Triazolinedione adduct, method for producing a triazolinedione adduct, method for producing an energy compound and method for analyzing an energy compound

Description

TECHNICAL FIELD

[0001] The present invention relates to a Cookson-type derivatization reagent consisting of a triazolinedione adduct, a method for producing the Cookson-type derivatization reagent, a method for producing an ene compound and a method for analyzing the ene compound.BACKGROUND ART

[0002] There has recently been an increased need for analysis of vitamin D and vitamin D metabolites in blood. With regard to an analysis method of vitamin D and the vitamin D metabolites, a method has been proposed in which the vitamin D and the vitamin D metabolites are derivatized using a Cookson-type derivatization reagent and subsequently the derivative is analyzed (see Patent Document 1 and Non-Patent Documents 1 to 4).

[0003] Specifically, the Cookson-type derivatization reagent causes a Diels-Alder reaction with a diene derivative such as vitamin D to proceed extremely rapidly under mild conditions, to give an ene compound quantitatively (see, Non-Patent Documents 1 to 4). Utilizing this reaction characteristic, vitamin D and the like, which are per se difficult to be quantitatively determined, are reacted with the Cookson-type derivatization reagent to convert vitamin D and the like to an ene compound having higher analysis sensitivity, and thereafter the ene compound is quantified.

[0004] Examples of the aforementioned Cookson-type derivatization reagent include triazolinedione compounds such as PTAD (4-phenyl-1,2,4-triazoline-3,5-dione) and DAPTAD (4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione).

[0005] Patent Document 2 discloses a method for derivatizing a s-cis-diene compound with a Cookson-type derivatization reagent, the method including adding, in a reaction-stopping step of stopping a derivatization reaction of the s-cis-diene compound, a decomposition inhibitor to inhibit decomposition of a derivative to be obtained. Patent Document 1: Japanese Unexamined Patent Application, Publication No. 2015-166740 Patent Document 2: EP 3301453 A Non-Patent Document 1: S. Ogawa,et al., Rapid Commun. Mass Spectrom, 27(2013) 2453-2460 Non-Patent Document 2: S. Ogawa,et al., Biomed. Chromatgr., 30(2016) 938-945 Non-Patent Document 3: S. Ogawa,et al., J. Pharm. Biomed. Anal., 136(2017) 126-133 Non-Patent Document 4: K. D. Bruycker, et al., Chem. Rev., 116(2016) 3919-3974 DISCLOSURE OF THE INVENTIONProblems to be Solved by the Invention

[0006] However, the triazolinedione compounds were unstable and accompanied by a high ratio of degradation during the process of isolation. The handling property as a compound was also low, because the stability after isolation was also low. Therefore, in order to handle a triazolinedione compound as a raw material for production or a quantitative reagent, a higher stability has been required.

[0007] The present invention was made in view of the above background and an object of the present invention is to provide a Cookson-type derivatization reagent consisting of a triazolinedione adduct having stability and a method for producing the same, as well as a method for producing an ene compound using the triazolinedione adduct and a method for analyzing a diene compound.

[0008] Another object of the present invention is to provide a method for producing an ene compound derived from a Cookson-type derivatization reagent consisting of a triazolinedione compound with high stability, using a triazolidinedione compound which is a precursor of the triazolinedione compound.Means for Solving the Problems

[0009] The triazolinedione compound is known to reversibly undergo Diels-Alder reaction with a polycyclic aromatic hydrocarbon such as anthracene to give an adduct (see, e.g., N. Roy, et al., Chem. Asian. J., 6 (2011) pp. 2419-2425; V. D. Kiseleva, et al., Russ. J. Phys. Chem. A, 88 (2014) pp. 2073-2080; and the like). That is, the triazolinedione adduct once obtained undergoes a retrospective reaction to the starting materials, that is, the triazolinedione compound and polycyclic aromatic hydrocarbon, by heating (retro-Diels-Alder reaction).

[0010] The present inventors have focused on the above characteristics and have found that the stability of a triazolinedione compound can be secured by converting an unstable triazolinedione compound into a stable adduct and carrying out reaction while reverting the adduct to the triazolinedione compound at the time of use, thus having completed the present invention.

[0011] Further, the present inventors have found that if the triazolinedione compound is generated in a system by using a triazolidinedione compound which is a precursor of the triazolinedione compound, and then the generated triazolinedione compound is immediately reacted with a diene, the ene compound can be produced in a highly stable manner, thereby completing the present invention.

[0012] That is, a first aspect of the present invention relates to a Cookson-type derivatization reagent consisting of a triazolinedione adduct represented by the following formula (2) or (3): in which R 1< represents a dialkylamino group or a dialkylaminoalkyl group; R 2< , R 3< , and R 4< each independently represent a substituent selected from the group consisting of an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted aralkyl group, an optionally substituted alkenyl group, an optionally substituted phenyl group, an optionally substituted acyl group, a halogen group, an optionally substituted amino group, a nitro group, and an optionally substituted heterocyclic group; l and m are each independently an integer of 0 to 4; and n is an integer of 0 to 2, in which R 1< represents a dialkylamino group or a dialkylaminoalkyl group; R 5< , R 6< , R 7< , and R 8< each independently represent a substituent selected from the group consisting of an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted aralkyl group, an optionally substituted alkenyl group, an optionally substituted phenyl group, an optionally substituted acyl group, a halogen group, an optionally substituted amino group, a nitro group, and an optionally substituted heterocyclic group; p is an integer of 0 to 4; q is an integer of 0 to 2; and r and s are integers of 0 or 1.

[0013] In an embodiment of the first aspect, the Cookson-type derivatization reagent is represented by the following formula (2-1) or (2-2): wherein Me represents a methyl group and Ph represents a phenyl group.

[0014] In an embodiment of the first aspect, the Cookson-type derivatization reagent is represented by the following formula (3-1): wherein Ph represents a phenyl group.

[0015] In a second aspect of the invention, there is provided a method for producing the Cookson-type derivatization reagent according to the first aspect or an embodiment thereof, comprising subjecting a triazolinedione compound and a compound having an anthracene ring to a Diels-Alder reaction to obtain the triazolinedione adduct, wherein the triazolinedione compound is represented by the following formula (4): wherein R 1< represents a 4-dialkylaminophenyl group or a 4-dialkylaminoalkylphenyl group.

[0016] In an embodiment of the second aspect, the method further comprises a purification step of purifying the triazolinedione adduct.

[0017] In a third aspect of the invention, there is provided a method for producing the Cookson-type derivatization reagent according to the first aspect or an embodiment thereof, comprising reacting a triazolidinedione compound with a compound having an anthracene ring in the presence of an oxidizing agent to obtain the triazolinedione adduct, wherein the triazolidinedione compound is represented by the following formula (7): wherein R 1< represents a 4-dialkylaminophenyl group or a 4-dialkylaminoalkylphenyl group.

[0018] In an embodiment of the third aspect, the oxidizing agent is a hypervalent iodine compound.

[0019] In an embodiment of the third aspect, the oxidizing agent is a hypervalent iodine compound represented by the following formula (8): wherein X and Y together represent an oxygen atom, or X and Y each independently represent a group selected from the group consisting of a hydroxy group, an alkoxy group, an acyloxy group, an acylamino group, a tosylamino group, a mesylamino group, a sulfonyloxy group, and a halogen group; and Ar represents a group selected from the group consisting of a phenyl group, a heterocyclic group, and a phenyl group substituted with an alkyl group, an alkoxy group, a halogen group, etc.

[0020] In an embodiment of the third aspect, the method further comprises a purification step of purifying the triazolinedione adduct.

[0021] In a fourth aspect of the invention, there is provided a method for producing an ene compound, comprising reacting a Cookson-type derivatization reagent according to the first aspect or an embodiment thereof with a diene compound to obtain the ene compound, wherein the Cookson-type derivatization reagent according to the first aspect or an embodiment thereof is reacted with vitamin D3 or 25-hydroxyvitamin D3 at a temperature of 20 to 350°C.

[0022] In a fifth aspect of the invention there is provided a method for analyzing an ene compound, comprising reacting a Cookson-type derivatization reagent according to the first aspect or an embodiment thereof with a diene compound to obtain the ene compound and analyzing the ene compound, wherein the Cookson-type derivatization reagent according to the first aspect or an embodiment thereof is reacted with vitamin D3 or 25-hydroxyvitamin D3 at a temperature of 20 to 350°C.

[0023] In an embodiment of the fifth aspect, the analysis uses a high performance liquid chromatography.Effects of the Invention

[0024] The triazolinedione adduct of the Cookson-type derivatization reagent of the present invention can be reacted while being reverted to a triazolinedione compound at the time of use and the triazolinedione adduct can maintain its stability until the time of use. Therefore, it is possible to improve the stability of unstable triazolinedione compounds and to improve the handling property of the triazolinedione compounds.

[0025] Further, the triazolinedione adduct of the Cookson-type derivatization reagent of the present invention easily returns to the triazolinedione compound by heating, and the triazolinedione compound reacts with a diene compound to generate an ene compound having high analytical sensitivity. Therefore, the triazolinedione adduct is a very meaningful compound as a reagent for quantifying diene compounds.

[0026] Further, since the triazolinedione adduct of the Cookson-type derivatization reagent of the present invention is an equivalent of a triazolinedione compound, the triazolinedione adduct can be used for producing various kinds of ene compounds, in addition to use as the above-described reagent for quantifying diene compounds.

[0027] Further, by using the triazolidinedione compound which is a precursor of the triazolinedione compound, feeding the triazolidinedione compound together with a diene, and performing an oxidation reaction while generating the triazolinedione compound in the system, it is possible to make the diene immediately react with the triazolinedione compound to obtain an ene compound. Thereby, it is possible to reduce the time during which an unstable triazolinedione compound exists and to obtain an ene compound derived from the triazolinedione compound in high yield.PREFERRED MODE FOR CARRYING OUT THE INVENTION

[0028] Hereinafter, embodiments of the present invention will be described. The embodiments described below do not limit the present invention.[Production Method (1) of Triazolinedione Adduct]

[0029] A first method of the present invention for producing the Cookson-type derivatization reagent, consisting of the triazolinedione adduct, is a method in which a triazolinedione compound represented by the following formula (4) and a compound having an anthracene ring are subjected to a Diels-Alder reaction to obtain the triazolinedione adduct.

[0030] In the formula (4), R 1< represents a 4-dialkylaminophenyl group or a 4-dialkylaminoalkylphenyl group.[Triazolinedione Compound]

[0031] R 1< in the formula (4) is preferably a group selected from the group consisting of 4-dimethylaminophenyl group and 4-dimethylaminomethylphenyl group.

[0032] As the triazolinedione compound used in the first method of the present invention for producing the triazolinedione adduct, a compound selected from the group consisting of 4-(4'-diethylaminophenyl)-1,2,4-triazoline-3,5-dione (DEAPTAD), 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), and 4-(4'-dimethylaminomethylphenyl)-1,2,4-triazoline-3,5-dione is particularly preferred. These compounds can be a Cookson-type derivatization reagent.

[0033] Further, R 1< is most preferably a 4-dimethylaminophenyl group or a 4-dimethylaminomethylphenyl group. That is, the most preferred triazolinedione compound used in the first production method of the present invention for producing a triazolinedione adduct is 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) or 4-(4'-dimethylaminomethylphenyl)-1,2,4-triazoline-3,5-dione.[Compound Having An Anthracene Ring]

[0034] In the present invention, a part of hydrogen atoms included in the compound having an anthracene ring may be substituted with a substituent selected from the group consisting of an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted aralkyl group, an optionally substituted alkenyl group, an optionally substituted phenyl group, an optionally substituted acyl group, a halogen group, an optionally substituted amino group, a nitro group, and an optionally substituted heterocyclic group. Examples of the substituent which may be included in the substituent selected from the group consisting of an alkyl group, an aralkyl group, an alkenyl group, a phenyl group, an acyl group, an amino group, and a heterocyclic group include: alkyl groups such as a methyl group, an ethyl group, a propyl group, a butyl group, an isobutyl group, a t-butyl group, a chloromethyl group, a 2-chloroethyl group, a 3-chloropropyl group, a 2-chloropropyl group, a 1-chloropropyl group, a bromomethyl group, a 2-bromoethyl group, a 3-bromopropyl group, a 2-bromopropyl group, and 1-bromopropyl group; aralkyl groups such as a benzyl group, a phenethyl group, a 3-phenylpropyl group, a 4-methylbenzyl group, and a 4-methylphenethyl group; alkenyl groups such as a vinyl group, an allyl group, a propenyl group, a cyclopropenyl group, a 2-chlorovinyl group, a 3-chloroallyl group, a cinnamyl group, and a styryl group; phenyl groups such as a phenyl group, a tolyl group, a xylyl group, and a trityl group; acyl groups such as a formyl group, an acetyl group, a propionyl group, a benzoyl group, an acryloyl group, and a methacryloyl group; halogen groups such as a chloro group, a bromo group, a fluoro group, and an iodo group; amino groups such as an amino group, a methylamino group, a dimethylamino group, an ethylamino group, and a diethylamino group; a nitro group; heterocyclic groups such as a quinolyl group, a pyridyl group, a pyrrolidyl group, a pyrrolyl group, a furyl group, a thienyl group, and a lutidyl group; and the like.[Reaction Conditions](Reaction Solvent)

[0035] In the first production method of the triazolinedione adduct of the Cookson-type derivatization reagent, the reaction solvent to be used when subjecting the triazolinedione compound represented by the formula (4) and the compound having an anthracene ring to Diels-Alder reaction is preferably at least one selected from the group consisting of esters, halogen-containing hydrocarbons, aromatic hydrocarbons, ketones, amides, alkylnitriles, dialkyl ethers, and ureas. Note that the diene compound to be subjected to the reaction per se may be used as the solvent.

[0036] The Diels-Alder reaction of the triazolinedione compound represented by the formula (4) with a compound having an anthracene ring is a reversible reaction. For this reason, in order to shift equilibrium of the reaction towards the production system, a solvent capable of crystallizing the product is preferred. In other words, in the production of the triazolinedione adduct, it is preferable to select a solvent in which the adduct is crystallized and in the reaction of the triazolinedione adduct and a diene compound, it is preferable to select a solvent in which the adduct is dissolved.

[0037] Examples of the solvent include aprotic solvents such as ethyl acetate, methyl acetate, butyl acetate, isopropyl acetate, tetrahydrofuran (THF), 2-methyltetrahydrofuran (2-methyl THF), 1,4-dioxane, t-butyl methyl ether, 1,2-dimethoxyethane, diglyme, acetone, diethyl ketone, methyl ethylketone, methylene chloride, chloroform, carbon tetrachloride, 1,2-dichloroethane, chlorobenzene, toluene, xylene, mesitylene, dimethylformamide (DMF), dimethyl acetamide (DMA), dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), 1,3-dimethyl-2-imidazolidinone (DMI), and the like. These may be used alone or as a mixed solvent.

[0038] Among these, at least one type selected from the group consisting of methylene chloride, chloroform, acetonitrile, 1,2-dimethoxyethane, toluene, chlorobenzene, and ethyl acetate is preferably used from the viewpoint of product crystallinity, yield, stability, safety, and price.

[0039] The amount of the solvent used in the reaction is preferably 5 to 1,000 volumes with respect to 1 part by mass of the reaction substrate.(Reaction Temperature)

[0040] The temperature for the reaction of the triazolinedione compound represented by the formula (4) with the compound having an anthracene ring is preferably in the range of -10°C to 60°C, and more preferably in the range of 0°C to 40°C.(Reaction Time)

[0041] The reaction time during which the triazolinedione compound represented by the formula (4) is reacted with the compound having an anthracene ring is preferably 10 minutes to 48 hours, and is more preferably in the range of 1 to 10 hours.(Feed Amount)

[0042] In the reaction of the triazolinedione compound represented by the formula (4) with the compound having an anthracene ring, the used amount of the compound having an anthracene ring is preferably in the range of 1.0 to 10,000 equivalents with respect to 1 equivalent of the triazolinedione compound.(Others)

[0043] For the purpose of removing water generated by the reaction, a dehydrating agent such as a molecular sieve may be added to the reaction solvent to perform the reaction.[Crystallization Conditions]

[0044] The first method for producing the triazolinedione adduct of the Cookson-type derivatization reagent includes reacting a triazolinedione compound represented by the formula (4) with a compound having an anthracene ring to obtain a triazolinedione adduct. The obtained triazolinedione adduct may be crystallized and obtained as a solid.(Crystallization Solvent)

[0045] Examples of the solvent for crystallizing the obtained triazolinedione adduct include aprotic solvents such as methylene chloride, chloroform, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, acetonitrile, 1,2-dimethoxyethane, toluene, xylene, chlorobenzene, mesitylene, ethyl acetate, hexane, heptane, pentane, cyclopentane, cyclohexane, isohexane, isooctane, decane, and the like, and protic solvents such as water, methanol, ethanol, 2-propanol, 2-butanol, and butanol. These may be used alone or as a mixed solvent.

[0046] The amount of the solvent used for crystallization is preferably 5 to 1,000 volumes with respect to 1 part by mass of the obtained triazolinedione adduct.(Crystallization Temperature)

[0047] The temperature at which the triazolinedione adduct is crystallized is preferably in the range of -10 to 40°C.(Crystallization Time)

[0048] The time duration for crystallizing the triazolinedione adduct is preferably in the range from 30 minutes to 24 hours, and more preferably from 1 to 10 hours.[Purification Step]

[0049] The first method for producing the triazolinedione adduct of the Cookson-type derivatization reagent may further comprise a purification step of purifying the resulting triazolinedione adduct. The purification method is not particularly limited, and examples thereof include silica gel column chromatography. As the developing solvent, a mixed solvent of a low polarity solvent and a high polarity solvent can be used. Examples of the low polarity solvent include hexane, heptane, etc. and examples of the high polarity solvent include ethyl acetate, tetrahydrofuran, etc.<Production Method (2) of Triazolinedione Adduct>

[0050] The second method of the present invention for producing the Cookson-type derivatization reagent consisting of the triazolinedione adduct is a method including reacting a triazolidinedione compound represented by the following formula (7) with a compound having an anthracene ring in the presence of an oxidizing agent to obtain the triazolinedione adduct.

[0051] In the formula (7), R 1< represents a 4-dialkylaminophenyl group or a 4-dialkylaminoalkylphenyl group.[Triazolidinedione Compound]

[0052] The compound represented by the formula (7), which serves as a starting material in the second method for producing the triazolinedione adduct, is a triazolidinedione compound that includes a urazole group, i.e., a triazolidinedione compound which includes a 1,2,4-triazolidine-3,5-dione group.

[0053] R 1< in the formula (7) is preferably a group selected from the group consisting of a 4-diethylaminophenyl group, 4-dimethylaminophenyl group and 4-dimethylaminomethylphenyl group.

[0054] Among them, 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMU) or 4-(4'-dimethylaminomethylphenyl)-1,2,4-triazolidine-3,5-dione is the most preferable, as the triazolidinedione compound used in the second method of the present invention for producing the triazolinedione adduct of the Cookson-type derivatization reagent.[Oxidizing Agent]

[0055] The oxidizing agent used in the second method of the present invention for producing a triazolinedione adduct is not particularly limited as long as the oxidizing agent enables the triazolidinedione compound represented by the formula (7) to react with a compound having an anthracene ring and produce the triazolinedione adduct, but the oxidizing agent is preferably a hypervalent iodine compound. The hypervalent iodine compound enables a smooth conversion from a triazolidinedione compound to a triazolinedione adduct.

[0056] Moreover, among hypervalent iodine compounds, a compound represented by the following formula (8) is more preferred.

[0057] In the formula (8), X and Y together represent an oxygen atom, or X and Y each independently represent a group selected from the group consisting of a hydroxy group, an alkoxy group, an acyloxy group, an acylamino group, a tosylamino group, a mesylamino group, a sulfonyloxy group, and a halogen group; and Ar represents a group selected from the group consisting of a phenyl group, a heterocyclic group, and a phenyl group substituted with an alkyl group, an alkoxy group, a halogen group, etc.

[0058] Examples of the compound represented by the formula (8) include idosobenzene, iodobenzene diacetate, iodobenzene triflate, iodobenzene tosylate, iodobenzene mesylate, iodobenzene dichloride and iodobenzene dibromide, etc. Among these, iodosobenzene or iodobenzene diacetate is especially preferable from the viewpoint of yield, stability and price.

[0059] The used amount of an oxidizing agent is preferably set to 1 to 10 molar equivalents, more preferably 1 to 5 molar equivalents, with respect to the triazolidinedione compound serving as a starting material. A range of 1 to 5 molar equivalents is preferred, because the oxidizing agent which remains after the reaction and decomposition products thereof can be easily removed.[Compound Having An Anthracene Ring]

[0060] The compound having an anthracene ring which can be used in the second method for producing the triazolinedione adduct is the same as those used in the first method for producing the triazolinedione adduct described above.[Reaction Conditions](Reaction Solvent)

[0061] In the second method for producing the triazolinedione adduct of the Cookson-type derivatization reagent, a solvent used in reacting the triazolidinedione compound represented by the formula (7) with the compound having an anthracene ring in the presence of an oxidizing agent is the same as the solvent used in the first method for producing the triazolinedione adduct described above. The used amount thereof is the same as in the first method for producing a triazolinedione adduct described above.(Reaction Temperature)

[0062] The temperature at which the triazolidinedione compound represented by the formula (7) is reacted with a compound having an anthracene ring is the same as that of the first method for producing the triazolinedione adduct.(Reaction Time)

[0063] In addition, the reaction time during which the triazolidinedione compound represented by the formula (7) is reacted with a compound having an anthracene ring is the same as that in the first method for producing the triazolinedione adduct.(Feed Amount)

[0064] In the reaction of the triazolidinedione compound represented by the formula (7) with a compound having an anthracene ring, the amount of the fused ring compound used is the same as in the first method for producing the triazolinedione adduct described above.(Others)

[0065] In the same manner as in the first method for producing the triazolinedione adduct described above, a dehydrating agent such as a molecular sieve may be added to the reaction solvent for the purpose of removing water generated by the reaction to perform the reaction.[Crystallization Conditions]

[0066] It is also possible to crystallize the obtained triazolinedione adduct and obtain the triazolinedione as a solid in the second method for producing the triazolinedione adduct of the Cookson-type derivatization reagent, as in the first method for producing the triazolinedione adduct. The type of solvent used for crystallization, the amount used, the crystallization temperature, and the crystallization time are the same as those of the first method for producing the triazolinedione adduct.[Purification Step]

[0067] The second production method of the triazolinedione adduct of the Cookson-type derivatization reagent may further comprise a purification step of purifying the resulting triazolinedione adduct. The purification method is not particularly limited, and may be the same as in the purification method in the first method for producing the triazolinedione adduct described above.<Triazolinedione Adduct>

[0068] The triazolinedione adduct of the Cookson-type derivatization reagent of the present invention undergoes a reverse reaction (retro-Diels-Alder reaction) to a compound having an anthracene ring and a triazolinedione compound represented by the formula (4), which are starting materials, by heating. Therefore, the triazolinedione adduct of the Cookson-type derivatization reagent of the present invention can be reacted while being reverted to the triazolinedione compound, and thus the triazolinedione adduct remains in a stable state until the time of use.

[0069] In the reverse reaction, the temperature is in the range of -78 to 200°C, and the reaction time is in the range of 0.01 to 48 hrs. Examples of the most suitable solvents for producing the triazolinedione compound from the triazolinedione adduct by the retro-Diels-Alder reaction include acetonitrile, ethyl acetate, dioxane, acetone, and the like. In the retro-Diels-Alder reaction, when the concentration of the triazolinedione adduct is higher, the triazolinedione compound can be produced at a higher concentration, so that the concentration of the triazolinedione adduct is preferably 1 mmol / L or more, more preferably 5 mmol / L or more, and particularly preferably 10 mmol / L or more. Additionally, the retro-Diels-Alder reaction is an endothermic reaction, so that higher temperatures are favorable for the formation of the triazolinedione compound.

[0070] The triazolinedione adduct of the Cookson-type derivatization reagent of the present invention can be obtained by the method of the present invention for producing a triazolinedione adduct.

[0071] R 1< in the triazolinedione adduct is the same as R 1< in the triazolinedione compound represented by the formula (4), which is used in the method of the present invention for producing the triazolinedione adduct.

[0072] The triazolinedione adduct of the Cookson-type derivatization reagent of the present invention can be represented by the following formula (2).

[0073] In the formula (2), R 1< represents a dialkylamino group or a dialkylaminoalkyl group; R 2< , R 3< , and R 4< each independently represent a substituent selected from the group consisting of an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted aralkyl group, an optionally substituted alkenyl group, an optionally substituted phenyl group, an optionally substituted acyl group, a halogen group, an optionally substituted amino group, a nitro group, and an optionally substituted heterocyclic group; l and m are each independently an integer of 0 to 4; and n is an integer of 0 to 2.

[0074] In the above-mentioned R 2< , R 3< , and R 4< , examples of the substituents which may be included in the substituent selected from the group consisting of an alkyl group, an aralkyl group, an alkenyl group, a phenyl group, an acyl group, an amino group, and a heterocyclic group include: alkyl groups such as a methyl group, an ethyl group, a propyl group, a butyl group, an isobutyl group, a t-butyl group, a chloromethyl group, a 2-chloroethyl group, a 3-chloropropyl group, a 2-chloropropyl group, a 1-chloropropyl group, a bromomethyl group, a 2-bromoethyl group, a 3-bromopropyl group, a 2-bromopropyl group, and 1-bromopropyl group; aralkyl groups such as a benzyl group, a phenethyl group, a 3-phenylpropyl group, a 4-methylbenzyl group, and a 4-methylphenethyl group; alkenyl groups such as a vinyl group, an allyl group, a propenyl group, a cyclopropenyl group, a 2-chlorovinyl group, a 3-chloroallyl group, a cinnamyl group, and a styryl group; phenyl groups such as a phenyl group, a tolyl group, a xylyl group, and a trityl group; acyl groups such as a formyl group, an acetyl group, a propionyl group, a benzoyl group, an acryloyl group, and a methacryloyl group; halogen groups such as a chloro group, a bromo group, a fluoro group, and an iodo group; amino groups such as an amino group, a methylamino group, a dimethylamino group, an ethylamino group, and a diethylamino group; a nitro group; heterocyclic groups such as a quinolyl group, a pyridyl group, a pyrrolidyl group, a pyrrolyl group, a furyl group, a thienyl group, and a lutidyl group; and the like.

[0075] Alternatively, the triazolinedione adduct of the Cookson-type derivatization reagent of the present invention can be represented by the following formula (3).

[0076] In the formula (3), R 1< represents a dialkylamino group or a dialkylaminoalkyl group; R 5< , R 6< , R 7< , and R 8< each independently represent a substituent selected from the group consisting of an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted aralkyl group, an optionally substituted alkenyl group, an optionally substituted phenyl group, an optionally substituted acyl group, a halogen group, an optionally substituted amino group, a nitro group, and an optionally substituted heterocyclic group; p is an integer of 0 to 4; q is an integer of 0 to 2; and r and s are integers of 0 or 1.

[0077] In the above-mentioned R 5< , R 6< , R 7< , and R 8< , examples of the substituents which may be included in the substituent selected from the group consisting of an alkyl group, an aralkyl group, an alkenyl group, a phenyl group, an acyl group, an amino group, and a heterocyclic group include: alkyl groups such as a methyl group, an ethyl group, a propyl group, a butyl group, an isobutyl group, a t-butyl group, a chloromethyl group, a 2-chloroethyl group, a 3-chloropropyl group, a 2-chloropropyl group, a 1-chloropropyl group, a bromomethyl group, a 2-bromoethyl group, a 3-bromopropyl group, a 2-bromopropyl group, and 1-bromopropyl group; aralkyl groups such as a benzyl group, a phenethyl group, a 3-phenylpropyl group, a 4-methylbenzyl group, and a 4-methylphenethyl group; alkenyl groups such as a vinyl group, an allyl group, a propenyl group, a cyclopropenyl group, a 2-chlorovinyl group, a 3-chloroallyl group, a cinnamyl group, and a styryl group; phenyl groups such as a phenyl group, a tolyl group, a xylyl group, and a trityl group; acyl groups such as a formyl group, an acetyl group, a propionyl group, a benzoyl group, an acryloyl group, and a methacryloyl group; halogen groups such as a chloro group, a bromo group, a fluoro group, and an iodo group; amino groups such as an amino group, a methylamino group, a dimethylamino group, an ethylamino group, and a diethylamino group; a nitro group; heterocyclic groups such as a quinolyl group, a pyridyl group, a pyrrolidyl group, a pyrrolyl group, a furyl group, a thienyl group, and a lutidyl group; and the like.<Production Method (1) of Ene Compound>

[0078] The first method of the present invention for producing an ene compound comprises reacting a Cookson-type derivatization reagent with a diene compound to obtain the ene compound at a temperature of 20 to 350°C, wherein the Cookson-type derivatization reagent is according to the present invention and the diene compound is vitamin D3 or 25-hydroxyvitamin D3.[Reaction Conditions](Reaction Solvent)

[0079] As a solvent which can be used in reacting the triazolinedione adduct of the Cookson-type derivatization reagent of the present invention with vitamin D3 or 25-hydroxyvitamin D3, solvents which are similar to the solvents used in reacting the triazolinedione compound represented by the formula (4) with the compound having an anthracene ring in the first method of the present invention for producing the triazolinedione adduct can be used.

[0080] As described above, a Diels-Alder reaction of the triazolinedione compound represented by the formula (4) with the compound having an anthracene ring is a reversible reaction. For this reason, in order to shift equilibrium of the reaction towards the production system, a solvent capable of crystallizing the product is preferred. In other words, it is preferable to select a solvent in which the adduct is crystallized in the production of the triazolinedione adduct described above and it is preferable to select a solvent in which the adduct is dissolved in the reaction of the triazolinedione adduct of the Cookson-type derivatization reagent of the present invention with vitamin D3 or 25-hydroxyvitamin D3 to obtain an ene compound.

[0081] In the first method of the present invention for producing an ene compound, it is preferable to use at least one type selected from the group consisting of chloroform, acetonitrile, 1,2-dimethoxyethane, ethyl acetate, and dimethyl sulfoxide (DMSO), from the viewpoint of solubility, boiling point, yield, safety, and price.

[0082] The amount of the solvent used in the reaction is preferably 5 to 1,000 volumes with respect to 1 part by mass of the reaction substrate.(Reaction Temperature)

[0083] The temperature at which the triazolinedione adduct of the Cookson-type derivatization reagent of the present invention is reacted with vitamin D3 or 25-hydroxyvitamin D3 is in the range of 20 to 350°C, and preferably in the range of 40 to 200°C.(Reaction Time)

[0084] The reaction time during which the triazolinedione adduct of the Cookson-type derivatization reagent of the present invention is reacted with vitamin D3 or 25-hydroxyvitamin D3 is preferably 1 minute to 12 hours, and more preferably in the range of 1 minute to 8 hours.(Feed Amount)

[0085] In the reaction of the triazolinedione adduct of the Cookson-type derivatization reagent of the present invention with vitamin D3 or 25-hydroxyvitamin D3, the used amount of the vitamin D3 or 25-hydroxyvitamin D3 is preferably in the range of 1 to 10 equivalents of the vitamin D3 or 25-hydroxyvitamin D3, more preferably in the range of 1 to 5 equivalents, with respect to 1 equivalent of the triazolinedione adduct.<Analysis Method (1) of Ene Compound >

[0086] A first method of the present invention for analyzing an ene compound comprises reacting vitamin D3 or 25-hydroxyvitamin D3 with the triazolinedione adduct of the Cookson-type derivatization reagent of the present invention to obtain the ene compound, and analyzing the obtained ene compound.(Analysis Method)

[0087] The analysis method is not particularly limited, but a method using mass spectrometry or high performance liquid chromatography is preferred from the viewpoint of sensitivity, accuracy, and ease of measurement.(Diene Compound)

[0088] The diene compound applicable to the first analysis method of the present invention of an ene compound is vitamin D3 or 25-hydroxyvitamin D3 because the triazolinedione adduct can act as a Cookson type derivatization reagent.EXAMPLES

[0089] Subsequently, the Examples of the present invention will be described, but the present invention is not limited to these Examples.<Example 1>[Synthesis of Anthracene Adduct (DAP-A)]

[0090] In a light-shielding vessel, 81 mg (0.45 mmol) of anthracene was dissolved in 10 mL of dry acetonitrile to obtain an acetonitrile solution containing anthracene dissolved. Further, 98.2 mg (0.45 mmol) of 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) was added, and the mixture was stirred at 20°C for 4 hours to obtain a suspension.

[0091] The resultant suspension was filtered, and the solid was washed with hexane and dried to obtain a triazolinedione adduct. The resultant triazolinedione adduct was 5,10-dihydro-2-(4-dimethylaminophenyl)-5,10-[1',2']-benzeno-1H-[1,2,4] triazolo[1,2-b]phthalazine-1,3(2H)-dione (DAP-A), and the yield was 75 mg, 42%.[Reaction Scheme]

[0092] The reaction scheme carried out in Example 1 is shown below. [Evaluation of Physical Properties]

[0093] Various analyses were carried out on the resultant DAP-A. The results are shown below. Mp: 217 to 218°C IR (KBr): 1780, 1717 cm -1< 1< H-NMR (CDCl 3 ): δ 7.20-7.75 (m, 8H), 6.50-7.15 (m, 4H), 6.25 (s, 2H), 3.00 (s, 6H) <Example 2>[Synthesis of 9-Methylanthracene Adduct (DAP-MA)]

[0094] In a light-shielding vessel, 87 mg (0.45 mmol) of 9-methylanthracene was dissolved in 5 mL of dry acetonitrile to obtain an acetonitrile solution containing 9-methylanthracene dissolved. Further, 98.2 mg (0.45 mmol) of 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) was added, and the mixture was stirred at 20°C for 2 hours to obtain a suspension.

[0095] The resultant suspension was filtered, and the solid was washed with hexane and dried to obtain a triazolinedione adduct. The resultant triazolinedione adduct was 5,10-dihydro-2-(4-dimethylaminophenyl)-5-methyl-5,10-[1',2']-benzeno-1H-[1,2,4]triazolo[1,2-b]phthalazine-1,3(2H)-dione (DAP-MA), and the yield was 95 mg, 51.4%.[Reaction Scheme]

[0096] The reaction scheme carried out in Example 2 is shown below. [Evaluation of Physical Properties]

[0097] Various analyses were carried out on the resultant DAP-MA. The results are shown below. Mp: > 218°C IR (KBr): 1765, 1708 cm -1< 1< H-NMR (CDCl 3 ): δ 7.10-7.75 (m, 8H), 6.50-7.20 (m, 4H), 6.25 (s, 1H), 3.00 (s, 3H), 2.70 (s, 3H) <Example 3>[Synthesis of 9-Phenylanthracene Adduct (DAP-PA)]

[0098] In a light-shielding vessel, 0.50 g (1.97 mmol) of 9-phenylanthracene was dissolved in 50 mL of dry methylene chloride to obtain a methylene chloride solution containing 9-phenylanthracene dissolved. Further, 50 mg of molecular sieves 3Å (MS-3Å) and 0.426 g (1.95 mmol) of 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) were added, and the mixture was stirred at 20°C for 2 hours, to obtain a reaction solution.

[0099] The resultant reaction solution was filtered, then 100 mL of hexane was added to the filtrate, and the mixture was stirred for 2 hours in an ice bath, followed by filtration to filter out insolubles. To the resultant filtrate 200 mL of hexane was added, and the mixture was stirred for 30 minutes at room temperature. Precipitated crystals were filtered, and dried under reduced pressure to give a triazolinedione adduct. The resultant triazolinedione adduct was 5,10-dihydro-2-(4-dimethylaminophenyl)-5-phenyl-5,10[1',2']-benzeno-1H-[1,2,4]triazolo[1,2-b]phthalazine-1,3(2H)-dione (DAP-PA), and the yield was 0.54 g, 58.6%.[Reaction Scheme]

[0100] The reaction scheme carried out in Example 3 is shown below. [Evaluation of Physical Properties]

[0101] Various analyses were carried out on the resultant DAP-PA. The results are shown below. Mp: > 200°C IR (KBr): 1774, 1714 cm -1< 1< H-NMR (CDCl 3 ): δ 6.25-8.10 (m, 17H), 2.80 (s, 6H) <Example 4>[Synthesis of 9,10-Diphenylanthracene Adduct (DAP-DPA)]

[0102] In a light-shielding vessel, 0.75 g (2.27 mmol) of 9,10-diphenylanthracene was dissolved in 75 mL of toluene to obtain a toluene solution containing 9,10-diphenylanthracene dissolved. Further, 0.495 g (2.27 mmol) of 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) was added, and the mixture was stirred for 3 hours at room temperature, to obtain a reaction solution.

[0103] The resultant reaction solution was filtered, then the filtrate was purified by a silica gel column chromatography (toluene to toluene / ethyl acetate = 10 / 1), to give a triazolinedione adduct. The resultant triazolinedione adduct was 12-dihydro-2-(4-dimethylaminophenyl)-6,11-diphenyl-5,12-etheno-1H-benzo[g][1,2,4]triazolo[1,2-b]phthalazine-1,3(2H)-dione (DAP-DPA), and the yield was 43 mg, 3.4%.[Reaction Scheme]

[0104] The reaction scheme executed in Example 4 is shown below. [Evaluation of Physical Properties]

[0105] Various analyses were carried out on the resultant DAP-DPA. The results are shown below. Mp: >200°C IR (KBr): 1764, 1705 cm -1< 1< H-NMR (DMSO-d 6 ): δ 7.30-8.00 (m, 16H), 6.70-7.30 (m, 4H), 5.50-5.80 (m, 2H), 2.90 (s, 6H) <Example 5 (Comparative)>[Reaction of 9-methylanthracene adduct (DAP-MA) and TTB (synthesis of ene compound)]

[0106] 10 mg (0.024 mmol) of 9-methylanthracene adduct (DAP-MA) and 10 mg (0.048 mmol) of trans,trans-diphenylbutadiene (TTB) were dissolved in 0.2 g of dimethylsulfoxide (DMSO) to obtain a dimethylsufoxide solution.

[0107] Upon stirring the obtained DMSO solution at 120°C for 3 hours, a TTB-DAPTAD adduct, which is an ene compound, i.e., cis-2-(4-dimethylaminophenyl)-5,8-diphenyl-1H-[1,2,4]triazolo[1,2-a]pyridazine-1,3(2H)-dione(DAPTAC) was generated. With respect to a conversion ratio, high performance liquid chromatography (HPLC) analysis was performed under the following conditions to find the conversion ratio as being 60%.(Analysis Conditions)

[0108] Sample concentration: 0.05% Injection volume: 1.0 µL Wavelength: 254 nm Flow rate: 1.0 mL / min. Mobile phase: 0 to 15 min. (CH 3 CN:water = 50:50 to CH 3 CN:water = 100:0) 15 to 20 min. (CH 3 CN:water = 100:0) Column temperature: 30°C Packing material: X Bridge C18 5 µm (4.6 × 150) Retention time: DAPTAC: 7.2 min. TTB: 11.4 min.

[0109] The reaction solution was concentrated and the concentrated residue was purified by a silica gel column chromatography (eluting solvent: ethyl acetate) to obtain a pure product of DAPTAC.[Reaction Scheme]

[0110] The reaction scheme carried out in Example 5 is shown below. [Evaluation of Physical Properties]

[0111] Various analyses were carried out on the resultant DAPTAC. The results are shown below. Mp: 174 to 177°C IR (KBr): 1772, 1700 cm -1< 1< H-NMR (CDCl 3 ): δ 7.10-7.75 (m, 12H), 6.50-6.85 (m, 2H), 6.00 (s, 2H), 5.51 (s, 2H), 2.90 (s, 6H) <Example 6>[Reaction of 9-phenylanthracene adduct (DAP-PA) and vitamin D3 (synthesis of ene compound)]

[0112] 50 mg (0.106 mmol) of 9-phenylanthracene adduct (DAP-PA) and 40 mg (0.103 mmol) of vitamin D3 were dissolved in 5 mL of 1,2-dimethoxyethane (DME) to obtain a DME solution. The obtained DME solution was stirred at 70°C for 5 hours to obtain a reaction solution.

[0113] The obtained reaction solution was analyzed using a high performance liquid chromatography (HPLC) under the same conditions as in Example 5, and as result, it was found that 51.4 mg (yield 82.7%) of a vitamin D3 adduct, an ene compound, that is, (5S,7S)-2-[4-(dimethylamino)phenyl]-5-[(E)-[(1R,3aS,7aR)-1-[(1R)-1,5-dimethylhexyl]octahydro-7a-methyl-4H-indene-4-ylidene]methyl]-5,6,7,8,9,10-hexahydro-7-hydroxy-1H-[1,2,4]triazolo[1,2-b]phthaldine-1,3(2H)-dione was contained. Further, 2.44 mg of vitamin D3 was contained (recovery rate: 6.1%).

[0114] The obtained reaction solution was concentrated under reduced pressure and the concentrated residue was purified by a silica gel column chromatography (eluting solvent: hexane / ethyl acetate = 4 / 1→2 / 1) to isolate the vitamin D3 adduct.[Reaction Scheme]

[0115] The reaction scheme carried out in Example 6 is shown below. [Evaluation of Physical Properties]

[0116] Various analyses were carried out on the resultant vitamin D3. The results are shown below. Mp: 125 to 129°C IR (KBr): 1762, 1697 cm -1< 1< H-NMR (CDCl 3 ): δ 7.25 (d, J = 15 Hz, 2H), 6.75 (d, J = 15 Hz, 2H), 4.75 to 5.25 (m, 2H), 3.75 to 4.30 (m, 2H), 3.00 (s, 6H), 0.25 to 2.25 (m, 4OH) <Example 7>[Reaction of 9, 10-diphenylanthracene adduct (DAP-DPA) and vitamin D3 (synthesis of ene compound)]

[0117] 50 mg (0.106 mmol) of 9, 10-diphenylanthracene adduct (DAP-DPA) and 40 mg (0.103 mmol) of vitamin D3 were dissolved in 5 mL of 1,2-dimethoxyethane (DME) to obtain a DME solution. The obtained DME solution was stirred at 70°C for 5 hours to obtain a reaction solution.

[0118] The obtained reaction solution was analyzed using a high performance liquid chromatography (HPLC) under the following conditions, and as result, it was found that 51.4 mg (yield 82.7%) of a vitamin D3 adduct, an ene compound, that is, (5S,7S)-2-[4-(dimethylamino)phenyl]-5-[(E)-[(1R,3aS,7aR)-1-[(1R)-1,5-dimethylhexyl]octahydro-7a-methyl-4H-indene-4-ylidene]methyl]-5,6,7,8,9,10-hexahydro-7-hydroxy-1H-[1,2,4]triazolo[1,2-b]phthaldine-1,3(2H)-dione was contained. Incidentally, 2.44 mg of vitamin D3 was contained (recovery rate: 6.1%).(Analysis Conditions)

[0119] Sample concentration: 0.05% Injection volume: 1.0 µL Wavelength: 254 nm Flow rate: 1.0 mL / min. Mobile phase: 0 to 15 min. (CH 3 CN:water = 50:50 to CH 3 CN:water = 100:0) 15 to 20 min. (CH 3 CN:water = 100:0) Column temperature: 30°C Packing material: X Bridge C18 5 µm (4.6 × 150) Retention time: Vitamin D3 adduct: 13.2 min. Vitamin D3: 18.7 min. [Reaction Scheme]

[0120] The reaction scheme carried out in Example 7 is shown below. <Example 8>[Synthesis of 9-Phenylanthracene Adduct (DAP-PA)]

[0121] A reaction apparatus was assembled by attaching a mechanical stirrer and a thermometer to a four-necked 500 mL glass flask. To the flask, 4.00 g (18.2 mmol; 1.0 eq.) of 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMU), 4.00 g (18.2 mmol; 1.0 eq.) of iodosobenzene, 4.63 g (18.2 mmol; 1.0 eq.) of 9-phenylanthracene, 4.0 g of anhydrous magnesium sulfate, and 400 mL of ethyl acetate, from which dissolved oxygen had been removed by nitrogen-bubbling, were charged and stirred at room temperature. At this time, the reaction mixture changed to a purple suspension, and after 1 hour, it changed to a pink suspension.

[0122] After stirring for 24 hours or more, 400 mL of heptane was added to the reaction mixture, and the mixture was concentrated to about 210 g with an evaporator. At this time, precipitation of the adduct occurred and the reaction mixture changed to a slurry-like state.

[0123] The obtained slurry was filtered through a Kiriyama funnel (φ 60 mm, No. 5A) and washed with 200 mL of hexane. The solid remaining on the filter paper was collected by dissolving in 200 mL of dichloromethane, and the obtained dichloromethane solution was concentrated with an evaporator to obtain a purple solid, and vacuum drying was carried out at room temperature for 15 hours or more to obtain a triazolinedione adduct. The obtained purple solid triazolinedione adduct was a DAPTAD-phenylanthracene adduct (DAP-PA). The amount obtained was 6.30 g, the yield was 73%, and the HPLC purity was 96.0%.<Example 9>[Synthesis of 9-Phenylanthracene Adduct (DAP-PA)]

[0124] A reaction apparatus was assembled by attaching a mechanical stirrer and a thermometer to a four-necked 1,000 mL glass flask. To the flask, 10.00 g (45.4 mmol; 1.0 eq.) of 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMU), 9.99 g (45.4 mmol; 1.0 eq.) of iodosobenzene, 11.55 g (45.4 mmol; 1.0 eq.) of 9-phenylanthracene, 10.0 g of anhydrous magnesium sulfate and 1,000 mL of ethyl acetate from which dissolved oxygen had been removed by nitrogen-bubbling were charged and stirred at room temperature. At this time, the reaction mixture changed to a purple suspension, and after 1 hour, it changed to a pink suspension.

[0125] After stirring for 24 hours or more, 1,000 mL of heptane was added to the reaction mixture, and the mixture was concentrated to about 500 g with an evaporator. At this time, precipitation of the adduct occurred and the reaction mixture changed to a slurry-like state.

[0126] The obtained slurry was filtered through a Kiriyama funnel (φ 60 mm, No. 5A) and washed with 500 mL of hexane. The solid remaining on the filter paper was collected by dissolving in 500 mL of dichloromethane, and the obtained dichloromethane solution was concentrated with an evaporator to obtain a purple solid, and vacuum drying was carried out at room temperature for 15 hours or more to obtain a triazolinedione adduct. The obtained purple solid triazolinedione adduct was a DAPTAD-phenylanthracene adduct (DAP-PA). An amount obtained was 16.52 g, a yield was 77%, and a HPLC purity was 96.4%.<Example 10>[Synthesis of 9-Methylanthracene Adduct (DAP-MA)]

[0127] A reaction apparatus was assembled by attaching a mechanical stirrer and a thermometer to a four-necked 500 mL glass flask. To the flask, 5.00 g (22.7 mmol; 1.0 eq.) of 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMU), 5.00 g (22.7 mmol; 1.0 eq.) of iodosobenzene, 4.36 g (22.7 mmol; 1.0 eq.) of 9-methylanthracene, 5.0 g of anhydrous magnesium sulfate, and 500 mL of ethyl acetate from which dissolved oxygen had been removed by nitrogen-bubbling were charged and stirred at room temperature. At this time, the reaction mixture changed to a purple suspension, and after 1 hour, it changed to a pale reddish-purple suspension.

[0128] After stirring for 24 hours or more, 500 mL of heptane was added to the reaction mixture, and the mixture was concentrated to about 250 g with an evaporator. At this time, precipitation of the adduct occurred and the reaction mixture changed to a slurry-like state.

[0129] The obtained slurry was filtered through a Kiriyama funnel (φ 60 mm, No. 5A) and washed with 250 mL of hexane. The solid remaining on the filter paper was collected by dissolving in 250 mL of dichloromethane, and the obtained dichloromethane solution was concentrated with an evaporator to obtain a red solid, and vacuum drying was carried out at room temperature for 15 hours or more to obtain a triazolinedione adduct. The obtained red solid triazolinedione adduct was a DAPTAD-methylanthracene adduct (DAP-MA). An amount obtained was 6.62 g, a yield was 71%, and a HPLC purity was 96.3%.<Example 11>[Synthesis of 9-Chloromethylanthracene Adduct (DAP-CMA)]

[0130] A reaction apparatus was assembled by attaching a mechanical stirrer and a thermometer to a four-necked 500 mL glass flask. To the flask, 5.00 g (22.7 mmol; 1.0 eq.) of 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMU), 5.00 g (22.7 mmol; 1.0 eq.) of iodosobenzene, 5.15 g (22.7 mmol; 1.0 eq.) of 9-chloromethylanthracene, 5.0 g of anhydrous magnesium sulfate, and 500 mL of ethyl acetate from which dissolved oxygen had been removed by nitrogen-bubbling were charged and stirred at room temperature. At this time, the reaction mixture changed to a purple suspension, and after 1 hour, it changed to a pale reddish-purple suspension.

[0131] After stirring for 24 hours or more, 500 mL of heptane was added to the reaction mixture, and the mixture was concentrated to about 250 g with an evaporator. At this time, precipitation of the adduct occurred and the reaction mixture changed to a slurry-like state.

[0132] The obtained slurry was filtered through a Kiriyama funnel (φ 60 mm, No. 5A) and washed with 250 mL of hexane. The solid remaining on the filter paper was collected by dissolving in 250 mL of dichloromethane, and the obtained dichloromethane solution was concentrated with an evaporator to obtain a reddish-purple solid, and vacuum drying was carried out at room temperature for 15 hours or more to obtain a triazolinedione adduct. The obtained reddish-purple solid triazolinedione adduct was a DAPTAD-chloromethylanthracene adduct (DAP-CMA). An amount obtained was 8.79 g, a yield was 87%, and a HPLC purity was 97.1%.<Example 12>[Synthesis of 9-Bromoanthracene Adduct (DAP-BA)]

[0133] A reaction apparatus was assembled by attaching a mechanical stirrer and a thermometer to a four-necked 500 mL glass flask. To the flask, 5.00 g (22.7 mmol; 1.0 eq.) of 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMU), 5.00 g (22.7 mmol; 1.0 eq.) of iodosobenzene, 5.84 g (22.7 mmol; 1.0 eq.) of 9-bromomethylanthracene, 5.0 g of anhydrous magnesium sulfate, and 500 mL of ethyl acetate from which dissolved oxygen had been removed by nitrogen-bubbling, were charged and stirred at room temperature. At this time, the reaction mixture changed to a purple suspension, and after 1 hour, it changed to a pale reddish-purple suspension.

[0134] After stirring for 24 hours or more, 500 mL of heptane was added to the reaction mixture, and the mixture was concentrated to about 250 g with an evaporator. At this time, precipitation of the adduct occurred and the reaction mixture changed to a slurry-like state.

[0135] The obtained slurry was filtered through a Kiriyama funnel (φ 60 mm, No. 5A) and washed with 250 mL of hexane. The solid remaining on the filter paper was collected by dissolving in 250 mL of dichloromethane, and the obtained dichloromethane solution was concentrated with an evaporator to obtain a reddish-purple solid, and vacuum drying was carried out at room temperature for 15 hours or more to obtain a triazolinedione adduct. The obtained reddish-purple solid triazolinedione adduct was a DAPTAD-bromoanthracene adduct (DAP-BA). An amount obtained was 8.09 g, a yield was 75%, and a HPLC purity was 96.4%.<Example 13>[Synthesis of 9-Acetylanthracene Adduct (DAP-AcA)]

[0136] A reaction apparatus was assembled by attaching a mechanical stirrer and a thermometer to a four-necked 500 mL glass flask. To the flask, 5.00 g (22.7 mmol; 1.0 eq.) of 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMU), 5.00 g (22.7 mmol; 1.0 eq.) of iodosobenzene, 5.00 g (22.7 mmol; 1.0 eq.) of 9-acetylanthracene, 5.0 g of anhydrous magnesium sulfate, and 500 mL of ethyl acetate, from which dissolved oxygen had been removed by nitrogen-bubbling, were charged and stirred at room temperature. At this time, the reaction mixture changed to a purple suspension, and after 1 hour, it changed to a pale reddish-purple suspension.

[0137] After stirring for 24 hours or more, 500 mL of heptane was added to the reaction mixture, and the mixture was concentrated to about 250 g with an evaporator. At this time, precipitation of the adduct occurred and the reaction mixture changed to a slurry-like state.

[0138] The obtained slurry was filtered through a Kiriyama funnel (φ 60 mm, No. 5A) and washed with 250 mL of hexane. The solid remaining on the filter paper was collected by dissolving in 250 mL of dichloromethane, and the obtained dichloromethane solution was concentrated with an evaporator to obtain a reddish-purple solid, and vacuum drying was carried out at room temperature for 15 hours or more to obtain a triazolinedione adduct. The obtained reddish-purple solid triazolinedione adduct was a DAPTAD-acetylanthracene adduct (DAP-AcA). An amount obtained was 9.36 g, a yield was 94%, and a HPLC purity was 96.7%.<Example 14>[Synthesis of [9-(4'-Dimethylaminophenyl) Anthracene Adduct (DAP-DMAP)]

[0139] A reaction apparatus was assembled by attaching a mechanical stirrer and a thermometer to a four-necked 500 mL glass flask. To the flask, 5.00 g (22.7 mmol; 1.0 eq.) of 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMU), 5.00 g (22.7 mmol; 1.0 eq.) of iodosobenzene, 6.75 g (22.7 mmol; 1.0 eq.) of 9-(4'-dimethylaminophenyl) anthracene, 5.0 g of anhydrous magnesium sulfate, and 500 mL of ethyl acetate, from which dissolved oxygen had been removed by nitrogen-bubbling, were charged and stirred at room temperature. At this time, the reaction mixture changed to a purple suspension, and after 1 hour, it changed to a pale reddish-purple suspension.

[0140] After stirring for 24 hours or more, 500 mL of heptane was added to the reaction mixture, and the mixture was concentrated to about 250 g with an evaporator. At this time, precipitation of the adduct occurred and the reaction mixture changed to a slurry-like state.

[0141] The obtained slurry was filtered through a Kiriyama funnel (φ 60 mm, No. 5A) and washed with 250 mL of hexane. The solid remaining on the filter paper was collected by dissolving in 250 mL of dichloromethane, and the obtained dichloromethane solution was concentrated with an evaporator to obtain a yellow solid, and vacuum drying was carried out at room temperature for 15 hours or more to obtain a triazolinedione adduct. The obtained yellow solid triazolinedione adduct was a DAPTAD-dimethylaminophenylanthracene adduct (DAP-DMAPA). An amount obtained was 7.96 g, a yield was 68%, and a HPLC purity was 96.9%.<Example 15>[Synthesis of [9-(4'-nitrophenyl)anthracene Adduct (DAP-NPA)]

[0142] A reaction apparatus was assembled by attaching a mechanical stirrer and a thermometer to a four-necked 500 mL glass flask. To the flask, 5.00 g (22.7 mmol; 1.0 eq.) of 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMU), 5.00 g (22.7 mmol; 1.0 eq.) of iodosobenzene, 6.80 g (22.7 mmol; 1.0 eq.) of 9-(4'-nitrophenyl) anthracene, 5.0 g of anhydrous magnesium sulfate, and 500 mL of ethyl acetate, from which dissolved oxygen had been removed by nitrogen-bubbling, were charged and stirred at room temperature. At this time, the reaction mixture changed to a purple suspension, and after 1 hour, it changed to a pale reddish-purple suspension.

[0143] After stirring for 24 hours or more, 500 mL of heptane was added to the reaction mixture, and the mixture was concentrated to about 250 g with an evaporator. At this time, precipitation of the adduct occurred and the reaction mixture changed to a slurry-like state.

[0144] The obtained slurry was filtered through a Kiriyama funnel (φ 60 mm, No. 5A) and washed with 250 mL of hexane. The solid remaining on the filter paper was collected by dissolving in 250 mL of dichloromethane, and the obtained dichloromethane solution was concentrated with an evaporator to obtain an orange solid, and vacuum drying was carried out at room temperature for 15 hours or more to obtain a triazolinedione adduct. The obtained orange solid triazolinedione adduct was a DAPTAD-nitrophenylanthracene adduct (DAP-NPA). An amount obtained was 8.46 g, a yield was 72%, and a HPLC purity was 97.2%.<Example 16>[Purification of 9-Phenylanthracene Adduct (DAP-PA)]

[0145] A glass column (φ 5.4 cm × 60 cm) was wet-packed with 150 g of silica gel (Wakosil C-300; Fuji Film Wako). Hexane / ethyl acetate=7 / 3 was used as a packing solvent.

[0146] 3.00 g of a purple solid DAPTAD-phenylanthracene adduct (DAP-PA), obtained in Example 9, was dissolved in 10 mL of chloroform and charged onto a column. Thereafter, development was carried out using hexane / ethyl acetate = 7 / 3.

[0147] A fraction of 400 mL containing a spot of the target adduct was concentrated with an evaporator, then vacuum drying was carried out for 15 hours or more to obtain the DAPTAD-phenylanthracene adduct (DAP-PA) as a white solid. The amount obtained was 1.02 g, the yield was 34%, and the HPLC purity was 98.2%.<Example 17>[Purification of 9-phenylanthracene Adduct (DAP-PA)]

[0148] A glass column (φ 5.4 cm × 60 cm) was wet-packed with 150 g of silica gel (Wakosil C-300; Fuji Film Wako). Hexane / tetrahydrofuran = 5 / 5 was used as a packing solvent.

[0149] 3.00 g of a purple solid DAPTAD-phenylanthracene adduct (DAP-PA), obtained in Example 9, was dissolved in 10 mL of chloroform and charged onto a column. Thereafter, development was carried out using hexane / tetrahydrofuran = 5 / 5.

[0150] A fraction of 600 mL containing a spot of the target adduct was concentrated with an evaporator, then vacuum drying was carried out for 15 hours or more to obtain the DAPTAD-phenylanthracene adduct (DAP-PA) as a white solid. The obtained amount was 1.55 g, yield was 52%, and a HPLC purity was 98.8%.<Example 18 (Comparative)>[Obtainment of DAPTAC by adding PIDA to Mixed Solution of DMU and TTB]

[0151] 0.10 g (0.45 mmol) of 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMU) and 0.09 g (0.43 mmol) of trans,trans-diphenylbutadiene (TTB) were added to 100 mL of 1,2-dimethoxyethane (DME) to obtain a DME suspension.

[0152] To the obtained suspension, 0.15 g (0.46 mmol) of iodobenzene diacetate (PIDA) was added at 20°C and stirred at the same temperature for 4 hours to obtain a reaction solution containing cis-2-(4-dimethylaminophenyl)-5,8-diphenyl-1H-[1,2,4]triazolo[1,2-a]pyridazine-1,3(2H)-dione(DAPTAC).

[0153] With respect to the obtained reaction solution, a high performance liquid chromatography (HPLC) analysis was carried to calculate an assay yield of DAPTAC under the following conditions. The assay yield of DAPTAC was 76.8%.(Analysis conditions)

[0154] Sample concentration: 50% Injection volume: 1.0 µL Wavelength: 254 nm Flow rate: 1.0 mL / min. Mobile phase: 0 to 15 min. (CH 3 CN:water = 50:50 to CH 3 CN:water = 100:0) 15 to 20 min. (CH 3 CN:water = 100:0) Column temperature: 30°C Packing material: X Bridge C18 5 µm (4.6 × 150) Retention time: DMU: 2.1 min. DAPTAC: 7.2 min. TTB: 11.4 min. [Reaction Scheme]

[0155] The reaction scheme carried out in Example 18 is shown below. [Isolation of DAPTAC]

[0156] The obtained reaction solution was successively washed with a 5% aqueous sodium bicarbonate solution, followed by water, and concentrated under reduced pressure. The concentrated residue was purified by a silica gel column chromatography (eluting solvent: ethyl acetate). After concentrating the fraction containing the product under reduced pressure, the concentrated residue was heated and dispersed with 10 mL of ethyl acetate and cooled at room temperature to precipitate crystals. The precipitated crystals were filtered, washed with hexane, and dried under reduced pressure to obtain DAPTAC in a solid state.[Evaluation of Physical Properties]

[0157] Various analyses were carried out on the resultant DAPTA. The results are shown below. Mp: 174 to 177°C IR (KBr): 1772, 1700 cm -1< 1< H-NMR (CDCl 3 ): δ 7.10-7.75 (m, 12H), 6.50-6.85(m, 2H), 6.00 (s, 2H), 5.51 (s, 2H), 2.90 (s, 6H) <Example 19 (Comparative)>[Obtainment of Vitamin D3 Adduct by adding PIDA to Mixed Solution of DMU and Vitamin D3]

[0158] 0.01 g (0.045 mmol) of 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMU) and 0.017 g (0.044 mmol) of vitamin D3 were added to 10 mL of 1,2-dimethoxyethane (DME) to obtain a DME suspension.

[0159] To the obtained suspension, 0.015 g (0.045 mmol) of iodobenzene diacetate (PIDA) was added at 20°C and stirred at the same temperature for 3 hours to obtain a reaction solution containing (5S,7S)-2-[4-(dimethylamino)phenyl]-5-[(E)-[(1R,3aS,7aR)-1-[(1R)-1,5-dimethylhexyl]octahydro-7a-methyl-4H-indene-4-ylidene]methyl]-5,6,7,8,9,10-hexahydro-7-hydroxy-1H-[1,2,4]triazolo[1,2-b]phthalazine-1,3(2H)-dione (vitamin D3 adduct).

[0160] With respect to the obtained reaction solution, a high performance liquid chromatography (HPLC) analysis was carried out to calculate an assay yield of the vitamin D3 adduct under the following conditions. The assay yield of the vitamin D3 adduct was 84.3%.(Analysis conditions)

[0161] Sample concentration: 50% (Measurement was made by diluting the reaction solution with the same weight of THF.) Injection volume: 1.0 µL Wavelength: 254 nm Flow rate: 1.0 mL / min. Mobile phase: 0 to 15 min. (CH 3 CN:water = 50:50 to CH 3 CN:water = 100:0) 15 to 20 min. (CH 3 CN:water = 100:0) Column temperature: 30°C Packing material: X Bridge C18 5 µm (4.6 × 150) Retention time: DMU: 2.1 min. Vitamin D3 adduct: 13.2 min. Vitamin D3: 18.8 min. [Reaction Scheme]

[0162] The reaction scheme carried out in Example 19 is shown below. [Isolation of Vitamin D3 Adduct]

[0163] The obtained reaction solution was successively washed with a 5% aqueous sodium bicarbonate solution, followed by water, and concentrated under reduced pressure. The concentrated residue was purified by a silica gel column chromatography (eluting solvent: hexane / ethyl acetate = 4 / 1→2 / 1) to obtain the vitamin D3 adduct.[Evaluation of Physical Properties]

[0164] Various analyses were carried out on the resultant vitamin D3 adduct. The results are shown below. Mp: 125 to 129°C IR (KBr): 1762, 1697 cm -1< 1< H-NMR (CDCl 3 ): δ 7.25 (d, J = 15Hz, 2H), 6.75 (d, J = 15Hz, 2H), 4.75-5.25 (m, 2H), 3.75-4.30 (m, 2H), 3.00 (s, 6H), 0.25-2.25 (m, 40H) <Comparative Example 1>[Obtainment of DAPTAC by obtaining DAPTAD Solution from DMU and PIDA and adding TTB thereto]

[0165] 0.30 g (1.36 mmol) of 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMU) was added to 10 mL of 1,2-dimethoxyethane (DME) to obtain a DME suspension.

[0166] To the obtained DME suspension, 0.44 g (1.36 mmol) of iodobenzen diacetate (PIDA) was added at 20°C and stirred at the same temperature for 3 hours to obtain a reaction solution containing 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD).

[0167] To the obtained reaction solution containing DAPTAD, 0.28 g (1.36 mmol) of trans, trans-diphenylbutadiene (TTB) was added at 20°C and stirred at the same temperature for 2 hours to generate DAPTAC.

[0168] The obtained reaction solution was subjected to the same HPLC analysis as in Example 18, and the assay yield of DAPTAC was found to be 31.1%.[Reaction Scheme]

[0169] The reaction scheme carried out in Comparative Example 1 is shown below. <Example 20>[Obtainment of Anthracene Adduct by adding PIO to Mixed Solution of DMU and Anthracene]

[0170] 81 mg (0.45 mmol) of anthracene was weighed into a light-shielding vessel, and dry acetonitrile (10 mL) was added thereto to dissolve the anthracene. Further, 100 mg (0.45 mmol) of 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMU) was added, then 110 mg (0.50 mmol) of iodosobenzene was added and the mixture was stirred at room temperature for 4 hours. The obtained suspension was filtered, and the obtained crystals were washed with hexane and dried to obtain an anthracene adduct (DAP-A). An amount of the obtained anthracene adduct (DAP-A) was 75 mg and the yield was 41.7%.[Reaction Scheme]

[0171] The reaction scheme carried out in Example 20 is shown below. [Evaluation of Physical Properties]

[0172] Various analyses were carried out on the resultant anthracene adduct (DAP-A). The results are shown below. Mp: 217 to 218°C IR (KBr): 1780, 1717 cm -1< 1< H-NMR (CDCl 3 ): δ 7.20-7.75 (m, 8H), 6.50-7.15 (m, 4H), 6.25 (s, 2H), 3.00 (s, 6H)

Claims

1. A Cookson-type derivatization reagent consisting of a triazolinedione adduct represented by the following formula (2) or (3): wherein R1 represents a dialkylamino group or a dialkylaminoalkyl group; R2, R3, and R4 each independently represent a substituent selected from the group consisting of an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted aralkyl group, an optionally substituted alkenyl group, an optionally substituted phenyl group, an optionally substituted acyl group, a halogen group, an optionally substituted amino group, a nitro group, and an optionally substituted heterocyclic group; l and m are each independently an integer of 0 to 4; and n is an integer of 0 to 2, wherein R1 represents a dialkylamino group or a dialkylaminoalkyl group; R5, R6, R7, and R8 each independently represent a substituent selected from the group consisting of an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted aralkyl group, an optionally substituted alkenyl group, an optionally substituted phenyl group, an optionally substituted acyl group, a halogen group, an optionally substituted amino group, a nitro group, and an optionally substituted heterocyclic group; p is an integer of 0 to 4; q is an integer of 0 to 2; and r and s are integers of 0 or 1.

2. The Cookson-type derivatization reagent according to claim 1, wherein the Cookson-type derivatization reagent is represented by the following formula (2-1) or (2-2): wherein Me represents a methyl group and Ph represents a phenyl group.

3. The Cookson-type derivatization reagent according to claim 1, wherein the Cookson-type derivatization reagent is represented by the following formula (3-1): wherein Ph represents a phenyl group.

4. A method for producing the Cookson-type derivatization reagent according to any one of claims 1 to 3, comprising subjecting a triazolinedione compound and a compound having an anthracene ring to a Diels-Alder reaction to obtain the triazolinedione adduct, wherein the triazolinedione compound is represented by the following formula (4): wherein R1 represents a 4-dialkylaminophenyl group or a 4-dialkylaminoalkylphenyl group.

5. The method for producing a Cookson-type derivatization reagent according to claim 4, wherein the method further comprises a purification step of purifying the triazolinedione adduct.

6. A method for producing the Cookson-type derivatization reagent according to any one of claims 1 to 3, comprising reacting a triazolidinedione compound with a compound having an anthracene ring in the presence of an oxidizing agent to obtain the triazolinedione adduct, wherein the triazolidinedione compound is represented by the following formula (7): wherein R1 represents a 4-dialkylaminophenyl group or a 4-dialkylaminoalkylphenyl group.

7. The method for producing a Cookson-type derivatization reagent according to claim 6, wherein the oxidizing agent is a hypervalent iodine compound.

8. The method for producing a Cookson-type derivatization reagent according to claim 6 or 7, wherein the oxidizing agent is a hypervalent iodine compound represented by the following formula (8): wherein X and Y together represent an oxygen atom, or X and Y each independently represent a group selected from the group consisting of a hydroxy group, an alkoxy group, an acyloxy group, an acylamino group, a tosylamino group, a mesylamino group, a sulfonyloxy group, and a halogen group; and Ar represents a group selected from the group consisting of a phenyl group, a heterocyclic group, and a phenyl group substituted with an alkyl group, an alkoxy group, a halogen group, etc.

9. The method for producing a Cookson-type derivatization reagent according to any one of claims 6 to 8, wherein the method further comprises a purification step of purifying the triazolinedione adduct.

10. A method for producing an ene compound, comprising reacting a Cookson-type derivatization reagent according to any one of claims 1 to 3 with a diene compound to obtain the ene compound, wherein the Cookson-type derivatization reagent according to any one of claims 1 to 3 is reacted with vitamin D3 or 25-hydroxyvitamin D3 at a temperature of 20 to 350°C.

11. A method for analyzing an ene compound, comprising reacting a Cookson-type derivatization reagent according to any one of claims 1 to 3 with a diene compound to obtain the ene compound and analyzing the ene compound, wherein the Cookson-type derivatization reagent according to any one of claims 1 to 3 is reacted with vitamin D3 or 25-hydroxyvitamin D3 at a temperature of 20 to 350°C.

12. The method for analyzing an ene compound according to claim 11, wherein the analysis uses a high performance liquid chromatography.