Novel crystals of triazoline dione adducts and methods for producing the same

Abstract

This invention provides novel crystals of triazoline dione adducts and methods for producing them, methods for producing and analyzing ene compounds using the novel crystals, and derivatization reagents for mass spectrometry and derivatization reagent kits containing the novel crystals. [Solution] The novel crystal according to the present invention is a crystal of 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 (DAPTAD-PA) represented by the following formula (1), and has a single exothermic peak in the range of 175 to 185°C in differential scanning calorimetry. TIFF2026064145000011.tif42134

Description

Technical Field

[0001] The present invention relates to a novel crystal of a triazoline dione adduct, a method for producing the same, a method for producing and analyzing an ene compound using the novel crystal, and a derivatization reagent for mass spectrometry and a derivatization reagent kit for mass spectrometry containing the novel crystal.

Background Art

[0002] Vitamin D is thought to be related not only to maintaining bone health but also to many diseases such as hypertension, periodontal disease, and multiple sclerosis. Therefore, it is required to quantitatively analyze vitamin D metabolites in blood.

[0003] As a method for analyzing vitamin D metabolites, a method has been proposed in which vitamin D metabolites are derivatized using a Cookson-type derivatization reagent and then the derivatives are analyzed. The Cookson-type derivatization reagent rapidly reacts (Diels-Alder reaction) with a compound having an s-cis-diene structure and quantitatively gives an ene compound. By taking advantage of this reaction characteristic and reacting a vitamin D metabolite that is difficult to quantify as it is with a Cookson-type derivatization reagent to convert it into an ene compound with high analysis sensitivity, it becomes possible to accurately quantify the vitamin D metabolite.

[0004] As the Cookson-type derivatization reagent, triazoline dione compounds such as 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (hereinafter also referred to as "DAPTAD") represented by the following formula (3) are known (for example, see Non-Patent Document 1). However, triazoline dione compounds are very unstable and have a problem that they are difficult to handle as reagents.

[0005]

Chemical formula

[0006] Therefore, in recent years, triazolindione adducts stabilized by protecting the N=N double bond of the triazolindione compound with a polycyclic aromatic compound have been proposed (see, for example, Patent Documents 2 and 3). When such a triazolindione adduct is heated, the polycyclic aromatic compound is released via a reverse Diels-Alder reaction, resulting in the active triazolindione compound. By utilizing this property, vitamin D metabolites can be derivatized by generating the triazolindione compound from the triazolindione adduct in a reaction system containing vitamin D metabolites.

[0007] An example of a triazoline dione adduct is 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 (hereinafter also referred to as "DAPTAD-PA"), represented by the following formula (1). In DAPTAD-PA, the N=N double bond of DAPTAD is protected by 9-phenylanthracene.

[0008] [ka] [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] International Publication No. 2019 / 240143 [Patent Document 2] International Publication No. 2022 / 019305 [Non-patent literature]

[0010] [Non-Patent Document 1] S.Ogawa,et al., Rapid Commun.Mass Spectrom, 27(2013) 2453-2460 [Overview of the project] [Problems that the invention aims to solve]

[0011] Conventionally, DAPTAD-PA was manufactured by methods described in Patent Documents 1 and 2, etc. However, the crystalline form of DAPTAD-PA had not been investigated at all.

[0012] The present invention aims to provide a novel crystal of DAPTAD-PA, a triazoline dione adduct, and a method for producing the same; a method for producing and analyzing an ene compound using the novel crystal; and a derivatization reagent for mass spectrometry and a derivatization reagent kit for mass spectrometry containing the novel crystal. [Means for solving the problem]

[0013] The following embodiments are specific means for solving the above problems. <1> A crystal of 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 represented by the following formula (1), A crystal exhibiting a single exothermic peak in the 175-185°C range in differential scanning calorimetry. [ka]

[0014] <2> Powder X-ray diffraction shows peaks at 2θ = 7.6±0.1°, 10.0±0.1°, 13.0±0.1°, 13.6±0.1°, and 18.9±0.1°. <1> The crystal described above.

[0015] <3> <1> or <2> A method for producing crystals as described above, A production method comprising obtaining the crystals by a reprecipitation method in which an alcohol-based solvent is added to a solution of 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 dissolved in a halogen-based solvent.

[0016] <4> The production method according to <3>, wherein the halogen-based solvent is at least one selected from chloroform and dichloromethane.

[0017] <5> The production method according to <3> or <4>, wherein the alcohol-based solvent is at least one selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, and tert-butanol.

[0018] <6> A production method of an en compound, comprising adding the crystal according to <1> or <2> and a compound having an s-cis-diene structure to a reaction system to obtain an en compound.

[0019] <7> An analysis method of an en compound, comprising adding the crystal according to <1> or <2> and a compound having an s-cis-diene structure to a reaction system to obtain an en compound, and then analyzing the en compound.

[0020] <8> The analysis method according to <7>, wherein the en compound is analyzed by liquid chromatography-mass spectrometry.

[0021] <9> The analysis method according to <7> or <8>, wherein the compound having an s-cis-diene structure is vitamin D or a vitamin D metabolite.

[0022] <10> A derivatization reagent for mass spectrometry containing the crystal according to <1> or <2>.

[0023] <11> A derivatization reagent kit for mass spectrometry containing the crystal according to <1> or <2>. [Effects of the Invention]

[0024] According to the present invention, it is possible to provide a novel crystal of DAPTAD-PA, which is a triazoline dione adduct, a method for producing the same, a method for producing and analyzing an ene compound using the novel crystal, and a derivatization reagent for mass spectrometry and a derivatization reagent kit for mass spectrometry containing the novel crystal. [Brief explanation of the drawing]

[0025] [Figure 1] This figure shows the 1H NMR spectrum of DAPTAD-PA obtained in Example 1. [Figure 2] This figure shows the 1H NMR spectrum of DAPTAD-PA obtained in Reference Example 1. [Figure 3] This figure shows the infrared absorption spectra of DAPTAD-PA obtained in Example 1 and Reference Example 1. [Figure 4] This figure shows the differential scanning calorimetry results of DAPTAD-PA obtained in Example 1 and Reference Example 1. [Figure 5] This figure shows the powder X-ray diffraction measurement results of DAPTAD-PA obtained in Example 1 and Reference Example 1. [Figure 6] This figure shows the differential scanning calorimetry results of DAPTAD-PA obtained in Reference Example 2. [Figure 7] This figure shows the powder X-ray diffraction measurement results of DAPTAD-PA obtained in Reference Example 2. [Figure 8A] This figure shows the calibration curve for 25(OH)D3 in Example 8. [Figure 8B] This figure shows the calibration curve for 25(OH)D2 in Example 8. [Figure 8C] This figure shows the calibration curve for 24,25(OH)2D3 in Example 8. [Figure 8D] This figure shows the calibration curve for 3-epi-25(OH)D3 in Example 8. [Figure 9A] This figure shows the SRM chromatogram of 25(OH)D3 in Example 8. [Figure 9B] It is a diagram showing the SRM chromatogram of 25(OH)D2 in Example 8. [Figure 9C] It is a diagram showing the SRM chromatogram of 24,25(OH)2D3 in Example 8. [Figure 9D] It is a diagram showing the SRM chromatogram of 3-epi-25(OH)D3 in Example 8.

Mode for Carrying Out the Invention

[0026] Hereinafter, specific embodiments to which the present invention is applied will be described. In this specification, unless otherwise specified, the notation "x to y" using numerical values x and y means "x or more and y or less". In such a notation, when a unit is attached only to the numerical value y, the unit is also applied to the numerical value x.

[0027] <Novel Crystal of DAPTAD-PA> The novel crystal of DAPTAD-PA according to this embodiment is a crystal of DAPTAD-PA represented by the following formula (1), and has a single exothermic peak in the range of 175 to 185°C in differential scanning calorimetry. This exothermic peak is a decomposition peak accompanying the decomposition of the crystal.

[0028]

Chemical formula

[0029] When the present inventors performed differential scanning calorimetry on the solid of DAPTAD-PA obtained by the method described in Patent Document 2, two exothermic peaks were observed in the range of 155 to 175°C. In contrast, the novel crystal of DAPTAD-PA according to this embodiment has a single exothermic peak in the range of 175 to 185°C as described above, and its thermal stability is improved compared to the conventional solid. Therefore, the novel crystal of DAPTAD-PA according to this embodiment is considered to be superior in storage stability compared to the conventional crystal.

[0030] The differential scanning calorimetry of the crystals of DAPTAD-PA shall be carried out under the conditions described in the examples below.

[0031] Also, the novel crystal of DAPTAD-PA according to this embodiment is significantly different from the crystals of DAPTAD-PA obtained by the methods described in Patent Documents 1 and 2 in the powder X-ray diffraction pattern. That is, the novel crystal of DAPTAD-PA according to this embodiment is characterized in that in powder X-ray diffraction using Cu-Kα rays, it has main peaks at 2θ = 7.6 ± 0.1 degrees, 10.0 ± 0.1 degrees, 13.0 ± 0.1 degrees, 13.6 ± 0.1 degrees, and 18.9 ± 0.1 degrees.

[0032] The powder X-ray diffraction measurement of the crystals of DAPTAD-PA shall be carried out under the conditions described in the examples below.

[0033] <Method for Producing Novel Crystal of DAPTAD-PA> The novel crystal of DAPTAD-PA according to this embodiment can be produced by a reprecipitation method in which an alcohol-based solvent (poor solvent) is added to a solution in which DAPTAD-PA is dissolved in a halogen-based solvent (good solvent). According to such a reprecipitation method, the novel crystal of DAPTAD-PA according to this embodiment can be obtained with high purity and high recovery rate. Also, according to the reprecipitation method, it is possible to suppress the coloring of the obtained crystals.

[0034] The halogen-based solvent is not particularly limited as long as it can dissolve DAPTAD-PA. Examples of the halogen-based solvent include at least one selected from chloroform and dichloromethane. The halogen-based solvent may be used alone or in combination of two or more.

[0035] The amount of the halogen-based solvent in the solution in which DAPTAD-PA is dissolved is not particularly limited as long as it can completely dissolve DAPTAD-PA. The amount of the halogen-based solvent is preferably 4 to 40 mL, more preferably 4 to 10 mL, per 1 g of DAPTAD-PA.

[0036] The temperature of the solution in which DAPTAD-PA is dissolved is not particularly limited as long as it does not promote the reverse Diels-Alder reaction of DAPTAD-PA, for example, a range of 0 to 40°C is preferred, and a range of 10 to 30°C is more preferred.

[0037] The alcoholic solvent is not particularly limited as long as it has low solubility for DAPTAD-PA. Examples of alcoholic solvents include at least one selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, and tert-butanol. Among these, methanol, ethanol, isopropanol, and tert-butanol are preferred, and ethanol and isopropanol are more preferred. The alcoholic solvent may be used alone or as a mixture of two or more.

[0038] The amount of alcohol-based solvent added is not particularly limited, as long as it is an amount sufficient for the precipitation of DAPTAD-PA crystals. The amount of alcohol-based solvent added is preferably 10 to 25 mL, and more preferably 10 to 15 mL, per 1 g of DAPTAD-PA.

[0039] To precipitate high-purity DAPTAD-PA crystals, it is preferable to slowly add an alcohol-based solvent over 10 minutes to 2 hours while stirring the solution containing the dissolved DAPTAD-PA. Furthermore, it is preferable to continue stirring for 30 minutes to 2 hours after the addition of the alcohol-based solvent is complete.

[0040] The alcohol-based solvent may be added in its entirety at once, but it is preferable to add it in multiple stages. This tends to facilitate the precipitation of high-purity DAPTAD-PA crystals. For example, it is preferable to slowly add 1 / 5 to 1 / 3 of the total amount of alcohol-based solvent over 10 minutes to 1 hour, then continue stirring for 30 minutes to 2 hours to generate crystal nuclei, and then slowly add the remaining alcohol-based solvent over 30 minutes to 2 hours, continuing stirring for 30 minutes to 2 hours to grow the crystal nuclei.

[0041] After the DAPTAD-PA crystals have precipitated, it is preferable to filter the crystals, wash them, and then dry them. For washing the crystals, for example, an alcohol-based solvent can be used. The alcohol-based solvent may be the same as the solvent used to precipitate the DAPTAD-PA crystals, or it may be a different solvent. Among alcohol-based solvents, at least one selected from the group consisting of methanol, ethanol, isopropanol, and tert-butanol is preferred because it is easy to remove by drying. The drying method and drying temperature after washing are not particularly limited as long as they do not promote the reverse Diels-Alder reaction of DAPTAD-PA. Examples of drying methods include natural drying, forced-air drying, and vacuum drying. The drying temperature is preferably around room temperature, specifically 10 to 30°C.

[0042] [Method for manufacturing DAPTAD-PA] The method for producing DAPTAD-PA for reprecipitation is not particularly limited, and any production method described in Patent Documents 1, 2, etc., can be employed. For example, DAPTAD-PA can be produced by reacting DAPTAD with 9-phenylanthracene. Alternatively, DAPTAD-PA can also be produced by reacting 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (hereinafter also referred to as "DAPTAD precursor"), represented by the following formula (2), with 9-phenylanthracene in the presence of an oxidizing agent.

[0043] [ka]

[0044] Among these manufacturing methods, the method using a DAPTAD precursor is preferred because DAPTAD is highly unstable. This manufacturing method will be described below.

[0045] In the production of DAPTAD-PA, a DAPTAD precursor and 9-phenylanthracene are added to a solvent, and then an oxidizing agent is added to carry out the reaction.

[0046] The amount of 9-phenylanthracene is preferably 1.0 to 1.5 mol, and more preferably 1.0 to 1.2 mol, per 1 mol of DAPTAD precursor.

[0047] The solvent is not particularly limited as long as it can dissolve the resulting DAPTAD-PA, but halogenated solvents are preferred. By using a halogenated solvent, the solution containing DAPTAD-PA obtained after the reaction can be directly subjected to the reprecipitation method without solvent substitution. Examples of halogenated solvents include at least one selected from chloroform and dichloromethane. The solvent may be used alone or as a mixture of two or more.

[0048] The amount of solvent is not particularly limited as long as it is sufficient to completely dissolve DAPTAD-PA. Preferably, the amount of solvent is 9 to 25 mL, and more preferably 9 to 15 mL, per 1 g of DAPTAD precursor. If the amount of solvent is large, it is necessary to remove the solvent using an evaporator before reprecipitation. Therefore, a smaller amount of solvent is preferable.

[0049] Hypervalent iodine compounds can be suitably used as oxidizing agents. Examples of hypervalent iodine compounds include iodosylbenzene, iodobenzene diacetate, iodobenzene ditriflate, iodobenzene ditosylate, iodobenzene dimesylate, iodobenzene dichloride, and iodobenzene dibromide. Among these, iodobenzene diacetate is preferred from the viewpoint of yield, stability, and cost.

[0050] The amount of oxidizing agent added is preferably 0.8 to 1.2 mol, and more preferably 0.9 to 1.0 mol, per 1 mol of DAPTAD precursor.

[0051] The reaction temperature for producing DAPTAD-PA is not particularly limited as long as it does not promote the reverse Diels-Alder reaction of DAPTAD-PA. For example, a range of 0 to 40°C is preferred, and a range of 10 to 30°C is more preferred. The reaction time is preferably 10 minutes to 4 hours, and more preferably 30 minutes to 2 hours.

[0052] Furthermore, the solution containing DAPTAD-PA obtained after the reaction may be filtered to remove impurities before being subjected to reprecipitation.

[0053] <Methods for producing and analyzing ene compounds> The novel DAPTAD-PA crystals according to this embodiment can be used in methods for producing and analyzing ene compounds, similar to conventionally known DAPTAD-PA. For example, an ene compound can be obtained by adding the novel DAPTAD-PA crystals according to this embodiment and a compound having an s-cis-diene structure to a reaction system. Furthermore, by analyzing the obtained ene compound, it is possible to accurately quantify the amount of the compound having an s-cis-diene structure.

[0054] Examples of compounds having an s-cis-diene structure include steroids, vitamin A, vitamin D, and vitamin D metabolites. Among these, vitamin D or vitamin D metabolites are preferred.

[0055] Steroids are not limited to naturally occurring compounds, but may also be synthetic products, such as 7-dehydrocholesterol, ergosterol, and conjugated linoleic acid.

[0056] Examples of vitamin D metabolites include 25-hydroxyvitamin D3 (25(OH)D3), 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3), 25-hydroxyvitamin D2 (25(OH)D2), 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), 1α,25-dihydroxyvitamin D2 (1α,25(OH)2D2), and 23,25-dihydroxyvitamin D3 (23,25(OH)2D3). Examples include 25,26-dihydroxyvitamin D3 (25,26(OH)2D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), 4β,25-dihydroxyvitamin D3 (4β,25(OH)2D3), 25-hydroxyvitamin D3-26,23-lactone (25(OH)D3-lactone), and 1α,25-dihydroxyvitamin D3-26,23-lactone (1α,25(OH)2D3-lactone). Vitamin D metabolites may also be conjugates such as sulfate conjugates and glucuronide conjugates. When using vitamin D metabolites as compounds having an s-cis-diene structure, the vitamin D metabolites may contain multiple molecular species.

[0057] For the analysis of ene compounds, it is preferable to employ liquid chromatography-mass spectrometry (LC-MS), which combines high-performance liquid chromatography (HPLC) and mass spectrometry. Among LC-MS methods, LC-MS / MS using a tandem mass spectrometer is preferred.

[0058] The detector for a high-performance liquid chromatograph is not particularly limited as long as it can measure the ene compound to be analyzed. Examples of detectors include ultraviolet-visible spectrometers, photodiode array detectors, fluorescence detectors, differential refraction detectors, and evaporative light scattering detectors.

[0059] The column used in the high-performance liquid chromatography (HHL) is not particularly limited as long as it can measure the target ene compound. Due to its versatility, a column using chemically bonded porous spherical silica gel modified with octadecylsilyl groups is preferred.

[0060] For high-performance liquid chromatography, common solvents such as organic solvents including methanol, ethanol, isopropanol, acetonitrile, and tetrahydrofuran; distilled water; and aqueous solutions such as trifluoroacetic acid aqueous solution, formic acid aqueous solution, acetic acid aqueous solution, ammonium bicarbonate aqueous solution, and phosphoric acid aqueous solution can be used as eluents. As aqueous solutions, buffer solutions such as acetic acid buffer solution containing acetic acid and sodium acetate, or phosphate buffer solution containing phosphoric acid, potassium dihydrogen phosphate, and disodium hydrogen phosphate may be used. These solvents may be used individually or in mixtures of two or more. From the viewpoint of ease of adjusting the measurement time, it is preferable to use a combination of distilled water or an aqueous solution with an organic solvent or an organic solvent to which an organic acid such as formic acid or acetic acid has been added. For example, combinations such as distilled water / acetonitrile, distilled water / methanol, formic acid aqueous solution / acetonitrile, formic acid aqueous solution / methanol, formic acid aqueous solution / acetonitrile with formic acid, and formic acid aqueous solution / methanol with formic acid are preferred.

[0061] A commercially available mass spectrometer can be used without any particular restrictions.

[0062] Examples of ionization methods for mass spectrometers include electron ionization (EI), chemical ionization (CI), electrolytic desorption (FD), fast atomic collision (FAB), matrix-assisted laser desorption ionization (MALDI), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), real-time direct analysis (DART), ion deposition, and inductively coupled plasma (ICP). Among these, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are preferred because they are easily combined with high-performance liquid chromatography.

[0063] Examples of the mass separation method of the mass spectrometer include, for example, magnetic field deflection type, quadrupole type, ion trap type, time-of-flight type, Fourier transform ion cyclotron resonance type, etc.

[0064] <Derivatization reagent for mass spectrometry and derivatization reagent kit for mass spectrometry> The novel crystal of DAPTAD-PA according to this embodiment can be used as a derivatization reagent for mass spectrometry, similar to the conventionally known DAPTAD-PA. Further, the novel crystal of DAPTAD-PA according to this embodiment can also be made into a derivatization reagent kit for mass spectrometry in combination with a decomposition inhibitor or the like for preventing the decomposition of the obtained compound. Examples of the decomposition inhibitor include ammonia, trimethylamine, triethylamine, dimethylamine, methylamine, diethylamine, ethylamine, etc.

Example

[0065] Hereinafter, the present invention will be described more specifically by way of examples, but the present invention is not limited by these examples. <00​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0067] < 1 H NMR measurement> Each sample in Example 1 and Reference Examples 1 and 2 1 ¹H NMR spectra were measured using a Fourier transform nuclear magnetic resonance spectrometer (JNM-ECZL600G, JEOL Ltd.) with a sample concentration of 10 mg / mL. Chloroform-d (CDCl3) containing 0.05% (v / v) tetramethylsilane was used as the solvent.

[0068] <Infrared absorption spectrum measurement> The infrared absorption spectra of each sample in Example 1 and Reference Examples 1 and 2 were measured using a Fourier transform infrared spectrophotometer (Spectrum One, PerkinElmer) by total internal reflection (ATR) measurement.

[0069] <Differential Scanning Calorimetry> The differential scanning calorimetry of each sample in Examples 1-7 and Reference Examples 1 and 2 was performed under the following conditions. Device: Thermo plus EVO2 DSC8230 (Rigaku Corporation) Sample cell: Aluminum pan Sample amount: 5 mg Measurement range: 30~200℃ Heating rate: 10℃ / min Nitrogen gas flow rate: 50 mL / min

[0070] <Powder X-ray diffraction measurement> Powder X-ray diffraction measurements of each sample in Examples 1-7 and Reference Examples 1 and 2 were performed under the following conditions. Equipment: Smart Lab (Rigaku Corporation) Measurement method: ASC6 BB Dtex X-ray tube current: 30mA X-ray tube voltage: 40kV Wavelength:CuKα / 1.541862Å Scanning range: 2θ = 5.0~35.0° Step: 2θ = 0.02° Scan speed: 10° / min

[0071] <Example 1> A reaction apparatus was assembled in a 1L four-necked glass flask, equipped with a mechanical stirrer, temperature sensor, and dropping funnel. 20.00g (90.8 mmol; 1.0 eq.) of DAPTAD precursor, 23.10g (90.8 mmol; 1.0 eq.) of 9-phenylanthracene, and 180mL of chloroform were charged at room temperature and stirred in a water bath at 150 rpm. Stirring was continued for more than 30 minutes to completely dissolve the phenylanthracene. At this point, the DAPTAD precursor remained undissolved and was in the form of a pink slurry. Subsequently, 29.25g (90.8 mmol; 1.0 eq.) of iodobenzene diacetate (PIDA) was added all at once. The reaction system changed to a dark purple color and the temperature rose from 20°C to 27°C. The DAPTAD precursor disappeared in about 10 minutes, and the mixture became a homogeneous solution. After 30 minutes of reaction, the reaction mixture was sampled and analyzed by HPLC, confirming the progress of the reaction.

[0072] The reaction was carried out for 2 hours, and 60 mL of isopropanol (3 v / w) was slowly added over 20 minutes from a dropping funnel (nucleation). After about 10 minutes, daptad-PA was observed to gradually precipitate, and stirring was continued for 1 hour. Subsequently, 440 mL of isopropanol (22 v / w) was slowly added over 2 hours from a dropping funnel (nuclear growth). At this time, the stirring speed was increased to 180 rpm. After stirring for another hour, the mixture was filtered through a Kiriyama funnel (φ95 mm, filter paper: No. 5B) and washed with 300 mL of isopropanol. Finally, the mixture was vacuum-dried at room temperature for more than 12 hours to obtain the target daptad-PA as a beige solid. The yield was 34.66 g, the yield was 80.8%, and the HPLC purity was 99.3%.

[0073] <Reference example 1> In Reference Example 1, DAPTAD-PA was synthesized according to the method of Example 1 in Patent Document 2.

[0074] A reaction apparatus was assembled in a 2L round-bottom flask by attaching a stirrer piece and a thermometer. 15.1g (68.6 mmol; 1.0 eq.) of DAPTAD precursor, 17.4g (68.6 mmol; 1.0 eq.) of 9-phenylanthracene, 15.1g (68.6 mmol; 1.0 eq.) of iodosylbenzene, and 15.1g of anhydrous magnesium sulfate were placed in the flask. After shaking the flask to homogenize the solids, 750mL of ethyl acetate was added and the mixture was stirred. At this point, the DAPTAD precursor did not dissolve and was in the form of a beige slurry, but it quickly changed to a dark purple color. After the reaction was carried out for more than 20 hours, it became a pink slurry. A sample of the reaction mixture was taken and analyzed by HPLC to confirm the progress of the reaction.

[0075] Next, the reaction mixture was transferred to another 2 L round-bottom flask, 750 mL of heptane was added, and the mixture was concentrated to approximately 500 mL using an evaporator (bath temperature: 20°C). At this point, a solid precipitate formed, resulting in a pink slurry. The mixture was filtered through a Kiriyama funnel (φ95 mm, filter paper: No. 5B) and washed with 750 mL of hexane to obtain a purple solid. The filtrate was orange at this point. The receiving flask was replaced, and while under suction, the mixture was washed with 500 mL of dichloromethane. The solid on the filter paper redissolved to form a purple solution. A brown solid (magnesium sulfate) remained on the filter paper. The obtained purple solution was concentrated using an evaporator (bath temperature: 20°C) to obtain a purple solid. Vacuum drying was performed at room temperature for 5 hours to obtain DAPTAD-PA as a purple solid. The yield was 26.5 g, the yield was 81.7%, and the HPLC purity was 93.1%.

[0076] Next, 26.5 g (56.1 mmol) of DAPTAD-PA (crude) and 500 mL of acetone were placed in a 1 L round-bottom flask, and the mixture was stirred using a stirrer piece to perform reslurry treatment. After reslurry treatment for more than 20 hours, the solution was filtered through a Kiriyama funnel (φ60 mm, filter paper: No. 5C), washed with 100 mL of acetone, and a pink solid was obtained. The receiving flask was changed, and while under suction, the solution was washed with 400 mL of dichloromethane. The solid on the filter paper redissolved to form a pink solution. The obtained pink solution was concentrated using an evaporator (bath temperature: 20°C) to obtain a solid. Vacuum drying was performed at room temperature for 5 hours to obtain DAPTAD-PA as a pink solid. The yield was 19.1 g, the recovery rate was 72.1%, and the HPLC purity was 98.4%.

[0077] Next, 19.1 g (40.4 mmol) of DAPTAD-PA (single-purified product) and 300 mL of acetone were charged into a 1 L round-bottom flask, and the mixture was stirred using a stirrer piece to perform reslurry treatment. After reslurry treatment for more than 20 hours, the solution was filtered through a Kiriyama funnel (φ60 mm, filter paper: No. 5C), washed with 50 mL of acetone, and a pale pink solid was obtained. The receiving flask was changed, and while under suction, the solution was washed with 400 mL of dichloromethane. The solid on the filter paper redissolved to form a pink solution. The obtained pink solution was concentrated using an evaporator (bath temperature: 20°C) to obtain a solid. Vacuum drying was performed at room temperature for 5 hours to obtain DAPTAD-PA as a pale pink solid. The yield was 14.4 g, the recovery rate was 75.4%, and the HPLC purity was 99.4%.

[0078] <Rating> [ 1 H NMR measurement] The DAPTAD-PA obtained in Example 1 1 The 1H NMR spectrum obtained in Reference Example 1 is shown in Figure 1. 1 The 1H NMR spectra are shown in Figure 2. As shown in Figures 1 and 2, no impurity peaks were observed in either DAPTAD-PA, and the integration ratios showed very good agreement. From these results, it can be concluded that the two are the same compound.

[0079] [Infrared absorption spectrum measurement] Figure 3 shows the infrared absorption spectra of DAPTAD-PA obtained in Example 1 and Reference Example 1. A detailed examination of the infrared absorption spectra reveals that at 1200 cm⁻¹... -1 Nearby and 3000cm -1 A minute difference was observed in the vicinity. This result suggests that the crystal forms of the two materials are different.

[0080] [Differential Scanning Calorimetry] Figure 4 shows the differential scanning calorimetry results for DAPTAD-PA obtained in Example 1 and Reference Example 1. As shown in Figure 4, the DAPTAD-PA obtained in Example 1 showed a single exothermic peak around 177°C. On the other hand, the DAPTAD-PA obtained in Reference Example 1 showed two exothermic peaks around 160°C and 169°C. In addition, for both DAPTAD-PA samples, an endothermic peak associated with the melting of 9-phenylanthracene was observed around 156°C during the second heating. Thus, the differences in the thermal decomposition temperature and peak shape based on the reverse Diels-Alder reaction suggest that the crystal forms of the two samples are different.

[0081] [Powder X-ray diffraction measurement] Figure 5 shows the results of powder X-ray diffraction measurements of DAPTAD-PA obtained in Example 1 and Reference Example 1. As shown in Figure 5, although both were crystalline, their diffraction patterns were significantly different. Specifically, the DAPTAD-PA obtained in Example 1 showed major peaks at 2θ = 7.6 degrees, 10.0 degrees, 13.0 degrees, 13.6 degrees, 15.1 degrees, and 18.9 degrees. On the other hand, the DAPTAD-PA obtained in Reference Example 1 showed major peaks at 2θ = 7.8 degrees, 10.3 degrees, 13.1 degrees, 13.4 degrees, 14.1 degrees, 17.8 degrees, 18.5 degrees, 19.0 degrees, and 20.5 degrees. From these results, it can be seen that the crystal forms of the two are different.

[0082] <Reference example 2> 4.30 g (9.1 mmol) of DAPTAD-PA with an HPLC purity of 98.5%, synthesized in the same manner as in Example 1, was dissolved with stirring in 26 mL of dichloromethane. The resulting solution was concentrated using an evaporator (bath temperature: 20°C) to obtain a solid. Vacuum drying was performed at room temperature for more than 12 hours to obtain DAPTAD-PA as a beige solid. The yield was 4.26 g, the recovery rate was 99.1%, and the HPLC purity was 98.4%.

[0083] <Rating> [Differential Scanning Calorimetry] Figure 6 shows the differential scanning calorimetry results for DAPTAD-PA obtained in Reference Example 2. As shown in Figure 6, two exothermic peaks were observed in the DAPTAD-PA obtained in Reference Example 2, around 163°C and around 172°C.

[0084] [Powder X-ray diffraction measurement] Figure 7 shows the results of powder X-ray diffraction measurements of DAPTAD-PA obtained in Reference Example 2. As shown in Figure 7, the DAPTAD-PA obtained in Reference Example 2 showed a diffraction pattern similar to that of Reference Example 1. Specifically, the DAPTAD-PA obtained in Reference Example 2 showed major peaks at 2θ = 7.7°, 10.1°, 12.9°, 13.3°, 14.0°, 17.6°, 18.4°, 18.8°, and 20.4°.

[0085] <Example 2> DAPTAD-PA crystals were obtained in the same manner as in Example 1, except that the alcoholic solvent used for nucleation and growth of DAPTAD-PA in the reprecipitation method was changed from isopropanol to methanol. The yield was 18.42 g, the yield was 85.8%, and the HPLC purity was 98.6%.

[0086] <Example 3> DAPTAD-PA crystals were obtained in the same manner as in Example 1, except that the alcoholic solvent used for nucleation and growth of DAPTAD-PA in the reprecipitation method was changed from isopropanol to ethanol. The yield was 18.01 g, the yield was 83.9%, and the HPLC purity was 99.1%.

[0087] <Example 4> DAPTAD-PA crystals were obtained in the same manner as in Example 1, except that the alcoholic solvent used for nucleation and growth of DAPTAD-PA in the reprecipitation method was changed from isopropanol to tert-butanol. The yield was 17.94 g, the yield was 83.6%, and the HPLC purity was 99.3%.

[0088] <Example 5> DAPTAD-PA crystals were obtained in the same manner as in Example 1, except that the halogenated solvent used to dissolve DAPTAD-PA (the solvent used in the synthesis of DAPTAD-PA) was changed from chloroform to dichloromethane in the reprecipitation method. The yield was 17.11 g, the yield was 79.7%, and the HPLC purity was 99.3%.

[0089] <Example 6> DAPTAD-PA crystals were obtained in the same manner as in Example 1, except that the alcoholic solvent used for nucleation of DAPTAD-PA in the reprecipitation method was changed from isopropanol to methanol. The yield was 18.13 g, the yield was 84.5%, and the HPLC purity was 99.0%.

[0090] <Example 7> DAPTAD-PA crystals were obtained in the same manner as in Example 1, except that the alcoholic solvent used for nucleation and growth of DAPTAD-PA was changed from isopropanol to a methanol / tert-butanol = 1 / 1 (v / v) mixed solvent in the reprecipitation method. The yield was 17.97 g, the yield was 83.7%, and the HPLC purity was 98.5%.

[0091] <Rating> [Differential Scanning Calorimetry] Differential scanning calorimetry was performed on the DAPTAD-PA obtained in Examples 2 to 7 in the same manner as in Example 1. As a result, a single exothermic peak associated with crystal decomposition was observed in the range of 175 to 185°C in all cases. The decomposition temperatures of the DAPTAD-PA obtained in Examples 2 to 7 are shown in Table 1 below.

[0092] [Powder X-ray diffraction measurement] Powder X-ray diffraction measurements were performed on the DAPTAD-PA obtained in Examples 2 to 7 in the same manner as in Example 1. As a result, major peaks were observed at 2θ = 7.6±0.1°, 10.0±0.1°, 13.0±0.1°, 13.6±0.1°, and 18.9±0.1° in all cases, confirming that they were novel crystal forms similar to those in Example 1.

[0093] [Table 1]

[0094] <Example 8: LC-MS / MS quantitative analysis of vitamin D metabolites in pooled serum using DAPTAD-PA> Using DAPTAD-PA synthesized in Example 1, vitamin D metabolites (25(OH)D3, 25(OH)D2, 24,25(OH)2D3, and 3-epi-25(OH)D3) contained in pooled serum were quantitatively analyzed by LC-MS / MS. The procedure for quantitative analysis is as follows.

[0095] (1) Preparation of calibrator solution and internal standard (IS) solution The calibrator solution and IS solution were prepared using the JeoQuant Kit for VD Metabolite (JEOL Ltd.). The calibrator concentrations are shown in Table 2 below.

[0096] [Table 2]

[0097] (2) Sample pretreatment Sample preprocessing was performed using the following procedure.

[0098] (2-1) SLE (Supported Liquid Extraction) Extraction 1) A mixed solution was prepared by mixing 50 μL of serum or calibrator solution with 250 μL of IS solution. 2) 300 μL of the mixed solution was loaded onto an ISOLUTE SLE+ column (Biotage). 3) Add 600 μL of ethyl acetate:hexane = 1:1 (v / v) solvent and elute the sample (this procedure was repeated three times). 4) The eluted sample was thoroughly dried with nitrogen gas.

[0099] (2-2) Derivatization 1) DAPTAD-PA was dissolved in ethyl acetate to a solution of 0.2 mg / mL. 2) The DAPTAD-PA solution was activated by heating it at 80°C for 15 minutes. 3) 100 μL of activated DAPTAD-PA solution was added to the sample extracted in (2-1) above, and the mixture was reacted at room temperature for 15 minutes. 4) After the reaction was complete, the mixture was dried with nitrogen gas. 5) 50 μL of 50% (v / v) acetonitrile solution was added to the dried sample to dissolve it, and this was used as the sample for measurement.

[0100] (3)LC analysis conditions The LC analysis conditions were as follows: Equipment: ACQUITY UPLC I-Class (Waters Corporation) Analytical column: CAPCELL CORE C18 (2.7 μm, 2.1 mm I.D. × 75 mm) (Osaka Soda Co., Ltd.) Elution conditions: flow rate 0.5mL / min Solvent A: 0.1% formic acid-water Solvent B: 0.1% formate-acetonitrile The details of the elution conditions are shown in Table 3 below.

[0101] [Table 3]

[0102] (4)MS / MS analysis conditions The MS / MS analysis conditions were as follows: Equipment: Triple quadrupole mass spectrometer Xevo TQ-S micro (Waters) Ionization conditions: ESI positive ion mode The SRM parameters were as shown in Table 4 below.

[0103] [Table 4]

[0104] Calibration curves for each vitamin D metabolite are shown in Figures 8A to 8D. SRM chromatograms of each vitamin D metabolite in pooled serum are shown in Figures 9A to 9D. Analysis revealed the following concentrations of vitamin D metabolites in pooled serum: 25(OH)D3: 14.6 ng / mL, 25(OH)D2: 0.42 ng / mL, 24,25(OH)2D3: 0.76 ng / mL, and 3-epi-25(OH)D3: 0.83 ng / mL.

Claims

1. A crystal of 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, represented by the following formula (1): A crystal exhibiting a single exothermic peak in the 175–185°C range in differential scanning calorimetry. 【Chemistry 1】

2. The crystal according to claim 1, having peaks at 2θ = 7.6±0.1 degrees, 10.0±0.1 degrees, 13.0±0.1 degrees, 13.6±0.1 degrees, and 18.9±0.1 degrees in powder X-ray diffraction.

3. A method for producing crystals according to claim 1 or 2, A method for producing the crystals, comprising obtaining the crystals by a reprecipitation method in which an alcohol-based solvent is added to a solution in which 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 is dissolved in a halogen-based solvent.

4. The manufacturing method according to claim 3, wherein the halogenated solvent is at least one selected from chloroform and dichloromethane.

5. The manufacturing method according to claim 3 or 4, wherein the alcohol-based solvent is at least one selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, and tert-butanol.

6. A method for producing an ene compound, comprising adding the crystal described in claim 1 or 2 and a compound having an s-cis-diene structure to a reaction system to obtain an ene compound.

7. A method for analyzing an ene compound, comprising adding the crystal described in claim 1 or 2 and a compound having an s-cis-diene structure to a reaction system to obtain an ene compound, and then analyzing the ene compound.

8. The analytical method according to claim 7, wherein the aforementioned ene compound is analyzed by liquid chromatography-mass spectrometry.

9. The analytical method according to claim 7 or 8, wherein the compound having the s-cis-diene structure is vitamin D or a vitamin D metabolite.

10. A derivatization reagent for mass spectrometry comprising the crystal described in claim 1 or 2.

11. A derivatization reagent kit for mass spectrometry comprising the crystal described in claim 1 or 2.

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