100 mL of DMF (10 mL per g of resin) for 15 min each at RT. Fmoc-Glu(OtBu)-OH (1.95 g, 4.40 mmol, 2.0 equiv.) and OxymaPure (0.969 g, 6.82 mmol, 3.1 equiv.) were dissolved in 100 mL of DMF (10 mL per g of resin). To this solution, 886 μL of DIC (5.72 mmol, 2.6 equiv.) was added, and the solution was stirred at RT for 15 min. The solution was then added to the pre-swollen compound 611-SP. After a 20-minute reaction time at RT, an additional 443 μL of DIC (2.86 mmol, 1.3 equiv.) was added to the reaction mixture, which was then stirred at RT for 1.5 h. After removing the liquid portion from the reaction mixture by draining, an additional coupling step using Fmoc-Glu(OtBu)-OH was carried out (so-called recoupling). To this solution, 1.95 g (4.40 mmol, 2.0 equiv.) of Fmoc-Glu(OtBu)-OH and 0.969 g (6.82 mmol, 3.1 equiv.) of OxymaPure were dissolved in 100 mL of DMF (10 mL of DMF per 1 g of resin). To this solution, 886 μL (5.72 mmol, 2.6 equiv.) of DIC was added, and the solution was stirred at RT for 15 min, after which it was added to the reaction mixture. After a reaction time of 20 min at RT, an additional 443 μL (2.86 mmol, 1.3 equiv.) of DIC was added to the reaction mixture, which was then stirred at RT for 16 h. The liquid portion was removed, and the remaining resin material was washed with 20 mL of DMF. Then, for Fmoc deprotection, 20 vol.% piperidine in DMF (10 mL per gram of resin) was used three times: 5 minutes for the first treatment, 10 minutes for the second treatment, and 60 minutes for the third treatment. After this Fmoc deprotection step, the resin material was washed three times alternately with 670 mL of DMF and 670 mL of IPA, followed by a final wash with 100 mL of IPA three times and then 670 mL of DMF twice. The washed resin material was dried under vacuum at room temperature for 3 days to yield 1.0 g of compound 612-SP1.

[0193] For analytical purposes, a small-scale test cleavage was performed, which is described in Example D-07-1.

[0194] Example C-07-1: Synthesis of solid-phase conjugate compound 613-SP [ka]

[0195] 2.00 g (0.88 mmol, 0.44 mmol / g) of compound 611-SP was pre-swollen twice with 20 mL of DMF (10 mL per g of resin) for 15 min each at RT. 0.781 g (1.76 mmol, 2.0 equiv.) of Fmoc-Glu(OtBu)-OH and 0.388 g (2.728 mmol, 3.1 equiv.) of OxymaPure were dissolved in 20 mL of DMF (10 mL per g of resin). To this solution, 354 μL (2.288 mmol, 2.6 equiv.) of DIC was added, and the solution was stirred at RT for 15 min. After this, the DIC was added to the pre-swollen compound 611-SP. After a 20-minute reaction time at RT, an additional 177 μL (1.144 mmol, 1.3 equiv.) of DIC was added to the reaction mixture and stirred at RT for 5 hours. After draining the liquid portion of the reaction mixture, an additional coupling step with Fmoc-Glu(OtBu)-OH was performed (recombination). To this solution, 0.781 g (1.76 mmol, 2.0 equiv.) of Fmoc-Glu(OtBu)-OH and 0.388 g (2.728 mmol, 3.1 equiv.) of OxymaPure were dissolved in 20 mL of DMF (10 mL per 1 g of resin). To this solution, 354 μL (2.288 mmol, 2.6 equiv.) of DIC was added. The solution was stirred at RT for 15 minutes and then added to the reaction mixture. After a 20-minute reaction time at room temperature, 177 μL (1.144 mmol, 1.3 equiv.) of DIC was added to the reaction mixture and stirred at room temperature for 15 hours. The liquid portion was removed, and the resin material was washed with 20 mL of DMF and treated three times with 20 vol.% piperidine in DMF (10 mL per g of resin): 5 minutes for the first treatment, 10 minutes for the second treatment, and 60 minutes for the third treatment. Then, 1.76 g (1.760 mmol, 2.0 equiv.) of compound 506 and 0.388 g (2.728 mmol, 3.1 equiv.) of OxymaPure were dissolved in 20 mL of DMF (10 mL per g of resin). 354 μL (2.288 mmol, 2.6 equiv.) of DIC was added to the solution. The solution was stirred at room temperature for 15 minutes, and then added to the reaction mixture. After a reaction time of 20 min at RT, an additional 177 μL of DIC (177 μL, 1.144 mmol, 1.3 equiv) was added to the reaction mixture and stirred at RT for 22 h.The liquid portion was removed, and the resin material was washed three times with DMF / IPA alternatingly and three times with IPA (10 mL per 1 g of resin for each washing step). The washed resin material was dried under high vacuum at RT for 24 h to give compound 613-SP (=N-Lys(tBuO-CO-(CH)). 16 2.22 g of -CO-gamma-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotrityl resin]) (SEQ ID NO: 18) was obtained.

[0196] For analytical purposes, a small-scale test cleavage was performed, which is described in Example D-08-1.

[0197] Example C-08-1: Synthesis of solid-phase conjugate compound 614-SP [ka]

[0198] 100 mg of compound 613-SP (0.21 mmol / g, 0.022 mmol) was pre-swollen twice in DMF (10 mL per g of resin) for 15 min each at RT. 31.5 mg of tris(2-carboxyethyl)phosphine (TCEP, 0.11 mmol, 5.0 equiv.) was dissolved in 250 μL of 10 vol.% aqueous DMF (2.5 mL per g of resin), and 18.8 μL of DIPEA (0.11 mmol, 5.0 equiv.) was added. This solution was then added to the pre-swollen compound 613-SP. The reaction mixture was then stirred at RT for 3 h and then at 50 °C for 16 h. The liquid portion was removed, and the resin material was washed three times with 10 vol.% aqueous DMF, three times with alternating DMF / IPA, and three times with IPA (10 mL per g of resin in each wash step). A portion of the resin material was further treated without drying as in Example C-09-1. A portion of the resin material was dried under high vacuum at RT for 24 hours to give compound 614-SP (=H-Lys(tBuO-CO-(CH) 16-CO-gamma-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxyacetyl])-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotrityl resin]) (SEQ ID NO: 19) was obtained.

[0199] For analytical purposes, a small-scale test cleavage was performed, which is described in Example D-02-2.

[0200] Example C-08-2: Synthesis of solid-phase conjugate compound 614-SP Compound 617-SP (100 mg, 0.022 mmol, 0.21 mmol per g of resin) was pre-swollen twice in DMF (10 mL per g of resin) for 10 min at RT. The pre-swollen compound 617-SP was treated twice with 20 vol.% piperidine in DMF (10 mL per g of resin), for 20 min in the first treatment and 60 min in the second treatment. The resin material was then washed three times with alternating DMF / IPA and three times with IPA (10 mL per g of resin in each wash). The washed resin material was dried under high vacuum at RT for 24 h to give compound 614-SP.

[0201] For analytical purposes, a small-scale test cleavage was performed, which is described in Example D-02-3.

[0202] Example C-08-3: Synthesis of solid-phase conjugate compound 614-SP 200 mg of compound 618-SP (0.21 mmol, 0.042 mmol per g of resin) was pre-swollen twice in DMF (10 mL per g of resin) for 10 min at RT. The pre-swollen resin material was treated twice with 2 vol.% DBU in 20 vol.% piperidine in DMF solution (10 mL per g of resin), for 20 min in the first treatment and 60 min in the second treatment. The resin material was then washed three times with alternating DMF / IPA and three times with IPA (10 mL per g of resin for each wash step). A portion of the washed peptide material was further processed without drying according to Example C-09-3. A portion of the washed peptide material was dried under high vacuum at RT for 24 h to obtain compound 614-SP.

[0203] For analytical purposes, a small-scale test cleavage was performed, which is described in Example D-02-4.

[0204] Example C-08-4: Synthesis of solid-phase conjugate compound 614-SP 200 mg of compound 619-SP (0.21 mmol, 0.042 mmol per g of resin) was pre-swollen twice in DMF (10 mL per g of resin) for 10 min at RT. The pre-swollen resin material was treated twice with 2 vol.% DBU in 20 vol.% piperidine in DMF solution (10 mL per g of resin), for 20 min in the first treatment and 60 min in the second treatment. The resin material was then washed three times with alternating DMF / IPA and three times with IPA (10 mL per g of resin for each wash step). A portion of the washed peptide material was further processed without drying according to Example C-09-4. A portion of the washed peptide material was dried under high vacuum at RT for 24 h to obtain compound 614-SP.

[0205] For analytical purposes, a small-scale test cleavage was performed, which is described in Example D-02-5.

[0206] Example C-08-5: Synthesis of solid-phase conjugate compound 614-SP 200 mg of compound 620-SP (0.21 mmol, 0.042 mmol per g of resin) was pre-swollen twice in DMF (10 mL per g of resin) for 10 min at RT. The pre-swollen resin material was treated twice with 20 vol.% piperidine in DMF solution (10 mL per g of resin), for 20 min in the first treatment and 60 min in the second treatment. The resin material was then washed three times with alternating DMF / IPA and three times with IPA (10 mL per g of resin for each wash step). A portion of the washed peptide material was further processed without drying according to Example C-09-5. A portion of the washed peptide material was dried under high vacuum at RT for 24 h to obtain compound 614-SP.

[0207] For analytical purposes, a small-scale test cleavage was performed, which is described in Example D-02-6.

[0208] Example C-09-1: Synthesis of solid-phase conjugate compound 616-SP [ka]

[0209] Compound 616-SP(=H-Ala-Lys(tBuO-CO-(CH2) 16 -CO-gamma-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotrityl resin]) (SEQ ID NO: 27) was obtained from compound 614-SP obtained in Example C-08-1 according to General Procedure 3 to give Fmoc-protected intermediate compound 615-SP (=Fmoc-Ala-Lys(tBuO-CO-(CH 16-CO-gamma-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotrityl resin]) (SEQ ID NO: 20). [ka]

[0210] For analytical purposes, a small-scale test cleavage was performed, which is described in Example D-04-2.

[0211] Example C-09-2: Synthesis of solid-phase conjugate compound 616-SP Compound 616-SP was synthesized from 100 mg (0.022 mmol, 0.22 mmol per 1 g of resin) of compound 614-SP obtained in Example C-08-2 according to General Procedure 3 via the Fmoc-protected intermediate compound 615-SP.

[0212] For analytical purposes, a small-scale test cleavage was performed, which is described in Example D-04-3.

[0213] Example C-09-3: Synthesis of solid-phase conjugated compound 616-SP Compound 616-SP was synthesized from 100 mg (0.022 mmol, 0.22 mmol per 1 g of resin) of compound 614-SP obtained in Example C-08-3 according to General Procedure 3 via the Fmoc-protected intermediate compound 615-SP.

[0214] For analytical purposes, a small-scale test cleavage was performed, which is described in Example D-04-4.

[0215] Example C-09-4: Synthesis of solid-phase conjugated compound 616-SP Compound 616-SP was synthesized from 100 mg (0.022 mmol, 0.22 mmol per 1 g of resin) of compound 614-SP obtained in Example C-08-4 according to General Procedure 3 via the Fmoc-protected intermediate compound 615-SP.

[0216] For analytical purposes, a small-scale test cleavage was performed, which is described in Example D-04-5.

[0217] Example C-09-5: Synthesis of solid-phase conjugated compound 616-SP Compound 616-SP was synthesized from 100 mg (0.022 mmol, 0.22 mmol per 1 g of resin) of compound 614-SP obtained in Example C-08-5 according to General Procedure 3 via the Fmoc-protected intermediate compound 615-SP.

[0218] For analytical purposes, a small-scale test cleavage was performed, which is described in Example D-04-6.

[0219] Example C-10-1: Synthesis of solid-phase conjugate compound 617-SP [ka]

[0220] 200 mg (0.044 mmol, 0.22 mmol per g of resin) of compound 612-SP and 105 mg (0.088 mmol, 2.0 equivalents) of compound 501 were reacted according to General Procedure 1 to give compound 617-SP (=Fmoc-Lys(tBuO-CO-(CH) 16 -CO-gamma-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotrityl resin]) (SEQ ID NO: 22) was obtained.

[0221] For analytical purposes, a small-scale test cleavage was performed, which is described in Example D-09-1.

[0222] Example C-11-1: Synthesis of solid-phase conjugate compound 618-SP [ka]

[0223] 200 mg (0.044 mmol, 0.22 mmol per g of resin) of compound 612-SP and 105 mg (0.088 mmol, 2.0 equivalents) of compound 507 were reacted according to General Procedure 1 to give compound 618-SP (=Psc-Lys(tBuO-CO-(CH) 16 -CO-gamma-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotrityl resin]) (SEQ ID NO: 24) was obtained.

[0224] For analytical purposes, a small-scale test cleavage was performed, which is described in Example D-10-1.

[0225] Example C-12-1: Synthesis of solid-phase conjugated compound 619-SP [ka]

[0226] 200 mg (0.044 mmol, 0.22 mmol per g of resin) of compound 612-SP and 105 mg (0.088 mmol, 2.0 equivalents) of compound 504 were reacted according to General Procedure 1 to give compound 619-SP (=Nsc-Lys(tBuO-CO-(CH) 16-CO-gamma-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotrityl resin]) (SEQ ID NO: 25) was obtained.

[0227] For analytical purposes, a small-scale test cleavage was performed, which is described in Example D-01-2.

[0228] Example C-13-1: Synthesis of solid-phase conjugate compound 620-SP [ka]

[0229] 200 mg (0.044 mmol, 0.22 mmol per g of resin) of compound 612-SP and 105 mg (0.088 mmol, 2.0 equivalents) of compound 508 were reacted according to General Procedure 1 to give compound 620-SP (=Bsmoc-Lys(tBuO-CO-(CH) 16 -CO-gamma-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotrityl resin]) (SEQ ID NO: 23) was obtained.

[0230] For analytical purposes, a small-scale test cleavage was performed, which is described in Example D-11-1.

[0231] D) Cleavage of solid-phase conjugates Example D-01-1: Synthesis of Compound 701 [ka]

[0232] Compound 701(=Nsc-Lys(HO-CO-(CH2) 16 —CO-gamma-Glu-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl]-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH) (SEQ ID NO: 1) could be obtained by deprotection reaction of compound 603-SP using, for example, a cleavage composition of TFA / EDT / HO / TIS (90 / 5 / 2.5 / 2.5 in vol.% based on the volume of the cleavage composition).

[0233] Example D-01-2: Synthesis of Compound 701 A small sample of dried compound 619-SP from Example C-12-1 was cleaved according to General Procedure 2 to give a solid containing compound 701 (SEQ ID NO: 1).

[0234] HPLC Method 2: 45.7 area% + 27.8 area%, retention time 18.42 min + 19.39 min / One of the peaks was presumed to be compound 701 and the other was presumed to be a degradation product. It was not known whether the degradation occurred during the TFA-based cleavage, the TFA-containing analytical HPLC Method 2, or both. The TFA sensitivity of Nsc-protected compound 504 was also observed in Example B-12-8.

[0235] Figure 1 shows the complete HPLC-UV chromatogram, and Figure 2 shows an excerpt from the complete HPLC-UV chromatogram.

[0236] Example D-02-1: Synthesis of Compound 702 [ka]

[0237] Compound 702(=H-Lys(HO-CO-(CH2) 16—CO-gamma-Glu-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl]-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH) (SEQ ID NO: 2) could be obtained by deprotection reaction of compound 606-SP using, for example, a cleavage composition of TFA / EDT / HO / TIS (90 / 5 / 2.5 / 2.5 in vol.% based on the volume of the cleavage composition).

[0238] Example D-02-2: Synthesis of Compound 702 A small sample of dried compound 614-SP from Example C-08-1 was cleaved according to General Procedure 2 to give a solid containing compound 702 (SEQ ID NO:2). HPLC method 2: 80.9 area%, retention time 16.65 minutes.

[0239] Figure 3 shows the complete HPLC-UV chromatogram, and Figure 4 shows an excerpt from the complete HPLC-UV chromatogram.

[0240] Example D-02-3: Synthesis of Compound 702 A small sample of dried compound 614-SP from Example C-08-2 was cleaved according to General Procedure 2 to give a solid containing compound 702 (SEQ ID NO:2). HPLC method 2: 76.2 area%, retention time 16.65 minutes.

[0241] Figure 5 shows the complete HPLC-UV chromatogram, and Figure 6 shows an excerpt from the complete HPLC-UV chromatogram.

[0242] Example D-02-4: Synthesis of Compound 702 A small sample of dried compound 614-SP from Example C-08-3 was cleaved according to General Procedure 2 to give a solid containing compound 702 (SEQ ID NO:2). HPLC method 2: 76.4 area%, retention time 16.66 minutes.

[0243] Figure 7 shows the complete HPLC-UV chromatogram, and Figure 8 shows an excerpt from the complete HPLC-UV chromatogram.

[0244] Example D-02-5: Synthesis of Compound 702 A small sample of dried compound 614-SP from Example C-08-4 was cleaved according to General Procedure 2 to give a solid containing compound 702 (SEQ ID NO:2). HPLC method 2: 81.4 area%, retention time 16.66 minutes.

[0245] Figure 9 shows the full HPLC-UV chromatogram, and Figure 10 shows an excerpt from the full HPLC-UV chromatogram.

[0246] Example D-02-6: Synthesis of Compound 702 A small sample of dried compound 614-SP from Example C-08-5 was cleaved according to General Procedure 2 to give a solid containing compound 702 (SEQ ID NO:2). HPLC method 2: 81.1 area%, retention time 16.66 minutes.

[0247] Figure 11 shows the complete HPLC-UV chromatogram, and Figure 12 shows an excerpt from the complete HPLC-UV chromatogram.

[0248] Example D-03-1: Synthesis of Compound 703 [ka]

[0249] Compound 703(=Fmoc-Ala-Lys(HO-CO-(CH2) 16—CO-gamma-Glu-2-[2-(2-aminoethoxy)-ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH) (SEQ ID NO: 3) could be obtained by deprotection reaction of compound 605-SP using, for example, a cleavage composition of TFA / EDT / HO / TIS (90 / 5 / 2.5 / 2.5 in vol.% based on the volume of the cleavage composition).

[0250] Example D-04-1: Synthesis of Compound 704 [ka]

[0251] Compound 704(=H-Ala-Lys(HO-CO-(CH2) 16 —CO-gamma-Glu-2-[2-(2-aminoethoxy)-ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH) (SEQ ID NO: 4) could be obtained by deprotection reaction of compound 608-SP using, for example, a cleavage composition of TFA / EDT / HO / TIS (90 / 5 / 2.5 / 2.5 in vol.% based on the volume of the cleavage composition).

[0252] Example D-04-2: Synthesis of Compound 704 A small sample of dried compound 616-SP from Example C-09-1 was cleaved according to general procedure 2 to give compound 704 (SEQ ID NO: 4). HPLC method 2: 73.6 area%.

[0253] Figure 13 shows the complete HPLC-UV chromatogram, and Figure 14 shows an excerpt from the complete HPLC-UV chromatogram. High-resolution mass spectrometry: From calculations [C 105 H 175 N 24 O 28 ] 3+m / z 740.0997, measured value 740.1031.

[0254] Example D-04-3: Synthesis of Compound 704 A small sample of dried compound 616-SP from Example C-09-2 was cleaved according to General Procedure 2 to give compound 704 (SEQ ID NO: 4). HPLC method 2: 74.6 area%, retention time 16.76 minutes.

[0255] Figure 15 shows the complete HPLC-UV chromatogram, and Figure 16 shows an excerpt from the complete HPLC-UV chromatogram. High-resolution mass spectrometry: From calculations [C 105 H 175 N 24 O 28 ] 3+ m / z 740.0997, measured value 740.1021.

[0256] Example D-04-4: Synthesis of Compound 704 A small sample of dried compound 616-SP from Example C-09-3 was cleaved according to general procedure 2 to give compound 704 (SEQ ID NO: 4). HPLC method 2: 74.9 area %, retention time 16.75 minutes.

[0257] Figure 17 shows the full HPLC-UV chromatogram, and Figure 18 shows an excerpt from the full HPLC-UV chromatogram.

[0258] Example D-04-5: Synthesis of Compound 704 A small sample of dried compound 616-SP from Example C-09-4 was cleaved according to General Procedure 2 to give compound 704 (SEQ ID NO: 4). HPLC method 2: 80.7 area%, retention time 16.75 minutes.

[0259] Figure 19 shows the full HPLC-UV chromatogram, and Figure 20 shows an excerpt from the full HPLC-UV chromatogram.

[0260] Example D-04-6: Synthesis of Compound 704 A small sample of dried compound 616-SP from Example C-09-5 was cleaved according to General Procedure 2 to give compound 704 (SEQ ID NO: 4). HPLC method 2: 78.2 area%, retention time 16.76 minutes.

[0261] Figure 21 shows the full HPLC-UV chromatogram, and Figure 22 shows an excerpt from the full HPLC-UV chromatogram.

[0262] Example D-05-1: Synthesis of Compound 705 H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(HO-CO-(CH2) 16 -CO-gamma-Glu-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (SEQ ID NO: 5) (705)

[0263] Compound 705(=H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(HO-CO-(CH2) 16 -CO-gamma-Glu-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (SEQ ID NO: 5), respectively semaglutide (CAS number 910463-68-2)) (SEQ ID NO: 5) could be obtained by deprotection reaction of compound 607-SP using, for example, a cleavage composition of TFA / EDT / HO / TIS (90 / 5 / 2.5 / 2.5 in vol.% based on the volume of the cleavage composition).

[0264] Example D-06-1: Synthesis of Compound 706 [ka]

[0265] A small sample of dried compound 611-SP from Example C-05-1 was cleaved according to general procedure 2 to give compound 706 (SEQ ID NO: 6). HPLC method 2: 88.4 area%, retention time 11.18 minutes.

[0266] Figure 23 shows the full HPLC-UV chromatogram, and Figure 24 shows an excerpt from the full HPLC-UV chromatogram.

[0267] Example D-07-1: Synthesis of Compound 707 [ka]

[0268] A small sample of dried compound 612-SP from Example C-06-1 was cleaved according to general procedure 2 to give compound 707 (SEQ ID NO: 7). HPLC method 2: 84.0 area%, retention time 11.65 minutes.

[0269] Figure 25 shows the full HPLC-UV chromatogram, and Figure 26 shows an excerpt from the full HPLC-UV chromatogram.

[0270] Example D-08-1: Synthesis of Compound 708 [ka]

[0271] A small sample of dried compound 613-SP from Example C-07-1 was cleaved according to general procedure 2 to give compound 708 (SEQ ID NO: 8). HPLC method 2: 81.6 area%, retention time 21.64 minutes.

[0272] Figure 27 shows the full HPLC-UV chromatogram, and Figure 28 shows an excerpt from the full HPLC-UV chromatogram.

[0273] Example D-09-1: Synthesis of Compound 709 [ka]

[0274] A small sample of dried compound 617-SP from Example C-10-1 was cleaved according to general procedure 2 to give compound 709 (SEQ ID NO: 9). HPLC method 2: 80.2 area%, retention time 21.72 minutes.

[0275] Figure 29 shows the full HPLC-UV chromatogram, and Figure 30 shows an excerpt from the full HPLC-UV chromatogram.

[0276] Example D-10-1: Synthesis of Compound 710 [ka]

[0277] A small sample of dried compound 618-SP from Example C-11-1 was cleaved according to general procedure 2 to give compound 710 (SEQ ID NO: 10). HPLC method 2: 76.4 area%, retention time 19.26 minutes.

[0278] Figure 31 shows the full HPLC-UV chromatogram, and Figure 32 shows an excerpt from the full HPLC-UV chromatogram.

[0279] Example D-11-1: Synthesis of Compound 711 [ka]

[0280] A small sample of dried compound 620-SP from Example C-13-1 was cleaved according to general procedure 2 to give compound 711 (SEQ ID NO: 11). HPLC method 2: 81.0 area%, retention time 19.45 minutes.

[0281] Figure 33 shows the full HPLC-UV chromatogram, and Figure 34 shows an excerpt from the full HPLC-UV chromatogram.

[0282] E) comparison The results obtained when small samples were cleaved from the solid phase conjugate in section D) are correlated in Table E-1 with the reactions carried out with the solid phase conjugate in section C).

[0283] [Table 7]

[0284] footnote: a) Comparison b) The present invention c) To quantify the effectiveness of the solid-phase reaction, cleavage of the solid-phase conjugation reaction product from the resin was carried out and the area fraction of the resulting compound in the HPLC-UV chromatogram was determined. d) Different amino protecting groups have different UV absorption properties at 220 nm and require some caution in interpretation, hence the parenthetical placement. e) Only one of the observed peaks at 45.7 area % or 27.8 area % is compound 701 and the other is a degradation product f) For each amino protecting group, optimized deprotection conditions are used: phosphine reduction for N3, 20 vol.% piperidine in DMF for Fmoc and Bsmoc, 2 vol.% DBU and 20 vol.% piperidine in DMF for Psc and Nsc. g) Due to the different deprotection conditions in step (d), it is not possible to formally compare the combined effect of the solid-phase reaction in step (c) and step (d) directly - however, the average effect of cycle I is a confirmation of the condensation effect in step (c) on the one hand, and the removal effect of the amino protecting group in step (d) on the other hand. h) Decomposition of compound 702, unable to calculate average i) The three dots represent the step of removing the Fmoc group in cycle II. i) Condensation with Fmoc-Ala-OH and removal of Fmoc using 20 vol.% piperidine in DMF k) Note that the starting resin materials are not similar, i.e., the synthesis in the previous cycle I was carried out using compounds 501, 504, 506, 507 or 508, and the deprotection conditions are partially different for each amino protecting group involved. l) Average across Cycle I and Cycle II based on the area % of compound 702 cleaved and fully deprotected and the area % of compound 704 cleaved and fully deprotected

[0285] From the results in Table E-1, - depending on the amino protecting group of the lysine derivative used as one of the reactants in the condensation step (c) in the solid-phase cycle I, the effectiveness of the cycle I, as indicated by the area fraction of the cleaved and fully deprotected compound 702, and the effectiveness of the subsequent solid-phase cycle II, as indicated by the area fraction of the cleaved and deprotected compound 704, are affected, as indicated by the area fraction of the peptide cleaved from the solid phase; -Based on area ratio, Nsc showed the greatest efficacy, closely followed by Bsmoc, while N3 was next, followed by Psc and Fmoc; - taking into account that the reasonably estimable influence of the steric hindrance of the amino protecting groups in lysine derivatives is not conclusive, i.e., N3 is the least hindered, followed by Psc / Nsc with two methylene units after the oxy-carbonyl unit, then Fmoc / Bsmoc with one methylene unit after the oxy-carbonyl unit; - The high sensitivity of compound 701 with Nsc as the amino protecting group to TFA, thus confirming the high sensitivity of Nsc as the amino protecting group in compound 504 to exposure to TFA (and to aqueous sodium bicarbonate), as shown in Example B-12-8. is shown.

Claims

1. 1. A method for producing peptide P, comprising: (c) an alpha amino acid derivative S-am having one unprotected alpha amino group or one unprotected alpha imino group, Compound of formula Pr-L 【Chemical 1】 (In the formula, R Ｌ－Ｏ－１ and R Ｌ－Ｏ－２ are each independently a carboxylic acid protecting group. to obtain peptide Pr-L-S. Including, R Ｌ－Ｎ－１ is an amino protecting group of formula Bsmoc 【Chemistry 2】 (In the formula, ＊ indicates a bond to a nitrogen atom) (Bsmoc) That's the method.

2. The alpha amino acid derivative S-am has the formula SI-am or S-II-am 【Chemistry 3】 is a compound of During the ceremony, R ＳＩ－１ Is - (AA ｎｘ ) ｍ -O-[Resin-1], -(AA ｎｘ ) ｍ -N-[Resin-2], -O-[Resin-1], -N-[Resin-2], -(AA ｎｘ ) ｍ -OR ＳＩ－１－１ , OR ＳＩ－１－１ or NH ２ and R ＳＩ－２ is H, C １－６ Alkyl or OR ＳＩ－２－１ , S.R. ＳＩ－２－２ , S.C.H. ３ , N.R. ＳＩ－２－３ R ＳＩ－２－４ , CO-OR ＳＩ－２－５ , CO-NR ＳＩ－２－６ R ＳＩ－２－７ , N'-R ＳＩ－２－８ -N"-R ＳＩ－２－９ -guanidino, phenyl, para-(R ＳＩ－２－１０ O)-phenyl, 1-R ＳＩ－２－１１ -imidazol-4-yl or 1-R ＳＩ－２－１２ -indol-3-yl monosubstituted C １－６ is alkyl, - (AA ｎｘ ) ｍ - denotes m condensed alpha amino acid residues AA ｎｘ wherein each x is an integer and x varies from 1 to m; Each condensed alpha amino acid residue AA ｎｘ are independently selected, and when a side chain bearing a functional group is present, said functional group is unprotected or protected by a protecting group, provided that interfering functional groups are protected; m is an integer from 1 to 30, R ＳＩ－１－１ is H or a carboxylic acid protecting group, R ＳＩ－２－１ is H or a hydroxy protecting group, R ＳＩ－２－２ is a thiol protecting group, R ＳＩ－２－３ and R ＳＩ－２－４ are H, an amino protecting group, or when neither is H, together form an amino protecting group; R ＳＩ－２－５ is H or a carboxylic acid protecting group, R ＳＩ－２－６ and R ＳＩ－２－７ is H or an amide protecting group, R ＳＩ－２－８ and R ＳＩ－２－９ is H or, if both are not H, a guanidino protecting group; R ＳＩ－２－１０ is a protecting group for an aromatic hydroxy group, R ＳＩ－２－１１ is H or a protecting group for the imidazole nitrogen atom, R ＳＩ－２－１２ is H or a protecting group for the indole nitrogen atom, R ＳＩＩ－１ is R ＳＩ－１ as defined in The peptide Pr-L-S has the formula Pr-L-S-I or Pr-L-S-II 【Chemistry 4】 is a compound of During the ceremony, R Ｌ－Ｏ－１ , R Ｌ－Ｏ－２ and R Ｌ－Ｎ－１ is as defined in formula Pr-L, R ＳＩ－１ , R ＳＩ－２ and R ＳＩＩ－１ is as defined in formula SI-am or S-II-am, The method of claim 1.

3. Process (d) (d) removing the amino protecting group R Ｌ－Ｎ－１ to remove the compound of formula L-SI-am or L-S-II-am 【Chemistry 5】 A step of obtaining a compound of Including, During the ceremony, R Ｌ－Ｏ－１ , R Ｌ－Ｏ－２ , R ＳＩ－１ , R ＳＩ－２ and R ＳＩＩ－２ is as defined in formula Pr-LS-I or Pr-LS-II.

4. Process (e) (e) reacting said compound of formula L-SI-am or LS-II-am with an alpha-amino acid derivative of formula T R Ｔ－Ｎ－１ -(AA ｐｙ ) ｑ -OH (T) (In the formula, R Ｔ－Ｎ－１ is an amino protecting group, - (AA ｐｙ ) ｑ - denotes q condensed alpha amino acid residues AA ｐｙ wherein each y is an integer and y varies from 1 to q; Each condensed alpha amino acid residue AA ｐｙ are independently selected, and when a side chain bearing a functional group is present, said functional group is unprotected or protected by a protecting group, provided that interfering functional groups are protected; q is an integer from 1 to 30. to form a compound of formula TLS-I or TLS-II 【Chemistry 6】 (In the formula, R Ｌ－Ｏ－１ , R Ｌ－Ｏ－２ , R ＳＩ－１ , R ＳＩ－２ and R ＳＩＩ－２ is as defined in formula L-SI-am or LS-II-am, R Ｔ－Ｎ－１ and -(AA ｐｙ ) ｑ - is as defined in formula T) The process of obtaining The method of claim 3, comprising:

5. and one or more further condensation cycles applied to said compound of formula TLS-I or TLS-II, said one or more further condensation cycles each comprising: the amino-protecting group R of the compound of formula TLS-I or TLS-II, which is the amino-protecting group of the alpha amino group of the N-terminal amino acid residue of the peptide resulting from the previous condensation cycle. Ｔ－Ｎ－１ to give the relevant peptide with one unprotected amino group; a second step of condensing the unprotected amino group of the related peptide obtained in the first step with an alpha amino acid derivative having an alpha amino group protected by an amino-protecting group at its N-terminal amino acid residue and one unprotected alpha carboxylic acid group located at its C-terminal amino acid residue to obtain a peptide resulting from the condensation cycle; The method of claim 4, comprising:

6. The alpha amino acid derivative S-am is covalently bound to a resin, and step (x) (x) cleaving the peptide obtained from the last condensation step from the resin with a cleavage composition to obtain the cleaved peptide. The method according to any one of claims 1 to 5, comprising:

7. Said alpha amino acid derivative S-am of step (c) is a derivative of formula SI-am, where: R ＳＩ－１ Is - (AA ｎｘ ) １０ -O-[resin-1], A.A. ｎ１ is Phe, and AA ｎ２ is Ile and AA ｎ３ is Ala, and AA ｎ４ is Trp with a protected side chain, and AA ｎ５ is Leu, and AA ｎ６ is Val and AA ｎ７ is Arg with a protected side chain, and AA ｎ８ is Gly, and AA ｎ９ is Arg with a protected side chain, and AA ｎ１０ is Gly, Resin-1 is a 2-chlorotritylamidomethyl resin or a 2-chlorotrityl resin; R ＳＩ－２ is 2-(tert-butyloxycarbonyl)ethyl; The method according to any one of claims 2 to 6.

8. Process (y) (y) removing any remaining protecting groups from the peptide resulting from the last condensation step to obtain a peptide free of protecting groups. The method according to any one of claims 1 to 7, comprising:

9. R Ｌ－Ｏ－１ and R Ｌ－Ｏ－２ The method according to any one of claims 1 to 8, wherein each of is independently tert-butyl or 3-methyl-pent-3-yl.

10. The method of any one of claims 1 to 9, wherein in step (c) said compound of formula Pr-L is activated by a condensing agent of step (c).

11. The method of any one of claims 1 to 10, wherein in step (c), a coupling additive of step (c) is present.

12. 12. The method of claim 10 or 11, wherein the condensing agent in step (c) is a carbodiimide derivative and the coupling additive in step (c) is cyano-hydroxyimino-acetic acid ethyl ester.

13. 13. The method of any one of claims 1 to 12, wherein step (c) is carried out in a solvent for step (c), and the solvent for step (c) comprises N,N-dimethylformamide.

14. Compound of formula Pr-L 【Chemistry 7】 (In the formula, R Ｌ－Ｏ－１ and R Ｌ－Ｏ－２ are each independently a carboxylic acid protecting group, R Ｌ－Ｎ－１ is an amino protecting group of formula Bsmoc 【Chemistry 8】 (In the formula, ＊ indicates a bond to a nitrogen atom) (Bsmoc) (It is).

15. Use of a compound of formula Pr-L as defined in claim 1 in the synthesis of a peptide P.

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