Method for producing peptide compound having ring structure

Abstract

The present invention provides a novel ring-closing metathesis reaction for constructing a peptide structure having a medium ring. The present invention provides a method for performing the ring-closing metathesis reaction, wherein the ring-closing metathesis reaction is carried out at a low dilution condition (for example, a concentration of 0.1 mol / L). The present invention provides a method for performing a ring-closing metathesis reaction suitable for mass production.

Description

In an aspect, the present invention relates to a method for producing a compound represented by formula (3) or a salt thereof. The method comprises the step of hydrogenating the compound represented by formula (1) or a salt thereof, which is obtained in the abovedescribed production method. [Formula 10] wherein R1, R2, R3, R4, n and X have the same meaning as R1, R2, R3, R4, n and X in the above [1], respectively.

[0112] The hydrogenation step can be performed by those skilled in the art, and examples thereof include a method in which the reaction mixture is stirred for 0.5 to 12 hours, and preferably for 1 to 6 hours, at a reaction temperature of 10 to 40°C in a hydrogen atmosphere with or without a solvent such as 2-methyltetrahydrofuran, methanol or ethyl acetate using a catalyst such as Pd / C, Pd(OH)2 / C or PtO2.

[0113] Method for producing compound represented by formula (4) or salt thereof In an aspect, the present invention relates to a method for producing a compound represented by formula (4) or a salt thereof. The method comprises the step of protecting the amino group of the compound represented by formula (1) or a salt thereof, which is obtained in the above-described production method, with a carbamate protective group. [Formula 11] wherein R2, R3, R4, n and X have the same meaning as R2, R3, R4, n and X in the above [1], respectively, and X1 is a carbamate protective group.

[0114] The step of protecting with a carbamate protective group can be performed by a method known to those skilled in the art, and examples thereof include a method in which the reaction mixture is stirred for 1 to 4 hours at a reaction temperature of 10 to 40°C with or without a solvent such as 2-methyltetrahydrofuran, acetonitrile or toluene using a carbamation reagent such as di-tert-butyl dicarbonate, N-carbobenzoxyoxysuccinimide or 9-fluorenylmethyl chloroformate.

[0115] In an aspect, the present invention relates to a method for producing (1S,4S,10S,13S,17S,20S,26S,28R,32S,38S,42Z)-20-cyclopentyl-28-ethoxy-32-[2-[3-methoxy-4-(trifluoromethyl)phenyl]ethyl]-N,N,2,14,18,21,24,36-octamethyl-10-[(1S)-1-methylpropyl]-3,9,12,15,19,22,25,31,34,37,45-undecaoxo-13-propyl-38-[[4-(trifluoromethyl)phenyl]methyl]spiro[2,8,11,14,18,21,24,30,33,36,39- undecazatetracyclo[37.5.1.04,8.026,30]pentatetracont-42-ene-23,1'-cyclobutane]-17-carboxamide or a salt thereof. The method includes a method in which the amino group of the compound represented by formula (4) or a salt thereof, which is obtained in the above-described production method, is synthesized according to International Publication No. WO 2023 / 214576.

[0116] Method for producing compound represented by formula (5) or salt thereof In an aspect, the present invention relates to a method for producing a compound represented by formula (5) or a salt thereof. The method comprises the step of protecting the amino group of the compound represented by formula (2) or a salt thereof, which is obtained in the above-described production method, with a carbamate protective group. [Formula 12] wherein R2, R3, R4, n and X have the same meaning as R2, R3, R4, n and X in the above [1], respectively, and X1 is a carbamate protective group.

[0117] The step of protecting with a carbamate protective group can be performed by a method known to those skilled in the art, and examples thereof include a method in which the reaction mixture is stirred for 1 to 4 hours at a reaction temperature of 10 to 40°C with or without a solvent such as 2-methyltetrahydrofuran, acetonitrile or toluene using a carbamation reagent such as di-tert-butyl dicarbonate, N-carbobenzoxyoxysuccinimide or 9-fluorenylmethyl chloroformate.

[0118] The following is description of each symbol used in the structural formula of the compounds represented by formulae (1) to (7).

[0119] R1 is hydrogen.

[0120] R2 is hydrogen or C1-C6 alkyl.  R2 is preferably hydrogen or methyl.

[0121] R3 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted halo-C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C14 aryl, optionally substituted 5- to 14membered heteroaryl, optionally substituted C7-C14 aralkyl, optionally substituted 3- to 14membered heterocyclyl, optionally substituted 5- to 10-membered heteroaryl-C1-C6 alkyl, optionally substituted C1-C6 alkoxy-C1-C6 alkyl, optionally substituted C1-C6 alkylsulfanyl-C1-C6 alkyl, optionally substituted C1-C6 alkylsulfinyl-C1-C6 alkyl, optionally substituted carboxy-C1-C6 alkyl, optionally substituted C7-C14 aralkoxy-C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl-C1-C6 alkyl, optionally substituted C3-C8 cycloalkoxy-C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclyl-C1-C3 alkyl, optionally substituted 5- to 10-membered heteroaryl-C1-C6 alkoxy-C1-C6 alkyl, or optionally substituted aminocarbonyl (the relevant amino is selected from the group consisting of -NH2, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, N-C1-C6 alkyl-N-C2-C6 alkenylamino, N-C1-C6 alkyl-N-C1-C6 alkoxy-C1-C6 alkylamino, and 4- to 9-membered cyclic amino).  R3 is preferably hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C7-C14 aralkyl, optionally substituted 5- to 10-membered heteroaryl-C1-C6 alkyl, or optionally substituted 3- to 14membered heterocyclyl, more preferably hydrogen, C1-C6 alkyl, or C7-C14 aralkyl optionally substituted with halo-C1-C3 alkyl or C1-C6 alkyl, and still more preferably hydrogen, 2-methylpropyl, p-(trifluoromethyl)benzyl, or p-methylbenzyl.

[0122] R4 is OR5, NHR5', an amino acid residue, or a peptide chain containing 1 to 20 amino acid residues, the amino acid residue and the peptide chain may have a protective group, and R5 is a protective group for the carboxy group.  R4 is preferably OR5, NHR5', an amino acid residue, or a peptide chain containing 2 to 13 amino acid residues.  R5 is preferably an alkyl ester-type protective group, a benzyl ester-type protective group, a substituted alkyl ester-type protective group, or an alkenyl ester-type protective group, more preferably a methyl group, an ethyl group, a t-Bu group, a benzyl group, a trityl group, a cumyl group, a methoxytrityl group, a 2-(trimethylsilyl)ethyl group, a 2,2,2-trichloroethyl group, or an allyl group, and still more preferably tert-butyl, or benzyl.  R5' is a phenylamide-type protective group, an alkylamide- type protective group, an alkenylamide-type protective group, a benzylamide-type protective group, or an alkoxyalkylamide-type protective group, more preferably a phenyl group, a t-Bu 41 group, an allyl group, a benzyl group, a 4-methoxybenzyl group, a trityl group, a cumyl group, a methoxymethyl group, or a benzyloxymethyl group, and still more preferably phenyl. The amino acid residue is preferably glycine, alanine, isoleucine, leucine, valine, methionine, phenylalanine, tyrosine, proline, N-methylglycine, N-methylalanine, N-methylisoleucine, N-methylleucine, N-methylvaline, N-methylmethionine, N-methylphenylalanine, N-methyltyrosine, or N-methylproline. The amino acid residue is more preferably glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, N-methylglycine, N-methylalanine, N-methylisoleucine, N-methylleucine, N-methylmethionine, N-methylphenylalanine, or N-methyltyrosine.

[0123] n is an integer of 1 to 4. n is an integer of 1 or 2. n is most preferably 2.

[0124] X is optionally substituted C1-C3 alkylene, -CH2OCH2-, or -CH2SCH2-. X is preferably optionally substituted C1-C3 alkylene, or -CH2OCH2-, more preferably optionally substituted C1-C3 alkylene, still more preferably C1-C3 alkylene, and most preferably methylene.

[0125] X1 is one selected from the group consisting of an Fmoc group, a Cbz group, a Troc group, an Alloc group, a Teoc group, a TSoc group, a BIBSoc group, an IPCSoc group, a BBSoc group, a CHBSoc group, a CDBSoc group, and a Boc group. X1 is preferably one selected from the group consisting of an Fmoc group, a Cbz group, and a Boc group. X1 is most preferably a Fmoc group.

[0126] X2 is one selected from the group consisting of an Fmoc group, a Cbz group, a Troc group, an Alloc group, a Teoc group, a TSoc group, a BIBSoc group, an IPCSoc group, a BBSoc group, a CHBSoc group, a CDBSoc group, and a Boc group. X2 is preferably one selected from the group consisting of an Fmoc group, a Cbz group, and a Boc group.  X2 is most preferably a Fmoc group.

[0127] As a combination of X1 and X2, it is preferred that each of both X1 and X2 be one selected from the group consisting of an Fmoc group, a Cbz group, a Troc group, an Alloc group, a Teoc group, a TSoc group, a BIBSoc group, an IPCSoc group, a BBSoc group, a CHBSoc group, a CDBSoc group, and a Boc group. More preferably, each of both X1 and X2 is one selected from the group consisting of an Fmoc group, a Cbz group, and a Boc group. Most preferably, each of both X1 and X2 is an Fmoc group.

[0128] Method for producing cyclic peptide compound or salt thereof In an aspect, the present invention relates to a method for producing a cyclic peptide compound or a salt thereof. The method includes a method in which the compound represented by each of formulae (1) to (5) or a salt thereof, which is obtained in the above-described production method, itself or a peptide compound obtained by chemically converting the compound or a salt thereof is cyclized.

[0129] In an aspect, the cyclic peptide compound is produced by amide cyclization of an N-terminal amino group and a C-terminal carboxy group of a peptide compound. The amide cyclization can be performed using, for example, a condensation reagent and / or a base. The condensation reagent and the base used in amide cyclization are not particularly limited. It is possible to use a condensation reagent and a base which are commonly used in peptide synthesis. The amide cyclization can also be performed by a liquid-phase synthesis method. The solvent used in the liquid-phase synthesis method is not particularly limited. It is possible to use a solvent which is commonly used in peptide synthesis.

[0130] In an aspect, the cyclic peptide compound is produced by thioether cyclization of N-terminal chloroacetyl and a C-terminal cysteine side chain of a peptide compound.

[0131] In an aspect, a cyclic peptide compound or a salt thereof produced by the method of the present invention contains 8 to 20, preferably 11 to 14, more preferably 11 to 13, most preferably 11 amino acid residues.

[0132] In an aspect, a cyclic peptide compound or a salt thereof produced by the method of the present invention can contain at least one, at least two, at least three, at least four, or at least five non-natural amino acid residues. In certain embodiments, the non-natural amino acid residues contained in the cyclic peptide compound or a salt thereof produced by the method of the present invention are N-methylamino acid residues.

[0133] In an aspect, a cyclic peptide compound produced by the method of the present invention may comprise a cyclic structure (cyclic portion) as its moiety structure. Specific examples of the cyclic structure include those in which the side chain of one amino acid residue is linked to a side chain of another amino acid residue, those in which the N-substituent of one amino acid residue is linked to the side chain of another amino acid residue, and those in which the N-substituent of one amino acid residue is linked to the N-substituent of another amino acid residue. The two amino acid residues involved in the linkage for the cyclic structure may be adjacent, or any number of amino acid residues, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 43 13, 14, 15, 16, 17, 18, or 19 amino acid residues may be present therebetween. Examples of the size of the ring formed by the cyclic structure include, but are not intended to be particularly limited to, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8membered ring, a 9-membered ring, a 10-membered ring, an 11-membered ring, a 12-membered ring, a 13-membered ring, a 14-membered ring, a 15-membered ring, a 16-membered ring, a 17membered ring, an 18-membered ring, a 19-membered ring, a 20-membered ring, a 21membered ring, a 22-membered ring, a 23-membered ring, a 24-membered ring, a 25-membered ring, a 26-membered ring, a 27-membered ring, a 28-membered ring, a 29-membered ring, a 30membered ring, a 31-membered ring, a 32-membered ring, a 33-membered ring, a 34-membered ring, and a 35-membered ring. Preferably, the ring formed by the cyclic structure is a 5membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, a 9-membered ring, a 10-membered ring, an 11-membered ring, a 12-membered ring, a 13-membered ring, a 14-membered ring, a 15-membered ring, a 16-membered ring, a 17-membered ring, an 18membered ring, a 19-membered ring, or a 20-membered ring, more preferably a 10-membered ring, an 11-membered ring, a 12-membered ring, a 13-membered ring, a 14-membered ring, a 15-membered ring, a 16-membered ring, a 17-membered ring, or an 18-membered ring, most preferably an 11-membered ring, a 12-membered ring, a 13-membered ring, or a 14-membered ring. When a cyclic structure is present in the peptide compound, the number of cyclic structures is not limited, but it is preferable that one, two, three, four, or five cyclic structures be present.

[0134] In an aspect, a cyclic peptide compound or a salt thereof produced by the method of the present invention can contain a cyclic portion composed of 4 or more, 6 or more, 8 or more, or 11 or more amino acid residues. In an embodiment, a cyclic peptide compound or a salt thereof produced by the method of the present invention consists of a cyclic portion composed of 4 to 14, preferably 6 to 14, further preferably 8 to 14, most preferably 11 to 14 amino acid residues. In an embodiment, a cyclic peptide compound or a salt thereof produced by the method of the present invention consists of a cyclic portion composed of 11 amino acid residues. In these aspects, the amide bond with which the amino group of the first amino acid or peptide and the carboxy group of the second amino acid or peptide are linked is contained in the cyclic portion at 1, 2, 3, 4, 5, 6, or 7 locations.

[0135] In an aspect, the cyclic peptide compound or a salt thereof produced by the method of the present invention is (1S,4S,10S,13S,17S,20S,26S,28R,32S,38S,42Z)-20-cyclopentyl-28-ethoxy-32-[2-[3-methoxy-4-(trifluoromethyl)phenyl]ethyl]-N,N,2,14,18,21,24,36-octamethyl-10-[(1S)-1-methylpropyl]-3,9,12,15,19,22,25,31,34,37,45-undecaoxo-13-propyl-38-[[4- 44 (trifluoromethyl)phenyl]methyl]spiro[2,8,11,14,18,21,24,30,33,36,39- undecazatetracyclo[37.5.1.04,8.026,30]pentatetracont-42-ene-23,1'-cyclobutane]-17-carboxamide or a salt thereof.

[0136] In an aspect, the compound represented by formula (1) or a salt thereof, which is produced by the method of the present invention, may be a compound represented by formula (1a) or a salt thereof. [Formula 13] wherein Y is a hydroxyl group, optionally substituted C1-C10 alkoxy, optionally substituted C6-C16 aryloxy, optionally substituted C7-C14 alkoxy, or optionally substituted 3- to 12-membered cyclic aminooxy.

[0137] In an aspect, Y in formula (1a) is preferably C1-C8 alkoxy, C6 aryloxy, C10 aryloxy, C7-C16 aralkoxy, or 3- to 12-membered cyclic aminooxy, more preferably methoxy, ethoxy, tertbutoxy, phenoxy, 1-naphthyloxy, tolyloxy, benzyloxy, phenethyloxy, 1-pyrrolidyloxy, 1-piperidyloxy, or 1-piperazyloxy, and most preferably tert-butoxy.

[0138] In an aspect, the compound represented by formula (1a) or a salt thereof is tert-butyl N-methyl-N-((S)-2-((S)-3-(methylamino)-2-oxo-2,3,4,7-tetrahydro-1H-azepin-1-yl)-3-(4-(trifluoromethyl)phenyl)propanoyl)glycinate or a hydrochloride thereof.

[0139] In an aspect, the compound represented by formula (1) or a salt thereof, which is produced by the method of the present invention, may be a compound represented by formula (1b) or a salt thereof. [Formula 14] wherein R6 is methyl or trifluoromethyl, and Z is a hydroxyl group, optionally substituted C1-C10 alkoxy, optionally substituted C6-C16 aryloxy, optionally substituted C7-C14 alkoxy, or optionally substituted 3- to 12-membered cyclic aminooxy.

[0140] In an aspect, Z in formula (1b) is preferably C1-C8 alkoxy, C6 aryloxy, C10 aryloxy, C7-C16 aralkoxy, or 3- to 12-membered cyclic aminooxy, more preferably methoxy, ethoxy, tertbutoxy, phenoxy, 1-naphthyloxy, tolyloxy, benzyloxy, phenethyloxy, 1-pyrrolidyloxy, 1-piperidyloxy, or 1-piperazyloxy, and most preferably tert-butoxy.

[0141] In an aspect, R6 in formula (1b) is preferably methyl. In another aspect, R6 in formula (1b) is preferably trifluoromethyl.

[0142] In an aspect, the compound represented by formula (1b) or a salt thereof is tert-butyl N-methyl-N-((S)-2-((S,Z)-3-(methylamino)-2-oxo-3,4,7,8-tetrahydroazocin-1(2H)-yl)-3-(p-tolyl)propanoyl)glycinate or a hydrochloride thereof.

[0143] In an aspect, the compound represented by formula (1b) or a salt thereof is tert-butyl N-methyl-N-((S)-2-((S,Z)-3-(methylamino)-2-oxo-3,4,7,8-tetrahydroazocin-1(2H)-yl)-3-(4-(trifluoromethyl)phenyl)propanoyl)glycinate or a hydrochloride thereof.

[0144] In an aspect, the compound represented by formula (1b) or a salt thereof is tert-butyl N-methyl-N-((S)-2-((S,Z)-3-(methylamino)-2-oxo-2,3,4,5,8,9-hexahydro-1H-azonin-1-yl)-3-(p-tolyl)propanoyl)glycinate or a hydrochloride thereof.

[0145] In an aspect, the compound represented by formula (1b) or a salt thereof is tert-butyl (S)-N,N-dimethyl-2-((S,Z)-3-(methylamino)-2-oxo-3,4,7,8-tetrahydroazocin-1(2H)-yl)-3-(4-(trifluoromethyl)phenyl)propaneamide or a hydrochloride thereof. 46

[0146] In an aspect, the method for producing a compound represented by formula (1) or a salt thereof comprises the step (metathesis step) of bringing a compound represented by formula (2) or a salt thereof into contact with a catalyst. Formation of an impurity is suppressed as compared to a case where R1 in formulae (1) and (2) is a carbamate protective group.

[0147] In an aspect, the method for producing a compound represented by formula (1) or a salt thereof comprises the step (metathesis step) of bringing a compound represented by formula (2) or a salt thereof into contact with a catalyst. Formation of a dimer is suppressed as compared to a case where R1 in formulae (1) and (2) is a carbamate protective group.

[0148] In an aspect, the purity of the compound represented by formula (1) or a salt thereof is 90% or more, preferably 95% or more, more preferably 98% or more, and most preferably 99% or more as determined by a UVArea value at 210 nm from HPLC analysis.

[0149] In an aspect, the content ratio of an impurity contained in the compound represented by formula (1) or a salt thereof is less than 10%, preferably less than 5%, more preferably less than 1%, and most preferably less than 0.5% or an undetectable level as determined by a UVArea value at 210 nm from HPLC analysis.

[0150] Examples of the impurity as used herein may include a dimer in which compounds represented by formula (2) are intermolecularly linked. As such a dimer, a chain compound obtained by an intermolecular olefin metathesis reaction proceeding at one position (e.g., a compound represented by the following formula (8) in Examples 1 to 6) or a cyclic compound obtained by the reaction proceeding at two positions (e.g., a compound represented by the following formula (9) in Examples 1 to 6). [Formula 15] The impurity may include, in addition to compounds represented by formulae (8) and (9), compounds in which the olefin metathesis reaction occurs at a different position, and compounds having a different E / Z form of the double bond.

[0152] All references cited herein, including the following references, with inclusion of patent applications and publications, are incorporated herein by reference in their entirety: International Publication No. WO 2013 / 100132; International Publication No. WO 2018 / 225851; International Publication No. WO 2018 / 225864; International Publication No. WO 2019 / 117274; International Publication No. WO 2020 / 111238; International Publication No. WO 202012 / 2182; International Publication No. WO 2021 / 075478; International Publication No. WO 2021 / 090856; International Publication No. WO 2021132545; International Publication No. WO 2021246471; International Publication No. WO 2022 / 097540; International Publication No. WO 2022 / 138891; International Publication No. WO 20221 / 45444; International Publication No. WO 2022 / 234864; International Publication No. WO 2023 / 127869; International Publication No. WO 2022 / 234853; and International Publication No. WO 2023 / 214576. [Examples]

[0153] Hereinafter, the present disclosure will be described in more detail based on Examples, but the present disclosure is not limited to the following Examples.

[0154] In the following Examples, high performance liquid chromatography (HPLC) analysis was performed using any of the analytical conditions described below. Detection of each compound was performed using a photodiode array detector or mass spectrometer, although other techniques such as evaporative light scattering detection may also be used.

[0155] HPLC analysis conditions: method 1 Apparatus: Waters ACQUITY UPLC H-Class + ACQUITY QDA Column: Ascentis Express 90A C18 (Sigma-Aldrich Co. LLC), 2.1 mm ID x 50 mm, 2.7 ^m Mobile phase: 0.05% TFA / water (A), 0.05% TFA / MeCN (B) Elution method: B) 5% (0 min) ^ 100% (5 min) ^ 5% (5.1 min) ^ 5% (7 min) Flow rate: 0.5 mL / min Column temperature: 35°C Detection wavelength: 210 nm (PDA)

[0156] HPLC analysis condition method 2 Apparatus: Waters ACQUITY UPLC H-Class Column: Ascentis Express 90A C18 (Sigma-Aldrich Co. LLC), 2.1 mm ID x 50 mm, 2.7 pm Mobile phase: 0.05% TFA / water (A), 0.05% TFA / MeCN (B) Elution method: B): 5% (0 min) > 100% (5 min) > 5% (5.1 min) > 5% (7 min) Flow rate: 0.5 mL / min Column temperature: 35°C Detection wavelength: 210 nm (PDA)

[0157] HPLC analysis condition method 3 Apparatus: Waters ACQUITY UPLC H-Class + ACQUITY QDA Column: Ascentis Express 90A C18 (Sigma-Aldrich Co. LLC), 2.1 mm ID x 50 mm, 2.7 pm Mobile phase: 0.05% TFA / water (A), 0.05% TFA / MeCN (B) Elution method: B) 50% (0 min)>100% (3 min) > 100% (5 min) > 50% (5.1 min) > 50% (7 min) Flow rate: 0.5 mL / min Column temperature: 35°C Detection wavelength: 220 nm (PDA)

[0158] HPLC analysis condition method 4 Apparatus: Waters ACQUITY UPLC H-Class Column: Ascentis Express RP-amide(Sigma-Aldrich Co. LLC), 3.0 mm ID x 50 mm, 2.7 pm Mobile phase: 0.05% TFA / water (A), 0.05% TFA / MeCN (B) Elution method: B): 5% (0 min) > 100% (5 min) > 5% (5.1 min) > 5% (7 min) Flow rate: 0.5 mL / min Column temperature: 35°C Detection wavelength: 210 nm (PDA)

[0159] HPLC analysis condition method 5 Apparatus: Waters ACQUITY UPLC H-Class Column: Ascentis Express 90A C18 (Sigma-Aldrich Co. LLC), 2.1 mm ID x 50 mm, 2.7 pm Mobile phase: 0.05% TFA / water (A), 0.05% TFA / MeCN (B) Elution method: B) 5% (0 min) >100% (5 min) > 100% (7 min) > 5% (7.1 min) > 5% (9 min) Flow rate: 0.5 mL / min Column temperature: 35°C Detection wavelength: 210 nm (PDA)

[0160] The conversion ratio of the reaction mixture from HPLC analysis and the ratio of a dimer formed through the intermolecular reaction to the product were calculated as follows. Conversion ratio (%): {area value of product / (area value of product + area value of starting material)} x 100 Ratio of dimer formed through intermolecular reaction to product: (sum of area values of all dimers / area value of product) x 100

[0161] 1H-NMR spectra were measured using a nuclear magnetic resonance device ECX500II (manufactured by JEOL Ltd.) and referenced to the deuterium lock signal from the sample solvent. As the sample solvent, commercially available deuterated solvents were used, depending on the purpose of measurement. The chemical shift of tetramethylsilane used as an internal standard was set to 0 ppm, and the chemical shift of the signal of the compound to be analyzed was expressed in ppm. Abbreviations of a signal were expressed as follows: s = singlet, brs = broad singlet, d = doublet, t = triplet, q = quartet, dd = double doublet, and m = multiplet. The splitting width of a signal was expressed as a J value (Hz). The integral value of signals was calculated on the basis of a ratio of the signal area intensity of each signal.

[0162] The measuring method by qNMR was performed by dissolving a residue containing a target compound and an internal standard substance in DMSO-d6 and subjecting to the following analysis conditions. Measuring apparatus: JNM-ECZ500R Internal standard: 3,5-bis(trifluoromethyl)benzoic acid Measurement conditions (1H-NMR): DMSO-d6, pulse angle 90°C, digital resolution 0.25 Hz, relaxation time 60 seconds, no spin, cumulative number 8 times

[0163] Example 1-1 Synthesis of (S)-2-(but-3-en-1-ylamino)-3-(4-(trifluoromethyl)phenyl)propionic acid (compound 1) [Formula 16] To a reaction vessel filled with nitrogen gas, acetonitrile (822 mL), 4-bromo-1-butene (235.07 g), and triethylamine (17.66 g) were added at an external temperature of 25°C, and the mixture was stirred for 1 hour. Subsequently, (S)-2-amino-3-(4-(trifluoromethyl)phenyl)propionic acid (135.46 g), water (676 mL), and triethylamine (158.19 g) were added, the external temperature was raised to 70°C, and the mixture was stirred for 3.5 hours. The reaction mixture was cooled to 25°C, and the precipitated solid was collected by filtration, and washed with a mixture of acetonitrile and water (v:v = 1:1, 676 mL). Subsequently, the obtained wet solid was further washed with acetonitrile (676 mL). The obtained wet solid was dried under reduced pressure at 40°C to afford compound 1 as a white solid (124.73 g). LCMS (ESI) of compound 1, retention time: 2.34 minutes, m / z = 288 [M+H]+ (HPLC analysis conditions: method 1)

[0165] Example 1-2 Synthesis of tert-butyl (S)-N-(2-(but-3-en-1-ylamino)-3-(4-(trifluoromethyl)phenyl)propanoyl)-N-methylglycinate hydrochloride (compound 2-HCl) [Formula 17]

[0166] To a first reaction vessel filled with nitrogen gas, compound 1 (107.26 g) synthesized in Example 1-1, sarcosine tert-butyl ester hydrochloride (102.00 g), acetonitrile (751 mL) and DBU (233.03 g) were added at an external temperature of 25°C, and the mixture was stirred for 10 minutes. After the solution became homogeneous, the external temperature was set to 2°C and a 50% solution of T3P in 2-MeTHF (309.03 g) was added dropwise over 2 hours and 14 minutes. After the reaction was completed, toluene (751 mL) and a 1 mol / L aqueous sodium hydroxide solution (536 mL) were added to the reaction mixture. After the resulting mixture was stirred for 30 minutes, the aqueous layer was discharged. The organic layer was stored at approximately 25°C overnight. After storage, a 5% aqueous sodium carbonate solution (536 mL) was added to the organic layer, which was stirred for 10 minutes, and the aqueous layer was then discharged. Subsequently, a 5% aqueous sodium dihydrogen phosphate solution (751 mL) was added to the organic layer, which was stirred for 10 minutes, and the aqueous layer was then 51 discharged. Further, a 5% aqueous sodium dihydrogen phosphate solution (751 mL) was added to the organic layer, which was stirred for 10 minutes, and the aqueous layer was then discharged. Subsequently, a 5% aqueous sodium chloride solution (751 mL) was added to the organic layer, which was stirred for 10 minutes, and the aqueous layer was then discharged. The organic layer was stored at an external temperature of 5°C overnight. After storage, the organic layer was concentrated under reduced pressure at 40°C until the liquid volume of the organic layer reached about 215 mL. Toluene (215 mL) was added to the concentrated solution, the mixture was concentrated under reduced pressure at 40°C to about 215 mL, and this operation was repeated two more times. The precipitated inorganic salt was filtered, and toluene (276.6 mL) was added to the obtained filtrate. In a second reaction vessel, pyridine hydrochloride (43.28 g) and acetonitrile (148 mL) were added, and the prepared solution was added dropwise over 45 minutes to the solution of compound 2 in toluene at an external temperature of 25°C.  Precipitation of a crystal was confirmed during the dropwise addition. The second reaction vessel was rinsed using acetonitrile (74 mL), and the resulting solution was added to the mixture containing compound 2. The resulting mixture was stirred for 1 hour. Thereafter, toluene (1.7 L) was added, and the mixture was stirred for 1 hour, the external temperature was then lowered to 0°C, and the mixture was further stirred for 2 hours. The obtained crystal was filtered, and washed twice with toluene (296 mL) which was cooled to 0°C. The obtained wet solid was stored at an external temperature of 5°C for a weekend. After storage, the wet solid was dried under reduced pressure at the external temperature of 40°C to afford compound 2-HCl as a white solid (98.66 g). LCMS (ESI) of compound 2, retention time: 3.03 minutes, m / z = 415 [M+H]+ (HPLC analysis conditions: method 1)

[0167] Example 1-3 Synthesis of (5S,8S)-5-allyl-7-(but-3-en-1-yl)-1-(9H-fluoren-9-yl)-4,10-dimethyl-3,6,9-trioxo-8-(4-(trifluoromethyl)benzyl)-2-oxa-4,7,10-triazadodecane-12-acid tert-butyl (compound Fmoc-3) [Formula 18]

[0168] The compound 2-HCl (2.70 kg, 6.00 mol) synthesized in the above Example 1-2 was added to a reaction vessel. Then, (S)-2-(((9-H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-pentenoic acid (2.74 kg), acetonitrile (6.34 kg) and MeTHF (5.74 kg) were added, the external temperature of the reaction vessel was set to 15°C, and DIPEA (3.86 kg) was added. Then, a solution obtained by dissolving TCFH (3.35 kg) in acetonitrile (7.58 kg) was added to the reaction vessel, the external temperature of the reaction vessel was set to 25°C, and the reaction mixture was then stirred for 5 hours. To the reaction vessel, MeTHF (5.74 kg) was added, the external temperature of the reaction vessel was then set to 15°C, a 5% aqueous potassium carbonate solution (13.45 kg) was then added, and the reaction mixture was then stirred for 30 minutes. The aqueous layer was discharged, and the obtained organic layer was then washed with 5% sulfuric acid (13.45 kg). To the reaction vessel, MeTHF (5.74 kg) was added, and the organic layer was then washed with a 5% aqueous potassium carbonate solution (13.45 kg). The obtained organic layer was concentrated under reduced pressure, acetonitrile (16.91 kg) was added, and the mixture was further concentrated to afford a residue containing compound Fmoc-3. LC retention time of compound Fmoc-3: 5.442 minutes (HPLC analysis conditions: method 2) LCMS (ESI) of compound Fmoc-3, retention time: 5.491 minutes, m / z = 770 [M+Na]+ (HPLC analysis conditions: method 1)

[0169] Example 1-4 Synthesis of tert-butyl N-((S)-2-((S)-N-(but-3-en-1-yl)-2-(methylamino)pent-4-enamide)-3-(4-(trifluoromethyl)phenyl)propanoyl)-N-methylglycinate hydrochloride (compound 3-HCl) [Formula 19]

[0170] The residue containing compound Fmoc-3 obtained in the above Example 1-3 was added to a reaction vessel. DBU (2.59 kg) was then added at 15°C, and the resulting mixture was stirred for 1 hour. Triethylamine (2.29 kg) and sodium hydrogen sulfite (2.12 kg) were then added, and the reaction mixture was stirred at 25°C for 1 hour. To the reaction vessel, toluene (18.38 kg) and 10% aqueous ammonia (33.92 kg) were added, the mixture was stirred for 10 minutes, and the aqueous layer was then discharged. The obtained organic layer was washed three times with 10% aqueous ammonia (33.92 kg), and then with a 5% aqueous sodium chloride 53 solution (33.92 kg). The obtained organic layer was concentrated under reduced pressure. To the obtained residue, toluene (7.7 kg) was added, and the mixture was concentrated under reduced pressure. The addition of toluene (7.7 kg) to the obtained residue and the operation of concentration under reduced pressure were repeated twice to afford a residue containing compound 3. To the obtained residue, toluene (0.86 kg) was added, and a solution obtained by dissolving pyridine hydrochloride (0.65 kg) in acetonitrile (3.17 kg) was then added at 25°C. The resulting mixture was stirred for 1 hour, MTBE (11.92 kg) was then added, and the mixture was stirred. The resulting precipitate was collected by filtration under reduced pressure, and the obtained wet solid was washed twice with MTBE (3.97 kg). The obtained solid was dried under reduced pressure to afford compound 3-HCl (2.60 kg, 4.63 mol, yield in two steps from compound 2-HCl: 77.5%). UV intensity ratio: 100% (Detection wavelength 210 nm, retention time 3.145 minutes, HPLC analysis conditions: method 2) LCMS (ESI) of compound 3, retention time: 3.213 minutes, m / z = 526 [M+H]+ (HPLC analysis conditions: method 1)

[0171] Example 1-5 Synthesis of compound Fmoc-4 by ring-closing metathesis of compound Fmoc-3 [Formula 20]

[0172] To the residue (55 ml) containing compound Fmoc-3 obtained in the above Example 1-3, acetonitrile (20 mL) was added, and azeotropic dehydration under reduced pressure was repeated twice. The resultant residue was purified by silica gel column chromatography (mobile phase: heptane / ethyl acetate, volume ratio 0 : 100 ^ 40 : 60). The collected solution was concentrated, and MTBE (20 mL) was added to the obtained concentrated mixture to precipitate compound Fmoc-3. The obtained solid was filtered to afford Fmoc-3 (4.90 g) as a white solid. To a first reaction vessel, Fmoc-3 (1.00 g, 1.34 mmol) was added, and dissolved with acetone (7 mL) and HFIP (4.46 g, 2.8 mL) to prepare a solution of Fmoc-3. To a second reaction vessel, Hoveyda-Grubbs catalyst ([1-[2,4,6-trimethylphenyl]-3,5,5-trimethyl-3-phenyl-54 2-pyrrolinylidene]dichloro(2-isopropoxybenzylidene)ruthenium (II)) (8.5 mg, 0.013 mmol) and acetone (5 mL) were added. After the second reaction vessel was filled with nitrogen gas, the solution of compound Fmoc-3 prepared as described above was added dropwise over 2 hours while the solution in the second reaction vessel was heated and refluxed at 65°C.  Then, the first reaction vessel was washed with acetone (1 mL), and the obtained wash solution was added dropwise over 10 minutes to the solution in the second reaction vessel. After completion of the dropwise addition, the reaction mixture was stirred for 4 hours, and the reaction mixture was sampled, and analyzed by HPLC. It was confirmed that the conversion ratio was 95.2%, and the ratio of the dimer formed through the intermolecular reaction to the product (Fmoc-4) was 361%. LCMS (ESI) of compound Fmoc-4, retention time: 2.784 minutes, m / z = 742 [M+Na]+ (HPLC analysis conditions: method 3)

[0173] Example 1-6 Synthesis of compound 4-HCl by ring-closing metathesis of compound 3-HCl [Formula 21]

[0174] To a reaction vessel, compound 3-HCl (50.2 mg) obtained in the above Example 1-4 was added, and dissolved with acetone (0.35 mL) and HFIP (0.14 mL). Separately, a catalyst solution was prepared by dissolving Hoveyda-Grubbs catalyst ([1-[2,4,6-trimethylphenyl]-3,5,5-trimethyl-3-phenyl-2-pyrrolinylidene]dichloro(2-isopropoxybenzylidene)ruthenium (II)) (2.8 mg) in acetone (0.25 mL), and the resulting catalyst solution was added to the reaction vessel. The resulting reaction mixture was heated and refluxed at 65°C with stirring for 5 hours. After the reaction, the reaction mixture was sampled, and analyzed by HPLC. It was confirmed that the conversion ratio was 99.3%, and the ratio of the dimer formed through the intermolecular reaction to the product (4-HCl) was 0.42%. LCMS (ESI) of compound 4, retention time: 2.975 minutes, m / z = 498 [M+H]+ (HPLC analysis conditions: method 1)

[0175] Table 1 shows comparison between the above Examples 1-5 and 1-6. [Formula 22]

[0176] [Table 1] PG Reaction conditions Conversion ratio (%) Dimer ratio (%) H (HCl salt) 5 mol % catalyst* Acetone (12 v / w) HFIP (2.8 v / w) reflux, 5 h 99.3 0.42 Fmoc 1 mol % catalyst* Acetone (13 v / w) HFIP (2.8 v / w) reflux, 4h 95.2 361 * Hoveyda-Grubbs catalyst ([1-[2,4,6-trimethylphenyl]-3,5,5-trimethyl-3-phenyl-2-pyrrolinylidene]dichloro(2-isopropoxybenzylidene)ruthenium (II))

[0177] It was confirmed that the use of the unprotected compound 3-HCl allowed the reaction to proceed in an intramolecularly selective manner as compared to the use of the Fmoc-protected compound Fmoc-3 as shown in Table 1.

[0178] Example 1-7 Synthesis of tert-Butyl N-methyl-N-((S)-2-((S,Z)-3-(methylamino)-2-oxo-3,4,7,8-tetrahydroazocin-1(2H)-yl)-3-(4-(trifluoromethyl)phenyl)propanoyl)glycinate hydrochloride (compound 4-HCl) [Formula 23]

[0179] To a first reaction vessel, compound 3-HCl (0.64 kg) obtained in the above Example 1-4 was added, and the mixture was dissolved with acetone (3.52 kg) and HFIP (2.88 kg) to prepare a solution of the compound 3-HCl. In a second reaction vessel, a catalyst solution was prepared using Hoveyda-Grubbs catalyst ([1-[2,4,6-trimethylphenyl]-3,5,5-trimethyl-3-phenyl-2-pyrrolinylidene]dichloro(2-isopropoxybenzylidene)ruthenium (II)) (7.12 g) and acetone (2.51 kg), and then the second reaction vessel was filled with nitrogen gas. Then, the compound 3-HCl solution prepared as described above was added to the second reaction vessel dropwise over 1.5 hours while the catalyst solution in the second reaction vessel was heated and refluxed at 65°C. After completion of dropwise addition, the reaction mixture was stirred for 1 hour, allowed to cool to 25°C, and then stirred for another hour. The resulting precipitate was collected by filtration, and the obtained wet solid was rinsed twice with acetone (2.52 kg). The obtained solid was dried under reduced pressure to afford 0.55 kg of compound 4-HCl (yield 90.7%). UV intensity ratio: 99.1% (Detection wavelength 210 nm, retention time 2.820 minutes, HPLC analysis conditions: method 2) LCMS (ESI) of compound 4, retention time: 2.859 minutes, m / z = 498 [M+H]+ (HPLC analysis conditions: method 1) 1H-NMR (500 MHz, DMSO-d6, detected as a mixture of rotamers) 5 9.74 (brs, 1H), 8.63 (brs, 1H), 7.60-7.39 (m, 4H), 5.78-5.39 (m,3H), 4.53-4.48 (m, 1H), 4.18-3.74 (m, 3H), 3.61-3.50 (m, 1H), 3.11-2.83 (m,6H), 2.45-2.41 (m, 1H), 2.30-2.12 (m, 2H), 1.96 (s, 1H), 1.87 (s, 2H), 1.40 (s,3H), 1.39 (s, 6H)

[0180] Example 1-8 Study on additives in ring-closing metathesis of compound 3-HCl To a reaction vessel, the compound 3-HCl obtained in the above Example 1-4, acetone (10 volumes based on the weight of compound 3-HCl), additives (2 volumes based on the weight of compound 3-HCl) and second-generation Hoveyda-Grubbs catalyst (5 mol%) were added, and then the reaction vessel was filled with nitrogen gas. The resulting mixture was stirred for 33.5 hours with an external temperature of 55°C. The resulting reaction mixture was sampled and analyzed by HPLC to determine a conversion ratio. Table 2 below shows a list of the additives used and conversion ratios. [Formula 24]

[0181] [Table 2] Additive Conversion ratio (%) HFIP 76.0 2-propanol 79.5 MeOH 60.9 TFE 79.5 AcOH 96.0

[0182] It was confirmed that the target 4-HCl was generated with a conversion ratio of 70% or more even when a plurality of weak acids and alcohols were used as additives as shown in Table 2.

[0183] Example 1-9 Synthesis of tert-butyl N-((S)-2-((S,Z)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-2-oxo-3,4,7,8-tetrahydroazocin-1(2H)-yl)-3-(4-(trifluoromethyl)phenyl)propanoyl)-N-methylglycinate (compound Fmoc-4) [Formula 25]

[0184] A reaction vessel was filled with nitrogen gas, and compound 4-HCl (3.14 kg, 5.88 mol) prepared in the same manner as in the above Example 1-7, acetonitrile (7.34 kg), MeTHF (2.54 kg) and DIPEA (1.90 kg) were added to the reaction vessel. Then, a solution obtained by dissolving 9-fluorenylmethyl chloroformate (1.60 kg) in MeTHF (5.36 kg) was added to the reaction vessel, and the mixture was stirred at 15°C for 1 hour. To the reaction vessel, N,N-dimethylethylenediamine (0.10 kg) was added, and the mixture was stirred at 25°C for 0.5 hours. Then, 4% sulfuric acid (31.35 kg) was added at 15°C, the mixture was stirred, and the aqueous 58 layer was discharged. To the obtained organic layer, MeTHF (5.38 kg) and 4% sulfuric acid (31.47 kg) were added, the mixture was stirred, and the aqueous layer was discharged. To the obtained organic layer, MeTHF (5.38 kg) and a 5% aqueous sodium carbonate solution (31.39 kg) were added, the mixture was stirred, and the aqueous layer was discharged. To the obtained organic layer, MeTHF (5.38 kg) and a 5% aqueous sodium chloride solution (31.43 kg) were added, the mixture was stirred, and the aqueous layer was discharged. The obtained organic layer was concentrated, and azeotropic dehydration with the addition of MeTHF was performed to afford a concentrated solution containing compound Fmoc-4. LC retention time of compound Fmoc-4: 4.980 minutes (HPLC analysis conditions: method 4)

[0185] In the ring-closing metathesis of Fmoc-3, compounds derived from intermolecular reactions are main products. On the other hand, Fmoc-4 was successfully obtained as a main product via a sequence of ring-closing metathesis of 3-HCl and subsequent Fmoc protection of the N-terminus.

[0186] Example 1-10 Synthesis of tert-Butyl N-methyl-N-((S)-2-((S)-3-(methylamino)-2-oxoazocan-1-yl)-3-(4-(trifluoromethyl)phenyl)propanoyl)glycinate hydrochloride (compound 5-HCl) [Formula 26]

[0187] The compound 4-HCl (240 mg, 0.45 mmol) obtained in the above Example 1-7, methanol (4.8 mL) and 10% Pd / C (water content 54%, 51.8 mg) were added to a reaction vessel, and the reaction vessel was then filled with hydrogen gas. The mixture was stirred at approximately 25°C for 75 minutes, the reaction mixture was then filtered, and palladium carbon separated by filtration was washed with methanol (3 mL). The obtained filtrate was dried under reduced pressure to afford compound 5-HCl (221 mg, 0.41 mmol, yield 91.9%) as a white solid. UV intensity ratio: 99.5% (Detection wavelength 210 nm, retention time 2.808 minutes, HPLC analysis conditions: method 1, m / z = 500 [M+H]+ (ESI))

[0188] The compound obtained by ring-closing metathesis was successfully converted to the corresponding lactam 5-HCl by hydrogenation of the double bond into a single bond.

[0189] Example 1-11 Synthesis of (S)-2-(but-3-en-1-ylamino)-3-(p-tolyl)propanoic acid (compound 6) [Formula 27]

[0190] (S)-2-amino-3-(p-tolyl)propanoic acid (3.45 g, 19.3 mmol), ethanol (20.7 mL), water (17.3 ml), triethylamine (8.05 mL, 57.8 mmol), and 4-bromo-1-butene (5.82 mL, 57.8 mmol) were added to a reaction vessel, and the resulting mixture was stirred at 70°C for 4 hours. The reaction mixture was cooled to 25°C, the resulting precipitate was then collected by filtration, and the obtained solid was dried under reduced pressure to afford compound 6 (2.40 g, yield 53%) as a white solid. UV intensity ratio: 99.0% (Detection wavelength 210 nm, retention time 1.983 minutes, HPLC analysis condition method 2)

[0191] Example 1-12 Synthesis of tert-butyl (S)-N-(2-(but-3-en-1-ylamino)-3-(p-tolyl)propanoyl)-N-methylglycinate (compound 7) [Formula 28]

[0192] Compound 6 (2.05 g, 8.79 mmol) obtained in the above Example 1-11, sarcosine tertbutyl hydrochloride (3.20 g, 17.6 mmol), acetonitrile (20 mL) and DIPEA (6.91 mL, 39.5 mmol) were added at approximately 25°C to a reaction vessel. After the reaction vessel was filled with nitrogen gas, chlorotripyrrolidinophosphonium hexafluorophosphate (5.60 g, 13.3 mmol) was then added, and the mixture was stirred at 72°C for 5 hours. The reaction mixture was cooled to 25°C, a 10% aqueous potassium carbonate solution (10 mL) was then added, and the mixture was stirred for 6 hours. The aqueous layer was discharged, and the obtained organic layer was washed with a 10% aqueous sodium hydrogen sulfate solution (16 mL x 2 times). To the obtained organic layer, a 1 mol / L aqueous sodium hydroxide solution (10 mL) and MeTHF (10 mL) were added, the mixture was stirred, and the aqueous layer was discharged. To the obtained organic layer, toluene (10 mL) was added, and the mixture was washed with water (10 mL x 2) and a 5% aqueous sodium chloride solution (10 mL). The obtained organic layer was concentrated, and azeotropic dehydration with the addition of toluene (20 mL) was performed to afford a residue (5.08 g) containing compound 7. LCMS (ESI) of compound 7, retention time: 2.756 minutes, m / z = 361 [M+H]+ (HPLC analysis conditions: method 1)

[0193] Example 1-13 Synthesis of tert-butyl N-((S)-2-((S)-N-(but-3-en-1-yl)-2-(methylamino)pent-4-enamide)-3-(p-tolyl)propanoyl)-N-methylglycinate (compound 8) [Formula 29]

[0194] The residue (5.75 g) containing compound 7 (10.6 mmol) obtained by the method of the above Example 1-12, (S)-2-(((9-H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-pentenoic acid (4.50 g, 12.8 mmol), acetonitrile (19 mL) and DIPEA (7.43 mL) were added at approximately 25°C to a reaction vessel. After the reaction vessel was filled with nitrogen gas, TCFH (5.97 g, 21.3 mmol) was then added, and the mixture was stirred at 25°C for 4 hours. Then, MTBE (19 mL), a 5% aqueous potassium carbonate solution (19 mL), and 1-methylimidazole (0.85 mL) were added, and the mixture was stirred at approximately 25°C for 10 minutes. The aqueous layer was discharged, and the obtained organic layer was washed 61 with a 10% aqueous sodium hydrogen sulfate solution (19 mL x 2) and a 5% aqueous sodium chloride solution (19 mL). The obtained organic layer was concentrated, DBU (4.81 mL) and acetonitrile (7.6 mL) were then added. The resulting mixture was stirred at approximately 25°C for 45 minutes. Then, sodium hydrogen sulfite (2.76 g), triethylamine (5.93 mL) and water (1.92 mL) were added, and the mixture was stirred at approximately 25°C for 2 hours. Then, toluene (19 mL), isopropyl acetate (19 mL) and 20% aqueous ammonia (36 mL) were added, and the mixture was stirred. The aqueous layer was discharged, and the obtained organic layer was then washed with 20% aqueous ammonia (36 mL x 3) and an aqueous sodium chloride solution (19 mL). The obtained organic layer was concentrated under reduced pressure, and subsequently, azeotropic dehydration with the addition of toluene (19 mL x 2) was performed to afford a residue (9.01 g) containing compound 8. LCMS (ESI) of compound 8, retention time: 3.038 minutes, m / z = 472 [M+H]+ (HPLC analysis conditions: method 1)

[0195] Example 1-14 Synthesis of tert-Butyl N-methyl-N-((S)-2-((S,Z)-3-(methylamino)-2-oxo-3,4,7,8-tetrahydroazocin-1(2H)-yl)-3-(p-tolyl)propanoyl)glycinate (compound 9) [Formula 30]

[0196] A purified product of compound 8 synthesized in the above Example 1-13 (purified with a silica gel column chromatography using dichloromethane / methanol as a mobile phase) (41.6 mg, 0.088 mmol) was added to a reaction vessel. Then, a toluene solution (0.45 mL) containing toluene (2.2 mL), boron trifluoride-tetrahydrofuran complex (9.7 ^L, 0.088 mmol) and first-generation Hoveyda-Grubbs catalyst (2.6 mg, 4.4 ^mol) was added to the reaction vessel. After the reaction vessel was filled with nitrogen gas, the resulting mixture was stirred at an external temperature of 40°C for 3 hours. The reaction mixture was then sampled, and analyzed by HPLC. It was confirmed that the conversion ratio was 74.8%, and the ratio of the dimers, formed through the intermolecular reactions, to the product (9) was 1.7%. LCMS (ESI) of compound 9, retention time: 2.492 minutes, m / z = 444 [M+H]+ (HPLC analysis conditions: method 1)

[0197] Example 1-15 Synthesis of (5S,8S)-5-allyl-7-(but-3-en-1-yl)-4,10-dimethyl-8-(4-methylbenzyl)-3,6,9-trioxo-1- phenyl-2-oxa-4,7,10-triazadodecane-12-acid tert-butyl (compound Cbz-8) [Formula 31]

[0198] A compound 7 (0.50 g, 1.39 mmol) synthesized by a method similar to that of the above Example 1-12 was added to a reaction vessel, and (S)-2-((benzyloxy)carbonyl)(methyl)amino)-4-pentenoic acid (0.43 g, 1.66 mmol), acetonitrile (2.5 mL) and DIPEA (1.09 mL) were then added. TCFH (0.78 g) was added at approximately 25°C, and the resulting mixture was stirred at approximately 25°C for 1 hour. Then, MeTHF (2.5 mL) and a 5% aqueous sodium hydrogen carbonate solution (5 mL) were added, the mixture was stirred, and the aqueous layer was then discharged. The organic layer was washed with a 5% aqueous sodium hydrogen carbonate solution (5 mL), a 5% aqueous potassium hydrogen sulfate solution (5 mL) and a 2.5% aqueous sodium chloride solution (5 mL) in this order. The obtained organic layer was concentrated to afford a residue containing compound Cbz-8. The residue was purified by silica gel column chromatography (mobile phase: heptane / ethyl acetate, volume ratio 100 : 0 ^ 80 : 20) to afford compound Cbz-8 (0.66 g). LCMS (ESI) of compound Cbz-8: retention time, 4.867 minutes, m / z = 628 [M+Na]+ (HPLC analysis conditions: method 1)

[0199] Example 1-16 Synthesis of tert-Butyl N-((S)-2-((S,Z)-3-(((benzyloxy)carbonyl)(methyl)amino)-2-oxo-3,4,7,8-tetrahydroazocin-1(2H)-yl)-3-(p-tolyl)propanoyl)-N-methylglycinate (compound Cbz-9) [Formula 32] Compound Cbz-8 (48.6 mg, 0.080 mmol) obtained in the above Example 1-15, toluene (2.67 mL), first-generation Hoveyda-Grubbs catalyst (2.3 mg, 3.9 ^mol) were added to a reaction vessel. After the reaction vessel was filled with nitrogen gas, the reaction mixture was stirred at an external temperature of 70°C for 2 hours. The reaction mixture was then sampled, and analyzed by HPLC. It was confirmed that the conversion ratio was 39.4%, and the ratio of the dimers, formed through the intermolecular reactions, to the product (Cbz-9) was 35.9%. LCMS (ESI) of compound Cbz-9: retention time, 1.877 minutes, m / z = 600 [M+Na]+ (HPLC analysis conditions: method 3)

[0201] Table 3 below shows comparison between the ring-closing metathesis of compound 8 in which the N-terminus is not protected (Example 1-14) and the ring-closing metathesis of the Cbz-protected compound Cbz-8 (Example 1-16). [Formula 33]

[0202] [Table 3] PG Additive Temperature Time Conversion ratio (%) Dimer ratio (%) H - 70°C then 100°C 1.5 h then 1 h 0 - H BF3THF  1 eq 40°C 3h 74.8 1.7 Cbz - 70°C 2h 39.4 35.8

[0203] It was confirmed that the use of the unprotected compound 8 allowed the ring-closing metathesis reaction to proceed with a higher conversion ratio and in an intramolecularly selective manner as compared to the use of the Cbz-protected compound Cbz-8.

[0204] Example 1-17 Synthesis of tert-butyl N-((S)-2-((S)-N-(but-3-en-1-yl)-2-(methylamino)pent-4-enamide)-3-(p-tolyl)propanoyl)-N-methylglycinate hydrochloride (compound 8-HCl) [Formula 34]

[0205] To the residue (9.01 g) containing the compound 8 obtained by the method of the above Example 1-13, a pyridine hydrochloride solution obtained by dissolving pyridine hydrochloride (0.86 g) in acetonitrile (1.9 mL) was added followed by an addition of MTBE (11.4 mL). The resulting mixture was stirred at approximately 25°C for 10 minutes, and MTBE (7.6 mL) was added. The reaction mixture was stirred for 1 hour, MTBE (19 mL) was then added, and the mixture was stirred for 1 hour, and then left to stand for 15 hours. The resulting precipitate was collected by filtration, and the obtained solid was dried under reduced pressure to afford 2.79 g of compound 8-HCl (content 97.5%, product of NMR content calculated by qNMR and UV intensity ratio) as a beige solid. UV intensity ratio: 99.1% (Detection wavelength 210 nm, retention time 3.042 minutes, HPLC analysis conditions: method 1, m / z = 472 [M+H]+ (ESI))

[0206] Example 1-18 Synthesis of tert-Butyl N-methyl-N-((S)-2-((S,Z)-3-(methylamino)-2-oxo-3,4,7,8-tetrahydroazocin-1(2H)-yl)-3-(p-tolyl)propanoyl)glycinate hydrochloride (compound 9-HCl) [Formula 35]

[0207] In a first reaction vessel, compound 8-HCl (1.02 g, content 97.5%) obtained in Example 1-17 was dissolved in acetone (5 mL) and HFIP (0.41 mL) to prepare a compound 8-HCl solution. To a second reaction vessel, second-generation Hoveyda-Grubbs catalyst (0.061 g), acetone (15 mL) and HFIP (1.8 mL) were added to prepare a catalyst solution. The second reaction vessel was filled with nitrogen gas, and the catalyst solution in the second vessel was heated and refluxed at an external temperature of 65°C. The compound 8-HCl solution prepared as described above was then added to the second reaction vessel dropwise over 1 hour. After completion of the dropwise addition, the reaction mixture was stirred for 1 hour, and the reaction mixture was sampled, and analyzed by HPLC. It was confirmed that the conversion ratio was 99.3%, and the ratio of the dimers, formed through the intermolecular reactions, to the product (9-HCl) was 1.7%. The reaction mixture was cooled to approximately 25°C, MTBE (10 mL) was added to the reaction mixture, and the resulting suspension was then filtered. Then, the obtained wet solid was washed twice with a mixed solution of acetone / MTBE (volume ratio 2 : 1, 5 mL). The obtained solid was dried under reduced pressure to afford 0.79 g of a compound 9-HCl (81.4%, content 97.0%, product of NMR content calculated by qNMR and UV intensity ratio) as a white solid. UV intensity ratio: 98.6% (Detection wavelength 210 nm, retention time 2.630 minutes, HPLC analysis conditions: method 1, m / z = 444 [M+H]+ (ESI)) 1H-NMR (500 MHz, DMSO-d6, detected as a mixture of rotamers) 5 8.93-9.54 (br, 1H), 8.298.93 (br, 1H), 7.03-7.14 (m, 4H),5.70-5.36 (m, 3H), 4.49-4.45 (m, 1H), 4.23-3.80 (m, 2H), 3.733.67 (m, 1H),3.59-3.49 (m, 1H), 2.96-2.78 (m, 6H), 2.44-2.40 (m, 1H), 2.28-2.09 (m, 5H),1.94 (s, 1.2H), 1.92 (s, 1.8H), 1.43 (s, 3.5H), 1.40 (s, 5.5H)

[0208] By using compound 8-HCl in which the N-terminus is not protected, a ring-closing metathesis reaction successfully proceeded to afford cyclic compound 9-HCl in >80% yield with an LC purity of >98%.

[0209] Example 1-19 Synthesis of (S)-2-(arylamino)-3-(4-(trifluoromethyl)phenyl)propanoic acid (compound 10) [Formula 36]

[0210] (S)-2-amino-3-(4-(trifluoromethyl)phenyl)propanoic acid (3.34 g, 14.3 mmol), acetonitrile (10 mL), water (8.3 ml), triethylamine (5.99 mL, 43.0 mmol), and allyl bromide (3.72 mL, 43.0 mmol) were added to a reaction vessel, and the reaction mixture was stirred at approximately 25°C for 1 hour. The resulting precipitate was collected by filtration, and the obtained solid was dried under reduced pressure to afford compound 10 (3.05 g, yield 77.9%) as a white solid. UV intensity ratio: 96.4% (Detection wavelength 210 nm, retention time 1.925 minutes, HPLC analysis conditions: method 1, m / z = 274 [M+H]+ (ESI))

[0211] Example 1-20 Synthesis of tert-butyl (S)-N-(2-(arylamino)-3-(4-(trifluoromethyl)phenyl)propanoyl)-N- methylglycinate (compound 11) [Formula 37]

[0212] To a reaction vessel, compound 10 (2.51 g, 9.19 mmol) obtained in the above Example 119, sarcosine tert-butyl hydrochloride (2.52 g, 13.9 mmol), acetonitrile (18 mL) and DBU (5.68 mL, 37.7 mmol) were added at approximately 25°C. T3P (50 wt% MeTHF solution, 7.38 mL) was then added to the reaction vessel at the external temperature of 0°C, and the resulting mixture was stirred at approximately 25°C for 1 hour. Toluene (20 mL) and a 1 mol / L aqueous sodium hydroxide solution (13 mL) were added, the mixture was stirred for 15 minutes, and the aqueous layer was discharged. The obtained organic layer was washed with a 5% aqueous sodium carbonate solution (13 mL), a 5% aqueous sodium dihydrogen phosphate solution (20 mL x 3 times) and a 10% aqueous sodium chloride solution (5 mL) in this order. The obtained organic layer was concentrated under reduced pressure, and azeotropic dehydration with the addition of toluene (20 mL) was performed. Filtration and concentration of the resulting mixture afforded a residue (5.80 g) containing compound 11. LCMS (ESI) of compound 11, retention time: 2.768 minutes, m / z = 401 [M+H]+ (HPLC analysis conditions: method 1)

[0213] Example 1-21 Synthesis of (5S,8S)-5,7-diallyl-1-(9H-fluoren-9-yl)-4,10-dimethyl-3,6,9-trioxo-8-(4-(trifluoromethyl)benzyl)-2-oxa-4,7,10-triazadodecane-12-acid tert-butyl (compound Fmoc-12) [Formula 38]

[0214] To a reaction vessel, a residue (5.41 g) containing compound 11 obtained by the same method as in the above Example 1-20, (S)-2-((9-H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-pentenoic acid (3.92 g), acetonitrile (17 mL), MeTHF (9 mL) and DIPEA (7.46 mL) were added at room temperature. After the reaction vessel was filled with nitrogen gas, TCFH (4.81 g) was added, and the mixture was stirred at approximately 25°C for 3.5 hours. Then, MeTHF (20 mL) and a 5% aqueous potassium carbonate solution (17 mL) were added, and the mixture was stirred at approximately 25°C.  The aqueous layer was discharged, and the obtained organic layer was washed with 5% sulfuric acid (17 mL) and a 5% aqueous potassium carbonate solution (17 mL). The obtained organic layer was concentrated under reduced pressure, acetonitrile (20 mL) was then added, and the mixture was concentrated. The resulting concentrated solution was cooled to 0°C, and the resulting precipitate was then collected by filtration. The obtained wet solid was washed with acetonitrile (10 mL), and dried under reduced pressure to afford compound Fmoc-12 (2.18 g) as a white solid. LC retention time of compound Fmoc-12: 5.231 minutes (HPLC analysis conditions: method 2)

[0215] Example 1-22 Synthesis of tert-butyl N-((S)-2-((S)-N-allyl-2-(methylamino)pent-4-enamide)-3-(4-(trifluoromethyl)phenyl)propanoyl)-N-methylglycinate hydrochloride (compound 12-HCl) [Formula 39]

[0216] Compound Fmoc-12 (2.18 g, 2.97 mmol) obtained in the above Example 1-21, acetonitrile (7 mL) and DBU (0.45 mL) were added at approximately 25°C to a reaction vessel, and the reaction mixture was stirred for 1.5 hours. Then, to the reaction mixture, water (0.54 mL), triethylamine (1.66 mL) and sodium hydrogen sulfite (0.77 g) were added, and the mixture 68 was stirred at approximately 25°C for 2 hours. To the reaction mixture, toluene (30 mL) and 10% aqueous ammonia (16 mL) were added, the mixture was stirred, and the aqueous layer was then discharged. The obtained organic layer was washed three times with 10% aqueous ammonia (16 mL), and then with a 5% aqueous sodium chloride solution (10 mL). The obtained organic layer was concentrated under reduced pressure, and azeotropic dehydration with the addition of toluene (20 mL) was performed 3 times. To the obtained residue, an acetonitrile solution (1 mL) containing pyridine hydrochloride (0.36 g) was added. MTBE (6 mL) and heptane (6 mL) were then added, and the resulting mixture was stirred. The resulting precipitate was collected by filtration, and the obtained wet powder was washed with heptane (5 mL). The obtained solid was dried under reduced pressure to afford 1.50 g of compound 12-HCl (2.74 mmol, yield 92.1%). UV intensity ratio: 99.5% (Detection wavelength 210 nm, retention time 3.030 minutes, HPLC analysis conditions: method 1, m / z = 512 [M+H]+ (ESI))

[0217] Example 1-23 Synthesis of tert-Butyl N-methyl-N-((S)-2-((S)-3-(methylamino)-2-oxo-2,3,4,7-tetrahydro-1H-azepin-1-yl)-3-(4-(trifluoromethyl)phenyl)propanoyl)glycinate hydrochloride (compound 13-HCl) [Formula 40]

[0218] Compound 12-HCl (75.8 mg, 0.14 mmol) obtained in the above Example 1-22, second-generation Hoveyda-Grubbs catalyst (4.44 mg), acetone (1.02 mL) and HFIP (0.68 mL) were added to a reaction vessel. After the reaction vessel was filled with nitrogen gas, the reaction mixture was then heated at the external temperature of 65°C for 1 hour. The reaction mixture was allowed to cool to approximately 25°C, and the reaction mixture was then sampled, and analyzed by HPLC. The results showed that the conversion ratio was 98.1%, and dimers formed by the intermolecular reactions were not detected. The reaction mixture was concentrated, acetone (1 mL) was then added, and the resulting precipitate was filtered. The obtained wet solid was washed with acetone (1 mL), and the obtained solid was dried under reduced pressure to afford compound 13-HCl (57.7 mg, yield 80.2%) as a white solid. UV intensity ratio: 98.9% (Detection wavelength 210 nm, retention time 2.733 minutes, HPLC analysis conditions: method 1, m / z = 484 [M+H]+ (ESI)) 1H-NMR (500 MHz, DMSO-d6) 5 9.44 (brs, 1H),8.69 (brs, 1H), 7.62-7.42 (m, 4H), 5.81-5.35 (m, 3H), 4.75-4.70 (m, 1H),4.38-4.29 (m, 1H), 4.11-3.87 (m, 2H), 3.83-3.60 (m, 1H), 3.14-2.81 (m, 5H),2.65-2.71 (m, 1H), 2.22-2.07 (m, 4H), 1.40 (s, 9H)

[0219] By using compound 12-HCl in which the N-terminus is not protected, a ring-closing metathesis reaction successfully proceeded to afford cyclic compound 13-HCl in >80% yield with an LC purity of >98%.

[0220] Example 1-24 Synthesis of tert-butyl (S)-N-(2-(but-3-en-1-ylamino)-3-(p-tolyl)propanoyl)-N-methylglycinate hydrochloride (compound 7-HCl) [Formula 41]

[0221] Compound 6 (1.10 g, 4.71 mmol) obtained in the above Example 1-11, 1,1'-carbonyldiimidazole (0.92 g, 5.66 mmol) and THF (16.5 mL) were added to a reaction vessel. After the reaction vessel was filled with nitrogen gas, trifluoromethanesulfonic acid (0.96 mL, 10.8 mmol) was added at 25°C, and the mixture was stirred at 50°C for 1.5 hours. The mixture was cooled to 25°C, a solution obtained by dissolving sarcosine tert-butyl hydrochloride (1.03 g, 5.66 mmol) in THF (2 mL), acetonitrile (3 mL) and DIPEA (3.8 mL, 21.7 mmol) was then added to the mixture. The resulting mixture was stirred for 2 hours. Then, a 5% aqueous potassium carbonate solution (11 mL) was added, the mixture was stirred for 5 minutes, and the aqueous layer was discharged. The obtained organic layer was washed with a 10% aqueous sodium hydrogen sulfate solution (11 mL) and a 5% aqueous sodium hydrogen sulfate solution (11 mL). Each aqueous layer was washed three times with MeTHF (11 mL). All organic layers were mixed, and concentrated under reduced pressure. To the obtained residue, acetonitrile (12 mL) and 2 M hydrochloric acid (5 mL) were added, and the mixture was concentrated under reduced pressure. The resulting precipitate was filtered, washed with water, and then dried under reduced pressure to afford 0.48 g of compound 7-HCl. LCMS (ESI) of compound 7, retention time: 2.689 minutes, m / z = 361 [M+H]+ (HPLC analysis conditions: method 1)

[0222] Example 1-25 Synthesis of tert-butyl N-((S)-2-((S)-N-(but-3-en-1-yl)-2-(methylamino)hex-5-enamide)-3-(p-tolyl)propanoyl)-N-methylglycinate hydrochloride (compound 14-HCl) [Formula 42]

[0223] Compound 7-HCl (0.89 g, 2.25 mmol) obtained by the same method as in the above Example 1-24, (S)-2-((((9-H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-5-hexenoic acid (1.07 g, 2.92 mmol), acetonitrile (4.5 mL), MeTHF (2.3 mL) and DIPEA (1.96 mL, 11.2 mmol) were added to a reaction vessel. To the reaction mixture, TCFH (1.29 g) was added, and the mixture was stirred at approximately 25°C for 2.5 hours. Then, MeTHF (18 mL) and a 5% aqueous potassium carbonate solution (9 mL) were added, the mixture was stirred, and the aqueous layer was discharged. The obtained organic layer was washed with 5% sulfuric acid (5.4 mL) and a 5% aqueous potassium carbonate solution (4.5 mL). The obtained organic layer was concentrated, acetonitrile (20 mL) was added, and azeotropic dehydration was performed. To the obtained residue, acetonitrile (2 mL) and DBU (0.51 mL) were added, and the mixture was stirred for 1 hour. DBU (0.34 mL) was added, and the mixture was stirred for 20 minutes. Then, to the reaction mixture, triethylamine (1.25 mL), water (0.41 mL) and sodium hydrogen sulfite (0.58 g) were added, and the mixture was stirred for 1 hour. To the reaction mixture, triethylamine (0.62 mL) was added, and the mixture was stirred for 45 minutes. Then, to the reaction mixture, toluene (15 mL) and 10% aqueous ammonia (20 mL) were added, the mixture was stirred, and the aqueous layer was discharged. The obtained organic layer was washed four times with 10% aqueous ammonia (10 mL), and then with a 5% aqueous sodium chloride solution (10 mL). The obtained organic layer was concentrated under reduced pressure, toluene (10 mL) was added, and azeotropic dehydration was performed. The obtained residue was filtered, the filtrate was then concentrated, and acetonitrile (1 mL) containing pyridine hydrochloride (0.27 g) was then added. To the mixture, MTBE (10 mL) was added, and the 71 resulting mixture was stirred at approximately 25°C for 1 hour. The mixture was cooled to 0°C, and the resulting precipitate was then filtered. The obtained wet solid was washed with MTBE (5 mL), and dried under reduced pressure to afford compound 14-HCl (0.81 g). UV intensity ratio: 99.3% (Detection wavelength 210 nm, retention time 3.150 minutes, HPLC analysis conditions: method 1, m / z = 486 [M+H]+ (ESI))

[0224] Example 1-26 Synthesis of tert-Butyl N-methyl-N-((S)-2-((S,Z)-3-(methylamino)-2-oxo-2,3,4,5,8,9-hexahydro-1H-azonin-1-yl)-3-(p-tolyl)propanoyl)glycinate hydrochloride (compound 15-HCl) [Formula 43]

[0225] Compound 14-HCl (53.3 mg, 0.10 mmol) obtained in the above Example 1-25, second-generation Hoveyda-Grubbs catalyst (3.1 mg, 0.005 mmol), acetone (1 mL) and HFIP (0.12 mL) were added to a reaction vessel. After the reaction vessel was filled with nitrogen gas, the reaction mixture was heated at an external temperature of 60°C for 1 hour. The reaction mixture was sampled, and analyzed by HPLC. The results showed that the conversion ratio was 97.8%, and the ratio of the dimers, formed through the intermolecular reactions, to the product (15-HCl) was 6.1%. The reaction mixture was further stirred for 1 hour, and allowed to cool to approximately 25°C, and MTBE (1 mL) was then added. The resulting precipitate was filtered, and the obtained wet powder was washed with a mixed solution of acetone and MTBE (0.4 mL, volume ratio 1 : 1), and then with MTBE (1 mL). The obtained solid was dried under reduced pressure to afford compound 15-HCl (19.7 mg, yield 39.1%). UV intensity ratio: 98.2% (Detection wavelength 210 nm, retention time 2.768 minutes, HPLC analysis condition method 2) LCMS (ESI) of compound 15, retention time: 2.696 minutes, m / z = 458 [M+H]+ (HPLC analysis conditions: method 1) 1H-NMR (500 MHz, DMSO-d6, detected as a mixture of rotamers) 5 9.29 (brs, 1H), 8.55 (brs, 1H), 7.15-6.99 (m, 4H), 5.97-5.55 (m,3H), 4.32-3.72 (m, 4H), 3.54-3.47 (m, 1H), 3.13-3.01 (m, 3H), 2.93-2.78 (m,2H), 2.27-1.71 (m, 9H), 1.67 (s, 1.8H), 1.52 (s, 1.2H), 1.46 (s, 3.2H), 1.37(s, 5.8H)

[0226] Example 1-27 Synthesis of (5S,8S)-5,7-di(but-3-en-1-yl)-1-(9H-fluoren-9-yl)-4,10-dimethyl-3,6,9-trioxo-8-(4-(trifluoromethyl)benzyl)-2-oxa-4,7,10-triazadodecane-12-acid tert-butyl (compound Fmoc-16) [Formula 44]

[0227] Compound 2-HCl (2.53 g, 5.61 mmol) obtained by the method of the above Example 1-2, (S)-2-((((9-H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-5-hexenoic acid (1.07 g, 7.03 mmol), acetonitrile (12.5 mL), MeTHF (6.3 mL) and DIPEA (4.89 mL) were added to a reaction vessel. Then, TCFH (3.20 g) was added to the reaction vessel, and the resulting mixture was stirred at approximately 25°C for 2.5 hours, and then left to stand for 23 hours. Then, MeTHF (25 mL) and a 5% aqueous potassium carbonate solution (18 mL) were added, the mixture was stirred, and the aqueous layer was discharged. The obtained organic layer was washed with 5% sulfuric acid (18 mL) and a 5% aqueous potassium carbonate solution (18 mL), and concentrated under reduced pressure. To the obtained residue, acetonitrile (20 mL) was added, and azeotropic dehydration was performed. The resulting precipitate was then filtered, and the obtained wet solid was washed with acetonitrile (10 mL). The obtained solid was dried under reduced pressure to afford compound Fmoc-16 (2.32 g). UV intensity ratio: 99.4% (Detection wavelength 210 nm, retention time 5.590 minutes, HPLC analysis condition method 5)

[0228] Example 1-28 Study on synthesis of tert-butyl N-((S)-2-((S,Z)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-2-oxo-2,3,4,5,8,9-hexahydro-1H-azonin-1-yl)-3-(4-(trifluoromethyl)phenyl)propanoyl)-N-methylglycinate (compound Fmoc-17) [Formula 45]

[0229] To a reaction vessel, compound Fmoc-16 (71.7 mg, 0.094 mmol) obtained in the above Example 1-27, second-generation Hoveyda-Grubbs catalyst (2.9 mg, 0.0045 mmol), acetone (0.94 mL) and HFIP (0.11 mL) were added. After the reaction vessel was filled with nitrogen gas, the reaction mixture was heated at 60°C for 1 hour. The reaction mixture was sampled, and analyzed by HPLC. The results showed that the conversion ratio was 77.6%, and the ratio of the dimers, formed through the intermolecular reactions, to the product (Fmoc-17) was 161%. LCMS (ESI) of compound Fmoc-17, retention time: 2.932 minutes, m / z = 756 [M+Na]+ (HPLC analysis conditions: method 3

[0230] Table 4 below shows comparison between the ring-closing metathesis of compound 14-HCl in which the N-terminus is not protected (Example 1-26) and the ring-closing metathesis of Fmoc-protected compound Fmoc-16 (Example 1-28). [Formula 46]

[0231] [Table 4] PG R Conversion ratio (%) Dimer ratio (%) H (HCl salt) CH3 97.8 6.1 Fmoc CF3 77.6 161

[0232] It was confirmed that the use of compound 14-HCl in which the N-terminus is not protected allowed the ring-closing metathesis reaction to proceed with a higher conversion ratio and in an intramolecularly selective manner as compared to the use of Fmoc-protected compound Fmoc-16.

[0233] Example 1-29 Synthesis of (S)-2-(but-3-en-1-ylamino)-N,N-dimethyl-3-(4-(trifluoromethyl)phenyl)propaneamide (compound 18) [Formula 47]

[0234] To a reaction vessel, compound 1 (2.21 g, 7.69 mmol) obtained in the above Example 11, dimethylamine (2 M THF solution, 7.69 mL, 15.4 mmol), acetonitrile (6.6 mL) and DBU (3.48 mL) were added at approximately 25°C.  Then, T3P (50 wt% MeTHF solution, 6.18 mL) was added to the reaction mixture at the external temperature of 0°C, and the resulting mixture was stirred at approximately 25°C for 40 minutes. T3P (50 wt% MeTHF solution, 3 mL) and dimethylamine (2M THF solution, 3.0 mL, 6.0 mmol) were added to the reaction mixture, and the mixture was stirred for 45 minutes. Then, to the reaction mixture, MeTHF (22 mL) and a 1 mol / L aqueous sodium hydroxide solution (11 mL) were added. After discharging the aqueous layer, the obtained organic layer was washed twice with a 1 mol / L aqueous sodium hydroxide solution (11 mL), twice with a 5% aqueous sodium dihydrogen phosphate solution (11 mL), with a 1 mol / L aqueous sodium hydroxide solution (11 mL), and with a 5% aqueous potassium carbonate solution (11 mL). The obtained organic layer was concentrated, MeTHF (20 mL) was added, and azeotropic dehydration was performed. The obtained residue was filtered, and concentrated to afford a residue (2.98 g) containing compound 18. LCMS (ESI) of compound 18, retention time: 2.207 minutes, m / z = 315 [M+H]+ (HPLC analysis conditions: method 1)

[0235] Example 1-30 Synthesis of (S)-N-(but-3-en-1-yl)-N-((S)-1-(dimethylamino)-1-oxo-3-(4-(trifluoromethyl)phenyl)propan-2-yl)-2-(methylamino)pent-4-eneamide hydrochloride (compound 19-HCl) [Formula 48]

[0236] To a reaction vessel, a residue (2.98 g) containing the compound 18 obtained in the above Example 1-29, (S)-2-((9-H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-pentenoic acid (2.70 g), acetonitrile (9.6 mL) and DIPEA (4.0 mL) were added. To the resulting mixture, TCFH (3.21 g) was added, and the mixture was then stirred at approximately 25°C for 2.5 hours. Then, to the reaction mixture, MeTHF (30 mL) and a 5% aqueous potassium carbonate solution (12 mL) were added, the mixture was then stirred, and the aqueous layer was discharged. The obtained organic layer was washed twice with 5% sulfuric acid (12 mL) and a 5% aqueous potassium carbonate solution (12 mL). The resulting organic layer was concentrated under reduced pressure. To the resulting mixture, MeTHF (30 mL) was added, and azeotropic dehydration was performed. To the obtained residue, acetonitrile (10 mL) and DBU (1.39 mL) were added. The reaction mixture was stirred at approximately 25°C for 75 minutes, triethylamine (4.29 mL), water (1.39 mL) and sodium hydrogen sulfite (1.99 g) were added, and the mixture was stirred for 2 hours. Then, to the reaction mixture, toluene (40 mL) and 10% aqueous ammonia (40 mL) were added, the mixture was stirred, and the aqueous layer was discharged. The obtained organic layer was washed three times with 10% aqueous ammonia (40 mL), and with a 5% aqueous sodium chloride solution (20 mL), and the organic layer was concentrated under reduced pressure. Azeotropic dehydration of the residue with toluene (20 mL) was repeated three times. The obtained slurry was filtered, and concentrated. To the residue, a solution prepared by dissolving pyridine hydrochloride (0.81 g) in acetonitrile (3 mL) was added. After an addition of MTBE (20 mL), the resulting mixture was stirred for 1 hour. The resulting precipitate was collected by filtration, and the obtained wet solid was washed with MTBE (5 mL). The obtained solid was dried under reduced pressure to afford compound 19-HCl (2.05 g). UV intensity ratio: 98.9% (Detection wavelength 210 nm, retention time 2.560 minutes, HPLC analysis conditions: method 1, m / z = 426 [M+H]+ (ESI))

[0237] Example 1-31 Synthesis of (S)-N,N-dimethyl-2-((S,Z)-3-(methylamino)-2-oxo-3,4,7,8-tetrahydroazocin-1(2H)- yl)-3-(4-(trifluoromethyl)phenyl)propaneamide hydrochloride (compound 20-HCl) [Formula 49]

[0238] To a reaction vessel, compound 19-HCl (98.5 mg, 0.21 mmol) obtained in the above Example 1-30, second-generation Hoveyda-Grubbs catalyst (6.6 mg, 0.011 mmol), acetone (1.5 mL) and HFIP (0.5 mL) were added. After the reaction vessel was filled with nitrogen gas, the reaction mixture was heated at an external temperature of 65°C for 1 hour. The reaction mixture was then sampled and analyzed by HPLC. The results showed that the conversion ratio was 89.8%, and the ratio of the dimers, formed through the intermolecular reactions, to the product (20-HCl) was 4.6%. After the reaction mixture was allowed to cool to approximately 25°C, the resulting precipitate was filtered, and the obtained wet solid was washed with acetone (2 mL). The obtained solid was dried under reduced pressure to afford compound 20-HCl (46.9 mg, yield 50.7%). UV intensity ratio: 98.2% (Detection wavelength 210 nm, retention time 2.135 minutes, HPLC analysis conditions: method 1, m / z = 398 [M+H]+ (ESI)) 1H-NMR(DMSO-d6,500 MHz) 5: 9.55 (brs, 1H), 8.57 (brs, 1H), 7.59 (d, J= 8.1 Hz, 2H), 7.50(d, J = 8.1 Hz, 2H), 5.72 (dd, J = 9.7, 5.5 Hz, 1H),5.62-5.58 (m, 1H), 5.42-5.37 (m, 1H), 4.50 (t, J = 8.0 Hz, 1H), 3.79-3.73 (m,1H), 3.59-3.53 (m, 1H), 3.11-3.01 (m, 2H), 2.88-2.81 (m, 7H), 2.462.41 (m,1H), 2.22-2.06 (m, 2H), 1.86 (s, 3H)

[0239] Cyclic dipeptide 20-HCl was successfully obtained with a purity of >98% through a ringclosing metathesis reaction.

[0240] Example 1-32 Synthesis of tert-butyl N-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-N-(but-3-en-1-yl)pent-4-eneamide)-3-(4-(trifluoromethyl)phenyl)propanoyl)-N-methylglycinate (compound Fmoc-21) [Formula 50]

[0241] Compound 2-HCl (5.01 g, 11.1 mol) synthesized in Example 1-2 was added to a reaction vessel. Then, to the reaction vessel, (S)-2-(((9-H-fluoren-9-yl)methoxy)carbonyl)amino)-4-pentenoic acid (4.74 g), 1,3-dimethyl-2-imidazolidinone (25 mL) and 2,4,6-trimethylpyridine (7.35 mL) were added. Then, TCFH (4.93 g) was added to the mixture, and the resulting mixture was stirred at an external temperature of 25°C for 7 hours, and the reaction mixture was then left to stand for 16 hours. To the reaction vessel, a 5% aqueous potassium carbonate solution (25 mL) and MeTHF (30 mL) were added, the mixture was stirred, and the aqueous layer was discharged. The obtained organic layer was washed with 5% sulfuric acid (25 mL) and a 5% aqueous potassium carbonate solution (25 mL) in this order. The obtained organic layer was concentrated under reduced pressure, and azeotropic dehydration with the addition of MeTHF (20 mL) was repeated twice to afford a residue containing compound Fmoc-21. The obtained residue was purified by reversed phase column chromatography (mobile phase: water / acetonitrile volume ratio 40 : 60 ^ 0 : 100). The collected solution was concentrated, and azeotropic dehydration with the addition of acetonitrile was repeated to afford Fmoc-21 (5.00 g) as a white solid. UV intensity ratio: 97.9% (Detection wavelength 210 nm, retention time 5.182 minutes, HPLC analysis conditions: method 1, m / z = 756 [M+H]+ (ESI))

[0242] Example 1-33 Synthesis of tert-butyl N-((S)-2-((S)-2-amino-N-(but-3-en-1-yl)pent-4-enamide)-3-(4-(trifluoromethyl)phenyl)propanoyl)-N-methylglycinate (compound 21) [Formula 51]

[0243] Compound Fmoc-21 (3.02 g) obtained in Example 1-32 was dissolved with acetonitrile (15 mL) in a reaction vessel. DBU (0.62 mL) was then added, and the resulting mixture was stirred at an external temperature of 25°C for 30 minutes. Then, to the reaction mixture, triethylamine (2.29 mL), sodium hydrogen sulfite (1.06 g) and water (0.74 mL) were added, and the reaction mixture was stirred at an external temperature of 25°C for 1.5 hours. To the reaction vessel, toluene (40 mL) and 10% aqueous ammonia (24 mL) were added, the obtained mixture was stirred for 10 minutes, and the aqueous layer was then discharged. The obtained organic layer was washed with 10% aqueous ammonia (24 mL), and then with a 10% aqueous sodium chloride solution (24 mL). The obtained organic layer was concentrated under reduced pressure. Azeotropic dehydration with the addition of toluene (20 mL) was performed to afford a residue containing compound 21. The obtained residue was purified by reversed phase column chromatography (mobile phase: water / acetonitrile volume ratio 80 : 20 ^ 40 : 60). The collected solution was concentrated, and azeotropic dehydration with the addition of acetonitrile was repeated to afford 1.36 g of compound 21 (content 91.6%, product of NMR content calculated by qNMR and UV intensity ratio) as a pale yellow oil. UV intensity ratio: 93.7% (Detection wavelength 210 nm, retention time 3.171 minutes, HPLC analysis conditions: method 2)

[0244] Example 1-34 Synthesis of tert-Butyl N-((S)-2-((S,Z)-3-(((benzyloxy)carbonyl)amino)-2-oxo-3,4,7,8-tetrahydroazocin-1(2H)-yl)-3-(4-(trifluoromethyl)phenyl)propanoyl)-N-methylglycinate (compound Cbz-22) [Formula 52]

[0245] Compound 21 (130.8 mg, content 91.6%, 0.23 mmol) synthesized in Example 1-33, acetone (2.34 mL), HFIP (0.24 mL), boron trifluoride-diethyl ether complex (44.5 pL, 0.35 mmol), and second-generation Hoveyda-Grubbs catalyst (10.3 mg, 16.4 mmol) were added to a reaction vessel. After the reaction vessel was filled with nitrogen gas, the mixture in the reaction vessel was stirred at an external temperature of 65°C for 1 hour. The reaction mixture 79 was sampled and analyzed by HPLC. It was confirmed that the conversion ratio was 95.1%, and the ratio of the dimers, formed through the intermolecular reactions, to the product (22) was 7.4%. The reaction mixture was allowed to cool to 25°C, triethylamine (52.3 p.L) was then added, and the mixture was stirred. To the reaction mixture, MTBE (3 mL) and a 5% aqueous potassium carbonate solution (1 mL) were added, the mixture was stirred, and the aqueous layer was then discharged. The obtained organic layer was concentrated. Thereafter, to the obtained residue, acetonitrile (1 mL), N-carbobenzoxyoxysuccinimide (58.7 mg) and DIPEA (40.8 ^L) were added, and the resulting mixture was stirred at an external temperature of 25°C for 30 minutes. The reaction mixture was concentrated under reduced pressure, and MTBE (3 mL) and a 1 M aqueous sodium hydroxide solution (1 mL) were then added. The resulting mixture was stirred, and the aqueous layer was then discharged. The obtained organic layer was washed with a 10% aqueous potassium hydrogen sulfate solution (1 mL), and then with a 5% aqueous potassium carbonate solution (1 mL). The obtained organic layer was concentrated under reduced pressure, and the obtained residue was purified twice by reversed phase column chromatography (mobile phase: water / acetonitrile, first: volume ratio 45 : 55 ^ 20 : 80, second: volume ratio 45 : 55 ^ 25 : 75). The collected solution was concentrated, and azeotropic dehydration with the addition of acetonitrile was repeated to afford 47.2 mg of compound Cbz-22 (yield: 32.6%) as a white solid. UV intensity ratio: 98.3% (Detection wavelength 210 nm, retention time 4.398 minutes, HPLC analysis conditions: method 1, m / z = 640 [M+H]+ (ESI)) 1H-NMR (500 MHz, DMSO-d6, detected as a mixture of rotamers) 5 7.54 (t, J = 7.8 Hz, 2H), 7.46-7.18 (m, 8H), 5.66-5.59 (m, 1.6H), 5.50-5.45 (m, 0.6H), 5.36-5.29 (m, 0.8H), 5.05-5.01 (m, 0.9H), 4.95-4.91 (m, 1.1H), 4.74-4.68 (m, 1H), 4.24 (d, J = 18.5 Hz, 0.4H), 3.98 (d, J = 17.0 Hz, 0.6H), 3.83-3.42 (m, 3H), 3.18-3.08 (m, 1H), 2.86-2.74 (m, 4H), 2.64-2.58 (m, 1H), 2.51-2.36 (m, 1H), 2.31-2.08 (m, 2H), 1.46-1.21 (m, 9H)

[0246] Example 1-35 Synthesis of tert-butyl (S)-(1-oxo-1-(phenylamino)propan-2-yl)carbamate (compound Boc-23) [Formula 53] Boc-23

[0247] To a reaction vessel, N-(tert-butoxycarbonyl)-L-alanine (3.98 g), acetonitrile (4 mL), aniline (2.11 mL) and N-methylmorpholine (6.94 mL) were added. HATU (8.96 g) was then added to the reaction mixture at an external temperature of 0°C, and the resulting mixture was stirred at an external temperature of 25°C for 1.5 hours. Then, to the reaction mixture, a 5% aqueous potassium carbonate solution (16 mL) and 4-methyltetrahydropyran (40 mL) were added, and the mixture was stirred. The aqueous layer was discharged, and the obtained organic layer was then washed twice with 10% aqueous ammonia (20 mL), and then twice with a 5% aqueous sodium dihydrogen phosphate solution (20 mL). The obtained organic layer was concentrated under reduced pressure, and azeotropic dehydration with the addition of acetonitrile was repeated twice. To the obtained concentrated product, MTBE (10 mL) was added, the mixture was then stirred. The resulting solid was collected by filtration, and the obtained wet solid was washed with MTBE (5 mL). The obtained wet solid was dried under reduced pressure to afford compound Boc-23 (3.64 g) as a white solid. UV intensity ratio: 99.6% (Detection wavelength 210 nm, retention time 2.732 minutes, HPLC analysis conditions: method 1, m / z = 287 [M+H]+ (ESI))

[0248] Example 1-36 Synthesis of (S)-2-(amino)-N-phenylpropanamide (compound 23) [Formula 54]

[0249] Compound Boc-23 (3.64 g) obtained in Example 1-35 and acetonitrile (15 mL) were added to a reaction vessel. Then, to the mixture, methanesulfonic acid (2.68 mL) was added. The resulting mixture was then stirred at an external temperature of 25°C for 3 hours. To the reaction mixture, 8 M aqueous sodium hydroxide solution (6.89 mL) and 4-methyltetrahydropyran (15 mL) were added at an external temperature of 0°C, and the mixture was then stirred. The organic layers were collected, and the aqueous layer was extracted with 4-methyltetrahydropyran (20 mL). The obtained organic layers were mixed and concentrated under reduced pressure. Azeotropic dehydration with the addition of 4-methyltetrahydropyran (20 mL) was performed twice. The obtained residue was filtered and washed with 4-methyltetrahydropyran (10 mL). The filtrate and the wash solution were mixed, and the mixture was then concentrated to afford a residue (3.13 g) containing compound 23. LCMS (ESI) of compound 23, retention time: 1.118 minutes, m / z = 165 [M+H]+ (HPLC analysis conditions: method 1)

[0250] Example 1-37 Synthesis of ((S)-1-(allyl((S)-1-oxo-1-(phenylamino)propan-2-yl)amino)-1-oxopent-4-en-2-yl)(methyl)carbamic acid (9H-fluoren-9-yl)methyl (compound Fmoc-24) [Formula 55]

[0251] The residue (2.83 g) containing compound 23 obtained in Example 1-36 and DMF (6.1 mL) were added to a reaction vessel. Then, to the mixture, DIPEA (3.25 mL) and allyl bromide (1.29 ml) were added. The mixture was then stirred at external temperature of 25°C for 2 hours. Then, to the reaction mixture, acetonitrile (6.1 mL), (S)-2-(((9-H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-pentenoic acid (3.49 g), DIPEA (5.2 mL) and TCFH (4.14 g) were added. The resulting mixture was then stirred at an external temperature of 25°C for 3 hours. To the reaction mixture, a 5% aqueous potassium carbonate solution (20 mL) and MTBE (30 mL) were added, the mixture was stirred, and the aqueous layer was then discharged. The obtained organic layer was washed with a 5% aqueous potassium carbonate solution (20 mL), 5% sulfuric acid (20 mL x 2 time) and a 5% aqueous potassium carbonate solution (20 mL) in this order. The obtained organic layer was concentrated under reduced pressure, and azeotropic dehydration with the addition of acetonitrile (20 mL) was performed twice. The obtained residue was purified by reversed phase column chromatography (mobile phase: water / acetonitrile volume ratio 50 : 50 ^ 15 : 85). The collected solution was concentrated, and azeotropic dehydration with the addition of acetonitrile was repeated to afford compound Fmoc-24 (2.63 g) as a white solid. UV intensity ratio: 97.9% (Detection wavelength 210 nm, retention time 4.475 minutes, HPLC analysis conditions: method 1, m / z = 560 [M+H]+ (ESI))

[0252] Example 1-38 Synthesis of (S)-N-allyl-2-(methylamino)-N-((S)-1-oxo-1-(phenylamino)propane-2-yl)pent-4-eneamide hydrochloride (compound 24-HCl) [Formula 56]

[0253] Compound Fmoc-24 (2.63 g) obtained in Example 1-37 and acetonitrile (8 mL) were added to a reaction vessel. Then, to the mixture, DBU (0.37 mL) was added, and the resulting mixture was stirred at an external temperature of 25°C for 75 minutes. To the reaction mixture, MTBE (26 mL) and a 5% aqueous potassium carbonate solution (13 mL) were added, the mixture was then stirred, and the aqueous layer was discharged. To the obtained organic layer, 0.5 M hydrochloric acid (15 mL) was added, the mixture was then stirred, and the organic layer was discharged. To the obtained aqueous layer, potassium carbonate (1.40 g) was added, and the resulting mixture was further stirred. To the mixture, 4-methyltetrahydropyran (25 mL) was added, and the mixture was further stirred. The resulting aqueous layer was discharged, and the obtained organic layer was then concentrated under reduced pressure. Azeotropic dehydration with the addition of 4-methyltetrahydropyran (20 mL) was performed. The obtained residue was then filtered, and the filtrate was concentrated. To the obtained residue, acetonitrile (20 mL) and 6 M hydrochloric acid (0.73 mL) were added, and the mixture was concentrated under reduced pressure. To the obtained residue, acetonitrile (5 mL) and MTBE (10 mL) were added, and the mixture was stirred until precipitation of a solid. MTBE (20 mL) was added, the mixture was further stirred, and the resulting solid was collected by filtration. The obtained wet solid was washed with MTBE (10 mL). The obtained wet solid was dried under reduced pressure to afford compound 24-HCl (1.21 g) as a white solid. UV intensity ratio: 99.3% (Detection wavelength 210 nm, retention time 1.991 minutes, HPLC analysis conditions: method 1, m / z = 316 [M+H]+ (ESI))

[0254] Example 1-39 Synthesis of (S)-2-((S)-3-(methylamino)-2-oxo-2,3,4,7-tetrahydro-1H-azepin-1-yl)-N-phenylpropanamide (compound 25) [Formula 57]

[0255] Compound 24-HCl (86.4 mg, 0.25 mmol) obtained in Example 1-38, second-generation Hoveyda-Grubbs catalyst (7.69 mg, 0.012 mmol), acetone (2.5 mL) and HFIP (0.28 mL) were added to a reaction vessel. After the reaction vessel was filled with nitrogen gas, the mixture in the reaction vessel was heated at an external temperature of 65°C for 2 hours. The reaction mixture was sampled and analyzed by HPLC. The results showed that the conversion ratio was 98.8%, and the dimers, formed through the intermolecular reactions, were undetected. The reaction mixture was allowed to cool to approximately 25°C, and then concentrated under reduced pressure. To the obtained residue, MTBE (3 mL) and 0.5 M hydrochloric acid (3 mL) were added, the mixture was then stirred, and the organic layer was discharged. To the obtained aqueous layer, potassium carbonate (0.23 g) was added. The mixture was stirred, and extraction with the addition of 4-methyltetrahydropyran (3 mL) was repeated three times. The obtained organic layers were mixed and concentrated under reduced pressure. Azeotropic dehydration with the addition of 4-methyltetrahydropyran (10 mL) was performed. The obtained residue was purified by reversed phase column chromatography (mobile phase: water / acetonitrile volume ratio 95 : 5 ^ 65 : 35). The collected solution was concentrated, and azeotropic dehydration with the addition of acetonitrile was repeated to afford compound 25 (39.8 mg, yield: 56.4%) as a brown oil. UV intensity ratio: 98.8% (Detection wavelength 210 nm, retention time 1.643 minutes, HPLC analysis conditions: method 1, m / z = 288 [M+H]+ (ESI)) 1H-NMR (500 MHz, DMSO-d6) 8 9.75 (s, 1H), 7.54 (d, J = 7.6 Hz, 2H), 7.28 (t, J = 7.6 Hz, 2H), 7.03 (t, J = 7.6 Hz, 1H), 5.73-5.62 (m, 2H), 5.09 (q, J = 7.0 Hz, 1H), 4.27-4.23 (m, 1H), 3.823.73 (m, 2H), 2.47-2.23 (m, 5H), 2.13-1.91 (m, 1H), 1.29 (d, J = 7.0 Hz, 3H) [Industrial Applicability]

[0256] The present invention provides a method for efficiently producing a peptide compound having a peptide structure with a medium ring. The production method of the present invention enables reducing costs in production of the peptide compound and also reducing the environmental loads, and therefore, the production method of the present invention is particularly useful for synthesis of the peptide on a large scale. 84

Claims

[Document Name] Claims

1. A method for producing a compound represented by formula (1) or a salt thereof, comprising the step (metathesis step) of bringing a compound represented by formula (2) or a salt thereof into contact with a catalyst: [Formula 1]whereinR1 is hydrogen,R2 is hydrogen or C1-C6 alkyl;R3 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted halo-C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C14 aryl, optionally substituted 5- to 14-membered heteroaryl, optionally substituted C7-C14 aralkyl, optionally substituted 3- to 14-membered heterocyclyl, optionally substituted 5- to 10-membered heteroaryl-C1-C6 alkyl, optionally substituted C1-C6 alkoxy-C1-C6 alkyl, optionally substituted C1-C6 alkylsulfanyl-C1-C6 alkyl, optionally substituted C1-C6 alkylsulfinyl-C1-C6 alkyl, optionally substituted C1-C6 alkylsulfonyl-C1-C6 alkyl, optionally substituted carboxy-C1-C6 alkyl, optionally substituted C7-C14 aralkoxy-C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl-C1-C6 alkyl, optionally substituted C3-C8 cycloalkoxy-C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclyl-C1-C3 alkyl, optionally substituted 5- to 10-membered heteroaryl-C1-C6 alkoxy-C1-C6 alkyl, or optionally substituted aminocarbonyl (the relevant amino is selected from the group consisting of -NH2, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, N-C1-C6 alkyl-N-C2-C6 alkenylamino, N-C1-C6 alkyl-N-C1-C6 alkoxy-C1-C6 alkylamino, and 4- to 9-membered cyclic amino),R4 is OR5, NHR5', an amino acid residue, or a peptide chain containing 1 to 20 amino acid residues, where the amino acid residue and the peptide chain may have a protective group, R5 is a protective group for a carboxy group,R5' is a protective group for an amide group,n is an integer of 1 to 4, andX is optionally substituted C1-C3 alkylene, -CH2OCH2-, or -CH2SCH2-.

2. A method for producing a compound represented by formula (3) or a salt thereof, comprising the step of hydrogenating a compound represented by formula (1) or a salt thereof, which is obtained by the method of claim 1: [Formula 2]wherein R1, R2, R3, R4, n and X have the same meaning as R1, R2, R3, R4, n and X in [1], respectively.

3. The method according to claim 1 or 2, wherein the catalyst is a metal-alkylidene complex.

4. The method according to any one of claims 1 to 3, wherein the compound represented by formula (2) or a salt thereof is a salt of a compound represented by formula (2).

5. The method according to any one of claims 1 to 3, wherein the compound represented by formula (2) or a salt thereof is a compound represented by formula (2), and an acid is used in the metathesis step.

6. The method according to any one of claims 1 to 5, wherein alcohols and / or Bronsted acids are further used as an additive in the metathesis step, and the pKa of the additive is 4 to 17.

7. The method according to any one of claims 1 to 6, wherein the metathesis step is performed by a liquid-phase synthesis method.

8. The method according to claim 7, wherein the solvent used in the liquid-phase synthesis method is one or more selected from the group consisting of acetone, methyl t-butyl ether, dimethyl carbonate, ethyl acetate, toluene, and dichloromethane.

9. The method according to claim 7 or 8, wherein a concentration of the compound represented by formula (2) or a salt thereof in the liquid-phase synthesis method is 0.01 to 0.5 mol / L.

10. The method according to any one of claims 1 to 9, wherein R2 is hydrogen or linear C1-C3 alkyl.

11. The method according to any one of claims 1 to 10, wherein R3 is hydrogen, C1-C6 alkyl, or C7-C14 aralkyl optionally substituted with halo-C1-C3 alkyl or C1-C6 alkyl.

12. The method according to any one of claims 1 to 11, wherein R4 is OR5, NHR5', an amino acid residue, or a peptide chain containing 2 to 13 amino acid residues,R5 is a protective group for a carboxy group,R5' is a protective group for an amide group, andthe 2 to 13 amino acid residues are one or more selected from the group consisting of glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, N-methylglycine, N-methylalanine, N-methylisoleucine, N-methylleucine, N-methylmethionine, N-methylphenylalanine, and N-methyltyrosine.

13. The method according to any one of claims 1 to 12, wherein n is an integer of 1 or 2.

14. The method according to any one of claims 1 to 13, wherein X1 is one selected from the group consisting of an Fmoc group, a Cbz group, a Troc group, an Alloc group, a Teoc group, a TSoc group, a BIBSoc group, an IPCSoc group, a BBSoc group, a CHBSoc group, a CDBSoc group, and a Boc group.

15. The method according to any one of claims 1 to 14, wherein X is optionally substituted C1-C3 alkylene.