JP7812157B2 - Method for producing cyclic amide - Google Patents

Description

The present invention relates to a method for producing cyclic amides.

Cyclic amides, such as β-lactams and γ-lactams, are the 15th and 42nd most common structures, respectively, among the 351 types of ring structures contained in commercially available pharmaceuticals (see, for example, Non-Patent Document 1). Among cyclic amides, cyclic peptides with multiple amide bonds are known to have higher metabolic stability and target selectivity than non-cyclic peptides, making them useful as drugs such as antibacterial agents, immunosuppressants, and antitumor agents. For this reason, there is a demand for simple methods for synthesizing cyclic amides and cyclic peptides.

A relatively simple method for synthesizing unfunctionalized cyclic amides is known, involving the condensation of linear aminocarboxylic acids with inexpensive Lewis acids (see, for example, Non-Patent Document 2). However, condensation methods using inexpensive Lewis acids generally require long reaction times under high-temperature conditions, making them unsuitable for synthesizing cyclic amides functionalized with unstable functional groups, such as cyclic peptides. Therefore, there is a need for an excellent method for synthesizing cyclic amides that can be applied to a wide range of cyclic amides, including cyclic peptides.

On the other hand, it is known that using a phosphonium peptide condensing agent such as (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) as a method for synthesizing cyclic peptides makes it easier to obtain the desired peptide while avoiding side reactions in head-to-tail cyclization reactions (see, for example, Non-Patent Document 3). However, such phosphonium peptide condensing agents are expensive compared to other common condensing agents, and suffer from the problem of generating large amounts of impurities derived from the condensing agent. Therefore, there is a need for a method for synthesizing cyclic peptides using an inexpensive, highly active condensing agent that leaves few impurities behind after the reaction.

The present inventors have reported that using inexpensive, highly active triphosgene as a condensing agent in a method for synthesizing cyclic peptides can produce cyclic peptides in higher yields than conventional synthesis methods (see, for example, Non-Patent Document 4). However, the method in Non-Patent Document 4 cannot produce cyclic peptides in sufficient yield depending on the substrate. For this reason, it cannot be said to be an excellent method for synthesizing cyclic peptides that can cyclize a wide variety of peptides.

In addition, there have been reports of methods for synthesizing cyclic peptides using ethyl chloroformate as a condensing agent, which is cheaper than triphosgene (see, for example, Non-Patent Documents 5 and 6). However, Non-Patent Documents 5 and 6 only report that certain peptides can be cyclized, and none of the cyclic peptides were obtained in sufficiently high yields. Other reports have also been reported of methods for synthesizing cyclic peptides using isobutyl chloroformate as a condensing agent (see, for example, Non-Patent Documents 7-9). Non-Patent Document 8 reports the synthesis of certain cyclic peptides in higher yields than when ethyl chloroformate is used, but Non-Patent Document 9 makes it clear that when conditions similar to Non-Patent Document 8 are applied to other substrates, the yield drops significantly. Furthermore, Non-Patent Documents 5-9 merely attempt to slightly improve yield by performing the cyclization reaction over a long period of time under low-temperature conditions, but do not allow for the efficient synthesis of cyclic peptides in a short period of time.

As such, a method for obtaining a variety of cyclic amides, including cyclic peptides, by cyclization in a short period of time using an inexpensive condensing agent has not yet been established.

J. Med. Chem. 2014, 57, 5845-5859. Tetrahedron Lett. 1988, 29, 3049-3052. Org. Process Res. Dev., 2018, 22, 760-772. Chem. Eur. J. 2021, 27, 7525-7532. Liebigs Ann. Chem., 1968, 713, 201-208. Helv. Chim. Acta, 1952, 35, 2229-2235. Helv. Chim. Acta, 1987, 70, 1012-1016. Int. J. Peptide Protein Res. 1988, 32, 527-535. J. Med. Chem. 1999, 42, 1705-1713.

The present invention was made in consideration of the current state of the prior art described above, and its main objective is to provide a method for producing a variety of cyclic amides by cyclization in a short period of time using an inexpensive condensing agent.

The inventors have conducted extensive research to achieve the above-mentioned objectives. As a result, they have discovered that by selecting a specific condensing agent and a specific tertiary amine compound, it is possible to cyclize a variety of acyclic compounds having terminal amino and carboxyl groups using an inexpensive condensing agent in a short period of time to obtain a variety of cyclic amides. Based on this finding, the inventors have conducted further research and completed the present invention. Specifically, the present invention encompasses the following features:

Item 1. General formula (1):

[In the formula, R represents an optionally substituted divalent organic group. R may form a ring together with the adjacent nitrogen atom. _R represents a hydrogen atom or a hydrocarbon group.]
A method for producing a cyclic amide represented by the formula:
General formula (2):

[In the formula, R and R _A are the same as above.]
and a cyclic amide precursor represented by general formula (3):

[wherein X represents a halogen atom, and Y represents an optionally substituted alkyl group] with a condensing agent represented by general formula (4):

[In the formula, R ¹ , R ² and R ³ are the same or different and represent an optionally substituted alkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group. Alternatively, two or more of R ¹ , R ² and R ³ may be linked to form a ring (excluding a pyridine ring) which may have one or more heteroatoms or substituents.]
with a tertiary amine compound represented by the following formula (excluding tributylamine, diisopropylethylamine, and triethylamine):

Item 2. The manufacturing method according to Item 1, wherein R is a divalent hydrocarbon group which may have an amide bond and/or a substituent.

Item 3. The cyclic amide is represented by general formula (1a):

[Wherein, _RA is the same as defined above. Two or more ^RA may be the same or different. _RC , _RN , and _RP may be the same or different and represent a divalent organic group. _RC , _RN , and _RP may each form a ring together with the adjacent nitrogen atom. n represents an integer of 0 or greater. However, when n is an integer of 2 or greater, n _RPs may be the same or different.]
A cyclic peptide represented by the formula:
The cyclic amide precursor is represented by the general formula (2a):

[In the formula, R _A , R _C , R _N , R _P and n are the same as defined above.]
Item 3. The method according to Item 1 or 2, wherein the cyclic peptide precursor is represented by the formula:

Item 4. The production method according to Item 3, wherein R _C , R _N and R _P are the same or different and each represents a divalent hydrocarbon group which may have a substituent.

Item 5. The manufacturing method described in Item 3 or 4, wherein n is an integer of 2 or more.

Item 6. The manufacturing method described in any one of Items 1 to 5, wherein X is a chlorine atom.

Item 7. The manufacturing method described in any one of Items 1 to 6, wherein Y is a branched alkyl group.

Item 8. The manufacturing method described in any one of Items 1 to 7, wherein Y is an isopropyl group.

Item 9. The production method according to any one of Items 1 to 8, wherein at least one of R ¹ , R ² and R ³ is a methyl group.

Item 10. The production method according to any one of Items 1 to 9, wherein at least two of R ¹ , R ² and R ³ are methyl groups.

Item 11. The production method described in any one of Items 1 to 10, wherein the reaction step is carried out in a solvent containing an aprotic polar solvent.

Item 12. The manufacturing method described in any one of Items 1 to 11, wherein the reaction temperature in the reaction step is 0 to 100°C.

Item 13. The manufacturing method described in any one of Items 1 to 12, wherein the reaction time of the reaction step is less than 10 minutes.

Item 14. The manufacturing method described in any one of Items 1 to 13, wherein the reaction step is carried out by a flow method.

Item 15. The manufacturing method described in any one of Items 1 to 14, wherein the reaction step is carried out by a microflow method.

According to the present invention, a variety of cyclic amides can be synthesized in a short period of time by reacting a cyclic amide precursor with a specific condensing agent and a specific tertiary amine compound.

FIG. 1 is a schematic diagram showing a general configuration of a microflow reactor. Chromatograms of cyclic peptide 16 (Versicotide D analog; L-MeAla-L-Phe-L-MeAla-L-Phe-L-Ala) consisting of the amino acid sequence represented by SEQ ID NO: 6 obtained in Example 9-1 and its epimer, cyclic peptide 17 (Versicotide D analog epimer) represented by SEQ ID NO: 7 obtained in Example 9-2 (Column: GLscience Inertsil™ ODS-3 5 μm, 4.6 mm × 75 mm, Solvent: Methanol + 0.1% formic acid/H ₂ O + 0.1% formic acid (0-13 min: 0-100%, 13-16 min: 100%, 16-17 min: 0%, 17-22 min: 0%), Flow rate: 1.0 mL/min, Detection wavelength: 254 nm, Temperature: 40°C).

In this specification, the term "containing" is a concept that encompasses all of "comprise," "consist essentially of," and "consist only of."

In addition, in this specification, the notation of a numerical range such as "A to B" means "greater than or equal to A and less than or equal to B."

The method for producing a cyclic amide of the present invention includes a step of reacting a cyclic amide precursor with a condensing agent and a tertiary amine compound.

In addition, in the method of the present invention, the reaction between the condensing agent and the tertiary amine compound is thought to generate highly reactive active species, which allows for rapid activation of the terminal carboxyl group of the cyclic amide precursor (hereinafter referred to as the "C-terminus" in this specification). Furthermore, the subsequent cyclization reaction between the activated C-terminal carboxyl group and the amino group at the other terminal (hereinafter referred to as the "N-terminus" in this specification) is also thought to proceed rapidly. In this case, since the reaction can be carried out under near-neutral conditions in a short time, the method of the present invention can be applied to a variety of substrates. Furthermore, the method of the present invention can avoid epimerization and dimerization that accompany cyclization.

In addition, the method of the present invention makes it possible to carry out the reaction inexpensively and with high yield, especially when isopropyl chloroformate is used as the condensing agent, and it is also easy to remove impurities that form after the reaction.

Furthermore, in the method of the present invention, particularly when a compound having one or more methyl groups bonded to the nitrogen atom is used as the tertiary amine compound, the C-terminus of the cyclic amide precursor can be selectively activated, thereby making it possible to obtain cyclic amides in high yield.

Furthermore, when using the microflow method in the method of the present invention, by appropriately selecting the condensing agent and base, it is possible to quantitatively obtain cyclic peptides in a yield (>99%) that is unthinkable with conventional peptide synthesis methods.

1. Cyclic Amide The cyclic amide of the present invention is represented by the general formula (1):

[In the formula, R represents an optionally substituted divalent organic group. R may form a ring together with the adjacent nitrogen atom. _R represents a hydrogen atom or a hydrocarbon group.]
It is a compound represented by the formula:

In general formula (1), the group represented by R is an optionally substituted divalent organic group.

The divalent organic group represented by R is preferably a divalent hydrocarbon group which may have an amide bond and/or a substituent. Examples of the hydrocarbon group include an optionally substituted alkyl group and an optionally substituted aryl group.

The alkyl group represented by R is not particularly limited and includes, for example, linear alkyl groups having 1 to 10 carbon atoms (especially 1 to 6), such as methyl, ethyl, n-propyl, n-butyl, and n-pentyl groups; branched alkyl groups having 3 to 10 carbon atoms (especially 3 to 6), such as isopropyl, isobutyl, sec-butyl, and tert-butyl groups; and cyclic alkyl groups having 3 to 10 carbon atoms (especially 5 to 8), such as cyclopentyl and cyclohexyl groups.

The aryl group represented by R is not particularly limited, and can be any of a monocyclic aryl group, a fused-ring aryl group, and a polycyclic aryl group. Examples include aryl groups having 6 to 18 carbon atoms (particularly 6 to 14 carbon atoms), such as a phenyl group, a naphthyl group, an anthracenyl group, a phenanthrenyl group, a biphenyl group, a terphenyl group (all of an o-terphenyl group, an m-terphenyl group, and a p-terphenyl group are also acceptable; the same applies below), a fluorenyl group, a pyrenyl group, and a triphenylenyl group.

The amide bond in the hydrocarbon group represented by R may have a hydrogen atom substituted.

The hydrocarbon group represented by R may have a substituent. Examples of the substituent include, but are not limited to, a hydroxyl group, a halogen atom (such as a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), the alkyl group, the aryl group, an alkoxy group, a carboxyl group, and an amino group. When the hydrocarbon group has a substituent, the number of substituents is not particularly limited, but is preferably 1 to 6, and more preferably 1 to 3.

The substituent on the hydrocarbon group represented by R may have a protecting group. Any known protecting group can be used as the protecting group, for example, alkyl-type protecting groups such as triphenylmethyl (Trt) group; silyl-type protecting groups such as tert-butyldimethylsilyl (TBS) group; aryl-type protecting groups such as p-methoxybenzyl (PMB) group and p-methoxyphenyl (PMP) group; amide-type protecting groups such as formyl group and acetyl (Ac) group; phthalimide-type protecting groups such as phthaloyl (Phth) group; benzyloxycarbonyl (Cbz) group and tert-amyl (Cbz) group. Examples of protecting groups include carbamate-type protecting groups such as oxycarbonyl group (Aoc), 9-fluorenylmethoxycarbonyl group (Fmoc), tert-butyloxycarbonyl group (Boc), allyloxycarbonyl group (Alloc), and 2,2,2-triethoxycarbonyl group (Troc); and sulfonamide-type protecting groups such as 3-nitro-2-pyridinesulfenyl group (Npys), 2-nitrobenzenesulfonyl group (Ns), and (2-trimethylsilyl)-ethanesulfonyl group (SES). The present invention has the advantage that even protecting groups that are unstable to heat or acid can be used.

When protective groups are present, the number of protective groups is not particularly limited, but 1 to 6 is preferred, and 1 to 3 is more preferred.

In general formula (1), the group represented by R 1 _A is a hydrogen atom or a hydrocarbon group.

Examples of the hydrocarbon group represented by R 1 _A include an alkyl group and an aryl group.

The alkyl group represented by R _A is not particularly limited and examples thereof include linear alkyl groups having 1 to 10 carbon atoms (particularly 1 to 6), such as a methyl group, an ethyl group, an n-propyl group, an n-butyl group, and an n-pentyl group, as well as branched alkyl groups having 3 to 10 carbon atoms (particularly 3 to 6), such as an isopropyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group.

The aryl group represented by R _A is not particularly limited, and any of a monocyclic aryl group, a fused ring aryl group, and a polycyclic aryl group can be used. Examples thereof include aryl groups having 6 to 18 carbon atoms (particularly 6 to 14 carbon atoms), such as a phenyl group, a naphthyl group, an anthracenyl group, a phenanthrenyl group, a biphenyl group, a terphenyl group, a fluorenyl group, a pyrenyl group, and a triphenylenyl group.

In particular, when the cyclic amide of the present invention is a cyclic peptide, the cyclic amide of the present invention is represented by the general formula (1a):

[Wherein, _RA is the same as defined above. Two or more ^RA may be the same or different. _RC , _RN , and _RP may be the same or different and represent a divalent organic group. _RC , _RN , and _RP may each form a ring together with the adjacent nitrogen atom. n represents an integer of 0 or greater. However, when n is an integer of 2 or greater, n _RPs may be the same or different.]
It is a compound represented by the formula:

In formula (1a), the groups represented by R _C , R _N and R _P are the same or different and are divalent organic groups.

The divalent organic groups represented by R _C , R _N and R _P are preferably divalent hydrocarbon groups which may have a substituent, and examples of the hydrocarbon group include an optionally substituted alkyl group and an optionally substituted aryl group.

The alkyl group represented by R _C , R _N and R _P is not particularly limited and examples thereof include linear alkyl groups having 1 to 10 carbon atoms (particularly 1 to 6), such as a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, etc.; branched alkyl groups having 3 to 10 carbon atoms (particularly 3 to 6), such as an isopropyl group, an isobutyl group, a sec-butyl group, an tert-butyl group, etc.; and cyclic alkyl groups having 3 to 10 carbon atoms (particularly 5 to 8), such as a cyclopentyl group, a cyclohexyl group, etc.

The aryl group represented by R _C , R _N and R _P is not particularly limited, and any of a monocyclic aryl group, a fused ring aryl group and a polycyclic aryl group can be used. Examples thereof include aryl groups having 6 to 18 carbon atoms (particularly 6 to 14 carbon atoms), such as a phenyl group, a naphthyl group, an anthracenyl group, a phenanthrenyl group, a biphenyl group, a terphenyl group, a fluorenyl group, a pyrenyl group and a triphenylenyl group.

The hydrocarbon groups represented by R _C , R _N and R _P may have a substituent. Examples of the substituent include, but are not particularly limited to, a hydroxyl group, a halogen atom (such as a fluorine atom, a chlorine atom, a bromine atom or an iodine atom), the alkyl group, the aryl group, an alkoxy group, a carboxyl group, an amino group, etc. When the hydrocarbon group has a substituent, the number of the substituents is not particularly limited, and is preferably 1 to 6, and more preferably 1 to 3.

The substituents possessed by the hydrocarbon groups represented by R _C , R _N and R _P may have a protecting group. Any known protecting group can be used as the protecting group, and examples thereof include alkyl-type protecting groups such as triphenylmethyl group (Trt); silyl-type protecting groups such as tert-butyldimethylsilyl group (TBS); aryl-type protecting groups such as p-methoxybenzyl group (PMB) and p-methoxyphenyl group (PMP); amide-type protecting groups such as formyl group and acetyl group (Ac); phthalimide-type protecting groups such as phthaloyl group (Phth); benzyloxycarbonyl group (Cbz), tert-amyl Examples of protecting groups include carbamate-type protecting groups such as oxycarbonyl group (Aoc), 9-fluorenylmethoxycarbonyl group (Fmoc), tert-butyloxycarbonyl group (Boc), allyloxycarbonyl group (Alloc), and 2,2,2-triethoxycarbonyl group (Troc); and sulfonamide-type protecting groups such as 3-nitro-2-pyridinesulfenyl group (Npys), 2-nitrobenzenesulfonyl group (Ns), and (2-trimethylsilyl)-ethanesulfonyl group (SES). The present invention has the advantage that even protecting groups that are unstable to heat or acid can be used.

When protective groups are present, the number of protective groups is not particularly limited, but 1 to 6 is preferred, and 1 to 3 is more preferred.

When the cyclic amide that is the target of the present invention is a cyclic peptide, the method of the present invention is intended to synthesize a cyclic peptide using a non-cyclic peptide as a cyclic peptide precursor, and the groups represented by R _C , R _N and R _P correspond to the groups represented by R _C , R _N and R _P that the non-cyclic peptide has. Therefore, the groups represented by R _C , R _N and R _P constitute amino acids together with the adjacent amino group and carboxyl group, and are preferably amino acid residues.

In this specification, an amino acid residue refers to a divalent group obtained by removing the amino group and carboxyl group from an amino acid having an amino group and a carboxyl group in one molecule.

In general formula (1a), the amino acid residues represented by R _C , R _N and R _P are derived from the C-terminal amino acid residue, N-terminal amino acid residue and non-terminal amino acid residue of the cyclic peptide precursor described below, respectively. Note that, since a cyclic peptide is composed of a repeating structure of amino acid residues and peptide bonds that are equivalent to each other, the amino acid residues represented by R _C , R _N and R _P are equivalent to each other. In other words, in a cyclic peptide, determining the amino acid residues represented by R _C , R _N and R _P is equivalent to determining the position of the peptide bond formed by cyclization by the method of the present invention. For this reason, it is preferable to appropriately select the amino acid residues represented by R _C , R _N and R _P taking into account the reactivity of the C-terminus and N-terminus of the derived cyclic peptide precursor (i.e., ease of cyclization). Specifically, from among the amino acid residues of the cyclic peptide, an amino acid residue that is easily activated when used as the C-terminal amino acid residue of the cyclic peptide precursor can be designated as R _C , and an N-terminal amino acid residue that is easily cyclized by attacking the activated C-terminal amino acid residue can be designated as R _N.

In general formula (1a), the amino acid residues represented by R _C , R _N and R _P can be any of natural amino acid residues and synthetic amino acid residues, and examples thereof include glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (ILE), phenylalanine (Phe), serine (Ser), threonine (Thr), lysine (Lys), 5-hydroxylysine (Hyl), arginine (Arg), aspartic acid (Asp), asparagine (Asn), glycine (Gly ... Examples of such amino acid residues include glutamic acid (Glu), glutamine (Gln), cysteine (CySH), cystine (Cyss), cysteic acid (Cya), methionine (Met), tyrosine (Tyr), thyroxine (Thy), proline (Pro), hydroxyproline (Hyp), tryptophan (Trp), histidine (His), β-alanine, N-methyl-β-alanine, sarcosine, γ-aminobutyric acid, kainic acid, and derivatives thereof. These amino acid residues may be used alone or in combination of two or more.

Furthermore, in general formula (1a), the amino acid residues represented by R _C , R _N , and R _P can be amino acid residues having a protecting group, depending on the type of functional group to be protected. When the amino acid residue has an amino group or a carboxyl group in its side chain, an amino acid residue having a protecting group is preferred from the viewpoint of avoiding side reactions. On the other hand, when the amino acid residue consists only of amino acid residues having functional groups other than amino groups and carboxyl groups in their side chains, an amino acid residue not having a protecting group is preferred from the viewpoints of eliminating the deprotection step, improving atom efficiency, and reducing waste. According to the method of the present invention, even if the amino acid residue does not have a protecting group, side reactions derived from the side chain are unlikely to occur.

The protecting groups possessed by the amino acid residues described above can be any known protecting group, including, for example, alkyl-type protecting groups such as triphenylmethyl (Trt) groups; silyl-type protecting groups such as tert-butyldimethylsilyl (TBS) groups; aryl-type protecting groups such as p-methoxybenzyl (PMB) groups and p-methoxyphenyl (PMP) groups; amide-type protecting groups such as formyl groups and acetyl (Ac) groups; phthalimide-type protecting groups such as phthaloyl (Phth) groups; benzyloxycarbonyl (Cbz) groups and tert-butyldimethylsilyl (TBS) groups. Examples of protecting groups include carbamate-type protecting groups such as t-amyloxycarbonyl group (Aoc), 9-fluorenylmethoxycarbonyl group (Fmoc), tert-butyloxycarbonyl group (Boc), allyloxycarbonyl group (Alloc), and 2,2,2-triethoxycarbonyl group (Troc); and sulfonamide-type protecting groups such as 3-nitro-2-pyridinesulfenyl group (Npys), 2-nitrobenzenesulfonyl group (Ns), and (2-trimethylsilyl)-ethanesulfonyl group (SES).

In general formula (1a), n is an integer of 1 or more, and from the viewpoint of structural stability of the cyclic peptide and improving the yield, n is preferably 2 to 100, more preferably 3 to 40. When n is an integer of 2 or more, the n R _Ps may be the same or different. According to the method of the present invention, it is possible to provide cyclic tetrapeptides and cyclic pentapeptides, which are known to be usually difficult to synthesize.

The above-mentioned cyclic peptides tend to have high metabolic stability and high cell membrane permeability. Therefore, they can be used as ingredients in, for example, antibacterial agents, immunosuppressants, antitumor agents, brain function improvers, hair quality improvers, antipruritics, and other pharmaceuticals, as well as skin care products, quasi-drugs, dietary supplements, and cosmetics.

From the above, examples of the cyclic amide include:

etc.

Furthermore, examples of the above-mentioned cyclic peptides include the following: cyclic peptide 11 (L-Ala-L-Tyr(OBn)-L-Leu-L-Ala-Gly) consisting of the amino acid sequence represented by SEQ ID NO: 1; cyclic peptide 12 (L-Trp-L-Phe-L-Leu-L-Ala-Gly) consisting of the amino acid sequence represented by SEQ ID NO: 2; cyclic peptide 13 (L-Phe-L-Pro-L-Ser(OBn)-L-Phe-Gly) consisting of the amino acid sequence represented by SEQ ID NO: 3; and cyclic peptide 14 (L-Phe-L-Pro-L-Ser-L-Phe-Gly) consisting of the amino acid sequence represented by SEQ ID NO: 4. Gly), cyclic peptide 15 (L-Pro-L-Val-L-Tyr(OBn)-L-Ala-Gly) consisting of the amino acid sequence represented by SEQ ID NO: 5, cyclic peptide 16 (L-MeAla-L-Phe-L-MeAla-L-Phe-L-Ala) consisting of the amino acid sequence represented by SEQ ID NO: 6, cyclic peptide 17 (L-MeAla-L-Phe-L-MeAla-L-Phe-D-Ala) consisting of the amino acid sequence represented by SEQ ID NO: 7, and cyclic peptide 18 (L-MeAla-L-Leu-L-MePhe-Gly) consisting of the amino acid sequence represented by SEQ ID NO: 8.

(2) Cyclic Amide Precursor The cyclic amide precursor used in the present invention is represented by the general formula (2):

[In the formula, R and R _A are the same as above.]
It is a compound represented by the formula:

In general formula (2), when the group represented by R _A is a hydrocarbon group rather than a hydrogen atom, it is generally considered that the cyclization reaction does not proceed easily. However, the method of the present invention has the advantage that the cyclization reaction can proceed easily even when the group represented by R _A is a hydrocarbon group.

In particular, when the cyclic amide of the present invention is a cyclic peptide, the cyclic amide precursor is represented by the general formula (2a):

[In the formula, R _A , R _C , R _N , R _P and n are the same as defined above.]
It is a cyclic peptide precursor represented by the formula:

The cyclic amide precursor used in the present invention can be a cyclic amide precursor that has previously been cyclized, or a cyclic amide precursor that has not previously been cyclized. According to the method of the present invention, cyclic amide precursors that have previously been cyclized can be cyclized in high yield, and a variety of cyclic amide precursors that have not previously been cyclized can be cyclized. Therefore, the method of the present invention makes it possible to provide amide libraries and peptide libraries for drug discovery screening.

From the above, examples of the cyclic amide precursor include:

etc.

Furthermore, examples of the cyclic peptide precursor include the following: cyclic peptide precursor 21 (H-L-Ala-L-Tyr(OBn)-L-Leu-L-Ala-Gly-OH) consisting of the amino acid sequence represented by SEQ ID NO: 1; cyclic peptide precursor 22 (H-L-Trp-L-Phe-L-Leu-L-Ala-Gly-OH) consisting of the amino acid sequence represented by SEQ ID NO: 2; cyclic peptide precursor 23 (H-L-Phe-L-Pro-L-Ser(OBn)-L-Phe-Gly-OH) consisting of the amino acid sequence represented by SEQ ID NO: 3; and cyclic peptide precursor 24 (H-L-Phe-L-Pro-L-Ser-L-Phe-Gly-OH) consisting of the amino acid sequence represented by SEQ ID NO: 4. ly-OH), cyclic peptide precursor 25 (H-L-Pro-L-Val-L-Tyr(OBn)-L-Ala-Gly-OH) consisting of the amino acid sequence represented by SEQ ID NO: 5, cyclic peptide precursor 26 (H-L-MeAla-L-Phe-L-MeAla-L-Phe-L-Ala-OH) consisting of the amino acid sequence represented by SEQ ID NO: 6, cyclic peptide precursor 27 (H-L-MeAla-L-Phe-L-MeAla-L-Phe-D-Ala-OH) consisting of the amino acid sequence represented by SEQ ID NO: 7, and cyclic peptide precursor 28 (H-L-MeAla-L-Leu-L-MePhe-Gly-OH) consisting of the amino acid sequence represented by SEQ ID NO: 8.

(3) Condensing Agent The condensing agent that can be used in the present invention is a compound represented by the general formula (3):

[In the formula, X represents a halogen atom. Y represents an optionally substituted alkyl group.]

In general formula (3), the group represented by X is a halogen atom.

Examples of halogen atoms represented by X include fluorine atoms, chlorine atoms, bromine atoms, and iodine atoms. From the standpoints of yield, economy, safety, etc., chlorine atoms or bromine atoms are preferred, with chlorine atoms being more preferred.

In general formula (3), the alkyl group represented by Y is not particularly limited and includes linear alkyl groups having 1 to 10 carbon atoms (particularly 1 to 6), such as n-propyl, n-butyl, and n-pentyl, as well as branched alkyl groups having 3 to 10 carbon atoms (particularly 3 to 6), such as isopropyl, sec-butyl, and tert-butyl. From the standpoint of reactivity with the cyclic amide precursor and the tertiary amine compound, the alkyl group represented by Y is preferably a branched alkyl group having 3 to 10 carbon atoms, more preferably a branched alkyl group having 3 to 6 carbon atoms, and even more preferably an isopropyl group.

From the above, the condensing agent used in the present invention is, for example,

etc.

These condensation agents can be used alone or in combination of two or more.

The amount of condensing agent used is not particularly limited and can be selected appropriately from a wide range. From the standpoint of suppressing the formation of impurities and improving yield, an amount of condensing agent is preferably about 0.1 to 10 moles, more preferably about 0.2 to 5 moles, and even more preferably about 0.5 to 3 moles, per mole of cyclic amide precursor.

(4) Tertiary amine compound The tertiary amine compound that can be used in the present invention is represented by the general formula (4):

[In the formula, R ¹ , R ² and R ³ are the same or different and represent an optionally substituted alkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group. Alternatively, two or more of R ¹ , R ² and R ³ may be linked to form a ring (excluding a pyridine ring) which may have one or more heteroatoms or substituents.]
(However, tributylamine, diisopropylethylamine, and triethylamine are excluded.) Here, "excluding tributylamine" means excluding tri-n-butylamine, tri-s-butylamine, tri-t-butylamine, and triisobutylamine.

In general formula (4), the alkyl group represented by R ¹ , R ² and R ³ is not particularly limited, and examples thereof include linear alkyl groups having 1 to 10 carbon atoms (particularly 1 to 6), such as a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, and the like, as well as branched alkyl groups having 3 to 10 carbon atoms (particularly 3 to 6), such as an isopropyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group.

In general formula (4), the aryl group represented by R ¹ , R ² and R ³ is not particularly limited, and any of a monocyclic aryl group, a fused ring aryl group and a polycyclic aryl group can be used. Examples thereof include aryl groups having 6 to 18 carbon atoms (particularly 6 to 14 carbon atoms), such as a phenyl group, a naphthyl group, an anthracenyl group, a phenanthrenyl group, a biphenyl group, a terphenyl group, a fluorenyl group, a pyrenyl group and a triphenylenyl group.

In general formula (4), the heteroaryl group represented by R ¹ , R ² , and R ³ is not particularly limited, and either a monocyclic heteroaryl group or a polycyclic heteroaryl group can be employed. Examples thereof include a pyrrolidyl group, a pyrrolyl group, a tetrahydrothienyl group, a thienyl group, an oxolanyl group, a furanyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an oxazolyl group, a piperidyl group, a pyridyl group, a pyrazyl group, an indolyl group, an isoindolyl group, a benzimidazolyl group, a quinolyl group, an isoquinolyl group, and a quinoxalyl group.

The alkyl group, aryl group, and heteroaryl group represented by R ¹ , R ² , and R ³ may have a substituent. Examples of the substituent include, but are not particularly limited to, a hydroxyl group, a halogen atom (such as a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), the alkyl group, the aryl group, an alkoxy group, and an amino group. When the group has a substituent, the number of the substituents is not particularly limited, and is preferably 1 to 6, and more preferably 1 to 3.

Two or more of R ¹ , R ² and R ³ may be linked to form a ring (excluding a pyridine ring) which may have one or more heteroatoms or substituents. The ring is not particularly limited, and examples thereof include an aziridine ring, an azirine ring, a diaziridine ring, a diazirine ring, an azetidine ring, an azeto ring, a diazeto ring, a pyrrolidine ring, a pyrrole ring, an imidazolidine ring, an imidazole ring, a piperidine ring, a piperazine ring and a morpholine ring.

In particular, from the viewpoint of improving yield, it is preferable that at least one of R ¹ , R ² and R ³ is a methyl group, and it is more preferable that at least two of R ¹ , R ² and R ³ are methyl groups.

From the above, the tertiary amine compounds used in the present invention include, for example,

etc.

Among these, from the viewpoint of improving yield, the tertiary amine compounds that can be used in the present invention are preferably dimethylbenzylamine (Me ₂ NBn), trimethylamine (Me ₃ N), dimethylethylamine (Me ₂ NEt), dimethyl n-hexylamine (Me ₂ Nn-Hex), and 1-methylpyrrolidine, and more preferably dimethylbenzylamine (Me ₂ NBn), trimethylamine (Me ₃ N), dimethylethylamine (Me ₂ NEt), and dimethyl n-hexylamine (Me ₂ Nn-Hex).

These tertiary amine compounds can be used alone or in combination of two or more.

The amount of tertiary amine compound used is not particularly limited and can be selected appropriately from a wide range. From the standpoint of the yield of the desired cyclic amide and economic efficiency, an amount of approximately 0.1 to 10 moles per mole of cyclic amide precursor is preferred, approximately 0.2 to 5 moles is more preferred, and approximately 0.5 to 3 moles is even more preferred. Note that while the yield of the desired cyclic amide improves as the amount of tertiary amine compound used increases, using an excess of tertiary amine compound does not improve the yield of the desired cyclic amide at all. For this reason, it is preferable to appropriately adjust the amount of tertiary amine compound used, taking economic efficiency into consideration.

5. Reaction Solvent The reaction step of the present invention is not particularly limited and can be carried out in, for example, any organic solvent in which the substrate is soluble, water, or a mixed solvent thereof. From the viewpoint of the solubility of the substrate, however, it is preferable to carry out the reaction step in a solvent containing an aprotic polar solvent.

The aprotic polar solvent used in the reaction step of the present invention is not particularly limited, but includes ketone solvents such as acetone and methyl ethyl ketone; ether solvents such as 1,4-dioxane, tetrahydrofuran, and diethyl ether; amide solvents such as N,N-dimethylformamide and N,N-diethylformamide; sulfoxide solvents such as dimethyl sulfoxide; nitrile solvents such as acetonitrile; and mixtures thereof. Of these, from the standpoints of substrate solubility, low reactivity, ease of removal, and environmental impact, ether solvents and nitrile solvents are preferred, with 1,4-dioxane, tetrahydrofuran, acetonitrile, etc. being more preferred. These solvents can be used alone or in combination of two or more.

In particular, as a solvent for dissolving cyclic amides (precursors) or cyclic peptides (precursors) that are difficult to dissolve in organic solvents alone, it is preferable to use a mixed solvent of water and an aprotic polar solvent, and more preferably a mixed solvent of water and acetonitrile, water and 1,4-dioxane, or water and tetrahydrofuran. The mixing ratio of the above mixed solvents (water: aprotic polar solvent) is preferably approximately 10:90 to 90:10 by volume, more preferably approximately 75:25 to 25:75, and even more preferably 50:50.

6. Method for Producing Cyclic Amides The cyclic amides of the present invention can be produced by reacting the cyclic amide precursor, the condensing agent, and the tertiary amine compound. The reaction is not particularly limited, and all components can be added simultaneously or sequentially.

The reaction process of the present invention can be carried out by either a batch method or a flow method. From the viewpoints of high safety and ease of scale-up, the flow method is more preferable, and from the viewpoints of precise control of reaction temperature and reaction time, the microflow method is even more preferable.

In this specification, the term "batch method" refers to a batch operation, specifically a series of operations in which the substances to be reacted are loaded into a reaction vessel or reactor all at once, reacted, and then removed once equilibrium or a certain reaction rate is reached. In this specification, the term "flow method" refers to a continuous operation, specifically an operation in which raw materials and substances required for their processing are continuously fed into and discharged from an apparatus at a certain rate (flow rate).

In this specification, the microflow method means a flow method (continuous operation) using a microflow reactor.

In particular, the reaction process of the present invention can be carried out using a microflow reactor. A microflow reactor includes, for example, a flow path for transporting a fluid containing a raw material or intermediate used in the reaction, a pump for feeding the fluid into the flow path, and a mixer for mixing the fluid.

Regarding the use of a microflow reactor, for example, the reaction between a condensing agent and a tertiary amine compound may be carried out in a microflow reactor, or the reaction between a cyclic amide precursor and a highly reactive active species produced by the reaction between a condensing agent and a tertiary amine compound may be carried out in a microflow reactor. From the standpoint of the stability of the condensing agent and the highly reactive active species and reaction efficiency, it is preferable to use a microflow reactor to mix and react a fluid containing a cyclic amide precursor and a tertiary amine compound with a fluid other than the fluid containing a condensing agent.

The method for producing a cyclic amide of the present invention is not limited to being carried out using a microflow reactor; for example, a batch vessel with a small volume that allows for a high stirring speed may also be used. The volume of the mixing section of the batch vessel may be 1 to 100 mL, or may be 5 to 50 mL.

The configuration of the microflow reactor and the method for producing a cyclic amide of the present invention using the same will be described below with reference to Figure 1. However, it goes without saying that the production method of the present invention is not limited to the embodiment shown in Figure 1.

Figure 1 is a schematic diagram showing the general configuration of a microflow reactor 1. The microflow reactor 1 can include a tank 11 containing a first liquid, a tank 12 containing a second liquid, and a tank 13 containing a third liquid.

As one example, the first liquid may contain a cyclic amide precursor, the second liquid may contain a tertiary amine compound, and the third liquid may contain a condensing agent. As another example, the first liquid may contain a cyclic amide precursor and a tertiary amine compound, the second liquid may contain a condensing agent, and the third liquid may contain a reaction terminator. Regarding the use of a microflow reactor, for example, at least the mixture of the first liquid and the second liquid and the third liquid may be mixed in a microflow reactor, or further, the first liquid and the second liquid may be mixed in a microflow reactor.

Microflow reactor 1 can include flow paths f1, f2, f3, f4, and f5 for transporting fluids. The inner diameter of the flow paths can be, for example, 0.1 to 10 mm, or 0.3 to 1 mm. Microflow reactor 1 can also include mixers 31 and 32 for mixing fluids. The mixers are not particularly limited, but can include, for example, V-mixers, T-mixers, and other devices that allow fluids to be mixed by introducing them. In such mixers, the entrainment flow generated within the flow paths increases the interfacial area between the two liquids, making it easier to mix two or more reaction liquids within a few milliseconds. The inner diameter of the flow paths within the mixers can be, for example, 0.1 to 10 mm, or 0.2 to 1 mm.

The inner diameter of the above-mentioned flow path can be the diameter of the inner portion of the flow path (the portion through which the fluid passes) in the cross section of the flow path in a direction perpendicular to the longitudinal direction of the flow path. If the shape of the inner portion of the flow path is not a perfect circle, the inner diameter of the above-mentioned flow path can be the diameter when the shape of the inner portion of the flow path is converted into a perfect circle based on area.

Tanks 11, 12, 13 and 14, mixers 31 and 32, and flow paths f1, f2, f3, f4 and f5 may be formed, for example, from resins such as plastic or elastomer, glass, metal, ceramic, etc.

Tank 11 is connected to pump 21, and when pump 21 is operated, the first liquid contained in tank 11 moves through flow path f1 and flows into mixer 31. Tank 12 is connected to pump 22, and when pump 22 is operated, the second liquid contained in tank 12 moves through flow path f2 and flows into mixer 31. The first and second liquids are then mixed by mixer 31 to form a first mixed liquid, which can be sent to flow path f4. Meanwhile, tank 13 is connected to pump 23, and when pump 23 is operated, the liquid contained in tank 13 moves through flow path f3 and flows into mixer 32, where it is mixed with the first mixed liquid to form a second mixed liquid, which can be sent to flow path f5. The second mixed liquid can be stored in tank 14.

The microflow reactor 1 described above makes it easy to increase the area for heat exchange per volume of reaction solution. In addition, it is easy to control the reaction time by adjusting the flow rate and the length of the flow path. This makes it easy to precisely control the reaction solution, which in turn makes it easy to minimize the progression of undesired side reactions and improve the yield of the target product.

As such, the method for producing cyclic amides of the present invention can be carried out using a liquid phase method, making it easy to scale up.

After the reaction using a microflow reactor is completed, the desired compound, the cyclic amide, can be obtained, if necessary, through conventional isolation and purification processes.

In addition, while conventional peptide synthesis requires complicated isolation and purification processes, the present invention uses a condensing agent that produces few impurities after the reaction and is easy to remove, making it easy to obtain cyclic peptides in high yields.This means that these processes can be omitted or simplified, and the peptides can be directly subjected to crystallization and analysis.

7. Other Conditions In addition to the above components, additives may be used as appropriate within the scope of the present invention, as long as the effects of the present invention are not impaired. Examples of such additives include surfactants, peptide coupling agents, crown ethers, and metal salts.

The reaction temperature in the reaction step of the present invention is preferably 0 to 100°C, more preferably 20 to 80°C, and even more preferably 40 to 80°C, from the viewpoints of improving cyclization efficiency, suppressing epimerization, suppressing dimerization, and shortening the reaction time. The reaction time can be the time required for the cyclization reaction to proceed, and from the viewpoints of improving the cyclization yield and suppressing epimerization, it is preferably 10 seconds to 12 hours, more preferably 10 seconds to 6 hours, and even more preferably 10 seconds to 1 hour. When the reaction step of the present invention is carried out by a microflow method, the reaction time can be preferably less than 10 minutes, more preferably less than 5 minutes, and even more preferably less than 1 minute.

8. Reaction Mechanism In the method of the present invention, it is believed that a tertiary amine compound reacts with a condensing agent to produce an acylammonium cation (compound 5 in Scheme 1 below).

According to Makromol. Chem., Macromol. Symp. 1992, 54/55, 397-412, it has been reported that the following reaction is promoted by electrostatic interactions between ionic nucleophiles (e.g., hydroxide ions, phenoxide anions) and ionic acylammonium cations:

Based on the above reports, it is believed that the reaction of the present invention proceeds via a similar reaction mechanism. First, a neutral tertiary amine compound reacts with a neutral compound to produce an ionic acylammonium cation, a highly reactive species. This ionic acylammonium cation readily reacts with an ionic carboxylate. The reason why the method of the present invention can easily improve the yield of cyclic amides and cyclic peptides is that the acylammonium cation obtained by the reaction of a condensing agent with a tertiary amine compound selectively activates the C-terminus of the cyclic amide precursor, and the subsequent cyclization reaction also proceeds rapidly. The method of the present invention can be applied to a variety of substrates because it allows the reaction to proceed under near-neutral conditions in a short time. Furthermore, because the reaction proceeds via the above reaction mechanism, epimerization and dimerization associated with cyclization can be suppressed, and preferably, epimerization and dimerization associated with cyclization can be substantially avoided.

Regarding the above reaction mechanism, only Brunelle et al. mentioned the possibility of the above-mentioned ionic interactions in their research on the synthesis of cyclic carbonates, and no other reports have been published to date. This is the first time that the reaction process proceeds according to the above reaction mechanism has been discovered in the case of amidation.

The following examples and comparative examples will be presented to further clarify the features of the present invention, but the present invention is not limited to the following examples.

[Example 1]
Examination of tertiary amine compounds and solvents (model substrates)
The method for producing cyclic amides of the present invention proceeds in two steps: (i) activation of the C-terminus of the cyclic amide precursor and (ii) cyclocondensation. Therefore, to explore conditions that enable selective activation of the C-terminus of the cyclic amide precursor, we first investigated using model substrates with simple structures as models for each terminal. The C-terminal model substrate, potassium salt 3-phenylpropionic acid 1 (0.100 M, 1.0 molar equivalent), the N-terminal model substrate, 2-phenylethylamine 2 (0.100 M, 1.0 molar equivalent), and a tertiary amine compound (0.100 M, 1.0 molar equivalent) were dissolved in a mixed solvent of solvent X and water, and isopropyl chloroformate (0.050 M, 1.0 molar equivalent) was dissolved in solvent Y. A mixed solvent solution of potassium salt of 3-phenylpropionic acid 1 and 2-phenylethylamine 2 was injected into a V-shaped mixer at a flow rate of 2.40 mL/min, and a solvent Y solution of isopropyl chloroformate was injected at a flow rate of 4.80 mL/min, and the reaction was allowed to proceed at 60°C for 30 seconds (Table 1). The reaction was then terminated by injecting the reaction mixture and an excess of solvent Y solution of hexylamine 7 (1.000 M, 10.00 molar equivalents) into a T-shaped mixer. The product yield was determined by HPLC-UV analysis. The formation of amide 3 indicates the desired activation of the C-terminus, while the formation of carbamate 4 indicates the undesired reaction with the N-terminus. No reaction product between potassium salt of 3-phenylpropionic acid 1 and hexylamine 7 was detected.

The reaction selectivity (3/4) (hereinafter referred to as "C-terminal selectivity") between amide 3 and carbamate 4 was high when acetone, 1,4-dioxane, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), and dimethylformamide (DMF) were used (Table 1, Examples 1-1 to 1-5). In the following examples, we investigated the base using acetonitrile (Example 1-9), which has high solubility in a wide range of substrates, a low risk of side reactions with the side chain functional groups of the cyclic amide precursor, a relatively low boiling point for easy removal, and is environmentally friendly.

Without a base, the undesired carbamate 4 was obtained in 77% yield, with no desired amide 3 at all (Comparative Example 1-19). When heterocyclic amines such as pyridine and 4-dimethylaminopyridine (DMAP) were used, little or no desired amide 3 was obtained. Interestingly, however, the use of a tertiary alkylamine containing two or more methyl groups improved C-terminal selectivity (Examples 1-6 to 1-9). In particular, when dimethylbenzylamine (Me ₂ NBn) was used, the yield of the desired amide 3 was approximately three times higher than the yield of carbamate 4. The use of a tertiary alkylamine containing one methyl group reduced C-terminal selectivity (Examples 1-11 to 1-14), and the use of a bulky tertiary alkylamine without any methyl groups yielded almost no desired amide 3 (Comparative Examples 1-17 and 1-18). From these results, we speculate that the nucleophilicity of the alkylamine, rather than its basicity, significantly affected the C-terminal selectivity of the reaction. In fact, diethylmethylamine (Et ₂ NMe) (pKaH = 10.3) and triethylamine (Et ₃ N) (pKaH = 10.8) have almost the same basicity, but the yields of the desired amide 3 are significantly different (21% and 0%, respectively). Interestingly, pyridine and 4-dimethylaminopyridine (DMAP) showed low C-terminal selectivity (Comparative Examples 1-15 and 1-16). The detailed reasons are currently unknown, but it is thought that the amount of acylpyridinium cation produced with pyridine is too small, and that the reactivity of the resulting cation with 4-dimethylaminopyridine (DMAP) is insufficient due to its stabilization.

N-phenethyl-3-phenylpropamide (Table 1, amide 3):
White solid, ¹ H NMR (400 MHz, CDCl ₃ ):δ7.35-7.14 (m, 8H), 7.14-7.05 (m, 2H), 5.45 (brs, 1H), 3.46 (td, J = 6.8 Hz, 2H), 2.93 (t, J = 8.0 Hz, 2H), 2.72 (t, J = 6.8 Hz, 2H), 2.41 (t, J = 6.8 Hz, 2H) ppm; ¹³ C NMR (100 MHz, CDCl ₃ ): δ172.1, 141.0, 139.0, 128.8, 128.7, 128.6, 128.5, 126.6, 126.3, 40.7, 38.6, 35.8, 31.8 ppm; LCMS: calcd for [C ₁₇ H ₁₉ NO+H] ⁺ 254.15, found 254.20.

Isopropyl phenethyl carbamate (Table 1, Carbamate 4):
White solid, ¹ H NMR (400 MHz, CDCl ₃ ):δ7.35-7.10 (m, 5H), 4.98-4.83 (m, 1H), 4.65 (brs, 1H), 3.41 (dt, J = 7.2 Hz, 2H), 2.80 (t, J = 6.8 Hz, 2H),1.20 (d, J = 6.4 Hz, 6H) ppm; ¹³ C NMR (100 MHz, CDCl ₃ ): δ156.3, 139.0, 128.9, 128.7, 126.5, 68.0, 42.1, 36.3, 22.3 ppm; LCMS: calcd for [C ₄₀ H ₄₃ N ₃ O ₆ +H] ⁺ 208.13 found 208.20.

[Example 2]
Ionic interaction studies (model substrates)
In Example 1, dimethylbenzylamine (Me ₂ NBn) was used to investigate the reactivity improvement achieved by the combination of the above-mentioned ionic electrophile and nucleophile (Table 2). Specifically, we added lithium chloride to the reaction of potassium 3-phenylpropionic acid 1 with 2-phenylethylamine 2 and isopropyl chloroformate under the same concentration, equivalents, temperature, and time conditions as those used for the base study in Table 1, and observed the change in C-terminal selectivity. If ionic interactions are important for improving C-terminal selectivity, we hypothesized that the addition of lithium chloride might inhibit these interactions and reduce C-terminal selectivity. As a result, we observed a dose-dependent decrease in C-terminal selectivity with the addition of lithium chloride. This suggests that ionic interactions between the electrophile and nucleophile likely play an important role in C-terminal selectivity. To our knowledge, only Brunelle et al. have mentioned the possibility of the above-mentioned ionic interactions in the synthetic studies of cyclic carbonates. However, there have been no other reports, and this is the first finding in amidation.

[Example 3]
Examination of substrate applicability range (model substrate)
To investigate the substrate applicability of the identified reaction conditions, model substrates 5 and 6, derived from amino acids more closely resembling the C- and N-terminal structures of cyclic peptides, were used for further investigation. Activation of the desired C-terminal model 5 yielded C-terminal products 7 and 8 (C-terminal product 8 was generated by the reaction of 5 with n-hexylamine (H N-n-Hex ₎ , a reaction terminator, in the slow amidation reaction between C-terminal model 5 and N-terminal model 6). Conversely, activation of N-terminal model 6 yielded the undesired N-terminal product 9. The C-terminal selectivity (7 + 8)/9 was calculated from the ratio of the combined yield of C-terminal products 7 and 8 to the yield of N-terminal product 9 (Table 2). Moderate to high C-terminal selectivity was observed in the reaction of C-terminal model 5a derived from glycine (Gly) with N-terminal models 6a and 6c derived from Gly and alanine (Ala) (Examples 3-1 and 3-3). As expected, high C-terminal selectivity was observed in the reaction of 5a with the N-terminal model 6d derived from the even more bulky N-methylalanine (MeAla), resulting in the production of a large amount of C-terminal product 7 (Example 3-4). On the other hand, the reaction of 5a with the N-terminal model 6b derived from sarcosine (Sar) resulted in decreased C-terminal selectivity (Example 3-2). This is thought to be due to the high nucleophilicity of the secondary amine moiety of Sar. Furthermore, unexpectedly high C-terminal selectivity was observed in the reaction of the C-terminal model 5b derived from Ala with the N-terminal model 6a derived from Gly (Example 3-5). This suggests that selective activation of the C-terminus is actually possible in cyclic peptide synthesis.

Benzyl (2-((2-(benzylamino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (Table 2, C-terminal product 7a):
White solid, ¹ H NMR (400 MHz, CDCl ₃ ): δ7.45-7.20 (m, 10H), 6.68 (brs, 1H), 6.47 (brs, 1H), 5.38 (brs, 1H), 5.05 (s, 2H), 4.44 (d, J = 6.0 Hz, ^13C NMR (100 MHz, DMSO): δ170.1, 169.3, 157.1, 139.8, 137.5,128.9, 128.8, 128.3, 128.3, 127.7, 127.3, 66.1, 44.2, 42.5, 31.2 ppm; LCMS: calcd for [C ₁₉ H ₂₁ N ₃ O ₄ +H] ⁺ 356.10 found 356.25.

Benzyl (2-(hexylamino)-2-oxoethyl)carbamate (Table 2, C-terminal product 8a):
White solid, ¹ H NMR (400 MHz, CDCl ₃ ): δ7.38-7.31 (m, 5H), 5,96 (brs, 1H), 5.41 (brs, 1H), 5.13 (s, 2H), 3.83 (d, J = 5.2 Hz, 2H), 3.24 (td, J = ¹³ C NMR (100 MHz, CDCl ₃ ): δ169.1, 156.8, 136.2, 128.6, 128.3, 128.1, 67.1, 44.6, 39.6, 31.5, 29.4, 26.6, 22.6, 14.1 ppm.; LCMS: calcd for [C ₁₆ H ₂₄ N ₂ O ₃ +H] ⁺ 293.18, found 293.25.

Isopropyl(2-(benzylamino)-2-oxoethyl)carbamate (Table 2, N-terminal product 9a):
White solid, ¹ H NMR (400 MHz, CDCl ₃ ): δ7.35-7.29 (m, 5H), 6.27 (brs, 1H), 5.14 (brs, 1H), 4.93-4.83 (m, 1H), 4.47 (d, J = 5.6 Hz, 2H), 3.87 (d, J = 6.0 Hz, 2H), 1.22 (d, J = 6.4 Hz, 6H) ppm; ¹³ C NMR (100 MHz, CDCl ₃ ): δ169.3, 156.7, 138.0, 128.8, 127.8, 127.7, 69.1, 44.7, 43.6, 22.2 ppm; HRMS (ESI): calcd for [C ₁₃ H ₁₈ N ₂ O ₃ +Na] ⁺ 273.1210, found 273.1156.

Benzyl (2-((2-(benzylamino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)carbamate (Table 2, C-terminal product 7b): Rotamers were observed. The following is the major isomer.
White solid, ¹ H NMR (400 MHz, CDCl ₃ ): δ7.35-7.24 (m, 10H), 6.38 (brs, 1H), 5.65 (brs, 1H), 5.09 (s, 2H), 4.43 (d, J = 5.6 Hz, 2H), 4.06 (s, 4H), 3.10 (s, 3H) ppm; ¹³ C NMR (100 MHz, CDCl ₃ /CD ₃ OD): δ169.8, 168.7, 157.0, 137.8, 136.1, 128.2, 128.1, 127.7, 127.5,127.1, 126.9, 66.6, 51.1, 42.8, 42.0, 35.2 ppm; HRMS (ESI): calcd for [C ₂₀ H ₂₃ N ₃ O ₄ +Na] ⁺ 392.1581, found 392.1579.

Isopropyl(2-(benzylamino)-2-oxoethyl)(methyl)carbamate (Table 2, N-terminal product 9b): Rotamers were observed. The following is the major isomer.
White solid, ¹ H NMR (400 MHz, CDCl ₃ ): δ7.38-7.18 (m, 5H), 6.47 (brs, 1H), 4.96-4.80 (m, 1H), 4.48 (d, J = 6.0 Hz, 2H), 3.95 (s, 2H), 2.98 (s, ^HRMS _​ (ESI): calcd for [C ₁₄ H ₂₀ N ₂ O ₃ +Na] ⁺ 287.1366 found 287.1338.

Benzyl (S)-(2-((1-(benzylamino)-1-oxopropan-2-yl)amino)-2-oxoethyl)carbamate (Table 2, C-terminal product 7c)
White solid; ¹ H NMR (400 MHz, CD ₃ OD): δ7.34-7.22 (m, 10H), 5.03 (s, 2H), 4.44-4.37 (m, 3H), 3.79 (s 2H), 1.36 (d, J = 7.6 Hz, 3H) ppm; ¹³ C NMR (100 MHz, DMSO): δ172.0, 168.7, 156.5, 139.3, 137.0, 128.4, 128.3, 127.8, 127.7, 127.0, 126.7, 65.5, 48.1, 43.5, 41.8, 18.3 ppm; HRMS (ESI): calcd for [C ₂₀ H ₂₃ N ₃ O ₄ +Na] ⁺ 392.1581, found 392.1469.

Isopropyl (S)-(1-(benzylamino)-1-oxopropan-2-yl)carbamate (Table 2, N-terminal product 9c):
White solid; ¹ H NMR (400 MHz, CDCl ₃ ): δ7.38-7.18 (m, 5H), 6.43 (brs, 1H), 5.07 (brs, 1H), 4.95-4.78 (m, 1H), 4.45 (d, J = 6.0 Hz, 2H), ¹³ C NMR (100 MHz, CDCl ₃ ): δ172.5, 156.0, 138.1, 128.8, 127.7, 127.6, 68.9, 50.5, 43.6, 31.1, 22.2, 18.7 ppm; HRMS (ESI): calcd for [C ₁₄ H ₂₀ N ₂ O ₃ +Na] ⁺ 287.1366 found 287.1338.

Benzyl (S)-(2-((1-(benzylamino)-1-oxopropan-2-yl)(methyl)amino)-2-oxoethyl)carbamate (Table 2, C-terminal product 7d): Rotamers were observed. The following is the major isomer.
White solid; ¹ H NMR (400 MHz, CDCl ₃ ): δ7.40-7.15 (m, 10H), 6.54 (brs, 1H), 5.73 (brs, 1H), 5.20-5.05 (m, 1H), 5.08 (s, 2H), 4.46-4.28 (m, ¹³ C NMR (100 MHz, CDCl ₃ ): δ170.4, 169.4, 156.3, 138.3, 136.3, 128.6, 128.5, 128.2, 128.0, 127.5, 127.4, 66.9, 52.3, 43.3, 42.9, 29.6, 13.7 ppm; HRMS (ESI): calcd for [C ₂₁ H ₂₅ N ₃ O ₄ +Na] ⁺ 406.1737, found 406.1737.

Isopropyl (S)-(1-(benzylamino)-1-oxopropan-2-yl)(methyl)carbamate (Table 2, N-terminal product 9d): Rotamers were observed. The following is the major isomer. Colorless oil, ¹ H NMR (400 MHz, CDCl ₃ ): δ7.38-7.20 (m, 5H), 6.49 (brs, 1H), 4.95-4.86 (m, 1H), 4.77 (brs, 1H), 4.48 (dd, J = 5.6, 14.8 Hz, 1H), ¹³ C NMR (100 MHz, CDCl ₃ ): δ171.4, 156.7, 138.3, 128.5, 127.4, 126.4, 69.4, 53.8, 43.3, 29.3, 22.1, 13.8 ppm; HRMS (ESI): calcd for [C ₁₅ H ₂₂ N ₂ O ₃ +Na] ⁺ 301.1523, found 301.1522.

Benzyl (S)-(1-((2-(benzylamino)-2-oxoethyl)amino)-1-oxopropan-2-yl)carbamate (Table 2, C-terminal product 7e):
White solid, ¹ H NMR (400 MHz, CDCl ₃ ): δ7.40-7.17 (m, 10H), 7.02 (brs, 2H), 5.50 (d, J = 5.6 Hz 1H), 4.98 (d, J = 12.0 Hz, 1H), 4.87 (d, J = 12.0, 1H), 4.50-4.30 (m, 2H), 4.25-4.10 (m, 1H), 3.98 (dd, J = 4.4, 16.0 Hz, 1H), 3.88 (dd, J = 4.4, 16.0 Hz, 1H), 1.33 (d, J = 6.8 Hz, 3H) ppm; ^13C NMR (100 MHz, CDCl ₃ ): δ173.3, 168.8, 156.4, 138.0, 136.1, 128.7, 128.7, 128.4, 128.2, 127.9, 127.6, 67.2, 51.1, 43.5, 43.3, 18.2 ppm; LCMS: calcd for [C ₂₀ H ₂₃ N ₃ O ₄ +H] ⁺ 370.17 found 370.25.

Benzyl (1-(hexylamino)-1-oxopropan-2-yl)carbamate (Table 2, C-terminal product 8b):
White solid, ¹ H NMR (400 MHz, CDCl ₃ ): δ7.40-7.23 (m, 5H), 6.08 (brs, 1H), 5.37 (brs, 1H), 5.10 (s, 2H), 4.27-4.10 (m, 1H), 3.22 (t, J = 6.4 Hz, ¹³ C NMR (100 MHz, CDCl ₃ ): δ172.3, 156.1, 136.3, 128.6, 128.3, 128.0, 67.0, 50.6, 39.6, 31.5, 29.5, 26.6, 22.6, 18.9, 14.1 ppm; LCMS: calcd for [C ₁₇ H ₂₆ N ₂ O ₃ +Na] ⁺ 307.19 found 307.30.

[Example 4]
Synthesis of Cyclic Peptides (Protected Stellarin G)
The reaction conditions for cyclic peptide synthesis were investigated (Table 4). Stellarin G, a five-residue cyclic peptide known as a Chinese medicinal herb, was selected as a model substrate for cyclic peptides. Cyclic peptide precursor 21 (H-L-Ala-L-Tyr(OBn)-L-Leu-L-Ala-Gly-OH·HCl, 0.010 M, 1.0 molar equivalent) consisting of the amino acid sequence represented by SEQ ID NO: 1, potassium hydroxide (0.020 M, 2.0 molar equivalents), and a tertiary amine compound (1.0 molar equivalent) in a 1:1 mixture of acetonitrile and water (flow rate: 2.40 mL/min), and an acetonitrile solution of isopropyl chloroformate 6 (1.0 molar equivalent) (flow rate: 4.80 mL/min) were introduced into a V-shaped mixer and reacted at 60°C for 30 seconds. The yield of cyclic peptide 11 (protected Stellarin G; L-Ala-L-Tyr(OBn)-L-Leu-L-Ala-Gly) consisting of the amino acid sequence represented by SEQ ID NO: 1 was calculated by HPLC-UV analysis. When 1.0 molar equivalent of dimethylbenzylamine (Me ₂ NBn) was used, the yield was moderate at 62%, but by adding an excess of 2.0 molar equivalents, a high yield of 99% was successfully obtained (Table 4, Examples 4-1 and 4-2). Furthermore, even when the equivalent amount of base was increased, cyclic peptide 11 was successfully obtained quantitatively (Table 4, Example 4-3).

To confirm the effect of the base Me ₂ NBn, the reaction was examined using diisopropylethylamine (i-Pr ₂ NEt), which did not show C-terminal selectivity in the study in Table 1. As expected, no cyclic peptide was obtained (Table 4, Comparative Example 4-4).

When a similar cyclic peptide 11 and its precursor 21 were examined in a batch (stirring speed: 1000 rpm) under conditions that allowed the production of cyclic peptides in high yield in a microflow reactor (Table 4, Example 4-3), the HPLC-UV yield was 99%.

From the above, it is believed that the cyclization in the method of the present invention proceeds via a reaction mechanism similar to that when the model substrate described above is used. The highly reactive species formed from dimethylbenzylamine and isopropyl chloroformate are extremely reactive and can rapidly activate the C-terminus of the cyclic peptide precursor. The subsequent reaction is also rapid, which is likely why cyclic peptide 11 was successfully obtained in high yield.

Protection Stellarin G:
White solid, 1H NMR (400 MHz, DMSO): δ 8.45-8.31 (m, 3H), 8.17 (d, J = 5.6 Hz, 1H), 8.08 (brs, 1H ), 7.48-7.25 (m, 5H), 7.11 (d, J = 7.6 Hz, 2H), 6.88 (d, J = 7.6 Hz, 2H), 5.05 (s, 2H), 4.35 (q, J = 6.8 Hz, 1H), 4.30-4.15 (m, 1H), 4.13-4.03 (m, 2H), 3.95-3.85 (m, 1H), 3.50-3.40 (m, 1H), 2.93 (d, J = 5.6 Hz, 2H), 1.70-1.60 (m, 1H), 1.45-1.30 (m, 2H), 1.22 (d, J = 6.8 Hz, 3H), 1.12 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 5.6 Hz, 3H), 0.78 (d, J = 5.6 Hz, 3H) ppm; 13C NMR (100 MHz, DMSO): δ172.4, 171.9, 171.6, 170.5, 168.9, 157.0, 137.2, 130.1, 129.5 128.4, 127.8, 127.6, 114.4, 69.1, 55.8, 53.9, 49.8, 48.4, 43.2, 36.0, 24.3, 22.9, 21.4, 17.3 ppm; HRMS (ESI):calcd for [C ₃₀ H ₃₉ N ₅ O ₆ +H] ⁺ 588.2793, found 588.2792.

[Example 5]
Synthesis of cyclic peptides (Stellarin G analogues)
To investigate whether the present invention can be applied to different cyclic amides and cyclic peptides, a Stellarin G analog was selected as a model substrate for cyclic amides and cyclic peptides. Specifically, the tyrosine (Tyr) residue of the protected Stellarin G in Example 4 was replaced with a phenylalanine (Phe) residue, and the alanine (Ala) residue was replaced with a tryptophan (Trp) residue to prepare a cyclic peptide as follows. A cyclic peptide precursor 22 (H-L-Trp-L-Phe-L-Leu-L-Ala-Gly-OH.TFA, 0.010 M, 1.0 molar equivalent) consisting of the amino acid sequence represented by SEQ ID NO: ₂ , potassium hydroxide (0.020 M, 2.0 molar equivalents), and a 1:1 mixture of acetonitrile and water (flow rate 2.40 mL/min) of dimethylbenzylamine (Me NBn, 0.020 M, 2.0 molar equivalents) and an acetonitrile solution of isopropyl chloroformate (0.020 M, 2.0 molar equivalents) (flow rate 4.80 mL/min) were introduced into a V-shaped mixer at 60°C using a syringe pump. The reaction mixture was introduced into a reaction tube (inner diameter 0.8 mm, length 2388 mm, volume 1200 μL, reaction time 10 seconds) at the same temperature. After reaching a steady state over 60 seconds, the reaction mixture was poured into a test tube at room temperature for 240 seconds. After stirring for 2 minutes, the mixture was mixed with 2 mL of ethyl acetate (EtOAc). The organic layer was washed with 1 M aqueous HCl and saturated brine, dried over magnesium sulfate ( _MgSO ), filtered, and concentrated under vacuum at room temperature. The mixture was then purified by recrystallization from dichloromethane (CH ₂ Cl ₂ )/hexane. The isolated yield of cyclic peptide 12 (Stellarin G analog; L-Trp-L-Phe-L-Leu-L-Ala-Gly) consisting of the amino acid sequence represented by SEQ ID NO: 2 was 86%.

Stellarin G analogues:
White solid, mp: > 245 ℃ (decomposed and change to black solid); IR (neat): 3730, 3058, 2954, 1651, 1534, 740 cm ^-1 ; [α] ²⁵ _D = -130.7 (c 0.12, MeOH); ¹ H NMR (400 MHz, CD ₃ OD): δ 7.62 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.25-7.00 (m, 8H), 4.61 (dd, J = 6.8 Hz, 1H), 4.30-4.20 (m, 2H), 4.13 (d, J = 14.8 Hz, 1H), 4.02 (dd, J = 10.8, 4.8 Hz, 1H), 3.37 (d, J = 14.8 Hz, 1H), 3.24 (dd, J = 15.2, 6.8 Hz, 1H), 3.07 (dd, J = 15.2, 8.0 Hz, 1H), 3.03 (d, J = 9.2 Hz, 2H), 1.89-1.80 (m, 1H), 1.55-1.45 (m, 1H), 1.48 (d, J = 4.8 Hz, 3H), 1.33-1.30 (m, 1H), 0.89 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H) ppm; ^13C NMR (100MHz, CD ₃ OD): δ 174.9, 174.2, 173.6, 172.1, 138.3, 138.1, 130.2, 129.5, 128.6, 127.7, 124.3, 122.5, 119.8, 119.4, 112.3, 110.9, 58.7, 56.5, 56.0, 50.6, 44.6, 40.9, 37.4, 28.2, 25.8, 23.5, 21.6, 17.3 ppm; HRMS (ESI): calcd for [C ₃₁ H ₃₈ N ₆ O ₅ +Na] ⁺ 597.2796, found 597.2796.

[Example 6]
Synthesis of Cyclic Peptides (Protected Dianthin I)
Next, to examine whether the present invention can be applied to other cyclic amides and cyclic peptides, Dianthin I, a five-residue cyclic peptide known as an anticancer drug, was selected as a model substrate for cyclic amides and cyclic peptides. A cyclic peptide precursor 23 (H-L-Phe-L-Pro-L-Ser(OBn)-L-Phe-Gly-OH·TFA, 0.01 M, 1.0 molar equivalent) consisting of the amino acid sequence represented by SEQ ID NO: 3, potassium hydroxide (0.02 M, 2.0 molar equivalents), and dimethylbenzylamine (Me ₂ NBn, 0.03 M, 3 molar equivalents) in a 1:1 mixture of acetonitrile and water (flow rate: 2.40 mL/min), and an acetonitrile solution of isopropyl chloroformate (0.015 M, 3 molar equivalents) (flow rate: 4.80 mL/min) were introduced into a V-shaped mixer at 60°C using a syringe pump. The reaction mixture was introduced into a reaction tube (inner diameter 0.8 mm, length 2388 mm, volume 1200 μL, reaction time 10 seconds) at the same temperature. After reaching a steady state over 30 seconds, the reaction mixture was poured into a test tube at room temperature for 120 seconds. After stirring for 2 minutes, the mixture was mixed with 2 mL of ethyl acetate (EtOAc). The organic layer was washed with 1 M aqueous HCl and saturated brine, dried over magnesium sulfate (MgSO ₄ ), filtered, and concentrated under vacuum at room temperature. The mixture was then purified by gel permeation chromatography (GPC). The isolated yield of cyclic peptide 13 (protected Dianthin I; L-Phe-L-Pro-L-Ser(OBn)-L-Phe-Gly) consisting of the amino acid sequence represented by SEQ ID NO: 3 was 72%.

Protection Dianthin I:
21.6 mg, 0.034 mmol, 72%.
White solid, ¹ H NMR (400 MHz, CDCl3): (broad NMR signals were observed probably due to existence of conformational isomers) δ 7.99-7.80 (m, 1H), 7.70-7.55 (m, 1H), 7.40-7.00 (m, 17H), 4.85-4.60 (m, 2H), 4.48-4.23 (m, 3H), 4.20-4.00 (m, 1H), 3.80-3.75 (m, 1H), 3.65-3.45 (m, 3H), 3.41-3.21 (m, 4H), 3.09-2.95 (m, 2H), 1.89-1.20 (m, 4H) ppm; ^13C NMR (100 MHz, CDCl3): δ 172.4, 171.0, 170.4, 169.8, 169.7, 138.3, 137,4, 136.4, 129.5, 129.4, 128.7, 128.5,128.5, 128.0, 127.8, HRMS (ESI): calcd for [C ₃₅ H ₃₉ N ₅ O ₆ +H] ⁺ 648.2793, found 648.2796.

[Example 7]
Synthesis of Cyclic Peptides (Dianthin I)
We investigated whether the present invention can be applied to unprotected cyclic amides and cyclic peptides. The cyclic peptide Dianthin I was produced in the same manner as in Example 6, except that the protecting groups of the amino acid residues were removed, i.e., the benzyl protecting groups were replaced with hydrogen atoms. A cyclic peptide precursor 24 (H-L-Phe-L-Pro-L-Ser-L-Phe-Gly-OH.TFA, 0.010 M, 1.0 molar equivalent) consisting of the amino acid sequence represented by SEQ ID NO: 4, potassium hydroxide (0.020 M, 2.0 molar equivalents), and dimethylbenzylamine (Me ₂ NBn, 0.030 M, 3.0 molar equivalents) in a 1:1 mixture of acetonitrile and water (flow rate 2.40 mL/min), and an acetonitrile solution of isopropyl chloroformate (0.015 M, 3.0 molar equivalents) (flow rate 4.80 mL/min) were introduced into a V-shaped mixer at 80°C using a syringe pump. The reaction mixture was introduced into a reaction tube (inner diameter 0.8 mm, length 2388 mm, volume 1200 μL, reaction time 10 seconds) at the same temperature. After reaching a steady state over 30 seconds, the reaction mixture was poured into a test tube at room temperature for 480 seconds. The reaction mixture was mixed with 5 mL of 1 M aqueous HCl and stirred for 2 minutes. The mixture was poured into 50 mL of ethyl acetate (EtOAc). The organic layer was washed with 1 M aqueous HCl and saturated brine, dried over magnesium sulfate (MgSO ₄ ), filtered, and concentrated under vacuum at room temperature. The mixture was then purified by recrystallization from dichloromethane (CH ₂ Cl ₂ )/hexane. The isolated yield of cyclic peptide 14 (Dianthin I; L-Phe-L-Pro-L-Ser-L-Phe-Gly) consisting of the amino acid sequence represented by SEQ ID NO:4 was 62%. Thus, the present invention can also be applied to cyclic amides and cyclic peptide precursors without protecting groups.

Dianthin I (J. Nat. Prod. 2016, 79, 1769-1774.):
White solid,¹H NMR (400 MHz, pyridine-d₅): δ 10.82 (d, J = 7.2 Hz, 1H), 8.66 (d, J = 8.8 Hz, 1H), 8.60 (d, J = 6.8 Hz, 1H), 8.15 (d, J = 7.6 Hz, 1H), 7.50-7.35 (m, 5H), 7.32-7.17 (m, 5H), 5.39 (dd, J = 14.4, 8.4 Hz 1H), 5.13-5.00 (m, 1H), 4.84 (dd, J = 16.0, 9.2 Hz, 1H), 4.46 (d, J = 8.4 Hz, 1H), 4.35-4.29 (m, 1H), 4.28-4.21 (m, 2H), 4.00-3.85 (m, 2H), 3.82-3.65 (m, 3H), 3.50-3.40 (m, 2H), 2.13-2.03 (m, 1H), 1.85-1.70 (m, 2H), 1.55-1.50 (m, 1H) ppm.¹³C NMR (100 MHz, pyridine-d₅): δ 175.5, 173.7, 171.2, 171.0, 170.7, 140.0, 138.2, 130.0, 129.9, 128.7, 128.5, 126.7, 126.4, 61.4, 61.3, 57.9, 57.5, 55.6, 48.6, 43.3, 39.8, 35.1, 32.0, 21.6 ppm; HRMS (ESI): calcd for [C₂₈H₃₃N₅O₆+Na]⁺558.2323, found 558.2329.

[Example 8]
Synthesis of Cyclic Peptides (Protected Heterophyllin J)
Furthermore, to examine whether the present invention can be applied to other cyclic amides and cyclic peptides, Heterophyllin J, a five-residue cyclic peptide known as a traditional Chinese medicine for diuresis and torticollis, was selected as a model substrate for cyclic amides and cyclic peptides. A mixture of cyclic peptide precursor 25 (H-L-Pro-L-Val-L-Tyr(OBn)-L-Ala-Gly-OH·TFA, 0.010 M, 1.0 molar equivalent) consisting of the amino acid sequence represented by SEQ ID NO: 5, potassium hydroxide (0.020 M, 2.0 molar equivalents), and dimethylbenzylamine (Me ₂ NBn, 0.020 M, 2 molar equivalents) in acetonitrile and water (flow rate: 2.40 mL/min) and a solution of isopropyl chloroformate (0.010 M, 2 molar equivalents) in acetonitrile (flow rate: 4.80 mL/min) were introduced into a V-shaped mixer at 60°C using a syringe pump. The reaction mixture was introduced into a reaction tube (inner diameter 0.8 mm, length 2388 mm, volume 1200 μL, reaction time 10 seconds) at the same temperature. After 30 seconds to reach a steady state, the reaction mixture was poured into a test tube at room temperature for 150 seconds. After stirring for 2 minutes, the mixture was mixed with 2 mL of ethyl acetate (EtOAc). The organic layer was washed with 1 M aqueous HCl and saturated brine, dried over magnesium sulfate ( _MgSO ), filtered, and concentrated under vacuum at room temperature. The mixture was then purified by recrystallization from dichloromethane (CH ₂ Cl ₂ )/hexane. The isolated yield of cyclic peptide 15 (protected Heterophyllin J; L-Pro-L-Val-L-Tyr(OBn)-L-Ala-Gly) consisting of the amino acid sequence represented by SEQ ID NO:5 was 74%.

Protection Heterophyllin J:
20.5 mg, 0.035 mmol, 74%.
White solid, ¹ H NMR (400 MHz, CDCl3): δ 8.21 (d, J = 6.4 Hz, 1H ), 7.84 (brs, 1H), 7.54 (d, J = 8.8 Hz, 1H ), 7.45-7.28 (m, 5H), 7.12-7.05 (m, 3H), 6.84 (d, J = 8.4 Hz, 2H ), 4.96 (s, 2H), 4.55-4.48 (m, 1H),4.42-4.31 (m, 2H), 4.22 (dd, J = 5.2, 2.8 Hz, 1H), 4.111 (t, J = 8.8 Hz, 1H), 4.02-3.95 (m, 1H), 3.65-3.45 (m, 2H), 3.20 (dd, J = 8.8, 8.4 Hz, 1H), 3.11 (dd, J = 7.6, 6.4 Hz, 1H), 2.31-2.22 (m, 1H), 2.20-2.03 (m, 2H), 2.02-1.93 (m, 2H), 1.33 (d, J = 6.8 Hz, 3H), 0.84 (d, J = 2.8 Hz, 6H) ppm; ¹³ C NMR (100 MHz, CDCl3): δ 173.6, 172.4,172.4, 172.3, 168.7, 157.8, 137.2, 130.5, 129.1, 128.7, 128.0, 127.6, 114.9, 70.1, 62.6, 60.0, 57.9, 49.8, 47.1, 42.2, 36.4, 30.0, 29.8, 25.2, 19.5, 18.8, 16.1 ppm. (ESI): calcd for [C ₃₁ H ₃₉ N ₅ O ₆ +H] ⁺ 600.2793, found 600.2796.

The RME (Reaction Mass Efficiency) of the present invention is higher than that of conventional technology. RME is calculated using the following formula:

[Example 9]
Synthesis of Cyclic Peptides (Versicotide D Analogues and Their Epimers)
We investigated whether the present invention can be applied to cyclic amide precursors and cyclic peptide precursors in which the free amino group is replaced with a methyl group. A five-residue cyclic peptide, a Versicotide D analog, was selected as a model substrate for cyclic amides and cyclic peptides. A solution of cyclic peptide precursor 26 (H-L-MeAla-L-Phe-L-MeAla-L-Phe-L-Ala-OH·TFA, 0.010 M, 1.0 molar equivalent) consisting of the amino acid sequence represented by SEQ ID NO:6, dimethylbenzylamine (Me ₂ NBn, 0.020 M, 2.0 molar equivalents) in acetonitrile (flow rate: 2.40 mL/min), and a solution of isopropyl chloroformate (0.010 M, 2.0 molar equivalents) in acetonitrile (flow rate: 4.80 mL/min) were introduced into a V-shaped mixer at 60°C using a syringe pump. The reaction mixture was introduced into a reaction tube (inner diameter 0.8 mm, length 2388 mm, volume 1200 μL, reaction time 10 seconds) at the same temperature. After reaching a steady state over 30 seconds, the reaction mixture was poured into a test tube at room temperature for 180 seconds. The reaction mixture was mixed with 5 mL of 1 M aqueous HCl and stirred for 2 minutes. The mixture was poured into 50 mL of ethyl acetate (EtOAc). The organic layer was washed with 1 M aqueous HCl and saturated brine, dried over magnesium sulfate (MgSO ₄ ), filtered, and concentrated under vacuum at room temperature. The mixture was then purified by GPC. The isolated yield of cyclic peptide 16 (Versicotide D analog; L-MeAla-L-Phe-L-MeAla-L-Phe-L-Ala) consisting of the amino acid sequence represented by SEQ ID NO:6 was 41% (Table 5, Example 9-1).

Conventionally, it has been common knowledge that linear peptides with an N-methyl amino acid at the N-terminus are not suitable for cyclization reactions because bulky amino groups have low nucleophilicity. However, by using the method of the present invention, it is possible to cyclize linear peptides with an N-methyl amino acid at the N-terminus.

Versicotide D analogues:
White solid, mp: 102-106℃; IR (neat): 3298, 2933, 1632, 1534, 1454, 1287, 1121, 701 cm ^-1 ; [α] ²⁶ _D = -69.0 (c 0.13, CHCl ₃ ); ¹ H NMR (400 MHz, CDCl _{3 )} ): δ 8.18 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 7.6 Hz, 1H), 7.40-7.12 (m, 10H), 5.85 (d, J = 6.4 Hz, 1H), 4.98-4.85 (m, 2H), 4.52 (d, J = 4.4Hz, 1H), 3.75-3.65 (m, 1H), 3.66-3.46 (m, 1H), 3.40-3.33 (m, 1H), 3.27-3.15 (m, 1H), 3.12-3.05 (m, 1H), 3.01 (s, 3H), 2.95-2.85 (m, 1H), 2.66 (s, 3H), 1.78 (d, J = 8.0 Hz, 3H), 1.29 (d, J = 6.0 Hz, 3H), 1.19 (d, J = 6.0 Hz, 3H) ppm; ¹³ C NMR (100 MHz, CDCl ₃ ): δ 173.1, 173.0, 172.7, 170.0, 169.8, 136.21, 136.15, 129.6, 129.3, 128.7, 127.7, 127.4, 67.5, 62.0, 54.3, 50.8, 47.0, 39.3, 38.8, 38.3, 37.2, 17.5, 16.4, 12.7 ppm; HRMS (ESI): calcd for [C ₂₉ H ₃₇ N ₅ O ₅ +Na] ⁺ 558.2687, found 558.2706.

We investigated whether the present invention can suppress epimerization. The epimer of the Versicotide D analog produced in Example 9-1 was selected as a model substrate for cyclic amides and cyclic peptides. Specifically, cyclic peptide 17 was produced in the same manner as in Example 9-1, except that cyclic peptide 27 (H-L-MeAla-L-Phe-L-MeAla-L-Phe-D-Ala-OH·TFA), an epimer of cyclic peptide precursor 26, was used. The isolation yield of cyclic peptide 17 (Versicotide D analog epimer; L-MeAla-L-Phe-L-MeAla-L-Phe-D-Ala), consisting of the amino acid sequence represented by SEQ ID NO: 7, was 47% (Table 5, Example 9-2).

Versicotide D analog epimer (Tetrahedron Lett. 2019, 60, 151281.):
White solid, mp: 105-109 ℃; IR (neat): 3293, 2933, 1651, 1523, 1454, 1538, 1454, 1123, 700 cm ^-1 ; [α] ²⁵ _D = -107.8 (c 0.13, CHCl ₃ ); ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.78 (d, J = 9.6 Hz, 1H), 7.59 (d, J = 5.6 Hz, 1H), 7.42-7.16 (m, 10H), 6.91 (d, J = 8.4 Hz, 1H), 4.98-4.85 (m, 2H), 4.70 (q, J = 7.2 Hz, 1H), 4.67-4.58 (m, 1H), 3.38 (q, J = 7.2 Hz, 1H), 3.22-3.12 (m, 4H), 3.10-2.93 (m, 3H), 2.75 (s, 3H), 1.59 (d, J = 7.2 Hz, 3H), 1.30 (d, J = 7.2 Hz, 3H), 1.21 (d, J = 6.4 Hz, 3H) ppm; ¹³ C NMR (100 MHz, CDCl ₃ ): δ 174.3, 172.5, 172.0, 171.8, 170.4, 137.1, 136.9, 129.9, 129.3, 128.4, 128.3, 126.8, 126.7 _, 66.0, 54.2, 54.1, 51.9, 46.0, 38.8, 38.5, 37.7, 30.9, 16.5, 15.3, _14.7 ppm. ₅ O ₅ +Na] ⁺ 558.2687, found 558.2702.

The D:L ratios of Examples 9-1 and 9-2 (Versicotide D analogs and their epimers) were evaluated by HPLC-UV analysis (column: GLscience Inertsil™ ODS-3 5 μm, 4.6 mm × 75 mm, solvent: methanol + 0.1% formic acid/H ₂ O + 0.1% formic acid (0-13 min: 0-100%, 13-16 min: 100%, 16-17 min: 0%, 17-22 min: 0%), flow rate: 1.0 mL/min, detection wavelength: 254 nm, temperature: 40°C). The results are shown in Table 5 and Figure 2. The retention time of cyclic peptide 16 was 11.8 min (Figure 2a), and the retention time of its epimer, cyclic peptide 17, was 12.2 min (Figure 2b). As shown in FIG. 2 , in Example 9-1, only cyclic peptide 16 (L-form) corresponding to cyclic peptide precursor 26 was obtained, and in Example 9-2, only cyclic peptide 17 (D-form) corresponding to cyclic peptide precursor 27 was obtained. As such, the present invention can substantially suppress epimerization during cyclization. As a result, in the present invention, it is possible to selectively produce optical isomers by selecting the corresponding cyclic amide precursor and cyclic peptide precursor. In the present invention, for example, a cyclic peptide substantially free of D-amino acids can be synthesized.

Cyclic peptide 16 (Versicotide D analog) was produced (Example 9-3) in the same manner as in Example 9-1, except that the reaction temperature was changed to 20°C. The present invention enables cyclization even at relatively low reaction temperatures, without detectable epimerization (Table 5).

[Comparative Example 9]
Synthesis of Cyclic Peptides by Conventional Techniques (Versicotide D Analogues and Their Epimers)
reported the first total synthesis of the Versicotide D analogue in Tetrahedron Lett. 2019, 60, 151281. The present applicant synthesized a Versicotide D analogue according to this report and investigated epimerization using conventional techniques. Specifically, macrocyclization was carried out in a batchwise manner in anhydrous acetonitrile at 20°C using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU, 1.5 molar equivalents), 4-dimethylaminopyridine (DMAP, 0.1 molar equivalents), and diisopropylethylamine (i-Pr ₂ NEt, 3 molar equivalents) in a dilute solution of 0.005 M (1.0 molar equivalent) of cyclic peptide precursor 26 (H-L-Ala(NMe)-L-Phe-L-Ala(NMe)-L-Phe-L-Ala-OH.TFA) (Table 5, Comparative Example 9-1). The reaction mixture was washed with 5% HCl, then with saturated aqueous sodium bicarbonate (NaHCO ₃ ), dried over magnesium sulfate (MgSO ₄ ), filtered, and concentrated under vacuum at room temperature. The NMR spectrum of the resulting cyclic peptide was consistent with that of the D-form of Versicotide D analog (corresponding to Example 9-2), which corresponds to cyclic peptide precursor 27, an epimer of cyclic peptide precursor 26. Furthermore, HPLC-UV analysis of the resulting cyclic peptide confirmed the results of the D-form of Versicotide D analog. Thus, the method of Posoda et al. revealed that cyclic peptide precursor 26 first underwent epimerization and then cyclization. Interestingly, the method of the present invention is able to avoid unwanted epimerization despite the use of a mixed acid anhydride with higher nucleophilicity.

A Versicotide D analog was produced by the method of Posoda et al. in the same manner as in Comparative Example 9-1, except that the reaction temperature was set to 60°C (Table 5, Comparative Example 9-2). A Versicotide D analog was produced by the method of Posoda et al. in the same manner as in Comparative Example 9-2, except that the solvent was changed to dichloromethane (Table 5, Comparative Example 9-3). As shown in Table 5, it was confirmed that the method of Posoda et al. produced the undesired D-form of Versicotide D analog in greater amounts than the desired L-form, regardless of the reaction temperature. Thus, epimerization was impossible to avoid with conventional technology.

[Example 10]
Synthesis of cyclic peptides (Dihydrotentoxin)
We investigated whether the present invention can be applied to four-residue cyclic peptides with large strains. We also investigated whether the present invention can suppress dimerization. Dihydrotentoxin, a four-residue cyclic peptide, was selected as a model substrate for cyclic peptides. A cyclic peptide precursor 28 (H-L-MeAla-L-Leu-L-MePhe-Gly-OH·TFA, 0.010 M, 1.00 molar equivalents) consisting of the amino acid sequence represented by SEQ ID NO: 8, diisopropylethylamine (i-Pr ₂ NEt, 0.020 M, 2.00 molar equivalents), and dimethylbenzylamine (Me ₂ NBn, 0.030 M, 3.00 molar equivalents) in acetonitrile (flow rate 2.40 mL/min), and an acetonitrile solution of isopropyl chloroformate (0.015 M, 3.00 molar equivalents) (flow rate 4.80 mL/min) were introduced into a V-shaped mixer at 60°C using a syringe pump. The reaction mixture was introduced into a reaction tube (inner diameter 0.8 mm, length 7164 mm, volume 3600 μL, reaction time 30 seconds) at the same temperature. After reaching a steady state over 30 seconds, the reaction mixture was poured into a test tube at room temperature for 120 seconds. 5 mL of 1 M HCl aqueous solution was added to the reaction mixture, stirred for 2 minutes, and then mixed with 50 mL of ethyl acetate (EtOAc). The organic layer was washed with 1 M HCl aqueous solution and saturated brine, dried over magnesium sulfate (MgSO ₄ ), filtered, and concentrated under vacuum at room temperature. The mixture was then purified by GPC. The isolated yield of cyclic peptide 18 (Dihydrotentoxin; L-MeAla-L-Leu-L-MePhe-Gly) consisting of the amino acid sequence represented by SEQ ID NO:8 was 50%. Thus, the present invention can also be applied to highly strained four-residue cyclic peptides.
Cyclic peptide 18 was also obtained by similar RME when 10 molar equivalents of cyclic peptide precursor 28 were used. Under any concentration conditions, no dimer of cyclic peptide precursor 28 was detected in the produced cyclic peptide 18. Thus, the present invention makes it possible to avoid dimerization associated with cyclization.

Dihydrotentoxin (Tetrahedron 2018, 74, 6173-6181):
White solid, ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.74 (d, J = 9.2 Hz, 1H), 7.35-7.15 (m, 5H), 4.94 (dd, J = 10.0, 5.2 Hz ,1H), 4.61 (dd, J = 7.6, 3.6 Hz, 1H), 4.26 (q, J = 6.8 Hz, 1H), 3.70 (d, J = 14.0 Hz, 1H), 3.50 (d, J = 14.8 Hz, 1H), 3.10-2.95 (m,1H), 2.84 (s, 3H), 2.78 (s, 3H), 1.68 (m, 1H), 1.55 (d, J = 6.8 ¹³ C NMR (100 MHz, CDCl ₃ ):δ 172.4, 172.0, 170.8, 170.2, 137.2, 129.1, 128.3, 127.2, 63.1, 57.4, 48.6, 44.6, 41.0, 34.4, 30.7, 30.2, 24.7, 22.7, 22.5, 15.7 ppm; HRMS (ESI): calcd for [C ₂₂ H ₃₂ N ₄ O ₄ +Na] ⁺ 439.2321, found 439.2324.

[Comparative Example 10]
Synthesis of cyclic peptides by conventional techniques (Dihydrotentoxin and its dimer)
reported the synthesis of the above-mentioned dihydrotentoxin by cyclization in Tetrahedron 2018, 74, 6173-6181. The present applicant synthesized dihydrotentoxin according to this report and investigated dimerization using conventional techniques. Specifically, chlorodiisopinocampheylborane (DIPCl, 3.2 molar equivalents), hydroxybenzotriazole (HOBt, 3.2 molar equivalents), and diisopropylethylamine (i-Pr 2 NEt, 4.0 molar equivalents) were added to an acetonitrile (MeCN) or dimethylformamide (DMF) solution of cyclic peptide precursor 28 (H-L-MeAla-L-Leu-L-MePhe-Gly-OH, 0.1 mM, 1.0 molar equivalent), and the mixture was stirred at ₂₂ °C or 30°C for 24 hours. The reaction mixture was purified by GPC. The yields of cyclic peptide 18 and cyclized dimer 19 contained in the residue were calculated by HPLC-UV analysis (column: GLscience Inertsil™ ODS-3 5 μm, 4.6 mm × 75 mm, solvent: methanol + 0.1% formic acid/H ₂ O + 0.1% formic acid (0-23 min: 0-70%, 23-23.5 min: 70-100%, 23.5-26 min: 100%, 26-26.5 min: 0%, 26.5-29 min: 0%), flow rate: 1.0 mL/min, detection wavelength: 254 nm, temperature: 40°C, retention time: 15.8 min (cyclic peptide 18; corresponding to Example 10) and 21.6 min (cyclized dimer 19)). The results are shown in Table 6 (Comparative Examples 10-1 to 10-4). As shown in Table 6, it was confirmed that the method of Sato et al. produced not only the target cyclized monomer 18 but also the cyclized dimer 19 (L-MeAla-L-Leu-L-MePhe-Gly-L-MeAla-L-Leu-L-MePhe-Gly) represented by SEQ ID NO: 9 as a by-product. As such, it was impossible to avoid dimerization with conventional techniques.

Dihydrotentoxin dimer:
White solid, mp 169-172 ℃; ¹ H NMR (400 MHz, CDCl ₃ ): δ 9.05 (d, J = 8.4 Hz, 2H), 8.09 (d, J = 6.4 Hz, 2H), 7.40-7.10 (m, 10H), 4.92-4.80 (m, 2H), 4.57 (dd, J = 10.0, 7.2 Hz,2H), 4.50-4.32 (m, 2H), 3.60-3.40 (m, 4H), 3.60-3.45 (m, 4H), 3.23-3.17 (m, 2H), 3.14 (s, 6H), 3.11 (s, 6H), 1.90-1.80 (m, ¹³ C NMR (100 MHz, CDCl ₃ ):δ 174.9, 172.7, 171.0, 170.1, 135.8, 128.9, 128.5, 127.0, 63.1, 60.4, 50.9, 40.5, 37.6, 37.3, 35.1, 31.6, 25.1, 23.1, 20.7, 13.1 ppm; HRMS (ESI): calcd for [C ₄₅ H ₆₄ N ₈ O ₈ +Na] ⁺ 855.4739 found 855.4738.

Claims (15)

General formula (1):
[In the formula, R represents an optionally substituted divalent organic group. R may form a ring together with the adjacent nitrogen atom. _R represents a hydrogen atom or a hydrocarbon group.]
A method for producing a cyclic amide represented by the formula:
General formula (2):
[In the formula, R and R _A are the same as above.]
and a cyclic amide precursor represented by general formula (3):
[wherein X represents a halogen atom, and Y represents an optionally substituted alkyl group] with a condensing agent represented by general formula (4):
[In the formula, R ¹ , R ² and R ³ are the same or different and represent an optionally substituted alkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group. Alternatively, two or more of R ¹ , R ² and R ³ may be linked to form a ring (excluding a pyridine ring) which may have one or more heteroatoms or substituents.]
with a tertiary amine compound represented by the following formula (excluding tributylamine, diisopropylethylamine, and triethylamine): The manufacturing method described in claim 1, wherein R is a divalent hydrocarbon group which may have an amide bond and/or a substituent. The cyclic amide is represented by the general formula (1a):
[Wherein, _RA is the same as defined above. Two or more ^RA may be the same or different. _RC , _RN , and _RP may be the same or different and represent a divalent organic group. _RC , _RN , and _RP may each form a ring together with the adjacent nitrogen atom. n represents an integer of 0 or greater. However, when n is an integer of 2 or greater, n _RPs may be the same or different.]
A cyclic peptide represented by the formula:
The cyclic amide precursor is represented by the general formula (2a):
[In the formula, R _A , R _C , R _N , R _P and n are the same as defined above.]
The method according to claim 1 or 2, wherein the cyclic peptide precursor is represented by the formula: The method according to claim 3 , wherein R _C , R _N and R _P are the same or different and each represents a divalent hydrocarbon group which may have a substituent. The manufacturing method according to claim 3 , wherein n is an integer of 2 or more. The method according to claim 1 or 2 , wherein X is a chlorine atom. The method according to claim 1 or 2 , wherein Y is a branched alkyl group. The method according to claim 1 or 2 , wherein Y is an isopropyl group. The method according to claim 1 or 2 , wherein at least one of R ¹ , R ² and R ³ is a methyl group. The method according to claim 1 or 2 , wherein at least two of R ¹ , R ² and R ³ are methyl groups. The method according to claim 1 or 2 , wherein the reaction step is carried out in a solvent containing an aprotic polar solvent. The method according to claim 1 or 2 , wherein the reaction temperature in the reaction step is 0 to 100°C. The method according to claim 1 or 2 , wherein the reaction time of the reaction step is less than 10 minutes. The method according to claim 1 or 2 , wherein the reaction step is carried out by a flow method. The method according to claim 1 or 2 , wherein the reaction step is carried out by a microflow method.