Lysine salts and methods of making lysine derivatives

Abstract

The present invention relates to a Bsmoc protected lysine salt (1 X ) of formula 1 X , for example the hydrochloride salt (n = 1, HY = hydrochloric acid). The present invention also relates to a method for preparing a Bsmoc protected lysine derivative, which method employs a Bsmoc protected lysine salt of formula 1 X and comprises the step of reacting the epsilon-amino group of lysine with an activated carboxylic acid derivative. Some of the obtained Bsmoc protected lysine derivatives with side chain modifications are used as starting materials in solid phase peptide synthesis.

Description

Technical Field

[0001] This invention relates to a Bsmoc-protected lysine salt, wherein the α-nitrogen atom is protected by a 1,1-dioxobenzo[b]thiophene-2-ylmethoxycarbonyl group (Bsmoc). The invention also relates to a method for preparing Bsmoc-protected lysine derivatives, using the Bsmoc-protected lysine salt as a starting material and reacting its ε-nitrogen atom with an activated carboxylic acid derivative to form an amide group. Furthermore, the invention relates to the application of Bsmoc-protected lysine salts in the synthesis of peptide compounds. Background Technology

[0002] Smegglutide (CAS No.: 910463-68-2) is an active pharmaceutical ingredient, known as a glucagon-like peptide-1 receptor agonist. When represented by a three-letter peptide code, the molecular formula of smegglutide is as follows: 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-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu-Phe-Ile-Ala-Trp 25 -Leu-Val-Arg-Gly-Arg 30 -Gly-OH (SEQ ID NO:5).

[0003] Therefore, smegglutinin has a linear 31-peptide backbone, and in its lysine... 20 (lysine 20 The ε-nitrogen atom of lysine has a fatty acid side chain. 20 The ε-nitrogen atom is HO-CO-(CH2) 16 It is substituted by -CO-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl]-2-[2-(2-aminoethoxy)ethoxy]acetyl.

[0004] WO 2006-097537 A2 discloses the peptide drug semaglutide in Example 4. The method for synthesizing semaglutide involves adding lysine to a pre-formed linear peptide backbone. 20The ε-nitrogen atom undergoes an acylation reaction. Therefore, after the complete linear peptide backbone of smegraglutide is synthesized, the lysine atom then undergoes an acylation reaction. 20 Introduce fatty acid side chains.

[0005] CN 104356224 A discloses a method for synthesizing smegraglutide, which uses an Fmoc-protected lysine derivative as a building block, the derivative already containing fatty acid side chains. Therefore, during the synthesis of the linear peptide backbone of smegraglutide, lysine with pre-contained fatty acid side chains is introduced. 20 The Fmoc-protected lysine derivative used (CAS No.: 1662688-20-1) is shown below: .

[0006] CN 113461801 A discloses a solid-phase peptide synthesis method for the above-mentioned Fmoc-protected lysine derivative (CAS No.: 1662688-20-1) in Example 1. This method involves solid-phase coupling with an Alloc-protected lysine derivative (CAS No.: 2721349-46-6), the structure of which is shown below: .

[0007] Example 4 of CN 115677827 A discloses a method for synthesizing Boc-lysine (tert-butoxycarbonyl-(CH2)). 16 -Carboxyl-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-alanine-allyl ester (CAS No.: 2921565-74-2) is synthesized via a Boc-protected lysine derivative (CAS No.: 2921565-73-1), the structure of which is shown below: .

[0008] WO 96-25394 discloses 2-(4-nitrobenzenesulfonyl)ethoxycarbonyl (i.e., the Nsc group) as an amino protecting group in solid-phase peptide chemistry, and on page 18 it states that the Nsc group is completely resistant to the action of acidic reagents commonly used to cleave tert-butyl-type protecting groups.

[0009] A paper published in *Protein and Peptide Letters* (1997), Volume 4, Issue 5, pp. 307-312, compared the use of Fmoc and Nsc groups in automated solid-phase peptide synthesis. The study concluded that the high-performance liquid chromatography-ultraviolet (HPLC-UV) spectra obtained after comparative synthesis of three test peptides were extremely similar. On page 312, the paper noted that in analytical HPLC using a C18 column and a water / acetonitrile gradient containing 0.1% trifluoroacetic acid, the retention time of Nsc-phenylalanine-OH was 16 minutes, while that of Fmoc-phenylalanine-OH was 24 minutes. Given that only one retention time was observed, Nsc-phenylalanine-OH did not appear to have undergone decomposition.

[0010] A method for preparing Nsc-Lys(Boc)-OH from a trimethylsilyl derivative is disclosed on page 7822 of *Tetrahedron Letters* (1994), Vol. 35, No. 42, pp. 7821-7824. The yield of the homogeneous product after recrystallization is reported to be 83%. Extraction with a 5% aqueous sodium hydroxide solution is performed in the post-treatment process.

[0011] Organic Letters (2001), Vol. 3, No. 5, pp. 781-783, discloses a method for solid-phase peptide synthesis by protecting amino acids with α-azido groups, and in Table 2, it is noted that α-azido amino acids outperformed control Fmoc-amino acids in several tested peptide sequences.

[0012] Journal American Chemical Soc I The journal *Etty* (1997), Vol. 119, pp. 9915-9916, discloses a Bsmoc group, an abbreviation for 1,1-dioxobenzo[b]thiophene-2-methylmethoxycarbonyl, as an amino protecting group in peptide synthesis.

[0013] "Bulletin of Korean Chemical Soc I The journal "Etty" (1998), Volume 19, Issue 6, pp. 696-698, discloses the use of 2-(benzenesulfonyl)ethoxycarbonyl (i.e., the Psc group) in combination with the Boc group to orthogonally protect the ε-amino group of lysine, i.e., Boc-Lys(Psc)-OH, and its application in liquid-phase peptide synthesis.

[0014] RU 2196144 C1 discloses a peptide Psc-D-Phe-Cys(Bzm)-Phe-D-Trp(For)-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol(Psc)2 (SEQ ID NO:12) in its Example 11, wherein Psc represents 2-(benzenesulfonyl)-ethoxycarbonyl.

[0015] EP 3819308 A1 discloses a method for preparing protected amino acids with side-chain substitutions. In step 1f of its Example 1, Fmoc-Lys-OH is silanized with N-methyl-N-trimethylsilylacetamide, followed by reaction with activated tBuOOC-(CH2). 16 The reaction -CO-Glu(AEEA-AEEA)-OtBu yields Fmoc-Lys([tBuOOC-(CH2)]) 16 -CO-γ-Glu-OtBu)-AEEA-AEEA]-OH. In step 3b of Example 3, Fmoc-Lys-OH was also silanized with N-methyl-N-trimethylsilylacetamide, and then reacted with Pal-Glu(Osu)-OtBu to obtain Fmoc-Lys(Pal-Glu-OtBu)-OH.

[0016] International patent application PCT / EP2023 / 077884 discloses a method for preparing peptide P, which includes the steps of: condensing an α-amino acid derivative S-am with a protected α-amino acid derivative of formula Pr-L, wherein S-am has an unprotected α-amino group or an unprotected α-imino group. (Pr-L), Among them, amino protecting group R L-N-1 It is 1,1-dioxobenzo[b]thiophene-2-ylmethoxycarbonyl (Bsmoc). Furthermore, a method for preparing the α-amino acid derivative shown in formula Pr-L is also disclosed.

[0017] Generally, increasing the yield of condensation reactions is desirable, especially when the condensation reaction is part of a multi-step reaction system for synthesizing large molecules (e.g., molecules with molecular weights exceeding 1000 g / mol, which is common for (poly)peptides). This is because removing relatively large molecular weight byproducts generated due to inefficient condensation reactions from the target molecule is often very difficult. This is usually because these byproducts are generally similar in physical properties to the target molecule, thus requiring purification by preparative high-performance liquid chromatography (HPLC) for (poly)peptides. Therefore, the initial purity of the starting material containing the target macromolecule is crucial, directly affecting the efficiency of the condensation reaction. Furthermore, it is advantageous if the starting material used has sufficient, at least temporarily, resistance to degradation when exposed to alkalis or acids (e.g., in the field of peptide synthesis, when exposed to trifluoroacetic acid). "At least temporarily" means sufficient stability under common conditions (e.g., room temperature, exposure time up to one hour, and the presence of water). On the one hand, this makes the starting material generally readily available for synthesis, for example, in aqueous post-processing. In this context, the initial purity of the starting materials is equally important, thus avoiding the use of preparative liquid chromatography for purification. On the other hand, for (poly)peptides, the ability to analyze and monitor the starting materials using typical acidic analytical reversed-phase high-performance liquid chromatography (RP-HPLC) facilitates a deeper understanding and control of the condensation reaction. Another desirable aspect for (poly)peptides is that the condensation reaction allows for a base-catalyzed deprotection mode that retains the amino protecting group, which can then be used for further condensation reactions. Therefore, new active amino or imine groups can be generated for further condensation reactions to form amide bonds without disrupting the resin's acid-sensitive linking groups or the acid-sensitive protecting groups on the pre-existing condensed amino acid side chains in the peptide. Furthermore, it is sometimes desirable that the condensation reaction does not introduce heavy metals during the subsequent amino protecting group deprotection reaction.

[0018] To ensure good synthetic accessibility of the starting materials used, another important aspect is that the synthetic precursors of the starting materials should possess sufficient, at least temporarily, resistance to degradation. "At least temporarily" means that the resistance to degradation of the synthetic precursor is appropriate to its position in the synthetic pathway leading to the final used starting material. If the synthetic precursor is stored separately before the chemical reaction begins, or is post-processed to a separately storable form after the chemical reaction ends, then the synthetic precursor has a "temporary storage position." Conversely, if the synthetic precursor is not separated from the reaction mixture, then it has a "transient position" in the synthetic pathway leading to the final used starting material. Synthetic precursors with a "temporary storage position" preferably possess storage stability, allowing them to be stored separately at room temperature for extended periods. This allows for transport without strict temperature control and allows for storage within the production facility where the chemical reaction is carried out. Another advantage is that synthetic precursors with a "temporary storage position" also exhibit stability when dissolved in a solvent or suspended in a suspension medium. For example, this allows for the preparation of large quantities of synthetic precursor solutions with a "temporary storage position." The larger batches of solution can be stored in tanks and used in batches as needed for multiple different chemical reactions, which can be carried out at different times during production activities. Precursors with a "temporary storage location" are preferably used directly in the chemical reactions along the synthetic pathway without prior chemical conversion, or even if conversion is required, it can be performed simply before the desired chemical reaction.

[0019] Lysine derivatives whose α-nitrogen atom is protected by the Fmoc group and do not carry other base-sensitive protecting groups can typically achieve the exchange of protecting groups on the α-nitrogen atom through base-induced Fmoc deprotection and subsequent introduction of different protecting groups. However, it is preferable to adopt a synthetic route that directly uses the desired protecting group to protect the lysine α-nitrogen atom from the outset, thereby avoiding the cycle of deprotection and reprotection. Summary of the Invention

[0020] Surprisingly, it has been found that the stability of Bsmoc-Lys-OH is significantly lower than that of Fmoc-Lys-OH. This conclusion applies in particular to the isolated solid form of Bsmoc-Lys-OH, as well as to Bsmoc-Lys-OH dissolved in solution.

[0021] A formula 1 has now been discovered. X Compounds: (1 X ) in: n is 1 or 0.5, When n is 1, HY is hydrochloric acid, trifluoroacetic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, propanesulfonic acid, butyric acid, hydrobromic acid, hydroiodic acid, perchloric acid, trifluoromethanesulfonic acid, nitric acid, picric acid, trichloroacetic acid, camphorsulfonic acid, dichloroacetic acid, difluoroacetic acid, or phosphoric acid. When n is 0.5, HY is sulfuric acid.

[0022] Toluenesulfonic acid, for example, is p-toluenesulfonic acid (CAS No.: 104-15-4), m-toluenesulfonic acid (CAS No.: 617-97-0), o-toluenesulfonic acid (CAS No.: 88-20-0), or a mixture thereof. Preferably, the toluenesulfonic acid is p-toluenesulfonic acid. Propanesulfonic acid, for example, is 1-propanesulfonic acid (CAS No.: 5284-66-2), 2-propanesulfonic acid (CAS No.: 14159-48-9), or a mixture thereof. Preferably, the propanesulfonic acid is 1-propanesulfonic acid. Butyryl acid, for example, is 1-butyryl acid (CAS No.: 2386-47-2), 2-butyryl acid (CAS No.: 16794-12-0), 2-methyl-2-propanesulfonic acid (CAS No.: 16794-13-1), or a mixture thereof. Preferably, the butyryl acid is 1-butyryl acid. Camphor sulfonic acid is, for example, (-)-camphor sulfonic acid (also known as (1R,4S)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-methanesulfonic acid, CAS No.: 35963-20-3), (+)-camphor sulfonic acid (also known as (1S,4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-methanesulfonic acid, CAS No.: 3144-16-9) or mixtures thereof (also known as 7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-methanesulfonic acid, (+ / -)-camphor sulfonic acid, CAS No.: 5872-08-2). Preferably, the camphor sulfonic acid is (+)-camphor sulfonic acid.

[0023] Hydrochloric acid, trifluoroacetic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, propanesulfonic acid, butyric acid, hydrobromic acid, hydroiodic acid, perchloric acid, trifluoromethanesulfonic acid, nitric acid, picric acid, trichloroacetic acid, camphorsulfonic acid, dichloroacetic acid, difluoroacetic acid, phosphoric acid, and sulfuric acid all share a common pK value. a The values ​​are all below 2.3. For phosphoric acid, this refers to its first pK value. a The value is 2.15, while the first pK of sulfuric acid is... a Value and second pK a The values ​​are all below 2.3. HY has this low pK a The advantage of this value is that its associated anion Y- is quite inert in chemical reactions in which carboxylic acid derivatives react with molecules having unprotected amino or unprotected imine groups to form amide groups.

[0024] Preferably, Formula 1 X The compounds, wherein: When n is 1, HY is hydrochloric acid, trifluoroacetic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, propanesulfonic acid, butyric acid, hydrobromic acid, hydroiodic acid, perchloric acid, trifluoromethanesulfonic acid, nitric acid, picric acid, trichloroacetic acid, camphorsulfonic acid, dichloroacetic acid, or difluoroacetic acid. When n is 0.5, HY is sulfuric acid.

[0025] If the boiling point of HY is relatively low, in preparation formula 1 X When adding an excess of HY to a compound, it can be easily removed by evaporation. Preferably, the boiling point of HY at 101325 Pa is below 200 °C. Preferably, HY is hydrochloric acid (-85 °C), trifluoroacetic acid (72.4 °C), hydrobromic acid (-67 °C), hydroiodic acid (-35 °C), nitric acid (83 °C), trichloroacetic acid (198 °C), dichloroacetic acid (194 °C), or difluoroacetic acid (133 °C). More preferably, the boiling point of HY at 101325 Pa is below 140 °C. More preferably, HY is hydrochloric acid (-85 °C), trifluoroacetic acid (72.4 °C), hydrobromic acid (-67 °C), hydroiodic acid (-35 °C), nitric acid (83 °C), or difluoroacetic acid (133 °C). Very preferably, the boiling point of HY at 101325 Pa is below 90 °C. Very preferably, HY is hydrochloric acid (-85 °C), trifluoroacetic acid (72.4 °C), hydrobromic acid (-67 °C), hydroiodic acid (-35 °C) or nitric acid.

[0026] Preferred Formula 1 X The compounds, wherein: When n is 1, HY is hydrochloric acid, trifluoroacetic acid, p-toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, 2-propanesulfonic acid, 1-butanesulfonic acid, hydrobromic acid, hydroiodic acid, perchloric acid, trifluoromethanesulfonic acid, or nitric acid. When n is 0.5, HY is sulfuric acid.

[0027] Preferred Formula 1 X The compounds in which When n is 1, HY is hydrochloric acid, trifluoroacetic acid, p-toluenesulfonic acid, or methanesulfonic acid. When n is 0.5, HY is sulfuric acid.

[0028] Preferably, Formula 1 X The compound has been stored for at least 12 hours. More preferably, Formula 1 X The compound has been stored for a period of 12 hours to 1 month, very preferably 18 hours to 3 weeks, particularly 24 hours to 2 weeks, even more particularly 36 hours to 7 days, and very particularly 48 hours to 120 hours.

[0029] Preferred Formula 1 X The compound, wherein the compound has been stored for at least 12 hours. Preferably, Formula 1 X The compound has been stored at temperatures from 4 °C to 40 °C, more preferably from 7 °C to 38 °C, very preferably from 9 °C to 36 °C, particularly from 14 °C to 34 °C, even more particularly from 17 °C to 32 °C, very particularly from 19 °C to 30 °C, and especially from 21 °C to 28 °C.

[0030] Preferably, Formula 1 X The compound is in a solid state. More preferably, Formula 1 X The compound is in a solid state at temperatures ranging from 4 °C to 40 °C, very preferably in a solid state at temperatures ranging from 21 °C to 28 °C, and very preferably in a solid state at 23 °C. Here, "solid state" means that the compound contains formula 1. X In the composition of the compound, the compound of formula 1 X The compound constitutes 50 to 100% by weight. Therefore, for example, Formula 1 X The compound is 10% by weight of Formula 1. X The frozen solution of the compound is not considered a solid in this case, as per Formula 1. X Compounds.

[0031] Preferred Formula 1 X The compound is in the solid state.

[0032] Preferably, Formula 1 X The compound is in a dissolved or suspended state. More preferably, Formula 1 X The compound is dissolved or suspended in the solvent composition Solv I In -1, the composition comprises N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, N-octyl-2-pyrrolidone, methyl 5-(dimethylamino)-2-methyl-5-oxovalerate, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, nitromethane, acetonitrile, benzonitrile, acetone, methyl ethyl ketone, γ-butyrolactone, γ-valerolactone, triethyl phosphate, methanol, ethanol, propanol, butanol, diethyl ether, methyl tert-butyl ether, diisopropyl ether, benzene, toluene, xylene, chlorobenzene, pentane, hexane, heptane, octane, cyclohexane, water, or mixtures thereof.

[0033] Propyl acetate is n-propyl acetate, isopropyl acetate, or a mixture thereof, preferably isopropyl acetate. Butyl acetate is n-butyl acetate, sec-butyl acetate, isobutyl acetate, tert-butyl acetate, or a mixture thereof, preferably sec-butyl acetate. Propanol is n-propanol, isopropanol, or a mixture thereof, preferably isopropanol. Butanol is n-butanol, sec-butanol, isobutanol, tert-butanol, or a mixture thereof, preferably tert-butanol. Xylene is o-xylene, m-xylene, p-xylene, or a mixture thereof, preferably m-xylene. Hexane is n-hexane, 2-methylpentane, 3-methylpentane, 2,3-dimethylbutane, 2,2-dimethylbutane, or a mixture thereof, preferably n-hexane. Heptane is n-heptane, 2-methylhexane, 3-methylhexane, 2,2-dimethylpentane, 2,3-dimethylpentane, 2,4-dimethylpentane, 3,3-dimethylpentane, 3-ethylpentane, 2,2,3-trimethylbutane, or a mixture thereof, preferably n-heptane. Octane is, for example, n-octane, 2-methylheptane, 3-methylheptane, 4-methylheptane, 3-ethylhexane, 2,2-dimethylhexane, 2,2,4-trimethylpentane or mixtures thereof, preferably n-octane.

[0034] Preferably, the solvent composition Solv I -1 comprises N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, nitromethane, acetonitrile, benzonitrile, acetone, methyl ethyl ketone, γ-butyrolactone, methanol, ethanol, propanol, butanol, diethyl ether, methyl tert-butyl ether, diisopropyl ether, water, or mixtures thereof. More preferably, the solvent composition Solv... I -1 comprises N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, nitromethane, acetonitrile, benzonitrile, acetone, methyl ethyl ketone, γ-butyrolactone, methanol, ethanol, propanol, butanol, water, or mixtures thereof. Most preferably, the solvent composition Solv... I -1 comprises N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, nitromethane, acetonitrile, γ-butyrolactone, methanol, ethanol, propanol, butanol, water, or mixtures thereof. In particular, the solvent composition Solv... I-1 comprises N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, acetonitrile, methanol, ethanol, propanol, butanol, water, or mixtures thereof. More particularly, the solvent composition Solv... I -1 comprises N,N-dimethylformamide, water, or a mixture of water and N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, acetonitrile, methanol, ethanol, propanol, or butanol. Very particularly, the solvent composition Solv... I -1 comprises a mixture of N,N-dimethylformamide or water with 1,4-dioxane. The solvent composition is particularly composed of a mixture of N,N-dimethylformamide or water with 1,4-dioxane in a ratio of 1:2 to 1:4 (the volume ratio of water to 1,4-dioxane).

[0035] Preferably, Formula 1 X The compound in the solvent composition Solv I The concentration of -1 is between 5 mg / mL and 150 mg / mL, more preferably between 10 mg / mL and 120 mg / mL, very preferably between 20 mg / mL and 100 mg / mL, particularly between 30 mg / mL and 80 mg / mL, even more particularly between 35 mg / mL and 70 mg / mL, very particularly between 40 mg / mL and 60 mg / mL, especially 50 mg / mL.

[0036] Preferred Formula 1 X The compound is dissolved or suspended in the solvent composition Solv I In -1, the composition comprises N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, N-octyl-2-pyrrolidone, methyl 5-(dimethylamino)-2-methyl-5-oxovalerate, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, nitromethane, acetonitrile, benzonitrile, acetone, methyl ethyl ketone, γ-butyrolactone, γ-valerolactone, triethyl phosphate, methanol, ethanol, propanol, butanol, diethyl ether, methyl tert-butyl ether, diisopropyl ether, benzene, toluene, xylene, chlorobenzene, pentane, hexane, heptane, octane, cyclohexane, water, or mixtures thereof.

[0037] The above-mentioned compound and solvent composition Solv of Formula I I The description and preferred embodiments of -1 also apply to further embodiments of the present invention.

[0038] Another embodiment of the present invention is a method for preparing lysine derivative LYS. X The method includes step (a) I -1), (c I -1) and (d I -1), or includes step (a) I -2), (b I -2), (c I -2) and (d I -2): (a I -1) Provide Form 1 X compounds (1 X ) in: n is 1 or 0.5, When n is 1, HY is hydrochloric acid, trifluoroacetic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, propanesulfonic acid, butyric acid, hydrobromic acid, hydroiodic acid, perchloric acid, trifluoromethanesulfonic acid, nitric acid, picric acid, trichloroacetic acid, camphorsulfonic acid, dichloroacetic acid, difluoroacetic acid, or phosphoric acid. When n is 0.5, HY is sulfuric acid; (c I -1) Use the provided equation 1 X The compound is mixed with an activated carboxylic acid derivative, one of one or more carboxyl groups of which has been converted into a carbonyl group having a covalently linked leaving group. In the presence of multiple carboxyl groups, the other carboxyl groups are protected, and in the case where the activated carboxylic acid derivative has one or more amino or imine groups, the one or more amino or imine groups are protected. (d I -1) Make the provided equation 1 X The ε-nitrogen atom of the compound reacts with the carbonyl group of the activated carboxylic acid derivative to form a covalent bond between the ε-nitrogen atom and the carbonyl group, thereby generating an amide group; Among them, step (d) I -1) It may have been partially completed in step (c) I -1) Occurred during the period; or (a I -2) Provide, for example (a) I -1) Equation 1 as defined X Compounds; (b I-2) Use a silanizing agent to treat the provided formula 1 X The compound was silanized to obtain a silanized compound; (c I -2) Mixing a silanized compound with an activated carboxylic acid derivative, wherein one of one or more carboxyl groups of the derivative has been converted into a carbonyl group having a covalently linked leaving group, wherein in the presence of multiple carboxyl groups, the other carboxyl groups are protected, and wherein in the presence of one or more amino or imine groups of the activated carboxylic acid derivative, the one or more amino or imine groups are protected. (d I -2) The ε-nitrogen atom of the silanized compound reacts with the carbonyl group of the activated carboxylic acid derivative to form a covalent bond between the ε-nitrogen atom and the carbonyl group, thereby generating an amide group; Among them, step (d) I -2) It may have been partially completed in step (c) I -2) occurs during this period.

[0039] Method variant 1, which includes step (a) I -1), (c I -1) and (d I The method of -1) does not include step (a) I- 1) after and in step (c) I -1) and (d I -1) Formula 1 provided before or during silanization X The steps of the compound. Method variant 2, which includes step (a) I -2), (b I -2), (c I -2) and (d I -2) method, in its step (b) I -2) includes formula 1 X Silanization of the compound. Silanization occurs at least in formula 1. X The α-carboxyl group of the compound. Does silanization also occur on the α-carboxyl group of formula 1? X The ε-amino group of the compound is not yet fully defined. Silylation of the ε-amino group is stoichiometric with the addition of an excess molar amount of silanizing agent. An equilibrium may also exist between protonated ε-amino groups, unprotonated silanized ε-amino groups, and protonated silanized ε-amino groups. A solution using trimethylsilyl groups for silanization is described below: .

[0040] If an actual equilibrium exists, it may depend on the amount and type of silanizing agent, temperature, reaction medium, etc.

[0041] Step (b)I The silanizing agent in -2) is, for example, trimethylchlorosilane, N-methyl-N-trimethylsilylacetamide, N,O-bis(trimethylsilyl)acetamide, N,O-bis(trimethylsilyl)trifluoroacetamide, trimethylsilylmethanesulfonate, or trimethylsilylmethanesulfonate. Preferably, the silanizing agent is trimethylchlorosilane or N-methyl-N-trimethylsilylacetamide.

[0042] Step (b) I The silanization in -2) is preferably trimethylsilanization, and the silanizing agent is a trimethylsilanizing agent.

[0043] Step (b) I -2) The molar amount of the silanizing agent in the formula is preferably greater than that provided in Formula 1. X The molar amount of the compound. In step (b) I In -2), the provided formula 1 X The molar ratio between the compound and the silanizing agent is more preferably between 1:1.1 and 1:6, very preferably between 1:1.3 and 1:5, particularly between 1:1.5 and 1:4, even more particularly between 1:2 and 1:3.5, and very particularly between 1:3.

[0044] Step (b) I -2) The reaction temperature is preferably between 0 °C and 55 °C, more preferably between 10 °C and 50 °C, very preferably between 15 °C and 48 °C, particularly between 20 °C and 45 °C, and even more particularly between 22 °C and 40 °C.

[0045] Step (b) I The silanization in -2) is preferably trimethylsilanization, and the silanizing agent is a trimethylsilanizing agent.

[0046] Preferred preparation of lysine derivative LYS X The method includes step (a) I -2), (b I -2), (c I -2) and (d I -2), and in step (b) I In -2), the silanizing agent is trimethylchlorosilane, N-methyl-N-trimethylsilylacetamide, N,O-bis(trimethylsilyl)acetamide, N,O-bis(trimethylsilyl)trifluoroacetamide, trimethylsilylmethanesulfonate, or trimethylsilylmethanesulfonate.

[0047] Preferred preparation of lysine derivative LYS X The method includes step (a) I -1), (c I-1) and (d I -1), and in step (a) I -1) after and in step (c) I -1) and (d I -1) Before or during the period, not containing the formula 1 provided by silanization. X The steps of compounding.

[0048] Preferred preparation of lysine derivative LYS X The method includes step (a) I -2), (b I -2), (c I -2) and (d I -2).

[0049] In step (a) I In -1), the provided formula 1 X The compound is provided, for example, in a solid state, such as by grinding it into a powder. Another example is Formula 1. X The compound in the solvent composition Solv I -1 is a solution or suspension. This suspension is preferably sufficiently fine and homogeneous to allow for processing in step (c). I -1) Conduct reasonable measurements.

[0050] Including step (a) I -1), (c I -1) and (d I -1) Method variant 1 step (c I -1) and (d I -1) Not necessarily sensitive to the presence of water or protic solvents. This is because some amide coupling reactions can be carried out in water, in aqueous solvent compositions, in alcohols, or in alcohol-containing solvent compositions. This includes step (a) I -2), (b I -2), (c I -2) and (d I -2) Method variant 2 step (b I -2), (c I -2) and (d I -2) They are generally sensitive to the presence of water or protic solvents. This is because silanizing agents typically react with water and alcohols. Furthermore, silanizing compounds react in step (c) I -2) and steps (d) I -2) It also reacts with water and may react with alcohols to some extent.

[0051] In step (a) I In -2), formula 1 is provided. X The compound is provided, for example, in a solid state, such as by grinding it into a powder. Another example is Formula 1.X The compound in the anhydrous solvent composition Solv I -2 is the solution or suspension. This suspension is preferably sufficiently fine and homogeneous to allow for the reaction in step (b) I -2) Perform proper metering. Solvent composition Solv I -2 is anhydrous, meaning it contains no water and no alcohols. Solvent composition Solv I -1 and solvent composition Solv I The difference with -2 is that Solv I -1 may also contain methanol, ethanol, propanol, butanol or water.

[0052] Solvent composition Solv I -2 contains N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, N-octyl-2-pyrrolidone, methyl 5-(dimethylamino)-2-methyl-5-oxovalerate, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, nitromethane, acetonitrile, benzonitrile, acetone, methyl ethyl ketone, γ-butyrolactone, γ-valerolactone, triethyl phosphate, diethyl ether, methyl tert-butyl ether, diisopropyl ether, benzene, toluene, xylene, chlorobenzene, pentane, hexane, heptane, octane, cyclohexane, or mixtures thereof.

[0053] Propyl acetate is n-propyl acetate, isopropyl acetate, or a mixture thereof, preferably isopropyl acetate. Butyl acetate is n-butyl acetate, sec-butyl acetate, isobutyl acetate, tert-butyl acetate, or a mixture thereof, preferably sec-butyl acetate. Xylene is o-xylene, m-xylene, p-xylene, or a mixture thereof, preferably m-xylene. Hexane is n-hexane, 2-methylpentane, 3-methylpentane, 2,3-dimethylbutane, 2,2-dimethylbutane, or a mixture thereof, preferably n-hexane. Heptane is n-heptane, 2-methylhexane, 3-methylhexane, 2,2-dimethylpentane, 2,3-dimethylpentane, 2,4-dimethylpentane, 3,3-dimethylpentane, 3-ethylpentane, 2,2,3-trimethylbutane, or a mixture thereof, preferably n-heptane. Octane is, for example, n-octane, 2-methylheptane, 3-methylheptane, 4-methylheptane, 3-ethylhexane, 2,2-dimethylhexane, 2,2,4-trimethylpentane or mixtures thereof, preferably n-octane.

[0054] Preferably, the solvent composition Solv I-2 comprises N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, nitromethane, acetonitrile, benzonitrile, acetone, methyl ethyl ketone, γ-butyrolactone, diethyl ether, methyl tert-butyl ether, diisopropyl ether, or mixtures thereof. More preferably, the solvent composition Solv... I -2 comprises N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, nitromethane, acetonitrile, benzonitrile, acetone, methyl ethyl ketone, γ-butyrolactone, or mixtures thereof. Most preferably, the solvent composition Solv... I -2 comprises tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, acetonitrile, γ-butyrolactone, or mixtures thereof. In particular, the solvent composition Solv... I -2 comprises tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, acetonitrile, or mixtures thereof. More particularly, the solvent composition Solv... I -2 contains tetrahydrofuran or a mixture of tetrahydrofuran and acetonitrile. Very specifically, the solvent composition Solv... I -2 is composed of tetrahydrofuran or a mixture of tetrahydrofuran and acetonitrile in a ratio of 1:1 to 1:5 (the volume ratio of tetrahydrofuran to acetonitrile).

[0055] Preferred preparation of lysine derivative LYS X The method, in which, In step (a) I In equation 1, -1) X The compound in the solvent composition Solv IProvided in solution or suspension form as described in -1, the solvent composition comprises N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, N-octyl-2-pyrrolidone, methyl 5-(dimethylamino)-2-methyl-5-oxovalerate, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, nitromethane, acetonitrile, acetone, methyl ethyl ketone, γ-butyrolactone, γ-valerolactone, triethyl phosphate, methanol, ethanol, propanol, butanol, diethyl ether, methyl tert-butyl ether, diisopropyl ether, benzene, toluene, xylene, chlorobenzene, pentane, hexane, heptane, octane, cyclohexane, water, or mixtures thereof; and In step (a) I In -2), equation 1 X The compound in anhydrous solvent composition Solv I The solvent composition is provided in solution or suspension form as described in -2, comprising N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, N-octyl-2-pyrrolidone, methyl 5-(dimethylamino)-2-methyl-5-oxovalerate, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, nitromethane, acetonitrile, acetone, methyl ethyl ketone, γ-butyrolactone, γ-valerolactone, triethyl phosphate, diethyl ether, methyl tert-butyl ether, diisopropyl ether, benzene, toluene, xylene, chlorobenzene, pentane, hexane, heptane, octane, cyclohexane, or mixtures thereof.

[0056] Step (c) I -1) or (c I -2) The temperature is, for example, in the range of 0 °C to 40 °C, preferably in the range of 4 °C to 35 °C, more preferably in the range of 10 °C to 30 °C, very preferably in the range of 20 °C to 28 °C, and particularly in the range of 22 °C to 25 °C, in step (c I -1) and steps (c) I -2) is considered to be room temperature.

[0057] Step (d) I -1) or (d I -2) The temperature is, for example, in the range of 0 °C to 50 °C, preferably in the range of 4 °C to 40 °C, more preferably in the range of 10 °C to 30 °C, very preferably in the range of 20 °C to 28 °C, and particularly in the range of 22 °C to 25 °C, in step (d)I -1) and steps (d) I -2) is considered to be room temperature.

[0058] Adding a base that does not itself react with the activated carboxylic acid derivative to form a covalent bond can release the ε-nitrogen atom from its protonated state by acidic HY. Therefore, the nucleophilicity of the ε-nitrogen atom is significantly increased, and the process of reacting with the activated carboxylic acid derivative to form a covalent bond between the ε-nitrogen atom and the carbonyl group (which forms an amide group) is accelerated. If this is the case, adding such a base that does not itself react with the activated carboxylic acid derivative to form a covalent bond is done in step (c) of method variant 1. I -1) or step (d) I -1) is performed, and in step (c) of method variant 2. I -2) or step (d) I -2) is carried out. This base, which itself does not react with activated carboxylic acid derivatives to form covalent bonds, is preferably a base that does not contain unprotected amino or unprotected imino groups. If a very strong base is chosen, relative to Formula 1... X When the molar excess of the compound is only slightly excessive, racemization may occur at the α-carbon atom of the lysine core. However, in the (L-)lysine derivative LYS X Impurities of the D-lysine type are generally not required. The molar amount of the added base should, for example, be at least the amount provided in Formula 1. X The molar amount of the compound, preferably with the base added in excess at a molar equivalent of 1.05 to 1.5. The base, free of unprotected amino or unprotected imino groups, is, for example, diisopropylethylamine, triethylamine, N-methylmorpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene, or 1,5-diazabicyclo[4.3.0]non-5-ene. Preferably, the base is diisopropylethylamine.

[0059] Preferred preparation of lysine derivative LYS X The method, wherein, in step (c) I -1) or step (d) I In step (c), a base without unprotected amino or unprotected imino groups is added, and in step (c) I -2) or step (d) I -2) Add a base that does not contain unprotected amino or unprotected imine groups.

[0060] Activated carboxylic acid derivatives are obtained, for example, through a pre-activation reaction. In the pre-activation reaction, the carboxylic acid derivative reacts with the protonated form of the leaving group. For some leaving groups, the protonated form of the leaving group is also called a coupling additive. The pre-activation reaction is driven by a condensing agent (added to the pre-activation reaction). Examples of condensing agents for the pre-activation reaction are diisopropylcarbodiimide, dicyclohexylcarbodiimide, or 1-tert-butyl-3-ethylcarbodiimide. In some cases, the condensing agent and the leaving group are already combined in a single molecule, which reacts directly with the carboxylic acid derivative. An example of this is (benzotriazolyl)tetramethylureon tetrafluoroborate. The already activated carboxylic acid derivative avoids the reaction in step (c I -1) and (d I -1) during or in step (c) I -2) and (d I -2) Other carboxyl groups exist during this period, namely carboxyl groups other than the carboxyl group at the α-carbon atom of lysine (which is silanized in method variant 2).

[0061] Activated carboxylic acid derivatives, for example, are compound 201 (201), Compound 203 (203), compound act-carb-01 (act-carb-01), Or compound act-carb-02 (act-carb-02).

[0062] The leaving group (LG) of the activated carboxylic acid derivative is, for example (named after the protonated leaving group): N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, or pentafluorophenol. Preferably, the leaving group of the activated carboxylic acid derivative is N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxybenzotriazole, or 1-hydroxy-7-azabenzotriazole, more preferably N-hydroxysuccinimide or 1-hydroxybenzotriazole, and very preferably N-hydroxysuccinimide.

[0063] The unactivated carboxylic acid derivative is, for example, compound carb-1. (carb-1), compound carb-2 (carb-2), Palmitic acid, Compound 202 (202), compound carb-3 (carb-3), Compound 306 (306), compound carb-4 (carb-4), Or compound carb-5 (carb-5).

[0064] Lysine derivative LYS X Including Formula 2 X Structural units: (2 X ) in: This indicates a covalent bond with a carbon atom. Furthermore, in step (c) I -1) and steps (c) I In -2), the activated carboxylic acid derivative includes formula 3 X Structural units: (3 X ) in: This indicates a covalent bond with a carbon atom. LG is a leaving group.

[0065] It is understandable that in Equation 2 X Covalently bonded carbon atoms, and in Equation 3 X The carbon atoms that are covalently bonded are the same carbon atom.

[0066] Preferably, the carbon atom itself has a total of 4 covalent bonds, that is, the carbon atom is sp 3 A hybrid carbon atom, or one with three covalent bonds, meaning the carbon atom is sp. 2 Hybridized carbon atoms. In sp 3 At the hybridized carbon atom, one of the four covalent bonds is with formula 2X or formula 3X, and the other three are with three different atoms. In sp 2 At the hybridized carbon atom, one of the three covalent bonds is a covalent bond with formula 2X or formula 3X, and the other two covalent bonds are covalent bonds with two different atoms. These two preferred schemes are shown in the following formula 2. X-sp 3 and 3 X -sp 3 or 2 X -sp 2 and 3 X -sp 2 The Chinese side indicated that...

[0067] Preferably, the lysine derivative LYS X Including Formula 2 X -sp 3 or 2 X -sp 2 Structural units: (2 X -sp 3 ) (2 X -sp 2 ) in: Indicates covalent connection. And in step (c) I -1) and steps (c) I In -2), the activated carboxylic acid derivative includes formula 3 X -sp 3 Or 3 X -sp 2 Structural units: (3 X -sp 3 ) (3 X -sp 2 ) in: This indicates a covalent bond with a carbon atom. LG is a leaving group.

[0068] More preferably, the carbon atom itself has three covalent bonds, that is, the carbon atom is sp 3 Hybridized carbon atoms. More preferably, lysine derivatives LYS X Including Formula 2 X -sp 3 The structural unit, and in step (c) I -1) and steps (c) I In -2), the activated carboxylic acid derivative includes formula 3 X -sp 3 Structural units.

[0069] Preferred preparation of lysine derivative LYS X The method, in which the lysine derivative LYS X Including 4 X -A or 4 X -B structural unit: (4 X -A) (4 X -B) in: Indicates covalent connection. R 4X-B-N-1 It is an amino protecting group; Furthermore, in step (c) I -1) and steps (c) I In -2), the activated carboxylic acid derivative includes formula 5 X -A or 5 X -B structural unit (5 X -A) (5 X -B) in: Indicates covalent connection. LG is a leaving group. R 5X-B-N-1 It is an amino protecting group.

[0070] Preferred preparation of lysine derivative LYS X The method wherein the lysine derivative LYS X Including 6 X -Aa、6 X -Ab or 6 X -Ac's structural unit: (6 X -Aa) (6 X -Ab) (6 X -Ac) in: Indicates covalent connection. R 6X-A-b-O-1 It is a carboxylic acid protecting group; Furthermore, in step (c) I -1) and steps (c) IIn -2), the activated carboxylic acid derivative includes formula 7 X -Aa、7 X -Ab or 7 X -Ac's structural unit: (7 X -Aa) (7 X -Ab) (7 X -Ac) in: Indicates covalent connection. LG is a leaving group. R 7X-A-b-O-1 It is a carboxylic acid protecting group.

[0071] Lysine derivative LYS X For example, a compound with the formula Pr-L: (Pr-L), in: R L-O-1 and R L-O-2 Each of them independently represents a carboxylic acid protecting group. Examples of Pr-L compounds are as follows: Compound 508 (508); Compound 309 (309); Compound 310 (310); Compound 511 (511); Compound 514 (514); Or compound 517 (517).

[0072] Preparation of lysine derivative LYS X Examples of further steps in the method (which is a Pr- compound) include steps (prl-b) and (prl-c): (prl-b) De-lysine derivative LYS X The amino protecting group R on (a compound of formula Pr-A) A-N-1 (Pr-A), in: R A-N-1 It has a different amino protecting group than Bsmoc. Thus, a compound with the formula Pr-A-am was obtained. (Pr-A-am), (prl-c) Condensation of a compound of formula Pr-A-am with a compound of formula B. (B), in: R L-O-1 and R L-O-2 As defined by Pr-L Thus, compounds of the formula Pr-L are obtained.

[0073] Preferably, in step (prl-c), the compound of formula Pr-A-am is persilylated to obtain a persilylated intermediate compound, the compound of formula B is activated using the condensing agent of step (prl-c) to obtain an activated compound of formula B, the persilylated compound is mixed with the activated compound, and the two are reacted. Alternatively, the compound of formula Pr-A-am can also be used as a hydrochloride salt, i.e., compound 310. Compound 309 is a compound of formula Pr-A, wherein R A -N-1 It is tert-butyloxycarbonyl.

[0074] Preferably, the lysine derivative LYS X It is a compound of formula Pr-L, compound 309, compound 310, compound 511, compound 514, or compound 517. More preferably, the lysine derivative LYS X The compounds are 508, 309, 310, 511, 514, or 517. Most preferably, the lysine derivative is 508, 511, 514, or 517.

[0075] Preferred preparation of lysine derivative LYS X The method wherein the lysine derivative LYS X It is a compound of formula Pr-L, compound 309, compound 310, compound 511, compound 514 or compound 517.

[0076] The above refers to the compounds of formula I and the solvent composition Solv. I -1. And the preparation of lysine derivative LYS XThe description and preferred embodiments of the method also apply to further embodiments of the present invention.

[0077] Another embodiment of the present invention is a method for preparing peptide P X A method comprising step (c) X ): (c X The α-amino acid derivative S-am, having one unprotected α-amino group or one unprotected α-imino group, is combined with the lysine derivative LYS. X Condensation was performed to obtain the intermediate peptide Int-P X The lysine derivative LYS X A method for preparing lysine derivative LYS X The method is obtained by means of step (a) I -1), (c I -1) and (d I -1), or includes step (a) I -2), (b I -2), (c I -2) and (d I -2), (a I -1) Provide Form 1 X compounds (1 X ) in: n is 1 or 0.5, When n is 1, HY is hydrochloric acid, trifluoroacetic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, propanesulfonic acid, butyric acid, hydrobromic acid, hydroiodic acid, perchloric acid, trifluoromethanesulfonic acid, nitric acid, picric acid, trichloroacetic acid, camphorsulfonic acid, dichloroacetic acid, difluoroacetic acid, or phosphoric acid. When n is 0.5, HY is sulfuric acid; (c I -1) The provided equation 1 X The compound is mixed with an activated carboxylic acid derivative, one of which has been converted into a carbonyl group having a covalently linked leaving group. In the case of multiple carboxyl groups, the other carboxyl groups are protected, and in the case of the activated carboxylic acid derivative having one or more amino or imine groups, the one or more amino or imine groups are protected. (d I -1) Make the provided formula 1 X The ε-nitrogen atom of the compound reacts with the carbonyl group of the activated carboxylic acid derivative to form a covalent bond between the ε-nitrogen atom and the carbonyl group, thereby generating an amide group; Among them, step (d) I-1) It may have been partially completed in step (c) I -1) Occurred during the period; or (a I -2) Provide, for example (a) I -1) Equation 1 as defined X Compounds; (b I -2) Silanize the provided formula 1 with a silanizing agent. X The compounds were obtained by silanizing them; (c I -2) Mixing a silanized compound with an activated carboxylic acid derivative, wherein one of one or more carboxyl groups of the derivative has been converted into a carbonyl group having a covalently linked leaving group, wherein in the case of multiple carboxyl groups, the other carboxyl groups are protected, and in the case of the activated carboxylic acid derivative having one or more amino or imino groups, the one or more amino or imino groups are protected. (d I -2) The ε-nitrogen atom of the silanized compound reacts with the carbonyl group of the activated carboxylic acid derivative to form a covalent bond between the ε-nitrogen atom and the carbonyl group, thereby generating an amide group; Among them, step (d) I -2) It may have been partially completed in step (c) I -2) occurs during this period.

[0078] Peptide P X With lysine derivative LYS X The difference lies in the fact that it no longer has an α-carboxyl group at the lysine residue. Lysine derivative LYS X The carboxyl group has undergone condensation with an unprotected α-amino group or an unprotected α-imino group of the α-amino acid derivative S-am. Lysine derivative LYS X The original α-carboxyl carbonyl group is now the intermediate peptide Int-P. X Part of the amide bond, and if the intermediate peptide Int-P X With final peptide P X If they are different, then it is also peptide P. X Part of the amide bond.

[0079] If the intermediate peptide Int-P X Without further chemical modification, the intermediate peptide Int-P X It could be peptide P X Or if the intermediate peptide Int-P X Further chemical modifications were performed on the intermediate peptide Int-P. X It can be different from peptide P X .

[0080] Preparation of peptide PX An example of a method for preparing peptide P is a method that includes step (c): (c) An α-amino acid derivative S-am having an unprotected α-amino group or an unprotected α-imino group is condensed with a compound of formula Pr-L to obtain peptide Pr-LS.

[0081] (Pr-L), in: R L-O-1 and R L-O-2 Each of them represents a carboxylic acid protecting group independently.

[0082] Peptide P is preferably a compound of formula Pr-LS (described below). It is understood that the term "peptide P" refers to the designation of the synthesized peptide. The designation "P" may also be omitted or placed in parentheses without altering the meaning of the peptide as defined by its structural features. The term "Pr-LS" describes the structure as defined in the illustrated structure. It is intended to illustrate the structural unit described below. The designation "Pr-LS" may also be omitted or placed in parentheses without altering the meaning of the peptide as defined in the illustrated structure.

[0083] To combine an unprotected α-amino group or an unprotected α-imino group of the α-amino acid derivative S-am with the lysine derivative LYS X (A compound of preferred formula Pr-L) undergoes condensation to produce the lysine derivative LYS. X The carboxyl group of lysine in (preferred Pr-L compounds) is usually obtained by step (c) X (Preferred step (c)) activates the condensing agent. Step (c) X The condensing agent in (preferred step (c)) leads to the formation of an activated lysine derivative LYS. X (Preferably activated compounds of the formula Pr-L), whose carboxyl hydroxyl groups are activated by step (c) X The leaving group substitution in step (c) results in the activation of the lysine derivative LYS. X (Preferably activated compounds of the Pr-L formula) are more reactive to the unprotected α-amino or unprotected α-imino group of the α-amino acid derivative S-am. This depends on step (c). X (Preferred step (c)) The specific condensing agent is the activated lysine derivative LYS. X(Preferred activated compounds of the formula Pr-L) can be intermediates suitable for separation at room temperature or intermediates unsuitable for separation at room temperature. The condensing agent in step (cX) (preferably step (c)) is, for example, a carbodiimide derivative. This carbodiimide derivative is, for example, diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1-tert-butyl-3-ethylcarbodiimide, or 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. Very preferably, the carbodiimide derivative is diisopropylcarbodiimide. Preferably, the carbodiimide reacts with the condensing agent in step (cX)... X (Preferred step (c)) The coupling additive is applied together. Step (c) X The coupling additive in step (c) is, for example, ethyl cyano-hydroxyimino, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, 5-hydroxyimino-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, step (c) X The coupling additive in step (c) is ethyl cyano-hydroxyimino, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, or 2-hydroxypyridine N-oxide. More preferably, step (c) X The coupling additive in step (c) is ethyl cyano-hydroxyiminoacetate or 1-hydroxybenzotriazole. Most preferably, step (c) X (Preferred step (c)) The coupling additive is cyano-hydroxyiminoethyl acetate. In the case where the α-amino acid derivative S-am contains an unprotected carboxyl group, the lysine derivative LYS... X The activation of (preferred Pr-L compounds) must occur before contact with the α-amino acid derivative S-am. The α-amino acid derivative S-am contains substances that interfere with step (c). X (Preferred step (c)) condensation, and unlike the unprotected α-amino or unprotected α-imino functional group, the functional group is protected by a suitable protecting group. Preferably, the α-amino acid derivative S-am does not contain an unprotected guanidine group or an unprotected thiol group.

[0084] Preferred preparation of peptide P X (Preferred peptide P) method, wherein, in step (c) X In the preferred step (c), the lysine derivative LYS X (Compounds of preferred formula Pr-L) are processed using step (c) X(Preferred step (c)) activates the condensing agent.

[0085] Preferred preparation of peptide P X (Preferred peptide P) method, wherein, in step (c) X In the preferred step (c), there exists step (c) X (Preferred step (c)) coupling additive.

[0086] Preferred preparation of peptide P X (Preferred peptide P) method, wherein, in step (c) X In the preferred step (c), the lysine derivative LYS X (Compounds of preferred formula Pr-L) are processed using step (c) X (Preferred step (c)) activates the condensing agent (which is a carbodiimide derivative), and step (c) is present. X (Preferred step (c)) coupling additive (which is cyano-hydroxyiminoethyl acetate).

[0087] Step (c) X (Preferred step (c)) Preferred step (c) X The process (preferred step (c)) is carried out in the presence of a solvent. X (Preferred step (c)) Solvent dissolution of lysine derivative LYS X (A compound of the preferred formula Pr-L). In the case of an α-amino acid derivative S-am covalently linked resin, this step (c) X (Preferred step (c)) The solvent is preferably used to swell the resin. Step (c) X The solvent in step (c) is, for example, N,N-dimethylformamide, N-methyl-2-pyrrolidone, N-butylpyrrolidone, dimethyl isosorbide dimethylate, γ-valerol, dihydro-L-glucanone (e.g., commercially available Cyrene™), dimethyl sulfoxide, tetrahydropyran, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, 1,3-dioxapentane, ethyl acetate, dichloromethane, acetonitrile, toluene, water, and mixtures thereof. Preferably, step (c) X (Preferred step (c)) uses N,N-dimethylformamide as the solvent. More preferably, step (c) X The solvent in step (c) preferably comprises at least 20 vol.% N,N-dimethylformamide, most preferably 40 vol.%, especially 70 vol.%, more especially 90 vol.%, and very especially more than 95 vol.%. Preferably, step (c) X (Preferred step (c)) The solvent is N,N-dimethylformamide.

[0088] Preferred preparation of peptide P X (Preferred peptide P) method, wherein step (c) is carried out in the solvent of step (c) and the solvent contains N,N-dimethylformamide.

[0089] Preferably, step (c) X (Preferred 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, even more particularly between 19 °C and 28 °C, very particularly between 20 °C and 27 °C, and especially between 22 °C and 25 °C. For step (c) X (Preferred step (c)) For example, the temperature between 22 °C and 25 °C is defined as room temperature.

[0090] Preferred preparation of peptide P X (Preferred peptide P) method, wherein step (c) X (Preferred step (c)) is carried out at a temperature between 15 °C and 35 °C.

[0091] Preferably, in step (c) X In the preferred step (c), the lysine derivative LYS X (The preferred compound, Pr-L), is applied in a molar excess relative to the α-amino acid derivative S-am. This molar excess is, for example, the lysine derivative LYS. X The molar amount of (preferred Pr-L compound) is 1.05 to 3.5 times the molar amount of the α-amino acid derivative S-am. Preferably, this molar amount is between 1.3 and 3.0 times, more preferably between 1.5 and 2.5 times, very preferably between 1.7 and 2.3 times, particularly between 1.8 and 2.2 times, and even more particularly between 1.9 and 2.1 times.

[0092] Preferably, in step (c) X In the preferred step (c), the condensing agent is relative to the lysine derivative LYS X (The compound of preferred formula Pr-L) is applied in a molar excess. This molar excess is, for example, in step (c) X (Preferred step (c)) The molar amount of the condensing agent is the lysine derivative LYS. X (The compound of preferred formula Pr-L) is 1.05 to 3 times its molar amount. Preferably, this molar amount is between 1.3 and 2.5 times, more preferably between 1.5 and 2.3 times, very preferably between 1.7 and 2.2 times, particularly between 1.8 and 2.2 times, and even more particularly between 1.9 and 2.0 times. Step (c) X(Preferred step (c)) The total amount of condensing agent is, for example, a one-time addition of the lysine derivative LYS. X (The preferred compound of formula Pr-L) may be added in two or more batches. For example, in step (c) X (preferred step (c)) a portion of the condensing agent (e.g., step (c)) X (Preferred step (c)) 50 wt.% to 70 wt.% of the total amount of condensing agent is added to the dissolution step (c) X (Preferred step (c)) Lysine derivative LYS in solvent X (A compound of preferred formula Pr-L). The resulting solution is stirred for, for example, 5 to 30 minutes, and then added to the α-amino acid derivative S-am.

[0093] Preferably, step (c) X (Preferred step (c)) The coupling additive relative to the lysine derivative LYS X (The preferred compound of formula Pr-L) is applied in molar amounts, which are lysine derivatives LYS. X (The molar amount of the compound with the preferred formula Pr-L) is 0.5 to 3 times. This molar amount is, for example, that of the lysine derivative LYS. X (The molar amount of the compound of preferred formula Pr-L) is 0.8 to 2.5 times. Preferably, this molar amount is in the lysine derivative LYS X The molar amount of (preferred Pr-L compound) is between 0.9 and 2.2 times, more preferably between 1.1 and 2.0 times, very preferably between 1.3 and 1.7 times, and particularly between 1.4 and 1.6 times.

[0094] In this article, a peptide bond should be understood as a covalent bond between the α-amino or α-imino group of the first α-amino acid residue and the α-carboxyl group of the second α-amino acid residue. The α-amino acid derivative S in this article should be understood as a compound without a peptide bond, or a compound having at least one peptide bond. In the latter case, the α-amino acid derivative S is, for example, a dipeptide derivative, a tripeptide derivative, or a tetrapeptide derivative. In both of these compounds, there is an N-terminal α-amino group (which may also be an α-imino group) of the N-terminal amino acid residue and a C-terminal carboxyl or carboxamide group of the C-terminal amino acid residue. In compounds without a peptide bond, the N-terminal α-amino group (which may also be an α-imino group) and the C-terminal carboxyl or carboxamide group are substituents of the same α-carbon atom, or belong to the same α-amino acid residue. The α-amino acid derivative S-am is, for example, a linking resin. This resin comprises a linking group and a polymer carrier. The linking group is covalently attached to the polymer carrier. The linking group is used for the covalent attachment of the α-amino acid residue, wherein the covalent attachment of the α-amino acid residue is cleavable. Typically, C-terminal α-amino acid residues are covalently attached to a resin. This resin is, for example, resin-1 or resin-2. Resin-1 has a linking group, which is particularly suitable for the covalent attachment of the oxygen atom of the carboxyl group. The linking group of resin-1 (written as a di-substituent) is, for example, 2-chlorotriphenylmethyl-p-aminomethyl, 2-chlorotriphenylmethyl, or 4-(methyleneoxy)benzyl. Resin-2 has a linking group, which is particularly suitable for the covalent attachment of the nitrogen atom of the carboxylic acid amide group. The linking group of resin-2 (written as a di-substituent) is, for example, xanthon-3-(oxomethylene)-9-yl or α-(2,4-dimethoxyphenyl)-α-(4-(N-methyleneaminomethyloxy)phenyl)methyl. The polymer carrier of resin-1 or resin-2 is, for example, polystyrene, a copolymer containing polystyrene units and polyethylene oxide units, or polyethylene oxide. The α-amino acid derivative S-am is, for example, a single molecule. Preferably, the α-amino acid derivative S-Am is covalently linked to the resin. Preferably, the α-amino acid derivative S-Am is a single molecule. Preferably, the α-amino acid derivative S-Am does not contain an unprotected carboxyl group.

[0095] Peptide P includes structural units of formula L: (L), in: # and ## indicate covalent connection. R L-O-1 and R L-O-2The H or carboxylic acid protecting groups are represented independently of each other. The covalent link represented by # in Formula L is an α-nitrogen atom facing an α-amino acid residue, which is the N-terminal α-amino acid residue of the α-amino acid derivative S-am. The covalent link represented by ## in Formula L is, for example, an α-carbonyl group facing a hydrogen atom, an amino protecting group, or an α-amino acid residue, which is the C-terminal α-amino acid residue of another α-amino acid derivative.

[0096] S-am, an α-amino acid derivative having one unprotected α-amino group or one unprotected α-imino group, is preferably an α-amino acid derivative of the formula SI-am or S-II-am. (SI-am) (S-II-am), in: R SI-1 Yes - (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, R SI-2 It is H, C 1-6 Alkyl, or composed of OR SI-2-1 SR 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 -Guidino, phenyl, p-(R) SI-2-10 O)-Phenyl, 1-RS I-2-11 -imidazol-4-yl or 1-R SI-2-12 -Indole-3-yl monosubstituted C 1-6 alkyl, -(AA nx ) m - is composed of m condensed α-amino acid residues AA nx The substituents formed are each x, where x is an integer and its value ranges from 1 to m. Each condensed α-amino acid residue AA nxIndependent selection, and in cases where it has a functional group-containing side chain, the functional group is either unprotected or protected by a protecting group, provided that the interfering functional group is protected. m is an integer from 1 to 30. R SI-1-1 It is an H or carboxylic acid protecting group. R SI-2-1 It is an H or hydroxyl protecting group. R SI-2-2 It is a thiol protecting group. R SI-2-3 and R SI-2-4 It consists of H, an amino protecting group, or both forming an amino protecting group, provided that neither is an H. R SI-2-5 It is an H or carboxylic acid protecting group. R SI-2-6 and R SI-2-7 It is an H or amide protecting group. R SI-2-8 and R SI-2-9 It is either an H or a guanidine protecting group, provided that neither is an H group. R SI-2-10 It is a protecting group for aromatic hydroxyl groups. R SI-2-11 It is a protecting group for the nitrogen atom of imidazole. R SI-2-12 It is a protecting group for the indole nitrogen atom. R SII-1 For example, R SI-1 Defined.

[0097] The carboxylic acid protecting group is, for example, tert-butyl, benzyl, benzoylmethyl, 2-phenyl-isopropyl-2-yl, or methyl, preferably tert-butyl. More preferably, the carboxylic acid protecting group is tert-butyl or 3-methyl-pentan-3-yl. More preferably, the carboxylic acid protecting group is tert-butyl. Preferably, R L-O-1 and R L-O-2 It is a carboxylic acid protecting group, which is removed under acidic conditions (more preferably exposed to trifluoroacetic acid). Preferably, R L-O-1 and R L-O-2 Each is independently tert-butyl or 3-methyl-pentan-3-yl. More preferably, R L-O-1 and R L-O-2 The same, and is tert-butyl or 3-methyl-pentan-3-yl. Most preferably, R L-O-1 and R L-O-2It is tert-butyl. The hydroxyl protecting group is, for example, tert-butyl, benzyl, 2-bromobenzyloxycarbonyl, triphenylmethyl, or 2-chlorotriphenylmethyl, preferably tert-butyl. In the case of an α-amino acid residue containing a β-hydroxyl group, the hydroxyl protecting group is also, for example, 1,1-dimethyldiyl, which, together with the nitrogen atom at the α-position, the carbon atom at the α-position, and the oxygen atom at the β-hydroxyl group, forms an oxazolidine ring (so-called pseudoproline). The thiol protecting group is, for example, tert-butyl, 4-methylbenzyl, acetamidomethyl, triphenylmethyl, tert-butylthio, tetrahydropyran-2-yl, diphenylmethyl, or 2,4,6-trimethoxybenzyl. The amino protecting group is, for example, tert-butyloxycarbonyl, 9-fluorenyl-methoxycarbonyl, benzyloxycarbonyl, or allyloxycarbonyl. The amide protecting group is, for example, 9-H-xanthon-9-yl, 2,4,6-trimethoxybenzyl, dimethylcyclopropylmethyl, triphenylmethyl, or 3-methylpentan-3-yl. The guanidine protecting group is, for example, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-ylsulfonyl (=Pbf), 2,2,5,7,8-pentamethylchroman-6-ylsulfonyl, mesitylenesulfonyl, p-toluenesulfonyl, triphenylmethyl, or methoxytriphenylmethyl, preferably 2,2,4,6,7-pentamethyldihydrobenzofuran-5-ylsulfonyl. The aromatic hydroxyl protecting group is, for example, tert-butyl, benzyl, 2-chlorotriphenylmethyl, or 2-bromobenzyloxycarbonyl. The imidazole nitrogen protecting group is, for example, tert-butoxycarbonyl, 2,4-dinitrophenyl, benzyloxymethyl, triphenylmethyl, or methoxytriphenylmethyl, preferably tert-butoxycarbonyl. The protecting group of indole nitrogen is, for example, tert-butyloxycarbonyl or formyl, preferably tert-butyloxycarbonyl.

[0098] AA n1 The nitrogen atom of its α-amino group is covalently bonded to the carbon atom of the carbonyl group shown in formula SI-am or S-II-am. AA nm The carbonyl group is covalently bonded to an oxygen or nitrogen atom. The carbonyl group is preferably AA. nm The α-carbonyl group. Interference step (c) X (Preferred step (c)) An example of the condensed side chain functional group is, for example, an amino group. Therefore, the amino group is protected by a protecting group.

[0099] AA nxFor example, in its corresponding condensed form as an α-amino acid residue, it is glycine, alanine, serine, serine with a tert-butyl-protected side chain, threonine, threonine with a trityl-protected side chain, cysteine with a tert-butyl-protected side chain, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine with a tert-butyl-protected side chain, tryptophan with a tert-butoxycarbonyl-protected side chain, aspartic acid with a tert-butyl-protected side chain, glutamic acid with a tert-butyl-protected side chain, asparagine, asparagine with a trityl-protected side chain, glutamine, glutamine with a dimethylcyclopropylmethyl-protected side chain, histidine with a trityl-protected side chain, lysine with an allyloxycarbonyl-protected side chain or arginine with a 2,2,4,6,7-pentamethyldihydrobenzofuran-5-ylsulfonyl-protected side chain.

[0100] Substituent -(AA nx ) m - Examples (where m = 3, AA n1 = Gly, AA n2 = Arg(Pbf) and AA n3 = Gly) are -(Gly-Arg(Pbf)-Gly)-, as follows:

[0101] where and represent covalent linkages.

[0102] C 1-6 alkyl is, for example, methyl, 1-methylethyl, 1-methylpropyl or 2-methylpropyl. C SI-2-1 alkyl monosubstituted by OR 1-6 is, for example, hydroxymethyl, hydroxymethyl protected by R SI-2-1 , 1-hydroxyethyl or 1-hydroxyethyl protected by R SI-2-1 . C SI-2-2 alkyl monosubstituted by SR 1-6 is, for example, mercaptomethyl protected by R SI-2-2 . C 1-6 alkyl monosubstituted by SCH3 is, for example, 2-(methylthio)ethyl. C SI-2-3 R SI-2-4 alkyl monosubstituted by NR 1-6 is, for example, 4-aminobutyl protected by R SI-2-3 , R SI-2-4 or R SI-2-3 and R SI-2-4 . C SI-2-5 alkyl monosubstituted by CO-OR 1-6 is, for example, carboxymethyl protected by R SI-2-5 or carboxymethyl protected by R SI-2-5Protected 2-carboxyethyl. (By CO-NR) SI-2-6 R SI-2-7 Monosubstituted C 1-6 Alkyl groups, for example, are 2-amino-2-oxoethyl, or are R SI-2-6 R SI-2-7 Or R SI-2-6 and R SI-2-7 Protected 2-amino-2-oxoethyl. (Chemical name: CO-NR) SI-2-6 R SI-2-7 Monosubstituted C 1-6 Alkyl groups, for example, are also 3-amino-3-oxopropyl, or are R SI-2-6 R SI-2-7 Or R SI-2-6 and R SI-2-7 Protected 3-amino-3-oxopropyl. Enclosed by N'-R SI-2-8 -N''-R SI-2-9 - Guanidino monosubstituted C 1-6 Alkyl groups, for example, are formed by R SI-2-8 R SI-2-9 Or R SI-2-8 and R SI-2-9 Protected 3-guanidinopropyl. C-terminus monosubstituted with phenyl. 1-6 Alkyl groups, for example, are benzyl groups. (The following appears to be a separate, unrelated sentence fragment: "p-(R...") SI-2-10 O)-Phenyl monosubstituted C 1-6 Alkyl groups, for example, are formed by R SI-2-10 Protected 4-hydroxybenzyl group. (By 1-R) SI-2-11 -imidazolium-4-monosubstituted C 1-6 The alkyl group is, for example, imidazole-4-ylmethyl, or R-substituted at the 1-position of the imidazole ring. SI-2-11 Protected imidazole-4-ylmethyl. (By 1-R) SI-2-12 -Indole-3-yl monosubstituted C 1-6 The alkyl group is, for example, indole-3-ylmethyl, or R-substituted at the 1-position of the indole ring. SI-2-12 Protected indole-3-ylmethyl.

[0103] Preferably, R SI-1 Yes - (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 Yes - (AA) nx ) m -O-[resin-1], -(AA nx ) m -N-[resin-2], -O-[resin-1], or -N-[resin-2].

[0104] Preferably, R SI-1-1 It is a carboxylic acid protecting group. Preferably, R SI-2-5 It is a carboxylic acid protecting group. Preferably, all AA nx None of them contain unprotected carboxylic acids. More preferably, R SI-1-1 and R SI-2-5 Each is an independent carboxylic acid protecting group, and all AA nx None of them contain unprotected carboxylic acid groups. More preferably, the α-amino acid derivatives of formula SI-am or S-II-am do not contain unprotected carboxylic acid groups.

[0105] Preferably, R SI-2 Unlike H, more preferably, R SI-2 It is C 1-6 Alkyl, or OR SI-2-1 SR 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 -Guidino, phenyl, p-(R) SI-2-10 O)-Phenyl, 1-R SI-2-11 -imidazol-4-yl or 1-R SI-2-12 -Indole-3-yl monosubstituted C 1-6 Alkyl group. Very preferably, R SI-2 Is it OR SI-2-1 SR 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 -Guidino, phenyl, p-(R) SI-2-10 O)-Phenyl, 1-R SI-2-11 -imidazol-4-yl or 1- RSI-2-12 -Indole-3-yl monosubstituted C 1-6 Alkyl group. Specifically, R SI-2 It was CO-OR SI-2-5 or CO-NR SI-2-6 R SI-2-7 Monosubstituted C 1-6 alkyl.

[0106] 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, even more particularly from 3 to 11, and very particularly from 4 to 10.

[0107] Preferably, R SI-2 The α-carbon atom in the substituted formula SI-am is in the S-configuration, provided that R SI-2 Unlike H, it is preferably covalently linked to R. SII-1 The carbonyl-substituted S-II-am has an S-configuration α-carbon atom.

[0108] When the α-amino acid derivative S-am in step (c) is a compound of formula SI-am or S-II-am, the peptide Pr-LS obtained in step (c) is a compound of formula Pr-LSI or Pr-LS-II. (Pr-LSI) (Pr-LS-II), in: R L-O-1 and R L-O-2 As defined by Pr-L R SI-1 R SI-2 and R SII-1 As defined by formula SI-am or S-II-am.

[0109] Preferred preparation of peptide P X The method wherein the α-amino acid derivative S-am is a compound of formula SI-am or S-II-am.

[0110] A preferred method for preparing peptide P, wherein the α-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-LSI or Pr-LS-II.

[0111] Preferred preparation of peptide P X (Preferred peptide P) method, wherein step (c) X (Preferred step (c)) The α-amino acid derivative S-am is a compound of formula SI-am, wherein R SI-1 Yes - (AA) nx ) 10 -O-[resin-1], AA n1 It's Phe, AA n2 It's Ile, AA n3 It's Ala, AA n4 It is a Trp with protected sidechains, AA n5It's Leu, AA n6 It's Val, AA n7 It is Arg, AA with protected side chains. n8 It's Gly, AA n9 It is Arg, AA with protected side chains. n10 It is Gly, Resin-1 is 2-chlorotriphenylmethylaminomethyl resin, and R SI-2 It is 2-(tert-Butoxycarbonyl)ethyl.

[0112] Preferred preparation of peptide P X (Preferred peptide P) method, wherein the α-amino acid derivative S-am in step (c) is a compound of formula SI-am, wherein R SI-1 Yes - (AA) nx ) 10 -O-[resin-1], AA n1 It's Phe, AA n2 It's Ile, AA n3 It's Ala, AA n4 It is a Trp with protected sidechains, AA n5 It's Leu, AA n6 It's Val, AA n7 It is Arg, AA with protected side chains. n8 It's Gly, AA n9 It is Arg, AA with protected side chains. n10 It is Gly, resin-1 is a 2-chlorotriphenylmethyl resin, and R SI-2 It is 2-(tert-Butoxycarbonyl)ethyl.

[0113] A preferred method for preparing peptide P, wherein R L-O-1 and R L-O-2 They are either tert-butyl or 3-methyl-pent-3-yl, which are independent of each other.

[0114] Preferred preparation of peptide P X The method, in which lysine derivative LYS X These are compounds of the formula Pr-L, particularly compounds 508, 309, 511, 514, or 517.

[0115] A preferred method for preparing peptide P, wherein the compound of formula Pr-L is compound 508.

[0116] The chemical name of the molecular structure of Bsmoc is 1,1-dioxobenzo[b]thiophene-2-methoxycarbonyl, which is also commonly referred to as benzo[b]thiophene sulfone-2-methoxycarbonyl.

[0117] Preferably, peptide P is prepared X The method includes steps (d) X): From the intermediate peptide Int-P X Remove the Bsmoc- protecting group from the α-nitrogen atom.

[0118] Preferably, the method for preparing peptide P includes step (d): removing the amino protecting group R from the compound of formula Pr-LSI or Pr-LS-II. L-N-1 Remove, to obtain compounds of formula LSI-am or LS-II-am. (LSI-am) (LS-II-am), in: R L-O-1 R L-O-2 R SI-1 R SI-2 and R SII-2 As defined by Pr-LSI or Pr-LS-II.

[0119] Step (d) X Removal of the Bsmoc protecting group in step (d), or removal of the amino protecting group R in step (d). L-N-1 The removal is performed using step (d) X The deprotection composition of step (d) or step ( ) is carried out. Step (d) X The deprotected composition of step (d) is, for example, 20 vol.% piperidine in N,N-dimethylformamide. In the method for preparing peptide P, suitable selection of other protecting groups for compounds of formula Pr-LSI or Pr-LS-II results in the formation of compounds of formula LSI-am or LS-II-am, which contain a deprotected α-amino group and are free of other unprotected amino or imino groups, preferably also free of unprotected carboxylic acid groups.

[0120] Preferred preparation of peptide P X The method includes steps (d) X ): (d X ) intermediate peptide Int-P X Remove the Bsmoc-protecting group.

[0121] A preferred method for preparing peptide P includes step (d): (d) The amino protecting group R on the compound of formula Pr-LSI or Pr-LS-II L-N-1 Remove, and obtain a compound with the formula LSI-am or LS-II-am.

[0122] Preferably, peptide P is prepared X The method includes steps (e)X ): The result from step (d) X Bsmoc-deprotected intermediate Int-P X Condensation with α-amino acid derivatives of formula T R T-N-1 -(AA py ) q -OH (T) in: R T-N-1 It is an amino protecting group. -(AA py ) q - is composed of q condensed α-amino acid residues AA py The substituents formed are each y, where y is an integer and its value ranges from 1 to q. Each condensed α-amino acid residue AA py Independent selection, and in cases where it has a functional group-containing side chain, the functional group is either unprotected or protected by a protecting group, provided that the interfering functional group is protected. AA pq The carbon atom of its α-carbonyl group is covalently linked to the hydroxyl group. q is an integer from 1 to 30.

[0123] Preferably, the method for preparing peptide P includes step (e): condensing a compound of formula LSI-am or LS-II-am with an α-amino acid derivative of formula T. R T-N-1 -(AA py ) q -OH (T) in: R T-N-1 It is an amino protecting group. -(AA py ) q - is composed of q condensed α-amino acid residues AA py The substituents formed are each y, where y is an integer and its value ranges from 1 to q. Each condensed α-amino acid residue AA py Independent selection, and in cases where it has a functional group-containing side chain, the functional group is either unprotected or protected by a protecting group, provided that the interfering functional group is protected. AA pq The carbon atom of its α-carbonyl group is covalently linked to the hydroxyl group. q is an integer from 1 to 30. To obtain compounds of formula TLSI or TLS-II (TLSI) (TLS-II), in: R L-O-1 R L-O-2 R SI-1 R SI-2 and R SII-2 As defined by formula LSI-am or LS-II-am R T-N-1 and -(AA py ) q -As defined by equation T.

[0124] AA p1 The α-amino group is R T-N-1 Protection. In step (e) X In ), AA pq The carbon atom of its α-carbonyl group is covalently linked to the hydroxyl group shown in formula T. AA pq Through its α-carbonyl carbon atom and step (d) X Int-P, an intermediate for deprotection in ) X The α-nitrogen atoms are covalently linked. In step (e), AA pq The carbon atom of its α-carbonyl group is covalently linked to the nitrogen atom shown in formula TLSI or TLS-II. Compounds of formula T contain an unprotected carboxylic acid group. This unprotected carboxylic acid group is formed by the amino acid residue AA. pq The α-carbonyl group and the hydroxyl group shown in formula T are formed. Interference step (e) X An example of a side-chain functional group condensed in step (e) is an amino group. Therefore, this amino group is protected by a protecting group. AA py Examples include those AA listed in step (c). nx .

[0125] Examples of compounds of formula T (q=1, R) T-N-1 =9-fluorenylmethoxycarbonyl, and AA p1 =Alanine residue) is Fmoc-Ala-OH as shown below: .

[0126] 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, even more particularly from 1 to 8, very particularly from 1 to 5, especially from 1 to 3, even more particularly from 1 to 2, and q is very particularly from 1.

[0127] To condense an unprotected α-amino group of a compound of formula LSI-am or LS-II-am, the carboxylic acid group of the compound of formula T is typically activated with the condensing agent of step (e). The condensing agent of step (e) results in the formation of an activated compound of formula T, in which the hydroxyl group at the carboxylic acid group is replaced by a leaving group, making the activated compound of formula T more reactive to the unprotected α-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 can be an intermediate suitable for separation at room temperature or an intermediate unsuitable for separation at room temperature. In the case where the compound of formula LSI-am or LS-II-am contains an unprotected carboxylic acid group, the activation of the compound of formula T must occur before contact with the compound of formula LSI-am or LS-II-am. The condensing agent of step (e) is, for example, a carbodiimide. This carbodiimide is, for example, diisopropylcarbodiimide. Preferably, the carbodiimide is applied together with the coupling additive of step (e). The coupling additive in step (e) is, for example, cyano-hydroxyimino-ethyl acetate.

[0128] The preferred method includes step (e): (e) A compound of formula LSI-am or LS-II-am is condensed with an α-amino acid derivative of formula T to obtain a compound of formula TLSI or TLS-II.

[0129] Preferably, the method comprises one or more further condensation cycles after the condensation in step (c) to extend the peptide Pr-LS obtained in step (c) by one or more other α-amino acid residues. The condensation cycle comprises a first step: removing the amino protecting group of the α-amino group of the N-terminal α-amino acid residue of the peptide to be extended, obtaining an unprotected amino group on the peptide. The amino group of the α-amino group of the N-terminal α-amino acid residue means that an imino group is also included, for example, in the case where the N-terminal α-amino acid residue of the peptide to be extended is proline. The condensation cycle further comprises a second step: condensing the unprotected amino group obtained in the first step with an α-amino acid derivative having an α-amino group protected by an amino protecting group at its N-terminal amino acid residue and an unprotected α-carboxylic acid group at its C-terminal amino acid residue, obtaining a peptide derived from the condensation cycle. An example of the first step is step (d). An example of the second step is step (e).

[0130] Optionally, the condensation step is followed by a capping step, i.e., treatment of the reaction mixture containing the peptide obtained in the condensation step with acetic anhydride. The capping step is performed as part of the condensation cycle before the removal of the amino protecting group, for example, as the first step of the amino protecting group removal process. Therefore, any potentially residual amino or imino groups that were not reacted in the condensation step are acetylated and become unreactable in the next condensation step.

[0131] A preferred method comprises performing one or more further condensation cycles on a compound of formula TLSI or TLS-II, each of the one or more further condensation cycles comprising: Step 1: Remove the amino protecting group R from compounds of formula TLSI or TLS-II. T-N-1 The amino protecting group of the α-amino group of the N-terminal amino acid residue of the peptide obtained in the previous condensation cycle, and the related peptide with an unprotected amino group, and Step 2: Condense the unprotected amino group of the relevant peptide obtained in Step 1 with an α-amino acid derivative, which has an α-amino group protected by an amino protecting group at its N-terminal amino acid residue and an unprotected α-carboxylic acid group at its C-terminal amino acid residue, to obtain a peptide derived from the condensation cycle.

[0132] In the case of α-amino acid derivative S-am covalently linked resin, step (c) X The intermediate peptide Int-P obtained in (preferred step (c)) X (Preferred peptide Pr-LS) is also covalently linked to the resin. This also applies to elongated peptides obtained in one or more further condensation cycles. The peptide obtained in the final condensation step can be cleaved from the resin by a cleavage composition to obtain a cleaved peptide. Whether the protecting groups of the elongated peptide are retained, partially removed, or completely removed on the cleaved peptide depends on the cleavage composition, reaction conditions, and the specific protecting group.

[0133] Preferably, step (c) X α-amino acid derivatives of S-am are covalently linked to resins, and peptide P is prepared. X The method includes steps (x) X ): (x X The peptide obtained in the final condensation step is cleaved from the resin by cleaving the cleavage composition to obtain the cleaved peptide.

[0134] Preferably, the α-amino acid derivative S-am in step (c) is covalently linked to the resin, and the method for preparing peptide P includes step (x): (x) The peptide obtained in the final condensation step is cleaved from the resin by cleaving the cleavage composition to obtain the cleaved peptide.

[0135] In step (c) XIn the case where the α-amino acid derivative S-am of step (c) is covalently linked to the resin via the carbonyl group of its C-terminal α-amino acid residue, the cleaved peptide contains a carboxylic acid group at its C-terminal α-amino acid residue, which has an unprotected α-carboxylic acid group or an unprotected α-carboxamide group.

[0136] Preferred preparation of peptide P X The method, wherein step (c) X The method comprises the step (x) of covalently linking the α-amino acid derivative S-am to a resin. X ): (x X The peptide obtained in the final condensation step is cleaved from the resin by cleaving the cleavage composition to obtain the cleaved peptide.

[0137] A preferred method for preparing peptide P, wherein the α-amino acid derivative S-am from step (c) is covalently linked to a resin, and the method includes step (x): (x) The peptide obtained in the final condensation step is cleaved from the resin by cleaving the cleavage composition to obtain the cleaved peptide.

[0138] Preferably, peptide P is prepared X The method includes steps (y) X ): (y X Remove all remaining protecting groups from the peptide obtained in the final condensation step to obtain a peptide without protecting groups.

[0139] Preferably, the method for preparing peptide P includes step (y): (y) Remove all remaining protecting groups from the peptide obtained in the final condensation step to obtain a peptide without protecting groups.

[0140] The removal of protecting groups is carried out using a deprotection composition. If the protecting groups of the peptides obtained in the final condensation step can all be removed by the same deprotection composition (e.g., all protecting groups are unstable to trifluoroacetic acid), step (y) X The removal of protection in step (x) or step (y) requires only one deprotection composition. In the case of the peptide covalently linked resin obtained in the final condensation step, when step (x) X The pyrolysis composition of step (x) or step (y) is also used as the pyrolysis composition of step (y). X When the deprotecting composition of step (y) acts on all protecting groups of the peptide obtained in the final condensation step, for example, when all protecting groups are unstable to trifluoroacetic acid and the covalent bonds with the resin linking groups are also unstable to trifluoroacetic acid, step (y) X ) or step (y) and step (x) XStep (x) or step (x) can occur simultaneously.

[0141] Preferred preparation of peptide P X A method, wherein the method includes step (y) X ): (y X Remove all remaining protecting groups from the peptide obtained in the final condensation step to obtain a peptide without protecting groups.

[0142] A preferred method for preparing peptide P, wherein the method includes step (y): (y) Remove all remaining protecting groups from the peptide obtained in the final condensation step to obtain a peptide without protecting groups.

[0143] An example of peptide P without a protecting group is smegglutinin (CAS No.: 910463-68-2), represented by a three-letter code for the α-amino acid residues (except for the condensed L-lysine residues, whose fatty acid coupling motifs are covalently linked to their ε-nitrogen atoms, shown as structural units of formula L). The α-amino acid residues are numbered from 1, representing the N-terminal condensed L-histidine residue, to 31, representing the C-terminal condensed glycine residue (corresponding to SEQ ID NO: 5): .

[0144] The above information pertains to the preparation of peptide P. X The methods and steps described and preferred embodiments are also applicable to other embodiments of the present invention.

[0145] A further embodiment of the present invention is a lysine derivative LYS. X It is a compound of the formula Pr-L (Pr-L), in: R L-O-1 and R L-O-2 Each group independently represents a carboxylic acid protecting group; Compounds of formula Pr-A (Pr-A), in: R A-N-1 It is different from R L-N-1 The amino protecting groups of 1-(4,4-dimethyl-2,6-dioxocyclohexylene)ethyl and 1-(4,4-dimethyl-2,6-dioxocyclohexylene)-3-methylbutyl R L-N-1 The amino protecting group of formula Bsmoc:

[0146] (Bsmoc) in: Indicates the bond with a nitrogen atom; Compound 310 (as shown above); Compound 511 (as shown above); Compound 514 (as shown above) or Compound 517 (as shown above).

[0147] Preferably, R in formula (Pr-A) A-N-1 It is tert-butyloxycarbonyl, namely compound 309 (as shown above).

[0148] Lysine derivative LYS X Preferably, compounds 508, 309, 310, 511, 514, or 517 are used. More preferably, lysine derivative LYS is used. X It is compound 508, compound 511, compound 514 or compound 517.

[0149] Preferred lysine derivative LYS X It is a compound of formula Pr-L, wherein R L-O-1 and R L-O-2 They are either tert-butyl or 3-methyl-pent-3-yl, which are independent of each other.

[0150] Preferred lysine derivative LYS X It is a compound of formula Pr-L, wherein R L-O-1 and R L-O-2 It is tert-butyl.

[0151] Preferred lysine derivative LYS X It is a compound of formula Pr-L, which is compound 508.

[0152] A further embodiment of the present invention is a lysine derivative LYS. X In peptide P X Applications in synthesis of this lysine derivative LYS X It is a compound of formula Pr-L (Pr-L), in: R L-O-1 and R L-O-2 Each group independently represents a carboxylic acid protecting group; Compounds of formula Pr-A (Pr-A), in: R A-N-1It is an amino protecting group that is different from Bsmoc, 1-(4,4-dimethyl-2,6-dioxocyclohexylene)ethyl and 1-(4,4-dimethyl-2,6-dioxocyclohexylene)-3-methylbutyl; Compound 310 (as shown above); Compound 511 (as shown above); Compound 514 (as shown above) or Compound 517 (as shown above).

[0153] Preferably, the lysine derivative LYS X In the synthesis of peptide P, the formula is Pr-L R. A-N-1 Compounds.

[0154] A further embodiment of the present invention is preparation formula 1. X Methods for compound (1 X ) in: n is 1 or 0.5. When n is 1, HY is hydrochloric acid, trifluoroacetic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, propanesulfonic acid, butyric acid, hydrobromic acid, hydroiodic acid, perchloric acid, trifluoromethanesulfonic acid, nitric acid, picric acid, trichloroacetic acid, camphorsulfonic acid, dichloroacetic acid, difluoroacetic acid, or phosphoric acid. When n is 0.5, HY is sulfuric acid. It includes the following steps (aa): Steps (aa) X ): Provides compound 118 (118); Steps (ab) X Adding HY to the provided compound 118 yields Formula 1 X Compounds.

[0155] A further embodiment of the present invention is Formula 1 X Applications of these compounds in peptide synthesis: (1 X ) in: n is 1 or 0.5. When n is 1, HY is hydrochloric acid, trifluoroacetic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, propanesulfonic acid, butyric acid, hydrobromic acid, hydroiodic acid, perchloric acid, trifluoromethanesulfonic acid, nitric acid, picric acid, trichloroacetic acid, camphorsulfonic acid, dichloroacetic acid, difluoroacetic acid, or phosphoric acid. When n is 0.5, HY is sulfuric acid. This peptide, for example, is a lysine derivative, LYS. X or peptide P X . Attached Figure Description

[0156] Figure 1 The complete HPLC-UV chromatogram of D-02-1 is shown, in which one of the two peaks represents compound 701 and the other is the decomposition product. Both peaks are labeled and their area percentages are listed.

[0157] Figure 2 Show Figure 1 The HPLC-UV chromatogram of D-02-1 is a partially magnified chromatogram between approximately 14 and 20 minutes, in which two peaks are marked, one of which represents compound 701, and their area percentages are listed.

[0158] Figure 3 The complete HPLC-UV chromatogram of D-02-2 is shown, in which the peak representing compound 702 is labeled and its area percentage is listed.

[0159] Figure 4 Show Figure 3 The HPLC-UV chromatogram of D-02-2 is a partially magnified chromatogram between approximately 10 and 22 minutes, in which the peak representing compound 702 is labeled and its area percentage is listed.

[0160] Figure 5 The complete HPLC-UV chromatogram of D-02-3 is shown, in which the peak representing compound 702 is labeled and its area percentage is listed.

[0161] Figure 6 Show Figure 5 The HPLC-UV chromatogram of D-02-3 is a partially magnified chromatogram between approximately 14 and 24 minutes, in which the peak representing compound 702 is labeled and its area percentage is listed.

[0162] Figure 7 The complete HPLC-UV chromatogram of D-02-4 is shown, in which the peak representing compound 702 is labeled and its area percentage is listed.

[0163] Figure 8 Show Figure 7 The HPLC-UV chromatogram of D-02-4 is a partially magnified chromatogram between approximately 11 and 22 minutes, in which the peak representing compound 702 is labeled and its area percentage is listed.

[0164] Figure 9 The complete HPLC-UV chromatogram of D-02-5 is shown, in which the peak representing compound 702 is labeled and its area percentage is listed.

[0165] Figure 10 Show Figure 9 The HPLC-UV chromatogram of D-02-5 is a partially magnified chromatogram between approximately 14 and 20 minutes, in which the peak representing compound 702 is labeled and its area percentage is listed.

[0166] Figure 11 The complete HPLC-UV chromatogram of D-02-6 is shown, in which the peak representing compound 702 is labeled and its area percentage is listed.

[0167] Figure 12 Show Figure 11 The HPLC-UV chromatogram of D-02-6 is a partially magnified chromatogram between approximately 14 and 20 minutes, in which the peak representing compound 702 is labeled and its area percentage is listed.

[0168] Figure 13 The complete HPLC-UV chromatogram of D-04-2 is shown, in which the peak representing compound 704 is labeled and its area percentage is listed.

[0169] Figure 14 Show Figure 13 The HPLC-UV chromatogram of D-04-2 is a partially magnified spectrum between approximately 10 and 22 minutes, in which the peak representing compound 704 is labeled and its area percentage is listed.

[0170] Figure 15 The complete HPLC-UV chromatogram of D-04-3 is shown, in which the peak representing compound 704 is labeled and its area percentage is listed.

[0171] Figure 16 Show Figure 15 The HPLC-UV chromatogram of D-04-3 is a partially magnified chromatogram between approximately 14 and 24 minutes, in which the peak representing compound 704 is labeled and its area percentage is listed.

[0172] Figure 17 The complete HPLC-UV chromatogram of D-04-4 is shown, in which the peak representing compound 704 is labeled and its area percentage is listed.

[0173] Figure 18 Show Figure 17 The HPLC-UV chromatogram of D-04-4 is a partially magnified chromatogram between approximately 11 and 22 minutes, in which the peak representing compound 704 is labeled and its area percentage is listed.

[0174] Figure 19The complete HPLC-UV chromatogram of D-04-5 is shown, in which the peak representing compound 704 is labeled and its area percentage is listed.

[0175] Figure 20 Show Figure 19 The HPLC-UV chromatogram of D-04-5 is a partially magnified spectrum between approximately 14 and 20 minutes, in which the peak representing compound 704 is marked and its area percentage is listed.

[0176] Figure 21 The complete HPLC-UV chromatogram of D-04-6 is shown, in which the peak representing compound 704 is labeled and its area percentage is listed.

[0177] Figure 22 Show Figure 21 The HPLC-UV chromatogram of D-04-6 is a partially magnified chromatogram between approximately 14 and 22 minutes, in which the peak representing compound 704 is labeled and its area percentage is listed.

[0178] Figure 23 The complete HPLC-UV chromatogram of D-06-1 is shown, in which the peak representing compound 706 is labeled and its area percentage is listed.

[0179] Figure 24 Show Figure 23 The HPLC-UV chromatogram of D-06-1 is a partially magnified spectrum between approximately 9 and 14 minutes, in which the peak representing compound 706 is labeled and its area percentage is listed.

[0180] Figure 25 The complete HPLC-UV chromatogram of D-07-1 is shown, in which the peak representing compound 707 is labeled and its area percentage is listed.

[0181] Figure 26 Show Figure 25 The HPLC-UV chromatogram of D-07-1 is a partially magnified spectrum between approximately 10 and 14 minutes, in which the peak representing compound 707 is labeled and its area percentage is listed.

[0182] Figure 27 The complete HPLC-UV chromatogram of D-08-1 is shown, in which the peak representing compound 708 is labeled and its area percentage is listed.

[0183] Figure 28 Show Figure 27 The HPLC-UV chromatogram of D-08-1 is a partially magnified spectrum between approximately 10 and 22 minutes, in which the peak representing compound 708 is labeled and its area percentage is listed.

[0184] Figure 29 The complete HPLC-UV chromatogram of D-09-1 is shown, in which the peak representing compound 709 is labeled and its area percentage is listed.

[0185] Figure 30 Show Figure 29 The HPLC-UV chromatogram of D-09-1 is a partially magnified chromatogram between approximately 14 and 24 minutes, in which the peak representing compound 709 is labeled and its area percentage is listed.

[0186] Figure 31 The complete HPLC-UV chromatogram of D-10-1 is shown, in which the peak representing compound 710 is labeled and its area percentage is listed.

[0187] Figure 32 Show Figure 31 The HPLC-UV chromatogram of D-10-1 is a partially magnified chromatogram between approximately 11 and 22 minutes, in which the peak representing compound 710 is labeled and its area percentage is listed.

[0188] Figure 33 The complete HPLC-UV chromatogram of D-11-1 is shown, in which the peak representing compound 711 is labeled and its area percentage is listed.

[0189] Figure 34 Show Figure 33 The HPLC-UV chromatogram of D-11-1 is a partially magnified chromatogram between approximately 14 and 22 minutes, in which the peak representing compound 711 is labeled and its area percentage is listed.

[0190] Figure 35 The complete HPLC-UV chromatogram of B-29-1 is shown, in which the peak representing compound 509 is labeled and its area percentage is listed.

[0191] Figure 36 The complete HPLC-UV chromatogram of B-30-1 is shown, in which the peak representing compound 511 is labeled and its area percentage is listed.

[0192] Figure 37 The complete HPLC-UV chromatogram of B-32-1 is shown, in which the peak representing compound 514 is labeled and its area percentage is listed.

[0193] Figure 38 The complete HPLC-UV chromatogram of B-33-1 is shown, in which the peak representing compound 515 is labeled and its area percentage is listed.

[0194] Figure 39 The complete HPLC-UV chromatogram of B-34-1 is shown, in which the peak representing compound 517 is labeled and its area percentage is listed.

[0195] Figure 40 The complete HPLC-UV chromatogram of B-35-1 is shown, in which the peak representing compound 518 is labeled and its area percentage is listed.

[0196] Figure 41 The complete HPLC-UV chromatogram of B-36-1 is shown, in which the peak representing compound 520 is labeled and its area percentage is listed.

[0197] sequence list Compound 701 KEFIAWLVRGRG (SEQ ID NO:1) Position 1 K = 2-(p-nitrobenzenesulfonyl)ethoxycarbonyl-lysine (17-carboxy-heptadecanoyl-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl) Compound 702 KEFIAWLVRGRG (SEQ ID NO:2) K at position 1 = lysine (17-carboxy-heptadecanoyl-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl) Compound 703 AKEFIAWLVRGRG (SEQ ID NO:3) A at position 1 = 9-fluorenylmethoxycarbonyl-alanine K at position 2 = lysine (17-carboxy-heptadecanoyl-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl) Compound 704 AKEFIAWLVRGRG (SEQ ID NO:4) K at position 2 = lysine (17-carboxy-heptadecanoyl-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl) Compound 705 (smegglutinin) HXEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO:5) X at position 2 = Aib (α-aminoisobutyric acid) K at position 20 = lysine (17-carboxy-heptadecanoyl-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl) Compound 706 FIAWLVRGRG (SEQ ID NO:6) Compound 707 EFIAWLVRGRG (SEQ ID NO:7) Compound 708 KEFIAWLVRGRG (SEQ ID NO:8) K at position 1 = α-azido-lysine (17-carboxy-heptadecanoyl-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl) Compound 709 KEFIAWLVRGRG (SEQ ID NO:9) Position 1 K=9-fluorenylmethoxycarbonyl-lysine (17-carboxy-heptadecanoyl-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl) Compound 710 KEFIAWLVRGRG (SEQ ID NO:10) Position 1 K = 2-(benzenesulfonyl)ethoxycarbonyl-lysine (17-carboxy-heptadecanoyl-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl) Compound 711 KEFIAWLVRGRG (SEQ ID NO:11) K=1,1-dioxobenzo[b]thiophene-2-yl-methoxycarbonyl-lysine (17-carboxy-heptadecanoyl-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl) at position 1 Detailed Implementation Example A) Overview A-1) Abbreviation %: Unless otherwise specified, percentage values ​​refer to weight percentage. ACN: Acetonitrile AcOH: Acetic acid Aib: 2-Aminoisobutyric acid Bsmoc: 1,1-dioxobenzo[b]thiophene-2-ylmethoxycarbonyl (also commonly referred to as: benzo[b]thiophene sulfone-2-methoxycarbonyl) Boc: tert-Butoxycarbonyl CAD: Electro-fog detector d: sky DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene DEPBT: 3-(diethoxyphosphoryloxy)-3H-benzo[d][1,2,3]triazine-4-one DIC: N,N'-Diisopropylcarbodiimide DIPEA: Diisopropylethylamine DMF: N,N-dimethylformamide EDT: Ethane-1,2-dithiol EtOAc: Ethyl acetate Fmoc: 9-fluorenylmethoxycarbonyl HPLC: High Performance Liquid Chromatography HR-MS: High-resolution mass spectrometry IPA: Isopropyl alcohol IPC: Process Control IPE: Diisopropyl Ether MeTHF: 2-Methyltetrahydrofuran Mpe: 3-Methyl-pentan-3-yl MS: Mass Spectrometry MTBE: Methyl tert-butyl ether N3: Azide group NMP: N-methylpyrrolidone Nsc: 2-(p-nitrobenzenesulfonyl)ethoxycarbonyl OxymaPure (TM): Cyano-hydroxyimino-ethyl acetate Pbf: 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-ylsulfonyl Psc: 2-(benzenesulfonyl)ethoxycarbonyl Psi(Me,Me)pro: 1,1-Dimethylmethylene, covalently bonded to the β-oxygen atom of serine or threonine, and covalently bonded to the α-nitrogen atom of serine or threonine (pseudoproline). RT: Room temperature TBTU: (benzotriazolyl)tetramethylurea tetrafluoroborate tBu: tert-butyl TFA: Trifluoroacetic acid TIS: Triisopropylsilane TLC: Thin-layer chromatography Trt: Triphenylmethyl TsOH: 4-Toluenesulfonic acid UV: Ultraviolet vol.%: Volume percentage Unless otherwise specified, all α-amino acids except glycine are L-type. Room temperature in this text refers to a temperature between 22°C and 25°C.

[0198] A-2) Chemicals All reagents and solvents were obtained directly from the standard suppliers of raw materials for peptide synthesis.

[0199] A-3) Analytical Methods A-3-1: HPLC Method 1 HPLC Method 1 is an 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 at a flow rate of 0.4 mL / min. UV detection (220 nm) or CAD detection was performed at a column temperature of 25°C. Buffer A was an ACN / water (1:99 v / v) solution containing 0.05 vol.% TFA, and buffer B was an ACN solution containing 0.05 vol.% TFA.

[0200] In some embodiments, gradient procedures and detection methods are specified. In some instances, one of the following general modes A to F is specified: Mode A: 0 min 20% B - 5 min 100% B - 7 min 100% B, UV detection Mode B: 0 min 5% B - 5 min 100% B - 7 min 100% B, UV detection Mode C: 0 min 80% B - 5 min 100% B - 7 min 100% B, CAD inspection Mode D: 0 min 20% B - 5 min 100% B - 7 min 100% B, UV inspection and CAD inspection Mode E: 0 min 0% B - 5 min 100% B - 7 min 100% B, UV detection Mode F: 0 min 30% B - 3 min 72% B - 5 min 100% B - 9 min 100% B, UV detection A-3-2: HPLC Method 2 HPLC Method 2 is an 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 × 150 mm) column at a flow rate of 0.4 mL / min and UV detection (220 nm) at a column temperature of 50°C. For mass spectrometry analysis, MS analysis was performed on a UHPLC coupled with a Bruker™ maXis II™ spectrometer, which features ultra-high resolution QTOF technology and is equipped with electron transfer dissociation (ETD) functionality. Buffer A was an ACN / water (1:99 v / v) solution containing 0.05 vol.% TFA, and buffer B was an ACN solution containing 0.05 vol.% TFA.

[0201] Gradient program of HPLC method 2

[0202] A-3-3: HPLC Method 3 HPLC Method 3 is an analytical HPLC procedure performed on a Thermo Scientific™ Vanquish™ UHPLC system or a Dionex™ Ultimate™ 3000 RS UHPLC system using an AQUITY™ UPLC BEH C18, 130 A (1.7 μm, 2.1 × 150 mm) column at a flow rate of 0.4 mL / min and UV detection (220 nm) at a column temperature of 25°C. Buffer A is an ACN / water (1:99, v / v / v) solution containing 0.05 vol.% TFA, and buffer B is an ACN solution containing 0.05 vol.% TFA.

[0203] Gradient program of HPLC method 3

[0204] A-4) General Synthesis Procedures A-4-1: General Operating Procedures for Condensing Lysine Derivatives 1 200 mg of compound 612-SP (0.22 mmol / g, 0.044 mmol) (as shown in Example C-06-1) was pre-swollen twice in DMF (10 mL / g resin), each time for 15 minutes at room temperature. The corresponding lysine derivative (0.088 mmol, 2.0 eq.) and OxymaPure (19.4 mg, 0.136 mmol, 3.1 eq.) were dissolved in DMF (10 mL / g resin). 17.7 µL of DIC (0.114 mmol, 2.6 eq.) was added to this solution. The solution was stirred at room temperature for 15 minutes and then added to the reaction mixture. After reacting for 20 minutes at room temperature, another 8.8 µL of DIC (0.057 to 0.143 mmol, 1.3 eq.) was added to the reaction mixture, and the mixture was generally stirred at room temperature for 24 hours. After removing the liquid portion, the resin material was washed three times alternately with DMF / IPA, and then three times with IPA (10 mL / g resin per washing step). The washed peptide material was then dried under high vacuum at room temperature for 24 hours to obtain the corresponding product of the condensation of lysine derivative and compound 612-SP.

[0205] For analytical purposes, small-scale test lysis can be performed.

[0206] A-4-2: General Operating Procedures for Testing Pyrolysis 2 In a 2 mL plastic syringe (50 to 100 mg peptide resin), treat the peptide resin at room temperature for 2 hours with 0.5 to 1.0 mL of lysis mixture (TFA / TIS / H2O = 90 / 5 / 5, based on volume) to lyse the peptide from the resin. Then, precipitate the lysed peptide in solution in 5 to 10 mL of cold IPE (< -15°C), centrifuge, and wash twice more with cold IPE. The resulting crude peptide (usually a solid) is dried under high vacuum.

[0207] A-4-3: General operating procedure 3 for removing Fmoc after Fmoc-Ala-OH condensation The procedure is described using 200 mg of compound 614-SP as the starting material. This procedure can be adjusted (reduced or increased) depending on the amount of starting material.

[0208] 200 mg of compound 614-SP (0.21 mmol / g resin) (as shown in Example C-08-1) was pre-swelled twice in DMF (10 mL / g resin), each time for 15 minutes at room temperature. Pre-swelling can be omitted if compound 614-SP is already in contact with DMF. 27.7 mg of Fmoc-Ala-OH (0.084 mmol, 2.0 eq.) and 18.5 mg of OxymaPure (0.130 mmol, 3.1 eq.) were dissolved in 2.0 mL of DMF. 16.9 µL of DIC (0.109 mmol, 2.6 eq.) was added to this solution, and the solution was stirred at room temperature for 15 minutes before being added to the pre-swelled or DMF-contacted compound 614-SP. After reacting at room temperature for 20 minutes, 8.5 µL of DIC (0.059 mmol, 1.3 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature for 17 hours. The liquid fraction was removed, and the resin material was washed three times with alternating DMF / IPA, followed by three washes with IPA (10 mL / g resin per wash step) to obtain compound 615-SP (as shown in Example C-09-1).

[0209] Compound 615-SP was treated twice in DMF with 20 vol.% piperidine (10 mL / g resin), the first treatment lasting 20 minutes and the second treatment lasting 60 minutes. Afterwards, the resin material was washed three times alternately with DMF / IPA, and then three times with IPA (10 mL / g resin per wash). The washed resin material could be used for further reactions without drying, or it could be dried. A portion of the washed resin material was dried under high vacuum at room temperature for 24 hours to obtain compound 616-SP.

[0210] For analytical purposes, a small-scale cleavage test can be performed to obtain compound 704 (as shown in Example D-04-1).

[0211] B) Synthesis of lysine derivatives and some of their starting materials Example B-01-1: Synthesis of Compound 101 (101) For example, compound 101 can be derived from compound 102 (CAS No.: 21386-32-3). (102) With compound 103 (CAS No.: 7693-46-1) (103) The condensation reaction was obtained, referring to molecule II in International Journal Peptide Research (1975), Vol. 7, pp. 295-305.

[0212] Example B-02-1: Synthesis of Compound 104 (104) For example, compound 104 can be derived from compound 105 (CAS No.: 102093-85-6). (105) With compound 106 (CAS No.: 22325-27-5) (106) The condensation reaction was used to obtain molecule 10, as described in US Patent 3791830.

[0213] Example B-02-2: Synthesis of Compound 104 For example, compound 104 can be derived from compound 102 (CAS No.: 21386-32-3). (102) With compound 107 (CAS No.: 41840-26-0) (107) The condensation reaction was obtained, referring to Example 5 of US Patent 3936452.

[0214] Example B-03-1: Synthesis of Compound 108 (108) For example, compound 108 can be obtained by the condensation reaction of compound 105 (CAS No.: 102093-85-6) with N-hydroxyphthalimide, see molecule IVb in International Journal Peptide Research (1975), Vol. 7, pp. 295-305.

[0215] Example B-04-1: Synthesis of Compound 109 (109) Compound 109 is a hydrochloride salt (WO 2002-098903 A1, listed as Nsc-Lys-OH in Table 1 / Item 19), for example, through compound 110 (CAS No.: 160422-23-1). (110) It is obtained by deprotection reaction with dioxane hydrochloride solution.

[0216] Example B-05-1: Synthesis of Compound 110 (110) For example, compound 110 (CAS No.: 160422-23-1) can be obtained as described in Example 3f of Tetrahedron Letters (1994), Vol. 35, No. 42, pp. 7821-7824, i.e., by compound 111 (CAS No.: 2418-95-3). (111) A protecting reaction with chlorotrimethylsilane yielded compound 112, as shown in Scheme 1 in *Tetrahedron Letters* (1994), Vol. 35, No. 42, pp. 7821-7824. (112), Compound 112 was then reacted with compound 105 (CAS No.: 102093-85-6).

[0217] Example B-05-2: Synthesis of Compound 110 For example, compound 110 (CAS No.: 160422-23-1) can be obtained by the protective reaction of compound 111 (CAS No.: 2418-95-3) with compound 101, see International Journal Peptide Research (1975), Vol. 7, pp. 295-305, p. 302 / General Procedure B.

[0218] Example B-05-3: Synthesis of Compound 110 For example, compound 110 (CAS No.: 160422-23-1) can be obtained by a protective reaction of compound 111 (CAS No.: 2418-95-3) with compound 104, as described in Example 26 of US Patent 3936452.

[0219] Example B-05-4: Synthesis of Compound 110 For example, compound 110 (CAS No.: 160422-23-1) can be obtained by the protective reaction of compound 111 (CAS No.: 2418-95-3) with compound 108, according to the solid-phase synthesis method of Example 7 of WO 2004-065412.

[0220] Example B-06-1: Synthesis of Compound 201 (201) For example, compound 201 can be derived from compound 202 (CAS No.: 1069067-08-8). (202) It was obtained by condensation reaction with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide, as described in Example 3c of CN113121627 A.

[0221] Example B-06-2: Synthesis of Compound 201 3.27 g of compound 202 (8.0 mmol) was dissolved in 12 mL of MeTHF. 1.02 g of N-hydroxysuccinimide (8.9 mmol) was added to the solution. Then, a solution of 1.36 g of DIC (8.8 mmol) dissolved in 4 mL of MeTHF was added. The resulting solution was stirred at room temperature for 7 hours to obtain a solution containing compound 201. This solution was used directly as a pre-activated carboxyl compound for the coupling reaction in Example B-28-1.

[0222] Example B-07-1: Synthesis of Compound 301 (301) For example, referring to step 3b of EP 3819308 A, compound 113 is obtained by the protective reaction of compound 109 with chlorotrimethylsilane. (113), Following step 3b of EP 3819308 A, compound 113 is reacted with compound 201 to prepare compound 301.

[0223] Example B-08-1: Synthesis of Compound 302 (302) Compound 302 is a hydrochloride salt, which can be obtained, for example, by deprotection reaction of compound 301 with dioxane hydrochloride solution.

[0224] Example B-09-1: Synthesis of Compound 303 (303) Compound 303 is a hydrochloride salt, for example, that can be obtained from compound 304 (CAS No.: 1662688-18-7). (304) It is obtained by deprotection reaction with dioxane hydrochloride solution.

[0225] Example B-10-1: Synthesis of Compound 501 (501) For example, compound 501 (CAS No.: 1662688-20-1) can be prepared as described in Example 9 of CN104356224 A, i.e., by compound 308 (CAS No.: 1662688-19-8). (308) With compound 203 (CAS No.: 1188328-22-4) (203) It is prepared by coupling reaction.

[0226] Example B-10-2: Synthesis of Compound 501 For example, compound 501 (CAS No.: 1662688-20-1) can be obtained by reacting compound 303 (a hydrochloride salt) with trichloromethylsilane to obtain compound 305. (305) [Note: The structure of compound 305 is shown as -NH2x HCl, but there may also be compounds of 305 containing -NH-Si(CH3)3 or -NH-Si(CH3)3x HCl—this issue remains unresolved.] Compound 305 was then reacted with compound 203 (CAS No.: 1188328-22-4) to prepare compound 501 (CAS No.: 1662688-20-1), following step 3b of EP 3819308 A1.

[0227] Example B-10-3: Synthesis of Compound 501 For example, compound 501 (CAS No.: 1662688-20-1) can be prepared as described in Example 6 of WO 2021-205388 A2.

[0228] Example B-10-4: Synthesis of Compound 501 4.1 mL of ACN, 4.1 mL of 1,4-dioxane, and 1.0 mL of trichloromethylsilane (7.9 mmol) were added to 2.0 g of compound 304 (80% purity, 2.1 mmol). The reaction mixture was heated to 40°C and maintained for 15 minutes to obtain a solution containing compound 312. (312).

[0229] 3.0 mL of a 5.7 mol / L solution of 1,4-dioxane hydrochloride (17 mmol) was added to the solution. The resulting reaction mixture was kept at 40°C for 1.5 hours. The solvent was then removed under vacuum, yielding an oily residue, which was then removed twice by co-evaporation with 20 mL of ACN to remove excess HCl. The resulting oily residue was dissolved in 20 mL of a 1:1 mixture of ACN and 1,4-dioxane. This solution contained the compound... (313) [Note: The structure of compound 313 is shown as -NH2x HCl, but there may also be compounds of 313 containing -NH-Si(CH3)3 or -NH-Si(CH3)3x HCl—this issue is still unresolved.] Compound 313 is a silylated amine compound used in subsequent coupling reactions.

[0230] For the coupling reaction, 1.24 g of compound 203 (1.9 mmol) and 0.4 mL of DIPEA (2.3 mmol) were added to a solution containing a silylamine compound at room temperature. The reaction mixture was stirred for 17 hours, followed by the addition of 0.6 mL of DIPEA (3.4 mmol) and 0.07 g of compound 203 (0.1 mmol). Stirring was continued for another 4 hours. The reaction mixture was diluted with 12 mL of IPE and 3 mL of water, and acidified with 1 mL of 5.5 N hydrochloric acid. The phases were separated, and the organic phase was washed four times with 3 mL of water each time, and concentrated under vacuum to obtain a viscous oil. This oil was dissolved three times with 20 mL of EtOAc and concentrated again under reduced pressure to give 1.72 g (68% yield) of compound 501 as a viscous resin.

[0231] HPLC Method 1 Mode F: 91.3% area, retention time 8.19 min.

[0232] Mass spectrometry (MS): Mass-to-charge ratio m / z = 1196.7374 [M+H] + .

[0233] Example B-11-1: Synthesis of Compound 502 (502) For example, compound 502 (CAS No.: 2682856-38-6) can be prepared as described in molecule 10 of scheme 3 in Organic Process Research and Development (2021), 25(7), pp. 1598-1611, i.e., compound 306 (CAS No.: 1118767-16-0). (306) With compound 114 (CAS No.: 21512-99-2) (114) A coupling reaction was performed to obtain compound 503 (CAS No.: 2682856-37-5). (503), Compound 503 was then treated with tetrasodium ethylenediaminetetraacetic acid (EDTA) to induce dechelation.

[0234] Example B-11-2: Synthesis of Compound 502 For example, compound 502 (CAS No.: 2682856-38-6) can be obtained by deprotecting compound 501 (CAS No.: 1662688-20-1) with piperidine.

[0235] Example B-11-3: Synthesis of Compound 502 100 g of compound 501 (84 mmol) was dissolved in 600 mL of ACN (40°C), and the clear solution was cooled to room temperature. 33 mL of piperidine was added to the solution, and the resulting suspension was stirred for 30 minutes. Then, 100 mL of ACN was added, and stirring 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, followed by two times with 300 mL of ACN. The solid was dried under vacuum overnight at 35°C. The resulting crude product (78 g) was dissolved at 50°C in a mixture of 600 mL of ACN and 85 mL of water. 200 mL of the resulting solution was evaporated under reduced pressure using a rotary evaporator. 400 mL of ACN was added to the formed emulsion, and the same volume was evaporated under reduced pressure. This addition / evaporation cycle was repeated twice. The resulting suspension was then diluted with 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. 73.6 g (76 mmol, 90%) of compound 502 was obtained as a white solid.

[0236] HPLC Method 1 (including CAD detection): 98.7% area, retention time 6.19 minutes.

[0237] Gradient program of HPLC method 1

[0238] Example B-12-1: Synthesis of Compound 504 (504) For example, compound 504 can be obtained by a protective reaction of compound 502 (CAS No.: 2682856-38-6) and compound 105 (CAS No.: 102093-85-6), as described in Example 1 of US Patent 5616788.

[0239] Example B-12-2: Synthesis of Compound 504 For example, compound 504 can be obtained by reacting compound 502 (CAS No.: 2682856-38-6) with trichlorosilane to obtain compound 505. (505), Compound 505 was then reacted with compound 105 (CAS No.: 102093-85-6) according to Example 3 of US Patent US6165590.

[0240] Example B-12-3: Synthesis of Compound 504 For example, compound 504 can be synthesized from compound 502 (CAS No.: 2682856-38-6) and compound 115 (CAS No.: 122865-54-7). (115) The protective reaction was obtained by solid-phase synthesis as described in Example 7 of WO 2004-065412.

[0241] Example B-12-4: Synthesis of Compound 504 For example, compound 504 can be obtained by the protective reaction of compound 502 (CAS No.: 2682856-38-6) with compound 101, see International Journal Peptide Research (1975), Vol. 7, pp. 295-305, p. 302 / General Procedure B.

[0242] Example B-12-5: Synthesis of Compound 504 For example, compound 504 can be obtained by a protective reaction of compound 502 (CAS No.: 2682856-38-6) with compound 104, as described in Example 26 of US Patent 3936452.

[0243] Example B-12-6: Synthesis of Compound 504 For example, compound 504 can be obtained by the protective reaction of compound 502 (CAS No.: 2682856-38-6) with compound 108, according to the solid-phase synthesis method in Example 7 of WO 2004-065412.

[0244] Example B-12-7: Synthesis of Compound 504 For example, compound 504 can be obtained by reacting compound 302 (a hydrochloride salt) with trichloromethylsilane to obtain compound 307. (307), [Note: The structure of compound 307 is shown as -NH2x HCl, but there may also be compounds of 307 containing -NH-Si(CH3)3 or -NH-Si(CH3)3x HCl—this issue remains unresolved.] Compound 307 was then reacted with compound 203 (CAS No.: 1188328-22-4) according to step 3b of EP 3819308 A1.

[0245] Example B-12-8: Synthesis of Compound 504 19.5 g of compound 502 (20 mmol, 1.0 eq.) was suspended in 60 mL of ACN, and then 8.1 mL of N-methyl-N-trimethylsilylacetamide (50 mmol, 2.5 eq.) was added. The mixture was heated to 40°C to form a clear solution, and then cooled to room temperature. 300 mL of DMF and 1.6 mL of pyridine (20 mmol, 1.0 eq.) were added to the solution, followed by 8.5 g of compound 101 (20 mmol, 1.0 eq.) over 30 minutes. After 5 hours, 0.8 mL of pyridine (10 mmol, 0.5 eq.) and 8.5 g of compound 101 (20 mmol, 1.0 eq.) were added, and the mixture was stirred continuously for 17 hours. 120 mL of 0.25 N dilute hydrochloric acid was added to the mixture, and the solvent was evaporated under reduced pressure at 45°C. The resulting thin oily substance was dissolved in 250 mL of EtOAc and transferred to a separatory funnel. 50 mL of water was added, and the pH was adjusted to 1–2 with a 5.5 N hydrochloric acid aqueous solution. The phases were separated; the organic phase was washed three times with 50 mL of water and then concentrated under reduced pressure to obtain a viscous oily substance. The purity of this viscous oily substance was 88.1% by area, according to HPLC method 1 and the gradient procedure described below. The purity would have been even lower with extraction using an ideal sodium bicarbonate aqueous solution (the latter has been omitted). It is speculated that the Nsc protecting group has a high base sensitivity in the current molecular structure, which was already apparent upon contact with pyridine and exacerbated during the initial attempt at extraction with sodium bicarbonate aqueous solution. Purification of the viscous oily substance using a first preparative reversed-phase HPLC (C4 column, 0.1% TFA, gradient from 3 vol.% ACN / water to 100 vol.% ACN) resulted in partial decomposition during freeze-drying after collection and combining of fractions. It is speculated that the TFA content has an adverse effect. The lyophilized material was purified using a second, TFA-free preparative HPLC (C4 column, TFA-free, gradient from 3 vol.% ACN / water to 100 vol.% ACN). The fractions were collected and combined, and then lyophilized to give 2.1 g of compound 504 (1.7 mmol, 9% yield).

[0246] HPLC Method 1 (including CAD detection): 99.4% area, retention time 5.14 minutes.

[0247] Gradient program of HPLC method 1

[0248] Example B-13-1: Synthesis of Compound 117 (117) For example, compound 117 can be derived from compound 116 (CAS No.: 20611-21-6). (116) With compound 103 (CAS No.: 7693-46-1) (103) The condensation reaction was obtained, referring to molecule II in International Journal Peptide Research (1975), Vol. 7, pp. 295-305.

[0249] Example B-14-1: Synthesis of Compound 506 (506) Preparation of azide reagents 23.35 g of sodium azide (359 mmol, 10 eq.) was dissolved in 82 mL of water and cooled in an ice bath. While maintaining an internal temperature below 5°C, 12.1 mL of trifluoromethanesulfonic anhydride (72 mmol, 2.0 eq.) dissolved in 135 mL of cyclohexane was slowly added to the cooled solution over 35 minutes. The reaction mixture was stirred in an ice bath for 2 hours 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 phases were used as the azide reagent for subsequent azide reactions.

[0250] Azide reaction 35.0 g of compound 502 (36 mmol, 1.0 eq.) and 8.99 g of potassium bicarbonate (90 mmol, 2.5 eq.) were suspended in a mixture of 65 mL water and 130 mL methanol. The mixture was heated to 35°C until the remaining solid dissolved. 90 mg of CuSO4·5H2O (0.4 mmol, 0.01 eq.) was added at 20°C, followed by the addition of the azide reagent solution to the mixture over 30 minutes. The resulting emulsion was diluted with 56 mL of dioxane and stirred for 20 hours. 130 mL of cyclohexane and 50 mL of dioxane were added, and the phases were separated in a separatory funnel. The aqueous phase was extracted with 3 x 240 mL IPE and 3 x 250 mL MTBE. 400 mL of 5-methyltetrahydrofuran and 200 mL of 5% sodium bicarbonate aqueous solution were added to the aqueous phase. The phases were separated, and the organic phase was repeatedly extracted with 5% sodium bicarbonate aqueous solution, dilute hydrochloric acid aqueous solution, and water. The organic phase was evaporated under reduced pressure until a white oily substance was obtained, which was then dissolved in 250 mL of 5-methyl-tetrahydrofuran and evaporated again under reduced pressure. This operation was repeated four times to give a yellow viscous oily substance, which was dried under vacuum overnight. 29.5 g of compound 506 (29.5 mmol, yield 82%) was obtained as a yellow viscous oily substance.

[0251] HPLC Method 1 (including CAD detection): 94.8% area, retention time 7.40 minutes.

[0252] Gradient program of HPLC method 1

[0253] Example B-15-1: Synthesis of Compound 507 (507) Following the preparation method of compound 508 in Example B-16-1, compound 507 was prepared from 19.5 g of compound 502, 9.6 mL of N-methyl-N-trimethylsilylacetamide (60 mmol, 3.0 eq.), and 8.3 g of compound 117 (20 mmol, 1.0 eq.). 22.8 g of compound 507 (19.2 mmol, 96% yield) in a viscous oily state was obtained.

[0254] HPLC Method 1 (including CAD detection): 99.3% area, retention time 7.11 minutes.

[0255] Gradient program of HPLC method 1

[0256] Example B-16-1: Synthesis of Compound 508 (508) 15.4 g of compound 502 (15.8 mmol, 1.0 eq.) was suspended in 47 mL of ACN, and then 6.3 mL of N-methyl-N-trimethylsilylacetamide (39.5 mmol, 2.5 eq.) was added. The mixture was heated to 40°C and held for 2 hours to form a clear solution, and then cooled to room temperature. 107 mL of ACN and 2.5 mL of pyridine (36.3 mmol, 2.3 eq.) were added to the solution, and the mixture was cooled to 0°C. While maintaining the internal temperature at 0°C, 4.71 g of 1,1-dioxobenzo[b]thiophene-2-ylmethoxycarbonyl chloride (CAS No.: 135204-19-2, 18.2 mmol, 1.15 eq.) dissolved in 178 mL of CAN was added over 30 minutes. Next, 0.4 mL of pyridine (5 mmol, 0.3 eq.) and 0.6 g of 1,1-dioxobenzo[b]thiophene-2-ylmethoxycarbonyl chloride (CAS No.: 135204-19-2, 2.3 mmol, 0.15 eq., also known as Bsmoc-Cl) dissolved in 26 mL of CAN were added, and the mixture was stirred at 0°C for 15 hours. The reaction mixture was then transferred to a separatory funnel, 1400 mL of MTBE and 320 mL of water were added, and the mixture was acidified with 1 N hydrochloric acid aqueous solution. The phases were separated, and the organic phase was washed three times with 350 mL of dilute hydrochloric acid aqueous solution, followed by washing with 320 mL of water. The organic phase was concentrated under reduced pressure to obtain a crude product that was a viscous oil. The crude product was dissolved at 35°C in a mixture of 170 mL of water, 170 mL of ACN, and 170 mL of IPE, and the solution was transferred to a separatory funnel. Add 31 mL of 10% sodium bicarbonate aqueous solution to separate the phases. Wash the aqueous phase with a mixture of 340 mL of IPE ether and ACN, separate the phases, acidify the aqueous phase with 5.5 N hydrochloric acid aqueous solution, and then extract with 1200 mL of MTBE. Separate the phases, and wash the organic phase with 250 mL of dilute hydrochloric acid aqueous solution and 250 mL of water. Evaporate the organic phase under reduced pressure, and dry the resulting oil under vacuum to give 10.6 g of compound 508 (8.8 mmol, yield 56%) as a very viscous oil.

[0257] HPLC Method 1 (including CAD detection): 97.7% area, retention time 7.16 minutes.

[0258] Gradient program of HPLC method 1

[0259] Example B-16-2: Synthesis of Compound 508 50 mL of 1,4-dioxane and 2.0 mL of trichlorosilane were added to 3.5 g of compound 310 (5.0 mmol) at room temperature. The mixture was heated to 40°C with stirring. After 1 hour, the reaction mixture was cooled to 20°C. The reaction mixture contained compound 311. (311) [Note: The structure of compound 311 is shown as -NH2x HCl, but there may also be compounds with the structure -NH-Si(CH3)3 or -NH-Si(CH3)3x HCl—this issue is still unresolved.] Compound 311 is a silylated amine compound used in subsequent coupling reactions.

[0260] For the coupling reaction, 2.68 mL of compound 203 (4.0 mmol) and 3.5 mL of DIPEA (20 mmol) were added to the reaction mixture containing the silylamine compound. Another 2.25 mL of DIPEA (13 mmol) was added over 4 hours. The reaction mixture was stirred overnight at room temperature. The reaction mixture was then diluted with 250 mL of MTBE and quenched with 30 mL of 0.5 N hydrochloric acid aqueous solution. 100 mL of ACN was added to separate the phases. The organic phase was washed with 3 x 25 mL of deionized water and concentrated under vacuum to give 5.5 g of crude product as a viscous oil. The crude product was purified by 75 g silica gel column chromatography (eluent changes from EtOAc to EtOAc / MeOH 4:1 and then to EtOAc / MeOH / H2O 5:4:1) to give 2.66 g (54% yield) of compound 508 as a white foam.

[0261] HPLC Method 1 Mode D: 98.1 area % (220 nm) and 92.2 area % (CAD), retention times 7.11 min (220 nm) and 7.19 min (CAD).

[0262] MS: m / z = 1196.6659 [M+H] + , 2392.3167 [2M+H] + .

[0263] Example B-17-1: Synthesis of Compound 118 (118) 68.4 g of compound 111 (278 mmol, CAS No.: 2418-95-3, also known as H-Lys(Boc)-OH) was suspended in 1000 mL of water, and 29.4 g of Na2CO3 (278 mmol) and 550 mL of acetone were added to the suspension. At room temperature, 89.0 g of compound 119 (264 mmol, CAS No.: 197244-91-0, also known as Bsmoc-OSu) was added in portions over 2 hours. (119), The solution was stirred overnight, and the resulting suspension was filtered through a G4 suction filter. The clarified filtrate was concentrated using a rotary evaporator to remove acetone, and 1.7 L MeTHF / 340 mL water / 550 mL 1N HCl aqueous solution was added to separate the phases. The organic phase was washed with 680 mL water / 100 mL 1N HCl aqueous solution, followed by washing with 850 mL water. The resulting organic phase was concentrated under vacuum to a thin oily state, and 4 x 1 L MeTHF was added, with each addition being concentrated to a thin oily state, yielding 161.3 g (124%; crude product containing residual solvent) of a beige foamy compound 118 (CAS No.: 197245-37-7, also known as Bsmoc-Lys(Boc)-OH). This material was used directly in the subsequent deprotection step.

[0264] HPLC method 1, Model A: 96.9% area, retention time 3.17 min.

[0265] MS: m / z = 469.1660 [M+H] + , 954.3512 [2M+NH4] + .

[0266] Example B-18-1: Synthesis of Compound 120 (120) 1.6 L of 3.4 M HCl / EtOAc solution was added to 159 g of compound 118 (up to 278 mmol, also known as Bsmoc-Lys(Boc)-OH) from Example B-17-1. The resulting suspension was stirred at room temperature for 2 hours, then filtered through a G3 suction filter and washed with 3 x 250 mL EtOAc. The resulting solid was dried under vacuum overnight to give 94.3 g (88%) of compound 120 (also known as Bsmoc-Lys-OH x HCl) as a white powder; compound 120 is a hydrochloride salt.

[0267] HPLC Method 1 Mode B: 98.6% area, retention time 2.60 min.

[0268] MS: m / z = 369.1111 [M+H] + 737.2152 [2M+H] + .

[0269] Example B-18-1: Synthesis of Compound 122 (122) 1.0 g of compound 120 (2.0 mmol, also known as Bsmoc-Lys-OH x HCl) was passed through an ion exchange column containing a strongly basic ion exchange resin (acetate form). Elution with 20% aqueous acetic acid yielded a solution containing compound 121 (also known as Bsmoc-Lys-OH x AcOH), which is an acetate. (121).

[0270] The solution was freeze-dried, the freeze-dried product was dissolved in 30 mL of deionized water, and then freeze-dried a second time to obtain 0.82 g of compound 122 (also known as Bsmoc-Lys-OH) as a white solid.

[0271] The chloride test was negative, and the residual acetic acid content was 5.2 wt.%.

[0272] HPLC Method 1 Mode B: 93.5% area, retention time 2.60 min.

[0273] Example B-19-1: Synthesis of Compound 123 (123) 5.0 g of compound 124 (10.7 mmol, CAS No.: 71989-26-9, also known as Fmoc-Lys(Boc)-OH) was added. (124) Dissolved in 50 mL of 3.4 M HCl / EtOAc solution. After stirring at room temperature for 2 hours, the resulting suspension was filtered through a G3 suction filter and washed with 3 x 25 mL EtOAc. The resulting solid was dried under vacuum overnight to give 4.0 g (92%) of compound 123 (CAS No.: 139262-23-0, also known as Fmoc-Lys-OH x HCl) as a white powder. Compound 123 is a hydrochloride salt.

[0274] HPLC Method 1 Mode A: 99.6% area, retention time 2.57 min.

[0275] Example B-20-1: Synthesis of Compound 125 (125) 1.0 g of compound 123 (2.0 mmol, CAS No.: 139262-23-0, also known as Fmoc-Lys-OH xHCl) was passed through an ion exchange column containing a strongly basic ion exchange resin (acetate form). Elution with 20% aqueous acetic acid yielded a solution containing compound 126 (also known as Fmoc-Lys-OH x AcOH), which is an acetate. (126), The solution was freeze-dried, the freeze-dried product was suspended in 30 mL of deionized water and freeze-dried a second time to obtain 0.92 g of compound 125 (CAS No.: 105047-45-8, also known as Fmoc-Lys-OH) as a white solid.

[0276] The chloride test was negative, and the residual acetic acid content was 2.2 wt.%.

[0277] HPLC Method 1 Mode B: 99.3% area, retention time 3.34 min.

[0278] Example B-21-1: Synthesis of Compound 127 (127) 10.0 g of compound 120 (22 mmol, also known as Bsmoc-Lys-OH x HCl) was dissolved in 40 mL of deionized water and passed through an ion exchange column containing the strongly basic Amberlite IRN 78 ion exchange resin (acetate form). Eluting with deionized water yielded 172 g of an aqueous solution containing compound 122 (also known as Bsmoc-Lys-OH). This aqueous solution was divided into four equal portions.

[0279] 0.54 mL of TFA (7.0 mmol) was added to one (43 g) aqueous solution containing compound 122. The resulting solution was freeze-dried to give 2.73 g (100%) of compound 127 (also known as Bsmoc-Lys-OHx TFA) as a white powder.

[0280] HPLC Method 1 Mode B: 98.9% area, retention time 2.61 min.

[0281] Example B-22-1: Synthesis of Compound 128 (128) 1.35 g of p-toluenesulfonic acid (7.0 mmol, CAS No.: 104-15-4) was added to one (43 g) aqueous solution containing compound 122 (also known as Bsmoc-Lys-OH) from Example B-21-1. The resulting solution was freeze-dried to give 3.20 g (100%) of compound 128 (also known as Bsmoc-Lys-OH x TsOH) as a white powder, which is either p-toluenesulfonate or 4-toluenesulfonate.

[0282] HPLC Method 1 Mode B: 98.8% area, retention time 2.61 min.

[0283] In the stability test of F), compound 128 of this quality was used.

[0284] To crystallize, 1.0 g of compound 128 (prepared previously, also known as Bsmoc-Lys-OH x TsOH) was suspended in 20 mL of deionized water and heated to 45°C to obtain a clear solution. The water was evaporated under reduced pressure to a final weight of 3 g. 20 mL of IPA was added to the resulting white suspension, and the solvent was partially evaporated. The addition / evaporation cycle was repeated twice. The resulting suspension was diluted with 10 mL of IPA, cooled to 4°C, and then filtered. The filter cake was washed with 10 mL of IPA and 2 x 10 mL of IPE and dried under vacuum to give 0.73 g (73%) of crystalline compound 128 (also known as Bsmoc-Lys-OH x TsOH) in white crystalline form.

[0285] The crystals can be clearly observed under a microscope.

[0286] HPLC Method 1 Mode B: 99.6% area, retention time 2.60 min.

[0287] Example B-23-1: Synthesis of Compound 129 (129) 1.35 g of methanesulfonic acid (7.0 mmol, CAS No.: 75-75-2) was added to one (43 g) aqueous solution containing compound 122 (also known as Bsmoc-Lys-OH) from Example B-21-1. The resulting solution was freeze-dried to give 2.60 g (100%) of compound 129 (also known as Bsmoc-Lys-OH x MeSO3H) as a translucent solid resin, which is a methanesulfonate.

[0288] HPLC Method 1 Mode B: 98.9% area, retention time 2.61 min.

[0289] Example B-24-1: Synthesis of Compound 130 (130) 0.39 mL of sulfuric acid (7.0 mmol, CAS No.: 7664-93-9) was added to one (43 g) aqueous solution containing compound 122 (also known as Bsmoc-Lys-OH) from Example B-21-1. The resulting solution was freeze-dried to give 2.61 g (100%) of compound 130 (also known as Bsmoc-Lys-OH x 0.5 H2SO4) as a translucent solid resin, which is a hemisulfate.

[0290] HPLC Method 1 Mode B: 98.9% area, retention time 2.61 min.

[0291] Example B-25-1: Synthesis of Compound 203 (203) 20.0 g of 17-(tert-butoxycarbonyl)-heptadecanoyl-Glu-OtBu (36.0 mmol, CAS No.: 1188328-39-3) and 4.97 g of N-hydroxysuccinimide (43.2 mmol) were dissolved in a mixture of 120 mL THF and 60 mL toluene. While maintaining an internal temperature of 25°C, 5.85 mL of DIC (37.8 mmol) dissolved in 20 mL THF was added to the resulting clear solution over 1.5 hours. The temperature was then raised to 35°C, and the reaction mixture was stirred at this temperature overnight. Next, 0.72 mL of DIC (4.6 mmol) dissolved in 2 mL THF was added, and the mixture was stirred continuously at 35°C for 5.5 hours. The resulting suspension was filtered through a G4 glass suction filter, and the filtrate was concentrated under reduced pressure. 3 x 100 mL of toluene was added to the resulting oil, and the mixture was evaporated under reduced pressure each time. Then, 100 mL of IPE was added, and the resulting white suspension was filtered through a G3 glass suction filter. The filter cake was washed with toluene and IPE, and then dried under vacuum at 35°C to give 19.6 g (83%) of compound 203 as a white solid (CAS No.: 1188328-22-4, also known as 17-(tert-butoxycarbonyl)-heptadecanoyl-Glu(OSu)-OtBu).

[0292] HPLC Method 1 Mode C: 97.9% area, retention time 3.66 min.

[0293] Example B-26-1: Synthesis of Compound 309 (309) 5 mL of THF and 2 mL of trichlorotrimethylsilane (18 mmol) were added to 2.31 g of compound 120 (5.0 mmol, also known as Bsmoc-Lys-OH x HCl) at room temperature. The mixture was heated to 40°C with stirring. 15 mL of ACN was added. After 1 hour, the resulting heterogeneous mixture was cooled to 20°C. This heterogeneous mixture contained compound 131. (131) [Note: The structure of compound 131 is shown as -NH2x HCl, but there may also be structures of compound 131 containing -NH-Si(CH3)3 or -NH-Si(CH3)3x HCl—this issue is still unresolved.] Compound 131 is a silylated amine compound used in subsequent coupling reactions.

[0294] At room temperature, 2.1 g of compound 202 (5.0 mmol, CAS No.: 1069067-08-8) was dissolved in 15 mL of ACN. 1.6 g of TBTU (5.0 mmol) and 0.65 mL of DIPEA were added, and the mixture was stirred for 10 minutes. The pre-activated carboxyl compound was used for subsequent coupling reactions.

[0295] For the coupling reaction, a pre-activated carboxyl compound solution was added to a mixture containing a silylamine compound. 2.8 mL of DIPEA was added to the resulting mixture. After 1.5 hours, 100 mL of EtOAc was added to the reaction mixture, which was then transferred to a separatory funnel. The solution was washed with 20 mL of 1N HCl aqueous solution and 3 x 20 mL of semi-saturated NaCl aqueous solution. The organic phase was separated and concentrated under vacuum to give 4.4 g (116%; crude product containing residual solvent) of compound 309 as a beige foam. Compound 309 was used directly in the subsequent deprotection step.

[0296] HPLC Method 1 Mode B: 94.5% area, retention time 3.47 min.

[0297] MS: m / z = 759.3137 [M+H] + .

[0298] Example B-27-1: Synthesis of Compound 310 (310) 3.6 g of compound 309 (4.7 mmol) was dissolved in 16 mL of EtOAc. 50 mL of 3.4 N HCl / EtOAc solution was added to the resulting solution. A viscous residue was formed immediately, and the supernatant was discarded after 30 minutes. This viscous residue was ground with 3 x 50 mL MTBE. It was then vacuum dried at 30°C to give 3.70 g (112%; crude product containing residual solvent) of compound 310, a beige resinous salt, which was used directly in the subsequent deprotection step.

[0299] HPLC Method 1 Mode B: 95.6% area, retention time 2.75 min.

[0300] MS: m / z = 659.2590 [M+H] + , 1317.5111 [2M+H] + .

[0301] Example B-28-1: Synthesis of Compound 304 (304) At room temperature, 3.2 mL of THF and 3.2 mL of N-methyl-N-trimethylsilylacetamide (20 mmol) were added to 3.24 g of compound 123 (8.0 mmol, also known as Fmoc-Lys-OH x HCl). The mixture was heated to 40°C with stirring. After 2 hours, the resulting solution was cooled to 20°C. This solution contained compound 132. (132) [Note: The structure of compound 132 is shown as -NH2x HCl, but there may also be structures of compound 132 containing -NH-Si(CH3)3 or -NH-Si(CH3)3x HCl—this issue is still unresolved.] Compound 132 is a silylated amine compound used in subsequent coupling reactions.

[0302] For the coupling reaction, the pre-activated carboxyl compound solution from Example B-06-2 was added to the solution containing the silanized amine compound. 1.39 mL of DIPEA (8.0 mmol) was added to the resulting mixture. After stirring at room temperature for 3 hours, 8 mL of deionized water was added, and the mixture was acidified to pH 1 by adding 5.5 N hydrochloric acid aqueous solution. The phases were separated, and the organic phase was washed with 3 x 10 mL of water and then evaporated under reduced pressure. The resulting yellow oil was dissolved in 60 mL of MeTHF. 12 mL of THF and 12 mL of water were added. The phases were separated, and the organic phase was washed with 3 x 12 mL of water. The washed organic phase was then diluted with 30 mL of hexane and extracted with 20 mL of 10% sodium bicarbonate aqueous solution. The aqueous phase was washed with 2 x 20 mL of IPE. Then 50 mL of MeTHF was added, and the mixture was acidified to pH 1 with 5.5 N hydrochloric acid aqueous solution. The phases were separated, the organic phase was washed with 3 x 10 m of water and then evaporated under reduced pressure to give 2.8 g (46%) of compound 304 in a viscous foamy state.

[0303] HPLC Method 1 Mode E: 83.3% area, retention time 5.58 min.

[0304] MS: m / z = 759.380 [M+H] + .

[0305] Example B-29-1: Synthesis of Compound 509 (509) In a 500 mL temperature-controlled double-jacketed reactor equipped with a mechanical stirrer, 10 g of compound 510 (CAS No.: 1491158-62-3) was added. (510) The sample was dissolved in 100 mL THF and stirred at 25°C for 2 minutes to obtain a clear solution. 15.4 mL of dodecyl mercaptan and 1.88 mL of DBU were added, and the solution was stirred at 25°C for 1 hour to obtain a clear gel-like solution. TLC and HPLC process control showed complete conversion of the feedstock. Then, 50 mL of water and 50 mL of n-heptane were added to the reaction mixture, the mixture was stirred, the phases were separated, and the organic layer was discarded. 50 mL of n-heptane, 50 mL of THF, and 10 mL of water were added to the separated aqueous layer. The mixture was stirred, the phases were separated, and the organic layer was discarded. 100 mL of MeTHF and 2.4 mL of 5.5N hydrochloric acid aqueous solution were added to the separated aqueous layer to adjust the pH to 5.1, the mixture was stirred, the phases were separated, and the aqueous layer was discarded. The separated organic layer was concentrated to a volume of 50 mL using a rotary evaporator at a bath temperature of 40°C. Water was removed by two azeotropic distillations with 100 mL of MeTHF, and the solution was then concentrated to dryness. 150 mL of IPE was added to the residue, and the resulting suspension was stirred at 40°C for 30 minutes. The suspension was cooled to room temperature and filtered. The resulting solid was resuspended and washed twice with 20 mL of IPE. The washed solid was dried under reduced pressure at 40°C for 16 hours to give 6.37 g (88.5%) of compound 509 (CAS No.: 1962161-20-1) as a white solid.

[0306] HPLC method 3: 72.4 area.

[0307] Figure 35 The complete HPLC-UV chromatogram is shown.

[0308] Example B-30-1: Synthesis of Compound 511 (511) In a 25 mL round-bottom flask equipped with a magnetic stirrer, 500 mg of compound 510 (CAS No.: 1491158-62-3) was suspended in 5 mL of THF. 352 µL of N-methyl-N-trimethylsilylacetamide was added. The reaction mixture was stirred at 40°C for 1 hour to obtain a clear solution. The solution was cooled to room temperature, and 162.4 µL of pyridine dissolved in 3.5 mL of THF was added. The clear solution was cooled to 5°C. 261.3 mg of 1,1-dioxobenzo[b]thiophene-2-ylmethoxycarbonyl chloride (CAS No.: 135204-19-2, also known as Bsmoc-Cl) dissolved in 6 mL of THF was added dropwise over 5 minutes. The reaction mixture was stirred at 0°C for 1 hour. TLC and HPLC process control showed complete conversion of the starting material. The reaction mixture was concentrated to a volume of 50 mL using a rotary evaporator at a bath temperature of 25°C. The residue was dissolved in 5 mL of Et₂O, then 1 mL of water was added, and the pH was adjusted to 7 with 4.8 mL of 2.5% sodium bicarbonate aqueous solution. The phases were then separated, and the organic layer was discarded. 5 mL of Et₂O was added to the separated aqueous layer. The mixture was stirred, the phases were separated, and the organic layer was discarded. 5 mL of MTBE and 0.4 mL of 5.5 N hydrochloric acid aqueous solution were added to the separated aqueous layer to adjust the pH to 2.0. The mixture was stirred, the phases were separated, and the aqueous layer was discarded. The separated organic layer was concentrated to dryness using a rotary evaporator at a bath temperature of 40°C. 3 mL of Et₂O and 8 mL of n-hexane were added to the resulting residue, and the resulting suspension was concentrated to dryness again. This process of adding only 8 mL of n-hexane and concentrating to dryness was repeated. The resulting residue was dried under reduced pressure at 30°C for 16 hours to give 432 mg (62.2%) of compound 511 as a white solid.

[0309] HPLC method 3: 90.7% area.

[0310] Figure 36 The complete HPLC-UV chromatogram is shown.

[0311] HR-MS: Calculated value 791.4391 m / z, measured value 792.4413 m / z [M+H] + .

[0312] Example B-31-1: Synthesis of Compound 512 (512) In a 50 mL round-bottom flask equipped with a magnetic stirrer, 2.0 g of compound 513 (CAS No.: 201004-46-8) was added. (513) The sample was dissolved in 20 mL THF and stirred at 25°C for 7 minutes to obtain a clear solution. 4.0 mL of dodecanethiol and 492 µL of DBU were added. A solid formed immediately upon the addition of DBU. The white suspension was stirred at 25°C for 1 hour. TLC and HPLC process control showed complete conversion of the starting material. The suspension was filtered, and the solid was washed with 10 mL THF. The washed solid was dried under reduced pressure at 40°C for 64 hours to give 1.50 g (theoretical yield 100%, calculated yield 118%) of compound 512 (CAS No.: 59012-43-0) as a pale pink solid. This solid appeared to be insoluble or sparingly soluble in common organic solvents. HPLC or TLC analysis appeared difficult to perform.

[0313] Example B-32-1: Synthesis of Compound 514 (514) In a 10 mL round-bottom flask equipped with a magnetic stirrer, 100 mg of compound 512 (obtained as in Example B-31-1, CAS No.: 59012-43-0) was suspended in 2 mL of THF. 104.4 µL of N-methyl-N-trimethylsilylacetamide was added, and the reaction mixture was stirred at 40°C for 1 hour. The slightly turbid solution was cooled to 25°C, and 48.2 µL of pyridine dissolved in 0.6 mL of THF was added. After cooling to 5°C, 77.2 mg of 1,1-dioxobenzo[b]thiophene-2-ylmethoxycarbonyl chloride (CAS No.: 135204-19-2, also known as Bsmoc-Cl) dissolved in 1.2 mL of THF was added dropwise over 5 minutes. The reaction mixture was stirred at 0°C for 2 hours and concentrated to dryness using a rotary evaporator at a bath temperature of 25°C. Add 8 mL of Et₂O and 3 mL of water to the residue, and adjust the pH to 7 with 1 mL of 2.5% sodium bicarbonate aqueous solution. A two-phase mixture is formed. Separate the phases, discard the organic layer, and repeat the extraction with 2 x 5 mL of Et₂O. Dilute the aqueous layer with 15 mL of MTBE and adjust the pH to 2.5 with 30 µL of 5.5 N hydrochloric acid aqueous solution. Separate the phases, discard the aqueous layer, and concentrate the organic layer to dryness using a rotary evaporator at 40°C. Co-evaporate the residue with 2 x 10 mL of MeTHF, 1 x 10 mL of IPE, and 1 x 5 mL of IPE. Dry the resulting residue under reduced pressure at 30°C for 16 hours to give 68 mg (43.1%) of compound 514 as a white solid.

[0314] HPLC method 3: 68.9% area.

[0315] Figure 37 The complete HPLC-UV chromatogram is shown.

[0316] HR-MS: Calculated value 606.3339 m / z, measured value 607.3417 m / z [M+H] + .

[0317] Example B-33-1: Synthesis of Compound 515 (515) In a 100 mL round-bottom flask equipped with a magnetic stirrer, 5.0 g of compound 516 (CAS No.: 2460751-66-8) was added. (516) Dissolve in 50 mL THF. After stirring at 25°C for 1 minute, a clear solution is obtained. Add 5.0 mL dodecylthiol and 609 µL DBU, and stir at 25°C to obtain a colorless, clear solution for 1 hour. TLC and HPLC process control showed complete conversion of the starting material. Dilute with 20 mL IPE, 20 mL water, and 20 mL THF, separate the phases, and discard the organic layer. Extract the aqueous layer twice with 20 mL IPE and 50 mL THF. Add 100 mL MeTHF and 30 mL water to the aqueous layer, adjust the pH to 5 with 720 µL 5.5 N hydrochloric acid aqueous solution, separate the phases, and discard the aqueous layer. Concentrate the organic layer to dryness using a rotary evaporator at 40°C. Co-evaporate with 3 x 50 mL MeTHF and 1 x 50 mL ACN, then add 50 mL ACN. Stir the resulting suspension at 25°C for 15 minutes and filter. Wash the resulting solid with 2 x 5 mL ACN. The washed solid was dried under reduced pressure at 40°C for 64 hours to give 2.15 g (52.5%) of compound 515 (CAS No.: 2931515-16-9) as a white solid.

[0318] HPLC method 3: 97.9% area.

[0319] Figure 38 The complete HPLC-UV chromatogram is shown.

[0320] Example B-34-1: Synthesis of Compound 517 (517) In a 100 mL three-necked round-bottom flask equipped with a magnetic stirrer, 1.0 g of compound 515 (CAS No.: 2931515-16-9) was suspended in 25 mL of THF. 401 µL of N-methyl-N-trimethylsilylacetamide was added, and the reaction mixture was stirred at 40°C for 1 hour. The clear solution was cooled to 25°C, and 185 µL of pyridine dissolved in 5 mL of THF was added. After cooling to 5°C, 297 mg of 1,1-dioxobenzo[b]thiophene-2-ylmethoxycarbonyl chloride (CAS No.: 135204-19-2, also known as Bsmoc-Cl) dissolved in 20 mL of THF was added dropwise over 30 minutes. The reaction mixture was stirred at 0°C for 1.5 hours. TLC process control showed that the starting material had been completely converted. After concentrating to dryness using a rotary evaporator at 25°C, 60 mL of Et₂O and 10 mL of water were added to the residue, and the pH was adjusted to 7 with 8 mL of 2.5% sodium bicarbonate aqueous solution, forming a two-phase solution. The phases were separated, and the organic layer was discarded. The aqueous layer was extracted with 2 x 25 mL Et₂O. 150 mL of MTBE was added to the aqueous layer, and the pH was adjusted to 2.0 with 600 µL of 5.5 N hydrochloric acid aqueous solution. The phases were separated, and the aqueous layer was discarded. Then, the organic layer was extracted with 50 mL of water. The organic layer was concentrated to dryness using a rotary evaporator at 40°C. The residue was dissolved in 50 mL of ACN and co-evaporated with 3 x 50 mL ACN and 30 mL of IPE. After solvent evaporation, the residue was redissolved in 20 mL of ACN. The dilute suspension was filtered, and the filtrate containing the product was concentrated to dryness. The residue was dried under reduced pressure at 30°C for 16 hours to give 1.05 g (85.8%) of compound 517 as a colorless viscous solid.

[0321] HPLC method 3: 86.2 area.

[0322] Figure 39 The complete HPLC-UV chromatogram is shown.

[0323] HR-MS: Calculated value 1223.6862 m / z, measured value 1224.6961 m / z [M+H] + .

[0324] Example B-35-1: Synthesis of Compound 518 (518) In a 50 mL round-bottom flask equipped with a magnetic stirrer, 2.0 g of compound 519 (CAS No.: 1931109-48-6) was added. (519) Dissolve in 20 mL THF. After stirring at 25°C for 10 minutes, a clear solution is obtained. Add 2.7 mL dodecylthiol and 329.4 µL DBU, and stir the yellow solution at 25°C for 1 hour. TLC and HPLC process control showed complete conversion of the starting material. Add 20 mL n-heptane, 20 mL water, and 20 mL THF to the reaction mixture, separate the phases, and discard the organic layer. Extraction of the aqueous layer is repeated twice: the second extraction uses 10 mL n-heptane, 20 mL water, and 20 mL THF, and the third extraction uses 10 mL n-heptane and 10 mL THF. Add 50 mL MeTHF to the aqueous layer, adjust the pH to 5 with 250 µL of 5.5 N hydrochloric acid aqueous solution, separate the phases, and discard the aqueous layer. Concentrate the organic layer to 10 mL using a rotary evaporator at a bath temperature of 40°C. Co-evaporate the mixture with 3 x 50 mL MeTHF, and then concentrate the solution to dryness. 10 mL of ACN was added to the residue, and the resulting suspension was concentrated to dryness. After dilution with 10 mL of ACN, the mixture was stirred at 25°C for 15 minutes and the suspension was filtered. The resulting solid was washed with 2 mL of ACN. The washed solid was dried under reduced pressure at 40°C for 64 hours to give 720 mg (47.7%) of compound 518 as a white solid.

[0325] HPLC method 3: 72.2 area.

[0326] Figure 40 The complete HPLC-UV chromatogram is shown.

[0327] Example B-36-1: Synthesis of Compound 520 (520) In a 50 mL round-bottom flask equipped with a magnetic stirrer, 500 mg of compound 518 was suspended in 5 mL of ACN. 293.7 µL of N-methyl-N-trimethylsilylacetamide was added to the suspension. The reaction mixture was stirred at 40°C for 1 hour. The clear solution was cooled to 25°C. 135.3 µL of pyridine dissolved in 2.5 mL of ACN was added. After cooling to 5°C, 217.5 mg of 1,1-dioxobenzo[b]thiophene-2-ylmethoxycarbonyl chloride (CAS No.: 135204-19-2, also known as Bsmoc-Cl) dissolved in 5 mL of ACN was added dropwise over 6 minutes. The reaction mixture was stirred at 0°C for 1 hour. TLC and HPLC process control showed complete conversion of the starting material. The reaction mixture was concentrated to dryness using a rotary evaporator at a bath temperature of 25°C. Add 60 mL of Et₂O and 25 mL of water to the residue, and adjust the pH to 7 with 5 mL of 2.5% sodium bicarbonate aqueous solution. A two-phase solution is formed; separate the phases and discard the organic layer. Repeat extraction with 2 x 50 mL Et₂O. Dilute with 50 mL MTBE, and adjust the pH to 2.0 with 200 µL of 5.5 N hydrochloric acid aqueous solution. Then, separate the phases, discard the aqueous layer, and concentrate the organic layer to dryness using a rotary evaporator at 40°C. Dissolve the residue in 10 mL of MeTHF and co-evaporate with 3 x 20 mL MeTHF and IPE / MeTHF / n-heptane (45 mL, 2:1:6 v / v / v). After concentrating to dryness, stir the residue in 20 mL of n-heptane at 25°C for 30 minutes and filter. The washed solid was dried under reduced pressure at 30°C for 16 hours to obtain 451 mg (68.1%) of compound 520 as a white solid.

[0328] HPLC method 3: 75.3 area.

[0329] Figure 41 The complete HPLC-UV chromatogram is shown.

[0330] HR-MS: Calculated value 905.5071 m / z, measured value 906.5197 m / z [M+H] + .

[0331] C) Synthesis of solid-phase coupling compounds Example C-01-1: Synthesis of solid-phase coupling compound 601-SP Fmoc-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethylaminomethyl resin] (601-SP) (SEQ ID NO:13) For example, compound 601-SP can be obtained by solid-phase synthesis starting from compound 602-SP. H-Gly-[2-Chlorotriphenylmethylaminomethyl resin] (602-SP).

[0332] After the coupling cycle of compound 602-SP with Fmoc-Arg(Pbf)-OH, the 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 were carried out in sequence.

[0333] The coupling cycle includes the deprotection reaction of the N-terminal α-amino group of the solid-phase coupling intermediate, unless the N-terminal α-amino group is already in an unprotected state. The deprotection reaction of Fmoc is carried out using a 20 vol.% piperidine DMF solution, or, if necessary, a 20 vol.% piperidine NMP solution, for a typical reaction time of 0.5 to 4.0 hours. The coupling cycle also includes the coupling reaction of the solid-phase coupling intermediate with its unprotected N-terminal α-amino group, i.e., the acylation of the unprotected N-terminal α-amino group by the activated α-carboxyl group of the respective amino acid derivative, activated by DIC / OxymaPure, TBTU / DIPEA, or DEPBT / DIPEA. The typical duration of the acylation reaction is 1.5 to 24 hours. The coupling cycle also includes one or more washings of the solid phase after the acylation reaction. DMF or IPA is used as the solvent for one or more washings. The coupling cycle also includes, optionally, an acetylation reaction with acetic anhydride after the acylation reaction. The coupling cycle also includes, optionally, one or more washings following the acetylation of acetic anhydride. DMF or IPA is used as the solvent for the optional one or more washings following the acetylation of acetic anhydride.

[0334] Example C-02-1: Synthesis of solid-phase coupling compound 603-SP

[0335] -Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethylaminomethyl resin] (603-SP) (SEQ ID NO:26) For example, compound 603-SP (= Nsc-Lys(tBuO-CO-(CH2)) 16 -CO-γ-Glu(OtBu)-2-[2-(2-aminoethoxy)-ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethylaminomethyl resin])(SEQ ID NO:26) can be obtained by coupling and cycling with compound 504 applied to compound 601-SP, i.e., by deprotecting compound 601-SP with a 20 vol.% piperidine DMF solution to obtain compound 604-SP.

[0336] -Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethylaminomethyl resin] (604-SP) (SEQ ID NO:14), Compound 604-SP was then reacted with compound 504 and DIC / OxymaPure.

[0337] As used in this article, the residue tBu-O-CO-(CH2) 16 -CO-γ-Glu-2-[2-(2-aminoethoxy)-ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl can also be specified as O-tert-butyl-17-carboxy-heptadecanoyl-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl.

[0338] As used in this article, the residue HO-CO-(CH2) 16 -CO-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl can also be specified as 17-carboxy-heptadecanoyl-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl.

[0339] Example C-03-1: Synthesis of solid-phase coupling compound 605-SP

[0340] -Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethylaminomethyl resin] (605-SP) (SEQ ID NO:15) For example, compound 605-SP (= Fmoc-Ala-Lys(tBuO-CO-(CH2)) 16 -CO-γ-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethylaminomethyl resin]) (SEQ ID NO:15) can be obtained by coupling cycle with Fmoc-Ala-OH applied to compound 603-SP, i.e., by deprotection reaction of compound 603-SP with a 20 vol.% piperidine DMF solution to obtain compound 606-SP.

[0341] -Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethylaminomethyl resin] (606-SP) (SEQ ID NO:28), Compound 606-SP was then reacted with Fmoc-Ala-OH and DIC / OxymaPure.

[0342] Example C-04-1: Synthesis of solid-phase coupling 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-γ-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethylaminomethyl resin] (607-SP) (SEQ ID NO:16) For example, compound 607-SP (= a protected and solid-phase coupled derivative of smegglutinin (CAS No.: 910463-68-2) can be obtained through multiple coupling cycles, starting with a coupling cycle with Fmoc-Ala-OH applied to compound 605-SP, i.e., deprotecting compound 605-SP with a 20 vol.% piperidine DMF solution to obtain compound 608-SP.

[0343] -Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethylaminomethyl resin] (608-SP) (SEQ ID NO:29), Compound 608-SP was then reacted with Fmoc-Ala-OH and DIC / OxymaPure.

[0344] Subsequently, it is coupled in sequence with 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(1-Trt)-Aib-OH.

[0345] The coupling cycle is performed as described in Example C-01-1.

[0346] Example C-05-1: Synthesis of solid-phase coupling compound 611-SP

[0347] -Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin] (611-SP) (SEQ ID NO:17) Compound 611-SP (=H-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-2-chlorotriphenylmethyl 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-chlorotriphenylmethyl resin, 66.7 g, 0.45 mmol / g resin, 30 mmol), i.e., as shown... H-Gly-[2-Chlorotriphenylmethyl resin] (610-SP) As shown, pre-swelling was performed in DMF (10 mL / g resin) for two cycles, each cycle lasting 30 minutes. The first Fmoc-protected amino acid, Fmoc-Arg(Pbf)-OH, was pre-activated for 3 minutes with TBTU / DIPEA (1.8 eq. / 3.0 eq.) and added to the resin. The reaction mixture was stirred at room temperature for 2 hours. The subsequent Fmoc-protected amino acid derivative (2.0 eq.) was pre-activated for 15 minutes with DIC / OxymaPure (2.6 eq. / 3.1 eq., pre-activated with Fmoc-Arg(Pbf)-OH for 5 minutes) and added to the resin. After a 20-minute reaction time, DIC (1.3 eq.) was added to the reaction mixture (total reaction volume 670 mL), and the coupling step continued. The total reaction times for the coupling steps were: 1.5 hours for Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, and Fmoc-Ala-OH; 2 hours for Fmoc-Arg(Pbf)-OH; and 5 hours for Fmoc-Ile-OH and Fmoc-Phe-OH. At the end of each coupling step, the resin was drained, and an acetylation reaction was carried out using a solution of 0.05 M acetic anhydride / 17 mM 2,4,6-trimethylpyridine / 1.6 mM OxymaPure (670 mL) dissolved in DMF. This acetylation was used to acetylate any remaining unreacted amino groups and to prevent them from reacting in subsequent coupling steps (so-called end-capping). Subsequently, the resin was filtered and washed with DMF, followed by two or three Fmoc deprotection treatments using 670 mL of 20 vol.% piperidine in DMF (for Fmoc-Trp(Boc)-OH and Fmoc-Ala-OH coupling, the first treatment was 5 minutes, and the second treatment was 10 minutes; for Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, and Fmoc-Phe-OH coupling, the first treatment was 5 minutes, the second treatment was 10 minutes, and the third treatment was 15 minutes). After Fmoc deprotection, the resin was washed with 670 mL DMF, 670 mL IPA, and twice with 670 mL DMF. After Fmoc-Phe-OH coupling, acetylation, and Fmoc deprotection, the resin material was washed three times alternately with 670 mL DMF and 670 mL IPA, and then three times with 670 mL IPA. The washed resin material was vacuum dried at room temperature for three days to obtain 132 g of dried compound 611-SP.

[0348] For analytical purposes, a small-scale cleavage test was performed on compound 611-SP as described in Example D-06-1.

[0349] Example C-06-1: Synthesis of solid-phase coupling compound 612-SP

[0350] -Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin] (612-SP) (SEQ ID NO:21) 10.00 g of compound 611-SP (2.2 mmol, 0.22 mmol / g) was pre-swollen twice in 100 mL DMF (10 mL / g resin), each time for 15 minutes at room temperature. 1.95 g of Fmoc-Glu(OtBu)-OH (4.40 mmol, 2.0 eq.) and 0.969 g of OxymaPure (6.82 mmol, 3.1 eq.) were dissolved in 100 mL DMF (10 mL / g resin). 886 µL of DIC (5.72 mmol, 2.6 eq.) was added to this solution, and the mixture was stirred at room temperature for 15 minutes. This solution was then added to the pre-swollen compound 611-SP. After reacting for 20 minutes at room temperature, a further 443 µL of DIC (2.86 mmol, 1.3 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature for 1.5 hours. After removing the liquid portion of the reaction mixture by drainage, a recoupling step (so-called recoupling) was performed using Fmoc-Glu(OtBu)-OH. Therefore, 1.95 g of Fmoc-Glu(OtBu)-OH (4.40 mmol, 2.0 eq.) and 0.969 g of OxymaPure (6.82 mmol, 3.1 eq.) were dissolved in 100 mL of DMF (10 mL DMF / g resin). 886 µL of DIC (5.72 mmol, 2.6 eq.) was added to this solution, and the mixture was stirred at room temperature for 15 minutes before being added to the reaction mixture. After reacting at room temperature for 20 minutes, a further 443 µL of DIC (2.86 mmol, 1.3 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature for 16 hours. After removing the liquid fraction, the remaining resin material was washed with 20 mL of DMF, and then subjected to three Fmoc deprotection treatments with a 20 vol.% piperidine DMF solution (10 mL / g resin): 5 min for the first treatment, 10 min for the second, and 60 min for the third. Following this Fmoc deprotection step, the resin material was washed three times alternately with 670 mL of DMF and 670 mL of IPA, then three times with 100 mL of IPA, and finally twice with 670 mL of DMF. The washed resin material was then vacuum-dried at room temperature for three days to obtain 11.0 g of compound 612-SP.

[0351] For analytical purposes, a small-scale test lysis was performed, as described in Example D-07-1.

[0352] Example C-07-1: Synthesis of solid-phase coupling compound 613-SP

[0353] -Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin] (613-SP) (SEQ ID NO:18) 2.00 g of compound 611-SP (0.88 mmol, 0.44 mmol / g) was pre-swollen twice in 20 mL DMF (10 mL / g resin), each time for 15 minutes at room temperature. 0.781 g Fmoc-Glu(OtBu)-OH (1.76 mmol, 2.0 eq.) and 0.388 g OxymaPure (2.728 mmol, 3.1 eq.) were dissolved in 20 mL DMF (10 mL / g resin). 354 µL of DIC (2.288 mmol, 2.6 eq.) was added to this solution, and the mixture was stirred at room temperature for 15 minutes. This solution was then added to the pre-swollen compound 611-SP. After reacting for 20 minutes at room temperature, a further 177 µL of DIC (1.144 mmol, 1.3 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature for 5 hours. After removing the liquid portion of the reaction mixture by drainage, a recoupling step (so-called recoupling) was performed using Fmoc-Glu(OtBu)-OH. Therefore, 0.781 g Fmoc-Glu(OtBu)-OH (1.76 mmol, 2.0 eq.) and 0.388 g OxymaPure (2.728 mmol, 3.1 eq.) were dissolved in 20 mL DMF (10 mL / g resin). 354 µL DIC (2.288 mmol, 2.6 eq.) was added to this solution, and the solution was stirred at room temperature for 15 minutes before being added to the reaction mixture. After reacting at room temperature for 20 minutes, 177 µL DIC (1.144 mmol, 1.3 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature for 15 hours. After removing the liquid portion, the resin material was washed with 20 mL of DMF and treated three times with a 20 vol.% piperidine DMF solution (10 mL / g resin): 5 min for the first treatment, 10 min for the second, and 60 min for the third. Subsequently, 1.76 g of compound 506 (1.760 mmol, 2.0 eq.) and 0.388 g of OxymaPure (2.728 mmol, 3.1 eq.) were dissolved in 20 mL of DMF (10 mL / g resin). 354 µL of DIC (2.288 mmol, 2.6 eq.) was added to this solution, and the mixture was stirred at room temperature for 15 min before being added to the reaction mixture. After reacting at room temperature for 20 min, a further 177 µL of DIC (177 µL, 1.144 mmol, 1.3 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature for 22 h.After removing the liquid portion, the resin material was washed three times alternately with DMF / IPA, and then three times with IPA (10 mL / g resin per washing step). The washed resin material was dried under high vacuum at room temperature for 24 hours to obtain 2.22 g of compound 613-SP (= N3-Lys(tBuO-CO-(CH2)). 16 -CO-γ-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin]) (SEQ ID NO:18).

[0354] For analytical purposes, a small-scale test lysis was performed, as described in Example D-08-1.

[0355] Example C-08-1: Synthesis of solid-phase coupling compound 614-SP

[0356] -Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin] (614-SP) (SEQ ID NO:19) 100 mg of compound 613-SP (0.21 mmol / g, 0.022 mmol) was pre-swelled twice in DMF (10 mL / g resin), each time for 15 minutes at room temperature. 31.5 mg of tris(2-carboxyethyl)phosphine (= TCEP, 0.11 mmol, 5.0 eq.) was dissolved in 250 µL of 10 vol.% aqueous DMF solution (2.5 mL / g resin), and 18.8 µL of LDIPEA (0.11 mmol, 5.0 eq.) was added. This solution was then added to the pre-swollen compound 613-SP. The reaction mixture was then stirred at room temperature for 3 hours, followed by stirring at 50°C for 16 hours. After removing the liquid fraction, 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 / g resin per wash). A portion of the resin material was further processed in Example C-09-1 without drying. Another portion of the resin material was dried under high vacuum at room temperature for 24 hours to obtain compound 614-SP (= H-Lys(tBuO-CO-(CH2)). 16 -CO-γ-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin])(SEQ ID NO:19).

[0357] For analytical purposes, a small-scale test lysis was performed, as described in Example D-02-2.

[0358] Example C-08-2: Synthesis of solid-phase coupling compound 614-SP 100 mg of compound 617-SP (0.022 mmol, 0.21 mmol / g resin) was pre-swollen twice in DMF (10 mL / g resin), each time for 10 minutes at room temperature. The pre-swollen compound 617-SP was then treated twice with a 20 vol.% piperidine DMF solution (10 mL / g resin), the first treatment lasting 20 minutes and the second 60 minutes. Subsequently, the resin material was washed three times alternately with DMF / IPA, followed by three washes with IPA (10 mL / g resin per wash). The washed resin material was then dried under high vacuum at room temperature for 24 hours to obtain compound 614-SP.

[0359] For analytical purposes, a small-scale test lysis was performed, as described in Example D-02-3.

[0360] Example C-08-3: Synthesis of solid-phase coupling compound 614-SP 200 mg of compound 618-SP (0.21 mmol / g resin, 0.042 mmol) was pre-swelled twice in DMF (10 mL / g resin), each time for 10 minutes at room temperature. The pre-swelled resin material was treated twice with a DMF solution (10 mL / g resin) containing 2 vol.% DBU in 20 vol.% piperidine, the first treatment for 20 minutes and the second for 60 minutes. Subsequently, the resin material was washed three times with alternating DMF / IPA, followed by three washes with IPA (10 mL / g resin per wash). 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 room temperature for 24 hours to obtain compound 614-SP.

[0361] For analytical purposes, a small-scale test lysis was performed, as described in Example D-02-4.

[0362] Example C-08-4: Synthesis of solid-phase coupling compound 614-SP 200 mg of compound 619-SP (0.21 mmol / g resin, 0.042 mmol) was pre-swollen twice in DMF (10 mL / g resin), each time for 10 minutes at room temperature. The pre-swollen resin material was treated twice with a DMF solution (10 mL / g resin) containing 2 vol.% DBU in 20 vol.% piperidine, the first treatment for 20 minutes and the second for 60 minutes. Subsequently, the resin material was washed three times with alternating DMF / IPA, followed by three washes with IPA (10 mL / g resin per wash). 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 room temperature for 24 hours to obtain compound 614-SP.

[0363] For analytical purposes, a small-scale test lysis was performed, as described in Example D-02-5.

[0364] Example C-08-5: Synthesis of solid-phase coupling compound 614-SP 200 mg of compound 620-SP (0.21 mmol / g resin, 0.042 mmol) was pre-swollen twice in DMF (10 mL / g resin), each time for 10 minutes at room temperature. The pre-swollen resin material was treated twice with a 20 vol.% piperidine DMF solution (10 mL / g resin), the first treatment for 20 minutes and the second for 60 minutes. Subsequently, the resin material was washed three times with alternating DMF / IPA, followed by three washes with IPA (10 mL / g resin per wash). 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 room temperature for 24 hours to obtain compound 614-SP.

[0365] For analytical purposes, a small-scale test lysis was performed, as described in Example D-02-6.

[0366] Example C-09-1: Synthesis of solid-phase coupling compound 616-SP

[0367] -Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin] (616-SP) (SEQ ID NO:27) Compound 616-SP (= H-Ala-Lys(tBuO-CO-(CH2)) 16 -CO-γ-Glu(OtBu)-2-[2-(2-aminoethoxy)-ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin])(SEQ ID NO:27) is an intermediate compound 615-SP (= Fmoc-Ala-Lys(tBuO-CO-(CH2)) protected by Fmoc according to general operation step 3. 16 -CO-γ-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin]) (SEQ ID NO:20)

[0368] -Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin] (615-SP) (SEQ ID NO:20) It was synthesized from compound 614-SP obtained in Example C-08-1.

[0369] For analytical purposes, a small-scale test lysis was performed, as described in Example D-04-2.

[0370] Example C-09-2: Synthesis of solid-phase coupling compound 616-SP Compound 616-SP was synthesized from 100 mg of compound 614-SP (0.022 mmol, 0.22 mmol / g resin) obtained in Example C-08-2, via Fmoc-protected intermediate compound 615-SP, according to general operation step 3.

[0371] For analytical purposes, a small-scale test lysis was performed, as described in Example D-04-3.

[0372] Example C-09-3: Synthesis of solid-phase coupling compound 616-SP Compound 616-SP was synthesized from 100 mg of compound 614-SP (0.022 mmol, 0.22 mmol / g resin) obtained in Example C-08-3, via Fmoc-protected intermediate compound 615-SP, according to general operation step 3.

[0373] For analytical purposes, a small-scale test lysis was performed, as described in Example D-04-4.

[0374] Example C-09-4: Synthesis of solid-phase coupling compound 616-SP Compound 616-SP was synthesized from 100 mg of compound 614-SP (0.022 mmol, 0.22 mmol / g resin) obtained in Example C-08-4, via Fmoc-protected intermediate compound 615-SP, according to general operation step 3.

[0375] For analytical purposes, a small-scale test lysis was performed, as described in Example D-04-5.

[0376] Example C-09-5: Synthesis of solid-phase coupling compound 616-SP Compound 616-SP was synthesized from 100 mg of compound 614-SP (0.022 mmol, 0.22 mmol / g resin) obtained in Example C-08-5, via Fmoc-protected intermediate compound 615-SP, according to general operation step 3.

[0377] For analytical purposes, a small-scale test lysis was performed, as described in Example D-04-6.

[0378] Example C-10-1: Synthesis of solid-phase coupling compound 617-SP

[0379] -Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin] (617-SP) (SEQ ID NO:22) Following general procedure 1, 200 mg of compound 612-SP (0.044 mmol, 0.22 mmol / g resin) and 105 mg of compound 501 (0.088 mmol, 2.0 eq.) were reacted to obtain compound 617-SP (= Fmoc-Lys(tBuO-CO-(CH2)). 16 -CO-γ-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin]) (SEQ ID NO:22).

[0380] For analytical purposes, a small-scale test lysis was performed, as described in Example D-09-1.

[0381] Example C-11-1: Synthesis of solid-phase coupling compound 618-SP

[0382] -Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin] (618-SP) (SEQ ID NO:24) Following general procedure 1, 200 mg of compound 612-SP (0.044 mmol, 0.22 mmol / g resin) and 105 mg of compound 507 (0.088 mmol, 2.0 eq.) were reacted to obtain compound 618-SP (= Psc-Lys(tBuO-CO-(CH2)). 16 -CO-γ-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin]) (SEQ ID NO:24).

[0383] For analytical purposes, a small-scale test lysis was performed, as described in Example D-10-1.

[0384] Example C-12-1: Synthesis of solid-phase coupling compound 619-SP

[0385] -Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin] (619-SP) (SEQ ID NO:25) Following general procedure 1, 200 mg of compound 612-SP (0.044 mmol, 0.22 mmol / g resin) and 105 mg of compound 504 (0.088 mmol, 2.0 eq.) were reacted to obtain compound 619-SP (= Nsc-Lys(tBuO-CO-(CH2)). 16 -CO-γ-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin]) (SEQ ID NO:25).

[0386] For analytical purposes, a small-scale test lysis was performed, as described in Example D-01-2.

[0387] Example C-13-1: Synthesis of solid-phase coupling compound 620-SP

[0388] -Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin] (620-SP) (SEQ ID NO:23) Following general procedure 1, 200 mg of compound 612-SP (0.044 mmol, 0.22 mmol / g resin) and 105 mg of compound 508 (0.088 mmol, 2.0 eq.) were reacted to obtain compound 620-SP (= Bsmoc-Lys(tBuO-CO-(CH2)). 16 -CO-γ-Glu(OtBu)-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-[2-chlorotriphenylmethyl resin]) (SEQ ID NO:23).

[0389] For analytical purposes, a small-scale test lysis was performed, as described in Example D-11-1.

[0390] D) Pyrolysis of solid-phase coupling compounds Example D-01-1: Synthesis of Compound 701

[0391] -Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (701) (SEQ ID NO:1) For example, compound 701 (= Nsc-Lys(HO-CO-(CH2)) 16-CO-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH) (SEQ ID NO:1) can be obtained by deprotection reaction of compound 603-SP with a pyrolysis composition of TFA / EDT / H2O / TIS (90 / 5 / 2.5 / 2.5 vol.%, based on the volume of the pyrolysis composition).

[0392] Example D-01-2: Synthesis of Compound 701 According to general operating procedure 2, a small sample of dried compound 619-SP from Example C-12-1 was pyrolyzed to obtain a solid containing compound 701 (SEQ ID NO:1).

[0393] HPLC Method 2: 45.7 area % + 27.8 area %, retention time 18.42 min + 19.39 min / One of the two peaks is identified as compound 701, and the other is a decomposition product. It is unclear whether the decomposition occurs during the TFA-based cleavage process, during the TFA-containing analytical HPLC Method 2 process, or both. TFA sensitivity of Nsc-protected compound 504 was also observed in Example B-12-8.

[0394] Figure 1 The complete HPLC-UV chromatogram is shown. Figure 2 A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0395] Example D-02-1: Synthesis of Compound 702

[0396] -Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (702) (SEQ ID NO:2) For example, compound 702 (= H-Lys(HO-CO-(CH2)) 16-CO-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH) (SEQ ID NO:2) can be obtained by deprotection reaction of compound 606-SP with a pyrolysis composition of TFA / EDT / H2O / TIS (90 / 5 / 2.5 / 2.5 vol.%, based on the volume of the pyrolysis composition).

[0397] Example D-02-2: Synthesis of Compound 702 According to general operating procedure 2, a small sample of dried compound 614-SP from Example C-08-1 was pyrolyzed to obtain a solid containing compound 702 (SEQ ID NO:2).

[0398] HPLC Method 2: 80.9% area, retention time 16.65 min.

[0399] Figure 3 The complete HPLC-UV chromatogram is shown. Figure 4 A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0400] Example D-02-3: Synthesis of Compound 702 According to general operating procedure 2, a small sample of dried compound 614-SP from Example C-08-2 was pyrolyzed to obtain a solid containing compound 702 (SEQ ID NO:2).

[0401] HPLC Method 2: 76.2% area, retention time 16.65 min.

[0402] Figure 5 The complete HPLC-UV chromatogram is shown. Figure 6 A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0403] Example D-02-4: Synthesis of Compound 702 According to general operating procedure 2, a small sample of dried compound 614-SP from Example C-08-3 was pyrolyzed to obtain a solid containing compound 702 (SEQ ID NO:2).

[0404] HPLC Method 2: 76.4% area, retention time 16.66 min.

[0405] Figure 7 The complete HPLC-UV chromatogram is shown. Figure 8 A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0406] Example D-02-5: Synthesis of Compound 702 According to general operating procedure 2, a small sample of dried compound 614-SP from Example C-08-4 was pyrolyzed to obtain a solid containing compound 702 (SEQ ID NO:2).

[0407] HPLC Method 2: 81.4% area, retention time 16.66 min.

[0408] Figure 9 The complete HPLC-UV chromatogram is shown. Figure 10 A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0409] Example D-02-6: Synthesis of Compound 702 According to general operating procedure 2, a small sample of dried compound 614-SP from Example C-08-5 was pyrolyzed to obtain a solid containing compound 702 (SEQ ID NO:2).

[0410] HPLC Method 2: 81.1% area, retention time 16.66 min.

[0411] Figure 11 The complete HPLC-UV chromatogram is shown. Figure 12 A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0412] Example D-03-1: Synthesis of Compound 703

[0413] -Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (703) (SEQ ID NO:3) For example, compound 703 (= Fmoc-Ala-Lys(HO-CO-(CH2)) 16-CO-γ-Glu-2-[2-(2-aminoethoxy)-ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH) (SEQ ID NO:3) can be obtained by deprotection reaction of compound 605-SP with a pyrolysis composition of TFA / EDT / H2O / TIS (90 / 5 / 2.5 / 2.5 vol.%, based on the volume of the pyrolysis composition).

[0414] Example D-04-1: Synthesis of Compound 704

[0415] -Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (704) (SEQ ID NO:4) For example, compound 704 (= H-Ala-Lys(HO-CO-(CH2)) 16 -CO-γ-Glu-2-[2-(2-aminoethoxy)-ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH) (SEQ ID NO:4) can be obtained by deprotection reaction of compound 608-SP with a pyrolysis composition of TFA / EDT / H2O / TIS (90 / 5 / 2.5 / 2.5 vol.%, based on the volume of the pyrolysis composition).

[0416] Example D-04-2: Synthesis of Compound 704 According to general operating procedure 2, a small sample of dried compound 616-SP from Example C-09-1 was pyrolyzed to obtain compound 704 (SEQ ID NO:4).

[0417] HPLC Method 2: 73.6% area.

[0418] Figure 13 The complete HPLC-UV chromatogram is shown. Figure 14 A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0419] High-resolution mass spectrometry: [C 105 H 175 N 24 O28 ] 3+ The calculated value of m / z is 740.0997, and the measured value is 740.1031.

[0420] Example D-04-3: Synthesis of Compound 704 According to general operating procedure 2, a small sample of dried compound 616-SP from Example C-09-2 was pyrolyzed to obtain compound 704 (SEQ ID NO:4).

[0421] HPLC Method 2: 74.6% area, retention time 16.76 min.

[0422] Figure 15 The complete HPLC-UV chromatogram is shown. Figure 16 A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0423] High-resolution mass spectrometry: [C 105 H 175 N 24 O 28 ] 3+ The calculated value of m / z is 740.0997, and the measured value is 740.1021.

[0424] Example D-04-4: Synthesis of Compound 704 According to general operating procedure 2, a small sample of dried compound 616-SP from Example C-09-3 was pyrolyzed to obtain compound 704 (SEQ ID NO:4).

[0425] HPLC Method 2: 74.9% area, retention time 16.75 min.

[0426] Figure 17 The complete HPLC-UV chromatogram is shown. Figure 18 A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0427] Example D-04-5: Synthesis of Compound 704 According to general operating procedure 2, a small sample of dried compound 616-SP from Example C-09-4 was pyrolyzed to obtain compound 704 (SEQ ID NO:4).

[0428] HPLC Method 2: 80.7% area, retention time 16.75 min.

[0429] Figure 19 The complete HPLC-UV chromatogram is shown. Figure 20A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0430] Example D-04-6: Synthesis of Compound 704 According to general operating procedure 2, a small sample of dried compound 616-SP from Example C-09-5 was pyrolyzed to obtain compound 704 (SEQ ID NO:4).

[0431] HPLC Method 2: 78.2% area, retention time 16.76 min.

[0432] Figure 21 The complete HPLC-UV chromatogram is shown. Figure 22 A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0433] 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-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (705) (SEQ ID NO:5) For example, 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-γ-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, namely smegglutinin (CAS No.: 910463-68-2) (SEQ ID NO: 5), can be obtained by deprotection reaction of compound 607-SP with a pyrolysis composition of TFA / EDT / H2O / TIS (90 / 5 / 2.5 / 2.5 vol.%, based on the volume of the pyrolysis composition).

[0434] Example D-06-1: Synthesis of Compound 706

[0435] -Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (706) (SEQ ID NO:6) According to general operating procedure 2, a small sample of dried compound 611-SP from Example C-05-1 was pyrolyzed to obtain compound 706 (SEQ ID NO:6).

[0436] HPLC Method 2: 88.4% area, retention time 11.18 min.

[0437] Figure 23 The complete HPLC-UV chromatogram is shown. Figure 24 A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0438] Example D-07-1: Synthesis of Compound 707

[0439] -Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (707) (SEQ ID NO:7) According to general operating procedure 2, a small sample of dried compound 612-SP from Example C-06-1 was pyrolyzed to obtain compound 707 (SEQ ID NO:7).

[0440] HPLC Method 2: 84.0% area, retention time 11.65 min.

[0441] Figure 25 The complete HPLC-UV chromatogram is shown. Figure 26 A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0442] Example D-08-1: Synthesis of Compound 708

[0443] -Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (708) (SEQ ID NO:8) According to general operating procedure 2, a small sample of dried compound 613-SP from Example C-07-1 was pyrolyzed to obtain compound 708 (SEQ ID NO:8).

[0444] HPLC Method 2: 81.6% area, retention time 21.64 min.

[0445] Figure 27 The complete HPLC-UV chromatogram is shown. Figure 28 A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0446] Example D-09-1: Synthesis of Compound 709

[0447] -Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (709) (SEQ ID NO:9) According to general operating procedure 2, a small sample of dried compound 617-SP from Example C-10-1 was pyrolyzed to obtain compound 709 (SEQ ID NO:9).

[0448] HPLC Method 2: 80.2% area, retention time 21.72 min.

[0449] Figure 29 The complete HPLC-UV chromatogram is shown. Figure 30 A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0450] Example D-10-1: Synthesis of Compound 710

[0451] -Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (710) (SEQ ID NO:10) According to general operating procedure 2, a small sample of dried compound 618-SP from Example C-11-1 was pyrolyzed to obtain compound 710 (SEQ ID NO:10).

[0452] HPLC Method 2: 76.4% area, retention time 19.26 min.

[0453] Figure 31 The complete HPLC-UV chromatogram is shown. Figure 32 A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0454] Example D-11-1: Synthesis of Compound 711

[0455] -Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (711) (SEQ ID NO:11) According to general operating procedure 2, a small sample of dried compound 620-SP from Example C-13-1 was pyrolyzed to obtain compound 711 (SEQ ID NO:11).

[0456] HPLC Method 2: 81.0% area, retention time 19.45 min.

[0457] Figure 33 The complete HPLC-UV chromatogram is shown. Figure 34 A partially magnified view of the complete HPLC-UV chromatogram is shown.

[0458] E) Comparison of solid-phase coupling reactions The results of cleavage of small samples by solid-phase coupling compounds in Part D are correlated with the reactions of solid-phase coupling compounds in Part C, and are listed in Table E-1.

[0459] Table E-1

[0460] footnote: a) Comparative Example b) This invention c) To quantify the effectiveness of the solid-phase reaction, the solid-phase coupling reaction products were cleaved from the resin, and the area percentage of the resulting compounds in the HPLC-UV chromatogram was determined. d) Different amino protecting groups exhibit different UV absorption characteristics at 220 nm. Care should be taken when interpreting these characteristics; therefore, they are enclosed in parentheses. e) Of the observed peaks (45.7 area % or 27.8 area %), only one is compound 701, and the other is a decomposition product. f) Optimized deprotection conditions were applied for each amino protecting group: reduction of N3 phosphine; 20 vol.% DMF solution of piperidine for Fmoc and Bsmoc; and 2 vol.% DBU solution of piperidine in DMF solution for Psc and Nsc. g) Due to the different deprotection conditions in step (d), it is not possible to directly compare the overall effectiveness of the solid-state reactions in steps (c) and (d). However, the average effectiveness of cycle I confirms both the condensation effectiveness of step (c) and the effectiveness of amino protecting group removal in step (d). h) The decomposition of compound 702 made it impossible to calculate the average value. i) The three dots represent the steps in cycle II for removing the Fmoc group. j) Condensation with Fmoc-Ala-OH followed by removal of Fmoc using a 20 vol.% piperidine DMF solution. k) It should be noted that the starting resin materials are not the same; that is, the synthesis of the previous cycle I was carried out using compounds 501, 504, 506, 507, or 508, and the deprotection conditions are also partially different due to the different amino protecting groups involved. l) The average values ​​for Cycle I and Cycle II are based on the area % of cleaved and completely deprotected compound 702 and the area % of cleaved and completely deprotected compound 704. The results in Table E-1 show that: The effectiveness of Cycle I, as indicated by the area percentage of the amino protecting group of the lysine derivative used as one of the reactants in solid-phase Cycle I condensation step (c), such as the area percentage of the cleaved and completely deprotected compound 702, and the effectiveness of subsequent solid-phase Cycle II, as indicated by the area percentage of the cleaved and deprotected compound 704, are both influenced by the area percentage of the peptide cleaved from the solid phase. -Nsc offers the highest effectiveness based on area percentage, followed by Bsmoc, then N3, then Psc and Fmoc; - The reasonable assumption that the steric hindrance of the amino protecting group on the lysine derivative has an effect is not the decisive factor, i.e., considering the N3 with the smallest steric hindrance, followed by Psc / Nsc with two methylene groups after the oxygen carbonyl unit, and then Fmoc / Bsmoc with one methylene group after the oxygen carbonyl unit. Compound 701, with Nsc as the amino protecting group, exhibits high sensitivity to TFA, thus confirming the high sensitivity of compound 504, with Nsc as the amino protecting group, to TFA (and aqueous sodium bicarbonate solution), as shown in Examples B-12-8.

[0461] F) Stability Testing Example F-01-1: Comparison of compound 122 (Bsmoc-Lys-OH) and compound 125 (Fmoc-Lys-OH) in the solid state at 80°C The respective solid raw material samples were stored at 80°C and analyzed after 4 hours and 24 hours. The HPLC area percentage was determined by HPLC method 1, mode B. The results are shown in Table F-01-1-1.

[0462] Table F-01-1-1

[0463] The results in Table F-01-1-1 indicate that: Solid compound 122 (Bsmoc-Lys-OH) exhibits very low stability at 80°C and decomposes almost completely within 24 hours, while solid compound 125 (Fmoc-Lys-OH) is quite stable under the same conditions.

[0464] Example F-02-1: Comparison of compound 122 (Bsmoc-Lys-OH) and compound 125 (Fmoc-Lys-OH) in the solid state at 23°C The respective solid raw material samples were stored at 23°C and analyzed after 4 hours, 24 hours, and 168 hours. The HPLC area percentage was determined by HPLC method 1, mode B. The results are shown in Table F-02-1-1.

[0465] Table F-02-1-1

[0466] The results in Table F-02-1-1 indicate that: Solid compound 122 (Bsmoc-Lys-OH) exhibits limited stability at 23°C, particularly after 168 hours (7 days) of storage, while solid compound 125 (Fmoc-Lys-OH) is quite stable under the same conditions.

[0467] Example F-03-1: Comparison of compound 120 (Bsmoc-Lys-OH x HCl) and compound 123 (Fmoc-Lys-OH x HCl) in the solid state at 23°C The respective solid raw material samples were stored at 23°C and analyzed after 1, 2, and 5 days. The HPLC area percentage was determined by HPLC method 1, mode B. The results are shown in Table F-03-1-1.

[0468] Table F-03-1-1

[0469] footnote: a) This invention b) Comparative Example The results in Table F-03-1-1 indicate that: Solid compound 120 (Bsmoc-Lys-OH x HCl) was stable after 5 days of storage at 23°C, and its stability was comparable to that of solid compound 123 (Fmoc-Lys-OH x HCl) under the same conditions.

[0470] Example F-04-1: Comparison of compound 122 (Bsmoc-Lys-OH) with various compounds (Bsmoc-Lys-OH x acid) in the solid state at 23°C The respective solid raw material samples were stored at 23°C and analyzed after 1, 2, and 5 days. The HPLC area percentage was determined by HPLC method 1, mode B. The results are shown in Table F-04-1-1.

[0471] Table F-04-1-1

[0472] footnote: a) Comparative Example b) This invention c) Not measured d) Measured after 7 days e) The decrease in the percentage of area between t = 0 and t = 5 d f) The decrease in the percentage of area between t = 0 and t = 7 days. The results in Table F-04-1-1 indicate that: - Compared to the less stable compound 122 (Bsmoc-Lys-OH), solid compounds 120, 127, 128, 129 and 130 (Bsmoc-Lys-OH x acid) are quite stable after being stored at 23°C for 5 days; Compared with compounds 128 (Bsmoc-Lys-OH x TsOH), 129 (Bsmoc-Lys-OH x MeSO3H), and 130 (Bsmoc-Lys-OH x 0.5 H2SO4), solid compounds 120 (Bsmoc-Lys-OH x HCl) and 127 (Bsmoc-Lys-OH x TFA) are more stable after being stored at 23°C for 5 days. -Compared to the area percentages of compounds 120, 127, 128, 129 and 130 (Bsmoc-Lys-OH x acid) at t = 0, the area percentage of compound 122 (Bsmoc-Lys-OH) at t = 0 is relatively low. For details, please refer to Table F-09-1-2 in Example F-09-1.

[0473] Example F-05-1: Comparison of compound 122 (Bsmoc-Lys-OH) and compound 125 (Fmoc-Lys-OH) in DMF solution at 23°C A DMF solution (50 mg / mL) of compound 122 (Bsmoc-Lys-OH) was stored at 23°C and analyzed at 15 min, 4 h, and 24 h. Preparation of a DMF solution of compound 125 (Fmoc-Lys-OH) (target concentration 50 mg / mL) failed because compound 125 (Fmoc-Lys-OH) proved insoluble / insufficiently soluble for the purposes of this experiment under these conditions. HPLC area percentage was determined by HPLC method 1, mode B. Results are shown in Table F-05-1-1.

[0474] Table F-05-1-1

[0475] footnote: a) Given that compound 122 (Bsmoc-Lys-OH) was observed to decompose rapidly once dissolved in DMF, the area percentage of the solid feedstock used is listed. b) Undissolved / insufficiently soluble for the purposes of this experiment. The results in Table F-05-1-1 indicate that: - The DMF solution of compound 122 (Bsmoc-Lys-OH) exhibits very low stability at 23°C and decomposes significantly within 15 minutes of solution preparation and HPLC analysis. Since compound 125 (Fmoc-Lys-OH) cannot be dissolved in DMF under the same conditions as compound 122 (Bsmoc-Lys-OH), its stability cannot be measured.

[0476] Example F-06-1: Comparison of compound 120 (Bsmoc-Lys-OH x HCl) and compound 123 (Fmoc-Lys-OH x HCl) in DMF solution at 23°C The DMF solutions (50 mg / mL) of each raw material were stored at 23°C and analyzed after 1, 2, and 5 days. The HPLC area percentage of compound 120 (Bsmoc-Lys-OH x HCl) was determined by HPLC method 1, mode B. The HPLC area percentage of compound 123 (Fmoc-Lys-OH x HCl) was determined by HPLC method 1, mode A. The results are shown in Table F-06-1-1.

[0477] A portion of the DMF solution (50 mg / mL) of each raw material prepared at t = 0 in Example F-06-1 was taken for Example F-07-1.

[0478] Table F-06-1-1

[0479] footnote: a) This invention b) Comparative Example The results in Table F-06-1-1 indicate that: - A DMF solution of compound 120 (Bsmoc-Lys-OH x HCl) was stable after 5 days of storage at 23°C, and its stability was comparable to that of compound 123 (Fmoc-Lys-OH x HCl) under the same conditions.

[0480] Example F-07-1: Comparison of Compound 120 (Bsmoc-Lys-OH x HCl) + DIPEA and Compound 123 (Fmoc-Lys-OH x HCl) + DIPEA in DMF solution at 23°C At 23°C, a small amount of DIPEA (diisopropylethylamine) was added to a portion of the DMF solution (50 mg / mL) prepared from the respective raw materials taken at t = 0 in Example F-06-1 until a drop of the mixture showed a pH of approximately 7 on moistened pH paper. Samples were taken shortly after the addition of DIPEA, and then again after 1 and 2 days. The HPLC area percentage of compound 120 (Bsmoc-Lys-OHx HCl) + DIPEA was determined by HPLC method 1, mode B. The HPLC area percentage of compound 123 (Fmoc-Lys-OHxHCl) + DIPEA was determined by HPLC method 1, mode A. The results are shown in Table F-07-1-1.

[0481] Table F-07-1-1

[0482] footnote: a) This invention b) Comparative Example c) From Example F-06-1, at t = 0, i.e. before adding DIPEA. d) Samples were taken shortly after the addition of DIPEA. e) No precipitation was observed f) Precipitation was observed after adding DIPEA. g) Precipitation persists The results in Table F-07-1-1 indicate that: - At 23°C, the addition of base diisopropylethylamine to a DMF solution of compound 120 (Bsmoc-Lys-OH x HCl) leads to decomposition, but the decomposition rate does not appear to be as fast as when compound 122 (Bsmoc-Lys-OH) is directly dissolved in DMF in Example F-05-1; At 23°C, the addition of base diisopropylethylamine to a DMF solution of compound 123 (Fmoc-Lys-OH x HCl) resulted in precipitation, which is consistent with the low solubility of compound 125 (Fmoc-Lys-OH) in DMF in Example F-05-1. The low area percentages at 1 and 2 days after adding base diisopropylethylamine to a DMF solution of compound 123 (Fmoc-Lys-OH x HCl) at 23°C can be inferred in part to be due to: depletion of compound 125 (Fmoc-Lys-OH) in the solution, aging / maturation of the initial precipitate over time, and difficulty in extracting a homogeneous HPLC sample from the mixture.

[0483] Example F-08-1: Comparison of compound 122 (Bsmoc-Lys-OH) and compound 125 (Fmoc-Lys-OH) in THF:H2O = 3:1 (v / v) solution at 23°C The THF:H₂O = 3:1 (v / v) solutions (50 mg / mL) of each raw material were stored at 23°C and analyzed after 16 hours and 70 hours. The HPLC area percentage was determined by HPLC method 1, mode B. The results are shown in Table F-08-1-1.

[0484] Table F-08-1-1

[0485] The results in Table F-08-1-1 indicate that: -Compound 122 (Bsmoc-Lys-OH) exhibits limited stability after storage at 23°C for 70 hours in a THF : H2O = 3 : 1 (v / v) solution, compared to compound 125 (Fmoc-Lys-OH) which is quite stable under the same conditions.

[0486] Example F-09-1: Comparison of compound 120 (Bsmoc-Lys-OH x HCl) and compound 123 (Fmoc-Lys-OH x HCl) in THF : H2O = 3 : 1 (v / v) solution at 23°C The THF:H₂O = 3:1 (v / v) solutions (50 mg / mL) of each raw material were stored at 23°C and analyzed after 16 hours and 70 hours. The HPLC area percentage was determined by HPLC method 1, mode B. The results are shown in Table F-09-1-1.

[0487] Table F-09-1-1

[0488] footnote: a) This invention b) Comparative Example The results in Table F-09-1-1 indicate that: Compound 120 (Bsmoc-Lys-OH x acid) is stable after being stored at 23°C for 70 hours in a THF : H2O = 3 : 1 (v / v) solution, and its stability is comparable to that of compound 123 (Fmoc-Lys-OH x HCl) under the same conditions.

[0489] Example F-10-1: Comparison of compound 122 (Bsmoc-Lys-OH) with each compound (Bsmoc-Lys-OH x acid) in THF: H2O = 3 : 1 (v / v) solution at 23°C The THF:H₂O = 3:1 (v / v) solutions (50 mg / mL) of each raw material were stored at 23°C and analyzed after 16 hours and 70 hours. The HPLC area percentage was determined by HPLC method 1, mode B. The results are shown in Tables F-10-1-1 and F-10-1-2.

[0490] Table F-10-1-1

[0491] footnote: a) Comparative Example b) This invention c) The decrease in the percentage of area between t = 0 and t = 70 h The results in Table F-10-1-1 indicate that: -Compared to the less stable compound 122 (Bsmoc-Lys-OH), compounds 120, 127, 128, 129 and 130 (Bsmoc-Lys-OH x acid) are fairly stable after being stored at 23°C for 70 hours in a THF : H2O = 3 : 1 (v / v) solution; Compared with compounds 128 (Bsmoc-Lys-OH x TsOH), 129 (Bsmoc-Lys-OH x MeSO3H), and 130 (Bsmoc-Lys-OH x 0.5 H2SO4), compounds 120 (Bsmoc-Lys-OH x HCl) and 127 (Bsmoc-Lys-OH x TFA) showed similar stability after storage at 23°C for 70 hours in a THF : H2O = 3 : 1 (v / v) solution.

[0492] Table F-10-1-2

[0493] footnote: a) Comparative Example b) This invention c) Total number of drying cycles: One cycle is the drying of compound 120 (Bsmoc-Lys-OH x HCl), which serves as the common starting material for all other compounds, followed by individual drying (freeze-drying) of each compound, until the corresponding solid compounds are obtained. d) Obtaining compound 121 (Bsmoc-Lys-OH x AcOH) by first-stage separate drying (freeze-drying) and compound 122 (Bsmoc-Lys-OH) by second-stage separate drying (freeze-drying). e) Given that compound 122 (Bsmoc-Lys-OH) was observed to decompose once dissolved in THF : H2O = 3 : 1, the area percentage of the solid raw materials used is listed. f) t = 0 includes the time interval between the preparation of the solution and the measurement of the solution by HPLC. g) The decrease in area percentage of solid raw material and raw material dissolved in THF:H2O = 3:1 at t = 0. The results in Table F-10-1-2 indicate that: - The area percentage of compounds 120, 127, 128, 129 and 130 (Bsmoc-Lys-OH x acid) in THF : H2O = 3 : 1 (v / v) solution was comparable to the area percentage of the dissolved starting material, while a difference was observed in compound 122 (Bsmoc-Lys-OH); A comparison of the area percentages of compounds 120, 127, 128, 129, and 130 (Bsmoc-Lys-OH x acid) shows that drying from the aqueous solution of the tested acidic salt structures does not change the area percentages, in contrast to compound 122 (Bsmoc-Lys-OH), which has an inner salt structure.

[0494] G) Overview of Stability Testing In the solid state, compound 122 (Bsmoc-Lys-OH) exhibits relative instability compared to compound 125 (Fmoc-Lys-OH) [→ Example F-01-1, Example F-02-1], while compound 120 (Bsmoc-Lys-OH x HCl) shows similar stability to compound 123 (Fmoc-Lys-OH x HCl) at 23°C [→ Example F-03-1-1]. Compounds 120, 127, 128, 129, and 130 (Bsmoc-Lys-OH x acid) show similar stability at 23°C [→ Example F-04-1].

[0495] In DMF solution, compound 122 (Bsmoc-Lys-OH) showed great instability after dissolving at 23°C, while compound 125 (Fmoc-Lys-OH) did not dissolve [→ Example F-05-1]. In contrast, compounds 120 (Bsmoc-Lys-OH x HCl) and 123 (Fmoc-Lys-OH x HCl) showed similar stability [→ Example F-06-1]. Adding diisopropylethylamine to a DMF solution of compound 120 (Bsmoc-Lys-OH x HCl) resulted in an unstable state, while adding diisopropylethylamine to a DMF solution of compound 123 (Fmoc-Lys-OH x HCl) resulted in precipitation, which complicated the analysis [→ Example F-07-1].

[0496] In a THF:H₂O = 3:1 (v / v) solution, compound 122 (Bsmoc-Lys-OH) showed relative instability compared to compound 125 (Fmoc-Lys-OH) [→ Example F-08-1], while compound 120 (Bsmoc-Lys-OH x HCl) had similar stability to compound 123 (Fmoc-Lys-OH x HCl) at 23°C [→ Example F-09-1-1]. Compounds 120, 127, 128, 129, and 130 (Bsmoc-Lys-OH x acid) had similar stability at 23°C [→ Example F-10-1].

[0497] It should be noted that compound 120 (Bsmoc-Lys-OH x HCl) was used as a starting material [→ Example B-26-1]. It has been demonstrated that compound 120 (Bsmoc-Lys-OH x HCl) can be sufficiently silanized to allow for coupling with the activated carboxylic acid derivative to form an amide group. Therefore, the use of compound 122 (Bsmoc-Lys-OH) as a starting material was avoided. Furthermore, the chloride ions released from compound 120 (Bsmoc-Lys-OH x HCl) act as counter anions and do not interfere with the coupling reaction.

[0498] It was subsequently demonstrated that compound 123 (Fmoc-Lys-OH x HCl) could also be sufficiently silanized to allow coupling with activated carboxylic acid derivatives to form amide groups [→ Example B-28-1]. Therefore, compound 125 (Fmoc-Lys-OH) described in the literature as a silanization starting material (e.g., EP 3819308 A1, Example 1 / Step 1f, Example 3 / Step 3b) could be replaced by compound 123 (Fmoc-Lys-OH x HCl).

Claims

1. A formula 1 X compounds (1 X ) in: n is 1 or 0.5, When n is 1, HY is hydrochloric acid, trifluoroacetic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, propanesulfonic acid, butyric acid, hydrobromic acid, hydroiodic acid, perchloric acid, trifluoromethanesulfonic acid, nitric acid, picric acid, trichloroacetic acid, camphorsulfonic acid, dichloroacetic acid, difluoroacetic acid, or phosphoric acid. When n is 0.5, HY is sulfuric acid.

2. The compound according to claim 1, characterized in that, When n is 1, HY is hydrochloric acid, trifluoroacetic acid, p-toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, 2-propanesulfonic acid, 1-butanesulfonic acid, hydrobromic acid, hydroiodic acid, perchloric acid, trifluoromethanesulfonic acid, or nitric acid. When n is 0.5, HY is sulfuric acid.

3. The compound according to claim 1 or 2, characterized in that, When n is 1, HY is hydrochloric acid, trifluoroacetic acid, p-toluenesulfonic acid, or methanesulfonic acid. When n is 0.5, HY is sulfuric acid.

4. The compound according to any one of claims 1 to 3, characterized in that, The compound has been stored for at least 12 hours.

5. The compound according to any one of claims 1 to 4, characterized in that, The compound is in a solid state.

6. The compound according to any one of claims 1 to 4, characterized in that, The compound is dissolved or suspended in the solvent composition Solv. I In -1, the solvent composition comprises N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, N-octyl-2-pyrrolidone, methyl 5-(dimethylamino)-2-methyl-5-oxovalerate, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, nitromethane, acetonitrile, benzonitrile, acetone, methyl ethyl ketone, γ-butyrolactone, γ-valerate, triethyl phosphate, methanol, ethanol, propanol, butanol, diethyl ether, methyl tert-butyl ether, diisopropyl ether, benzene, toluene, xylene, chlorobenzene, pentane, hexane, heptane, octane, cyclohexane, water, or mixtures thereof.

7. A method for preparing lysine derivative LYS X The method is characterized by, The method includes step (a) I -1), (c I -1) and (d I -1), or includes step (a) I -2), (b I -2), (c I -2) and (d I -2), (a I -1) Provide Form 1 X compounds (1 X ) in: n is 1 or 0.5, When n is 1, HY is hydrochloric acid, trifluoroacetic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, propanesulfonic acid, butyric acid, hydrobromic acid, hydroiodic acid, perchloric acid, trifluoromethanesulfonic acid, nitric acid, picric acid, trichloroacetic acid, camphorsulfonic acid, dichloroacetic acid, difluoroacetic acid, or phosphoric acid. When n is 0.5, HY is sulfuric acid; (c I -1) Use the provided equation 1 X The compound is mixed with an activated carboxylic acid derivative, wherein one of one or more carboxylic acid groups of the activated carboxylic acid derivative has been converted into a carbonyl group having a covalently linked leaving group, wherein in the presence of multiple carboxylic acid groups, the other carboxylic acid groups are protected, and wherein in the presence of one or more amino or imino groups of the activated carboxylic acid derivative, the one or more amino or imino groups are protected. (d I -1) Make the provided equation 1 X The ε-nitrogen atom of the compound reacts with the carbonyl group of the activated carboxylic acid derivative to form a covalent bond between the ε-nitrogen atom and the carbonyl group, thereby forming an amide group; Among them, step (d) I -1) It may have been partially completed in step (c) I -1) Occurs during this period; or (a I -2) Provide, for example (a) I Equation 1 defined in -1) X Compounds; (b I -2) Use a silanizing agent to treat the provided formula 1 X The compound is silanized to obtain a silanized compound; (c I -2) The silanized compound is mixed with an activated carboxylic acid derivative, wherein one of one or more carboxylic acid groups of the activated carboxylic acid derivative has been converted into a carbonyl group having a covalently linked leaving group, wherein in the presence of multiple carboxylic acid groups, the other carboxylic acid groups are protected, and wherein in the presence of one or more amino or imino groups of the activated carboxylic acid derivative, the one or more amino or imino groups are protected. (d I -2) React the ε-nitrogen atom of the silanized compound with the carbonyl group of the activated carboxylic acid derivative to form a covalent bond between the ε-nitrogen atom and the carbonyl group, thereby forming an amide group; Among them, step (d) I -2) It may have been partially completed in step (c) I -2) occurs during this period.

8. The method according to claim 7, characterized in that, The lysine derivative LYS X Including 4 X -A or 4 X -B structural unit (4 X -A) (4 X -B) in: Indicates covalent connection. R 4X-B-N-1 It is an amino protecting group; And in step (c) I -1) and steps (c) I -2), the activated carboxylic acid derivative comprises formula 5 X -A or 5 X -B structural unit (5 X -A) (5 X -B) in: Indicates covalent connection. LG is a leaving group. R 5X-B-N-1 It is an amino protecting group.

9. The method according to claim 8, characterized in that, The lysine derivative LYS X Including 6 X -Aa、6 X -Ab or 6 X -Ac structural unit (6 X -A-a) (6 X -A-b) (6 X -A-c) in: Indicates covalent connection. R 6X-A-b-O-1 It is a carboxylic acid protecting group; And in step (c) I -1) and steps (c) I In -2), the activated carboxylic acid derivative comprises formula 7 X -Aa、7 X -Ab or 7 X -Ac structural unit (7 X -A-a) (7 X -A-b) (7 X -A-c) in: Indicates covalent connection. LG is a leaving group. R 7X-A-b-O-1 It is a carboxylic acid protecting group.

10. The method according to any one of claims 7 to 9, characterized in that, The lysine derivative LYS X Compounds of formula Pr-L (Pr-L), in: R L-O-1 and R L-O-2 Each group independently represents a carboxylic acid protecting group; Compound 309 (309)； Compound 310 (310)； Compound 511 (511)； Compound 514 (514)； Or compound 517 (517)。 11. The method according to any one of claims 7 to 10, characterized in that, In step (c) I -1) or step (d) I In step (c), a base that does not contain unprotected amino groups or unprotected imine groups is added, and in step (c) I -2) or step (d) I -2) Add a base that does not contain unprotected amino groups or unprotected imine groups.

12. The method according to any one of claims 7 to 11, characterized in that, In step (a) I In equation 1, -1) X The compound in the solvent composition Solv I Provided in solution or suspension form as described in -1, the solvent composition comprising N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, N-octyl-2-pyrrolidone, methyl 5-(dimethylamino)-2-methyl-5-oxovalerate, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, nitromethane, acetonitrile, acetone, methyl ethyl ketone, γ-butyrolactone, γ-valerolactone, triethyl phosphate, methanol, ethanol, propanol, butanol, diethyl ether, methyl tert-butyl ether, diisopropyl ether, benzene, toluene, xylene, chlorobenzene, pentane, hexane, heptane, octane, cyclohexane, water, or mixtures thereof; and In step (a) I In -2), equation 1 X The compound in anhydrous solvent composition Solv I The solvent composition is provided in solution or suspension form as described in -2, comprising N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, N-octyl-2-pyrrolidone, methyl 5-(dimethylamino)-2-methyl-5-oxovalerate, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, nitromethane, acetonitrile, acetone, methyl ethyl ketone, γ-butyrolactone, γ-valerolactone, triethyl phosphate, diethyl ether, methyl tert-butyl ether, diisopropyl ether, benzene, toluene, xylene, chlorobenzene, pentane, hexane, heptane, octane, cyclohexane, or mixtures thereof.

13. The method according to any one of claims 7 to 12, characterized in that, The method includes step (a) I -2), (b I -2), (c I -2) and (d I -2), and in step (b) I In -2), the silanizing agent is trimethylchlorosilane, N-methyl-N-trimethylsilylacetamide, N,O-bis(trimethylsilyl)acetamide, N,O-bis(trimethylsilyl)trifluoroacetamide, trimethylsilylmethanesulfonate or trimethylsilylmethanesulfonate.

14. A method for preparing peptide P X The method is characterized by, The method includes step (c) X ) (c X The α-amino acid derivative S-am having one unprotected α-amino group or one unprotected α-imino group is combined with the lysine derivative LYS obtained according to claim 7. X Condensation yields the intermediate peptide Int-P X .

15. Formula 1 according to any one of claims 1 to 6 X The application of compounds in peptide synthesis.

Images