JP7659541B2 - Method for producing amide bond-containing compound - Google Patents

Description

The present invention relates to a method for producing an amide bond-containing compound using a liquid phase method.

The extension of a compound by an amide bond reaction is carried out by a solid phase method or a liquid phase method. In the solid phase method, the extension of the compound is repeatedly carried out by an amide bond reaction in a state where the end of the compound is bound to a solid support, and the compound is cleaved from the support at the stage where the target compound is obtained. Therefore, it is possible to easily select unreacted active esters and the like by washing. However, the solid phase method requires a large excess of raw materials due to its low reactivity, and it is difficult to scale up. In contrast, the liquid phase method is highly reactive and easy to scale up, making it an industrially useful production method. On the other hand, in the liquid phase method, after the extension of the compound by the amide bond reaction is completed, an isolation operation such as crystallization is required to remove the remaining active esters and by-products. Therefore, there was a problem that the production process became complicated and the production took a very long time.
Therefore, there has been a demand for a method for producing an amide bond-containing compound by a liquid phase method that can remove the remaining active ester and by-products without an isolation procedure.

Patent Document 1 describes a method for synthesizing peptides in solution, in which an excess of an activated carboxyl component is used to acylate an amino component, followed by removal of residual activated carboxyl functional groups using a scavenger such as benzyl β-alaninate p-toluenesulfonate salt, and aqueous extraction.

JP 2003-55396 A

By the way, a scavenger is a compound having a group that reacts with an active ester, such as an amino group or a sulfanyl group, and an acidic or basic group, such as a sulfo group or a carboxy group, as a water-soluble group. By using such a scavenger, it is possible to decompose the active ester and remove the decomposition product by washing with water. However, it has been found that an active ester having an aromatic heterocycle to which a carbonyl group is bonded is more likely to be emulsified by the scavenger than other active esters, and therefore the decomposition product is not easily removed.
An object of the present invention is to provide a method capable of easily removing an activated acid raw material (such as an activated ester, an acid halide, or an acid anhydride) having an aromatic heterocycle bonded to a carbonyl group in the production of an amide bond-containing compound.

［式中、ｎは１以上５０以下の整数を示し、ＰｒｏはＣ末端の保護基を示し、Ｒａはアミノ酸残基を示す。ｎが２以上の場合、複数のＲａは同一であってもよく、異なっていてもよい。］で表される化合物と、
式（２）又は式（３）；

［式中、ｍは１又は２の整数を示し、Ｒｂはアミノ酸残基を示し、ｍが２の場合、２つのＲｂは同一であってもよく、異なっていてもよい。Ｒｂの少なくとも１つは式（４）で表されるアミノ酸残基である。ＱはＮ末端の保護基を示し、Ａｒ¹及びＡｒ²は、互いに独立に、カルボキシ基及びアミノ基を除く置換基を有していてもよい芳香族複素環を示す。式（２）で表される化合物が２つのＡｒ²を有する場合、２つのＡｒ²は、同一であってもよく、異なっていてもよい。＊は結合手を示す。］で表される化合物を活性エステル化、酸ハロゲン化、又は酸無水物化した化合物とを反応させて、式（５）又は式（６）；

［式中、Ｒａ、Ｒｂ、Ａｒ¹、Ｐｒｏ、Ｑ、ｎ、及びｍは、前記と同じである。］で表されるアミド結合含有化合物を合成する工程Ａ、
前記工程Ａで得られた式（５）又は式（６）で表されるアミド結合含有化合物を含む反応混合物に、式（７）；

［式中、Ａはアミノ基、又はスルファニル基を示し、Ｂは－ＳＯ₃Ｍ、－ＣＯ₂Ｍ、－ＰＯ₃Ｍ₂、又はジアルキルアミノ基を示し、Ｒ¹は炭素数１～４のアルキレン基を示す。Ｍは水素原子、又はアルカリ金属原子を示し、Ｍが複数存在する場合、それらは同一であってもよく、異なっていてもよい。］で表される化合物、及び
酸性基又は酸性基の塩を有さないモノアミン化合物を混合し、
前記式（２）又は式（３）で表される化合物を活性エステル化、酸ハロゲン化、又は酸無水物化した化合物と前記式（７）で表される化合物とを反応させる工程Ｂ、及び
前記工程Ｂで得られた反応混合物を塩基性及び／又は酸性水溶液で洗浄する工程Ｃを含むことを特徴とするアミド結合含有化合物の製造方法。
[２] 前記酸性基又は酸性基の塩を有さないモノアミン化合物がトリエチルアミン、トリブチルアミン、Ｎ，Ｎ－ジイソプロピルエチルアミン、又はＮ－メチルモルホリンである[１]に記載の製造方法。
[３] 前記式（７）中のＲ¹が炭素数２～３のアルキレン基である[１]又は[２]に記載の製造方法。
[４] 前記式（７）中のＢが－ＳＯ₃Ｍ、－ＣＯ₂Ｍ、又は－ＰＯ₃Ｍ₂［Ｍは、前記と同じである。］である[１]～[３]のいずれかに記載の製造方法。
[５] 前記式（７）で表される化合物がタウリンである[１]～[４]のいずれかに記載の製造方法。
[６] 前記塩基性水溶液が炭酸塩又は炭酸水素塩の水溶液である[１]～[５]のいずれかに記載の製造方法。
[７] 前記炭酸塩、炭酸水素塩がリチウム塩、ナトリウム塩、又はカリウム塩である[６]に記載の製造方法。
[８] 前記酸性水溶液が有機酸の水溶液である[１]～[７]のいずれかに記載の製造方法。
[９] 前記有機酸がクエン酸である[８]に記載の製造方法。
[１０] 前記式（５）又は式（６）で表されるアミド結合含有化合物がピロールイミダゾール（ポリ）アミド化合物である[１]～[９]のいずれかに記載の製造方法。
[１１] [１]～[１０]のいずれかに記載の製造方法で前記式（５）で表されるアミド結合含有化合物を製造し、得られた前記式（５）で表されるアミド結合含有化合物を単離することなく、Ｎ末端を脱保護し、続いて、前記工程Ａ、工程Ｂ、及び工程Ｃを行うことでアミド結合含有化合物の伸長を行うアミド結合含有化合物の製造方法。 The present invention, which has solved the above problems, is as follows.
[1] In a liquid phase amide bond formation reaction,
Formula (1);

[wherein n is an integer of 1 to 50, Pro is a protecting group for the C-terminus, and Ra is an amino acid residue. When n is 2 or more, multiple Ra's may be the same or different.]
Formula (2) or Formula (3);

[wherein m represents an integer of 1 or 2, Rb represents an amino acid residue, and when m is 2, the two Rb's may be the same or different. At least one of Rb's is an amino acid residue represented by formula (4). Q represents an N-terminal protecting group, and Ar ¹ and Ar ² each independently represent an aromatic heterocycle which may have a substituent other than a carboxy group and an amino group. When the compound represented by formula (2) has two Ar ^2's , the two Ar ² 's may be the same or different. * represents a bond.] is reacted with a compound which has been converted into an active ester, an acid halide, or an acid anhydride to obtain a compound represented by formula (5) or formula (6);

[wherein Ra, Rb, Ar ¹ , Pro, Q, n, and m are the same as defined above],
The reaction mixture containing the amide bond-containing compound represented by formula (5) or formula (6) obtained in step A is treated with a compound represented by formula (7);

[wherein A represents an amino group or a sulfanyl group, B represents _--SO3M , _--CO2M , _--PO3M2 , or _a dialkylamino group, and ^R1 represents an alkylene group having 1 to 4 carbon atoms. M represents a hydrogen atom or an alkali metal atom, and when a plurality of M's are present, they may be the same or different.] and a monoamine compound having no acidic group or a salt of an acidic group are mixed,
A method for producing an amide bond-containing compound, comprising: a step B of reacting a compound obtained by converting a compound represented by formula (2) or (3) into an active ester, an acid halide, or an acid anhydride with a compound represented by formula (7); and a step C of washing the reaction mixture obtained in the step B with a basic and/or acidic aqueous solution.
[2] The method according to [1], wherein the monoamine compound having no acidic group or a salt of an acidic group is triethylamine, tributylamine, N,N-diisopropylethylamine, or N-methylmorpholine.
[3] The method according to [1] or [2], wherein R ¹ in the formula (7) is an alkylene group having 2 to 3 carbon atoms.
[4] The method according to any one of [1] to [3], wherein B in the formula (7) is --SO ₃ M, --CO ₂ M, or --PO ₃ M ₂ [M is as defined above].
[5] The method according to any one of [1] to [4], wherein the compound represented by formula (7) is taurine.
[6] The method according to any one of [1] to [5], wherein the basic aqueous solution is an aqueous solution of a carbonate or a hydrogencarbonate.
[7] The method according to [6], wherein the carbonate or hydrogen carbonate is a lithium salt, a sodium salt, or a potassium salt.
[8] The method according to any one of [1] to [7], wherein the acidic aqueous solution is an aqueous solution of an organic acid.
[9] The method according to [8], wherein the organic acid is citric acid.
[10] The method according to any one of [1] to [9], wherein the amide bond-containing compound represented by the formula (5) or (6) is a pyrrole-imidazole (poly)amide compound.
[11] A method for producing an amide bond-containing compound represented by formula (5) by the production method according to any one of [1] to [10], and deprotecting the N-terminus of the obtained amide bond-containing compound represented by formula (5) without isolating it, and then carrying out the steps A, B, and C to elongate the amide bond-containing compound.

According to the present invention, in the production of an amide bond-containing compound, it is possible to easily remove an activated acid raw material such as an activated ester having an aromatic heterocycle to which a carbonyl group is bonded. It is also possible to obtain the desired amide bond-containing compound in high yield.

The method for producing an amide bond-containing compound of the present invention is an amide bond-forming reaction by a liquid phase method, and includes the following steps A to C.
[Step A] An amide bond-containing compound is synthesized by reacting a C-terminal protected amino acid or an amide bond-containing compound (1) (hereinafter, sometimes referred to as a reactive amine raw material) with a compound obtained by converting a carboxylic acid having an aromatic heterocycle to which a carbonyl group is bonded into an active ester, an acid halide, or an acid anhydride (hereinafter, sometimes referred to as an activated acid raw material);
[Step B] A step of mixing a specific derivatizing agent (sometimes referred to as a scavenger) and a monoamine compound with the reaction mixture containing the amide bond-containing compound obtained in Step A, and reacting the activated acid raw material remaining from the reaction in Step A with the derivatizing agent;
[Step C] The reaction mixture obtained in step B is washed with a basic and/or acidic aqueous solution.
The activated acid raw material has a problem that the reaction product with a derivatizing agent (scavenger) tends to become an emulsion because the aromatic heterocycle is bonded to a carbonyl group. By using a specific derivatizing agent (scavenger), the excess activated acid raw material can be removed without causing the emulsion problem.

［式中、ｎは１以上５０以下の整数を示し、ＰｒｏはＣ末端の保護基を示し、Ｒａはアミノ酸残基を示す。ｎが２以上の場合、複数のＲａは同一であってもよく、異なっていてもよい。］ <Step A>
<<Reactive amine raw material>>
The reactive amine starting material used in step A is represented by formula (1).

[In the formula, n represents an integer of 1 to 50, Pro represents a protecting group at the C-terminus, and Ra represents an amino acid residue. When n is 2 or more, multiple Ra's may be the same or different.]

Examples of amino acid residues represented by Ra include hydrocarbon groups having -NH- and -C(=O)-, and aromatic heterocycles. The hydrocarbon group may be bonded with a substituent other than a carboxy group and an amino group. In this specification, "substituents other than a carboxy group and an amino group" means a free (unsubstituted and unprotected) amino group and a substituent other than a carboxy group, and a carboxy group having a substituent (including a carboxy group protected by a protecting group) and an amino group having a substituent (including an amino group protected by a protecting group) are included in the substituent.

Examples of amino acid residues represented by Ra include α-amino acid residues such as glycine residue, alanine residue, valine residue, leucine residue, isoleucine residue, proline residue, phenylalanine residue, methionine residue, tryptophan residue, aspartic acid residue, glutamic acid residue, lysine residue, arginine residue, histidine residue, asparagine residue, glutamine residue, serine residue, threonine residue, cysteine residue, and tyrosine residue; β-amino acid residues such as β-alanine residue; and aliphatic amino acid residues such as γ-amino acid residues, such as γ-amino acid residues and α,γ-diaminobutyric acid residues.

When the α-amino acid residue has a reactive functional group (e.g., a hydroxyl group, a thiol group, a carboxyl group, an amino group, a guanidino group, etc.) in the side chain, the reactive functional group is preferably protected with a protecting group. Also, the amino group of a γ-amino acid having an amino group such as α,γ-diaminobutyric acid is preferably protected with a protecting group. Protective groups for protecting these reactive functional groups are preferably those which are not deprotected under acidic conditions for deprotecting the N-terminal amino group (Q described later). For example, protective groups for hydroxyl groups include benzyl (Bzl), 4-methylbenzyl (4-MeBzl), 4-methoxybenzyl (4-MeOBzl), etc.; protective groups for thiol groups include benzyl (Bzl), 4-methylbenzyl (4-MeBzl), 4-methoxybenzyl (4-MeOBzl), t-butyl (tBu), etc.; protective groups for carboxy groups include benzyloxy (OBzl), cyclohexyloxy (O-cHex), etc.; protective groups for amino groups include 2,4,6-trimethylbenzenesulfonyl (Mts), benzyloxycarbonyl (Z), nitro (NO ₂ ), p-toluenesulfonyl (Tos), benzyloxymethyl (BOM), 2,4-dinitrophenyl (Dnp), p-chlorobenzyloxycarbonyl (Cl-Z), and the like. Examples of the protecting group for the guanidino group include a 2,2,5,7,8-pentamethylchroman-6-sulfonyl group (Pmc group), and a 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group (Pbf group).

Further, examples of the amino acid residue represented by Ra include an aromatic heterocycle-containing amino acid residue represented by the following formula (4-1) (hereinafter, sometimes referred to as aromatic heterocycle-containing amino acid residue (4-1)). It is preferable that at least one Ra of the reactive amine raw material of formula (1) is an aromatic heterocycle-containing amino acid residue (4-1).

［式中、Ａｒ³はカルボキシ基及びアミノ基を除く置換基を有していてもよい芳香族複素環を示す。＊は結合手を示す。］
化合物（１）が２つ以上の芳香族複素環含有アミノ酸残基（４－１）を有する場合、複数のＡｒ³は、同一であってもよく、異なっていてもよい。
なお後述する様に、Ａｒ³の説明は、後述するＡｒ¹及びＡｒ²の説明で共通の内容として参照される。Ａｒ¹、Ａｒ²、Ａｒ³とで説明が異なる場合には、以下、都度、説明を補足する。

[In the formula, ^Ar3 represents an aromatic heterocycle which may have a substituent other than a carboxy group or an amino group. * represents a bond.]
When compound (1) has two or more aromatic heterocycle-containing amino acid residues (4-1), the multiple Ar ^3s may be the same or different.
As described later, the explanation of Ar ³ is referred to as the common content in the explanation of Ar ¹ and Ar ^2. When the explanations of Ar ¹ , Ar ² , and Ar ³ differ, the explanation will be supplemented below each time.

Examples of the heteroatom contained in the aromatic heterocycle represented by Ar ¹ to Ar ³ include a nitrogen atom, an oxygen atom, and a sulfur atom. The number of aromatic rings contained in the aromatic heterocycle is not particularly limited, and may be a single ring or a condensed ring having two or more aromatic rings. The number of carbon atoms in the aromatic heterocycle is preferably 2 to 10, more preferably 2 to 8, and even more preferably 2 to 5. Examples of the aromatic heterocycle include furan, thiophene, pyrrole, pyrazole, oxazole, thiazole, imidazole, pyridine, pyridazine, pyrimidine, pyrazine, purine, indole, isoindole, benzimidazole, quinoline, benzofuran, isobenzofuran, benzothiophene, indazole, benzoxazole, and benzothiazole, and thiophene, pyrrole, imidazole, thiazole, and indole are preferred. For Ar ¹ , thiophene, pyrrole, imidazole and thiazole are most preferred, and for Ar ² and Ar ³ , pyrrole and imidazole are most preferred.

Examples of the substituent that the aromatic heterocycle represented by Ar ¹ to Ar ³ may have include an amino group having a substituent, a halogen atom, a hydrocarbon group having 1 to 12 carbon atoms, an alkoxy group having 1 to 12 carbon atoms, an aryloxy group having 6 to 12 carbon atoms, and an aralkyloxy group having 7 to 12 carbon atoms, with a halogen atom and a hydrocarbon group being preferred. Examples of the amino group having a substituent include an amino group substituted with one or more selected from an alkyl group having 1 to 10 carbon atoms and an aryl group having 6 to 10 carbon atoms, such as a methylamino group, an ethylamino group, a dimethylamino group, an ethylmethylamino group, a phenylamino group, a methylphenylamino group, and a diphenylamino group.

Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and a chlorine atom is preferable.
The hydrocarbon group includes an alkyl group and an aromatic hydrocarbon group. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a (1-ethyl)propyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group. Examples of the aromatic hydrocarbon group include a phenyl group and a naphthyl group. The hydrocarbon group that may be substituted on the aromatic heterocycle represented by ^Ar3 is preferably an alkyl group having 1 to 3 carbon atoms, and more preferably a methyl group.

Examples of the alkoxy group include a methoxy group, an ethoxy group, a butoxy group, and a 2-methoxyethoxy group, and an alkoxy group having 1 to 6 carbon atoms is preferable.
Examples of the aryloxy group include a phenoxy group and a naphthoxy group.
Examples of the aralkyloxy group include a benzyloxy group and a 1-phenethyloxy group.

As the aromatic heterocycles represented by Ar ¹ to Ar ³ which may have a substituent, groups represented by the following formulae (A1-1) to (A1-5) are preferred.

［式中、＊１は－ＣＯ－との結合手を示す。＊２は、Ａｒ²又はＡｒ³のとき、－ＮＨ－との結合手を示し、Ａｒ¹のとき、水素原子またはハロゲン原子（好ましくは塩素原子）を示す。Ｒ¹⁰は、炭素数１～３のアルキル基を示し、好ましくはメチル基を示す。Ｘ¹⁰はハロゲン原子を示し、好ましくは塩素原子を示す。］
Ａｒ¹では式（Ａ１－１）、式（Ａ１－２）、式（Ａ１－３）、式（Ａ１－４）が最も好ましく、Ａｒ²又はＡｒ³では、式（Ａ１－１）、式（Ａ１－２）が最も好ましい。

[In the formula, *1 represents a bond to -CO-. *2 represents a bond to -NH- when it is ^Ar2 or ^Ar3 , and represents a hydrogen atom or a halogen atom (preferably a chlorine atom) when it is ^Ar1 . ^R10 represents an alkyl group having 1 to 3 carbon atoms, preferably a methyl group. ^X10 represents a halogen atom, preferably a chlorine atom.]
For Ar ¹ , the formula (A1-1), the formula (A1-2), the formula (A1-3) or the formula (A1-4) is most preferred, and for Ar ² or Ar ³ , the formula (A1-1) or the formula (A1-2) is most preferred.

As Ra, β-amino acid residues such as β-alanine residues; γ-amino acid residues such as γ-aminobutyric acid residues and α,γ-diaminobutyric acid residues; and aromatic heterocycle-containing amino acid residues (4-1) are preferred, and it is preferred that at least one Ra in the reactive amine raw material of formula (1) is an aromatic heterocycle-containing amino acid residue (4-1).

As the C-terminal protecting group represented by Pro, a protecting group for a carboxyl group used in general liquid phase synthesis can be appropriately used, and among the protecting groups described in Protective Groups in Organic Chemistry (4th Edition, published by Wiley-Interscience) pp. 533-646 by Theodora W. Greene and Peter G. M. Wuts, those that are not deprotected under acidic conditions for deprotecting the N-terminal amino group are preferred. Examples of -C(=O)-Pro include ester-based protecting groups such as methyl ester, ethyl ester, benzyl ester, 4-nitrobenzyl ester, and 4-methoxybenzyl ester; amide-based protecting groups such as amide, N,N-dimethylamide, N,N-(3-dimethylamino)propylamide, pyrrolidinylamide, piperidinylamide, and o-nitroanilide.

n may be, for example, 1 to 30, 1 to 10, or 1 to 5.

［式中、Ｑは、アミノ基の保護基を示し、後述する式（２）のＱと同じである。Ｒａ、ｎ、Ｐｒｏは前記と同じである。］ The reactive amine raw material can be produced by deprotecting a compound represented by formula (1x) (hereinafter, sometimes referred to as an amine raw material).

[In the formula, Q represents a protecting group for an amino group and is the same as Q in formula (2) described below. Ra, n, and Pro are the same as those described above.]

Deprotection can be carried out by known methods, for example, under acidic conditions such as hydrochloric acid or sulfonic acid.

［式中、ｍは１又は２の整数を示し、Ｒｂはアミノ酸残基を示し、ｍが２の場合、２つのＲｂは同一であってもよく、異なっていてもよい。Ｒｂの少なくとも１つは式（４）で表されるアミノ酸残基である。ＱはＮ末端の保護基を示し、Ａｒ¹及びＡｒ²は、互いに独立に、カルボキシ基及びアミノ基を除く置換基を有していてもよい芳香族複素環を示す。式（２）で表される化合物が２つのＡｒ²を有する場合、２つのＡｒ²は、同一であってもよく、異なっていてもよい。＊は結合手を示す。］ <<Activated acid raw material>>
The activated acid raw material is a compound obtained by converting a compound represented by formula (2) or (3) (carboxylic acid compound) into an active ester, an acid halide, or an acid anhydride.

[In the formula, m represents an integer of 1 or 2, Rb represents an amino acid residue, and when m is 2, the two Rb's may be the same or different. At least one of Rb's is an amino acid residue represented by formula (4). Q represents an N-terminal protecting group, and Ar ¹ and Ar ² each independently represent an aromatic heterocycle which may have a substituent other than a carboxy group and an amino group. When the compound represented by formula (2) has two Ar ^2's , the two Ar ² 's may be the same or different. * represents a bond.]

The activated acid raw material obtained from the carboxylic acid compound represented by formula (2) and formula (3) is highly reactive and is useful for synthesizing an amide bond-containing compound by combining with a reactive amine compound. However, the activated acid raw material is relatively stable even if it remains after the formation of the amide bond-containing compound because the aromatic heterocycle (Ar ¹ , Ar ² ) is combined with a carbonyl group, and cannot be easily decomposed and removed. Therefore, the remaining active oxygen raw material is removed using a derivatizing agent (scavenger), but the activated acid raw material having the aromatic heterocycle (Ar ¹ , Ar ² ) and a carbonyl group combined has a problem that it is prone to emulsion problems when decomposed and removed with a derivatizing agent (scavenger). According to the present invention, the problem of emulsion generation can be overcome by using a specific derivatizing agent (scavenger).

The amino acid residue represented by Rb is the same as that represented by Ra in the above formula (1), and the descriptions of Ar ¹ and Ar ² are the same as those of Ar ³ in the above formula (4-1).
When m is 2, it is preferable that one Rb is an amino acid residue represented by formula (4) (hereinafter, may be referred to as aromatic heterocycle-containing amino acid residue (4)), and the other Rb is an aromatic heterocycle-containing amino acid residue (4) or an aliphatic amino acid residue (preferably a β amino acid residue or a γ amino acid residue).

The N-terminal protecting group represented by Q is preferably an amino protecting group removable under acidic conditions, for example, a protecting group removable under acidic conditions described in Protective Groups in Organic Chemistry (4th Edition, published by Wiley-Interscience) pp. 696-926 by Theodora W. Greene and Peter G. M. Wuts, and more preferably a protecting group removable with sulfonic acid.
Examples of Q include carbamate-type protecting groups such as methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, tert-butoxycarbonyl (Boc), allyloxycarbonyl, benzyloxycarbonyl, phenoxycarbonyl, and 9-fluorenylmethoxycarbonyl (Fmoc); acyl-type protecting groups such as formyl, acetyl, trichloroacetyl, trifluoroacetyl, benzoyl, and p-nitrobenzoyl; alkoxymethyl-type protecting groups such as methoxymethyl (MOM), benzyloxymethyl, and pivaloyloxy; and phenylmethyl-type protecting groups such as triphenylmethyl, di(p-methoxyphenyl)methyl, and (p-methoxyphenyl)diphenylmethyl. From the viewpoint of ease of deprotection, carbamate-type protecting groups are preferred, and Boc and Fmoc are particularly preferred.

The term "active esterification" refers to, for example, converting the OH group of formula (2) or formula (3) into a -OR ^X group (R ^X represents a nitrogen atom-containing heterocyclic group such as a triazolidinone ring group, a triazole ring group, a succinimide ring group, or a dimethoxytriazyl ring group; an aryl halide group; a carboxylate group; a sulfonate group; an acyl group; or the like, and these groups may have a substituent).
R ^X is preferably a group represented by the following formula:

［式中、Ｒ^X1は、炭素数１～５のアルキル基を示す。＊は結合手を示す。］

[In the formula, ^R represents an alkyl group having 1 to 5 carbon atoms. * represents a bond.]

The active esterification can be carried out by reacting a carboxylic acid compound represented by formula (2) or formula (3) with an activating agent such as 1-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide (HOSu), ethyl cyano(hydroxyimino)acetate (Oxyma), or pentafluorophenol (HOPFP) in the presence of a known condensing agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

The amount of the activating reagent is, for example, 1.00 mol or more, preferably 1.05 mol or more, more preferably 1.10 mol or more, and for example, 1.5 mol or less, preferably 1.4 mol or less, more preferably 1.3 mol or less, relative to 1.00 mol of the carboxylic acid compound.
The amount of the condensing agent is, for example, 1.00 mol or more, preferably 1.05 mol or more, more preferably 1.10 mol or more, and for example, 1.5 mol or less, preferably 1.4 mol or less, more preferably 1.3 mol or less, relative to 1.00 mol of the carboxylic acid compound.

A solvent can be used appropriately in the active esterification reaction. After the active esterification reaction, it is preferable to remove the condensing agent by washing with an aqueous solution or water. The active ester obtained by the active esterification reaction may be concentrated before use, if necessary.

Examples of acid halides obtained by the acid halogenation reaction include acid fluorides, acid chlorides, acid bromides, and acid iodides, with acid chlorides being preferred. The acid halogenation reaction can be carried out using an electrophilic halogenating agent such as thionyl chloride, oxalyl chloride, phosphoryl chloride, sulfuryl chloride, phosphorus trichloride, or phosphorus pentachloride, preferably thionyl chloride or sulfuryl chloride. The acid bromide reaction can be carried out, for example, using phosphorus tribromide.

The acid anhydride obtained by the acid anhydridation reaction may be a dehydration condensation product (symmetric acid anhydride) of two molecules of a carboxylic acid compound represented by formula (2) or formula (3), or a condensation product of a carboxylic acid compound represented by formula (2) or formula (3) with another acid or its ester (such as a carbonate ester).

The activated acid raw material is preferably an activated ester obtained by an activated esterification reaction of a carboxylic acid compound represented by formula (2) or formula (3).
In addition, the activated acid raw material of the carboxylic acid represented by the formula (2) is preferable in that it can be used as a new reactive amine raw material in step A (i.e., in that the amide bond-containing compound can be elongated by forming an amide bond-containing compound together with a reactive amine raw material and performing steps B and C, and then removing the protecting group Q from the obtained amide bond-containing compound under acidic conditions (particularly, sulfonic acid conditions, etc.).
On the other hand, the activated acid raw material of the carboxylic acid represented by the formula (3) is preferable in that it can be used as a capping agent for terminating the elongation of the amide bond-containing compound.

The amount of the activated acid raw material used is, for example, 0.8 moles or more, preferably 0.9 moles or more, more preferably 1.0 moles or more, and even more preferably 1.1 moles or more per mole of the reactive amine raw material. The amount of the activated acid raw material used is, for example, 1.5 moles or less, preferably 1.4 moles or less, and more preferably 1.3 moles or less per mole of the reactive amine raw material.

<<Bases>>
The reaction between the reactive amine source and the activated acid source is preferably carried out in the presence of a base in order to accelerate the reaction.

Examples of bases include tertiary amines such as trimethylamine, triethylamine, tripropylamine, tributylamine, tripentylamine, trihexylamine, triheptylamine, trioctylamine, tridodecylamine, N-dodecyldimethylamine, N-hexyldibutylamine, N,N-diisopropylbutylamine, N,N-diisopropylethylamine, N,N-dimethylethylamine, N,N-dicyclohexylmethylamine, N,N-dimethylbenzylamine, N-methylpyrrolidine, N,N-dimethyl-4-aminopyridine, quinuclidine, N-methylmorpholine, 1,4-diazabicyclo[2,2,2]octane, and 1,8-diazabicyclo[5,4,0]-7-undecene, and preferably triethylamine, tributylamine, N,N-diisopropylethylamine, or N-methylmorpholine. These bases may be used alone or in combination of two or more kinds, and when they are mixed, there is no limitation on the mixing ratio.

The amount of base used is, for example, 0.5 moles or more, preferably 1.0 moles or more, more preferably 2 moles or more, and for example, 30 moles or less, preferably 20 moles or less, more preferably 10 moles or less, per mole of activated acid raw material.

<<Solvent>>
The reaction between the reactive amine raw material and the activated acid raw material is preferably carried out in the presence of a solvent (reaction solvent).

The reaction solvent is not particularly limited as long as it does not adversely affect the reaction, and examples of the reaction solvent that can be used include ether solvents such as diethyl ether, cyclopentyl methyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, etc.; halogenated hydrocarbon solvents such as methylene chloride, dichloroethane, etc.; nitrogen-containing solvents such as acetonitrile, dimethylformamide, dimethylacetamide, etc.; alcohol solvents such as methanol, ethanol, etc.; aromatic solvents such as benzene, toluene, xylene, pyridine, etc.; dimethyl sulfoxide; water, etc. The reaction solvent may be used alone or in combination of two or more kinds.
The amount of the reaction solvent used is, for example, about 0.1 to 20 parts by mass relative to 1 part by mass of the activated acid raw material.

The reaction solvent may also be used as a reaction solvent for other reactions described in this specification (e.g., the deprotection reaction of the protecting group Q, the reaction in step B, etc.), and the same reaction solvent may be used for the other reactions and the reaction in step A.

<Step B>
In the step A, an amide bond-containing compound represented by formula (5) or (6) is synthesized.

［式中、Ｒａ、Ｒｂ、Ａｒ¹、Ｐｒｏ、Ｑ、ｎ、及びｍは、前記と同じである。］
そして工程Ｂでは、前記アミド結合含有化合物と、未反応の活性酸原料とを含む反応混合物に、特定の誘導化剤とモノアミン化合物を混合し、残存した活性酸原料と誘導化剤とを反応させる。特定の誘導化剤を使用すれば、活性酸原料と誘導化剤との反応物を含む反応液がエマルジョンになるのを防止できる。

[In the formula, Ra, Rb, Ar ¹ , Pro, Q, n, and m are the same as defined above.]
In step B, a specific derivatizing agent and a monoamine compound are mixed into the reaction mixture containing the amide bond-containing compound and the unreacted active acid raw material, and the remaining active acid raw material is reacted with the derivatizing agent. The use of the specific derivatizing agent can prevent the reaction liquid containing the reaction product of the active acid raw material and the derivatizing agent from becoming an emulsion.

The amount of unreacted activated acid raw material in the reaction mixture is, for example, 0.0005 mol or more, preferably 0.005 mol or more, more preferably 0.01 mol or more, per 1 mol of the amide bond-containing compound, and is, for example, 1.0 mol or less, preferably 0.5 mol or less, more preferably 0.3 mol or less.

<<Derivatizing agent (scavenging agent)>>
The specific inducer can be specifically represented by formula (7).

［式中、Ａはアミノ基（－ＮＨ₂）、又はスルファニル基（－ＳＨ）を示し、Ｂは－ＳＯ₃Ｍ、－ＣＯ₂Ｍ、－ＰＯ₃Ｍ₂、又はジアルキルアミノ基を示し、Ｒ¹は炭素数１～４のアルキレン基を示す。Ｍは水素原子、又はアルカリ金属原子を示し、Ｍが複数存在する場合、それらは同一であってもよく、異なっていてもよい。］
前記ジアルキルアミノ基としては、ジメチルアミノ基、ジエチルアミノ基、ジプロピルアミノ基などのジＣ_1-4アルキルアミノ基が挙げられる。なお「Ｃ_1-4」と記載は、「炭素数１～４」を意味する。
前記Ｍとしては、リチウム、ナトリウム、カリウムなどが挙げられ、ナトリウム、カリウムが好ましく、ナトリウムがより好ましい。
Ｒ¹の炭素数は、誘導化剤（捕捉剤）の反応性と水溶性の観点から決定できる。炭素数は、好ましくは２～３である。
Ａ、Ｂは、Ｒ¹のアルキレン基の両末端（α位とω位）に結合していることが好ましい。
Ｂは、－ＳＯ₃Ｍ、－ＣＯ₂Ｍ、又は－ＰＯ₃Ｍ₂が好ましい。

[In the formula, A represents an amino group ( _-NH2 ) or a sulfanyl group (-SH), B represents _-SO3M , _-CO2M , _-PO3M2 or _a dialkylamino group, and ^R1 represents an alkylene group having 1 to 4 carbon atoms. M represents a hydrogen atom or an alkali metal atom, and when there are multiple M's, they may be the same or different.]
The dialkylamino group includes di-C _1-4 alkylamino groups such as dimethylamino group, diethylamino group, dipropylamino group, etc. Note that "C _1-4 " means "having 1 to 4 carbon atoms."
Examples of M include lithium, sodium, and potassium, with sodium and potassium being preferred, and sodium being more preferred.
The number of carbon atoms in ^R1 can be determined from the viewpoint of the reactivity and water solubility of the derivatizing agent (scavenger), and is preferably 2 to 3.
A and B are preferably bonded to both ends (α-position and ω-position) of the alkylene group of R ¹ .
B is preferably --SO ₃ M, --CO ₂ M, or --PO ₃ M ₂ .

Examples of the derivatizing agent include aliphatic amino acids such as glycine, alanine, β-alanine, and γ-aminobutyric acid, or their sodium salts; mercaptocarboxylic acids such as thioglycolic acid, 2-mercaptopropionic acid, and 3-mercaptopropionic acid, or their sodium salts; aminosulfonic acids such as aminomethanesulfonic acid, aminoethanesulfonic acid (e.g., taurine), and aminopropanesulfonic acid, or their sodium salts; mercaptosulfonic acids such as mercaptomethanesulfonic acid, 2-mercaptoethanesulfonic acid, and 3-mercaptopropanesulfonic acid, or their sodium salts; and diamines such as N,N-dimethylethanediamine and N,N-dimethylpropanediamine. As the derivatizing agent, from the viewpoint of achieving a better yield, aliphatic amino acids, aminosulfonic acids, and mercaptosulfonic acids are preferred, aminosulfonic acids and mercaptosulfonic acids are more preferred, and taurine is the most preferred.

The amount of the derivatizing agent used is, for example, 0.1 mol or more, preferably 0.5 mol or more, more preferably 1 mol or more, and, for example, 10 mol or less, preferably 7 mol or less, more preferably 5 mol or less, relative to 1 mol of the activated acid raw material used in step A. The amount of the derivatizing agent used is, for example, 1 mol or more, preferably 5 mol or more, more preferably 10 mol or more, relative to 1 mol of the activated acid raw material remaining after the reaction in step A, and, for example, 100 mol or less, preferably 50 mol or less, more preferably 30 mol or less.

The derivatizing agent reacts with the activated acid raw material in the presence of a monoamine compound. The monoamine compound is distinguished from the derivatizing agent in that it does not have an acidic group or a salt of an acidic group, and specific examples thereof include compounds similar to the tertiary amines exemplified as the base in step A. Preferred monoamine compounds include triethylamine, tributylamine, N,N-diisopropylethylamine, and N-methylmorpholine.
The amount of the monoamine compound used is, for example, 0.1 mol or more, preferably 0.5 mol or more, more preferably 0.7 mol or more, and for example, 10 mol or less, preferably 2 mol or less, more preferably 1.5 mol or less, relative to 1 mol of the derivatizing agent.
The amount (residual amount) of the unreacted activated acid raw material after the treatment with the derivatizing agent is, for example, 0.05 mol or less, preferably 0.01 mol or less, more preferably 0.001 mol or less, relative to 1 mol of the amide bond-containing compound. Note that the amount of the activated acid raw material does not need to be 0 mol, and may be, for example, 0.000001 mol or more.

<Step C>
In step C, the reaction mixture obtained in step B is washed with a basic aqueous solution and/or an acidic aqueous solution. Examples of the acidic aqueous solution include aqueous solutions of inorganic acids such as hydrochloric acid, sulfuric acid, sodium hydrogen sulfate, potassium hydrogen sulfate, phosphoric acid, sodium hydrogen phosphate, and potassium hydrogen phosphate, and aqueous solutions of organic acids (carboxylic acids) such as formic acid, acetic acid, and citric acid. As the acidic aqueous solution, an aqueous solution of an organic acid is preferred, and an aqueous solution of citric acid is more preferred.

Examples of basic aqueous solutions include aqueous solutions of alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, and aqueous solutions of carbonates or hydrogen carbonates, with aqueous solutions of carbonates or hydrogen carbonates being preferred. Examples of the carbonates and hydrogen carbonates include lithium salts, sodium salts, and potassium salts, with sodium salts being preferred.

The acid concentration of the acidic aqueous solution and the salt concentration of the basic aqueous solution are, for example, 1 mass % or more, preferably 3 mass % or more, and for example, 20 mass % or less, preferably 10 mass % or less.
When B in the formula (7) of the derivatizing agent is _-SO3M , _-CO2M , or _-PO3M2 , it is preferable to first wash with a basic aqueous solution, and then, if necessary, wash with _an acidic aqueous solution. The washing with the basic aqueous solution is, for example, once or more, preferably twice or more. The washing with the basic aqueous solution is, for example, five times or less, preferably four times or less, more preferably three times or less. The washing with the acidic aqueous solution that is then performed is, for example, once or more, for example, three times or less, preferably two times or less.

On the other hand, when B in formula (7) of the derivatizing agent is a dialkylamino group, it is preferable to first wash with an acidic aqueous solution, and then wash with a basic aqueous solution as necessary. The washing with the acidic aqueous solution is, for example, one or more times, preferably two or more times. The washing with the acidic aqueous solution is, for example, five or less times, preferably four or less times, more preferably three or less times. The washing with the basic aqueous solution is then, for example, one or more times, for example, three or less times, preferably two or less times.

After washing with the basic or acidic aqueous solution, the mixture is separated into an organic layer (preferably a layer containing the organic solvent used in step A) and an aqueous layer (liquid-liquid separation). The target product is distributed to the organic layer, and the decomposition product of the activated acid raw material is distributed to the aqueous layer, so that the activated acid raw material can be removed. The organic layer after washing with the basic or acidic aqueous solution may be washed with water and separated as necessary. The organic layer may also be dried with a drying agent such as sodium sulfate. The product may be further purified by known methods such as solvent distillation, crystallization, recrystallization, and column chromatography. However, when the N-terminal protecting group Q is removed from the product (amide bond-containing compound) and the product is used as a reactive amine raw material in step A (i.e., when the amide bond-containing compound is elongated), it is simpler and more preferable not to perform purification such as crystallization, recrystallization, and column chromatography.

The emulsion problem can also be solved by distilling off the organic solvent contained in the reaction mixture after washing with a basic aqueous solution or an acidic aqueous solution, precipitating the target product (amide bond-containing compound) in the remaining aqueous solvent, and subjecting it to solid-liquid separation (filtration, centrifugation, etc.). The target product recovered by solid-liquid separation may be further purified by known techniques such as crystallization, recrystallization, column chromatography, etc., but when the N-terminal protecting group Q is removed from the product (amide bond-containing compound) and the product is used as a reactive amine raw material in step A (i.e., when the amide bond-containing compound is elongated), it is simpler and more preferable not to perform purification such as crystallization, recrystallization, column chromatography, etc.
When the problems of the emulsion are solved by utilizing this solid-liquid separation, a compound other than the compound represented by the above formula (7) (for example, the derivatizing agent used in the Comparative Example described later) can also be used as the derivatizing agent (scavenger) as long as it is a compound having a group that reacts with an active ester, such as an amino group or a sulfanyl group, and an acidic group, such as a sulfo group or a carboxy group, or a basic group, such as a dialkylamino group, as a water-soluble group.

The yield of the target product (amide bond-containing compound) obtained in step C is, for example, 60 mol% or more, preferably 70 mol% or more, more preferably 80 mol% or more, and particularly preferably 85 mol% or more, and is, for example, 100 mol% or less, or 99 mol% or less.

The purity of the target product (amide bond-containing compound) obtained in step C is, for example, 70% or more, preferably 75% or more, more preferably 80% or more, even more preferably 85% or more, and particularly preferably 90% or more, and is, for example, 100% or less, or 98% or less.

The target product (amide bond-containing compound) purified in step C can be used as a raw material for further reactions. For example, after removing the amino protecting group Q at the N-terminus, it can be returned to step A as a reactive amine raw material, and then steps B and C can be carried out to elongate the amide bond-containing compound. In this case, the value of n in formula (1) is increased by one. The target product (amide bond-containing compound) can also be used as a raw material to carry out an elongation reaction of the amide bond-containing compound by a known method different from steps A to C. When the target product (amide bond-containing compound) purified in step C is used as a raw material for further reactions, it is preferable not to carry out isolation treatments such as crystallization, recrystallization, and column chromatography in step C. By using the reaction product (amide bond-containing compound) as a raw material for the next step without carrying out isolation treatment, the efficiency of the entire process can be improved.

The method for producing an amide bond-containing compound of the present invention and the obtained amide bond-containing compound are useful for producing pyrrole imidazole (poly)amide (PIPA). PIPA means an amide bond-containing compound whose main constituent units are pyrrole and imidazole, and compounds such as those represented by the following formulae (101), (102), and (103) are known. PIPA is a gene expression control compound that binds in a sequence-specific manner to the minor groove of double-stranded DNA to inhibit the transcription of target genes. Since it is resistant to nucleases and has nuclear membrane permeability, it is expected to be used in diagnostic applications for identifying DNA sequences and as a molecular probe, and is also expected to be used as a transcription control agent that inhibits the binding interaction between transcription factors and double-stranded DNA.

This application claims the benefit of priority based on Japanese Patent Application No. 2020-058966, filed on March 27, 2020. The entire contents of the specification of Japanese Patent Application No. 2020-058966, filed on March 27, 2020, are incorporated by reference into this application.

The present invention will be explained in more detail below with reference to examples. However, the present invention is not limited to the following examples, and it is of course possible to carry out the invention with appropriate modifications within the scope of the purpose described above and below, all of which are included in the technical scope of the present invention. In the following, unless otherwise specified, "parts" means "parts by mass" and "%" means "% by mass."

1. Measurement Method The HPLC conditions are as follows.
Measuring equipment: Agilent 1220 Infinity
Column: YMC Meteoric Core C18 (150 x 4.6 mm)
Flow rate: 1.5ml/ml
Detection wavelength: 210 nm
Injection volume: 10 μl
Mobile phase A: 0.1 wt% phosphoric acid aqueous solution Mobile phase B: acetonitrile Gradient conditions (mobile phase B concentration, time): 15% (0 min) → 80% (8 to 15 min) → 15% (15.1 to 20 min)

The HPLC purity of the product was calculated from the measurement results under the above-mentioned HPLC conditions using the following formula:
HPLC purity (% area) = product peak area / total peak area × 100
However, the total peak area was calculated by excluding the peaks from the blank analysis from the measurement results.

The amount of the remaining active ester was calculated from the measurement results under the above-mentioned HPLC conditions by the following formula:
Amount of remaining active ester (% area) = peak area of target compound / total peak area × 100
However, the total peak area was calculated by excluding the peaks from the blank analysis from the measurement results.

The amount of the product obtained was calculated from the amount of the synthesized compound and the HPLC purity of the product described above. The calculation formula is as follows.
Amount of product obtained = amount of synthesized compound × HPLC purity (%) / 100

2. Symbols The symbols used in the Examples section have the following meanings:
Boc: t-butoxycarbonyl group Bzl, Bn: benzyl group Py: N-methylpyrrole ring Im: N-methylimidazole ring HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine HOBt: 1-hydroxybenzotriazole HOAt: 1-hydroxy-7-azabenzotriazole Oxyma pure: ethyl cyanoglycosylate-2-oxime DMF: dimethylformamide

The active ester and amine raw materials used in the examples are as follows.
3. Active esters
Synthesis Example 1 [Production of Boc-Py-OOBt (C-1)]

To Boc-Py-OH (10.0 g, 41.6 mmol), dichloromethane (125 mL), HOOBt (8.2 g, 50.0 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter referred to as EDC hydrochloride) (9.6 g, 50.0 mmol) were added and stirred at room temperature for 1.5 hours. Next, EDC was removed by washing with H ₂ O (200 mL x 3). The obtained organic layer was dried using sodium sulfate, filtered, and distilled under reduced pressure at 40°C to obtain 16.6 g of a concentrate. Next, 50.0 g of ethyl acetate was added, heated to 70°C, and then cooled to 5°C while stirring. After 1 hour, the precipitate was collected by filtration and dried under vacuum at 50°C to obtain compound (C-1) (14.1 g, 36.6 mmol, yield 87.8%).

Synthesis Examples 2 to 10
Active esters (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), (C-9), and (C-10) were produced in the same manner as in Synthesis Example 1, except that carboxylic acid compounds (B-1), (B-2), (B-3), (B-4), (B-5), (B-6), and (B-7) were combined with activators shown in Table 1. The results are shown in Table 1.

4. Amine raw materials
Synthesis Example 11 [Production of Boc-Py-ONBn (A-1)]

To Boc-Py-OH (10.0 g, 41.5 mmol), DMF (100 mL), H ₂ O (17 mL), and Cs ₂ CO ₃ (7.2 g, 22.0 mmol) were added, and the mixture was stirred at 40° C. for 30 minutes. Then, 4-nitrobenzyl bromide (10.2 g, 47.3 mmol) was added, and the mixture was stirred for 29 hours. Next, the reaction solution was added to H ₂ O (50 mL) in an ice bath, and the mixture was stirred for 30 minutes. The resulting precipitate was collected by filtration and dried in vacuum at 50° C. to obtain the target compound (A-1) (11.3 g, 30.3 mmol, yield 73.0%).

Synthesis Example 12 [Production of H-Im-ONBn HCl salt (A-2)]

To Boc-Im-OH (10.0 g, 41.5 mmol), DMF (100 mL), H ₂ O (17 mL), and Cs ₂ CO ₃ (7.2 g, 22.0 mmol) were added, and the mixture was stirred at 40° C. for 30 minutes. Then, 4-nitrobenzyl bromide (10.2 g, 47.3 mmol) was added, and the mixture was stirred for 29 hours. Next, the reaction solution was added to H ₂ O (500 mL) in an ice bath, and the mixture was stirred for 30 minutes. The resulting precipitate was collected by filtration and dried in vacuum at 50° C. to obtain Boc-Im-ONBn (11.3 g, 30.3 mmol, yield 73.0%).

Next, the Boc-Im-ONBn (11.3 g, 30.0 mmol) obtained above was added to 14% HCl/dioxane (117.2 g, 450 mmol) and stirred at room temperature for 22 hours. Next, hexane (112.9 g) was added dropwise at room temperature and mixed. After stirring for an additional hour in an ice bath, the precipitate was filtered and dried in vacuum at 40°C to obtain the target compound (A-2) (9.6 g, 30.0 mmol, yield 100%).

5. Amide synthesis
Comparative Example 1
[Production of Boc-Py-Py-ONBn (D-1)]

Dichloromethane (0.8 mL) was added to Boc-Py-ONBn (A-1) (0.10 g, 0.27 mmol) to dissolve it, and then methanesulfonic acid (hereinafter referred to as MsOH) (0.09 g, 0.95 mmol) was added dropwise at 20° C. and mixed. After 30 minutes, triethylamine (0.15 g, 1.49 mmol) was added dropwise in an ice bath, and the temperature was raised to 20° C. Next, Boc-Py-OOBt (C-1) (0.12 g, 0.32 mmol) was added, and the mixture was stirred at 40° C. for 1 hour. Next, the mixture was washed with 5% aqueous sodium bicarbonate solution (1 mL x 3), 5% aqueous citric acid solution (1 mL x 1), and H ₂ O (1 mL x 1), dried over sodium sulfate, and filtered. The resulting solution was then distilled under reduced pressure at 30°C to obtain 0.14 g of a concentrated product of the desired product (D-1) (yield: 76.9%, HPLC purity: 71.4%, remaining active ester: 25.5%).
[Production of Boc-Py-Py-Py-ONBn (D-2)]

Dichloromethane (0.8 mL) was added to Boc-Py-Py-ONBn (D-1) (0.14 g) to dissolve it, and then MsOH (0.12 g, 1.28 mmol) was added dropwise at 20° C. and mixed. After 30 minutes, triethylamine (0.18 g, 1.81 mmol) was added dropwise in an ice bath, and the temperature was raised to 20° C. Then, Boc-Py-OOBt (C-1) (0.12 g, 0.32 mmol) was added, and the mixture was stirred at 40° C. for 16.5 hours. Next, the mixture was washed with 5% aqueous sodium bicarbonate solution (1 mL x 3), 5% aqueous citric acid solution (1 mL x 1), and H ₂ O (1 mL x 1), dried over sodium sulfate, and filtered. The resulting solution was then distilled under reduced pressure at 30°C to obtain 0.17 g of a concentrated product of the desired product (D-2) (yield from compound (A-1): 17.1%, HPLC purity: 16.7%, remaining active ester: 42.0%).

Example 1
[Production of Boc-Py-Py-ONBn (D-1)]

After adding dichloromethane (0.8 mL) to Boc-Py-ONBn (A-1) (0.10 g, 0.27 mmol) to dissolve it, MsOH (0.10 g, 1.03 mmol) was added dropwise at 20°C and mixed. After 60 minutes, triethylamine (0.16 g, 1.57 mmol) was added dropwise in an ice bath and the temperature was raised to 20°C. Then, Boc-Py-OOBt (C-1) (0.12 g, 0.32 mmol) was added and stirred at 40°C for 1 hour. Next, triethylamine (0.08 g, 0.80 mmol) and taurine (0.10 g, 0.80 mmol) were added and stirred overnight at 40°C. Then, washing was performed with 5% aqueous sodium hydrogen carbonate solution (1 mL x 3), 5% aqueous citric acid solution (1 mL x 1), and H ₂ O (1 mL x 1). The organic layer after washing was dried over sodium sulfate and filtered, and the resulting solution was distilled under reduced pressure at 30° C. to obtain 0.14 g of a concentrate of the target product (D-1) (yield: 96.8%, HPLC purity: 91.4%, remaining active ester: 0.1%).
[Production of Boc-Py-Py-Py-ONBn (D-2)]

After adding dichloromethane (0.8 mL) to Boc-Py-Py-ONBn (D-1) (0.14 g) to dissolve it, MsOH (0.12 g, 1.24 mmol) was added dropwise at 20 ° C. and mixed. After 30 minutes, triethylamine (0.18 g, 1.81 mmol) was added dropwise in an ice bath, and the temperature was raised to 20 ° C. Then, Boc-Py-OOBt (C-1) (0.12 g, 0.32 mmol) was added and stirred at 40 ° C. for 1 hour. Next, triethylamine (0.13 g, 1.33 mmol) and taurine (0.17 g, 1.33 mmol) were added, and the mixture was stirred overnight at 40 ° C. Then, washing was performed with 5% sodium bicarbonate aqueous solution (3 mL x 3), 5% citric acid aqueous solution (3 mL x 1), and H ₂ O (3 mL x 1). The liquid separation property during washing was good. The washed organic layer was dried over sodium sulfate and filtered, and the resulting solution was distilled under reduced pressure at 30° C. to obtain 0.15 g of a concentrate of the target product (D-2) (yield from compound (A-1) 73.1%, HPLC purity 79.5%, remaining active ester: 0.8%).

The yield and quality results of Boc-Py-Py-ONBn (D-1) and Boc-Py-Py-Py-ONBn (D-2) produced in Comparative Example 1 and Example 1 are shown in Table 2.

Example 2 [Preparation of Boc-Py-Py-ONBn (D-1)]

Dichloromethane (0.8 mL) was added to Boc-Py-ONBn (A-1) (0.10 g, 0.27 mmol) to dissolve it, and then MsOH (0.08 g, 0.88 mmol) was added dropwise at 20° C. and mixed. After 30 minutes, triethylamine (0.14 g, 1.41 mmol) was added dropwise in an ice bath, and the temperature was raised to 20° C. Then, Boc-Py-OOBt (C-1) (0.12 g, 0.32 mmol) was added and stirred at 40° C. for 2 hours. Next, triethylamine (0.08 g, 0.80 mmol) and sodium 3-mercaptopropanesulfonate (0.14 g, 0.80 mmol) were added, and the mixture was stirred at 40° C. overnight. Subsequently, washing was performed with a 5% aqueous sodium hydrogen carbonate solution (1 mL x 3), a 5% aqueous citric acid solution (1 mL x 1), and H ₂ O (1 mL x 1). Liquid separation during washing was good. The organic layer after washing was dried over sodium sulfate and filtered, and the resulting solution was distilled under reduced pressure at 30°C to obtain 0.16 g of a concentrate of the target product (D-1) (HPLC purity: 86.9%, remaining active ester: 0.2%).

Examples 3 to 6, Comparative Examples 2 and 3

The same procedure was followed as in Example 2, except that the inducer shown in Table 3 was used. The results are shown in Table 3 together with those of Comparative Example 1, Example 1, and Example 2.

Examples 7 to 13

The procedure was the same as in Example 2, except that the active esters and derivatizing agents shown in Table 4 were used. The results are shown in Table 4.

Example 14 [Preparation of Boc-Py-Im-ONBn (D-6)]

化合物（Ａ－２）（１２．５ｇ、４０．０ｍｍｏｌ）にピリジン（１２０ｍＬ）、ジイソプロピルエチルアミン（以下、ＤＩＰＥＡという、１５．５ｇ、１２０ｍｍｏｌ）を加えた。次に活性エステル（Ｃ－１０）（１３．１ｇ、３６．０ｍｍｏｌ）を添加し、混合液を４０℃で終夜攪拌した。次に、ＤＩＰＥＡ（１５．５ｇ、１２０ｍｍｏｌ）、タウリン（１５．０ｇ、１２０ｍｍｏｌ）を添加し、室温で終夜攪拌した。次に３０℃で減圧留去することでピリジンを除去し、ジクロロメタン（２００ｍＬ）及びＨ₂Ｏ（２００ｍＬ）を加えて洗浄した。続いて５％炭酸ナトリウム水溶液（２００ｍＬ×３）、５％ＫＨＳＯ₄水溶液（２００ｍＬｘ２）、２０％ＮａＣｌ水溶液（２００ｍＬ×１）で洗浄を行いった。洗浄時の分液性は良好であった。洗浄後の有機層を硫酸ナトリウムを用いて乾燥、ろ過後、得られた溶液を３０℃で減圧留去することにより、目的生成物（Ｄ－６）の濃縮物を１６．５ｇ取得した（ＨＰＬＣ純度：９５．４％、残存活性エステル：不検出）。

Pyridine (120 mL) and diisopropylethylamine (hereinafter referred to as DIPEA, 15.5 g, 120 mmol) were added to compound (A-2) (12.5 g, 40.0 mmol). Next, active ester (C-10) (13.1 g, 36.0 mmol) was added, and the mixture was stirred at 40 ° C. overnight. Next, DIPEA (15.5 g, 120 mmol) and taurine (15.0 g, 120 mmol) were added, and the mixture was stirred at room temperature overnight. Next, pyridine was removed by distillation under reduced pressure at 30 ° C., and dichloromethane (200 mL) and H ₂ O (200 mL) were added for washing. Next, washing was performed with a 5% sodium carbonate aqueous solution (200 mL × 3), a 5% KHSO ₄ aqueous solution (200 mL × 2), and a 20% NaCl aqueous solution (200 mL × 1). The liquid separation property during washing was good. The washed organic layer was dried over sodium sulfate and filtered, and the resulting solution was distilled under reduced pressure at 30° C. to obtain 16.5 g of a concentrated product of the target product (D-6) (HPLC purity: 95.4%, residual active ester: not detected).

Example 15

After adding dichloromethane (0.8 mL) to Boc-Py-ONBn (A-1) (0.10 g, 0.27 mmol) and dissolving it, MsOH (0.09 g, 0.97 mmol) was added dropwise at 20 ° C. and mixed. After 30 minutes, triethylamine (0.15 g, 1.51 mmol) was added dropwise in an ice bath and the temperature was raised to 20 ° C. Then, active ester (C-3) (0.10 g, 0.32 mmol) was added and stirred at 40 ° C. for 1 hour. Next, triethylamine (0.08 g, 0.80 mmol) and taurine (0.10 g, 0.80 mmol) were added and stirred overnight at 40 ° C. Then, 5% aqueous sodium bicarbonate solution (12 mL) was added, and dichloromethane was removed by distillation under reduced pressure at 30 ° C. to obtain a slurry liquid. The obtained slurry was heated to 50° C. and aged for 1 hour, then cooled to 20° C., filtered, and dried at 30° C. to obtain 0.10 g of the target product (D-7) (HPLC purity: 80.9%, residual active ester: 0.9%).

Example 16

Dichloromethane (0.8 mL) was added to Boc-Py-ONBn (A-1) (0.10 g, 0.27 mmol) to dissolve it, and then MsOH (0.09 g, 0.96 mmol) was added dropwise at 20 ° C. and mixed. After 30 minutes, triethylamine (0.15 g, 1.50 mmol) was added dropwise in an ice bath, and the temperature was raised to 20 ° C. Then, active ester (C-4) (0.11 g, 0.32 mmol) was added and stirred at 40 ° C. for 1 hour. Next, triethylamine (0.08 g, 0.80 mmol), taurine (0.10 g, 0.80 mmol), and DMF (0.5 mL) were added, and the mixture was stirred at 40 ° C. for 2.5 hours. Then, 5% aqueous sodium hydrogen carbonate solution (12 mL) and DMF (1 mL) were added, and dichloromethane was removed by distillation under reduced pressure at 30 ° C. to obtain a slurry liquid. The obtained slurry was heated to 50° C. and aged for 1 hour, then cooled to 20° C., filtered, and dried at 35° C. to obtain 0.12 g of the target product (D-8) (HPLC purity: 87.2%, residual active ester: not detected).

Claims (9)

In the liquid phase amide bond formation reaction,
Formula (1);

[wherein n is an integer of 1 or more and 30 or less, Pro is a protecting group for the C-terminus, the protecting group for the C-terminus being an ester-based protecting group or an amide-based protecting group, ^Ra is a β-amino acid residue, a γ-amino acid residue, or an aromatic heterocycle-containing amino acid residue represented by formula (4-1) . When n is 2 or more, multiple Ra's may be the same or different. Ar3 is an aromatic heterocycle which may have a substituent other than a carboxy group and an amino group. * represents a bond. ],
Formula (2) or Formula (3);

[wherein m represents an integer of 1 or 2, Rb represents an amino acid residue, and when m is 2, the two Rb's may be the same or different. At least one of Rb's is an amino acid residue represented by formula (4). Q represents an N-terminal protecting group, and the N-terminal protecting group is a carbamate type protecting group, an acyl type protecting group, an alkoxymethyl type protecting group, or a phenylmethyl type protecting group; Ar ¹ and Ar ² each independently represent an aromatic heterocycle which may have a substituent other than a carboxy group and an amino group, and the aromatic heterocycle is thiophene, pyrrole, imidazole, thiazole, or indole . When the compound represented by formula (2) has two Ar ^2's , the two Ar ² 's may be the same or different. * represents a bond. ] is reacted with a compound obtained by active esterification, acid halide, or acid anhydride conversion to obtain a compound represented by formula (5) or formula (6);

[wherein Ra, Rb, Ar ¹ , Pro, Q, n, and m are the same as defined above],
The reaction mixture containing the amide bond-containing compound represented by formula (5) or formula (6) obtained in step A is treated with a compound represented by formula (7);

[wherein A represents an amino group or a sulfanyl group, B represents _--SO3M or _--CO2M , and ^R1 represents an alkylene group having 2 to 3 carbon atoms. M represents a hydrogen atom or an alkali metal atom, and when there are a plurality of M's, they may be the same or different.] and a monoamine compound having no acidic group or a salt of an acidic group are mixed,
A method for producing an amide bond-containing compound, comprising: a step B of reacting a compound obtained by converting a compound represented by formula (2) or (3) into an active ester, an acid halide, or an acid anhydride with a compound represented by formula (7); and a step C of washing the reaction mixture obtained in the step B with a basic and/or acidic aqueous solution. The method according to claim 1, wherein the monoamine compound having no acidic group or no salt of an acidic group is triethylamine, tributylamine, N,N-diisopropylethylamine, or N-methylmorpholine. The method according to claim 1 or 2 , wherein the compound represented by formula (7) is taurine. 4. The method according to claim 1, wherein the basic aqueous solution is an aqueous solution of a carbonate or a hydrogen carbonate. The method according to claim 4 , wherein the carbonate or hydrogen carbonate is a lithium salt, a sodium salt, or a potassium salt. The method according to any one of claims 1 to 5 , wherein the acidic aqueous solution is an aqueous solution of an organic acid. The method according to claim 6 , wherein the organic acid is citric acid. The method according to any one of claims 1 to 7 , wherein the amide bond-containing compound represented by the formula (5) or (6) is a pyrrole-imidazole (poly)amide compound. A method for producing an amide bond-containing compound represented by formula (5) by the production method according to any one of claims 1 to 8 , and deprotecting the N-terminus of the obtained amide bond-containing compound represented by formula (5) without isolating it, and then carrying out the steps A, B, and C to elongate the amide bond-containing compound.