Method for producing pteroic acid amide compound

Abstract

[Problem] To provide a production method for obtaining a pteroic acid amide at a high yield without using a protecting group. [Solution] A method for producing a pteroic acid amid compound, the production method including using an amino compound and a condensing agent together with pteroic acid as a starting material in the presence of a basic compound in a solvent containing an organic solvent so as to thereby obtain a pteroic acid amide compound.

Description

Method for producing pteroinamide compounds

[0001] This invention relates to a method for producing pteroinamide compounds.

[0002] While folate receptor expression is limited to specific organs in normal tissues, overexpression has been reported in cancerous tissues, particularly in pancreatic cancer, brain tumors, and breast cancer. Therefore, folate receptors are attracting attention as a target molecule in cancer treatment. Studies are being conducted to introduce various target compounds into folate, which binds to folate receptors.

[0003] Patent Document 1 discloses that a complex composed of folic acid, which acts as a ligand, and a cationic peptide that interacts with siRNA to form a complex, can efficiently deliver nucleic acid drugs such as siRNA into cells.

[0004] Furthermore, Patent Document 2 discloses a FITC-fluorescein isothiocyanate conjugate that is conjugated to folic acid via a γ-carboxylated ethylenediamine bridge.

[0005] On the other hand, pteroinic acid is a component of folic acid and is a suitable starting material for producing the folic acid derivatives described above.

[0006] In the reaction of compounds obtained from pteroic acid by amide reactions, such as folic acid, it is known that pteroic acid protected with the protecting group trifluoroacetic acid (TFA) is used to introduce the compound (for example, Non-Patent Document 1).

[0007] International Publication No. 2021 / 080020, Japanese Patent Publication No. 2012-092097

[0008] Bioorganic & Medicinal Chemistry Letters Volume 14, Issue 9,3 May 2004, Pages 2313-2317

[0009] The use of protected pteroylglutamic acid requires the introduction of a protecting group and a deprotection step, which complicates the process. In addition, there may be limitations in synthesis due to functional groups that cannot withstand the deprotection conditions of the protecting group or the balance with other protecting groups.

[0010] Therefore, an object of the present invention is to provide a production method that is simple in process and can obtain pteroylglutamic acid amide in a high yield.

[0011] Preferred configurations of the method for producing a pteroylglutamic acid amide compound of the present invention are described in the following (1) to (13) and the like.

[0012] (1) A method for producing a pteroylglutamic acid amide compound, comprising obtaining a pteroylglutamic acid amide compound using an amino compound and a condensing agent with pteroylglutamic acid as a starting material in a solvent containing an organic solvent in the presence of a basic compound.

[0013] (2) In a solvent containing the organic solvent, in the presence of the basic compound, the pteroylglutamic acid and the condensing agent are reacted to obtain an active esterified product of pteroylglutamic acid, and further, the active esterified product of pteroylglutamic acid and the amino compound are reacted. The method for producing a pteroylglutamic acid amide compound according to (1).

[0014] (3) A method for producing a pteroylglutamic acid amide compound, comprising reacting pteroylglutamic acid and a condensing agent in a solvent containing an organic solvent in the presence of a basic compound to obtain an active esterified product of pteroylglutamic acid, and further, reacting the active esterified product of pteroylglutamic acid and an amino compound.

[0015] (4) A method for producing a pteroylglutamic acid amide compound according to (1), comprising reacting pteroylglutamic acid and an amino compound with a condensing agent in a solvent containing an organic solvent in the presence of a basic compound.

[0016] (5) The method for producing a pteroylglutamic acid amide compound according to any one of (1) to (4), wherein the amino compound is a compound having a structural unit derived from an amino acid.

[0017] (6) The method for producing a pteroic acid amide compound according to (5), wherein the amino compound is a compound having a structural unit derived from an amino acid at its terminal.

[0018] (7) The method for producing a pteroic acid amide compound according to any one of (1) to (6), wherein the amino compound is a compound having a structural unit derived from glutamic acid.

[0019] (8) The method for producing a pteroic acid amide compound according to any one of (1) to (7), wherein the basic compound is a quaternary ammonium salt.

[0020] (9) The method for producing a pteroic acid amide compound according to any one of (1) to (8), wherein the basic compound has a tetraalkylammonium structure.

[0021] (10) The method for producing a pteroic acid amide compound according to (9), wherein the basic compound is tetraalkylammonium hydroxide.

[0022] (10) The method for producing a pteroic acid amide compound according to any one of (1) to (9), wherein the condensing agent is a condensing agent that acts under basic conditions.

[0023] (11) The method for producing a pteroic acid amide compound according to any one of (1) to (10), wherein the condensing agent is at least one selected from the group consisting of uronium-based condensing agents, phosphonium-based condensing agents, triazine-based condensing agents, imidazolinium-based condensing agents, carbonate ester-based condensing agents, carbodiimidazole-based condensing agents, formamidinium-based condensing agents, borophosphorus-based condensing agents, acid anhydride-based condensing agents, and pyridinium-based condensing agents.

[0024] (12) The method for producing a pteroic acid amide compound according to any one of (1) to (11), wherein the amino compound is a polypeptide.

[0025] (13) The method for producing a pteroic acid amide compound according to any one of (1) to (12), wherein the organic solvent contains at least one selected from the group consisting of amide solvents and sulfoxide solvents.

[0026] Furthermore, preferred configurations of the method for introducing an amide bond to pteroic acid according to the present invention are described in (14) to (24) below.

[0027] (14) A method for introducing an amide bond to pteroic acid, comprising mixing a basic compound and pteroic acid in a solvent containing an organic solvent, further obtaining an active esterified product of pteroic acid with a condensing agent, and reacting the active esterified product of pteroic acid with an amino compound.

[0028] (15) The amino compound is a compound having structural units derived from an amino acid, a method for introducing an amide bond to pteroic acid as described in (14).

[0029] (16) The method for introducing an amide bond to pteroic acid as described in (15), wherein the amino compound is a compound having a structural unit derived from an amino acid at its terminal end.

[0030] (17) A method for introducing an amide bond to pteroic acid according to any one of (14) to (16), wherein the amino compound is a compound having a structural unit derived from glutamic acid.

[0031] (18) A method for introducing an amide bond to pteroic acid according to any one of (14) to (17), wherein the basic compound is a quaternary ammonium salt.

[0032] (19) A method for introducing an amide bond to a pteroic acid according to any one of (14) to (18), wherein the basic compound has a tetraalkylammonium structure.

[0033] (20) A method for introducing an amide bond to pteroic acid according to (19), wherein the basic compound is tetraalkylammonium hydroxide.

[0034] (21) A method for introducing an amide bond to pteroic acid according to any one of (14) to (20), wherein the condensing agent is a condensing agent that acts under basic conditions.

[0035] (22) A method for introducing an amide bond to pteroic acid according to any one of (14) to (21), wherein the condensing agent is at least one selected from the group consisting of uronium-based condensing agents, phosphonium-based condensing agents, triazine-based condensing agents, imidazolinium-based condensing agents, carbonate ester-based condensing agents, carbodiimidazole, formamidinium-based condensing agents, phosphalus-based condensing agents, acid anhydride-based condensing agents and pyridinium-based condensing agents.

[0036] (23) A method for introducing an amide bond to pteroic acid according to any one of (14) to (22), wherein the amino compound is a polypeptide.

[0037] (24) A method for introducing an amide bond to pteroic acid according to any one of (14) to (23), wherein the organic solvent comprises at least one selected from the group consisting of amide solvents and sulfoxide solvents.

[0038] This is a chromatogram of the reaction product obtained by the method using DIC / Oxyma (comparative example). This is a chromatogram of the reaction product obtained by the method of one embodiment of the present invention.

[0039] One embodiment of the present invention is a method for producing a pteroinamide compound, comprising using pteroic acid as a starting material, an amino compound, and a condensing agent in a solvent containing an organic solvent in the presence of a basic compound to obtain a pteroinamide compound. Another embodiment of the present invention is a method for producing a pteroinamide compound, comprising reacting pteroic acid and a condensing agent in a solvent containing an organic solvent in the presence of a basic compound to obtain an active ester of pteroic acid, and further reacting the active ester of pteroic acid with an amino compound.

[0040] Another embodiment of the present invention is a method for introducing an amide bond to pteroic acid, comprising mixing a basic compound and pteroic acid in a solvent containing an organic solvent, further obtaining an active ester of pteroic acid with a condensing agent, and reacting the active ester of pteroic acid with an amino compound.

[0041] According to the method for producing the pteroinic acid amide compound of this embodiment, or the method for introducing an amide bond to pteroic acid of this embodiment, it is not necessary to use a protecting group on pteroic acid, thus the process is simple and pteroinic acid amide can be obtained in high yield.

[0042] As described above, it is possible to react pteroic acid with amino compounds (e.g., polypeptides) by introducing a protecting group to pteroic acid. However, the introduction of a protecting group has various drawbacks, such as requiring a deprotection step or imposing constraints on the synthesis method due to deprotection. For example, when pteroic acid is protected with a TFA group, it is necessary to use a base (such as hydrazine) to remove the TFA group, increasing the number of processing steps, and there is a concern that functional groups sensitive to these conditions may cause side reactions. For this reason, the inventors attempted to form an amide bond with an amino compound using unprotected pteroic acid. Carboxylic acid derivatives used in amine-carboxylic acid condensation reactions include acid chlorides, acid anhydrides, and activated esters. As a result of various studies, the inventors found that an activated esterified product can be efficiently obtained from unprotected pteroic acid under basic conditions, and that the reaction with the amino group in the amino compound proceeds efficiently when using this activated esterified product. In the production method of the present invention, pteroic acid is converted to a pteroinate under basic conditions, and becomes soluble in organic solvents (particularly polar solvents). It is believed that this soluble pteroinate acts on the coupling agent, allowing for the efficient production of the activated esterified product.

[0043] Preferred embodiments of the present invention will be described below. However, the present invention is not limited to the embodiments described below. Furthermore, combinations of two or more of the individual preferred forms of the present invention described below are also preferred forms of the present invention.

[0044] In this specification, the range "X to Y" means "X or greater and Y or less," and "weight" and "mass" are treated as synonyms.

[0045] <Step 1: Step of reacting pteroic acid and a condensing agent in a solvent containing an organic solvent in the presence of a basic compound> In Step 1, it is preferable to mix the basic compound and pteroic acid in a solvent containing an organic solvent (hereinafter also simply referred to as the solvent). That is, it is preferable to mix the basic compound and pteroic acid in the solvent beforehand, before adding the condensing agent (before activating the pteroic acid). The order in which the basic compound and pteroic acid are mixed into the solvent is not particularly limited; the solvent, basic compound and pteroic acid may be mixed all at once, or the pteroic acid may be mixed into the solvent beforehand and then the basic compound may be added, or the basic compound may be mixed into the solvent beforehand and then the pteroic acid may be added. From the viewpoint of ease of operation, it is preferable to mix the pteroic acid into the solvent beforehand and then add the basic compound.

[0046] The mixing conditions are not particularly limited, and the temperature may be, for example, -20 to 150°C, 0 to 120°C, 10 to 100°C, or 15 to 50°C. Similarly, the mixing time is not particularly limited as long as the mixing proceeds uniformly, but may be, for example, 1 minute to 50 hours, 2 minutes to 10 hours, or 3 minutes to 1 hour. Stirring or shaking may be performed during mixing.

[0047] The amount of pteroic acid added to the solvent is, for example, 0.05 to 1 mol / L, considering its reactivity.

[0048] (Solvent) The activation of pteroic acid is carried out in a solvent containing an organic solvent. Considering the solubility of the organic amine involved in the condensation reaction and the reactivity of the condensation reaction, the solvent contains at least an organic solvent.

[0049] Organic solvents include amide solvents such as carboxylic acid amides (e.g., linear amides such as formamide, N-methylformamide, N,N-dimethylformamide, and cyclic amides such as N-methylpyrrolidone), phosphate amides (e.g., hexamethylphosphate), and acetamides (e.g., acetamide, N-methylacetamide, N,N-dimethylacetamide); sulfoxide solvents such as dimethyl sulfoxide (DMSO); nitrile solvents such as linear nitriles such as acetonitrile, propionitrile, and acrylonitrile, and cyclic nitriles such as benzonitrile; and linear ethers such as diethyl ether, ethylene glycol dimethyl ether, diethylene glycol dimethyl ether, and triethylene glycol dimethyl ether. Examples include ether solvents such as tetrahydrofuran (THF) and cyclic ethers such as dioxane; hydrocarbon solvents such as chain hydrocarbons such as n-hexane and aromatic hydrocarbons such as benzene, toluene, and p-xylene; halogen solvents such as haloalkanes such as dichloromethane, dichloroethane, and perfluorohexane, aryl halides such as chlorobenzene, and alkylarene halides such as trifluorotoluene and hexafluorometaxylene; urea solvents such as N,N-dimethylpropylene urea; ester solvents such as acetate esters; ketone solvents such as acetone and methyl ethyl ketone; and sulfone solvents such as sulfolane, ethyl isopropyl sulfone, dimethyl sulfone, and dinormal propyl sulfone. These organic solvents may be used individually or in combination of two or more.

[0050] As for the organic solvent, from the viewpoint of solubility of pteroic acid, it is preferably a polar solvent, preferably at least one selected from the group consisting of amide solvents, sulfoxide solvents, and sulfone solvents, more preferably at least one selected from the group consisting of amide solvents and sulfoxide solvents, and even more preferably an amide solvent. Furthermore, as the organic solvent, it is more preferably at least one selected from the group consisting of N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, and phosphate amide, even more preferably containing at least N-methylpyrrolidone and / or N,N-dimethylformamide, and particularly preferably containing at least N-methylpyrrolidone.

[0051] The solvent may further contain non-organic solvents such as water. The content of non-organic solvents in the solvent may be 10% by mass or less (lower limit 0% by mass), 5% by mass or less, 3% by mass or less, or 1% by mass or less. Alternatively, the content of non-organic solvents in the solvent may be 0.1 to 15% by mass, 0.1 to 10% by mass, 1 to 8% by mass, or 3 to 8% by mass. The basic compound may also be provided in the form of an aqueous solution, and therefore the solvent may be a mixed solvent of water and an organic solvent. Furthermore, the content of organic solvents in the solvent may be, for example, 30 to 100% by mass, 40 to 100% by mass, 50 to 100% by mass, 60 to 100% by mass, 70 to 100% by mass, 70 to 99% by mass, or 75 to 95% by mass.

[0052] (Basic compound) The basic compound is preferably one that can (partially) dissolve pteroic acid in the solvent. Specifically, it is preferably at least one selected from the group consisting of alkali metal salts, alkaline earth metal salts, cyclic amines, cyclic amidines, and quaternary ammonium salts. A quaternary ammonium salt is preferred because it readily dissolves pteroic acid in the solvent. In one embodiment, the basic compound is an organic basic compound due to its affinity for organic solvents.

[0053] Examples of alkali metal salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, lithium bicarbonate, sodium bicarbonate, potassium bicarbonate, cesium bicarbonate, lithium phosphate, sodium phosphate, potassium phosphate, cesium phosphate, dilithium hydrogen phosphate, dipotassium hydrogen phosphate, dicesium hydrogen phosphate, monolithium hydrogen phosphate, monosodium hydrogen phosphate, monopotassium hydrogen phosphate, monolithium hydrogen phosphate, lithium borate, sodium borate, potassium borate, cesium borate, lithium citrate, sodium citrate, potassium citrate, cesium citrate, lithium formate, sodium formate, potassium formate, cesium formate, lithium acetate, sodium acetate, potassium acetate, cesium acetate, lithium oxalate, sodium oxalate, potassium oxalate, cesium oxalate, lithium maleate, potassium maleate, cesium maleate, and others.

[0054] Examples of alkaline earth metal salts include magnesium hydroxide, calcium hydroxide, barium hydroxide, magnesium carbonate, calcium carbonate, barium carbonate, magnesium phosphate, calcium phosphate, barium phosphate, magnesium hydrogen phosphate, calcium hydrogen phosphate, barium hydrogen phosphate, dimagnesium hydrogen phosphate, dicalcium hydrogen phosphate, dibarium hydrogen phosphate, magnesium borate, calcium borate, barium borate, magnesium citrate, calcium citrate, barium citrate, magnesium formate, calcium formate, barium formate, magnesium acetate, calcium acetate, barium acetate, magnesium borate, calcium borate, barium borate, magnesium oxalate, calcium oxalate, barium oxalate, magnesium maleate, calcium maleate, and barium maleate.

[0055] Examples of cyclic amines include 1,4-diazabicyclo[2.2.2]octane (DABCO) and 1,8-bis(dimethylaminonaphthalene). Examples of cyclic amidines include diazabicycloundecene (DBU) and diazabicyclononene (DBN).

[0056] A quaternary ammonium salt is composed of a quaternary ammonium cation and an anion (counteranion). In a preferred embodiment, the quaternary ammonium salt is a tetraalkylammonium salt.

[0057] The above anions (counteranions) are not particularly limited, but for example, fluoride ions (F － ), chloride ions (Cl － ), bromide ions (Br － ) and iodide ions (I － ) and other halide ions; hydroxide ions (OH － Examples include the following. Among these, hydroxide ions are preferred.

[0058] Quaternary ammonium cations preferably have four alkyl groups (i.e., a tetraalkylammonium structure). Having four alkyl groups increases the affinity of quaternary ammonium cations to organic solvents, allowing the reaction to proceed more efficiently.

[0059] Here, the alkyl group is either substituted or unsubstituted, and the substituent may be an aryl group having 6 to 30 carbon atoms, or a phenyl group. It is preferable that the alkyl group is unsubstituted.

[0060] As for the alkyl group present in the tetraalkyl group, C1 to C16 alkyl groups are preferred, C1 to C8 alkyl groups are more preferred, C1 to C6 linear or branched alkyl groups are even more preferred, C1 to C4 linear or branched alkyl groups are even more preferred, propyl groups and butyl groups (more preferably linear propyl groups and butyl groups) are even more preferred, and butyl groups are particularly preferred, as they allow the reaction to proceed more efficiently. Of the four alkyl groups present in the tetraalkyl group, at least one (preferably at least two, more preferably at least three, and even more preferably all alkyl groups) is preferably a C1 to C16 alkyl group, more preferably a C1 to C8 alkyl group, more preferably a C1 to C6 linear or branched alkyl group, and even more preferably a C1 to C4 linear or branched alkyl group, and propyl groups and butyl groups are particularly preferred. The C1 to C8 alkyl group is not particularly limited and includes C1 to C8 linear, branched or cyclic alkyl groups. More specifically, examples of C1-C8 alkyl groups include linear, branched, or cyclic alkyl groups such as methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, isopentyl, neopentyl, cyclopentyl, n-hexyl, cyclohexyl, n-heptyl, n-octyl, and 2-ethylhexyl groups. Part of the alkyl group may be substituted, with examples of such substituents including hydroxyl and phenyl groups. The alkyl groups contained in the quaternary ammonium cation (quaternary ammonium salt) may be identical or different. It is preferable that the alkyl groups contained in the quaternary ammonium cation (quaternary ammonium salt) are identical.

[0061] In one embodiment, the quaternary ammonium salt may be composed of a quaternary ammonium cation having four linear or branched alkyl groups having 1 to 6 carbon atoms, and a hydroxide ion. In another embodiment, the quaternary ammonium salt may be composed of a quaternary ammonium cation having four linear or branched alkyl groups having 1 to 4 carbon atoms, and a hydroxide ion. In yet another embodiment, the quaternary ammonium salt may be composed of a quaternary ammonium cation having four butyl groups, and a hydroxide ion.

[0062] Examples of such quaternary ammonium salts (preferably having four alkyl groups) include tetramethylammonium hydroxide, trimethyl(2-hydroxyethyl)ammonium hydroxide, dimethylbis(2-hydroxyethyl)ammonium hydroxide, monomethyltris(2-hydroxyethyl)ammonium hydroxide, tetraethylammonium hydroxide, tetrapropylammonium hydroxide, tetrabutylammonium hydroxide, benzyltrimethylammonium hydroxide, tetrabutylammonium fluoride, and tetramethylammonium fluoride. In particular, from the viewpoint of reactivity, the basic compound is preferably tetraalkylammonium hydroxide, preferably at least one selected from the group consisting of tetramethylammonium hydroxide, trimethyl(2-hydroxyethyl)ammonium hydroxide, dimethylbis(2-hydroxyethyl)ammonium hydroxide, monomethyltris(2-hydroxyethyl)ammonium hydroxide, tetraethylammonium hydroxide, tetrapropylammonium hydroxide, tetrabutylammonium hydroxide, and benzyltrimethylammonium hydroxide, more preferably at least one selected from the group consisting of tetramethylammonium hydroxide, tetraethylammonium hydroxide, tetrapropylammonium hydroxide, and tetrabutylammonium hydroxide, even more preferably tetrapropylammonium hydroxide and / or tetrabutylammonium hydroxide, even more preferably tetra-n-propylammonium hydroxide and / or tetra-n-butylammonium hydroxide (TBAOH), and even more preferably tetra-n-butylammonium hydroxide (TBAOH).

[0063] Basic compounds may be used individually or in combination of two or more.

[0064] The lower limit of the pKa of a basic compound is, for example, 9, 10, 11, or 12. The upper limit of the pKa of a basic compound is, for example, 20, preferably 16, and more preferably 15. The pKa values ​​of basic compounds are, for example, 9 to 20, 10 to 16, 11 to 15, or 12 to 15. For pKa values ​​of basic compounds, refer to, for example, the Chemical Handbook (Basic Edition), 6th revised edition, edited by the Chemical Society of Japan (Maruzen Publishing), etc.

[0065] The amount of basic compound added is preferably set appropriately so that a salt of pteroic acid is formed. Pteroic acid is thought to form a salt in two steps, as shown in the structure below (in the following examples, the basic compound is given as A-OH).

[0066]

[0067] Therefore, theoretically, it is thought that a salt of pteroic acid can be formed by adding 2 molar equivalents of a basic compound to 1 mole of pteroic acid.

[0068] The amount of basic compound added to pteroic acid is, for example, 0.5 to 5 moles per mole of pteroic acid.

[0069] The basic compound may be added in the form of an aqueous solution. The solute concentration of the aqueous solution is, for example, 1 to 99% by mass.

[0070] (Condensing agent) A condensing agent is a compound that activates a carboxyl group and promotes a condensation reaction with an amino group in an amino compound.

[0071] Since the present invention involves dissolving / dispersing pteroic acid in a solvent using a basic compound, the condensing agent is preferably one that acts under basic conditions. The condensing agent is not particularly limited, but from the viewpoint of reactivity of the condensation reaction, it is preferably at least one selected from the group consisting of uronium-based condensing agents, phosphonium-based condensing agents, triazine-based condensing agents, imidazolinium-based condensing agents, carbonate ester-based condensing agents, carbodiimidazole-based condensing agents, formamidinium-based condensing agents, phosphalus-based condensing agents, acid anhydride-based condensing agents, and pyridinium-based condensing agents. It is more preferably at least one selected from the group consisting of uronium-based condensing agents, phosphonium-based condensing agents, and triazine-based condensing agents, and even more preferably a uronium-based condensing agent and / or a phosphonium-based condensing agent.

[0072] Examples of uronium-based bonding agents include O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU), and ((((1-cyano-2-ethoxy-2-oxoethylidene)amino)oxy)-4-morpholinomethylene)dimethylammonium hexafluorophosphate (O-(1-cyano-2-ethoxy-2-oxoethylideneaminooxy)-N,N-dimethylaminomorpholino-uronium hexafluorophosphate) (COMU).

[0073] Examples of phosphonium-based condensing agents include 1H-benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), (7-azabenzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), chlorotripyrrolidinophosphonium hexafluorophosphate (PyClOP), bromotris(dimethylamino)phosphonium hexafluorophosphate (Brop), and 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4(3H)-one (DEPBT).

[0074] Examples of triazine-based condensing agents include 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (n-hydrate) (DMT-MM).

[0075] Examples of imidazolinium-based condensing agents include 2-chloro-1,3-dimethylimidazolinium and 2-fluoro-1,3-dimethylimidazolinium.

[0076] Examples of carbonate ester-based condensing agents include N,N'-disuccinimidyl carbonate, bis(4-nitrophenyl) carbonate, and bis(pentafluorophenyl) carbonate.

[0077] Examples of carbodiimidazole-based condensing agents include 1,1'-carbonyldiimidazole (CDI), 1,1'-carbonyldi(1,2,4-triazole) (CDT), and 1,1'-oxalyldiimidazole.

[0078] Examples of formamidinium-based condensing agents include chloro-N,N,N',N'-tetramethylformamidinium, chloro-N,N,N',N'-bis(tetramethylene)formamidinium, chloro-N,N,N',N'-bis(pentamethylene)formamidinium, fluoro-N,N,N',N'-tetramethylformamidinium, and fluoro-N,N,N',N'-bis(tetramethylene)formamidinium.

[0079] Examples of phosphatidyl condensing agents include diphenylphosphoryl azide.

[0080] Examples of acid anhydride-based condensing agents include 2-methyl-6-nitrobenzoic anhydride.

[0081] Examples of pyridinium-based condensing agents include 2-chloro-1-methylpyridinium iodide, 2-chloro-1-methylpyridinium-p-toluenesulfonate, and 2-fluoro-1-methylpyridinium-p-toluenesulfonate.

[0082] Among these coupling agents, in terms of reactivity, the coupling agents are O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), ((((1-cyano-2-ethoxy-2-oxoethylidene)amino)oxy)-4-morpholinomethylene)dimethylammonium hexafluorophosphate (COMU), 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium=chloromethyluronium It is preferable that it is at least one selected from the group consisting of lido (n-hydrate) (DMT-MM), more preferably at least one selected from the group consisting of O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), and even more preferably O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU).

[0083] Furthermore, in a preferred embodiment, when the condensing agent is HATU, the organic solvent used is preferably an amide solvent, more preferably containing at least N-methylpyrrolidone and / or N,N-dimethylformamide, even more preferably containing at least N-methylpyrrolidone, and the organic solvent may consist only of N-methylpyrrolidone.

[0084] The condensing agent may be used alone or in combination of two or more types.

[0085] The amount of condensing agent added to pteroic acid is, for example, 0.5 to 5 moles per mole of pteroic acid.

[0086] (Reaction Conditions) The reaction temperature for obtaining the active esterified product is not particularly limited as long as the reaction proceeds. The reaction temperature may be, for example, -20 to 150°C, 0 to 120°C, 10 to 100°C, or 15 to 50°C. Similarly, the reaction time is not particularly limited as long as the reaction proceeds, but may be, for example, 1 minute to 50 hours, 1 minute to 10 hours, or 1 minute to 30 minutes. Stirring or shaking may be performed during the reaction.

[0087] <Step 2: Reaction of the activated esterified pteroic acid with an amino compound> In this step, the activated esterified pteroic acid is reacted with an amino compound.

[0088] Specifically, an amino compound is mixed with the solvent solution of the activated esterified pteroic acid obtained in step 1 above to carry out an amide condensation reaction.

[0089] (Reaction Conditions) The reaction temperature during the amide condensation reaction is not particularly limited as long as the reaction proceeds. The reaction temperature may be, for example, -20 to 150°C, 0 to 120°C, 10 to 100°C, or 15 to 50°C. Similarly, the reaction time is not particularly limited as long as the reaction proceeds, but may be, for example, 1 minute to 50 hours, 5 minutes to 24 hours, or 1 hour to 12 hours. Stirring or shaking may be performed during the reaction.

[0090] The mixing ratio of the active esterified form of pteroic acid and the amino compound is, for example, 5 to 0.1 mol of the amino compound per 1 mol of the active esterified form of pteroic acid.

[0091] (Amino compound) The amino compound is not particularly limited as long as it reacts with pteroic acid to form an amide compound, and is ammonia, a primary amine, or a secondary amine, each having the formula NH ３ , R-NH ２ , or RR'-NH (where R and R' each represent a substituent). Preferably, the amino compound contains R-NH ２ , and / or RR'-NH, and more preferably contains R-NH ２ .

[0092] The amino compound is preferably a compound having a structural unit derived from an amino acid, and more preferably a compound having a structure derived from an amino acid at the terminal. Further, the amino compound is preferably a compound having a structural unit derived from glutamic acid, and more preferably a compound having a structure derived from glutamic acid at the terminal.

[0093] Folic acid is composed of pteroic acid and glutamic acid as follows.

[0094]

[0095] Therefore, when the amino compound is a compound having a structure derived from glutamic acid at the terminal, an amide bond is formed by the condensation reaction between the carboxyl group of the activated pteroic acid and the amino group in the structure derived from glutamic acid, and a structure derived from folic acid (folic acid derivative) is formed.

[0096] When attempting to obtain folic acid derivatives by introducing compounds containing amino groups into folic acid, the low solubility of folic acid results in low reactivity. Furthermore, unreacted folic acid does not dissolve in the solvent during washing after the introduction reaction, making washing and separation difficult. Additionally, because folic acid contains two carboxyl groups, the amino group reacts with both carboxyl groups, resulting in the formation of positional isomers and preventing the acquisition of a single compound. This manufacturing method not only allows for the efficient introduction of pteroic acid, which is similarly poorly soluble as folic acid, but also solves the problems of washing and separation. Moreover, since folic acid is not used as a starting material, the separation of compounds containing structural units derived from folic acid is easy. Additionally, one of the carboxyl groups of glutamic acid can be easily protected with a detachable t-Bu group, making it easier to obtain a single compound.

[0097] Furthermore, examples of compounds having structural units derived from amino acids include polypeptides having structures derived from amino acids at their termini. Examples of compounds having structural units derived from glutamic acid include polypeptides having structures derived from glutamic acid at their termini. Polypeptides having structures derived from glutamic acid (amino acids) at their termini are not particularly limited, but for example, they are composed of 2 to 50, 3 to 45, or 4 to 40 amino acids (or structures derived from them). The compounds obtained in the examples described later are Glu-Gly-Gly-Gly-(Dab) ８ It is composed of a total of 12 amino acids (where Dab refers to 2,4-diaminobutyric acid). Furthermore, as a compound having a structural unit derived from glutamic acid, it may also be a polypeptide in which other amino acid structures are further bonded to the terminal glutamic acid structure of a polypeptide having a terminal glutamic acid structure. The compound obtained in the examples described later is Xaa-Glu-Gly-Gly-Gly-(Dab) ８ It is composed of a total of 12 amino acids (where Dab refers to 2,4-diaminobutyric acid, and Xaa is one of Phe, Pro, or Val).

[0098] In this specification, "amino acid" means a compound having a carboxyl group and an amino group. The type of amino acid is not particularly limited, and it may be a D-form, L-form, or racemic mixture. Furthermore, from the viewpoint of the relative positions of the carboxyl group and the amino group, it may be any of α-amino acids, β-amino acids, γ-amino acids, δ-amino acids, ω-amino acids, etc. Examples of amino acids include valine, leucine, isoleucine, alanine, arginine, glutamine, lysine, aspartic acid, glutamic acid, proline, cysteine, threonine, methionine, histidine, phenylalanine, tyrosine, tryptophan, asparagine, glycine, serine, L-2,3-diaminopropionic acid (Dap), L-2,4-diaminobutyric acid (Dab), L-ornithine (Orn), L-lysine (Lys), L-2-amino-3-guanidinopropionic acid (Agp), and L-2-amino-4-guanidinobutyric acid (Agb).

[0099] Furthermore, in this specification, "peptide" means a compound in which multiple amino acids are linked together via peptide bonds. Unless otherwise specified, the multiple amino acid units constituting a peptide may be of the same type, or they may be two or more different types of amino acid units. The number of amino acids constituting a peptide is not particularly limited, as long as it is two or more. In this specification, peptides of dipeptide or greater are referred to as "polypeptides" or "oligopeptides".

[0100] Peptides can be synthesized using known chemical synthesis methods for peptides. While not particularly limited, the synthesis method may be liquid-phase or solid-phase peptide synthesis. In solid-phase peptide synthesis, either Boc solid-phase synthesis or Fmoc solid-phase synthesis can be used, ligation methods may be used as needed, or a combination of solid-phase and liquid-phase methods may be employed. When synthesizing oligopeptides, considering the repeated extension steps, solid-phase peptide synthesis is preferable (it is simpler), and Fmoc solid-phase synthesis is even more preferable. Furthermore, individual amino acids constituting polypeptides can be produced by known methods, or commercially available products may be used. Solid-phase peptide synthesis can be performed using commercially available automated peptide synthesizers. Specifically, one cycle consists of Fmoc deprotection of the solid-phase synthesis resin, washing with a solvent (e.g., DMF), condensation of Fmoc amino acids, and washing with a solvent (e.g., dichloromethane). By repeating this cycle, the peptide chain can be extended.

[0101] Polypeptides having a glutamic acid-derived structure at the terminal end are not particularly limited, but for example, polypeptides having a cationic oligopeptide moiety disclosed in International Publication No. 2014 / 148620 and Bioorganic & Medical Chemistry 21 (2013) 1717-1723 can be used.

[0102] Specifically, the cationic oligopeptide constituting the cationic oligopeptide moiety may contain eight or more amino acid residues having an amino group or a guanidino group. The cationic oligopeptide may be an oligopeptide consisting of 2 to 20, 3 to 15, or 8 to 12 amino acids (or structures derived from them).

[0103] The cationic oligopeptide preferably contains a portion consisting of at least two consecutive amino acid residues having the structure of formula (I) below (hereinafter also simply referred to as the amino acid residue of formula (I)). In addition, the portion other than the consecutive amino acid residues having the structure of formula (I) below may contain other amino acid residues.

[0104]

[0105] [In equation (I), R １ is, base H ３ N ＋ -CH ２ -, or a group represented by formula (II), preferably the group H ３ N ＋ -CH ２ - and R ２ R １ is the base H ３ N ＋ -CH ２ - If it is absent (single bond), or it is an alkylene group with 1 to 3 carbon atoms (preferably a methylene group), R １ In the case of the group represented by formula (II), it is an alkylene group having 1 to 4 carbon atoms. In one cationic oligopeptide, R １ and R ２ They are all identical.

[0106]

[0107] In formula (II), R ３ , R ４ and R ５ These are either the same or different hydrogen atoms or methyl groups.

[0108] Examples of amino acids from which the amino acid residues of formula (I) are derived include L-2,3-diaminopropionic acid (Dap), L-2,4-diaminobutyric acid (2,4-diaminobutanoic acid) (Dab), L-ornithine (Orn), L-lysine (Lys), L-2-amino-3-guanidinopropionic acid (Agp), L-2-amino-4-guanidinobutyric acid (Agb), and L-arginine (Arg). Among these, 2,4-diaminobutyric acid is preferred as the amino acid from which the amino acid residues of formula (I) are derived. These amino acids may be used individually or in combination of two or more.

[0109] Furthermore, in the cationic oligopeptide, the number of amino acid residues in formula (I) may be, for example, 2 to 20, 4 to 18, or 6 to 12.

[0110] The other amino acid residues are preferably single, non-consecutive amino acid residues. Examples include glycine, L-alanine, L-proline, and amino acids having a proline skeleton, such as L-aminoproline and L-guanidinoproline. The other amino acid residues may be used individually or in combination of two or more.

[0111] In one embodiment, the cationic oligopeptide moiety may be a heteropolymer containing the above-mentioned "other amino acid residues." In another embodiment, the cationic oligopeptide moiety may be a homopolymer that does not contain the above-mentioned "other amino acid residues."

[0112] Considering the ease of synthesis of cationic oligopeptides and their stabilizing effects on siRNA and shRNA, it is preferable that the cationic oligopeptide moiety be a homopolymer composed of a single amino acid.

[0113] In this case, the amino acids as monomers may be either natural or unnatural. Furthermore, the amino acids may be either L-form or D-form, and both forms may be present within the oligopeptide molecule.

[0114] Therefore, cationic oligopeptides may have, for example, homopolymers of L-2,3-diaminopropionic acid (Dap), L-2,4-diaminobutyric acid (Dab), L-ornithine (Orn), L-lysine (Lys), L-2-amino-3-guanidinopropionic acid (Agp), L-2-amino-4-guanidinobutyric acid (Agb), or L-arginine (Arg) (preferably a polymer of L-2,4-diaminobutyric acid (Dab)) as partial structures.

[0115] Cationic oligopeptides may also be in the form of salts, and suitable salts include hydrochloride salts, acetate salts, trifluoroacetate salts, etc., but are not particularly limited.

[0116] Double-stranded nucleic acids have two distinct helical structures, type A and type B. DNA / DNA double strands have a type B double helix structure with a major groove width of 13–18 Å, while RNA / RNA double strands and DNA / RNA strands have a type A double helix structure with major groove widths of 7–14 Å and 8–15 Å, respectively. Cationic oligopeptides are useful for improving the stability of siRNA and can bind to type A double helix RNA / RNA strands.

[0117] In one specific embodiment, the cationic oligopeptide moiety can be an octamer of diaminobutyric acid-derived constituent units having the following structure. In the following structure, B － This refers to an anion, and is not particularly limited to it, but includes acetate ions (CH₂). ３ COO － ), trifluoroacetate ion (CF ３ COO － ), chloride ions (Cl － Examples include the following. Furthermore, in the case of pharmaceuticals, it is preferable that the anion is pharmaceutically acceptable.

[0118]

[0119] Cationic peptides having a glutamic acid-derived structure at their termini are obtained by bonding a cationic oligopeptide moiety (a moiety having amino acid residues of formula (I)) to glutamic acid. This bonding is preferably a covalent bond. The covalent bond can be directly attached to the N-terminus, C-terminus, or side chain of the cationic oligopeptide moiety, or via a linker. Any commonly used linker can be used as appropriate, and is not particularly limited; for example, a peptide linker consisting of amino acids such as glycine or serine can be used. The number of amino acids in this case is, for example, 1 to 4. Specifically, the peptide linker may be a peptide consisting of 1 to 4 glycine residues. A preferred embodiment is a linker consisting of 3 glycine residues.

[0120] Therefore, cationic peptides that have a glutamic acid-derived structure at their terminal end can be said to constitute a polypeptide as a whole.

[0121] Furthermore, an embodiment in which the amino compound is a polypeptide is preferred. In another embodiment, the amino compound may be a compound having a structural unit derived from an amino acid, and it is more preferable that the amino compound has a structure derived from an amino acid at its terminus. Examples of amino acids here include the amino acids described above, and among them, it is preferable that the amino acid be at least one selected from the group consisting of phenylalanine, proline, and valine. These amino acid-derived structures may be structures formed by bonding the glutamic acid-derived structure at the terminus of a cationic peptide having a glutamic acid-derived structure at its terminus, or they may be structures in which the amino group of the glutamic acid-derived structure at the terminus of the cationic peptide and the carboxyl group of an amino acid other than glutamic acid are linked by an amide bond.

[0122] When a polypeptide is obtained by solid-phase synthesis, the polypeptide can be subjected to an amide condensation reaction with an activated pteroic acid, followed by deprotection of various protecting groups (e.g., Boc group or t-Bu group) of the carboxyl and amino groups. Such deprotection can be easily carried out using known methods.

[0123] The effects of the present invention will be explained using the following examples and comparative examples. In the examples, the units "parts" or "%" may be used, but unless otherwise specified, they represent "parts by mass" or "mass%". Unless otherwise specified, each operation is performed at room temperature (25°C).

[0124] 1. H-Glu(OtBu)-Gly ３ -[Dab(Boc)] ８ Preparation of NH-resin: Using Rink amide resin (385 mg, 0.25 mmol, Merck), the peptide chain was extended according to the Fmoc solid-phase synthesis method using an automated microwave peptide synthesizer.

[0125] The removal of the Fmoc (9-fluorophenylmethoxycarbonyl) group (de-Fmoc) was performed by reacting 385 mg (0.25 mmol, Merck) of Rink amide resin with 8 mL of a mixture containing 20% ​​piperidine / N,N-dimethylformamide (DMF) and 0.1 M ethyl cyano(hydroxyimino)ethyl acetate (Oxyma) at 90°C for 1 minute.

[0126] The amino acid introduction reaction was carried out at 90°C for approximately 2 minutes using a mixture of Fmoc-AA-OH (5 eq relative to the amino groups on the resin), N,N'-diisopropylcarbodiimide (DIC, 5 eq), and Oxyma (5 eq) (solvent DMF) after washing the resin six times with 7 mL of DMF as described above. The amino acids subjected to condensation (Fmoc-AA-OH) were, in order, (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-((tert-butoxycarbonyl)amino)butanoic acid (product name: Fmoc-Dab(Boc)-OH, manufactured by Watanabe Chemical Industry Co., Ltd.), (((9H-Fluoren-9-yl)methoxy)carbonyl) glycine (product name: Fmoc-Gly- OH, manufactured by Watanabe Chemical Industry Co., Ltd.), (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (product name: Fmoc-Glu(OtBu)-OH・nH２ O (manufactured by Watanabe Chemical Industry Co., Ltd.) was used. Prior to the next amino acid reaction, the above-mentioned de-Fmoc and washing with DMF were performed in the same manner.

[0127] After the above extension reaction, the Fmoc removal was carried out in the same manner as above, and the resulting resin was washed four times with dichloromethane (approximately 10 mL), and then dried under reduced pressure. As a result, the following structure of H-Glu(OtBu)-Gly was obtained. ３ -[Dab(Boc)] ８ I obtained 830 mg of -NH-resin.

[0128]

[0129] A portion of this stock resin was extracted and used to investigate subsequent pteroic acid introduction reactions.

[0130] 2. Investigation of the introduction of pteroic acid <Method using DIC / Oxyma (comparative example)> The resin obtained above (H-Glu(OtBu)-Gly ３ -[Dab(Boc)] ８ 25 μmol of -NH-resin was extracted and transferred to a filter-equipped tube. The resin was swollen in dichloromethane (approximately 3 mL) for several minutes, and then washed three times with N,N-dimethylformamide (DMF).

[0131] Pteroic acid (15.5 mg, 50 μmol) was weighed into a separate container and suspended in N,N-dimethylformamide (DMF, 180 μL). 1 M Oxyma / DMF (50 μL) and 0.5 M DIC / DMF (100 μL) were added sequentially to the pteroic acid, and the mixture was shaken for 10 minutes. This suspension was added to the resin, and the reaction was allowed to proceed at room temperature for 2 hours. After filtering off the reaction solution, the resin was washed with DMF (1.5 mL) more than 20 times. 5% phenoxyacetic anhydride / DMF solution (0.30 mL) was added to the resin, and the reaction solution was shaken at room temperature for 1 hour to cap the unreacted amino ends. Subsequently, the resin was washed six times with DMF (approximately 1 mL) and five times with dichloromethane (approximately 1 mL). After drying, a portion of the obtained resin (approximately 5 mg) was removed, and a deprotection cocktail (trifluoroacetic acid:triisopropylsilane:purified water = 95:2.5:2.5 (mass ratio), 0.20 mL) was added and the mixture was reacted at room temperature for 2 hours. Approximately 10 times the volume of t-butyl methyl ether was added to precipitate the peptide, and the mixture was centrifuged. The resulting peptide precipitate was washed with t-butyl methyl ether (approximately 1 mL) and centrifuged (this step was repeated twice).

[0132] The obtained precipitate was dried and dissolved in 0.20 mL of purified water. The solution was filtered through a spin filter and analyzed by reverse-phase HPLC and mass spectrometry to obtain a chromatogram. The analytical conditions were as follows: Column: CAPCEL PAK C18MG2 Flow rate: 0.3 mL / min Column temperature: 30°C Phase A 0.1% (v / v) TFA / water Phase B 0.1% (v / v) TFA / ACN Gradient: 1–31% (15 min) UV detector wavelength: 220 nm The results of the analysis are shown in Figure 1. The peptide into which the target substance, pteroic acid, was introduced was not identified.

[0133] 3. Solubilization study with basic compounds DMF was added to pteroic acid to a concentration of 5% w / w and the suspension was shaken. The following bases were added to this suspension in a stepwise manner to the pteroic acid, and the state of dissolution was checked. With secondary and tertiary alkylamines, no dissolution of pteroic acid was observed (entries 1 and 2). When 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was used as the base, the solution changed color and the amount of suspended solid decreased slightly, suggesting that some dissolution occurred, but solid pteroic acid was still visible (entry 3). On the other hand, in the system in which tetraalkylammonium salts such as tetra-n-butylammonium hydroxide (TBAOH) were added, it was confirmed that the suspension state changed to (almost) a solution state. The results are summarized in Table 1.

[0134]

[0135] 4. Solvent Examination Pteroic acid was suspended in various solvents to a concentration of 5% w / v, and TBAOH (41.1% (w / w) aqueous solution) was added in a ratio of 2 moles of TBAOH to 1 mole of pteroic acid. The mixture was shaken at room temperature for approximately 10 minutes. The solution state was then checked. It was confirmed that pteroic acid dissolves in DMF, N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), and mixtures thereof. The results are summarized in Table 2.

[0136]

[0137] 5-1. Study on introduction using the condensing agent / base (TBAOH) method (invention) The above resin H-Glu(OtBu)-Gly ３ -[Dab(Boc)] ８ 25 μmol of -NH-resin was extracted and swollen in dichloromethane (approximately 3 mL) for several minutes. The resin was washed three times with the reaction solvent to be used in the next step, the pteroic acid condensation reaction.

[0138] Pteroic acid (15.5 mg, 50 μmol) was weighed into a separate container and suspended in various reaction solvents (500 μL). 63 mg of a 41.1% by mass TBAOH aqueous solution (2 moles of TBAOH per mole of pteroic acid) was added to the suspension, and the mixture was shaken at room temperature for approximately 10 minutes to dissolve the pteroic acid. Various activators (1 mole per mole of pteroic acid) were added to this solution, and the mixture was shaken at room temperature for 5 minutes to activate the pteroic acid.

[0139] This reaction solution (50 μmol of activated pteroic acid) was added to the resin (25 μmol) and shaken at room temperature for 24 hours.

[0140] To remove partially insoluble pteroic acid, the resin was washed with a 41.1% by mass TBAOH aqueous solution / acetic acid / N-methylpyrrolidone (NMP) = 5:0.45:100 v / v (approximately 0.50 mL), and then washed three times with NMP (approximately 1 mL).

[0141] Next, the resin was washed three times with DMF (approximately 1 mL), then 5% phenoxyacetic anhydride / DMF solution (0.30 mL) was added to the resin, and the reaction mixture was shaken at room temperature for 24 hours to cap the unreacted amino ends in the resin (phenoxyacetic acid capped product (unreacted material)). After that, the resin was washed six times with DMF (approximately 1 mL) and five times with dichloromethane (approximately 1 mL).

[0142] After drying the obtained resin under reduced pressure, a small amount of resin (approximately 5 mg) was taken out, and a deprotection cocktail (trifluoroacetic acid:triisopropylsilane:purified water = 95:2.5:2.5, 0.20 mL) was added. The mixture was reacted at room temperature for 2 hours to remove the resin and remove the protective components (t-Bu group, Boc group). Approximately 10 times the volume of t-butyl methyl ether was added to precipitate the peptide, which was then centrifuged. The resulting peptide precipitate was washed with t-butyl methyl ether (approximately 1 mL) and centrifuged (this step was repeated twice). After drying the resulting precipitate, it was dissolved in 0.20 mL of purified water. The solution was filtered through a spin filter, and then analyzed by reverse-phase HPLC and mass spectrometry to obtain a chromatogram. The analytical conditions were as follows: Column: CAPCEL PAK C18MG2 Flow rate: 0.30 mL / min Column temperature: 30°C Phase A 0.1% (v / v) TFA / water Phase B 0.1% (v / v) TFA / ACN Gradient: 1-31% (15 min) UV detector wavelength: 220 nm The reaction conversion rate was calculated by the ratio of the 220 nm absorbance area of ​​the phenoxyacetic acid capping product (unreacted product) and the target product. The results are shown in Table 3 below. As a result of the investigation, although the reaction proceeded without problems with each coupling agent, HATU gave the best results (Entry 5). In addition, when HATU was used as the coupling agent, it was found that NMP gave a higher conversion rate than DMF as the reaction solvent (Entry 6). The HPLC chart for Entry 6 is shown in Figure 2. Furthermore, this reaction yields folic acid derivatives from pteroic acid and polypeptide (Glu-Gly-Gly-Gly-Dab8) as follows.

[0143]

[0144]

[0145] *The conversion rate was calculated from the absorbance area of ​​the target product and the phenoxyacetic acid capping compound at a wavelength of 220 nm using the following formula: Conversion rate = Target product / (Target product + Phenoxyacetic acid capping compound) × 100. From the above results, it was shown that, according to the method of the present invention, pteroinic acid amide can be obtained with a high conversion rate of 70% or more without introducing a protecting group to pteroic acid.

[0146] 5-2: Example using other basic compounds 1. Stock resin obtained in 1. H-Glu(OtBu)-Gly ３ -[Dab(Boc)] ８ 25 μmol of -NH-resin was extracted and swollen in dichloromethane (approximately 3 mL) for several minutes. The resin was then washed three times with N-methylpyrrolidone (NMP).

[0147] Pteroic acid (15.5 mg, 50 μmol) was weighed into a separate container and suspended in NMP (450 μL). To the suspension, a 35.7% by mass aqueous solution of tetra-n-propylammonium hydroxide (TPAOH aqueous solution, 50 mg, 2 moles per mole of pteroic acid) was added, and the mixture was shaken at room temperature for about 10 minutes to dissolve the pteroic acid. To this solution, HATU (1 mole per mole of pteroic acid) was added, and the mixture was shaken at room temperature for 5 minutes to activate the pteroic acid.

[0148] This reaction solution (50 μmol of activated pteroic acid) was added to the resin (25 μmol) and shaken at room temperature for 3 hours.

[0149] To remove partially insoluble pteroic acid, the resin was washed with a 41.1% by mass TBAOH aqueous solution / acetic acid / N-methylpyrrolidone (NMP) = 5:0.45:100 v / v (approximately 0.50 mL), and then washed three times with NMP (approximately 1 mL).

[0150] Next, the resin was washed three times with DMF (approximately 1 mL), then 0.30 mL of 5% phenoxyacetic anhydride / DMF solution was added to the resin, and the reaction mixture was shaken at room temperature for 30 minutes to cap the unreacted amino ends in the resin (phenoxyacetic acid capped product (unreacted material)). After that, the resin was washed six times with DMF (approximately 1 mL) and five times with dichloromethane (approximately 1 mL).

[0151] After drying the obtained resin under reduced pressure, a small amount of resin (approximately 5 mg) was taken out, and a deprotection cocktail (trifluoroacetic acid:triisopropylsilane:purified water = 95:2.5:2.5, 0.20 mL) was added. The mixture was reacted at room temperature for 2 hours to remove the resin and remove the protective components (t-Bu group, Boc group). Approximately 10 times the volume of t-butyl methyl ether was added to precipitate the peptide, which was then centrifuged. The resulting peptide precipitate was washed with t-butyl methyl ether (approximately 1 mL) and centrifuged (this step was repeated twice). After drying the resulting precipitate, it was dissolved in 0.20 mL of purified water. The solution was filtered through a spin filter, and then analyzed by reverse-phase HPLC and mass spectrometry to obtain a chromatogram. The analytical conditions were as follows: Column: CAPCEL PAK C18MG2 Flow rate: 0.30 mL / min Column temperature: 30°C Phase A: TFA / water 0.1% Phase B: TFA / ACN 0.1% Gradient: 1-31% (15 min) The reaction conversion rate was calculated by the 220 nm absorbance ratio of the phenoxyacetic acid capping product (unreacted product) and the target product. As a result, it was confirmed that the target product was produced with a conversion rate of 95.4%.

[0152] 5-3: Examples of other amino compounds 1. H-Glu(OtBu)-Gly obtained ３ -[Dab(Boc)] ８ -NH-resin is an oligopeptide resin in which three amino acids (phenylalanine, proline, and valine) are amide-bonded to the glutamic acid at the terminal end of NH-resin (H-Xaa-Glu(OtBu)-Gly ３ -[Dab(Boc)] ８ The target product was obtained by carrying out the reaction in the same manner as in 5-1, using -NH-Rink amide resin (where Xaa is one of phenylalanine, proline, or valine), as described below. Specifically, the reaction was as follows.

[0153] 1. Stock resin obtained in step 1: H-Glu(OtBu)-Gly ３ -[Dab(Boc)] ８ 25 μmol of -NH-resin was extracted and swollen in dichloromethane (approximately 3 mL) for several minutes.

[0154] Various amino acids (((9H-Fluoren-9-yl)methoxy)carbonyl)-L-phenylalanine (product name: Fmoc-Phe-OH , manufactured by Watanabe Chemical Industry Co., Ltd.), ((9H-Fluoren-9-yl)methoxy)carbonyl)-L-proline (product name: Fmoc-Pro-OH・nH ２ 125 μmol each of (O, manufactured by Watanabe Chemical Industry Co., Ltd.) and (((9H-Fluoren-9-yl)methoxy)carbonyl)-L-valine (trade name: Fmoc-Val-OH, manufactured by Watanabe Chemical Industry Co., Ltd.) were weighed into separate containers, and 0.26 mL of 0.45 M HBTU / DMF solution was added and shaken until dissolved. 43 μL of N,N-diisopropylethylamine (DIEA) was added and shaken for 5 minutes to activate the amino acids. This solution was added to the resin and shaken at room temperature for 12 minutes. After washing the resin twice with DMF, 1 mL of 20% v / v piperidine / DMF solution was added to the resin and shaken at room temperature for 1 minute. After removing the solution, 1 mL of 20% v / v piperidine / DMF solution was added to the resin again and shaken at room temperature for 5 minutes to remove the Fmoc group. The resin is washed six times with DMF, and then three times with NMP, resulting in H-Xaa-Glu(OtBu)-Gly ３ -[Dab(Boc)] ８ - Resin was obtained (Xaa = Phe, Pro, Val).

[0155] Pteroic acid (15.5 mg, 50 μmol) was weighed into a separate container and suspended in NMP (500 μL). 63 mg of a 41.1% by mass TBAOH aqueous solution (2 moles of TBAOH per mole of pteroic acid) was added to the suspension, and the mixture was shaken at room temperature for approximately 10 minutes to dissolve the pteroic acid. HATU (1 mole per mole of pteroic acid) was added to this solution, and the mixture was shaken at room temperature for 5 minutes to activate the pteroic acid.

[0156] This reaction solution (50 μmol of activated pteroic acid) was added to each resin (25 μmol), and the mixture was shaken at room temperature for 3 hours.

[0157] To remove partially insoluble pteroic acid, the resin was washed with a 41.1% by mass TBAOH aqueous solution / acetic acid / N-methylpyrrolidone (NMP) = 5:0.45:100 v / v (approximately 0.50 mL), and then washed three times with NMP (approximately 1 mL).

[0158] Next, the resin was washed three times with DMF (approximately 1 mL), then 0.30 mL of 5% phenoxyacetic anhydride / DMF solution was added to the resin, and the reaction mixture was shaken at room temperature for 30 minutes to cap the unreacted amino ends in the resin (phenoxyacetic acid capped product (unreacted material)). After that, the resin was washed six times with DMF (approximately 1 mL) and five times with dichloromethane (approximately 1 mL).

[0159] After drying the obtained resin under reduced pressure, a small amount of resin (approximately 5 mg) was taken out, and a deprotection cocktail (trifluoroacetic acid:triisopropylsilane:purified water = 95:2.5:2.5, 0.20 mL) was added. The mixture was reacted at room temperature for 2 hours to remove the resin and remove the protective components (t-Bu group, Boc group). Approximately 10 times the volume of t-butyl methyl ether was added to precipitate the peptide, which was then centrifuged. The resulting peptide precipitate was washed with t-butyl methyl ether (approximately 1 mL) and centrifuged (this step was repeated twice). After drying the resulting precipitate, it was dissolved in 0.20 mL of purified water. The solution was filtered through a spin filter, and then analyzed by reverse-phase HPLC and mass spectrometry to obtain a chromatogram. The analytical conditions were as follows: Column: CAPCEL PAK C18MG2 Flow rate: 0.30 mL / min Column temperature: 30°C Phase A: TFA / water 0.1% Phase B: TFA / ACN 0.1% Gradient: 1-31% (15 min) The reaction conversion rate was calculated by the 220 nm absorbance ratio of the phenoxyacetic acid capping product (unreacted product) and the target product. The structure of the obtained target peptide was Pte-Xaa-Glu-Gly ３ -Dab ８ -NH ２ The results are shown in Table 4.

[0160]

[0161] From these results, we confirmed that the target product was produced with a conversion rate of 90% or more for every amino group.

[0162] This application is based on Japanese Patent Application No. 2024-203750, filed on 22 November 2024, and Japanese Patent Application No. 2025-176330, filed on 20 October 2025, the disclosures of which are referenced and incorporated in whole.

Claims

1. A method for producing a pteroinamide compound, comprising obtaining a pteroinamide compound using pteroic acid as a starting material, an amino compound, and a condensing agent in a solvent containing an organic solvent, in the presence of a basic compound.

2. A method for producing a pteroinic acid amide compound according to claim 1, comprising reacting the pteroic acid and the condensing agent in a solvent containing the organic solvent in the presence of the basic compound to obtain an active esterified product of pteroic acid, and further reacting the active esterified product of pteroic acid with the amino compound.

3. A method for producing a pteroinamide compound according to claim 1 or 2, wherein the amino compound is a compound having a structural unit derived from glutamic acid.

4. The method for producing a pteroinamide compound according to claim 1 or 2, wherein the basic compound is a quaternary ammonium salt.

5. The method for producing a pteroinamide compound according to claim 4, wherein the basic compound has a tetraalkylammonium structure.

6. The method for producing a pteroinamide compound according to claim 5, wherein the basic compound is tetraalkylammonium hydroxide.

7. The method for producing a pteroinamide compound according to claim 1 or 2, wherein the condensing agent is a condensing agent that acts under basic conditions.

8. The method for producing a pteroinamide compound according to claim 1 or 2, wherein the condensing agent is at least one selected from the group consisting of uronium-based condensing agents, phosphonium-based condensing agents, triazine-based condensing agents, imidazolinium-based condensing agents, carbonate ester-based condensing agents, carbodiimidazole-based condensing agents, formamidinium-based condensing agents, phosphatidyl-based condensing agents, acid anhydride-based condensing agents, and pyridinium-based condensing agents.

9. A method for producing a pteroinamide compound according to claim 1 or 2, wherein the amino compound is a polypeptide.

10. A method for producing a pteroinic acid amide compound according to claim 1 or 2, wherein the organic solvent comprises at least one selected from the group consisting of amide solvents and sulfoxide solvents.

11. A method for introducing an amide bond to pteroic acid, comprising: mixing a basic compound and pteroic acid in a solvent containing an organic solvent; further obtaining an active esterified product of pteroic acid with a condensing agent; and reacting the active esterified product of pteroic acid with an amino compound.

12. A method for producing a pteroinic acid amide compound according to claim 1, comprising reacting pteroinic acid and an amino compound with a condensing agent in a solvent containing an organic solvent in the presence of a basic compound.

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