KR20230074004A - Method for producing AGM peptide that specifically binds to nucleolin - Google Patents

Abstract

When following a production method of the present invention, AGM peptides that specifically bind to nucleolin can be obtained with high purity and high yield, and thus, it is possible to provide a method for mass synthesis of AGM peptides, which can be useful for diagnosis and targeted drug delivery in cancer treatment.

Description

Method for producing AGM peptide that specifically binds to nucleolin {Method for producing AGM peptide that specifically binds to nucleolin}

The present invention relates to a method for preparing an AGM peptide that specifically binds to nucleolin.

The use of cancer-specific ligands as pharmaceutical carriers enables tissue- or cell-specific delivery of chemotherapeutic agents relative to that achieved by conventional drugs, thereby reducing systemic toxicity (Allen TM. Nat Rev Cancer 2002;2: 750-63). Among cancer-specific ligands, nanoparticles and antibodies have been widely studied in clinical cancer diagnosis and treatment (Yao VJ, et al. J Control Release. 2016; 240: 267-86). Theragnostic nanoparticles and antibodies have great promise in the field of personalized medicine, as they can detect and monitor cancer in individual patients at an early stage and deliver anti-cancer drugs over a long period of time to increase the effectiveness of treatment. (Palmieri D, et al. Proc Natl Acad Sci USA. 2015; 112: 9418-23). Although nanoparticles are promising drug carrier systems, their practical application is limited due to circulatory instability, inadequate tissue distribution and cytotoxicity (Sukhanova A, et al. NanoscaleRes Lett. 2018; 13: 44). In addition, therapeutic antibodies have limitations in that delivery and diffusion to tumor tissues are slow due to their large size (Epenetos AA, et al. Cancer Res. 1986; 46: 3183-91).

As an alternative to classical diagnostic and treatment methods, cancer-specific peptides can be used to increase treatment efficiency and reduce side effects associated with nanoparticle and antibody cancer therapies (Mori T. Curr Pharm Des. 2004; 10: 2335- 43). Peptide ligands have many advantages, including facile large-scale synthesis, low immunogenicity, generation of non-toxic metabolites, and high in vivo biocompatibility (McGregor DP. Curr Opin Pharmacol. 2008; 8: 616-9).

As a preceding study, the present researchers invented a series of peptide ligands named AGM peptides that specifically bind to cancer cells and inhibit cancer growth when conjugated with anticancer drugs (Jae Il Kim, et al. Theranostics 2020. Vol. Issue 20 ).

The peptide is an AGM peptide or AGM peptide-PEG conjugate that specifically binds to cancer cells, an AGM peptide-PEG-drug conjugate that specifically binds to cancer cells and exhibits anticancer activity, and a cell-penetrating peptide that specifically binds to cancer cells. and a conjoined AGM-peptide-PEG-CPP fusion peptide.

The present researchers tried to develop a specific and optimized manufacturing method for the AGM peptide, which was invented as a prior study, and will be presented below.

Korean Registered Patent No. 1694190

An object of the present invention is to provide a method for preparing an AGM peptide that specifically binds to nucleolin.

The technical problem to be achieved by the present invention is not limited to the above-mentioned technical problem, and other technical problems not mentioned will be clearly understood by those skilled in the art from the following description.

Unless otherwise indicated herein, the abbreviations used for the designation of amino acids and protecting groups are based on terms recommended by the Commission of Biochemical Nomenclature of IUPAC-IUB (Biochemistry, 11:1726-1732 (1972)). (Pure & Appl. Chem., Vol. 56, No. 5, pp. 595-624, 1984).

As one aspect of the present invention, there is provided a method for producing an AGM peptide that specifically binds to nucleolin, comprising the following steps:

(a) obtaining a resin-attached peptide represented by Formula 20 by a solid-phase synthesis method; and

(b) removing a resin and a protecting group from the peptide obtained in step (a) to obtain a peptide represented by Formula 22:

[Formula 20]

A _m -dK _n -dC-O-Resin

[Formula 21]

Arg(R ¹ )-His(R ² )-Gly-Ala-Met-Val-Tyr(R ³ )-Leu-Lys(R ⁴ )-PEG _k -D-Lys{Arg(R ¹ )-His(R ² )-Gly-Ala-Met-Val-Tyr(R ³ )-Leu-Lys(R ⁴ )-PEG _k }

[Formula 22]

B _m -dK _n -dC-OH

[Formula 23]

Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG _k -D-Lys {Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG _k }

(In Chemical Formulas 20 to 23, A is represented by the following Chemical Formula 21, and B is represented by the following Chemical Formula 23,

R ¹ is a guanidine protecting group, R ² is an imidazole protecting group or a thio protecting group, R ³ is hydrogen or a hydroxy protecting group, and R ⁴ is hydrogen or an amine protecting group;

The dK and dC mean D-Lys and D-Cys, respectively,

wherein m and n are 1 and 0 or 2 and 1, respectively;

Wherein k is any one integer from 4 to 20).

Wherein R ¹ is tert-butyloxycarbonyl group (t-Butyloxycarbonyl), benzyloxycarbonyl group (Benzyloxycarbonyl), nitro group (Nitro), Pmc group (2,2,5,7,8-pentamethylchroman-6-sulfonyl), Mtr group (4-methoxy-2,3,6-trimethylbenzene sulfonyl), Mts group (2,3,6-trimethyl Benzenesulfonyl), Mtb group (trimethoxybenzenesulfonyl), Mds group (4-methoxy-2,6-dimethylbenzenesulfonyl), MIS group (1,2-Dimethylindole-3-sulfonyl), EDOT-2-sulfonyl group (3,4-ethylenedioxythiophene-2-sulfonyl), Pbf group (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl ) or Tos group (4-Toluenesulphonyl), and according to a preferred embodiment, it may be a Pbf group, but is not limited thereto.

Wherein R ² is a methyl group (Methyl), tert-butyloxycarbonyl group, (tert-Butyloxycarbonyl) triphenylmethyl group (Triphenylmethyl), Mmt group (4-Monomethoxytrityl), BOM group (Benzyloxymethylacetal), MBom group (3-methoxybebzyloxymethyl), or It may be an Mtt group (methyltrityl), and according to a preferred embodiment, it may be a triphenylmethyl group, but is not limited thereto.

Wherein R ³ is hydrogen, tert-butyl group (t-Butyl), triphenylmethyl group (triphenylmethyl), 2-chlorotriphenylmethyl group (2-chlorotriphenylmethyl) benzyl group (Benzyl), phenyl group (phenyl), allyl group (allyl) , methyl group (methyl), benzyl phospho group (benzyl phospho), SO3nP group (2,2-dimethylpropylsulfo), phospho group (phosphor), Clt group (2-chlorotrityl), DMAE group (dimethylaminoethyl), propargyl group (propargyl) Alternatively, it may be a PO(NMe ₂ ) ₂ ) group (bis-dimethylamino-phosphono), and according to a preferred embodiment, it may be a tert-butyl group (t-Butyl), but is not limited thereto.

R ⁴ is hydrogen, tert-butyloxycarbonyl group, triphenylmethyl group, Dde group ((4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl), Ddiv group ((4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbuty), Alloc group (Allyloxycarbonyl), methyl group (methyl), methyl, tert-Butyloxycarbonyl group (methyl, tert-Butyloxycarbonyl) , Dnp group (2,4-dinitrophenyl), hexadecanoyl group (hexadecanoyl), Mmt group (4-Monomethoxytrityl), Mtt group (methyltrityl), Mca group (7-methoxycoumarin-4-acetyl), 9-fluorenyl Methylcarbonyl group (9-Fluorenylmethylcarbonyl), benzyloxycarbonyl group, pNZ group (p-Nitrobenzyloxycarbonyl), azido group (Azido), acetyl group (Acetyl), Pryoc group (Propargyloxycarbonyl) or trifluoroacetyl group (Trifluoroacetyl) And, according to a preferred embodiment, it may be a tert-butyloxycarbonyl group, but is not limited thereto.

Protecting groups for these functional groups are described in detail in Protecting Groups in Organic Synthesis (Greene and Wuts, John Wiley & Sons, 1991).

The resin may be 2-Chlorotrityl, Trityl, 4-Methyltrityl or 4-Methoxytrityl, According to a preferred embodiment, it may be 2-chlorotrityl resin, but is not limited thereto.

The resin is dichloromethane, tetrahydrofurane, ethylacetate, acetone, and dimethylformamide. It may be mixed with one or more solvents selected from the group consisting of acetonitrile and dimethylsulfoxide, and according to a preferred embodiment, it may be mixed with dichloromethane (DCM), but is limited thereto no.

The step (a) may be a solid-phase synthesis method in which amino acids are sequentially linked. In this case, the step (a) may include loading the first amino acid into the resin, and in order to achieve high purity and high yield of the final peptide, selection of a specific first amino acid, equivalent weight relative to a specific resin, and specific loading It can have a specific reaction rate, specific reaction solution, specific addition step, etc.

The step (a) comprises the step of loading the first amino acid into the resin by reacting a first amino acid with one selected from the group consisting of the following base reagents in a resin mixed with DCM (Dichloromethane). , but is not limited to the base reagents listed below:

Pyridine, Imidazole, Pyrrolidine, Cyclohexylamine, Morpholine, Piperidine, 4-Methoxypyridine, 2 -Chloropyridine (2-Chloropyridine), 4-dimethylaminopyridine (4-Dimethylaminopyridine), aniline (Aniline), 4-Methoxyaniline (4-Methoxyaniline), 4-phenylenediamine (4-Phenylenediamine), ethylamine ( Ethylamine), diethylamine, triethylamine, DIPEA (N,N-Diisopropylethylamine) and DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene).

The step (a) further comprises loading the first amino acid on the resin, followed by capping by reacting with a solution containing any one selected from the group consisting of DCM, MeOH (Methanol), and the following base reagents. It may be a method of doing, but is not limited to the base reagents listed below:

Pyridine, Imidazole, Pyrrolidine, Cyclohexylamine, Morpholine, Piperidine, 4-Methoxypyridine, 2 -Chloropyridine (2-Chloropyridine), 4-dimethylaminopyridine (4-Dimethylaminopyridine), aniline (Aniline), 4-Methoxyaniline (4-Methoxyaniline), 4-phenylenediamine (4-Phenylenediamine), ethylamine ( Ethylamine), diethylamine, triethylamine, DIPEA (N,N-Diisopropylethylamine) and DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene).

In the capping step, DCM, MeOH, and any one selected from the group consisting of the following base reagents, (10 to 20): (1 to 5): volume ratio (v / v) of (1), (11 to 20) : (1 to 5): (1) volume ratio (v / v), (12 to 20): (1 to 5): (1) volume ratio (v / v), (13 to 20): (1 to 5): (1) volume ratio (v / v), (14 to 20): (1 to 5): (1) volume ratio (v / v), (15 to 20): (1 to 5): ( 1) volume ratio (v / v), (15 to 20): (1 to 4): (1) volume ratio (v / v) or (15 to 20): (1 to 3): (1) volume ratio It may be a method of reacting with a solution containing (v / v), but is not limited to the base reagents listed below and the above volume ratio:

Pyridine, Imidazole, Pyrrolidine, Cyclohexylamine, Morpholine, Piperidine, 4-Methoxypyridine, 2 -Chloropyridine (2-Chloropyridine), 4-dimethylaminopyridine (4-Dimethylaminopyridine), aniline (Aniline), 4-Methoxyaniline (4-Methoxyaniline), 4-phenylenediamine (4-Phenylenediamine), ethylamine ( Ethylamine), diethylamine, triethylamine, DIPEA (N,N-Diisopropylethylamine) and DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene).

Step (a) is dichloromethane, tetrahydrofurane, ethyl acetate, acetone, dimethylformamide. Acetonitrile (Acetonitrile) and dimethyl sulfoxide (Dimethylsulfoxide) can be carried out under the conditions of one or more solvents selected from the group consisting of, according to a preferred embodiment it can be carried out under dichloromethane solvent conditions, but is not limited thereto.

Step (a) is pyridine, imidazole, pyrrolidine, cyclohexylamine, morpholine, piperidine, 4-methoxypyridine ( 4-Methoxypyridine), 2-Chloropyridine, 4-Dimethylaminopyridine, Aniline, 4-Methoxyaniline, 4-phenylenediamine Phenylenediamine), Ethylamine, Diethylamine, Triethylamine, DIPEA (N,N-Diisopropylethylamine) and DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene) It may be performed under one or more base reagent conditions selected from the group consisting of, and according to a preferred embodiment, it may be performed under piperidine or DIPEA (or DIEA) base reagent conditions, but is not limited thereto.

In step (a), DCC (N, N′-Dicyclohexylcarbodiimide), DIC (N, N′-Diisopropylcarbodiimide), BOP (Benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate), PyBOP (Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate), HBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), TBTU(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate ), HATU(1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate), TATU(1-[Bis(dimethylamino)methylene]-1H -1,2,3-triazolo[4,5-b]pyridinium 3-Oxide Tetrafluoroborate), CDI (1,1'-Carbonyldiimidazole), HOBt (Hydroxybenzotriazole) and EDC HCl (1-Ethyl-3-(3- dimethylaminopropyl) carbodiimide), and according to a preferred embodiment, it may be performed under conditions of HBTU or HOBt binding reagent, but is not limited thereto.

According to a specific embodiment, the step (a) may include selecting D-Cys (R ² ) as the first amino acid and loading it into the resin, but depending on which reaction solution or other conditions are selected, may vary, but is not limited thereto.

Specifically, the step (a) may include loading the resin with the first amino acid in an amount of 0.1 to 0.5, 0.1 to 0.45, 0.1 to 0.4, 0.1 to 0.35, or 0.1 to 0.3 equivalents based on the number of moles of the resin. It is not limited.

Specifically, step (a) may include loading the first amino acid into the resin at a loading rate of 0.1 to 0.5, 0.1 to 0.45, 0.1 to 0.4, 0.1 to 0.35, or 0.1 to 0.3 mmol/g, It is not limited thereto.

The step (b) may be performed in the presence of an acidic solution.

Specifically, the step (b) is trifluoroacetic acid (TFA), triisopropylsilene (TIS), ethylenedioxydiesate acid thiol (DODT), dimethyl sulfide (DMS) and ammonium iodide (NH ₄ I) It may be performed in the presence of a mixed solution containing a combination of those selected from the group consisting of, and may include all of them according to a preferred embodiment, and may further include purified water according to the practice conditions of those skilled in the art, It is not limited thereto.

As a more specific embodiment, the step (b) includes trifluoroacetic acid (TFA), triisopropylsilene (TIS), ethylenedioxydiesatethiol (DODT), dimethylsulfide (DMS) and purified water (30 to 40): (1): (1): (1 to 5): (1 to 5) volume ratio (v/v), or (30 to 40): (1): (1): (1 to 3): (1 to 4) volume ratio (v / v), or (30 to 40): (1): (1): (1 to 3): (1 to 3) volume ratio (v / v) It may be performed in the presence, but is not limited thereto.

The peptide represented by Chemical Formula 22 may be specifically a peptide represented by Chemical Formula 24 or 25:

[Formula 24]

Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG ₆ -D-Lys (Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG ₆ )-D-Cys -OH

[Formula 25]

Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG ₆ -D-Lys (Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG ₆ )-D-Lys {Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG ₆ -D-Lys(Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG ₆ )}-D -Cys-OH.

The preparation method may further include a step (c) of reacting the peptide obtained in step (b) with a 3-maleimidopropionicacid-Paclitaxel (MPA-PTX) complex represented by Formula 3 below:

[Formula 3]

.

.

The preparation method may further include a step (d) of reacting the peptide obtained in step (b) with the MPA-AGM-130 (3-maleimidopropionicacid-AGM-130) complex represented by Formula 6 below. there is:

[Formula 6]

.

.

The preparation method may further include a step (e) of reacting the peptide obtained in step (b) with a cell penetrating peptide (CPP) represented by Formula 9 below:

[Formula 9]

.

.

In the reaction step, pH 7.0 to 10.0, 7.0 to 9.9, 7.0 to 9.8, 7.0 to 9.7, 7.0 to 9.6, 7.0 to 9.5, 7.0 to 9.4, 7.0 to 9.3, 7.0 to 9.2, 7.0 to 9.1, 7.0 to 9.0, 7.0 to 8.9, 7.0 to 8.8, 7.0 to 8.7, 7.0 to 8.6, 7.0 to 8.5, 7.0 to 8.4, 7.0 to 8.3, 7.0 to 8.2, 7.0 to 8.1, 7.0 to 8.0, 6.5 to 9.0, 6.5 to 8 .9, 6.5 to 8.8, 6.5 to 8.7, 6.5 to 8.6, 6.5 to 8.5, 6.5 to 8.4, 6.5 to 8.3, 6.5 to 8.2, 6.5 to 8.1 or 6.5 to 8.0. In terms of securing purity, it may be preferably carried out under pH 6.5 to 8.0 conditions, but is not limited thereto.

In the reaction step, dichloromethane, tetrahydrofurane, ethyl acetate, acetone, dimethylformamide. It may be performed under the condition of one or more solvents selected from the group consisting of acetonitrile and dimethylsulfoxide, and according to a preferred embodiment, it may be performed under the condition of acetonitrile, dichloromethane, or a combination thereof. , but is not limited thereto.

Hereinafter, reaction processes provided as an example will be briefly described. This is illustrative for the purpose of helping understanding of the embodiments to be described in the future, and the scope of the present invention is not limited thereto.

1. Manufacture of AGM-330d

Load the first amino acid into the 2-chlorotrityl chloride resin. Second, AGM-330d including a protecting group on the side chain is prepared by performing amide coupling sequentially according to the amino acid sequence including the protecting group on the side chain. Thereafter, the resin and the protecting group are removed using an acidic solution, and AGM-330d (Formula 1) is obtained by purification and lyophilization.

2. Manufacture of AGM-330t

Load the first amino acid into the 2-chlorotrityl chloride resin. Second, AGM-330t including a protecting group on the side chain is prepared by performing amide coupling sequentially according to the amino acid sequence including the protecting group on the side chain. Thereafter, the resin and the protecting group are removed using an acidic solution, and AGM-330t (Formula 2) is obtained by purification and freeze-drying.

3. Preparation of MPA-PTX

MPA-PTX (Formula 3) is synthesized through an esterification reaction between 3-maleimido-propionic acid (MPA; Formula 12) and Paclitaxel (PTX; Formula 13).

4. Preparation of MPA-AGM-130

As a first step, PMB-AGM-130 (Formula 15) is synthesized by introducing a PMB (p-Methoxybenzyl) protecting group into -OH of the oxime of AGM-130 (Formula 14). In the second step, MPA-PMB-AGM-130 (Formula 16) is synthesized through esterification of PMB-AGM-130 (Formula 15) and 3-Maleimido-propionic acid (Formula 12), and then PMB is deprotected in the last step. The reaction is carried out to synthesize MPA-AGM-130 (Formula 6).

5. Preparation of CPP

First, the first amino acid is loaded into the 2-chlorotrityl chloride resin. Second, amino acids containing a protecting group in the side chain are sequentially subjected to amide coupling according to the sequence to prepare CPP including a protecting group in the side chain. Thereafter, the resin and the protecting group are removed using an acidic solution, and CPP (Formula 9) is obtained by purification and lyophilization.

6. Manufacture of AGM-331d and AGM-331t

AGM-330d and AGM-330t, respectively, and MPA-PTX were subjected to 1,4-Michael addition reaction in PBS buffer to obtain AGM-331d and AGM-330t, respectively.

7. Manufacture of AGM-332d and AGM-332t

AGM-330d and AGM-330t, respectively, and MPA-AGM-130 were subjected to 1,4-Michael addition reaction in PBS buffer to obtain AGM-332d and AGM-332t.

8. Manufacture of AGM-380d and AGM-380t

AGM-330d and AGM-330t respectively and CPP were mixed with 20% ACN aq. AGM-380d and AGM-380t are respectively obtained through 1,4-Michael addition reaction at pH 7.0 (mCPP=monomeric CPP).

According to the production method of the present invention, AGM peptides that specifically bind to nucleolin can be obtained in high purity and yield, which can be usefully used for diagnosis and targeted drug delivery in cancer therapy. A method for mass synthesis of peptides can be presented.

However, it should be understood that it is not limited to the above effects, and includes all effects that can be inferred from the configuration of the invention described in the detailed description or claims.

1 schematically shows an AGM-330d manufacturing process.
Figure 2 schematically shows the AGM-330t manufacturing process.
Figure 3 schematically shows the manufacturing process of MPA-PTX.
Figure 4 schematically shows the manufacturing process of AGM-331d.
5 schematically shows the manufacturing process of AGM-331t.
Figure 6 schematically shows the manufacturing process of MPA-AGM-130.
Figure 7 schematically shows the manufacturing process of AGM-332d.
8 schematically shows the manufacturing process of AGM-332t.
9 schematically shows the manufacturing process of CPP.
10 schematically shows the manufacturing process of AGM-380d.
11 schematically shows the manufacturing process of AGM-380t.
12 is a MALDI-TOF mass analysis result for AGM-330d.
13 is a MALDI-TOF mass analysis result for AGM-330t.
14 is a MALDI-TOF mass analysis result for AGM-331d.
15 is a MALDI-TOF mass analysis result for AGM-331t.
16 is a MALDI-TOF mass analysis result for AGM-332t.
17 is a MALDI-TOF mass analysis result for AGM-380d.
18 is a MALDI-TOF mass analysis result for AGM-380t.
Figure 19 shows the crude purity (%) of AGM-330t according to the first amino acid loading rate.

Hereinafter, an example will be described in detail for a more detailed explanation. However, the following examples are illustrative, and the scope of the present invention is not limited thereto.

Example 1. Preparation of AGM-330d

1. Preparation of Fmoc-D-Cys(Trt)-O-Resin

(1) Swelling

248.2g of 2-chlorotrityl chloride resin (2-Chlorotrityl chloride resin; 350 mmol; Loading capacity: 1.41 mmol/g) was put into the reactor. Dichloromethane (DCM; 3.5L (10 L/mol) was added thereto, stirred at 25° C. for 30 minutes, and then the solvent was removed.

(2) Preparation of Fmoc-D-Cys(Trt)-O-Resin

Completely dissolve Fmoc-D-Cys(Trt)-OH (40.9 g, 0.2 eq.) and DIEA (N-N-Diisopropylethylamine; 24.4 mL, 0.4 eq.) in DCM (3.5 L). After adding this solution to the reactor, it is stirred at 25° C. for 1 hour. Remove the reaction solution and wash twice for 2 minutes with DCM (3.5 L). A capping solution (DCM: MeOH: DIEA = 85: 10: 5, v/v, 3.5 L) was added to the reactor and stirred twice at 25° C. for 15 minutes. Remove the reaction solution and wash the resin three times each for 2 minutes in the order of DCM (3.5 L) and DMF (Dimethylformamide; 3.5 L). The loading factor determined by sampling the resin after washing is 0.19 mmol/g.

2. Preparation of Fmoc-D-Lys(Fmoc)-D-Cys(Trt)-O-Resin

To remove Fmoc, 20% piperidine in DMF (3.5 L) dissolved in DMF was added to the reactor and stirred twice at 25° C. for 15 minutes. The reaction liquid is removed and the resin is washed 6 times for 2 minutes with DMF (3.5 L). 1.75 L of Fmoc-D-Lys(Fmoc)-OH (124.0 g, 3.0 eq.) in DMF, HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3 in DMF -tetramethyluronium hexafluorophosphate; 79.6 g, 3.0eq) 1.75L was added in turn, DIEA (36.6 mL, 3.0 eq.) was added, and the reaction was started at 25°C. After 1 hour, the reaction solution was removed and washed twice for 2 minutes with DMF (3.5 L).

3. Fmoc-PEG ₆ -D-Lys(Fmoc-PEG ₆ ) -D-Cys (Trt) -O-Resin Preparation

To remove Fmoc, 20% piperidine in DMF (3.5 L) dissolved in DMF was added to the reactor and stirred twice at 25° C. for 15 minutes. The reaction liquid is removed and the resin is washed 6 times for 2 minutes with DMF (3.5 L). 1.75 L of Fmoc-NH-PEG ₆ -COOH (161.2 g, 4.0 eq.) dissolved in DMF and 1.75 L of HBTU (106.2 g, 4.0 eq) dissolved in DMF were sequentially added and DIEA (48.8 mL, 4.0 eq.) After adding, the reaction starts at 25 °C. After 1 hour, the reaction solution was removed and washed twice for 2 minutes with DMF (3.5 L).

4. Fmoc-Lys(Boc)-PEG ₆ -D-Lys(Fmoc-Lys(Boc)-PEG ₆ ) -D-Cys (Trt) -O-Resin Preparation

To remove Fmoc, 20% piperidine in DMF (3.5 L) dissolved in DMF was added to the reactor and stirred twice at 25° C. for 15 minutes. The reaction liquid is removed and the resin is washed 6 times for 2 minutes with DMF (3.5 L). Fmoc-Lys(Boc)-OH (164.0 g, 5.0 eq.) and HOBt (Hydroxybenzotriazole; 93.0 g, 5.0 eq.) were dissolved in DMF (3.5 L) and placed in a reactor. After adding DIC (Diisopropylcarbodiimide; 55.0 mL, 5.0 eq.) to the reactor, the reaction is started at 25°C. After 3 hours, the reaction solution was removed and washed twice for 2 minutes with DMF (3.5 L).

5. Fmoc-Leu-Lys(Boc)-PEG ₆ -D-Lys(Fmoc-Leu-Lys(Boc)-PEG ₆ ) -D-Cys (Trt) -O-Resin Preparation

To remove Fmoc, 20% piperidine in DMF (3.5 L) dissolved in DMF was added to the reactor and stirred twice at 25° C. for 15 minutes. The reaction liquid is removed and the resin is washed 6 times for 2 minutes with DMF (3.5 L). Fmoc-Leu-OH (123.7 g, 5.0 eq.) and HOBt (93.0 g, 5.0 eq.) are dissolved in DMF (3.5 L) and placed in a reactor. DIC (55.0 mL, 5.0 eq.) is added to the reactor and the reaction is started at 25°C. After 3 hours, the reaction solution was removed and washed twice for 2 minutes with DMF (3.5 L).

6. Fmoc-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys(Fmoc-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ ) -D-Cys (Trt) -O-Resin Preparation

To remove Fmoc, 20% piperidine in DMF (3.5 L) dissolved in DMF was added to the reactor and stirred twice at 25° C. for 15 minutes. The reaction liquid is removed and the resin is washed 6 times for 2 minutes with DMF (3.5 L). Fmoc-Tyr(tBu)-OH (160.9 g, 5.0 eq.) and HOBt (93.0 g, 5.0 eq.) are dissolved in DMF (3.5 L) and placed in a reactor. DIC (55.0 mL, 5.0 eq.) is added to the reactor and the reaction is started at 25°C. After 3 hours, the reaction mixture was removed and washed twice for 2 minutes with DMF (3.5 L).

7. Fmoc-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys(Fmoc-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ ) -D-Cys (Trt) -O-Resin Preparation

To remove Fmoc, 20% piperidine in DMF (3.5 L) dissolved in DMF was added to the reactor and stirred twice at 25° C. for 15 minutes. The reaction liquid is removed and the resin is washed 6 times for 2 minutes with DMF (3.5 L). Fmoc-Val-OH (118.8 g, 5.0 eq.) and HOBt (93.0 g, 5.0 eq.) are dissolved in DMF (3.5 L) and placed in a reactor. DIC (55.0 mL, 5.0 eq.) is added to the reactor and the reaction is started at 25°C. After 3 hours, the reaction solution was removed and washed twice for 2 minutes with DMF (3.5 L).

8. Fmoc-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys(Fmoc-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ ) -D-Cys (Trt) -O-Resin Preparation

To remove Fmoc, 20% piperidine in DMF (3.5 L) dissolved in DMF was added to the reactor and stirred twice at 25° C. for 15 minutes. The reaction liquid is removed and the resin is washed 6 times for 2 minutes with DMF (3.5 L). Fmoc-Met-OH (130.1 g, 5.0 eq.) and HOBt (93.0 g, 5.0 eq.) are dissolved in DMF (3.5 L) and placed in a reactor. DIC (55.0 mL, 5.0 eq.) is added to the reactor and the reaction is started at 25°C. After 3 hours, the reaction solution was removed and washed twice for 2 minutes with DMF (3.5 L).

9. Fmoc-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys(Fmoc-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ ) -D-Cys (Trt) -O-Resin Preparation

To remove Fmoc, 20% piperidine in DMF (3.5 L) dissolved in DMF was added to the reactor and stirred twice at 25° C. for 15 minutes. The reaction liquid is removed and the resin is washed 6 times for 2 minutes with DMF (3.5 L). Fmoc-Ala-OH (109.0 g, 5.0 eq.) and HOBt (93.0 g, 5.0 eq.) are dissolved in DMF (3.5 L) and placed in a reactor. DIC (55.0 mL, 5.0 eq.) is added to the reactor and the reaction is started at 25°C. After 3 hours, the reaction solution was removed and washed twice for 2 minutes with DMF (3.5 L).

10. Fmoc-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys(Fmoc-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ ) -D-Cys (Trt) -O-Resin Preparation

To remove Fmoc, 20% piperidine in DMF (3.5 L) dissolved in DMF was added to the reactor and stirred twice at 25° C. for 15 minutes. The reaction liquid is removed and the resin is washed 6 times for 2 minutes with DMF (3.5 L). Fmoc-Gly-OH (104.1 g, 5.0 eq.) and HOBt (93.0 g, 5.0 eq.) are dissolved in DMF (3.5 L) and placed in a reactor. DIC (55.0 mL, 5.0 eq.) is added to the reactor and the reaction is started at 25°C. After 3 hours, the reaction solution was removed and washed twice for 2 minutes with DMF (3.5 L).

11. Fmoc-His(Trt)-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys(Fmoc-His(Trt)-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ ) -D-Cys (Trt) -O-Resin Preparation

To remove Fmoc, 20% piperidine in DMF (3.5 L) was added to the reactor and stirred twice at 25° C. for 15 minutes. The reaction liquid is removed and the resin is washed 6 times for 2 minutes with DMF (3.5 L). Fmoc-His(Trt)-OH (217.0 g, 5.0 eq.) and HOBt (93.0 g, 5.0 eq.) are dissolved in DMF (3.5 L) and placed in a reactor. DIC (55.0 mL, 5.0 eq.) is added to the reactor and the reaction is started at 25°C. After 3 hours, the reaction solution was removed and washed twice for 2 minutes with DMF (3.5 L).

12. Fmoc-Arg(Pbf)-His(Trt)-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys(Fmoc-Arg(Pbf)-His(Trt)-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ ) -D-Cys (Trt) -O-Resin Preparation

To remove Fmoc, 20% piperidine in DMF (3.5 L) was added to the reactor and stirred twice at 25° C. for 15 minutes. The reaction liquid is removed and the resin is washed 6 times for 2 minutes with DMF (3.5 L). Fmoc-Arg(Pbf)-OH (227.1 g, 5.0 eq.) and HOBt (93.0 g, 5.0 eq.) are dissolved in DMF (3.5 L) and placed in a reactor. DIC (55.0 mL, 5.0 eq.) is added to the reactor and the reaction is started at 25°C. After 3 hours, the reaction solution was removed and washed twice for 2 minutes with DMF (3.5 L).

To remove Fmoc, 20% piperidine in DMF (3.5 L) was added to the reactor and stirred twice at 25°C for 15 minutes. After removing the reaction solution, the resin was washed 6 times for 2 minutes with DMF (3.5 L) and 3 times with DCM (3.5 L) for 2 minutes, and dried with nitrogen.

13. Global cleavage for the manufacture of AGM-330d Crude represented by Formula 1

Cleavage cocktail (10.5 L, TFA: TIS: DODT: PW: DMS = 85: 2.5: 2.5: 5.0: 5.0, v:v) was added to the dried resin, stirred at room temperature for 2 hours, and purified water (PW) was added. ) was added to 10.0 mL of NH ₄ I (52.6 mmol, 7.6 g) dissolved in it, stirred for another 30 minutes, followed by IPC (In-process control), and after confirming that the reaction was complete, the reaction was terminated. Drain the Cleavage solution and slowly add it to 42.0L of cold ether and stir for 30 minutes. The precipitated solid was filtered under reduced pressure, washed twice with 8.0 L of ether, and then dried with nitrogen for more than 12 hours to obtain 215.0 g of AGM-330d Crude compound having a purity of 68.9%.

14. Purification process for manufacturing AGM-330d represented by Formula 1

After dissolving 215 g of AGM-330d Crude, it is filtered through a GF/C filter and a 0.45 μm HVHP membrane filter. The crude liquid was purified and lyophilized to obtain 144.2 g of AGM-330d represented by Formula 1 (or Formula 24) (Formula 24: Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG _6- D-Lys(Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG ₆ )-D-Cys-OH).

A schematic diagram of the AGM-330d manufacturing process is shown in FIG. 1, and the MALDI-TOF mass analysis result thereof is shown in FIG. 12.

[Formula 1]

Example 2. Preparation of AGM-330t

1. Preparation of Fmoc-D-Cys(Trt)-O-Resin

(1) Swelling

135.1 g of 2-chlorotrityl chloride resin (200 mmol, Loading capacity: 1.48 mmol/g) is put into the reactor. 2.0L of DCM is added here, and the solvent is removed after stirring at 20-30° C. for 30 minutes.

(2) Preparation of Fmoc-D-Cys(Trt)-O-Resin

Dissolve Fmoc-D-Cys(Trt)-OH (23.4 g, 0.2 eq.) and DIEA (13.9 mL, 0.4 eq.) in 2.0 L of DCM. After adding this solution to the reactor, it is stirred for 1 hour at 20-30°C. Remove the reaction solution and wash twice for 2 minutes each with 2.0 L of DCM. 2.0 L of a capping solution (DCM: MeOH: DIEA = 85: 10: 5, v/v) was added to the reactor and stirred at 20-30° C. for 15 minutes (repeated twice). Remove the reaction solution and wash the resin three times at 20-30 ° C for 2 minutes each in the order of DCM 2.0L and DMF 2.0L. The loading factor determined by sampling the resin after washing is 0.14 mmol/g.

2. Preparation of Fmoc-D-Lys(Fmoc)-D-Cys(Trt)-O-Resin

Add 20% piperidine (20% piperidine in DMF; 2.0L) dissolved in DMF and stir twice for 15 minutes at 20-30°C to remove Fmoc. Remove the reaction solution and wash 6 times with 2.0 L of DMF at 20-30 ° C for 2 minutes each. Dissolve Fmoc-D-Lys(Fmoc)-OH (43.1 g, 3.0 eq.) in 1.0 L of DMF. Dissolve HBTU (27.7 g, 3.0 eq.) and DIEA (12.7 mL, 3.0 eq.) in 1.0 L of DMF. The two solutions are put into a reactor and stirred at 20-30°C for 1 hour. Remove the reaction solution and wash twice for 2 minutes with 2.0 L of DMF.

3. Preparation of Fmoc-D-Lys(Fmoc)-D-Lys[Fmoc-D-Lys(Fmoc)]-D-Cys(Trt)-O-Resin

Add 20% piperidine (20% piperidine in DMF; 2.0L) dissolved in DMF and stir twice for 15 minutes at 20-30°C to remove Fmoc. Remove the reaction solution and wash 6 times with 2.0 L of DMF at 20-30° C. for 2 minutes each. Dissolve Fmoc-D-Lys(Fmoc)-OH (71.8 g, 5.0 eq.) in 1.0 L of DMF. Dissolve HBTU (46.1 g, 5.0 eq.) and DIEA (21.2 mL, 5.0 eq.) in 1.0 L of DMF. The two solutions are put into a reactor and stirred at 20-30°C for 2 hours. Remove the reaction solution and wash twice for 2 minutes with 2.0 L of DMF.

4. Fmoc-PEG ₆ -D-Lys{Fmoc-PEG ₆ }-D-Lys[Fmoc-PEG ₆ -D-Lys{Fmoc-PEG ₆ }] -D-Cys (Trt) -O-Resin Preparation

Add 20% piperidine (20% piperidine in DMF; 2.0L) dissolved in DMF and stir twice for 15 minutes at 20-30°C to remove Fmoc. Remove the reaction solution and wash 6 times with 2.0 L of DMF at 20-30 ° C for 2 minutes each. Dissolve Fmoc-NH-PEG6-COOH (112.0 g, 8.0 eq.) in 1.0 L of DMF. Dissolve HBTU (73.8 g, 8.0 eq.) and DIEA (33.9 mL, 8.0 eq.) in 1.0 L of DMF. The two solutions are put into a reactor and stirred at 20-30°C for 3 hours. Remove the reaction solution and wash twice with 2.0 L of DMF for 2 minutes each.

5. Fmoc-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-Lys(Boc)-PEG ₆ }-D-Lys[Fmoc-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-Lys(Boc)-PEG ₆ }] -D-Cys (Trt) -O-Resin Preparation

Add 20% piperidine (20% piperidine in DMF; 1.0L) dissolved in DMF and stir twice for 15 minutes at 20-30°C to remove Fmoc. Remove the reaction solution and wash 6 times with 1.0L of DMF at 20-30℃ for 2 minutes each. Fmoc-Lys(Boc)-OH (114.0 g, 10 eq.) and HOBt (16.4 g, 5 eq.) were dissolved in 1.0 L of DMF. After putting this solution in a reactor and adding DIC (37.7 mL, 10 eq.) to the reactor, the mixture was stirred at 20-30°C for 3 hours. Remove the reaction solution and wash twice for 2 minutes with 1.0 mL of DMF.

6. Fmoc-Leu-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-Leu-Lys(Boc)-PEG ₆ }-D-Lys[Fmoc-Leu-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-Leu-Lys(Boc)-PEG ₆ }] -D-Cys (Trt) -O-Resin Preparation

Add 20% piperidine (20% piperidine in DMF; 1.0L) dissolved in DMF and stir twice for 15 minutes at 20-30°C to remove Fmoc. Remove the reaction solution and wash 6 times with 1.0L of DMF at 20-30℃ for 2 minutes each. Fmoc-Leu-OH (86.0 g, 10 eq.) and HOBt (16.4 g, 5 eq.) were dissolved in 1.0 L of DMF. After putting this solution into a reactor and adding DIC (37.7 mL, 10 eq.) to the reactor, the mixture was stirred at 20-30°C for 3 hours. Remove the reaction solution and wash twice with 1.0 L of DMF for 2 minutes each.

7. Fmoc-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ }-D-Lys[Fmoc-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ }] -D-Cys (Trt) -O-Resin Preparation

Add 20% piperidine (20% piperidine in DMF; 1.0L) dissolved in DMF and stir twice for 15 minutes at 20-30°C to remove Fmoc. Remove the reaction solution and wash 6 times with 1.0L of DMF at 20-30℃ for 2 minutes each. Fmoc-Tyr(tBu)-OH (111.8 g, 10 eq.) and HOBt (16.4 g, 5 eq.) were dissolved in 1.0 L of DMF. After putting this solution into a reactor and adding DIC (37.7 mL, 10 eq.) to the reactor, the mixture was stirred at 20-30°C for 3 hours. Remove the reaction solution and wash twice for 2 minutes with 1.0 L of DMF.

8. Fmoc-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ }-D-Lys[Fmoc-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-Val-Tyr(tBu)-Leu(Boc)-Lys-PEG ₆ }] -D-Cys (Trt) -O-Resin Preparation

Add 20% piperidine (20% piperidine in DMF; 1.0L) dissolved in DMF and stir twice for 15 minutes at 20-30°C to remove Fmoc. Remove the reaction solution and wash 6 times with 1.0L of DMF at 20-30℃ for 2 minutes each. Fmoc-Val-OH (82.6 g, 10 eq.) and HOBt (16.4 g, 5 eq.) are dissolved in 1.0 L of DMF. After putting this solution into a reactor and adding DIC (37.7 mL, 10 eq.) to the reactor, the mixture was stirred at 20-30°C for 3 hours. Remove the reaction solution and wash twice with 1.0 L of DMF for 2 minutes each.

9. Fmoc-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ }-D-Lys[Fmoc-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ }] -D-Cys (Trt) -O-Resin Preparation

Add 20% piperidine (20% piperidine in DMF; 1.0L) dissolved in DMF and stir twice for 15 minutes at 20-30°C to remove Fmoc. Remove the reaction solution and wash 6 times with 1.0L of DMF at 20-30℃ for 2 minutes each. Fmoc-Met-OH (90.4 g, 10 eq.) and HOBt (16.4 g, 5 eq.) were dissolved in 1.0 L of DMF. After putting this solution into a reactor and adding DIC (37.7 mL, 10 eq.) to the reactor, the mixture was stirred at 20-30°C for 3 hours. Remove the reaction solution and wash twice with 1.0 L of DMF for 2 minutes each.

10. Fmoc-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ }-D-Lys[Fmoc-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ }] -D-Cys (Trt) -O-Resin Preparation

Add 20% piperidine (20% piperidine in DMF; 1.0L) dissolved in DMF and stir twice for 15 minutes at 20-30°C to remove Fmoc. Remove the reaction solution and wash 6 times with 1.0L of DMF at 20-30℃ for 2 minutes each. Fmoc-Ala-OH (75.7 g, 10 eq.) and HOBt (16.4 g, 5 eq.) were dissolved in 1.0 L of DMF. After putting this solution into a reactor and adding DIC (37.7 mL, 10 eq.) to the reactor, the mixture was stirred at 20-30°C for 3 hours. Remove the reaction solution and wash twice with 1.0 L of DMF for 2 minutes each.

11. Fmoc-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ }-D-Lys[Fmoc-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ }] -D-Cys (Trt) -O-Resin Preparation

Add 20% piperidine (20% piperidine in DMF; 1.0L) dissolved in DMF and stir twice for 15 minutes at 20-30°C to remove Fmoc. Remove the reaction solution and wash 6 times with 1.0L of DMF at 20-30℃ for 2 minutes each. Fmoc-Gly-OH (72.3 g, 10 eq.) and HOBt (16.4 g, 5 eq.) were dissolved in 1.0 L of DMF. After putting this solution into a reactor and adding DIC (37.7 mL, 10 eq.) to the reactor, the mixture was stirred at 20-30°C for 3 hours. Remove the reaction solution and wash twice with 1.0 L of DMF for 2 minutes each.

12. Fmoc-His(Trt)-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-His(Trt)-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ }-D-Lys[Fmoc-His(Trt)-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-His(Trt)-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ }] -D-Cys (Trt) -O-Resin Preparation

Add 20% piperidine (20% piperidine in DMF; 1.0L) dissolved in DMF and stir twice for 15 minutes at 20-30°C to remove Fmoc. Remove the reaction solution and wash 6 times with 1.0L of DMF at 20-30℃ for 2 minutes each. Fmoc-His(Trt)-OH (150.7 g, 10 eq.) and HOBt (16.4 g, 5.0 eq.) are dissolved in 1.0 L of DMF. After putting this solution into a reactor and adding DIC (37.7 mL, 10 eq.) to the reactor, the mixture was stirred at 20-30°C for 3 hours. Remove the reaction solution and wash twice for 2 minutes with 1.0 L of DMF.

13. Fmoc-Arg(Pbf)-His(Trt)-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-Arg(Pbf)-His(Trt)-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ }-D-Lys[Fmoc-Arg(Pbf)-His(Trt)-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ -D-Lys{Fmoc-Arg(Pbf)-His(Trt)-Gly-Ala-Met-Val-Tyr(tBu)-Leu-Lys(Boc)-PEG ₆ }] -D-Cys (Trt) -O-Resin Preparation

(1) Add 20% piperidine (20% piperidine in DMF; 1.0L) dissolved in DMF and stir twice for 15 minutes at 20-30°C to remove Fmoc. Remove the reaction solution and wash 6 times with 1.0L of DMF at 20-30℃ for 2 minutes each. Dissolve Fmoc-Arg(Pbf)-OH (157.8 g, 10 eq.) and HOBt (16.4 g, 5.0 eq.) in 1.0 L of DMF. After putting this solution into a reactor and adding DIC (37.7 mL, 10 eq.) to the reactor, the mixture was stirred at 20-30°C for 3 hours. Remove the reaction solution and wash twice with 1.0 L of DMF for 2 minutes each.

(2) Fmoc was removed by adding 20% piperidine (20% piperidine in DMF; 1.0L) dissolved in DMF and stirring twice for 15 minutes at 20-30°C. Remove the reaction solution and wash 6 times with 1.0L of DMF at 20-30℃ for 2 minutes each. Dry the resin under vacuum.

14. Cleavage reaction for manufacturing AGM-330t crude represented by Formula 2

Cooled cleavage cocktail 3.0 L (TFA : TIS : DODT : H ₂ O : DMS = 85 : 2.5 : 2.5 : 5 : 5, v/v, 10 mM NH ₄ I) was slowly added to the dried resin, and the temperature was 15-30℃. stirred for 3 hours. After 2 hours, NH ₄ I (4.4 g, 10 mM) was dissolved in 50.0 mL H ₂ O and added. After confirming the completion of the reaction by HPLC, the cleavage solution is drained and recovered. The cleavage solution was slowly added to 9.0 L of diethyl ether and stirred for 30 minutes. The precipitated solid was filtered under reduced pressure, washed with 3.0 L of diethyl ether, dried with nitrogen and vacuum dried to obtain 206.0 g of crude with a purity of 47.4%.

15. Purification process for manufacturing AGM-330t represented by Formula 2

After dissolving 206.6 g of AGM-330t Crude, it is filtered through a GF/C filter and a 0.45 μm HVHP membrane filter. The crude liquid was purified and lyophilized to obtain 73.1 g of AGM-330t represented by Formula 2 (or Formula 25) (Formula 25: Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG ₆ -D-Lys(Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG ₆ )-D-Lys{Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG _6- D-Lys(Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG ₆ )}-D-Cys-OH).

A schematic diagram of the AGM-330t manufacturing process is shown in FIG. 2, and the MALDI-TOF mass analysis result thereof is shown in FIG. 13.

[Formula 2]

Example 3. Preparation of AGM-331d

1. Preparation of MPA-PTX represented by Formula 3

After completely dissolving Paclitaxel (5.86 mmol, 5.0 g, 1.0 eq.) in 84 mL of anhydrous DCM, add DMAP (4-Dimethylaminopyridine; 0.0293 mmol, 3.6 mg, 0.005 eq.) and dissolve. After cooling the temperature to 0 ° C, MPA (3-Maleimidopropionic acid; 7.33 mmol, 1.24 g, 1.25 eq) and DIC (6.45 mmol, 997.5 μl, 1.1 eq.) were sequentially added and stirred at 25 ° C for 8 hours. 90 mL of DCM was added to the reaction mixture after the reaction was completed, and extraction was sequentially performed once each using 200 mL of a supersaturated NH ₄ Cl solution, 200 mL of a supersaturated NaHCO ₃ solution, and 200 mL of PW (Purified water). The DCM layer was treated with Na ₂ SO ₄ and concentrated by filtration. After adding 100 mL of DCM to the concentrated reaction mixture and completely dissolving it, the solution was slowly added dropwise to 500 mL of hexane to crystallize. The crystallized solid was filtered and dried to obtain 5.9 g of an MPA-PTX compound.

A schematic diagram of the manufacturing process of MPA-PTX is shown in FIG.

[Formula 3]

2. Preparation of AGM-331d represented by Formula 4

After completely dissolving AGM-330d (0.60 mmol, 1.81 g, 1.15 eq.) in PBS buffer 272 mL and PW (Purified water) 266 mL mixed solution (pH: ~7.4), MPA-PTX (0.52 mmol, A solution in which 520 mg, 1.0 eq.) was completely dissolved was added and stirred at room temperature for 6 hours. Upon completion of the reaction, purification and lyophilization were performed to obtain 2.1 g of AGM-331d.

A schematic diagram of the manufacturing process of AGM-331d is shown in FIG. 4, and the MALDI-TOF mass analysis result thereof is shown in FIG. 14.

[Formula 4]

Example 4. Preparation of AGM-331t

After completely dissolving AGM-330t (1.0 mmol, 6.0 g, 1.0 eq.) in a mixed solution (pH: ~7.4) of 640 mL of PBS buffer and 450 mL of PW (Purified water), MPA-PTX (1.0 mmol, 1.0 eq.) was added to 180 mL of ACN. g, 1.0 eq.) was completely dissolved and stirred at room temperature for 4 hours. Upon completion of the reaction, 2.7 g of AGM-331t represented by Chemical Formula 5 was obtained by purification and lyophilization.

A schematic diagram of the manufacturing process of AGM-331t is shown in FIG. 5, and the MALDI-TOF mass analysis result thereof is shown in FIG. 15.

[Formula 5]

Example 5. Preparation of AGM-332d

1. Preparation of MPA-AGM-130

(1) Manufacture of PMB-AGM-130

Dissolve AGM-130 (29.6 mmol, 10.0 g, 1.0 eq.) in 250 mL DMF in a 500 mL reactor, add K ₂ CO ₃ (59.1 mmol, 7.8 g, 2.0 eq.) and stir at 20-30 °C for 15 minutes. . After lowering the reaction temperature in an ice-bath, PMB-Cl (p-methoxybenzyl chloride) (44.3 mmol, 6.0 mL, 1.5 eq.) was slowly added for 10 minutes. After adding PMB-Cl, the ice-bath was removed, and the reaction temperature was raised to room temperature and stirred for 2 hours and 30 minutes. After completion of the reaction, 500 mL of H ₂ O was slowly added and stirred for 30 minutes. Filter the formed solid and wash twice with 250 mL of H ₂ O and 250 mL of diethyl ether. After that, vacuum drying for 16 hours. After vacuum drying, 11.5 g of PMB-AGM-130 was obtained.

(2) Manufacture of PMB-MPA-AGM-130

Dissolve MPA (3-Maleimidopropionic acid; 19.6 mmol, 3.3 g, 1.5 eq.) and HBTU (19.6 mmol, 7.5 g, 1.5 eq.) in 130 mL of DMF, and triethyl amine (19.6 mmol, 2.7 mL, 1.5 eq.) and stirred at 20-30°C for 16 hours. 150mL of diethyl ether was added and solidified. The solid was filtered and washed with 100 mL of ether to obtain 8.4 g. There are two other large impurities in this solid, so it is centrifuged twice through solvent crystallization using acetonitrile three to four times and centrifuged once by repeating the same process with DCM. After vacuum drying, 2.9 g of PMB-AGM-130 was obtained.

(3) Preparation of MPA-AGM-130 represented by Formula 6

Add 25 mL of a solution prepared in a DCM:TFA (2:1) ratio to MPA-PMB-AGM-130 (4.1 mmol, 2.5 g, 1.0 eq.) and stir at 20-30 °C for 2 hours. The reaction solution was concentrated under reduced pressure, and 10 mL of acetone was added thereto, and again concentrated under reduced pressure. After concentration, solidify with 20 mL of ether, and wash twice with 20 mL of ether. After vacuum drying, 1.68 g of MPA-AGM-130 was obtained.

A schematic diagram of the manufacturing process of MPA-AGM-130 is shown in FIG.

[Formula 6]

2. Preparation of AGM-332d represented by Formula 7

A schematic diagram of the manufacturing process of AGM-332d is shown in FIG.

[Formula 7]

Example 6. Preparation of AGM-332t

(1) Dissolve MPA-AGM-130 (5.1 mmol, 2.5 g, 3.0 eq.) in 860 mL DMF and stir for 15 minutes in an ice-bath. Dissolve AGM-330t (1.7 mmol, 10.4 g, 1.0 eq) in 860 mL PBS (pH: ~7.4) solution and add this solution to MPA-AGM-130 solution. Remove the ice-bath and stir for 21 hours at 20-30°C. Filter the reaction solution with 0.45 μm filter paper.

(2) To remove DMF in the reaction solution, the reaction solution is put into a column and only the main peak is separated to remove DMF. After separating the main peak, concentration is performed under reduced pressure to remove acetonitrile (ACN). After concentration, purification and salt-exchange processes were performed, and after lyophilization, 5.0 g of AGM-332t product represented by Formula 8 was obtained.

A schematic diagram of the manufacturing process of AGM-332t is shown in FIG. 8, and the MALDI-TOF mass analysis result thereof is shown in FIG. 16.

[Formula 8]

Example 7. Preparation of AGM-380d

1. Preparation of Cell Penetrating Peptide (CPP) represented by Chemical Formula 9

(1) Preparation of Fmoc-Arg(Pbf)-O-Resin

1) Swelling

142.0 g of 2-Chlorotrityl chloride resin (200 mmol, Loading capacity: 1.48 mmol/g) was put into the reactor. DCM (2.0 L) was added thereto, stirred at 20-30 ° C for 30 minutes, and then the solvent was removed.

2) Preparation of Fmoc-Arg(Pbf)-O-Resin

Dissolve Fmoc-Arg(Pbf)-OH (389.2 g, 3.0 eq.) and DIC (209.2 mL, 6.0 eq.) in 500 mL of DCM. After adding this solution to the reactor, it is stirred at 20-30°C for 3 hours. Remove the reaction solution and wash twice for 2 minutes with DCM (2.0 L). 2.0 L of a capping solution (DCM: MeOH: DIC = 85: 10: 5, v/v) was added to the reactor and stirred at 20-30° C. for 15 minutes (repeated twice). After removing the reaction solution, the resin is washed with DCM (2.0L) and DMF (2.0L) in that order at 20-30°C for 2 minutes twice each. Conduct an in-process test (measurement of substitution rate).

(2) Preparation of Fmoc-Ala-Arg(Pbf)-O-Resin

To remove Fmoc, 20% piperidine in DMF (2.0L) was added to the reactor and stirred twice at 20-30°C for 15 minutes. The reaction liquid is removed and the resin is washed with DMF (2.0 L) 6 times for 2 minutes at 20-30°C. Dissolve Fmoc-Ala-OH (186.8 g, 3.0 eq.) and HOBt (81.1 g, 3.0 eq.), DIC (93.9 mL, 3.0 eq.) in 2.0 L of DMF. After adding this solution to the reactor, it is stirred for 3 hours at 20-30°C. Remove the reaction solution and wash twice for 2 minutes with DMF (2.0 L).

(3) Preparation of Fmoc-Leu-Ala-Arg(Pbf)-O-Resin

To remove Fmoc, 20% piperidine in DMF (2.0L) was added to the reactor and stirred twice at 20-30°C for 15 minutes. The reaction solution was removed and the resin was washed 6 times with DMF (2.0 L) at 20-30° C. for 2 minutes. Dissolve Fmoc-Leu-OH (212.0 g, 3.0 eq.) and HOBt (81.1 g, 3.0 eq.), DIC (93.9 mL, 3.0 eq.) in 2.0 L of DMF. After adding this solution to the reactor, it is stirred for 3 hours at 20-30°C. Remove the reaction solution and wash twice for 2 minutes with DMF (2.0 L).

(4) Preparation of Fmoc-Lys(Boc)-Leu-Ala-Arg(Pbf)-O-Resin

To remove Fmoc, 20% piperidine in DMF (2.0L) was added to the reactor and stirred twice at 20-30°C for 15 minutes. The reaction liquid is removed and the resin is washed 6 times with DMF (2.0 L) at 20-30° C. for 2 minutes. Dissolve Fmoc-Lys(Boc)-OH (281.1 g, 3.0 eq.) and HOBt (81.1 g, 3.0 eq.), DIC (93.9 mL, 3.0 eq.) in 2.0 L of DMF. After adding this solution to the reactor, it is stirred for 3 hours at 20-30°C. Remove the reaction solution and wash twice for 2 minutes with DMF (2.0 L).

(5) Preparation of Fmoc-Leu-Lys(Boc)-Leu-Ala-Arg(Pbf)-O-Resin

To remove Fmoc, 20% piperidine in DMF (2.0L) was added to the reactor and stirred twice at 20-30°C for 15 minutes. The reaction liquid is removed and the resin is washed 6 times with DMF (2.0 L) at 20-30° C. for 2 minutes. Dissolve Fmoc-Leu-OH (212.0 g, 3.0 eq.) and HOBt (81.1 g, 3.0 eq.), DIC (93.9 mL, 3.0 eq.) in 2.0 L of DMF. After adding this solution to the reactor, it is stirred for 3 hours at 20-30°C. Remove the reaction solution and wash twice for 2 minutes with DMF (2.0 L).

(6) Preparation of Fmoc-Ile-Leu-Lys(Boc)-Leu-Ala-Arg(Pbf)-O-Resin

To remove Fmoc, 20% piperidine in DMF (2.0L) was added to the reactor and stirred twice at 20-30°C for 15 minutes. The reaction solution was removed and the resin was washed 6 times with DMF (2.0 L) at 20-30° C. for 2 minutes. Dissolve Fmoc-Ile-OH (212.0 g, 3.0 eq.) and HOBt (81.1 g, 3.0 eq.), DIC (93.9 mL, 3.0 eq.) in 2.0 L of DMF. After adding this solution to the reactor, it is stirred for 3 hours at 20-30°C. Remove the reaction solution and wash twice for 2 minutes with DMF (2.0 L).

(7) Preparation of Fmoc-Arg(Pbf)-Ile-Leu-Lys(Boc)-Leu-Ala-Arg(Pbf)-O-Resin

To remove Fmoc, 20% piperidine in DMF (2.0L) was added to the reactor and stirred twice at 20-30°C for 15 minutes. The reaction liquid is removed and the resin is washed with DMF (2.0 L) 6 times for 2 minutes at 20-30°C. Dissolve Fmoc-Arg(Pbf)-OH (389.2 g, 3.0 eq.) and HOBt (89.2 g, 3.3 eq.), DIC (93.9 mL, 3.0 eq.) in 2.0 L of DMF. After adding this solution to the reactor, it is stirred for 3 hours at 20-30°C. Remove the reaction solution and wash twice for 2 minutes with DMF (2.0 L).

(8) Preparation of Fmoc-Leu-Arg(Pbf)-Ile-Leu-Lys(Boc)-Leu-Ala-Arg(Pbf)-O-Resin

To remove Fmoc, 20% piperidine in DMF (2.0L) was added to the reactor and stirred twice at 20-30°C for 15 minutes. The reaction liquid is removed and the resin is washed 6 times with DMF (2.0 L) at 20-30° C. for 2 minutes. Dissolve Fmoc-Leu-OH (353.4 g, 5.0 eq.) and HOBt (148.6 g, 5.5 eq.), DIC (156.6 mL, 5.0 eq.) in 2.0 L of DMF. After adding this solution to the reactor, it is stirred for 3 hours at 20-30°C. Remove the reaction solution and wash twice for 2 minutes with DMF (2.0 L).

(9) Preparation of Fmoc-Ile-Leu-Arg(Pbf)-Ile-Leu-Lys(Boc)-Leu-Ala-Arg(Pbf)-O-Resin

1) To remove Fmoc, add 20% piperidine in DMF (2.0L) dissolved in DMF to the reactor and stir twice at 20-30°C for 15 minutes. The reaction liquid is removed and the resin is washed with DMF (2.0 L) 6 times for 2 minutes at 20-30°C. Dissolve Fmoc-Ile-OH (353.4 g, 5.0 eq.) and HOBt (148.6 g, 5.5 eq.), DIC (156.6 mL, 5.0 eq.) in 2.0 L of DMF. After adding this solution to the reactor, it is stirred for 3 hours at 20-30°C. Remove the reaction solution and wash twice for 2 minutes with DMF (2.0 L).

2) Dissolve Fmoc-Ile-OH (212.0 g, 3.0 eq.), HOBT (148.6 g, 5.5 eq.), and DIC (93.9 mL, 3.0 eq.) in 2.0 L of DMF. After adding this solution to the reactor, double coupling is performed by stirring at 20-30° C. for 3 hours. Remove the reaction solution and wash twice for 2 minutes with DMF (2.0 L).

(10) Preparation of Fmoc-Arg(Pbf)-Ile-Leu-Arg(Pbf)-Ile-Leu-Lys(Boc)-Leu-Ala-Arg(Pbf)-O-Resin

1) To remove Fmoc, add 20% piperidine in DMF (2.0L) dissolved in DMF to the reactor and stir twice at 20-30°C for 15 minutes. The reaction liquid is removed and the resin is washed 6 times with DMF (2.0 L) at 20-30° C. for 2 minutes. Dissolve Fmoc-Arg(Pbf)-OH (648.8 g, 5.0 eq.) and HOBt (148.6 g, 5.5 eq.), DIC (156.6 mL, 5.0 eq.) in 2.0 L of DMF. After adding this solution to the reactor, it is stirred for 3 hours at 20-30°C. Remove the reaction solution and wash twice for 2 minutes with DMF (2.0 L).

2) Dissolve Fmoc-Arg(Pbf)-OH (389.1 g, 3.0 eq.) and HOBt (148.6 g, 5.5 eq.), DIC (93.9 mL, 3.0 eq.) in 2.0 L of DMF. After adding this solution to the reactor, double coupling is performed by stirring at 20-30° C. for 3 hours. Remove the reaction solution and wash twice for 2 minutes with DMF (2.0 L).

(11) Preparation of Fmoc-Met-Arg(Pbf)-Ile-Leu-Arg(Pbf)-Ile-Leu-Lys(Boc)-Leu-Ala-Arg(Pbf)-O-Resin

To remove Fmoc, 20% piperidine in DMF (2.0L) was added to the reactor and stirred twice at 20-30°C for 15 minutes. The reaction solution was removed and the resin was washed 6 times with DMF (2.0 L) at 20-30° C. for 2 minutes. Dissolve Fmoc-Met-OH (371.5 g, 5.0 eq.) and HOBt (148.6 g, 5.5 eq.), DIC (156.6 mL, 5.0 eq.) in 500 mL of DMF. After adding this solution to the reactor, it is stirred for 3 hours at 20-30°C. Remove the reaction solution and wash twice for 2 minutes with DMF (2.0 L).

(12) Preparation of Fmoc-Ile-Met-Arg(Pbf)-Ile-Leu-Arg(Pbf)-Ile-Leu-Lys(Boc)-Leu-Ala-Arg(Pbf)-O-Resin

1) To remove Fmoc, add 20% piperidine in DMF (2.0L) dissolved in DMF to the reactor and stir twice at 20-30°C for 15 minutes. The reaction solution was removed and the resin was washed 6 times with DMF (2.0 L) at 20-30° C. for 2 minutes. Dissolve Fmoc-Ile-OH (353.4 g, 5.0 eq.) and HOBt (148.6 g, 5.5 eq.), DIC (156.6 mL, 5.0 eq.) in 2.0 L of DMF. After adding this solution to the reactor, it is stirred for 3 hours at 20-30°C. Remove the reaction solution and wash twice for 2 minutes with DMF (2.0 L).

2) Dissolve Fmoc-Ile-OH (353.4 g, 5.0 eq.) and HOBt (148.6 g, 5.5 eq.), DIC (156.6 mL, 5.0 eq.) in 2.0 L of 50% DMSO dissolved in DMF. After adding this solution to the reactor, double coupling is performed by stirring at 20-30° C. for 3 hours. Remove the reaction solution and wash twice for 2 minutes with DMF (2.0 L).

(13) Fmoc-Arg(Pbf)-Ile-Met-Arg(Pbf)-Ile-Leu-Arg(Pbf)-Ile-Leu-Lys(Boc)-Leu-Ala-Arg(Pbf)-O-Resin manufacturing

1) To remove Fmoc, add 20% piperidine in DMF (2.0L) dissolved in DMF to the reactor and stir twice at 20-30°C for 15 minutes. The reaction solution was removed and the resin was washed 6 times with DMF (2.0 L) at 20-30° C. for 2 minutes. Dissolve Fmoc-Arg(Pbf)-OH (648.8 g, 5.0 eq.) and HOBt (148.6 g, 5.5 eq.), DIC (156.6 mL, 5.0 eq.) in 2.0 L of DMF. After adding this solution to the reactor, it is stirred for 2 hours at 20-30°C. Remove the reaction solution and wash twice for 2 minutes with DMF (2.0 L).

2) Fmoc-Arg(Pbf)-OH (648.8 g, 5.0 eq.) and HOBt (148.6 g, 5.5 eq.), DIC (156.6 mL, 5.0 eq.) dissolved in 2.0 L of 50% DMSO dissolved in DMF do. After adding this solution to the reactor, double coupling is performed by stirring at 20-30° C. for 3 hours. Remove the reaction solution and wash twice for 2 minutes with DMF (2.0 L).

(14) 3-MPA-Arg(Pbf)-Ile-Met-Arg(Pbf)-Ile-Leu-Arg(Pbf)-Ile-Leu-Lys(Boc)-Leu-Ala-Arg(Pbf)-O- Resin manufacturing

To remove Fmoc, 20% piperidine in DMF (2.0L) was added to the reactor and stirred twice at 20-30°C for 15 minutes. The reaction solution was removed and the resin was washed 6 times with DMF (2.0 L) at 20-30° C. for 2 minutes. Dissolve MPA (3-Maleimidoproponic acid; 101.5 g, 3.0 eq.), HOBt (89.2 g, 3.3 eq.), and DIC (93.9 mL, 3.0 eq.) in 2.0 L of DMF. After adding this solution to the reactor, it is stirred for 3 hours at 20-30°C. After removing the reaction solution, washing twice for 2 minutes with DMF (2.0L) and twice for 2 minutes with DCM (2.0L), the resin is dried.

(15) Global cleavage reaction for producing CPP (TFA salt)

Cooled cleavage cocktail 7.0 L (TFA : TIS : H2O = 95 : 2.5 : 2.5 ) was slowly added to the dried resin and stirred at 15-30℃ for 3 hours. After confirming the completion of the reaction by HPLC, the cleavage solution is drained and recovered. The cleavage solution was slowly added to cooled diethyl ether (28.0L) and stirred for 30 minutes. In order to obtain the precipitated solid, it was filtered under reduced pressure, washed three times with 10 L of diethyl ether, dried with nitrogen and vacuum dried to obtain 266.3 g of CPP Crude.

(16) Purification process for producing CPP represented by Formula 9

After dissolving 226.3 g of the crude compound, it is filtered through a GF/C filter and a 0.45 μm HVHP membrane filter. After purification by injecting the crude liquid into a column, aliquots were collected and lyophilized to obtain 94.7 g of purified CPP (3-MPA-Arg-Ile-Met-Arg-Ile-Leu-Arg-Ile-Leu-Lys- Leu-Ala-Arg-OH).

A schematic diagram of the manufacturing process of CPP is shown in FIG.

[Formula 9]

2. Preparation of AGM-380d represented by Formula 10

After completely dissolving AGM-330d (6.2g, 2.04 mmol) in 20% acetonitrile aqueous solution (ACN aq; 620 mL, pH: ~ 7.0 adjust NH ₄ OH), CPP (5.5 g, 3.07 mmol) was added and the reaction proceeded. to terminate the reaction after overnight. The reaction solution was filtered through a GF/C filter and a 0.45 μm HVHP membrane filter, followed by purification and salt replacement, and lyophilization to obtain 6.57 g of AGM-330d-mCPP (AGM-380d) compound.

A schematic diagram of the manufacturing process of AGM-380d is shown in FIG. 10, and the MALDI-TOF mass analysis result thereof is shown in FIG. 17.

[Formula 10]

Example 8. Preparation of AGM-380t

AGM-330t (22.4 mmol, 136 g, 1.0 eq.) and CPP (33.7 mmol, 60.7 g, 1.5 eq.) are put into a reactor. Add 6800 mL of PBS (containing 25% acetonitrile, pH: ~ 7.0) and stir at 20-30°C for 2 hours. When the reaction is complete, the reaction solution is put into a rotary concentrator and concentrated under reduced pressure to remove acetonitrile, and then purification is performed. After purification, lyophilization was performed to obtain 101.4 g of AGM-380t represented by the final formula (11).

A schematic diagram of the manufacturing process of AGM-380t is shown in FIG. 11, and the MALDI-TOF mass analysis result thereof is shown in FIG. 18.

[Formula 11]

experimental example. Relationship between the loading rate and purity of the first amino acid

In general, when preparing a peptide compound, when loading the first amino acid into a resin in order to improve the final synthesis yield, 2.0 eq. By adding an excessive amount, the amino acid is loaded into the resin as much as possible. However, in the case of AGM-330d and AGM-330t, it was confirmed that as the loading ratio of the first amino acid to the resin increased, the synthesis purity was remarkably reduced due to amino acid aggregation and steric hindrance.

19 is a result showing the final product crude purity trend according to the loading ratio by adjusting the amount of the first amino acid for the resin when synthesizing AGM-330t Crude using an automatic synthesizer. As a result of the experiment, it was confirmed that the purity of AGM-330t Crude significantly decreased as the loading rate increased.

Based on the experimental results of FIG. 19, the loading ratio was set to 0.1 to 0.3 mmol/g, and the equivalent weight of amino acid relative to the number of moles of resin was set to 0.1 to 0.3 eq. When the amino acid equivalent is 0.1 eq, the crude purity is the highest at 37.0%, but considering the productivity of the product, mass synthesis of AGM-330d and AGM-330t was performed using 0.2 eq.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (20)

A method for producing an AGM peptide that specifically binds to nucleolin, comprising the following steps:
(a) obtaining a resin-attached peptide represented by Formula 20 by a solid-phase synthesis method; and
(b) removing a resin and a protecting group from the peptide obtained in step (a) to obtain a peptide represented by Formula 22:
[Formula 20]
A _m -dK _n -dC-O-Resin
[Formula 21]
Arg(R ¹ )-His(R ² )-Gly-Ala-Met-Val-Tyr(R ³ )-Leu-Lys(R ⁴ )-PEG _k -D-Lys{Arg(R ¹ )-His(R ² )-Gly-Ala-Met-Val-Tyr(R ³ )-Leu-Lys(R ⁴ )-PEG _k }
[Formula 22]
B _m -dK _n -dC-OH
[Formula 23]
Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG _k -D-Lys {Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG _k }
(In Chemical Formulas 20 to 23, A is represented by the following Chemical Formula 21, and B is represented by the following Chemical Formula 23,
R ¹ is a guanidine protecting group, R ² is an imidazole protecting group or a thio protecting group, R ³ is hydrogen or a hydroxy protecting group, and R ⁴ is hydrogen or an amine protecting group;
The dK and dC mean D-Lys and D-Cys, respectively,
wherein m and n are 1 and 0 or 2 and 1, respectively;
Wherein k is any one integer from 4 to 20).
The method according to claim 1, wherein R ¹ is tert-butyloxycarbonyl group (t-Butyloxycarbonyl), benzyloxycarbonyl group (Benzyloxycarbonyl), nitro group (Nitro), Pmc group (2,2,5,7,8-pentamethylchroman- 6-sulfonyl), Mtr group (4-methoxy-2,3,6-trimethylbenzene sulfonyl), Mts group (2,3,6-trimethyl Benzenesulfonyl), Mtb group (trimethoxybenzenesulfonyl), Mds group (4-methoxy-2, 6-dimethylbenzenesulfonyl), MIS group (1,2-Dimethylindole-3-sulfonyl), EDOT-2-sulfonyl group (3,4-ethylenedioxythiophene-2-sulfonyl), Pbf group (2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl) or Tos group (4-Toluenesulphonyl) method.
The method according to claim 1, wherein the R ² is a methyl group (Methyl), tert-butyloxycarbonyl group, (tert-Butyloxycarbonyl) triphenylmethyl group (Triphenylmethyl), Mmt group (4-Monomethoxytrityl), BOM group (Benzyloxymethylacetal), MBom group ( 3-methoxybebzyloxymethyl) or Mtt group (methyltrityl).
The method according to claim 1, wherein the R ³ is hydrogen, tert-butyl group (t-Butyl), triphenylmethyl group (triphenylmethyl), 2-chlorotriphenylmethyl group (2-chlorotriphenylmethyl) benzyl group (Benzyl), phenyl group (phenyl), Allyl group (allyl), methyl group (methyl), benzyl phospho group (benzyl phospho), SO3nP group (2,2-dimethylpropylsulfo), phospho group (phosphor), Clt group (2-chlorotrityl), DMAE group (dimethylaminoethyl), pro A method of propargyl or PO(NMe ₂ ) ₂ ) group (bis-dimethylamino-phosphono).
The method according to claim 1, wherein R ⁴ is hydrogen, tert-butyloxycarbonyl group (tert-Butyloxycarbonyl), triphenylmethyl group (triphenylmethyl), Dde group ((4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl), Ddiv group ((4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbuty), Alloc group (Allyloxycarbonyl), methyl group (methyl), methyl, tert-butyloxycarbonyl group (methyl , tert-Butyloxycarbonyl), Dnp group (2,4-dinitrophenyl), hexadecanoyl group (hexadecanoyl), Mmt group (4-Monomethoxytrityl), Mtt group (methyltrityl), Mca group (7-methoxycoumarin-4-acetyl), 9-Fluorenylmethylcarbonyl group, benzyloxycarbonyl group, pNZ group (p-Nitrobenzyloxycarbonyl), azido group, acetyl group (Acetyl), Pryoc group (Propargyloxycarbonyl) or trifluoroacetyl group (Trifluoroacetyl) method.
The method according to claim 1, wherein the resin is 2-chlorotrityl resin (2-Chlorotrityl), trityl resin (Trityl), 4-methyltrityl resin (4-Methyltrityl) or 4-methoxytrityl resin (4-Methoxytrityl ) method.
The method according to claim 1, wherein the step (a) is a step of loading the first amino acid into the resin by reacting the first amino acid with any one selected from the group consisting of the following base reagents in the resin mixed with DCM (Dichloromethane) How to include:
Pyridine, Imidazole, Pyrrolidine, Cyclohexylamine, Morpholine, Piperidine, 4-Methoxypyridine, 2 -Chloropyridine (2-Chloropyridine), 4-dimethylaminopyridine (4-Dimethylaminopyridine), aniline (Aniline), 4-Methoxyaniline (4-Methoxyaniline), 4-phenylenediamine (4-Phenylenediamine), ethylamine ( Ethylamine), diethylamine, triethylamine, DIPEA (N,N-Diisopropylethylamine) and DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene).
The method according to claim 1, wherein step (a) is capped by loading the first amino acid on the resin and then reacting with a solution containing any one selected from the group consisting of DCM, MeOH (Methanol) and the following base reagents The method further comprising the steps of:
Pyridine, Imidazole, Pyrrolidine, Cyclohexylamine, Morpholine, Piperidine, 4-Methoxypyridine, 2 -Chloropyridine (2-Chloropyridine), 4-dimethylaminopyridine (4-Dimethylaminopyridine), aniline (Aniline), 4-Methoxyaniline (4-Methoxyaniline), 4-phenylenediamine (4-Phenylenediamine), ethylamine ( Ethylamine), diethylamine, triethylamine, DIPEA (N,N-Diisopropylethylamine) and DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene).
The method according to claim 8, wherein the capping step comprises DCM, MeOH, and any one selected from the group consisting of the following base reagents in a volume ratio (v/v) of (10 to 20): (1 to 5): (1) Method of reacting with a solution of:
Pyridine, Imidazole, Pyrrolidine, Cyclohexylamine, Morpholine, Piperidine, 4-Methoxypyridine, 2 -Chloropyridine (2-Chloropyridine), 4-dimethylaminopyridine (4-Dimethylaminopyridine), aniline (Aniline), 4-Methoxyaniline (4-Methoxyaniline), 4-phenylenediamine (4-Phenylenediamine), ethylamine ( Ethylamine), diethylamine, triethylamine, DIPEA (N,N-Diisopropylethylamine) and DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene).
The method according to claim 1, wherein step (a) comprises loading D-Cys(R ² ) as the first amino acid into the resin.
Wherein step (a) comprises loading 0.1 to 0.3 equivalents of D-Cys (R ² ) to the resin relative to the number of moles of the resin.
The method according to claim 1, wherein step (a) comprises loading D-Cys(R ² ) into the resin at a loading rate of 0.1 to 0.3 mmol/g.
The method of claim 1, wherein step (b) is performed in the presence of an acidic solution.
The method according to claim 1, wherein step (b) is trifluoroacetic acid (TFA), triisopropylsilene (TIS), ethylenedioxydiesatethiol (DODT), dimethyl sulfide (DMS) and ammonium iodide (NH ₄ A method that is carried out in the presence of a mixed solution containing a combination of those selected from the group consisting of I).
The method according to claim 1, wherein R ¹ is a Pbf group, R ² is a triphenylmethyl group, R ³ is a tert-butyl group, and R ⁴ is a tert-butyloxycarbonyl group.
The method according to claim 1, wherein the peptide represented by Chemical Formula 22 is a peptide represented by Chemical Formula 24 or 25:
[Formula 24]
Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG ₆ -D-Lys (Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG ₆ )-D-Cys -OH
[Formula 25]
Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG ₆ -D-Lys (Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG ₆ )-D-Lys {Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG ₆ -D-Lys(Arg-His-Gly-Ala-Met-Val-Tyr-Leu-Lys-PEG ₆ )}-D -Cys-OH.
The method according to claim 1, further comprising the step (c) of reacting the peptide obtained in step (b) with a 3-maleimidopropionicacid-Paclitaxel (MPA-PTX) complex represented by Formula 3 below:
[Formula 3]

.
The method according to claim 1, further comprising the step (d) of reacting the peptide obtained in step (b) with the MPA-AGM-130 (3-maleimidopropionicacid-AGM-130) complex represented by Formula 6:
[Formula 6]

.
The method according to claim 1, further comprising the step (e) of reacting the peptide obtained in step (b) with a cell penetrating peptide (CPP) represented by Formula 9 below:
[Formula 9]

.
The method according to any one of claims 17 to 19, wherein the reacting is performed under conditions of pH 6.5 to 8.0.

Images