Compound, method for producing and method for storing compound, method for producing targeting agent, and composition

Abstract

In this production method, a ligand compound capable of being coordinated with a metal ion and a first compound having an atomic group capable of a click reaction are reacted to obtain a product comprising a second compound having in the structure thereof a ligand and an atomic group capable of a click reaction. Thereafter, an eluting solution which includes a water-soluble organic acid having prescribed physical properties and does not include a trifluoroacetic acid is used to subject the product to liquid chromatography to obtain the second compound which has been purified. The present invention also provides: a method for storing the second compound; a composition including the second compound; and a method for producing a targeting agent using the second compound.

Description

Compounds, methods for producing and storing compounds, methods for producing targeting agents, and compositions

[0001] The present invention relates to compounds, methods for making and storing compounds, methods for making targeting agents, and compositions.

[0002] Studies are currently being conducted on the manufacturing and storage conditions of compounds capable of coordinating to various metals, such as radioactive metals, with the aim of using them as reagents and diagnostic agents for detecting target molecules, or as pharmaceuticals for treating diseases.

[0003] Patent Document 1 discloses a method for purifying DOTAGA-DBCO obtained as the compound, and then using this to produce a polypeptide labeled with a radioactive metal.

[0004] In addition, Non-Patent Document 1 describes a method for producing radioactive metals. ６４ It is disclosed that in the production of nanoparticles labeled by coordinating a DOTA derivative to Cu, trifluoroacetic acid was used to purify the compound DO3A-DBCO.

[0005] International Publication No. 2019 / 125982

[0006] Zeng et al, ACS Nano. 2012, 6(6), 5209-5219.

[0007] Incidentally, when purifying a ligand compound containing a carboxyl group in its structure, such as DOTA or a derivative thereof, as a compound capable of coordinating to a metal ion, it is common in the technical field to use a solvent or eluent containing trifluoroacetic acid (hereinafter also referred to as "TFA" in this specification), as disclosed in Non-Patent Document 1. However, as shown in Comparative Example 1 described below, the present inventors have found that when the compound is purified and stored using a solvent or eluent containing TFA, the purity of the target compound may decrease over time. Based on this finding, a technology for improving the storage stability of the target compound has been desired.

[0008] Therefore, an object of the present invention is to improve the storage stability of the target compound.

[0009] The present invention provides a method for producing a compound, comprising reacting a ligand compound capable of coordinating to a metal ion with a first compound having a click-reactive atomic group to obtain a product containing a second compound having a click-reactive atomic group and a ligand in its structure, and then subjecting the product to liquid chromatography using an eluent containing a water-soluble organic acid that is liquid at 1 atmosphere and 20°C and not containing trifluoroacetic acid, thereby obtaining a purified second compound.

[0010] The present invention also provides a method for producing a targeting agent, comprising the steps of: coordinating a second compound obtained by the production method with a metal ion; and reacting the second compound with a targeting compound having a click-reactive atomic group via the click-reactive atomic groups, in this order or in reverse order.

[0011] The present invention also provides a composition comprising a compound having in its structure a ligand capable of coordinating to a metal ion and an atomic group capable of Click reaction, and a water-soluble organic acid that is liquid at 1 atmosphere and 20°C, but does not contain trifluoroacetic acid.

[0012] The present invention also provides a compound having, in its structure, a ligand capable of coordinating to a metal ion and an atomic group capable of click reaction, wherein the purity of the compound decreases by 5% or less after storage at −20° C. for one month, relative to the purity at the start of storage.

[0013] The present invention also provides a method for storing a compound, in which the second compound purified by the production method is stored frozen.

[0014] The present invention will be described below based on preferred embodiments thereof. The production method of the present invention comprises a step of reacting a ligand compound capable of coordinating to a metal ion with a first compound having a click-reactive atomic group to obtain a product containing a second compound having a click-reactive atomic group and a ligand in its structure (hereinafter also referred to as a synthesis step), and a step of purifying the product obtained via the synthesis step under predetermined conditions to obtain a purified second compound (hereinafter also referred to as a purification step).

[0015] In the synthesis step, a ligand compound and a first compound are reacted to obtain a product containing a second compound. From the viewpoint of reaction uniformity, the reaction of the two compounds is preferably carried out in a state in which both compounds are dissolved or dispersed in a reaction solvent. Details of the ligand compound, the first compound, the second compound, and the reaction conditions therefor will be described later.

[0016] The product obtained through the synthesis process contains, in addition to the second compound, which is the desired main product obtained by the reaction between the ligand compound and the first compound, unreacted ligand compound and the first compound, by-products other than the second compound such as positional isomers, and, if necessary, a reaction solvent. The second compound is an organic compound having two chemical structures: a ligand capable of coordinating to a metal ion derived from the chemical structure of the ligand compound, and an atomic group capable of Click reaction derived from the chemical structure of the first compound. That is, the second compound has, in its structure, a chemical structure capable of coordinating to a metal ion and a chemical structure capable of Click reaction.

[0017] One of the features of this production method is the adoption of specific conditions in the purification step of purifying the product containing the second compound. When purifying organic compounds having a ligand in their structure, solvents or eluents containing TFA have traditionally been used. However, it has become clear that under such conditions, the target compound may not be properly separated and purified, or the purity of the target compound may decrease over time when the purified compound is stored. The present inventors have intensively investigated the reason for this and speculated that TFA may unintentionally coordinate or bond to the ligand or unintentionally react with a highly reactive functional group. Based on this knowledge, they have unexpectedly found that by using an organic acid other than TFA in the purification step and purifying under conditions that do not include TFA, the target compound obtained by purification can be purified to a high purity, decomposition of the purified compound can be suppressed, and storage stability can be improved.

[0018] In the purification step, the product containing the second compound is subjected to liquid chromatography. The eluent used here preferably contains a water-soluble organic acid that is liquid at 1 atmosphere and room temperature and does not contain TFA. It is more preferable to use the water-soluble organic acid that is liquid at 1 atmosphere and room temperature and has a boiling point of 150°C or less at 1 atmosphere. By adopting such conditions, when the liquid composition containing the purified second compound is dried under reduced pressure or freeze-dried to obtain a solid second compound, the eluent can be evaporated without leaving any residue, resulting in increased purification efficiency and improved storage stability.

[0019] As used herein, the term "water-soluble organic acid" refers to an organic acid that dissolves in water at 1 atmosphere and 20°C. The water-soluble organic acid preferably has physical properties such that, when the organic acid is mixed with water, no layer separation is observed with the naked eye and the mixture is homogeneous. More preferably, the water-soluble organic acid used herein refers to an organic acid that dissolves in an amount of 3 mL or more in 100 mL of water. It is even more preferable that the water-soluble organic acid has physical properties such that, when the organic acid is mixed with water at a volume ratio of 1:10 (organic acid:water), no layer separation is observed with the naked eye and the mixture is homogeneous.

[0020] Furthermore, in this specification, "free from TFA" means that TFA is not intentionally contained in the reaction system or the obtained compound or composition, and it is acceptable for TFA present in trace amounts in raw materials or trace amounts of TFA remaining in a measuring instrument to be inevitably mixed in. Taking an eluent as an example, it means that TFA is not intentionally contained in the eluent, but it is acceptable for TFA derived from the raw materials to be inevitably mixed in the eluent. Not containing TFA means, for example, １９ This can be determined by the fact that no peaks derived from TFA are observed when analyzed by F-NMR.

[0021] The liquid chromatography used in the purification step may be, for example, at least one of column chromatography using columns or gels packed with various packing materials, high performance liquid chromatography, and medium pressure preparative liquid chromatography.

[0022] The water-soluble organic acid suitably used in the purification step preferably has a boiling point of 150°C or less at 1 atmosphere, and specific examples include formic acid (boiling point: 100.8°C), acetic acid (boiling point: 118°C), and propionic acid (boiling point: 141.2°C). These may be used alone or in combination. Of these, acetic acid is preferably used from the viewpoints of ease of handling, improved purification efficiency, suppression of unintended chemical reactions with the second compound, and improved storage stability of the purified second compound.

[0023] The concentration of the water-soluble organic acid in the eluent is preferably 0.001% by volume to 10% by volume, more preferably 0.01% by volume to 1.0% by volume, and even more preferably 0.1% by volume to 1.0% by volume. By keeping the concentration in this range, the purification efficiency can be further improved.

[0024] Examples of liquid components other than the water-soluble organic acid that constitute the eluent include water such as distilled water and ion-exchanged water; water-soluble protic solvents such as methanol and ethanol; water-soluble aprotic solvents such as acetonitrile, N,N-dimethylformamide (DMF), tetrahydrofuran, dimethyl sulfoxide, and acetone; and water-insoluble organic solvents such as hexane, toluene, and ethyl acetate. These solvents may be used alone or in combination. In this specification, water-soluble protic solvents and water-soluble aprotic solvents may be referred to as "polar organic solvents." Of these, from the viewpoint of dissolving all of the ligand compound, the first compound, and the second compound and facilitating purification, the liquid component of the eluent preferably contains at least one of water and a polar organic solvent, and more preferably contains at least one of water and acetonitrile.

[0025] From the viewpoints of shortening the purification time, improving the purification efficiency, and improving the ease of handling, it is preferable to carry out the purification step using a plurality of eluents. Specifically, when water and a polar organic solvent are selected as the liquid components of the eluent, a method can be exemplified in which an aqueous solution of the water-soluble organic acid not containing TFA is used as the first eluent, and then a polar organic solvent solution containing the water-soluble organic acid not containing TFA is used as the second eluent. The switching between the first eluent and the second eluent can be carried out as separate independent steps, or can be carried out within a single step by changing the concentration gradient thereof stepwise or continuously (so-called gradient).

[0026] A purified second compound can be obtained through the above steps. This second compound is preferably obtained in the form of a composition dissolved or dispersed in the eluent. That is, the composition contains a second compound having in its structure a ligand capable of coordinating to a metal ion and an atomic group capable of click reaction, as well as a water-soluble organic acid that is liquid at 1 atmosphere and 20°C, and does not contain TFA.

[0027] The purified second compound can be subjected to subsequent steps in the form of a liquid composition, or can be stored in the form of a composition. Alternatively, the composition can be subjected to a drying method such as vacuum drying or lyophilization (freeze drying) to remove the eluent, thereby obtaining a solid second compound. When dried, the second compound can be obtained as a simple substance (a single substance, in other words, a pure substance) or in the form of a solid composition with some of the organic acid remaining. The solid second compound can be dissolved in another solvent and subjected to subsequent steps, or can be stored in the solid state. Storage conditions can be at room temperature, refrigerated, or frozen. As used herein, "room temperature," "refrigerated," and "frozen" are not limited as long as they refer to the general range of temperatures that each term represents. For example, "room temperature" can be 15 to 25°C, "refrigerated" can be 0 to 10°C, and "frozen" can be -100 to 0°C.

[0028] Among these, from the viewpoints of suppressing decomposition of the second compound by the eluent and suppressing an increase in impurities, it is preferable to freeze-dry the purified second compound to obtain a solid second compound. Furthermore, regardless of whether the second compound is dried or not, it is also preferable to store the purified second compound frozen from the viewpoints of suppressing decomposition of the second compound and an increase in impurities.

[0029] The temperature during freeze-drying is set so that the product temperature of the target compound is preferably below 0° C., more preferably below −15° C., and even more preferably below −70° C. The external pressure during freeze-drying may be normal pressure or negative pressure.

[0030] Furthermore, the purity of the second compound purified through the above-mentioned steps decreases by preferably 5% or less, more preferably 3% or less, and even more preferably 2% or less after storage for one month, relative to the purity at the start of storage. The purity of the compound at the start of storage and after storage can be calculated by calculating the peak area by an automatic integration method using the results measured by high performance liquid chromatography, and then by an area percentage method.

[0031] Below, matters that are commonly applicable to the above-described embodiments will be described.

[0032] The ligand compound is not particularly limited as long as it is an organic compound capable of coordinating to a metal ion, and examples thereof include the following organic compounds and compounds containing structures derived from these compounds.

[0033] <CDTA or its derivatives> CB-TE2A (1,4,8,11-Tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid) CDTA (Cyclohexane-trans-1,2-diamine tetra-acetic acid) CDTPA (4-cyano-4-[[(dodecylthio)thioxomethyl]thio]-pentanoic acid)

[0034] <DOT A or its derivatives> DOT A (1,4,7,10 - Tetraazacyclododecane - 1,4,7,10 - tetraacetic acid) DOTMA ((1R,4R,7R,10R)-α,α’,α’’,α’’’ - tetramethyl - 1,4,7,10 - tetraazacyclododecane - 1,4,7,10 - tetraacetic acid) DOTAM (1,4,7,10 - tetrakis(carbamoylmethyl)-1,4,7,10 - tetraazacyclododecane) DOTAGA (α-(2 - Carboxyethyl)-1,4,7,10 - tetraazacyclododecane - 1,4,7,10 - tetraacetic acid) DOTP (((1,4,7,10 - Tetraazacyclododecane - 1,4,7,10 - tetrayl)tetrakis(methylene))tetraphosphonic acid) DOTMP (1,4,7,10 - Tetraazacyclododecane - 1,4,7,10 - tetrakis(methylenephosphonic acid)) DOT A - 4AMP (1,4,7,10 - tetraazacyclododecane - 1,4,7,10 - tetrakis(acetamidomethylenephosphonic acid) DO2P (Tetraazacyclododecane dimethanephosphonic acid) p - SCN - Bn - DOT A (2 -「(4 - isothiocyanatophenyl)methyl」-1,4,7,10 - Tetraazacyclododecane - 1,4,7,10 - tetraacetic acid)

[0035] <DTPA or its derivatives> DTPA (N,N - bis[2 - [bis(carboxymethyl)amino]ethyl]-glycine) DTPA - BMA (5,8 - Bis(carboxymethyl)-11 - [2 - (methylamino)-2 - oxoethyl]-3 - oxo - 2,5,8,11 - tetraazatridecane - 13 - oic acid)

[0036] <TETA, HEHA, PEPA or its derivatives> TETPA (1,4,8,11 - tetraazacyclotetradecane - 1,4,8,11 - tetrapropionic acid) TETA (1,4,8,11 - Tetraazacyclotetradecane - N,N’,N’’,N’’’ - tetraacetic acid) TTHA (3,6,9,12 - Tetrakis(carboxymethyl) - 3,6,9,12 - tetraazatetradecanedioic acid) HEHA (1,2,7,10,13 - hexaazacyclooctadecane - 1,4,7,10,13,16 - hexaacetic acid) 1,2 - HOPO (N,N’,N’’,N’’’ - tetra(1,2 - dihydro - 1 - hydroxy - 2 - oxopyridine - 6 - carbonyl) - 1,5,10,14 - tetraazatetradecane) PEPA (1,4,7,10,13 - pentaazacyclopentadecane - N,N’,N’’,N’’’,N’’’’ - penta - acetic acid) OTP (1,4,7 - Triazacyclononane - 1,4,7 - triyltris(methylenephosphonic acid)

[0037] <NOTA> NOTA (1,4,7 - Triazacyclononane - 1,4,7 - triacetic acid)

[0038] <Chain - like ligand compound> Deferoxamine (DFO) EDTA (2,2’,2’’,2’’’ - (ethane - 1,2 - diylbis(azanetriyl))tetraacetic acid)

[0039] <macropa or its derivatives> H2macropa (6 - (1,4,10,13 - tetraoxa - 7,16 - diazacyclooctadecane - N,N’ - methyl)picolinic acid) macropa - NH ２(4-氨基-6-[[16-[(6-羧基吡啶-2-基)甲基]-1,4,10,13-四氧杂-7,16-二氮杂环十八烷-7-基]甲基]吡啶-2-羧酸) 巨帕-NCS(6-[[16-[(6-羧基吡啶-2-基)甲基]-1,4,10,13-四氧杂-7,16-二氮杂环十八烷-7-基]甲基]-4-异硫氰酸根合吡啶-2-羧酸)

[0040] <Other ligand compounds> H4octapa(N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid) H2bispa2(6,6'-({9-hydroxy-1,5-bis(methoxycarbonyl)-2,4-di(pyridine-2-yl)-3,7-diazabicyclo[3.3.1]nonane-3,7-diy l}bis(-methylene))dipicolinic acid) H2dedpa(1,2-[{6-(carboxy)-pyridine-2-yl}-methylamino]ethane) H5decapa(N,N''-bis(6-carboxy-2-pyridylmethyl)-diethylenetriamine-N,N',N''-triacetic acid) H6phospa(N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridine-2-yl]-methyl-1,2-diaminoethane) HP-DO3A(Hydroxypropyltetraazacyclododecanetriacetic acid) HBED-CC(N,N'-Bis[2-hydroxy-5-(carboxyethyl)-benzyl]ethylenediamine-N,N'-diacetic acid)

[0041] Of these, from the viewpoint of achieving both simplicity of the subsequent production steps and improved complex formability, the ligand compound used in the synthesis step is preferably Macropa, Deferoxamine, HBED-CC, CDTA, DOTA, DTPA, NOTA, or a derivative thereof, and more preferably a compound containing a structure derived from DOTA or a derivative thereof. Examples of structures derived from DOTA include those shown in the following formula (1). These compounds may be anhydrides, hydrates, or acid anhydrides.

[0042]

[0043] In formula (1), R 11 , R 12 and R 13 are each independently -(CH ２ ) ｐ COOH, -(CH ２ ) ｐ C ５ H ４ N, -(CH ２ ) ｐ P.O. ３ H ２ , or -(CH ２ ) ｐ CONH ２ Each p is independently an integer of 0 or more and 3 or less.

[0044] In formula (1), R 14 or R 15 One of the groups is a hydrogen atom, -(CH ２ ) ｐ COOH, -(CH ２ ) ｐ C ５ H ４ N, -(CH ２ ) ｐ P.O. ３ H ２ , -(CH ２ ) ｐ CONH ２ , or -(CHCOOH)(CH ２ ) ｐ In formula (1), R 14 or R 15The other is -(CH ２ ) ｐ COOH, -(CH ２ ) ｐ C ５ H ４ N, -(CH ２ ) ｐ P.O. ３ H ２ , or -(CH ２ ) ｐ CONH ２ or an atomic group for linking to the first compound described below. Each p is independently an integer of 0 or more and 3 or less.

[0045] The first compound used in the synthesis step has an atomic group capable of undergoing a click reaction in its structure. Examples of such an atomic group include an alkynyl group or an azide group, or a diene or dienophile such as a 1,2,4,5-tetrazine or alkenyl group. These atomic groups are also preferably capable of being used in a metal-catalyst-free click reaction.

[0046] The click reaction occurs when an alkyne is combined with an azide, or when a diene such as 1,2,4,5-tetrazine is combined with an alkene and a dienophile. Specific examples of click reactions involving the combination of such atomic groups include the Huisgen cycloaddition reaction and the inverse electron demand Diels-Alder reaction.

[0047] Typically, the chemical structure produced by the click reaction of a combination of an alkyne and an azide contains a triazole skeleton, and the chemical structure produced by the click reaction of a combination of a diene and a dienophile, such as a combination of 1,2,4,5-tetrazine and an alkene, contains a pyridazine skeleton.

[0048] Specific examples of the atomic group capable of a click reaction include, as shown in the following formulas, an atomic group containing dibenzocyclooctyne (DBCO) as an alkyne (formula (5a)), an atomic group containing an azide group as an azide (formula (5b)), an atomic group containing 1,2,4,5-tetrazine (formula (5c)), or an atomic group containing trans-cyclooctene (TCO) as an alkene (formula (5d)).

[0049]

[0050]

[0051] In formula (5a), R １ represents a bonding site with another structure. ２ represents a bonding site with another structure. ３ and R ４ In formula (5d), one of R represents a bonding site to another structure, and the other represents a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group. ５ indicates the binding site with other structures.

[0052] From the viewpoint of facilitating introduction of the click-reactive atomic group into the target second compound and from the viewpoint of simplicity of the reaction conditions for the second compound, the click-reactive atomic group in the first compound is preferably dibenzocyclooctyne (DBCO).

[0053] As the first compound, various commercially available reagents may be used. Specifically, when an atomic group containing DBCO is introduced as the click-reactive atomic group, for example, DBCO reagents such as DBCO-C6-acid, DBCO-amine, DBCO-maleimide, DBCO-PEGacid, DBCO-PEG-NHS ester, DBCO-PEG-Alcohol, DBCO-PEG-amine, DBCO-PEG-NH-Boc, Carboxyrhodamine-PEG-DBCO, Sulforhodamine-PEG-DBCO, TAMRA-PEG-DBCO, DBCO-PEG-Biotin, DBCO-PEG-DBCO, DBCO-PEG-Maleimide, TCO-PEG-DBCO, and DBCO-mPEG can be used.

[0054] The reaction between the ligand compound and the first compound in the synthesis step is preferably carried out in a state in which both compounds are dissolved or dispersed in a reaction solvent. Examples of such a method include a method in which a solid ligand compound and a solid first compound are dissolved or dispersed in a reaction solvent, and a method in which one compound is added in solid or liquid form to a solution or dispersion of the other compound. If necessary, a compound other than the ligand compound and the first compound that can form a linker structure may be added to the reaction system. Examples of such other compounds include amino acids and polyethylene glycol (PEG).

[0055] As the reaction solvent used in the synthesis step, for example, the same solvents as those explained above for the eluent can be used alone or in combination.

[0056] When the reaction is carried out in a reaction solution in the synthesis step, the concentration of the ligand compound in the reaction solution can be appropriately changed depending on the type of compound, but from the viewpoint of improving the reaction yield, it is preferably 0.12 mol / L or more and 0.44 mol / L or less, more preferably 0.26 mol / L or more and 0.31 mol / L or less. From the same viewpoint, the concentration of the first compound in the reaction solution can be appropriately changed depending on the type of compound, but the lower limit of the concentration of the first compound in the reaction solution is preferably 0.10 mol / L or more, more preferably 0.20 mol / L or more, and the upper limit of the concentration of the first compound in the reaction solution is preferably 0.30 mol / L or less, more preferably 0.24 mol / L or less. From the viewpoint of improving the reaction yield, it is preferably 0.10 mol / L or more and 0.22 mol / L or less, more preferably 0.22 mol / L or more and 0.24 mol / L or less. From the same viewpoint, the molar ratio of the ligand compound to the first compound is preferably 1.2 or more and 3.0 or less, more preferably 1.2 or more and 2.0 or less, and particularly preferably 1.2 or more and 1.3 or less.

[0057] In the synthesis step, from the viewpoint of achieving a further improvement in yield in a short production time, it is preferable to carry out the reaction by heating the reaction solution. Here, heating refers to applying heat from outside the reaction system so that the temperature of the reaction solution becomes higher than 25° C., with 25° C. as the reference temperature. As a method for applying heat from outside the reaction system, any known method can be used as appropriate, and examples thereof include a water bath, an oil bath, a block heater, a mantle heater, etc.

[0058] When the reaction solution is heated to carry out the reaction, the reaction temperature is preferably 30° C. or more and 100° C. or less, more preferably 50° C. or more and 80° C. or less, from the viewpoint of simultaneously suppressing decomposition of the ligand compound and further improving the yield. The reaction time is preferably 2 hours or more and 24 hours or less, more preferably 2 hours or more and 4 hours or less, provided that the reaction temperature is as described above. The reaction pressure can be atmospheric pressure.

[0059] In the reaction between the ligand compound and the first compound, it is preferable to select conditions under which the chemical structure of the ligand compound capable of coordinating to a metal ion and the chemical structure of the click-reactive atomic group of the first compound are maintained without any particular modification, in order to enhance convenience in the subsequent production steps. Furthermore, it is preferable to select conditions under which a bond that is stable under ordinary synthesis conditions is formed, in order to suppress decomposition of the resulting compound and further improve the storage stability of the resulting compound.

[0060] A preferred example of a reaction method that can achieve both of the above-mentioned preferable conditions is a method in which a ligand compound and a first compound are subjected to an amidation reaction to bond the two compounds via an amide bond. It is also preferable that these reactions are carried out at the side chains of each compound. Specifically, a method can be mentioned in which a ligand compound containing a carboxy group in its structure and a first compound containing an amino group in its structure are used, and an amidation reaction is carried out between the carboxy group and the amino group to obtain a second compound having an amide bond in its structure. The carboxy group can be "-COOH" or "-COO － ". An amino group is a monovalent functional group represented by "-NH ２" or "-NH ３ ＋ " is a monovalent functional group represented by the formula:

[0061] Alternatively, the amidation reaction can be carried out using a ligand compound or first compound having an oxydicarbonyl group instead of a carboxy group. The oxydicarbonyl group is a divalent functional group represented by "-C(=O)OC(=O)-", and compounds having this functional group are called acid anhydrides or simply anhydrides. The ligand compound or first compound having an oxydicarbonyl group may be a symmetric anhydride having two identical acyl groups, or a mixed anhydride having different acyl groups. Alternatively, it may be a cyclic anhydride formed by dehydration condensation between carboxy groups present in the same molecule of a polycarboxylic acid. Of these, mixed anhydrides or cyclic anhydrides are preferred, and cyclic anhydrides are more preferred, from the standpoints of economy and synthesis efficiency.

[0062] The amidation reaction may be carried out by stirring in a reaction solvent at room temperature or under heating, and may be carried out with the addition of an amide condensation agent as needed. Examples of amide condensation agents include carbodiimide condensation agents such as N,N'-dicyclohexylcarbodiimide (DCC); condensation agents via acid azides such as diphenylphosphoric azide (DPPA); BOP reagents combining hexamethylphosphoric triamide (HMPA) and 1-hydroxybenzotriazole (HOBt); PyBOP, in which the dimethylamino group of a BOP reagent is substituted with a pyrrolidino group; uronium-type condensation agents such as O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), in which the phosphorus of a BOP reagent is substituted with a carbon atom; and triazole-based condensation agents such as DMT-MM. Furthermore, the amidation reaction may be carried out with the addition of a base such as triethylamine as needed.

[0063] Another preferred reaction method that can achieve both of the above-mentioned preferable conditions is a method in which a ligand compound and a first compound are bonded together by a reaction mode that forms a thiourea structure between them. These reactions are also preferably carried out at the side chains of each compound. Specifically, a method is exemplified in which a ligand compound containing an isothiocyanate group in its structure and a first compound containing an amino group in its structure are used, and the isothiocyanate group and the amino group are reacted to obtain a second compound having a thiourea structure in its structure. The isothiocyanate group is a functional group represented by "-N=C=S." The thiourea structure is a chemical structure represented by "-NH-C(=S)-NH-." Reaction conditions for forming a thiourea structure can be, for example, using a base such as N,N'-diisopropylethylamine or triethylamine and stirring at room temperature or under heating.

[0064] Examples of the second compound having an amide bond in its structure include, but are not limited to, compounds represented by the following formulas (7a) and (7b). Examples of the second compound having a thiourea structure in its structure include, but are not limited to, compounds represented by the following formulas (8a) and (8b). The second compounds represented by the following formulas (7a) and (7b) and (8a) and (8b) all maintain both a chemical structure capable of coordinating to a metal ion and the chemical structure of the click-reactive atomic group possessed by the first compound. The chemical structure of the second compound can be changed as appropriate depending on the types of ligand compound and first compound used.

[0065]

[0066]

[0067] From the viewpoint of obtaining a second compound having even higher storage stability and from the viewpoint of more efficiently carrying out the metal ion coordination reaction and click reaction in the subsequent steps, it is preferable that the ligand compound is DOTAGA or its anhydride, and the first compound is DBCO-amine. The second compound obtained by reacting these compounds is DOTAGA-DBCO represented by the above formula (7a). One embodiment of the reaction pathway is shown below.

[0068]

[0069] The second compound obtained through the purification step can be subjected to the subsequent steps in the form of a single compound or a composition, or in the form of a solution in a solvent or buffer solution, or in the form after a step of distilling off a predetermined eluent under reduced pressure or the like has been performed.

[0070] Examples of steps using the second compound obtained through the purification step include the following steps (a) and (b). These steps are one embodiment of a method for producing a targeting agent. The targeting agent contains, in its chemical structure, a metal complex and an atomic group that has directivity toward a target organ or tissue in a living body or specific binding ability to a target molecule.

[0071] Step (a): A step of forming a metal complex by coordinating a second compound (specifically, a ligand contained in the chemical structure of the second compound) with a metal ion (complex formation step). Step (b): A step of reacting the second compound with a targeting compound having a click-reactive atomic group via the click-reactive atomic groups (click reaction step).

[0072] The above-mentioned steps (a) and (b) may be performed in the order of steps (a) and (b), or alternatively, in the order of steps (b) and (a). Steps (a) and (b) may be performed consecutively in this order or in the reverse order, or alternatively, a step other than steps (a) and (b) may be interposed between steps (a) and (b).

[0073] Among the methods for producing a targeting agent, steps (a) and (b) are preferably performed in this order. Specifically, it is preferable to perform a step of coordinating the second compound obtained through the purification step with a metal ion to obtain a metal complex, and then perform a step of reacting the metal complex with a targeting compound having a click-reactive atomic group between the click-reactive atomic groups. Performing each step in this order is advantageous in that it increases the yield of the complex and allows the targeting agent to be subjected to subsequent steps without separating and purifying unreacted metal ions. Furthermore, it is possible to produce targeting agents with enhanced in vivo target organ or tissue specificity or enhanced specific binding to target molecules with high productivity. In particular, it is advantageous in that the use of a radioactive metal complex allows for effective treatment and detection of diseases.

[0074] A suitable method for producing a targeting agent will be described below, taking as an example a case where steps (a) and (b) are performed in this order. The second compound obtained through the purification step can be reacted with a metal ion, either as a single compound or as a composition, or dissolved in an aqueous solution such as a solvent or buffer solution, to form a metal complex (complex formation step). The metal complex obtained in this step is formed by coordinating a metal ion to a ligand contained in the chemical structure of the second compound. The metal to be coordinated may be a non-radioactive element or a radioactive isotope.

[0075] From the viewpoint of increasing the efficiency of complex formation, the metal to be reacted with the second compound is preferably used in the form of an ionizable metal compound, more preferably in the form of a metal ion (hereinafter, these forms are also collectively referred to as "metal source"). As the metal source, for example, a metal ion-containing liquid in which metal ions are dissolved in a solvent mainly composed of water can be used.

[0076] Furthermore, from the viewpoint of increasing the efficiency of complex formation without depending on the combination of the ligand and the metal in the structure of the second compound, it is preferable to heat the reaction system containing the compound and the metal ion during complex formation. The heating conditions are preferably 30°C or higher and 100°C or lower, more preferably 50°C or higher and 80°C or lower. The heating time can be appropriately changed depending on the type of metal used, but provided that the temperature is within the above-mentioned range, the lower limit is preferably 5 minutes or higher, more preferably 10 minutes or higher, even more preferably 15 minutes or higher, and even more preferably 30 minutes or higher, and the upper limit is preferably 150 minutes or lower, more preferably 120 minutes or lower, even more preferably 100 minutes or lower, and even more preferably 90 minutes or lower.

[0077] The volume of the reaction solution is not particularly limited, but from the viewpoint of practicality, a volume of 0.01 mL to 100 mL is practical at the start of this step. Furthermore, from the viewpoint of the yield of the target metal complex, it is preferable that the concentrations of the compound and metal ion in the reaction solution are each independently 1 μmol / L to 100 μmol / L at the start of this step.

[0078] Examples of the solvent used in the complex formation step include water, saline, and buffer solutions such as sodium acetate buffer, ammonium acetate buffer, phosphate buffer, phosphate-buffered saline, Tris buffer, HEPES buffer, and tetramethylammonium acetate buffer.

[0079] Examples of metals to be coordinated include alkali metals, alkaline earth metals, lanthanides, actinides, transition metals, and non-radioactive and radioactive elements of metals other than these metals, as well as their isotopes. Examples of such metal elements include Sc, Cr, Co, Fe, Ga, Cu, Sr, Zr, Y, Tc, Ru, In, Sm, Dy, Ho, Lu, Re, Au, Tl, Hg, Bi, Pb, Th, and Ac. From the viewpoint of enabling the metal complex or a composition containing the same to be used in the treatment, diagnosis, or detection of diseases, Zr, Lu, In, Y, Ga, Cu, or Ac is preferred, and a radioactive metal is more preferred. ８９ Zr, １７７ Lu, １１１ In,９０ Y. ６７ Ga, ６８ Ga, ６４ Cu or ２２５ These metals can be produced by a conventional method and obtained as a solution containing the metal in an ionized state.

[0080] The metal complex obtained through the complex formation step has a click-reactive atomic group derived from the chemical structure of the second compound. Therefore, the metal complex and a targeting compound having a click-reactive second atomic group are subjected to a click reaction to produce a targeting agent (click reaction step). This allows for the production of an agent with enhanced target organ or tissue specificity or enhanced binding specificity to a target molecule in vivo. The use of radioactive metal complexes is particularly advantageous in that disease treatment and detection can be effectively achieved.

[0081] In producing a targeting agent, it is preferable to carry out a click reaction between a click-reactive atomic group contained in the chemical structure of a metal complex and a click-reactive second atomic group contained in the chemical structure of a targeting compound. In one embodiment, when DOTAGA-DBCO is used as the second compound, the metal complex obtained using the second compound has DBCO as the click-reactive atomic group. Therefore, a targeting compound having an azide group as the click-reactive second atomic group can be used to carry out a click reaction between DBCO and the azide group. Known reaction conditions can be used for the click reaction, and from the viewpoint of preventing denaturation of the targeting compound, it is preferable to carry out the reaction under non-heated conditions.

[0082] The targeting compound preferably contains one or more atomic groups selected from linear peptides, cyclic peptides, or combinations thereof, proteins, antibodies or fragments thereof, peptide aptamers, growth factors, affibodies, unibodies, nanobodies, monosaccharides, polysaccharides, vitamins, antisense nucleic acids, siRNA, miRNA, nucleic acid aptamers, decoy nucleic acids, cPG oligonucleic acids, peptide nucleic acids, liposomes, micelles, carbon nanotubes, and nanoparticles. These atomic groups also preferably have a chemical structure capable of binding to the target molecule of interest. Furthermore, when a second atomic group capable of Click reaction is introduced into the targeting compound, known reagents can be used.

[0083] These targeting compounds may be directly bonded to the metal complex, or may be indirectly bonded via other known linker structures such as PEG. When the linker structure contains a structure derived from PEG, it is also preferable that the linker is indirectly bonded via a linker structure shown in formula (P) below. In formula (P), n is preferably an integer of 2 or more and 10 or less, more preferably an integer of 2 or more and 8 or less, and even more preferably an integer of 2 or more and 5 or less.

[0084]

[0085] When the targeting compound contains a peptide in its structure, the atomic group preferably includes a linear peptide, a cyclic peptide, or a combination thereof, a protein, an antibody, or a fragment thereof, which specifically binds to a specific molecule. Examples of such an atomic group include peptides consisting of three or more amino acid residues, such as antibodies (immunoglobulins) of the IgG, IgA, IgM, IgD, and IgE classes, antibody fragments such as Fab fragments and F(ab')2 fragments, and peptide aptamers. Furthermore, the amino acids that make up such targeting agents may be natural or synthetic. Furthermore, the molecular weight of the above-mentioned atomic group containing the peptide is not particularly limited.

[0086] The various peptides described above can be synthesized by conventionally known methods, such as liquid phase synthesis, solid phase synthesis, automated peptide synthesis, gene recombination, phage display, genetic code reprogramming, RaPID (Random Non-Standard Peptide Integrated Discovery), etc. When synthesizing the various peptides, the functional groups of the amino acids used may be protected as necessary.

[0087] When the targeting compound is an atomic group containing a nucleic acid in its structure, the atomic group is preferably an atomic group containing an antisense nucleic acid, siRNA, miRNA, nucleic acid aptamer, decoy nucleic acid, cPG oligonucleic acid, or peptide nucleic acid that specifically binds to a specific molecule. Furthermore, the nucleic acid base may be a natural one such as deoxyribonucleic acid or ribonucleic acid, or may be a synthetic one.

[0088] The atomic group containing the nucleic acid that can be used in the present invention can be produced by a conventionally known method. For example, in the case of a nucleic acid aptamer, a nucleic acid aptamer that specifically binds to a specific target substance such as a protein can be produced using the SELEX method (Systematic Evolution of Ligands by Exponential Enrichment).

[0089] The metal complex and targeting agent produced through the above-described steps are typically present in a dissolved state in the reaction solution. These solutions may be used independently as they are, or may be purified using a filtration filter, a membrane filter, a column filled with various packing materials, chromatography, or the like.

[0090] After the targeting agent is obtained, a further step can be carried out, for example, to prepare a drug containing the targeting agent as an active ingredient. The formulation step can be carried out by adding various additives such as pH adjusters such as citrate buffer, phosphate buffer, borate buffer, etc., solubilizers such as polysorbate, stabilizers, or antioxidants, or by adjusting the concentration of the agent by diluting with an isotonic solution such as water or physiological saline. The formulation step may also include a step of preparing an injectable preparation by sterilizing filtration using a membrane filter or the like after adding various additives or adjusting the concentration.

[0091] The ligand compound, the first compound, and the second compound in this specification may each independently be a compound labeled or isotopically substituted with an isotope of the same element as the element in the structure, and one or more atoms may be substituted with an atom having an atomic mass or mass number different from the naturally occurring atomic mass or mass number. Examples of the above-mentioned isotopes include: ２ H. ３ H. １１ C. １３ C. １４ C. １５ N. １７ O. １８ and isotopes of hydrogen, carbon, nitrogen, and oxygen, such as O. Pharmaceutically acceptable salts of each of the foregoing compounds that contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the present invention. ３ H and １４ Compounds incorporating radioactive isotopes such as C are useful in non-clinical or non-medical research, such as drug and / or substrate tissue distribution assays. In this case, when a metal complex is formed, the metal ion to be coordinated may be a non-radioactive element. ３ H and １４ A compound having at least one of C in its structure is preferred from the viewpoint of ease of preparation and detection. The isotopically labeled compounds described above can typically be prepared by using an isotopically labeled reagent instead of a non-isotopically labeled reagent.

[0092] The present invention will be described in more detail below with reference to examples. However, the scope of the present invention is not limited to such examples. All of the following examples were carried out at atmospheric pressure. In the following tables, a column marked with "-" indicates that no experiment was carried out.

[0093] [Examples 1 to 4] (Synthesis Process) DOTAGA anhydride was used as the ligand compound, and DBCO-amine was used as the first compound, and these were dissolved in DMF to obtain a reaction solution. The molar ratio of the ligand compound to the first compound was 3. This reaction solution was heated at 50°C for 3 hours to cause an amidation reaction between the carboxy group of the ligand compound and the amino group of the first compound, obtaining a product containing DOTAGA-DBCO as the second compound.

[0094] (Purification Step) Next, the product containing DOTAGA-DBCO was purified by medium-pressure preparative liquid chromatography using eluents. The eluents in the medium-pressure preparative liquid chromatography were a 0.1% by volume aqueous solution of acetic acid as the first eluent and acetonitrile containing 0.1% by volume acetic acid as the second eluent, and elution was performed under the following concentration gradient conditions. Neither eluent contained TFA.

[0095] <Medium-Pressure Preparative Liquid Chromatography Conditions> A first eluent (hereinafter also referred to as eluent 1) and a second eluent (hereinafter also referred to as eluent 2) were used, and the concentration gradient (volume %) was controlled and delivered in the following order (i) to (iii). The flow rate of each eluent was 12 mL / min. (i) 0 to 6 min: eluent 1:80, eluent 2:20 (ii) 6 to 34 min: eluent 1:80 to 58, eluent 2:20 to 42 (iii) 34 to 40 min: eluent 1:58 to 0, eluent 2:42 to 100. The column used was a Sfar C18 manufactured by Biotage, and detection was performed using a UV-visible absorption detector (measurement wavelength: 290 nm).

[0096] The liquid obtained through this process was evaporated under reduced pressure using an evaporator to remove the acetonitrile, yielding DOTAGA-DBCO as a purified second compound. This DOTAGA-DBCO was a composition dissolved in a 0.1% by volume aqueous solution of acetic acid.

[0097] This composition was then lyophilized to remove the 0.1% by volume aqueous acetic acid solution, yielding solid DOTAGA-DBCO. This was then placed in an amber vial and stored. The amber vials containing DOTAGA-DBCO were then placed and stored for a predetermined period under the following conditions: room temperature (Example 1: 21-25°C), high temperature (Example 2: 40°C), refrigeration (Example 3: 5°C), and freezing (Example 4: -20°C).

[0098] [Example 5] (Synthesis process) DOTAGA-DBCO was obtained by the method described in the synthesis process of Examples 1 to 4, except that DMSO was used as the organic solvent for dissolving DOTAGA and DBCO-amine, the molar ratio of the ligand compound to the first compound was 1.2, and the reaction temperature was 80°C.

[0099] (Purification Step) A product containing DOTAGA-DBCO was purified in the same manner as in the purification steps of Examples 1 to 4, except that formic acid was used as the water-soluble organic acid in the first and second eluents and elution was performed under the concentration gradient conditions shown below.

[0100] <Conditions for medium-pressure preparative liquid chromatography> A first eluent (hereinafter also referred to as eluent 1) and a second eluent (hereinafter also referred to as eluent 2) were used, and the concentration gradient (volume %) was controlled and delivered in the following order (i) to (v). The flow rate of each eluent was 12 mL / min. (i) 0 to 2.8 min: Eluent 1:95, Eluent 2:5 (ii) 2.8 to 5.7 min: Eluent 1:95-80, Eluent 2:5-20 (iii) 5.7 to 19.8 min: Eluent 1:80-40, Eluent 2:20-60 (iv) 19.8 to 22.7 min: Eluent 1:40-5, Eluent 2:60-95 (v) 22.7 to 25.5 min: Eluent 1:5, Eluent 2:95

[0101] The liquid obtained through this process was evaporated under reduced pressure using an evaporator to remove acetonitrile, yielding DOTAGA-DBCO as a purified second compound. This DOTAGA-DBCO was a composition dissolved in a 0.1% by volume aqueous solution of formic acid.

[0102] Subsequently, this composition was dried by lyophilization to remove the 0.1% by volume formic acid solution, yielding solid DOTAGA-DBCO. This was then placed in a colorless, transparent glass vial and stored. The colorless, transparent glass vial containing DOTAGA-DBCO was then placed at room temperature (Example 5: 19 to 23°C) and stored for a predetermined period of time.

[0103] [Comparative Examples 1 and 2] After carrying out a synthesis process in the same manner as in Examples 1 to 4, the product containing DOTAGA-DBCO was purified in the same manner as in Examples 1 to 4 using an eluent containing TFA. In these comparative examples, a 0.1 vol% TFA aqueous solution was used as the first eluent, and acetonitrile containing 0.1 vol% TFA was used as the second eluent. Therefore, the composition obtained in these comparative examples contained DOTAGA-DBCO and TFA. Subsequently, DOTAGA-DBCO obtained by drying in the same manner as in Examples 1 to 4 was placed in a brown vial, and the brown vial was placed at room temperature (Comparative Example 1: 21 to 25°C) and at a high temperature (Comparative Example 2: 40°C) and stored for a predetermined period of time.

[0104] [Evaluation of storage stability] The purity of DOTAGA-DBCO of the Examples and Comparative Examples was measured by the following measurement method at the start of storage, after 7 days of storage, after 1 month of storage, after 3 months of storage, and after 6 months of storage. The smaller the decrease in purity compared to the purity at the start of storage, the more excellent the storage stability. The results are shown in Table 1 below.

[0105] <Method for Measuring Purity> 1.0 mg of the target compound was precisely weighed, and for Examples 1 and 2 and Comparative Examples 1 and 2, a 10 mmol / L ammonium formate solution (pH 3.0) / liquid chromatography grade acetonitrile mixture (95:5) was added to make exactly 10 mL, to prepare the sample solution. For Examples 3 and 4, a 10 mmol / L ammonium formate solution (pH 3.0) / liquid chromatography grade methanol mixture (95:5) was added to make exactly 10 mL, and for Example 5, a 10 μL sample solution was added to make exactly 10 mL, to prepare the sample solution. 10 μL of each sample solution was measured by high-performance liquid chromatography under the following conditions. The peak areas of the sample solutions were measured using the automatic integration method, and the amounts of the compounds were calculated using the area percentage method. The sample solution was prepared once and measured three times. The arithmetic mean of the measurement results from the three measurements was taken as the purity (%). At the start of storage, both the sample solution was prepared once and measured once.

[0106] <High-performance liquid chromatography conditions> Detector (1): Ultraviolet absorption photometer (measurement wavelengths: 290 nm, 308 nm) Detector (2): ACQ-QDa (positive scan: cone voltage 15 V, capillary voltage 0.8 V; negative scan: cone voltage 15 V, capillary voltage 0.8 V, scanning range: m / z 50-1200) Column: A stainless steel tube with an inner diameter of 4.6 mm and a length of 10 cm packed with 3.5 μm octadecylsilylated silica gel for liquid chromatography (XBridge C18). Column temperature: Maintained at a constant temperature of approximately 25° C.

[0107] [Examples 1 to 5 and Comparative Examples 1 and 2] Mobile phase: (Mobile phase A) 10 mmol / L ammonium formate solution (pH 3.0), (Mobile phase B) acetonitrile were used, and the concentration gradient (volume %) was controlled in the following order (i) to (v). The flow rate of each mobile phase was 1 mL / min. (i) 0 to 12 min: Mobile phase A: 95 to 50, Mobile phase B: 5 to 50 (ii) 12 to 13 min: Mobile phase A: 50 to 5, Mobile phase B: 50 to 95 (iii) 13 to 20 min: Mobile phase A: 5, Mobile phase B: 95 (iv) 20 to 20.01 min: Mobile phase A: 5 to 95, Mobile phase B: 95 to 5 (v) 20.01 to 30 min: Mobile phase A: 95, Mobile phase B: 5

[0108]

[0109] As shown in Table 1, the compounds of each Example purified in the absence of TFA can be stored in a state where the purity at the start of storage is maintained even after one month of storage, compared to the compounds of the Comparative Examples. In particular, Examples 3 and 4 stored under refrigerated or frozen conditions can be stored in a state where the purity at the start of storage is maintained even after six months of storage, demonstrating excellent long-term storage stability.

Claims

1. React a ligand compound capable of coordinating with metal ions with a first compound having an atom group capable of click reaction to obtain a product containing a second compound having an atom group capable of click reaction and a ligand in its structure, and then subject the product to liquid chromatography using an eluent containing a water-soluble organic acid that is liquid at 1 atm and 20 °C and does not contain trifluoroacetic acid to obtain the purified second compound. A method for producing a compound.

2. The production method according to claim 1, wherein the water-soluble organic acid having a boiling point of 150 °C or lower at 1 atm is used.

3. The production method according to claim 1 or 2, wherein the water-soluble organic acid is acetic acid.

4. The production method according to any one of claims 1 to 3, wherein the atom group capable of click reaction is dibenzocyclooctyne.

5. The production method according to any one of claims 1 to 4, wherein the purified second compound is lyophilized.

6. The production method according to any one of claims 1 to 5, wherein the eluent further contains a polar organic solvent.

7. The production method according to any one of claims 1 to 6, wherein the ligand compound is Macropa, Deferoxamine, HBED-CC, CDTA, DOTA, DTPA, or NOTA or a derivative thereof.

8. The production method according to any one of claims 1 to 7, wherein the ligand compound and the first compound are reacted by an amidation reaction or in a reaction mode that forms a thiourea structure.

9. An amidation reaction is carried out between the carboxy group or oxydicarbonyl group of the ligand compound and the amino group of the first compound, or a reaction mode that forms a thiourea structure is carried out between the isothiocyanate group of the ligand compound and the amino group of the first compound. The production method according to any one of claims 1 to 7.

10. The production method according to any one of claims 1 to 9, wherein the ligand compound is DOTAGA or its anhydride, and the first compound is DBCO-amine.

11. A step of coordinating the second compound obtained by the production method according to any one of claims 1 to 10 with a metal ion, and a step of reacting the second compound with a targeting compound having an atom group capable of click reaction between the atom groups capable of click reaction. A method for producing a targeting agent, comprising these steps in this order or in the reverse order.

12. The production method according to claim 11, wherein ions of a radioactive metal are used as the metal ions.

13. A composition comprising a compound having a ligand capable of coordinating to a metal ion and an atom group capable of click reaction in the structure, and a water-soluble organic acid that is liquid at 1 atm and 20 °C and does not contain trifluoroacetic acid.

14. １９ The composition according to claim 13, wherein no peak derived from trifluoroacetic acid is observed when analyzed by F-NMR.

15. The composition according to claim 13 or 14, wherein the atom group capable of click reaction is dibenzocyclooctyne.

16. The composition according to any one of claims 13 to 15, wherein the water-soluble organic acid is acetic acid.

17. A compound having a ligand capable of coordinating to a metal ion and an atom group capable of click reaction in the structure, and having a purity reduction rate of 5% or less after storage at -20 °C for 1 month with respect to the purity at the start of storage.

18. A method for storing a compound, comprising storing the second compound purified by the production method according to any one of claims 1 to 10 under freezing conditions.