Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
  • C07B59/002—Heterocyclic compounds
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
  • B01D15/424—Elution mode
  • B01D15/426—Specific type of solvent
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
  • C07B2200/05—Isotopically modified compounds, e.g. labelled

Definitions

• the present invention relates to compounds, methods for producing and storing compounds, methods for producing targeting agents, and compositions.
• Patent Document 1 discloses a method of purifying DOTAGA-DBCO obtained as the above compound and then using it to produce a polypeptide labeled with a radioactive metal.
• Non-Patent Document 1 relates to a method for producing nanoparticles labeled by coordinating a DOTA derivative to a radioactive metal 64 Cu. is disclosed.
• an object of the present invention is to enhance the storage stability of the target compound.
• the present invention reacts a ligand compound capable of coordinating to a metal ion with a first compound having a click-reactive atomic group to obtain a first compound having a click-reactive atomic group and a ligand in its structure. obtaining a product containing two compounds, after which subjecting the product to a liquid chromatography method using an eluent containing a water-soluble organic acid that is liquid at 1 atm and 20° C. and free of trifluoroacetic acid to obtain the purified second compound; A method for producing a compound is provided.
• the present invention provides a step of coordinating the second compound obtained by the above production method to a metal ion;
• a method for producing a targeting agent comprising the step of reacting the second compound and a targeting compound having click-reactive atomic groups between the click-reactive atomic groups in this order or in reverse order. It provides
• the present invention also provides a compound having a ligand capable of coordinating to a metal ion and an atomic group capable of a click reaction in its structure,
• a composition is provided that contains a water-soluble organic acid that is liquid at 1 atmosphere and 20° C. and does not contain trifluoroacetic acid.
• the present invention has a ligand capable of coordinating to a metal ion and an atomic group capable of a click reaction in its structure, Provided is a compound whose purity decreases by 5% or less after storage at ⁇ 20° C. for one month relative to the purity at the start of storage.
• the present invention also provides a compound storage method for freezing and storing the second compound purified by the production method.
• a ligand compound capable of coordinating to a metal ion is reacted with a first compound having a click-reactive atomic group to form a click-reactive atomic group and a ligand in the structure.
• a step of obtaining a product containing the second compound hereinafter also referred to as a synthesis step
• a step of purifying the product obtained through the synthesis step under predetermined conditions to obtain a purified second compound hereinafter also referred to as purification step).
• the ligand compound and the first compound are reacted to obtain a product containing the second compound.
• the reaction of both 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 their reaction conditions will be described later.
• the product obtained through the synthesis step is the reaction of the ligand compound and the first compound, in addition to the second compound that is the main product of interest, the unreacted ligand compound and the first compound , positional isomers and other by-products other than the second compound, and, if necessary, the reaction solvent.
• the second compound has two chemical structures: a ligand capable of coordinating to the metal ion derived from the chemical structure of the ligand compound, and an atomic group capable of a click reaction derived from the chemical structure of the first compound. It is an organic compound. That is, the second compound has, in its structure, a chemical structure capable of coordinating with metal ions and a chemical structure capable of a click reaction.
• One of the characteristics of this production method is that predetermined conditions are employed in the purification step for purifying the product containing the second compound described above.
• Solvents or eluents containing TFA have conventionally been used to purify organic compounds having ligands in their structures.
• the target compound cannot be properly separated and purified, or the purity of the target compound may decrease over time when the purified compound is stored. It became clear that there is a possibility to As a result of intensive investigation by the present inventors on the reason for this, it was speculated that TFA unintentionally coordinated or bonded to the ligand or unintentionally reacted with a highly reactive functional group.
• the product containing the second compound is subjected to liquid chromatography.
• the eluent used at this time preferably contains a water-soluble organic acid that is liquid at 1 atm and room temperature and does not contain TFA. It is more preferable to use one having a boiling point of 150° C. or lower at 1 atm.
• Water-soluble organic acid as used herein means an organic acid that dissolves in water at 1 atm and 20°C.
• the water-soluble organic acid preferably has physical properties such that when the organic acid and water are mixed, no layer separation is visually observed and the mixture is uniform. More preferably, the water-soluble organic acid used herein means an organic acid that dissolves 3 mL or more in 100 mL of water.
• the water-soluble organic acid further has physical properties such that when the organic acid and water are mixed at a volume ratio of 1:10 (organic acid:water), layer separation is not visually observed and a uniform state is obtained. preferable.
• the phrase "not including TFA” means that TFA is not intentionally included in the reaction system or the resulting compound or composition. Alternatively, unavoidable contamination of a trace amount of residual TFA in the measuring instrument is allowed.
• TFA is not intentionally contained in the eluent, it is permissible for TFA derived from the raw material to be unavoidably mixed into the eluent, for example.
• the absence of TFA can be judged by, for example, no peak derived from TFA being observed when analyzed by 19 F-NMR.
• the liquid chromatography method used in the purification process includes, for example, at least one type of column chromatography using columns or gels filled with various fillers, high performance liquid chromatography, medium pressure preparative liquid chromatography, and the like.
• the water-soluble organic acid suitably used in the purification step preferably has a boiling point of 150°C or less at 1 atm.
• formic acid (boiling point: 100.8°C), acetic acid (boiling point: 118°C), propionic acid (boiling point: 141.2° C.) and the like. These can be used singly or in combination.
• acetic acid from the viewpoint of ease of handling, improvement of purification efficiency, suppression of unintended chemical reaction with the second compound, and improvement of storage stability of the purified second compound. .
• the concentration of the water-soluble organic acid in the eluent is preferably 0.001% by volume or more and 10% by volume or less, more preferably 0.01% by volume or more and 1.0% by volume or less, and even more preferably 0.1% by volume. It is more than 1.0 volume% or less. Purification efficiency can be further improved by using such a concentration range.
• liquid components other than the water-soluble organic acid constituting the eluent include water such as distilled water and ion-exchanged water; water-soluble protic solvents such as methanol and ethanol; acetonitrile, N,N-dimethylformamide (DMF ), water-soluble aprotic solvents such as tetrahydrofuran, dimethylsulfoxide and acetone; water-insoluble organic solvents such as hexane, toluene and ethyl acetate. These can be used singly or in combination. In this specification, water-soluble protic solvents and water-soluble aprotic solvents are sometimes referred to as "polar organic solvents”.
• the liquid component of the eluent preferably contains at least one of water and a polar organic solvent, More preferably, it contains at least one of water and acetonitrile.
• the purification process using multiple types of eluents.
• an aqueous solution of the above-described water-soluble organic acid that does not contain TFA is used as the first eluent, and then the second A method using a polar organic solvent solution containing the above-mentioned water-soluble organic acid and not containing TFA as an eluent in the above can be mentioned.
• Switching between the first eluent and the second eluent may be performed as separate steps, or may be performed while changing the concentration gradients stepwise or continuously in one step. good (so-called gradient).
• a purified second compound can be obtained through each of the above steps.
• This second compound is preferably obtained in the form of a composition dissolved or dispersed in the eluent described above. That is, the composition contains a second compound having a ligand capable of coordinating to a metal ion and an atomic group capable of a click reaction in its structure, and is a water-soluble organic acid that is liquid at 1 atm and 20°C. and TFA-free.
• 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.
• the composition can be subjected to a drying method such as vacuum drying or freeze-drying (freeze-drying) to remove the eluent to obtain a solid second compound.
• a drying method such as vacuum drying or freeze-drying (freeze-drying) to remove the eluent to obtain a solid second compound.
• the second compound When dried, the second compound may be obtained as a simple substance (a single substance, in other words, a pure substance), or may be in the form of a solid composition in which a portion of the organic acid remains.
• the solid second compound can be dissolved in another solvent and subjected to subsequent steps, or can be stored in a solid state. Storage conditions can be normal temperature, refrigeration, or freezing.
• Normal temperature can be 0-10°C and “Frozen” can be -100-0°C.
• the purified second compound is preferably stored frozen from the viewpoint of suppressing the decomposition of the second compound and the increase of impurities.
• the temperature in the freeze-drying is preferably less than 0°C, more preferably -15°C or less, and even more preferably -70°C or less as the product temperature of the target compound.
• the external pressure in freeze-drying may be normal pressure or negative pressure.
• the second compound purified through each of the above steps has a purity reduction rate after storage at -20 ° C. or lower for one month relative to the purity at the start of storage, which is preferably 5% or less, more preferably 3%. % or less, more preferably 2% or less.
• a purity reduction rate after storage at -20 ° C. or lower for one month relative to the purity at the start of storage which is preferably 5% or less, more preferably 3%. % or less, more preferably 2% or less.
• Each purity of the compound at the start of storage and after storage can be calculated by calculating the peak area by the automatic integration method using the results measured by high performance liquid chromatography, and then by the area percentage method.
• the ligand compound is not particularly limited as long as it is an organic compound capable of coordinating with a metal ion, and examples thereof include the following organic compounds and compounds containing structures derived from such compounds.
• CDTA or its derivative>
• 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)
• DOTA (1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid)
• DOTMA ((1R,4R,7R,10R)- ⁇ , ⁇ ', ⁇ '', ⁇ '''-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 ⁇ -(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))tetraphosphonicacid)
• 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-tetraazatridecan-13-oic acid
• 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-tetraazatradecanedioic 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-tetraazatradecane )
• PEPA (1,4,7,10,13-pentaazacyclopenta
• H2macropa (6-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-N,N'-methyl)picolinic acid) macropa-NH 2 (4-amino-6-[[16-[(6-carboxypyridin-2-yl)methyl]-1,4],10,13-tetraoxa-7,16-diazacyclooctadec-7-yl]methyl ) pyridine-2-carboxylic acid) macropa-NCS (6-[[16-[(6-carboxypyridin-2-yl)methyl]-1,4,10,13-t]]etraoxa-7,16-diazacycloctadec-7-yl)methyl]-4 -isothiocyanatopyridine-2-carboxylic acid)
• H4octapa N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid
• H5decapa N,N''-bis(6-carboxy-2-pyridylmethyl)-diethylenetriamine-N,N',N''-triacetic acid
• H6phospa N,N'-(methylenephosphonate)-N,N'-[6-(methoxy
• the ligand compound used in the synthesis step is Macropa, Deferoxamine, HBED-CC, CDTA, DOTA, DTPA, or NOTA or a derivative thereof is preferred, and a compound containing a structure derived from DOTA or a derivative thereof is more preferred.
• Structures derived from DOTA include, for example, those represented by the following formula (1). These compounds may be anhydrides, hydrates, or acid anhydrides.
• 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 PO 3 H 2 or a group consisting of —(CH 2 ) p CONH 2 .
• Each p is independently an integer of 0 or more and 3 or less.
• one of R 14 or R 15 is a hydrogen atom, —(CH 2 ) p COOH, —(CH 2 ) p C 5 H 4 N, —(CH 2 ) p PO 3 H 2 , — (CH 2 ) p CONH 2 or a group consisting of —(CHCOOH)(CH 2 ) p COOH.
• the other of R 14 or R 15 is —(CH 2 ) p COOH, —(CH 2 ) p C 5 H 4 N, —(CH 2 ) p PO 3 H 2 , or —( CH 2 ) p CONH 2 or an atomic group for linking with the first compound described later.
• Each p is independently an integer of 0 or more and 3 or less.
• the first compound used in the synthesis process has an atomic group capable of a click reaction in its structure.
• atomic groups include, for example, alkynyl or azide groups, or dienes or dienophiles such as 1,2,4,5-tetrazine or alkenyl groups. These are also preferably atomic groups that can be used in metal catalyst-free click reactions.
• a click reaction is a reaction that occurs with a combination of an alkyne and an azide, or a combination of a diene such as a 1,2,4,5-tetrazine and an alkene, and a dienophile.
• Specific examples of the click reaction by such a combination of atomic groups include Huisgen cycloaddition reaction, inverse electron demand type Diels-Alder reaction, and the like.
• the chemical structures produced by the click reaction with combinations of alkynes and azides are those containing triazole skeletons, and combinations of 1,2,4,5-tetrazines and alkenes as combinations of dienes and dienophiles.
• the chemical structure generated by the click reaction at contains a pyridazine backbone.
• click-reactive atomic groups include an atomic group containing dibenzocyclooctyne (DBCO) as alkyne (formula (5a)), and an atomic group containing an azide group as azide (formula ( 5b)), a group containing 1,2,4,5-tetrazine (formula (5c)), or a group containing trans-cyclooctene (TCO) as the alkene (formula (5d)).
• DBCO dibenzocyclooctyne
• an azide group as azide
• formula (5c) a group containing 1,2,4,5-tetrazine
• TCO trans-cyclooctene
• R 1 represents a binding site with another structure.
• R2 represents a binding site with another structure.
• one of R3 and R4 represents a bonding site with another structure, and the other represents a hydrogen atom, a methyl group, a phenyl group or a pyridyl group.
• R5 represents a binding site with another structure.
• the click-reactive atomic group in the first compound is dibenzocyclooctyne (DBCO).
• 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 reagents such as DBCO-PEG-Maleimide, TCO-PEG-DBCO, DBCO-mPEG can be used.
• the reaction between the ligand compound and the first compound in the synthesis step is preferably carried out with both compounds dissolved or dispersed in the reaction solvent.
• examples of this include a method of dissolving or dispersing a solid ligand compound and a solid first compound in a reaction solvent, or a method of adding one compound to a solution or dispersion of the other compound in a solid or liquid form.
• the aspect of adding is mentioned.
• a compound other than the ligand compound and the first compound and capable of forming a linker structure may be added to the reaction system.
• Such other compounds include amino acids, polyethylene glycol (PEG), and the like.
• reaction solvent used in the synthesis step for example, the same solvents as those described in the eluent above can be used singly or in combination.
• the concentration of the ligand compound in the reaction solution can be appropriately changed according to the type of the 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 point of view, the concentration of the first compound in the reaction solution can be appropriately changed according to 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.
• the upper limit of the first compound in the reaction solution is preferably 0.30 mol/L or less, more preferably 0.24 mol/L or less, and the reaction yield is improved. From the viewpoint, 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 1.2 or more. .3 or less.
• heating refers to applying heat from the outside of the reaction system so that the temperature of the reaction solution is higher than 25°C, with 25°C being the standard.
• a known method can be appropriately used, and examples thereof include a water bath, an oil bath, a block heater, a mantle heater and the like.
• the reaction temperature is preferably 30° C. or higher and 100° C. or lower, more preferably 50° C. or higher, from the viewpoint of achieving both suppression of decomposition of the ligand compound and further improvement of the yield.
• the reaction solution is heated to 80° C. or lower to react.
• 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.
• the chemical structure of the ligand compound capable of coordinating to the metal ion and the chemical structure of the click-reactive atomic group of the first compound are particularly It is preferable to select conditions under which both chemical structures are maintained without modification of , from the viewpoint of enhancing the convenience in subsequent production steps.
• the binding mode of the ligand compound and the first compound can be selected so that a stable bond is formed under normal synthesis conditions, thereby suppressing the decomposition of the resulting compound. It is preferable in that the storage stability can be further improved.
• a preferred example of a reaction method that can achieve both the above-mentioned suitable conditions is a method in which the ligand compound and the first compound are subjected to an amidation reaction to bind the two compounds via an amide bond. These reactions are also preferably carried out on the side chains of each compound. Specifically, 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 performed between the carboxy group and the amino group to obtain A method of obtaining a second compound having an amide bond is included.
• a carboxy group is a monovalent functional group represented by “—COOH” or “—COO ⁇ ”.
• An amino group is a monovalent functional group represented by “—NH 2 ” or “—NH 3 + ”.
• a ligand compound or first compound having an oxydicarbonyl group can be used for the amidation reaction.
• the ligand compound or first compound having an oxydicarbonyl group may be a symmetrical anhydride having two identical acyl groups or a mixed anhydride having different acyl groups. It may also be a cyclic anhydride produced by dehydration condensation of carboxy groups present in the same molecule of polyvalent carboxylic acid. Of these, mixed anhydrides or cyclic anhydrides are preferable, and cyclic anhydrides are more preferable, from the viewpoint of economy and synthesis efficiency.
• the amidation reaction may be carried out by stirring in a reaction solvent at room temperature or under heating, and if necessary, an amide condensing agent may be added.
• amide condensing agents include carbodiimide condensing agents such as N,N'-dicyclohexylcarbodiimide (DCC); condensing agents via acid azides such as diphenyl phosphate azide (DPPA); and hexamethylphosphoric acid triamide (HMPA).
• BOP reagent combined with 1-hydroxybenzotriazole HOBt
• PyBOP in which dimethylamino group of BOP reagent is substituted with pyrrolidino group
• O-(benzotriazol-1-yl)-N in which phosphorus of BOP reagent is substituted with carbon
• Uronium type condensing agents such as N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and triazole type condensing agents such as DMT-MM
• the amidation reaction may be carried out by adding a base such as triethylamine, if necessary.
• Another reaction method that can achieve both the above-mentioned suitable conditions is preferably a method of binding the ligand compound and the first compound in a reaction manner that forms a thiourea structure between the two compounds. These reactions are also preferably carried out on the side chains of each compound. Specifically, 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 method for obtaining a second compound having a thiourea structure is included.
• the reaction conditions for forming a thiourea structure can be, for example, conditions in which a base such as N,N'-diisopropylethylamine or triethylamine is used and stirred at room temperature or under heating.
• 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).
• Each of the second compounds represented by the following formulas (7a) and (7b) and formulas (8a) and (8b) has a chemical structure capable of coordinating to a metal ion and the first compound has a click reaction capable of the chemical structure of the same group is maintained together.
• the chemical structure of the second compound can be changed as appropriate according to the types of ligand compound and first compound used.
• the ligand compound is DOTAGA or its anhydride, from the viewpoint of obtaining a second compound having higher storage stability and from the viewpoint of more efficiently performing the coordination reaction and click reaction of metal ions in subsequent steps, 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.
• the second compound obtained through the purification process is either in the state of a single substance or in the form of a composition, or in the state of being dissolved in a solvent, buffer, or the like, or is subjected to a process such as distilling off a predetermined eluent under reduced pressure. It can be used for the subsequent steps in the state after washing.
• 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 of manufacturing a targeting agent.
• the targeting agent contains, in its chemical structure, a metal complex and an atomic group having directivity to a target organ or tissue in vivo or specific binding property to a target molecule.
• steps (a) and (b) may be performed in the order of (a) and (b), or alternatively, may be performed in the order of (b) and (a). Steps (a) and (b) may also be performed consecutively in this order or in reverse order, alternatively, between steps (a) and (b), step (a) ) and (b) may intervene.
• steps (a) and (b) it is preferable to carry out steps (a) and (b) in this order. Specifically, a step of coordinating the second compound obtained in the purification step to a metal ion to obtain a metal complex is performed, and then the metal complex and a targeting compound having an atomic group capable of a click reaction are subjected to a click reaction. It is preferred to carry out a step of reacting between possible atomic groups. Performing each step in this order is advantageous in that the yield of the complex can be increased and the targeting agent can be subjected to subsequent steps without separating and purifying unreacted metal ions. In addition, it is possible to obtain a targeting agent with enhanced directivity to target organs or tissues in vivo or specific binding to target molecules with high productivity. In particular, the use of radioactive metal complexes is advantageous in that it is possible to effectively treat and detect diseases.
• a preferred method for producing a targeting agent will be described below by taking as an example the case where steps (a) and (b) are performed in this order.
• the second compound obtained through the purification step can be reacted with metal ions in the form of a single substance or composition, or dissolved in an aqueous solution such as a solvent or buffer to form a metal complex.
• complex formation step The metal complex obtained in this step is formed by coordinating metal ions to the ligands contained in the chemical structure of the second compound.
• the metal to be coordinated may be a non-radioactive element or a radioactive isotope.
• 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 collectively referred to as Also referred to as “metal source”).
• the metal source for example, a metal ion-containing liquid dissolved in the form of metal ions in a solvent mainly composed of water can be used.
• the reaction system containing the compound and the metal ion is heated to react is preferred.
• 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 the lower limit is preferably 5 minutes or more, more preferably 10 minutes or more, and still more preferably 15 minutes, provided that the temperature is within the above-described temperature range. Above, it is more preferably 30 minutes or more, and the upper limit is preferably 150 minutes or less, more preferably 120 minutes or less, still more preferably 100 minutes or less, and still more preferably 90 minutes or less.
• the volume of the reaction liquid is not particularly limited, but from the viewpoint of practicality, 0.01 mL to 100 mL is realistic at the start of this step. Further, the concentrations of the compound and the metal ion in the reaction solution are each independently preferably 1 ⁇ mol/L to 100 ⁇ mol/L at the start of this step from the viewpoint of the yield of the desired metal complex. .
• Solvents used in the complex formation step include, for example, water, physiological saline, sodium acetate buffer, ammonium acetate buffer, phosphate buffer, phosphate-buffered saline, Tris buffer, HEPES buffer, or tetra
• a buffer such as a methylammonium acetate buffer can be used.
• metals to be coordinated include alkali metals, alkaline earth metals, lanthanides, actinides, transition metals, non-radioactive elements and radioactive elements of metals other than these metals, and isotopes thereof. Taking such metal elements as examples, Sc, Cr, Co, Fe, Ga, Cu, Sr, Zr, Y, Tc, Ru, In, Sm, Dy, Ho, Lu, Re, Au, Tl, Hg , Bi, Pb, Th or Ac. Zr, Lu, In, Y, Ga, Cu or Ac, more preferably a radioactive metal, from the viewpoint of making the metal complex or composition containing the same applicable to treatment, diagnosis or detection of diseases . Zr, 177 Lu, 111 In, 90 Y, 67 Ga, 68 Ga, 64 Cu, or 225 Ac. These metals can be produced according to a conventional method and obtained as a solution containing the metal in an ionized state.
• the metal complex obtained through the complex formation process has an atomic group capable of a click reaction derived from the chemical structure of the second compound. Therefore, the metal complex is click-reacted with a targeting compound having a click-reactive second atomic group to produce a targeting agent (click reaction step).
• a targeting compound having a click-reactive second atomic group to produce a targeting agent (click reaction step).
• This makes it possible to obtain an agent with enhanced directivity to target organs or tissues in vivo or specific binding to target molecules.
• the use of radioactive metal complexes is advantageous in that it is possible to effectively treat and detect diseases.
• the click reaction can be performed between the click-reactive atomic group contained in the chemical structure of the metal complex and the click-reactive second atomic group contained in the chemical structure of the targeting compound.
• the metal complex obtained using the second compound has DBCO as a click-reactive atomic group. Therefore, a targeting compound having an azide group as the click-reactive second atomic group can be used to cause a click reaction between DBCO and the azide group.
• Known conditions can be employed for the click reaction, and from the viewpoint of preventing denaturation of the targeting compound, the click reaction is preferably carried out in a non-heated state.
• Targeting compounds may be 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, It preferably contains one or more atomic groups selected from miRNA, nucleic acid aptamers, decoy nucleic acids, cPG oligonucleic acids, peptide nucleic acids, liposomes, micelles, carbon nanotubes, and nanoparticles. These atomic groups are also preferably chemical structures capable of binding to the target molecule of interest. Further, when introducing a second atomic group capable of a click reaction into the targeting compound, a known reagent can be used.
• 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 still more preferably an integer of 2 or more and 5 or less.
• the group preferably contains a linear peptide, a cyclic peptide, or a combination thereof, a protein, an antibody, or a fragment thereof that specifically binds to a particular molecule.
• atomic groups are peptides having three or more constituent amino acid residues, such as antibodies (immunoglobulins) and Fab fragments having classes of IgG, IgA, IgM, IgD and IgE. , antibody fragments such as F(ab')2 fragments, and peptide aptamers.
• the amino acids that constitute such targeting agents may be natural or synthetic.
• the molecular weight of the atomic group containing the peptide is not particularly limited.
• the various peptides described above can be prepared by conventionally known methods such as liquid-phase synthesis, solid-phase synthesis, automated peptide synthesis, genetic recombination, phage display, genetic code reprogramming, RaPID (Random non-standard peptide integrated It can be synthesized by a technique such as the Discovery method. In synthesizing various peptides, functional groups of amino acids used may be protected as necessary.
• the targeting compound is an atomic group containing a nucleic acid in its structure
• the atomic group includes antisense nucleic acids, siRNA, miRNA, nucleic acid aptamers, decoy nucleic acids, cPG oligonucleic acids, and peptide nucleic acids that specifically bind to specific molecules. It is preferably an atomic group.
• Nucleic acid bases may be natural ones such as deoxyribonucleic acid and ribonucleic acid, or synthetic ones.
• nucleic acid aptamers that specifically bind to specific target substances such as proteins can be produced using the SELEX method (Systematic Evolution of Ligands by Exponential Enrichment).
• the metal complex and targeting agent produced through the above steps typically exist in a dissolved state in the reaction solution.
• These solutions may be independently used as they are, or may be purified using filtration filters, membrane filters, columns filled with various fillers, chromatography, or the like.
• a formulation step for obtaining a drug containing the targeting agent as an active ingredient can be further performed.
• citrate buffers, phosphate buffers, pH adjusters such as borate buffers, solubilizers such as polysorbate, various additives such as stabilizers or antioxidants are added as appropriate, It can be carried out by adjusting the concentration of the agent by diluting it with an isotonic solution such as water or physiological saline.
• the formulation step may include a step of adding various additives or adjusting the concentration, followed by sterilizing filtration with a membrane filter or the like to prepare an injection.
• the ligand compound, the first compound and the second compound in the present specification may each independently be a compound labeled or isotopically substituted with the same element as the element in the structure.
• One or more atoms may be replaced by an atom having an atomic mass or mass number different from the naturally occurring atomic mass or mass number.
• the aforementioned isotopes include, for example, isotopes of hydrogen, carbon, nitrogen and oxygen such as 2H , 3H , 11C , 13C , 14C , 15N , 17O, 18O .
• Pharmaceutically acceptable salts of each of the foregoing compounds containing the above mentioned isotopes and/or other isotopes of other atoms are within the scope of this invention.
• radioactive isotopes such as 3 H and 14 C are incorporated are useful in research for non-clinical or non-medical purposes, such as drug and/or substrate tissue distribution assays.
• the metal ion to be coordinated may be a non-radioactive element.
• Compounds having at least one of 3 H and 14 C in their structure are preferred from the standpoint of their ease of preparation and detection.
• Isotopically-labeled compounds described above can typically be prepared by substituting an isotopically-labeled reagent for a non-isotopically-labeled reagent.
• the product containing DOTAGA-DBCO was then subjected to a medium pressure preparative liquid chromatography method using eluents for purification.
• the eluent in the medium pressure preparative liquid chromatography method uses 0.1% by volume acetic acid aqueous solution as the first eluent, and uses acetonitrile containing 0.1% by volume acetic acid as the second eluent, with the following concentrations Elution was performed under gradient conditions. None of the eluents contained TFA.
• eluent 1 a first eluent
• eluent 2 a second eluent
• the flow rate of each eluent was 12 mL/min.
• DOTAGA-DBCO is a composition dissolved in a 0.1% by volume aqueous solution of acetic acid.
• Example 1 21-25°C
• Example 2 40°C
• Example 3 5°C
• Example 4 -20°C
• Example 5 (Synthesis process) Example 1 except that DMSO was used as an 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. DOTAGA-DBCO was obtained by the method described in the synthesis steps of 1 to 4.
• eluent 1 a first eluent
• eluent 2 a second eluent
• the flow rate of each eluent was 12 mL/min.
• DOTAGA-DBCO is a composition dissolved in a 0.1% by volume formic acid aqueous solution.
• this composition was dried by lyophilization to remove the 0.1 vol% formic acid aqueous solution to obtain solid DOTAGA-DBCO.
• This was stored in a colorless and transparent glass vial. After that, the colorless transparent glass vial containing DOTAGA-DBCO was placed under room temperature conditions (Example 5: 19 to 23° C.) and stored for a predetermined period of time.
• DOTAGA-DBCO obtained by drying in the same manner as in Examples 1 to 4 was placed in a brown vial, and this brown vial was placed at room temperature (Comparative Example 1: 21 to 25 ° C.) and at high temperature (Comparative Example 2: 40° C.) and stored for a predetermined period.
• ⁇ Method for measuring purity> Accurately weigh 1.0 mg of the target compound, and for Examples 1 and 2 and Comparative Examples 1 and 2, add 10 mmol/L ammonium formate solution (pH 3.0)/acetonitrile mixture for liquid chromatography (95:5) The volume was adjusted to 10 mL exactly and used as a sample solution.
• a mixture of 10 mmol/L ammonium formate solution (pH 3.0)/methanol for liquid chromatography (95:5) was added to make exactly 10 mL, and for Example 5, methanol for liquid chromatography was added. was added to make exactly 10 mL and used as a sample solution. 10 ⁇ L of these sample solutions were measured by high performance liquid chromatography under the following conditions.
• the peak areas of the sample solutions were measured by the automatic integration method, and the amounts of those compounds were determined by the area percentage method.
• the sample solution was prepared once and measured three times. Purity (%) was defined as the arithmetic mean value of the measurement results of three measurements. At the start of storage, the sample solution was prepared once and measured once.
• Detector (1) UV absorption photometer (measurement wavelength: 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 to 1200)
• the compounds of each example purified in the absence of TFA were stored in a state in which the purity at the start of storage was maintained even after one month of storage, compared with those of the comparative examples. It turns out that it is done.
• 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 6 months of storage, and it can be seen that the long-term storage stability is excellent. .

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